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BIOACTIVE FOOD

AS DIETARY
INTERVENTIONS
FOR LIVER AND
GASTROINTESTINAL
DISEASE
ACKNOWLEDGMENTS FOR BIOACTIVE FOODS IN
CHRONIC DISEASE STATES
The work of editorial assistant, Bethany L. Stevens and the Oxford-based Elsevier
staff in communicatin with authors, workin with the manuscri!ts and the !ublisher
was critical to the successful com!letion of the book and is much a!!reciated. Their
daily res!onses to "ueries, and collection of manuscri!ts and documents were
extremely hel!ful. #artial su!!ort for $s Stevens% work, raciously !rovided by the
&ational 'ealth (esearch )nstitute as !art of its mission to communicate to scientists
about bioactive foods and dietary su!!lements, was vital
*htt!+,,www.naturalhealthresearch.or-. This was !art of their efforts to educate
scientists and the lay !ublic on the health and economic benefits of nutrients in the
diet as well as su!!lements. $ari Stoddard and .nnabelle &une/ of the .ri/ona
'ealth Sciences library were instrumental in findin the authors and their addresses in
the early staes of the book%s !re!aration.
BIOACTIVE FOOD
AS DIETARY
INTERVENTIONS
FOR LIVER AND
GASTROINTESTINAL
DISEASE
Edited by
RONALD ROSS WATSON AND
VICTOR R. PREEDY
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PREFACE) LIVER AND GASTROINTESTINAL
HEALTH+ L)IE( .&5 H.ST(O)&TEST)&.L 'E.LT'
O!timum functionin of the liver and astrointestinal systems is critical for health.
They are critical for the diestion and absor!tion of nutrients and foods to !roduce
rowth. &utrient and non-nutrients are im!ortant modulators of the liver function. The
sym!-toms related to liver dysfunction include both !hysical sins and sym!toms of
abnormal absor!tion of fat, chanes in blood suar, and altered metabolism. This
book !rovides evidence that foods and their com!ounds can modify some of these
diseases. Ex!ert reviews are !rovided on liver function as !eo!le mature and
mechanisms of fatty liver as modified wild and bioactive foods for he!ato-!rotection
and diestion. The data su!!ortin actions of bioactive, and es!ecially 6hinese foods,
to !revent and treat liver diseases are defined by ex!erts. S!ecific individual foods
and herbs have shown s!ecific liver disease benefits includin+ betal leaf, selected
)ndian herbs, ooseberries, and curcu -min.&on-botanical materials in reviews show
!romise, includin !robiotics. )n definin mechanisms includin antioxidant ca!acity
of antocyanins, extracts of !omeranate and medicinal !lants as well as s!ecifically
their carotenoids show benefits in modifyin liver function in reviews.
#hytochemicals% involvement in liver and astrointestinal health is concisely defined.
$ore diverse information is !rovided about bioactive foods in the thera!y of
astro-intestinal diseases and functions, which are many and im!ortant in health. )n
this book the astrointestinal focuses on the stomach and intestine. )t releases
hormones that hel! re-ulate the diestive !rocess and is subGect to many diseases and
!roblems. .n overview reviews functional assessment of astrointestinal tract
function and alkaline in diestive health. (eviews enerally define the !rotective
effects of bioactive botanical foods. The human microbiome diseases are defined in a
metaenomic a!!roach. S!ecific classes and ty!es of foods are reviewed for selected
astrointestinal diseases. ?or exam!le, a cha!-ter defines the role of milk bacteria in
astrointestinal alleries. Then selected reviews of !rebiotics and !robiotics
documented their value in irritable bowel syndrome, mucosal immunity, and viral
infections. Their lactic acid and its stimulation of folate !roduction are reviewed as
mechanisms of !robiotic astrointestinal health. The actions of non-bioactive fiber on
bowel health are reviewed. Several additional reviews focus on !oly-saccharides from
soy sauce and fiber from a!!les, sources readily available to the !ublic. 5ietary fibers
and cholelithiasis are shown to be im!ortant in li!id lowerin. S!ecific small
molecules and defined substances are im!ortant in astrointestinal health. Omea 9
fatty acids are shown to be an interestin story of biotechnoloy leadin to health.
One review describes fatty acids in inflammatory bowel diseases. Black !lum has a
lon research history, which is summari/ed on its !hytochemicals in health, as do
bioactive !oly!henols on other mucosal diseases of the lun. )ndian !lants have a
historical a!!li-cation to health such as s!ices in treatment of ulcerative colitis.
Hiner and basil are reviewed as an ancient remedy, while another ex!ert ives an
overview of medicinal !lants in astrointestinal diseases. ?inally not all bioactive
materials are safe. Therefore the daners of herbal weiht loss su!!lements and
alcohol on astrointestinal functions are reviewed. Bioactive foods however, as
reviewed, a!!ear to have a role in !reventin the e!idemic on non-communicable
diseases. 6learly bioactive herbs, foods and their extracts can !lay key roles in liver
function and astrointestinal health.
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CHAPTER >
Th( A,ai$( Way i$ Di"(sti+( H(ath
R. 5a''(
'ealth Studies 6olleium, .shburn, I., 8S.
The biochemical conse"uences of diet are the reatest influence on overall
metabolism for most !atients. ?ood choices clearly affect the course of common
!atho!hysioloical errors such as insulin resistance, metabolic syndrome, and their
se"uella. 'owever, these dynamics can also be considered a leverae !oint M an
o!!ortunity to reverse immune reactivity throuh !ractical interventions that !atients
can im!lement in their daily lives.
>. DIETARY FACTORS IN METABOLISM
The intestinal tract !lays a key !art in nutrient absor!tion, immune defense aainst
for-ein invaders, !hysioloic re!air from wear and tear, rowth, neurohormone
reulation and stress manaement. 5isorders anywhere in the astrointestinal system
can affect the function of the entire body and overall health. 5iestive com!etence
tends to !redict survival and the ca!acity to thrive years to decades later.
>.>. Pr!'i() M(tab!i& A&i/!sis as a Ma1!r Ca#s( !' Chr!$i&
Dis(as(
Toxin accumulation in the body can result from a diet that !romotes metabolic
acidosis *net acid excess after metabolism- as shown by low levels of bufferin
minerals such as !otassium and manesium. . number of lare research studies
involvin thousands of !artici!ants have re!orted about the association between
metabolic acidosis and insulin resistance *:affe and $ani, 144DE Souto et al., 1422-,
ty!e 1 diabetes *:affe and $ani, 144DE Schul/e et al., 1449-, cardiometabolic risk
*$urakami et al., 144C-, coronary heart disease *Liu et al., 1444-, and osteo!orosis
*:affe and Brown, 1444E :ehle et al., 144D-, as well as cancer *Tavani et al., 1444-. .
ty!ical .merican diet !rovides insufficient minerals and fiber to counter or buffer the
buildu! of metabolic acids and to hel! dis!lacement of toxic wastes. .s a result,
alkaline cellular reserves within the body reduce and de!lete as the intracellular
environment becomes !roressively acidic, mineral de!leted and !roton rich *Lim,
144<E Neidel and Seifter, 23CD-.
1.1.1. Associated signs and symptoms
The sym!toms associated with metabolic acidosis include malaise and fatiue,
metabolic syndrome and diabetes, osteo!enia and osteo!orosis, and de!ression.
$etabolic acidosis is associated with a broad rane of clinical conditions in the body
because of the biochemical reduction of the !roton radient, u!on which cell enery
de!ends. The ratio of .T#+ .5# is a measure of cell enery. . ratio of 244+2 is
healthy. . ratio less than C4 beins to shift cells from an elective !rotective,
!roactive, and !revention mode to a sur-vival mode.
2.2.2.2. ?atiue
Low enery is the maGor com!laint that !atients re!ort to their !rimary care
!hysician. Enery !roduction and the ability to remove toxins safely are com!romised
when even minor increases in acidity occur. $etabolic acidosis has also been linked
to chronic fa-tiue immune dysfunction syndrome *:affe and Brown, 1444-.
?ibromyalia and chronic muscle !ain that is unres!onsive to !ain medication have
been documented to !roduce acidic end !roducts that directly irritate and inflame
nerve muscle end !lates *5euster and :affe, 233C-. ;e observe restoration of vitality
and "uality of life when metabolic acidosis is corrected com!rehensively usin
!redictive tests com!ared to best outcome reference ranes thus incor!oratin
!ersonali/ed biochemical individuality into !rimary care.
2.2.2.1. Osteo!enia and osteo!orosis
Excess acid within the cells is also a key factor in osteo!orosis *$aurer et al.,
1449-. One of the best exam!les of this metabolic sensitivity is the influence of acidM
alkali balance on skeletal structure, health, and interity. Skeletal muscles are the
larest storehouse of available minerals in the body and are thus ex"uisitely sensitive
to small chanes in !'. Even a 24R reduction in !' increases osteoclastic activity
while inhibitin osteo-blastic function, inducin am!lified bone mineral loss *:ehle et
al., 144D-. ?or the !ast 14 years, we have consistently observed 1M24R new bone
rowth confirmed by 5EO. scores after Gust 1 years.
1.1.2. Relevant evaluations
One of the most useful assessments in the manaement of metabolic acidosis is
self-testin for !', which can be !erformed sim!ly by the !atient in their home. .fter
D h of rest, we find the urine !' is e"uilibrated with the urinary tract cells. 6ostin
!ennies !er day, this is a useful self-care test that motivates better com!liance with
healthier choices. .nother assessment involves laboratory testin for reactive food
antiens. )n tandem, these tests can be !ivotal in correctin metabolic acidosis and
re!air deficits often called inflamma-tion and their myriad se"uellae.
2.2.1.2. Self-evaluation+ Testin for !'
The ha/ard of metabolic acidosis is that it re"uires additional minerals to buffer
and remove excess acids from the body, stri!!in out needed minerals with !otential
harm to the kidneys and urinary tract. The role of metabolic acidosis in chronic
kidney disease has been extensively documented *Sahni et al., 1424-.
Fi"#r( >.> #icture of !' stri!s.
Fi"#r( >.? )nter!retation of first, mornin-urine measurements.
. !' assessment of the first mornin urine !rovides a clinically useful measure of
met-abolic acidosis risk. The urine !' is a !redictive indicator of the body%s mineral
reserves, as well as acid,alkaline status *;hitin and Bell, 1441-. Ty!ically !'
balance is restored durin slee! and rest when excess acids are excreted *Shafiee et
al., 1441-. This ca!acity varies widely based on the s!ecific toxic load and the
individual%s ability to make enery, deactivate toxins, and excrete those toxins as
re!orted by Ba/hin *144<- *see ?iure 2.2, !' stri!s and ?iure 2.1, reference rane
for urine measurement-.
. value of <.4 indicates a neutral state, a balance of acid, and alkaline elements.
The first mornin urine !' oal of D.0M<.0 shows healthy mineral balance. &eutral or
low-level acid excess reflected in lower !' values indicates that metabolic chemistry
is a!!ro!riately alkaline and that the small amounts of metabolic acids built u! from
daily metabolism have been easily concentrated and excreted. 6ell cyto!lasm or Scell
Guice% functions in an ex"uisitely narrow, slihtly alkaline o!timum functional !'
rane *5e =oun, 2337E Neidel and Seifter, 23CD-.
@N*BERINGALaboratory evaluation+ (educin immune reactivity
)mmune res!onses directly and indirectly enerate substantial amounts of acidic
!roducts. ?or the at-risk individual with im!aired dietary bufferin ca!acity, it is
es!ecially im-!ortant to avoid immune reactions due to antien reactivity or other
causes that can contribute to additional cell acidity in the system *:affe et al., 144D-. .
lym!hocyte re-s!onse assay *L(.- can identify delayed alleric reactivity.
Substitution of immune re-active substances lowers acid loads.
>.>.B Clinical interventions: the alkaline way @ALIGNMENTA
(eduction of hy!eracidity in the body can be achieved throuh a nutrient-rich alkaline
diet, tareted su!!lementation with alkaline nutrients, and the inclusion of buffered
fats.
2.2.9.2 .lkaline diet
The .lkaline ;ay diet is a health-!romotin, fiber-rich diet that consists !rimarily
of whole foods based on individual food tolerances and sensitivities. #reference is
iven to locally, vine-ri!ened, oranic, or biodynamic sources of foods. $ineral-rich
water is the !referred beverae. (educin the net excess cell acidity su!!orts a rane
of health benefits.
>.>.B.>.> E$ha$&i$" i..#$( /('($s(s .lkalini/in foods im!rove immune de-
fense and re!air functions *Lee and Shen, 144C- by reducin host hos!itality to
chronic infections. This reduced infectious challene results in lower levels of
inflammation, more resources for anticancer surveillance, and enhanced re!air
ca!acity. 6linical strat-eies that accom!any an alkaline diet include a rotation or a
substitution diet to reduce ex!osure to reactive foods cou!led with health-!romotin
food choices, fresh fruits and veetables, !ulses and rasses, whole rains, minimal
animal !rotein, and a !roram of individuali/ed nutritional su!!lements to fully meet
biochemical needs.
>.>.B.>.? B#''(ri$" &(#ar &h(.istry@FONT SICEA .
metabolically alkaline diet means that food has a bufferin or cell acid neutrali/in
effect on in vivo cellular chemistry, in vivo *Budde and 6renshaw, 1449-. The effects
of s!ecific food res!onses within the body can differ from that food%s test tube
chemistry *Honick et al., 23DC-. ?or exam!le, citrus fruits are alkalini/in in the body
because citrate, malate, succinate, and fumarate all !ro-mote the eneration of more
than twice as much bicarbonate as the acid contributed from the total amount of food
metaboli/ed *Brown and Trivieri, 144D-. This means that citrus fruits and similar
foods are acidic in a test tube environment, yet alkaline formin in the body.
?iure 2.9 reflects this real-time !ers!ective on metabolism M assessin nutrition for
in vivo efficacy rather than merely evaluatin the ash residue of the food as has been
historically !erformed in nutrient assays. The foods listed here are cateori/ed based
Fi"#r( >.B ?ood and chemical effects on acidic,alkaline body chemical balance
on an em!irical formula calculated from the actual com!osition of the foods% total
!ro-tein, fat, carbohydrates, minerals, cofactors, and fiber contents *:affe, 23C<-.
2.2.9.1 .lkaline nutrients
. diet hih in acidic foods tends to be less-nutrient-dense and fiber-rich than an
alkaline formin, whole foods, immune tolerant diet. Once mineral de!letion occurs,
cells be-come !roressively more acidic and less eneretic. The cell cyto!lasm !roton
radient is re"uired for the cellular !ower centers, mitochondria, to work effectively.
;hen the cell becomes acidic, the !roton radient is reduced and cells become
de!endent on anaerobic TsurvivalU metabolism. This is a less efficient form of enery
!roduction. Lower enery !roduction shifts cells into minimal function survival mode
until ade"uate mineral buffers are restored.
>.>.B.?.> B#''(ri$" .i$(ras $inerals are re"uired to activate en/yme catalysts
within cellsE lack of s!ecific minerals has been linked to numerous s!ecific ty!es of
en/yme deficits. Su!!lementation at maintenance levels includes a healthy balance of
cal-cium and manesium, as well as co!!er and /inc, and all of the divalent cations
that !er-form essential bufferin minerals needed for healthy function. These minerals
are re"uired su!!lements for individuals sufferin from metabolic acidosis *also
known as net acid excess- because buffered minerals neutrali/e metabolic acids to
maintain healthy !' homeostasis inside the cell.
>.>.B.?.? B#''(ri$" 'ats Short-chain and medium-chain fatty acids with less
than 2D carbons such as octanoate and decanoate are alkalini/in. ?ound in !alm
kernel oil, coconut oil, and hee *clarified butter-, these short and medium chain fatty
acids can acce!t acetate molecules.
1.1.4 Individual essential nutritional supplementation
.dditional functional strateies in clinical manaement include the reduction of
oxida-tive stress, su!!ort of detoxification !rocesses *throuh healthy methylation-,
and reduc-tion of risks such as homocysteine. ;e find a healthier, least risk oal
value for homocysteine to be VD mol,l. ;ith a combined 14-fold risk difference
between a ho-mocysteine of V1D versus VD mol,l, healthier homocysteine levels are
a maGor clinical o!!ortunity.
2.2.7.2 .ntioxidants+ .scorbate to /inc
.scorbates are the !rinci!al antioxidants in eukaryotic cells. .s one of but three
s!ecies that are unable to convert lucose to ascorbate, !eo!le are vulnerable to
chronic as well as acute scurvy. .scorbates uni"uely set the cell redox
electrochemical !otential. .scorbates recycle and reenerate vitamins E, taurine,
lutathione, al!ha li!oic acid and can even salvae mitochondrial cytochromes.
6umulative antioxidant deficits become re!air deficits observed clinically as
inflammation, in turn is associated with metabolic acidosis. .ntioxidant su!!lements
are !rovided to !rotect aainst oxidative damae, restore cell enery !roduction,
rehabilitate mitochondria, and reset homeostatic mechanisms *:affe and Brown,
1444-. ;e suest a functional !ersonali/ed assessment of ascorbate need
*www.#E(W8E.com-.
2.2.7.1 B-com!lex vitamins to su!!ort methylation
)m!aired methylation is commonly reflected in elevations in homocysteine above
the healthy value of VD Xmol l
-2
. #roblems with cell communication, detoxification,
and trans!ort result from such im!aired methylation. This reframes these common
states in !hysioloic rather than !atholoic terms, and offers interative a!!roaches to
care as evidence-based o!tions to be included as first-line com!rehensive care.
'ealthy alkaline cell balance is clinically assessed throuh first, mornin-urine !'
measurements. .n increase in alkali-formin foods and su!!lements is recommended
in !ro!ortion to individual need to reach the healthy oal rane of D.0M<.0 for first,
mornin-urine !'. 'ealthy methylation and detoxification is reflected in a !lasma
homocysteine of less than D m dl
-2
. Other measures of detoxification such as
lucarate, merca!turate, and hi!!urate urine excretion are discussed elsewhere *:affe,
144D-. . slow, steady u!take of lucose by manain lycemic load is another
im!ortant as!ect of this a!!roach.
?. GLYCEMIC LOAD AS A TOOL FOR BETTER DIGESTIVE
AND CARDIOVASC*LAR MANAGEMENT
Sim!le carbohydrates are easily taken u! and cause a ra!id rise of lucose in the
blood-stream, which re"uires the release of insulin to return blood suar levels to a
safer level. Onoin stress on the lucoseMinsulinMenery reulatory system
fre"uently leads to hih insulin levels of less functional insulin. 6hronically elevated
insulin is associated with a series of se"uella with adverse lon-term health effects. )t
is increasinly a!!arent that !roress in treatin chronic illness re"uires effective
manaement of food lucoseMinsulin interaction. ;e suest the .lkaline ;ay to
im!rove insulin sensitivity throuh cor-rected metabolic acidosis, antioxidant, and
mineral deficits and enhanced toxin removal.
?.>. Ass!&iat(/ Si"$s a$/ Sy.3t!.s
The consum!tion of a hih suar, low fiber diet invites the continuum of weiht
ain, obesity, metabolic syndrome and diabetes. .ssociated risks include !oor lucose
manae-ment, with effects such as li!oenesis, loss of insulin sensitivity,
develo!ment of insulin resistance, and a wide rane of cardiovascular and systemic
conse"uences.
Fi"#r( >.D 6om!arison of lycemic res!onse between 244R native whey meal and
standard 04 carbohydrate load from bread and Gam.
?.?. S('%(+a#ati!$
Two tools useful in lycemic manaement by !atients include the lycemic index
and calculation of lycemic load.
2.2.1. Glycemic inde: !lder and less use"ul
The lycemic index measures the effects of carbohydrates on blood suar levels
*.tkinson et al., 144C-. . measure of 244 on the index reflects the ty!ical metabolic
res!onse to white suar *based on research-determined norms-. ?oods rated 00 or
below on the index are identified as healthful because they re"uire lower levels of
insulin, defined as Sinsulin-s!arin% *?oster-#owell et al., 1441-. ?ructose, certain
!rocessed foods as well as the si/e, com!lexity and constituents of a meal, can
!rovide conflictin lycemic index results.
2.2.2. Glycemic load: #ewer and more use"ul
The lycemic load is a better measure of the im!act of carbohydrate consum!tion.
)t takes the lycemic index into account, but !rovides a fuller !icture than the index
alone *$urakami et al., 144<-. Hlycemic load indicates how ra!idly a s!ecific
carbohydrate food raises blood suar and factors in the actual amount of the !articular
carbohydrate bein consumed *see ?iure 2.7 for an ideal lycemic res!onse to a low
lycemic load meal that includes 244R whey-. This is evident since the ex!erimental
meal was 1.0 times the basic carbohydrate load and still showed a small chane in
blood lucose. The contrast with the blood lucose chane with 04 carbohydrate
*bread B Gam- was dramatic.
This cha!ter focuses on cellular metabolic acidosis and lycemic load as key
clinical as!ects of the .lkaline ;ay. )mmune tolerance and delayed alleries are also
discussed.
?.B. I$t(r+($ti!$) L!0 t! M!/(rat( Gy&(.i& Di(t
Hlycemic manaement involves the reduction of refined food !roducts in the diet
M foods that trier the release of hih levels of insulin, resultin in li!oenesis and
weiht ain. By definition, these foods are hih on the lycemic index and have a
hih lycemic load *:enkins 1447E (iccardi et al., 144C-. ?oods containin a lare
!ro!ortion of sim!le carbo-hydrates include white flour and other refined rains,
white rice, white !otatoes, and corn, as well as cane suar, corn syru!, fructose,
honey, ma!le syru!, and other sweet-eners. The o!timal diet is hih in fresh fruits and
veetables, whole rains, and !rotein *5e &atale et al., 1443E :enkins, 1447E (iccardi
et al., 144C-. &ote that this is essentially a more alkaline diet, so that the two systems
can be used in tandem by both clinicians and consumers. ?or other reasons, we
suest a minimum non-caloric sweeteners.
B NATIVE WHEY%BASED MEALS AND
GASTROINTESTINAL HEALTH
(esearch suests that hih amounts of animal !rotein can cause undesirable
astrointes-tinal issues. Excessive amounts of !rotein from meat and fish have the
!otential to in-crease disease risk as hih as threefold in the develo!ment of
inflammatory bowel disease and ulcerative colitis *:antchou et al., 1424-. . lare
)talian study of more than 24444 individuals demonstrated a sinificant association
between meat intake and the develo!ment of cancer *Tavani et al., 1444-.
)n selectin o!timal !rotein sources, a "uality whey !rotein !rovides essential
amino acids, such as lutamine and cysteine, and fatty acids, such as conGuated
linoleic acid *BelobraGdic et al., 1449-. The hih cysteine content of whey has been
found a su!erior means of su!!ortin lutathione levels and, therefore, antioxidant
function, a findin re!orted by Bounous *1444-.
?or healthy lycemic control, a balance of all food rou!s is essential with an
em!hasis on nutrients from fiber-rich whole foods that maintain stable blood lucose
levels.
4 FOOD ALLERGIES AND SENSITIVITIES
)mmune res!onses to s!ecific foods, chemicals, or contaminants can take the form
of classic alleries or of delayed sensitivities. ?or decades, the conventional a!!roach
to allery focused on histamine immunolobulin E or )E reactions, which can trier
a harmless case of hives or life-threatenin ana!hylactic shock. 'owever, it is now
widely reconi/ed that reactivity can be driven by ty!e 2 alleries *ty!ical skin tests
or radioallerosorbent *(.ST- )E reac-tions-, or by ty!e 1 antibody
reaction*).,TH,or )$ reactive antibodies-, ty!e 9immune com!lexes, and ty!e 7+
T-cell mediated res!onses. )n contrast, hel!ful neutrali/in anti-bodies are often
misunderstood when conventional EL)S. or E). )H tests are done.
D.>. Ass!&iat(/ Si"$s a$/ Sy.3t!.s
Eihty !ercent of food reactions are not )E-ty!e reactions M rather, they are
delayed ty!es of reactions. (eactive antibodies can cause sym!toms that become
a!!arent from hours to weeks later, so that they are fre"uently difficult to identify.
Hiven the rane of !otential antiens and the various ty!es of res!onses, a
com!rehensive food assessment is vital. )E tests do not measure delayed reactions.
4.1.1. $he link %etween allergies and digestive competence
?ood intolerance and alleries result from im!aired diestion. .ll of the followin
issues ty!ically develo! before food alleries or intolerances occur+
Maldigestion results in !artially diested diestive remnants that are
reconi/ed as for-ein antiens M thus evokin immune res!onses to neutrali/e
the forein invader.
Infectious and noninfectious foreign antigens (partially digested food) invade
the astrointes-tinal mucosa,are treated e"ually as invasive antiens. This
means that with increased !resence of diestive remnants, the individual has
fewer immune defense and re!air resources to defend aainst infection or
re!air from daily wear and tear. This results in a loss of immune tolerance and
emerence of delayed hy!ersensitivities with a shift from homeostasis to self-
attackin immune res!onses. The .lkaline ;ay restores homeostasis and
tolerance by !ersonali/ed dianostics and includin thera!eutic monitorin to
assess how much of the healthy oal states have been achieved.
Fi"#r( >.E ;heel of immune res!onse mechanisms.
4.1.2 &actose intolerance
;ith this ty!e of functional im!airment, the en/yme that diests milk suar M
lactase M is either not !roduced or available in such low levels that milk cannot be
com!letely diested. This intolerance is almost always ac"uired as a by!roduct of
maldiestion and entero!athy.
4.1.' Gluten or casein intolerance or sensitivity
6asein is one of the maGor !roteins in milk. Hluten is a !rotein from rains like
wheat. Both are amon the most difficult foods to diest. )ntolerance to casein or
luten can occur when diestive ability is im!aired. 8!!er abdominal discomfort and
alternatin consti!ation and diarrhea sometimes accom!any this condition. ?unc-
tional hy!ochlorhydria, maldiestion, dysbiosis, and entero!athy are ty!ically the
underlyin causes.
Hluten intolerance results from maldiestion, food intolerance, or delayed alleric
hy-!ersensitivity. .dvanced ex vivo L(.s can be em!loyed to measure all delayed
allery !athways.
D.B. E+a#ati!$) LRA by ELISAFACT T(sts
Testin for delayed, ac"uired sensitivities focuses on the ty!e of functional
antibody and the s!ecific reactive allerens. Sensitivities reflect the variety of immune
reactions !resent *see ?iure 2.0-. Older technoloies, such as )H antibody assays,
have !roven at best only tem!orarily hel!ful in the treatment of chronic autoimmune
conditions. The tests do not distinuish hel!ful from harmful antibodies. &ewer
functional tests such as L(.s are recommended because they detect only the reactive
antibodies. These antibodies are in-dications of a burdened immune system. L(. lab
tests can measures reactions Gust as they occur in the body *ex vivo-. This ty!e of
functional testin tarets antibodies that cannot be distinuished by standard antibody
detection systems.
.ssessin delayed allery or hy!ersensitivity to foods or other chemicals !rovides
an Simmunoloic finer!rint% of the foods, chemicals, and medications to which the
indi-vidual is tolerant and those which their lym!hocytes react aainst. Tests such as
the L(. by EL)S.,.6T
T$
can sensitively, s!ecifically, and !redictively identify
underlyin causes of immune burdens and increased hos!itality to infection, as well
as accelerate de-enerative, chronic, and autoimmune conditions with less than 9R
variance day to day *:affe and >ruesi, 2331-. These tests have been em!loyed to
identify the e!ienetic bur-dens on a iven individual%s immune system. The causes
of autoimmunity are ty!ically !rovoked by ex!osures to s!ecific foods or chemicals
to which the !erson has ac"uired hy!ersensitivity. Substitutin other food choices for
reactive items and minimi/in chemical ex!osure, while evokin human healin
res!onses is !art of the .lkaline ;ay. ;ith over 04,444 cases in our database, we
suest this technoloy hel!ful in
Tab( >.> $aGor &utrients &oted for Beneficial Effects in 5iestive
'ealth
N#tri($t M(tab!i& '#$&ti!$ Sy.3t!.s !' /('i&i($&y
Vita.i$s
Iitamin ., from mixed
natural carotenoids
includin beta carotene
which re"uires retinol
bindin !rotein and /inc
for trans!ort into cellsE
available as a s!ecific
su!!lement and in cod
liver oil
$aGor factor in the rowth
and healin of the
astrointestinal *H)- tract,
intestinal linin, and other
mucous membranes in the
bodyE !roduction of
!rotective mucus and S).
*the !rimary antibody
!rotectin the H) tract-E
antioxidantE antibacterialE
and antimicrobialE 5&.
synthesis
Beta carotene conversion
is reduced by intestinal
disease, diabetes,
hy!othyroidism, ex!osure
to toxic metals or
chemicals. Sym!toms
include inflammation and
slower healin, rouh skin
on elbows or underside of
forearms, reflux-like
sym!toms
Thiamine *B
2
- Builds red cellsE
antioxidant !rotectionE
mental focus
6onsti!ation and bloatinE
conitive chanes
(iboflavin *B
1
- Enery !roductionE
antioxidant activity
throuh lutathione
!roduction
.ltered iron metabolismE
maenta tonue
&iacin *B
9
- Lowers L5L cholesterol
and trilyceridesE reulates
ene ex!ression
6hronic diarrhea, cracked
li!s, and mental confusion
#antothenic acid *B
0
- .nti-inflammatory,
antiviral !ro!erties, and
wound-healin actionE
iven after abdominal
surery to restart bowel
motility
Sluish motility and
bloatinE headache,
fatiue, insomnia,
com!romised immune
defenses and re!air
#yridoxine *B
D
- .mino acid metabolism
and neurotransmitter
synthesisE reduces
homocysteine
)rritable Bowel Syndrome
*)BS-, hih blood suar,
and !rediabetesE
deficiencies can result in
lactose intolerance,
alleries, or internal
bleedinE incom!lete
conversion of amino acids
to neurotransmitters such
as serotonin, causin
de!ression or confusionE
macrocytic anemiaE reflux-
like sym!tomsE !eri!heral
neuro!athyE !otential liver
damae
Biotin *B
<
- Biosynthesis of fatty acids
and luconeoenesisE
5&. re!licationE
.ccelerated ain, hair
loss, brittle nailsE
accelerated rayinE
im!roves lucose muscle !ain
Tab( >.> $aGor &utrients &oted for Beneficial Effects in 5iestive
'ealthYcontZd
N#tri($t M(tab!i& '#$&ti!$ Sy.3t!.s !' /('i&i($&y
tolerance and decreases
insulin resistance
)nositol *B
C
-
*hexa!hos!hate-
Essential for fat trans!ortE
feeds brain cells
HastritisE alo!ecia
?olate *B
3
and B
22
- M low
folate can be caused by
low levels of activated
thyroid *T
9
-, selenium, and
trace minerals and by
irritation in the u!!er
duodenum due to wheat or
luten sensitivity, low
intake of fruits and
veetables, a enetic
disorder that interferes
with folate absor!tion,
malabsor!tion, or
microsco!ic !arasitesE
restored in !art by
su!!lementin selenium
and folic acidE available in
activated sublinual form
as folinic acid or as
inGectable folateE must be
su!!lemented at the same
time as B
21
E acts in concert
with riboflavin, niacin,
vitamin 6, /inc, and serine
)nstrumental in healin the
H) tractE minimi/es
!eriodontal infectionsE
su!!orts immune functionE
!rotective aainst yeast
and !arasitic infectionsE
strenthens connective
tissueE key in methylation
and moderation of
homocysteine levelsE 5&.
synthesisE su!!ort of T cell
!roduction healin
!rocesses
5eficiency can be caused
by im!aired absor!tion
and leaky ut syndrome.
6an !lay a role in chronic
H) inflammationE bleedin
umsE reflux-like
sym!toms, sinusitis, or
fre"uent colds. Low levels
can result in hih
homocysteine. .nemia
resultin in weakness or
fatiue, de!ression, loss of
a!!etite, and weiht lossE
foretfulness, irritabilityE
sore red tonue. #otential
liver damae.
Betaine, trimethyllycine
*B
24
and B
20
-
Es!ecially !rotective,
lowerin homocysteine by
recyclin it into
methionineE !revents fatty
liverE characteri/ed by
hih trilyceridesE acts as a
diestive aid
#rotein maldiestion and
hih homocysteineE
!otential liver damae
'ydroxocobalamin *B21-
M essential nutrient in
metabolism, absorbed in
the ileum and reduced by
ileitis, celiac disease,
bacterial and !arasitic
infections, chronic )BS,
6rohn%s disease, low
intrinsic factor *enetic
disorder-, or a strict vean
dietE available in meats or
)nstrumental in healin the
H) tract and a source
material for com!etent H)
defensesE key factor in
methylation, lowerin
homocysteine levelsE
essential for en/yme
!roduction and enery
!roduction, 5&.
synthesis, and health of the
nervous system
5eficiency can be caused
by im!aired absor!tion
and leaky ut syndrome.
Sym!toms include
im!aired !rotein diestion,
diestive disorders, hih or
low stomach acid, !e!tic
ulcers, fatiue, or
weakness, bloatin,
diarrhea, or anemia.
.dditional sym!toms+
throuh sublinual numbness, tinlin of
hands and feetE !ernicious
Tab( >.> $aGor &utrients &oted for Beneficial Effects in 5iestive
'ealthYcontZd
N#tri($t M(tab!i& '#$&ti!$ Sy.3t!.s !' /('i&i($&y
su!!lements or inGectionsE
must be su!!lemented at
the same time as folateE
acts in concert with
riboflavin, niacin, vitamin
6, /inc, and serine
anemia linked to
autoimmune conditionsE
smooth tonue, beet redE
!otential liver damaeE
!remature ain and a
form of irreversible
dementia
#ara amino ben/oic acid
*Bx-
$etabolism and utili/ation
of amino acidsE su!!orts
folates !roduction
)rritability, de!ression,
skin !roblems, ec/ema,
headaches, diestive
disorders, and hair
!rematurely ray
6holine as citrate )m!ortant in moderatin
homocysteine levels
5eficiency linked to hih
homocysteineE insomnia,
fatiueE in the extreme,
heart disease, liver and
kidney dysfunction
6holine citrate ;orks with inositol to
balance communication
between cells
)m!aired diestion of fatE
low acetylcholineE reduced
mental clarity
.scorbate *vitamin 6- as
buffered ascorbate
*calcium, manesium,
!otassium, /inc ascorbate-
6ontrollin inflammation
and immune resistance.
6ollaen and structural
!rotein formationE
antioxidantE lutathione
enhancedE
antihy!ertensiveE
detoxifyin aent for
heavy metals
)mmune su!!ressionE
inflamed and bleedin
ums, chronic H) infection
or inflammation,
es!ecially in the ut wallE
leaky ut, includin
bloatin, soreness,
malabsor!tion, toxicity,
delayed astric em!tyin,
and resultin free radical
damae
6holecalciferol *vitamin
5- M hormone adhesion
molecule
.nti-inflammatoryE
reulates calcium
homeostasis and
absor!tion, com!romisin
function of lands,
im!airin diestionE
su!!orts conitive healthE
vital in !roduction of
diestive en/ymes
)ncreased vulnerability to
infectionE im!aired mineral
metabolism, !articularly
calciumE com!romised
diestionE !oor blood
suar reulationE altered
bone and Goint healthE
diabetic conditions
Toco!herols and
tocotrienols *vitamins E-
#rimary backu! when
cellular antioxidants are
com!romised due to
inflammationE !rotects cell
membrane from oxidative
?at malabsor!tionE vitamin
E deficiency anemia in
infantsE !eri!heral
neuro!athy
damaeE antiatheroenic
and neuro!rotective
Su!!orts a!!ro!riate blood
clottin
5eficiencies rare, exce!t in
the case of heavy antibiotic
Tab( >.> $aGor &utrients &oted for Beneficial Effects in 5iestive
'ealthYcontZd
N#tri($t M(tab!i& '#$&ti!$ Sy.3t!.s !' /('i&i($&y
Iitamin >, fat-soluble
nutrients !roduced in the
lare intestine
usae, malabsor!tion, or
maGor damae to the
intestines
C!'a&t!rs
6oen/yme W
24
.ntioxidantE
cardio!rotectiveE
neuro!rotective
#eriodontal diseaseE
im!aired .T# enery
!roductionE statin
medication ty!ically
reduces 6oW
24
by 04R.
Fatty A&i/s
Omea 9 essential fatty
acids, E#.,5'. M
available in fish oil and in
those able to convert it, in
flax oil
(esistance to infectionE
anti-inflammation effectsE
su!!orts cell hydration and
functionality, !reventin
brittle cell wallsE reulates
calcium metabolismE turns
on inflammatory and anti-
inflammatory defenses at
the cellular level throuh
!rostalandinsE lower
trilycerides. E#. has
antithrombotic effectsE
5'. is vital for normal
brain develo!ment and
function
)ncreased susce!tibility to
infection and
inflammationE increased
alleriesE sym!toms from
dry skin, rashes, and hair
loss to incontinenceE
deficiencies can result in
either consti!ation or
diarrheaE a factor in
inflamed nerves and
neuroloical disorders
Omea Ds M a source of
linoleic acid, which is the
basis for amma linoleic
acid
(eulate metabolismE
!romote the rowth of skin
and hairE !lay a role in
brain function
5eficiency M increased
susce!tibility to infectionE
thinnin hair and dry skin,
dandruff or ec/emaE !oor
concentration, learnin
disabilities
Excess M contribute to
inflammatory conditions
due to the buildu! of
arachidonic acid
Short-chain fatty acids
such as butyrate M found in
butter and creamE also
made from starches and
!roteins by diestive flora
The !referred fuel for cells
in the colon linin and
maGor factor in diestive
health
)ncreased risk of chronic
colitis and eventually
cancer
H.B. 6alms and stabili/es
nervous system
Loss of mental clarityE
more aberrant thouht
Mi$(ras
6alcium M as the !rinci!le
mineral in the teeth,
essential
Essential to muscle rowth
and function, includin
heart
5eficiency M unformed
stools, increased risk of
colon cancerE
Tab( >.> $aGor &utrients &oted for Beneficial Effects in 5iestive
'ealthYcontZd
N#tri($t M(tab!i& '#$&ti!$ Sy.3t!.s !' /('i&i($&y
to mastication in order to
!re!are food for diestion
beatE transmission of nerve
im!ulses
weakened teeth and bones
Excess M muscle and
abdominal !ainE kidney
stones
6o!!er M trace mineral (educes hy!eracidityE
re"uired in various en/yme
!rocessesE assists in the
absor!tion of ironE
intracellular antioxidant
6an become deficient with
diet hih in sweets,
resultin in hy!eracidity,
indiestion and dys!e!siaE
astritis or ulcers
)ron M mineral (e"uired for the
!roduction of red blood
cells and the trans!ort of
oxyen to cellE resistance
to infectionE enery
!roduction
5eficiency Manemia
Excess M consti!ation,
abdominal cram!s, black
stools
$anesium M mineral Essential to a!!ro!riate
motility and in some cases,
an effective, inex!ensive
solution to chronic
consti!ation
6onsti!ationE reuritation
due to constriction of
lower eso!haus
$ananese M trace mineral 8seful in the treatment of
diestive disordersE
antioxidantE cofactor in
en/yme, lucose, and li!id
metabolism
5eficiency M a factor in the
develo!ment of diabetesE
the develo!ment of
myasthenia ravis *muscle
weakness- Excess M
hy!ertensionE sym!toms
resemblin #arkinson%s
Selenium as
selenomethionine M trace
minerals found in whole
rain breads and cereals
(educes hy!eracidityE
im!roves immune
resistance and healin of
all kinds of H) !roblemsE
su!!orts !roduction of
both folate and T9E
antioxidant
'y!eracidityE chronic
indiestionE increased risk
of astric cancer
Ninc M trace mineral #aired with vitamin .,
essential to the !roduction
of secretory ). *S).-,
the most !revalent
antibody that !rotects the
utE antioxidant
5eficiency M )ncreased
infection due to low S).
levels
Excess M hy!eracidity,
with indiestion and
dys!e!sia
A.i$! A&i/s
.mino acids M derived
from !rotein breakdownE
!rovide the basis of
Essential for effective
immune defensesE
intracellular antioxidant
To !erform antioxidant
functions, re"uires trace
minerals includin
!rotective antibodies selenium, /inc, co!!er, and
mananese
6arnitine M s!eciali/ed
amino !roduct, one of the
most
&ourish the H) tract and
ut lininE !rovide enery
to trans!ort micronutrients
into
6hronic bowel
irreularityE bloatinE
fatiue
Tab( >.> $aGor &utrients &oted for Beneficial Effects in 5iestive
'ealthYcontZd
N#tri($t M(tab!i& '#$&ti!$ Sy.3t!.s !' /('i&i($&y
!revalent aminos in
diestive tract
the bloodstreamE su!!orts
mitochondria in
conversion of fats into
enery within the cell
6ysteine M amino acid
derived from methionine,
from sulfurous veetables,
and animal !roteins
)ntracellular antioxidant To !erform antioxidant
functions, re"uires trace
minerals includin
selenium, /inc, co!!er, and
mananese
Hlutathione M !e!tide
manufactured from lycine
*a form of lutamine-,
lutamic acid, and cysteine
found in sulfurous
veetables and animal
!rotein
The !rimary antioxidant in
the bodyE a maGor cellular
defenseE essential to
enery !roductionE
detoxification of
carcinoensE buildin
5&. and !roteins
6hronic fatiueE hemolytic
anemiaE metabolic acidosis
Hlycine M metabolic
by!roduct of lutamine,
nucleic acid !recursor
One of the most im!ortant
factors in ut healthE
detoxificationE synthesis of
bile acids, and of
lutathioneE construction
of (&. and 5&.
5eficiency M !oor enery
!roduction Excess M
fatiue #eo!le with kidney
or liver disease should not
consume
L-lutamine M the amino
acid most !revalent in the
diestive tractE recycled by
#yridoxal .l!ha
>etolutarate *#.>-
&ourish the diestive tract
and ut linin, which
enhances nourishment
throuhout the body
6hronic bowel
irreularityE bloatinE
fatiue
$ethionine M amino acid
derived from sulfurous
veetables and animal
!roteins, which is
converted to cysteine, a
!recursor of lutathione
#rotein synthesisE
methylation *a !rimary
form of
detoxificationwhich occurs
in every cell of the body-E
essential in the !roduction
of adrenaline and creatine,
as well as choline and
carnitine
Excess M ;ith the intake
of animal !rotein,
methionine is converted
into excess homocysteine.
6hronic levels of
homocysteine increase the
risk for all maGor
deenerative diseases
Taurine .ntioxidant, role in
detoxificationE im!roves
insulin resistance by
increasin the excretion of
cholesterol
'ih homocysteineE
reduced bile acidsE
allstones, abdominal !ain,
intolerance of fat
Hlucosamine and other
suar aminos M !recursor
in the synthesis of
lycosylated !roteins and
li!ids
Enhances defenses by
bindin to and dis!osin
of undesirable microbes
5eficiency occurs with
ainE dietary sources
often inade"uate
#robiotics and related
factors ?iber M ideally C4R
soluble fiber and 14R
insoluble fiber
Lower carbohydrate
content to food, and
therefore, lower weiht
ainE cleansin
Slowed stool motility
Tab( >.> $aGor &utrients &oted for Beneficial Effects in 5iestive
'ealthYcontZd
N#tri($t M(tab!i& '#$&ti!$ Sy.3t!.s !' /('i&i($&y
functionE !revention of
!athoen adherence to the
ut wallE im!roved
cholesterol levelsE vitamin
activationE im!roved
eliminationE toxin
reduction
#rebiotics M fibers, soluble
and insoluble, ?OS
*fructooliosaccharides-,
non absorbable
carbohydrates
Su!!ort the rowth of
flora and lactic acidE
!romote beneficial
fermentation, reducin
ammonia by!roducts
Beneficial when sins of
ammonia intoxication are
!resent, such as headache
and fatiueE ?OS not well
tolerated by those with
sensitivities or are lactose
or carbohydrate
intolerance
#robiotics M beneficial
flora
#roduction of nutrients
such as butyrateE
!rotection aainst
!athoensE !roduction of
diestive en/ymesE
reduction of li!id levelsE
metabolism of nutrients
such as folate
Source+ Haby .. &utritional $edicine. 'endler, S.S., (orvik, 5.$., 1444. #5(
for &utritional Su!!lements, second ed. Thomson (euters, &ew =orkE :affe, (., 1424.
The .lkaline ;ay+ )nterative manaement of autoimmune conditions. Townsend
Letter for 5octors and #atients. &ovember, !!. 77M02E Shils, $.E., Shils, $., (oss,
..6., 6aballero, B., 6ousins, (.:., 1440. $odern &utrition in 'ealth and 5isease,
tenth ed. Li!!incott ;illiams and ;ilkins, #hiladel!hia, #..
all conditions where immune tolerance is lost. This includes all autoimmune and most
chronic, deenerative ills.
D.B. I$t(r+($ti!$) Hy3!a(r"($i& Di(t
By avoidin offendin food and chemical substances, followin an alkalini/in
diet, and utili/in tareted su!!lementation, the body can stimulate healin and
induce re!air. This conce!t has been successfully tested in controlled outcome studies
on diabetes *:affe et al., 1447, 144D-, as well as fibromyalia and chronic fatiue
syndrome *5euster and :affe, 233C-. 6linical data indicate that other autoimmune
conditions re-s!ond to this a!!roach as well. The .kaline ;ay includes the
identification of acute and delayed food and chemical alleries and intolerances. This
allows for a diet that can be diested, assimilated, and eliminated efficiently, while
enhancin the individual%s vitality and homeostatic com!etence. $ental and !hysical
exercises are as im!ortant for astrointestinal health as they are for any oran system.
. hih-nutrient, low-toxicity diet in the context of a lifestyle of mindfulness and
ratitude rounds out the .lkaline ;ay to sustainable health.
E. THE ROLE OF SPECIFIC N*TRIENTS IN DIGESTIVE
HEALTH
;hen chronic diestive disorders are !resent, the use of nutritional
su!!lementation can im!rove clinical outcome. Thousands of research studies
evaluatin nutritional su!!le-ments have shown the benefits of s!ecific nutrients in
treatin a wide rane of com!lex conditions. The followin table !rovides a brief
overview of maGor nutrients that have been found to !lay an im!ortant role in
diestive disorders. Su!!lementation with stan-dardi/ed inredients makes it !ossible
to !rovide nutrients at a level that will su!!ort ac-tive metabolic function, while
im!rovin com!romised diestion and assimilation based on individual re"uirements
revealed throuh their functional, interative a!!roach *Table 2.2-.
.G CONCL*SION
The combined effects of individuali/ed dietary !rorams, su!!lementation
tareted to the s!ecific needs of the !atient and reduction of allerenic stimulation
offer the !rovider viable tools for conservative and effective clinical manaement of
cardiovascular condi-tions. )n this context, treatment stratey includes a detailed
history of health, lifestyle, and diet. Laboratory assessment, develo!ment of a case-
s!ecific treatment !lan that includes diet and nutritional su!!lements, and a lon-term
!lan to su!!ort !atient motivation and com!liance, reinforced by onoin carin and
com!etence.
REFERENCES
.tkinson, ?.S., ?oster-#owell, >., Brand-$iller, :.6., 144C. )nternational tables of
lycemic index and lycemic load values. 5iabetes 6are 92 *21-, 11C2M11C9.
Ba/hin, &., 144<. #roton radient enery in the catalytic .T# synthesis. (eaction
>inetics and 6atalysis Letters 34, 742M747.
BelobraGdic, 5.#., $c)ntosh, H.'., Owens, :..., 1449. ;hey !roteins !rotect
more than red meat aainst a/oxymethane induced .6? in ;istar rats. 6ancer Letters
23C, 79M02.
Bounous, H., 1444. ;hey !rotein concentrate *;#6- and lutathione modulation
in cancer treatment. .nticancer (esearch 14, 7<C0M7<31.
Brown, S.E., Trivieri :r., L., 144D. The .cid .lkaline ?ood Huide+ . Wuick
(eference to ?oods and Their Effect on !' Levels. S"uare One #ublishers, Harden
6ity #ark, &=.
Budde, (..., 6renshaw, T.5., 1449. 6hronic metabolic acid load induced by
chanes in dietary electrolyte balance increased chloride retention but did not
com!romise bone in rowin swine. :ournal of .nimal Science C2, 23<M14C.
5e &atale, 6., .nnu//i, H., Bo//etto, L., et al., 1443. Effects of a !lant-based
hih-carbohydrate,hih-fiber diet versus hih-monounsaturated fat,low-carbohydrate
diet on !ost!randial li!ids in ty!e 1 diabetic !atients. 5iabetes 6are 91, 12DCM12<9.
5e =oun, L., 2337. $ayo 6linic 5iet $anual+ . 'andbook of &utrition
#ractices, seventh ed. $osby, St. Louis, $O.
5euster, #..., :affe, (., 233C. . novel treatment for fibromyalia im!roves
clinical outcomes in a commu-nity based study. :ournal of $usculoskeletal #ain D,
299M273.
?oster-#owell, >., 'olt, S.'..., Brand-$iller, :.6., 1441. )nternational table of
lycemic index and lycemic load values. .merican :ournal of 6linical &utrition <D,
00M0D.
Honick, '.6., Holdber, H., $ulcare, 5., 23DC. (eexamination of the acid-ash
content of several diets. .merican :ournal of 6linical &utrition 12, C3CM349.
:affe, (., 144D. $anain toxic minerals, biocides, hormone mimics, solvents and
chemical disru!tors. )n+ >ohlstadt, )., *Ed.-, &utrition for $usculoskeletal 'ealth.
$arcel 5ekker,6(6 #ress, &ew =ork *6ha!ter 94-.
:affe, (., Brown, S., 1444. .cidMalkaline balance and its effect on bone health.
)nternational :ournal of )nterative $edicine 1, <M2C.
:affe, (., >ruesi, O., 2331. The biochemical immunoloy window+ a molecular
view of !sychiatric case manaement. :ournal of .!!lied &utrition 77, 1DM79.
:affe, (., $ani, :., 5eIane, :., $ani, '., 144D. Tolerance loss in diabetics+
association with forein antien ex!osure. 5iabetic $edicine 19, 317M310.
:affe, (., $ani, :., Trocki, T., $ehl-$adrona, L., 1447. ?irst line com!rehensive
care+ the diabetes continuum of insulin, lucose, enery dysreulation+ better clinical
manaement of causes im!roves outcomes, reduces risks and vascular com!lications.
Oriinal )nternist 22, 22M1<.
:antchou, #., $orois, S., 6lavel-6ha!elon, ?., Boutron-(uault, $.6., 6arbonnel,
?., 1424. .nimal !rotein intake and risk of inflammatory bowel disease+ the E9&
!ros!ective study. .merican :ournal of Hastroenteroloy 240, 1230M1142.
:ehle, S., Nanetti, .., $user, :., 'ulter, '.&., >ra!f, (., 144D. #artial
neutrali/ation of the acidoenic western diet with !otassium citrate increases bone
mass in !ostmeno!ausal women with osteo!enia. :ournal of the .merican Society of
&e!hroloy 2<, 9129M9111.
:enkins, 5.:., >endall, 6., $archie, .., .uustin, L., 1447. Too much suar, too
much carbohydrate, or Gust too much[ .merican :ournal of 6linical &utrition <3,
<22M<21.
Lee, $.$., Shen, :.$., 144C. 5ietary !atterns usin Traditional 6hinese $edicine
!rinci!les in e!idemi-oloical studies. .sia #acific :ournal of 6linical &utrition 2<
*Su!!l. 2-, <3MC2.
Lim, S., 144<. $etabolic acidosis. .cta $edica )ndonesiana 93, 270M204.
Liu, S., ;illett, ;.6., Stamfer, $.:., et al., 1444. . !ros!ective study of dietary
lycemic load, carbohydrate intake, and risk of coronary heart disease in women.
.merican :ournal of 6linical &utrition <2, 2700M27D2.
$aurer, $., (iesen, ;., $user, :., 'ulter, '.&., >ra!f, (., 1449. &eutrali/ation
of ;estern diet inhibits bone resor!tion inde!endently of > intake and reduces
cortisol secretion in humans. .merican :ournal of #hysioloy. (enal #hysioloy 1C7,
?91M?74.
$urakami, >., Sasaki, S., Okubo, '., et al., 144<. 5ietary fiber intake, dietary
lycemic index and load, and body mass index+ a cross-sectional study of 9392
:a!anese women aed 2CM14 years. Euro!ean :ournal of 6linical &utrition D2, 3CDM
330.
$urakami, >., Sasaki, S., Takahashi, =., 8enishi, >., 144C. :a!an dietetic
students% study for nutrition and biomarkers rou!. .ssociation between dietary acidM
base load and cardiometabolic risk factors in youn :a!anese women. British :ournal
of &utrition 244, D71MD02.
(iccardi, H., (ivellese, ...., Hiacco, (., 144C. (ole of lycemic index and
lycemic load in the healthy state, in !rediabetes, and in diabetes. .merican :ournal
of 6linical &utrition C<, 1D3SM1<7S.
Sahni, I., (osa, (.$., Battle, 5., 1424. #otential benefits of alkali thera!y to
!revent H?( loss+ time for a !alatable Ssolution% for the manaement of 6>5. >idney
)nternational <C, 24D0M24D<.
Schul/e, $.B., $anson, :.E., ;illett, ;.6., 'u, ?.B., 1449. #rocessed meat
intake and incidence of ty!e 1 diabetes in youner and middle-aed women.
5iabetoloia 7D, 27D0M27<9.
Shafiee, $..., >amel, >.S., 'al!erin, $.L..., 1441. 6once!tual a!!roach to the
!atient with metabolic acidosis a!!lication to a !atient with diabetic ketoacidosis.
&e!hron 31 *Su!!l. 2-, 7DM00.
Souto, H., 5ona!etry, 6., 6alvi\o, :., .deva, $.$., 1422. $etabolic acidosis-
induced insulin resistance and cardiovascular risk. $etabolic Syndrome and (elated
5isorders 3 *7-, 17<M109.
Tavani, .., La Iecchia, 6., Halus, S., et al., 1444. (ed meat intake and cancer
risk+ a study in )taly. )nternational :ournal of 6ancer CD, 710M71C.
;hitin, S.:., Bell, :., 1441. ?irst mornin urine measured with !' !a!er stri!s
reflects acid excretion. #roceedins of the .merican Society for Bone and $ineral
(esearch. .merican Society for Bone and $ineral (esearch, ;ashinton, 56.
Neidel, $.L., Silva, #., Seifter, :.L., 23CD. )ntracellular !' reulation and !roton
trans!ort by rabbit renal medullary collectin duct cells. (ole of !lasma membrane
!roton adenosine tri!hos!hatase. :ournal of 6linical )nvestiation << *2-, 229M214.
RELEVANT WEBSITES
htt!+,,www.'ealthstudiescolleium.or
htt!+,,www.EL)S..6T.com
htt!+,,www.#E(W8E.com
htt!+,,www.#E(W8E;heyHuard.com
htt!+,,www.ncbi.nlm.nih.ov,!ubmed
CHAPTER ?
F#$&ti!$a Ass(ss.($t !'
Gastr!i$t(sti$a H(ath
R. 5a''(
'ealth Studies 6olleium, .shburn, I., 8S.
5iestive illness is fre"uently a cause of or dis!osition to maldiestion-induced
autoim-mune conditions, as well as a factor in chronic deenerative disorders such as
cancer and cardiovascular diseases. ;orldwide, more than 2.0 million children die
each year from diarrheal diseases *8&)6E?,;'O, 1443-.
)n the 8nited >indom 5e!artment of 'ealth, diestive disorders affect one of
every three !eo!le and are associated with one of every four sureries *5o',
1441,1449-. )n the 8nited States, diestive health issues affect D4M<4 million !eo!le
at a direct annual cost of ]3<.C billion *Everhart, 144C-. The association between
diestive disorders and health issues is reflected in conditions such as acute and
delayed alleries, insulin resis-tance, and metabolic syndrome, as well as diabetes, an
avoidable, costly, too common conse"uence. ?urthermore, ut malfunction and
!athoen overrowth are the common underlyin factors in numerous other chronic
conditions, as discussed below.
>. PHYSIOLOGY OF DIGESTION
5iestion is a series of so!histicated metabolic !rocesses that convert !lant
carbohydrates, !roteins, fats, and other nutrients into buildin blocks that the body
can utili/e for nour-ishment, rowth, and re!air when toxin load and stress hormones
!ermit. 'ealthy dies-tion !roduces molecular buildin blocks that su!!ort immune
system tolerance and enable !roactive re!air. $ulti!le mechanisms exclude tra! and
neutrali/e larer mole-cules that can be bioactive and sometimes immunoenic. ;ith
cumulative stress, toxin ex!osure and nutritional deficits, maldiestion re!laces
eudiestion. The erosion of di-estive defenses and the shift of immune res!onses
from tolerant to hy!er-reactive is a molecular ex!ression of what !eo!le feel when
they are chronically unwell althouh somewhat functional. )n the 23D4s, my
astroenteroloy !rofessors were alarmed at the ra!idly risin e!idemic of e!idemics
related to the many ex!ressions and comorbid-ities of maldiestion. 5iestive
metabolism involves chemical and mechanical functions that break down food so it
can be assimilated, utili/ed, and eliminated efficiently, safely, and effectively.
Essential nutrients released or manufactured by the body durin this !rocess must be
derived in sufficient amounts to meet individual enetic, e!ienetic, and sustainability
needs for the body to row, heal, and function well.
5iestion beins with the initial visual and ustatory contacts with food that tell
the brain and then the body which diestive Guices to secrete. The !rocess of seein
the food to be eaten, tastin it, and smellin the aromas stimulates the release of saliva
containin s!ecific en/ymes such as li!ase *which beins the !rocessin of fats- and
amylase *which o!ens u! and beins breakin down carbohydrates-. This favors the
locavore, Sslow food% a!!roach.
The stomach exerts remarkable com!etence in mechanically churnin diverse
food mixtures. This !rocess breaks u! and acidifies the stomach contents *to create a
mixture termed chyme-, while addin the diestive en/yme !e!sin to the stomach%s
contents. This ex!oses food molecules to en/ymes and hydrochloric acid that hydrate,
cleanse, and !rocess carbohydrates and !rotein-rich foods. The resultin !rediested
chyme, the consistency of oatmeal, is !assed from the stomach to the small intestine.
;hen suf-ficient hydrochloric acid is !resent, biosensors are triered to em!ty
contents into the duodenum. )f the chime is sufficiently acidified, bicarbonate and
!ancreatic diestive en-/ymes are then released. .de"uate stomach acid is essential
for healthy diestion. Block-in stomach acid !roduction dis!oses one to
maldiestion and its !ervasive conse"uences.
5iestion occu!ies D4R of the body%s enery !roduction and its consum!tion is
devoted to diestin food. )f the 14^24 ft of intestines were unfolded to create a flat
surface, the intestinal membrane surface covers an area the si/e of a tennis court, a re-
markable 1044 ft
1
or 1D4 m
1
of surface area.
?. CLINICAL ISS*ES IN DIGESTIVE HEALTH
The issues reviewed in this cha!ter im!act or are comorbidities for numerous
chronic health issues. .!!roximately a third of the astrointestinal *H)- com!laints
seen are truly disablin, whereas 1,9rds are a !art of an underlyin chronic issue. ;e
will also see how livin the .lkaline ;ay_ restores diestive health.
?.>. Pr!'i() Dysbi!sis
'ealthy flora are a maGor, increasinly a!!reciated as!ect of health. The contents
of the healthy human diestive tract ty!ically contain at least 2C44 different s!ecies of
flora that in total number in the trillions. The !resence of infection by a forein
!athoen or the overrowth of any resident s!ecies is termed dysbiosis, which can
result in !oor health and a rane of nondescri!t sym!toms *6ani and 5el/enne, 1424-.
Beneficial microflora minimi/e this ty!e of imbalance. 'ealthy bus in abundance
crowd out bad bus. #ath-oenic oranisms do not ive out their toxins until crowd
sinalin confirms that they are !resent in hih density.
2.1.1. Associated signs and symptoms
)n a healthy human body, there are ty!ically five to seven !ounds of bacteria, of
which more than 30R are anaerobes. .ntibiotic thera!y has been found to destroy
both harmful and beneficial bacteria in the body *6harteris et al., 233C-. ;hen healthy
flora is absent, food decom!osition is slowed or incom!lete, im!airin diestion and
reducin the level of nutrients available for absor!tion. Sym!toms and dianoses
associated with com!ro-mised flora and dysbiosis include diarrhea, consti!ation,
urinary tract infections, irritable bowel syndrome *)BS-, irritable bowel disease *)B5-,
6rohn%s disease, and even diabetes *Iaarala et al., 144C-. 5iestive health !rotects
and !romotes health. 5iestive ill health is a comorbidity in almost all autoimmune,
chronic, and deenerative illnesses.
2.1.2. (tiology
$ulti!le courses of antibiotics favor !athoenic bacterial overrowth *Es!osito et
al., 144<E $aGewski and $c6allum, 144<-, such as Clostridium difficile, and yeast
overrowth, such as the 6andida s!ecies. )n some cases, this !romotes antibiotic-
resistant strains of bac-teria or other !athoens to which the individual is ex!osed and
vulnerable. . diet hih in suars, milk, or meat !roducts can also result in the
overrowth of various bacterial s!ecies with adverse effects on health *:antchou et al.,
1424-. 'arder to diest foods like cow dairy and rains become sources of diestive
intolerance.
1.2.1.2. Sidebar+ initial !robiotic research
)n 234C, &obel !ri/e-winnin scientist Elie $etchnikoff of the #asteur )nstitute in
#aris !rovided the first evidence that microoranisms may be res!onsible for the
health-!romotin effects of fermented milks. .fter observin that Bularian !easants
live to ri!e old aes, $etchnikoff became convinced that their health and lonevity
were linked to the beneficial microbes in the cultured milk they drank co!iously. )n
his book, The Pro-longation of Life, $etchnikoff suests that disease-causin
bacteria were minimi/ed or elim-inated by inestin lare amounts of Bularian kefir
or yourt, which contained beneficial bacteria later identified as Lactobacillus
bulgaricus. These oranisms are members of the bacterial s!ecies Lactobacillus M
bacteria that !roduce lactic acid. ifidobacter and !treptococcus thermophilus are
other maGor beneficial !robiotic oranisms. ;e recommend 74M244 billion !robiotic
oranisms taken daily between fermented foods and bioactive su!!lements.
?.>.B. I$t(r+($ti!$) 3r!bi!ti& s#33(.($tati!$
(estoration of a healthy level of ut microflora hel!s !romote the balance toward
healthy and away from harmful microoranisms.
1.2.9.2 Benefits of microflora
Beneficial microflora !rovide a sur!risinly extensive rane of !rotective
functions in the body. #robiotic oranisms decom!ose food in both the small and
lare intestines to liberate nutrients to be assimilated and utili/ed for enery and
re!air.
?.>.B.>.>. @FONT SICEAPr!/#&ti!$ !' /i"(sti+( ($-y.(s by .i&r!'!ra
#robiotic bacteria normally found in a healthy ut su!!ort the !roduction of essential
en/ymes, which in-creases the availability of nutrients as food is more efficiently and
com!letely broken down *6ha!man et al., 1422-. ?or exam!le, the en/yme lactase,
!roduced by lactic acid bacteria, im!roves diestion, metabolism, and absor!tion of
milk suar *lactose-. 'eyman *1444- found that these bacteria also facilitate the action
of intestinal lactase, im!rovin overall diestion by reducin sym!toms such as
diarrhea.
?.>.B.>.?. R(/#&(/ i3i/ (+(s . variety of studies have shown that healthy !ro-
biotics at ade"uate levels can im!rove overall health, increasin metabolic breakdown
of certain li!id or li!o!hilic substances and reducin toxins, bindin toxins to
!rebiotic fiber that are contained in bile *Iaarala, 144C-. This is im!ortant, for
exam!le, in fat emulsification that occurs in the u!!er area of the small intestine
*duodenum-, where fats are mixed with bile durin diestion. )m!roved li!id
metabolism also reduces the reu!take of cholesterol and fatty acid !roducts, and this
has been associated with a 0M2<R reduction in serum cholesterol after Gust 2 month of
daily consum!tion of viable !robiotic oranisms in the 24M14 billion oranism,day
*colony-formin units, 6?8- rane *:ackson et al., 2333-.
?.>.B.>.B. I$hibiti!$ !' 3ath!"($s One of the maGor beneficial effects of
!robioticsis the su!!ression of harmful microoranisms *?uller and Hibson, 233<-.
;hen the mi-croflora are sinificantly de!leted, there is heihtened risk for intestinal
conditions such as viral astroenteritis *Biller et al., 2330-. (esearch by 6am!ieri and
Hionchetti *2333- suests that when there are sufficient numbers of healthy
!robiotics in the ut, the risk of inflammatory bowel disease,syndrome *)B5,)BS-, is
substantially reduced. 6onditions such as )B5 and )BS, ulcerative colitis, and reional
enteritis *6am!ieri and Hionchetti, 2333- have been reversed in individuals who have
develo!ed these sym!toms throuh the use of !robiotics as !art of com!rehensive
care. (ecent research also re!orts !robiotic mixtures beneficial in the treatment of
diarrhea, ut microbiota modulation, and "elico-bacter pylori infection, as well as
ato!ic disease and res!iratory tract infections *6ha!man et al., 1422-. )n our
ex!erience, an ounce of !rebiotic and !robiotic su!!lementation is worth a !ound of
diestive diseases cures.
1.2.9.1. #robiotic dosae
;hen flora are killed off by takin antibiotics, or from xenotoxins or distress,
beneficial bacteria levels can be restored with !robiotic su!!lements. 6urrent clinical
recommen-dations suest a maintenance intake of 24M04 billion bacteria daily from
a variety of mixed cultures. ;e !refer multi!le strains of human im!lantable
acido!hilus, bifidobac-ter, and healthy !# thermophilus.
?.>.B.?.>. Pr(+($ti+( a33i&ati!$s ;hen one is travelin, under stress, or recov-
erin from illness or disease, or !ost antibiotic consum!tion, the ideal dosae is a
!robiotic culture that contains 14M244 billion viable !robiotic oranisms consumed
daily for 1M9 months to restore diestive com!etence. #roducts are currently available
on the market that !rovide as many as 144 billion count in a sinle dose.
?.>.B.?.?. Th(ra3(#ti& i$t(r+($ti!$s #robiotics are recommended in cases of
bac-terial and yeast infection or overrowth *Hionchetta and 6am!ieri, 1444-. To
address these forms of dysbiosis, many researchers now advocate antibiotic,!robiotic
combina-tions of 14M144 billion 6?8 mixed flora for conditions such as consti!ation,
diarrhea, urinary tract infections, and infective endocarditis *6harteris et al., 233C-.
?.>.B.?.B. M(/i&a 3r!bi!ti&s #robiotics harvested in the lo !hase for o!timum
rowth and viability of 6?8 are recommended. $ulti!le strains, ty!ically containin
nine to ten different strains, are more effective in re!o!ulatin the ut. The level of
su!-!lementation is based on the severity of the !atient condition, their res!onse, and
other factors determined by the !hysician. Ty!ical re!lenishment needs are intakes of
14M244 billion oranisms,day for 1M9 monthsE 0M24 billion !er day for maintenance.
?.?. Pr!'i() Hy3(r3(r.iabiity @L(a,y G#t Sy$/r!.(A
. condition described as intestinal !ermeability or Sleaky ut% results whenever
the linin of the small intestine leaks its contents into the intestinal lym!hatics and
then the blood-stream *Solly et al., 1442-. ;hen the body is under stress or in shock
or nutritional deficit, !ores that line the H) tract o!en wide and release metabolic and
microbial toxins from the ut. These toxins are then !assed on to the liver *6ariello et
al., 1424-, the lym!hatic system, the bloodstream, and the immune system and
distributed throuhout the body and vasculatures. Leakae from the ut can also occur
in conditions such as 6rohn%s dis-ease, with the deterioration of tissue in the intestinal
wall. )ntestinal surfaces are also sus-ce!tible to erosion from mechanical action,
toxins, and the !roducts of !athoenic bacteria.
2.2.1. Associated signs and symptoms
Leaky ut is im!licated in chronic conditions with a broad rane of clinical
sym!toms *Liu et al., 1440-Emany include a direct inflammatory com!onent such as
)BS, or toxic reactions, such as certain ty!es of miraines. Leaky ut has also been
im!licated in both Ty!e 2 and Ty!e 1 diabetes *Secondulfo et al., 2333E Iisser et al.,
1443-.'y!er!ermeability is also im-!licated in skin conditions due to inflammation
*reconi/ed as re!air deficit- and may reflect the intake of foods that induce delayed
alleric reactions !resentin as ec/ema, !soriasis or any other autoimmune condition.
;hen diestion is incom!lete, diestive remnants accumulate in the H) tract and
increase inflammation *re!air deficit-. This cause atro!hy and subse-"uently
entero!athy.
Leaky ut also occurs whenever the body oes into shock in res!onse to inGury,
surery, or severe illness.
'y!er!ermeability can result from any number of insults to the body+
.lcohol abuse
?ood !oisonin, !arasitic infections, bacterial overrowth
?ull rane of H) conditions, includin astritis, colitis, and 6rohn%s disease
Eatin disorders *!articularly anorexia-
Shock, trauma, burns, or surery
6ancer and chemothera!y
6hronic he!atitis, !ancreatitis
&S.)5S and certain other medications
(heumatoid arthritis
Oenotoxins * toxic metals, !ersistent oranic !ollutants, solvents, endocrine
disru!tors-
5istress
;hen the immune system detects oversi/ed food molecules in the bloodstream,
these molecules are tareted as forein Sinvaders%. This results in one or more delayed
immune reaction that release !owerful and !otentially damain cytokines and other
am!lifiers.
2.2.2 Intervention: recycled glutamine supplementation
Leaky ut syndrome and damaed mucosa are usually associated with lutamine
defi-ciency. These conditions have been reversed throuh lutamine su!!lementation
*Byrne et al., 2330-. Hlutamine and butyrate are the !rinci!al fuels that eneri/e the
in-testinal linin cells. Henerally s!eakin, diestion and normal metabolic function
of the intestines are de!endent on ade"uate amounts of lutamine, abundant in health
and conditionally essential with res!ect to stress. The effects of lutamine have also
shown to maintain the interity of the ut barrier structure and decrease intestinal cell
wall dam-ae *;u et al., 144D-.
Throuh the action of lutamine on the kidneys, the body controls !' balance and
eliminates acids. (esearch indicates that lutamine can effectively enhance bowel
func-tion in !eo!le with short bowel syndrome and other H) conditions involvin
extensive intestinal surery, includin trans!lantation *Byrne et al., 2330-. #rovidin
L-lutamine and !yridoxal-al!ha-ketolutarate in combination !rovides clinically an
enhancement of lutamine u!take !resumably throuh recyclin. This a!!roach
allows full lutamine dosin without the risk of lutamate buildu!.
?.B. Pr!'i() A(r"i& R(a&ti!$s as a Ca#s( a$/ E''(&t !'
L(a,y G#t
(esearch and clinical ex!erience indicate that alleries and intestinal
hy!er!ermeability are linked *=amauchi et al., 144D-. )n an ae of increasinly
!ersonali/ed medicine, L(. by EL)S.,.6T tests !rovide insiht into individual
ac"uired delayed or late !hase alleries. 6om!rehensive !rorams have been tested
and found to sinificantly im!rove outcomes *:affe, 233C, 144D-. Either of these
conditions can serve as a cause or an effect. ;hile it may be useful to identify the
initial cause, such as luten sensitivity, in !ractical clinical terms, it is not always
!ossible to determine which factor is the cause and which is
Fi"#r( ?.> Lym!hocyte res!onse assays.
the effect. 6onse"uently, it is enerally advisable to treat both conditions at the same
time.
2.'.1. )yperpermea%ility as a cause o" reactivity
The likelihood of develo!in antien reactivity and food sensitivities is
exce!tionally hih in anyone already ex!eriencin leaky ut from any cause. )n the
case of food reac-tions, C4R of food reactions are not )E-ty!e reactions. (ather, they
are delayed reactions caused by )., )H, or )$. 6onse"uently, a com!rehensive
food assessment is vital, iven the fre"uency of delayed reaction. .n )E screen alone
will not usually !ick u! delayed reactions *?iure 1.2-.
2.'.2. Allergies as a cause o" hyperpermea%ility
6onsum!tion of antienically reactive foods can trier hy!er!ermeability, often
within a matter of minutes. )n addition, it is common for !atients to consume more
than one reactive foods in their daily diet. This causes a constant state of antienic
stimulation and burdens immune res!onses. 6hronic inflammation and immune
reactivity occurs when immune tolerance is lost. Leaky ut has been associated with a
wide rane of chronic disorders, e.., arthritis * Goint and connective tissue disorders-,
asthma, ec/ema, !soriasis, vascular diseases and diabetes, as well as anxiety,
de!ression, learnin disabilities, and some dementias. To manae these conditions
effectively, it is essential to address the relationshi! between delayed food alleries,
nutritional distress, and leaky ut.
?.D. Pr!'i() Ma/i"(sti!$ a$/ E$t(r!3athy
$aldiestion is one of the undera!!reciated causes of illness that is increasinly
common in industrial society. The causes are elusive because of the lon la from
antien ex!osure to sym!tomatic ex!ression.
2.4.1. Comor%idities
#revalent sym!toms of im!aired or incom!lete diestion include weiht
manaement issues, adult failure to thrive, lack of restorative slee!, skin disorders,
and alleries. )m-!aired diestive function can result from any number of functional
disorders, includin low levels of essential stomach acid *hy!ochlorhydria-,
insufficient !ancreatic diestive en/ymes, and bile salt deficiency. 5isorders of the
liver, kidneys, and !ancreas can all result from, or contribute to, maldiestion.
2.4.2. Cause and Conse*uences
1.7.1.2. Low en/yme levels
;hen !ancreatic en/yme levels decrease, the cause is usually functional
hy!ochlorhydria. Sym!toms such as bloatin, heartburn, consti!ation, diarrhea,
insomnia, muscle aches, !ain, and skin conditions that occur when the skin is used as
an accessory ouster of ex-cretes. 6ausal factors include an abundance of !rocessed
food in the diet and overuse of medications such as antibiotics and !ainkillers.
En/yme insufficiencies can be caused by enetic conditions or low levels of
!robio-tics, which result in a lack of the en/ymes needed for diestion. Two !otential
solutions include the su!!lementation of !robiotics *described in the section S#rofile+
$aldiestion and Entero!athy%- and en/ymes *5omPnue/-$u\o/ et al., 1440-. ;e
find im!lantable !robiotics, un!rocessed dietary fiber a whole food-based
immunocom!atible diet to re-store diestive and detox com!etence that in turn
restores neuro-hormonal balance of the immune system.
1.7.1.1. #oorly timed astric em!tyin
Early or delayed astric em!tyin are additional sins of incom!lete diestion.
These dis-turbances in the naturally orchestrated !rocesses of diestion com!romise
the nutrition available to the body *see the Section S#rofile+ $aldiestion and
Entero!athy% for a dis-cussion of interventions-.
1.7.1.9. Surical restructurin of the H) tract
Surery can induce maldiestion if !ortions of the lare or small bowel are
removed or if the stomach is reconstructed. )n some cases, these structural chanes
are deliberate, for exam!le, in cases of bariatric weiht loss in which surery is
intended to limit diestion. .ll metabolic manaement a!!roaches should be
!erformed before surery is evaluated.
1.7.1.7. $alabsor!tion
6hronically !oor diestion can lead to malabsor!tion. The individual does not
obtain sufficient nutrients from the diet and therefore ex!eriences health !roblems as
a result. )ncrease in intake of !rebiotics *74M244 ,day- and re!lenishment of
!robiotics *74M 244 billion,day- and essential nutrients for healthy diestion, such as
recycled lutamine, is recommended.
1.7.1.0. Entero!athy
Loss of diestive com!etence can occur as a result of atro!hy, a lack of essential
nutrients, or excess toxins such as heavy metals, biocides, and hormone disru!ters.
(e!air nutrients described above are recommended for a year or two it ty!ically takes
to restore and re-build diestive com!etence after entero!athy.
?.E. Tra$sit Ti.(
The s!eed at which diested food moves throuh the H) tract is described as the
transit time. This time is the interval between food consum!tion and the elimination
of diested waste.
2.+.1. Associated signs and symptoms
. number of factors affect transit time, includin diarrhea, consti!ation, and
metabolic toxicity. Even with different sections of the H) tract, the time re"uired for
food to move throuh the diestive !rocess is sinificantly affected by the
com!osition of the meal !ass-in throuh. ?ats, for exam!le, s!eed u! muscle
contraction and !eristalsis. Transit time is also influenced by factors such as
!sycholoical stress, ender, and re!roductive status *(iccardi and (ivellese, 2332-.
1.0.2.2. 5elayed transit time
The loner the transit time, the reater the !otential for !utrefaction and the
develo!-ment of dysbiosis. ;hen this occurs, unhealthy waste !roducts are fre"uently
reabsorbed and interfere with !ro!er metabolism or with the overrowth of s!ecific
ty!es of bacteria such as "elicobacter pylori$ Clostridium or s!ecies of %scherichia
coli *associated with hih concentrations of meat !roducts in the bowel-. The result is
!redis!osition toward intes-tinal or systemic illnesses or their exacerbation.
Tab( ?.> Transit Time Evaluation+ 6harcoal 5osaes
D!sa"( a&&!r/i$" t! 0(i"ht
V204 lbs D ca!sules
204M144 lbs C ca!sules
144M104 lbs 24 ca!sules
`104 lbs 21 ca!sules
1.0.2.1. (a!id transit time
Iery short transit times may not !rovide ade"uate o!!ortunity to diest and
assimilate the food consumed. Sym!toms and res!onse are always individual and
a!!ro!riate for dis-cussion with a health !rofessional. )t is recommended that transit
time be rechecked twice a month until healthy bowel movements and normali/ed
transit time are achieved.
2.+.2. (valuation: sel",test "or transit time
.lthouh various methods have been suested to track transit time, a sim!le
!rotocol can be used usin charcoal ca!sules. *6harcoal is also sometimes utili/ed for
sym!tomatic relief of intestinal as.- This !rotocol involves takin 2.0MD of
charcoal with C o/. of water on a s!ecific occasion and recordin the time of
consum!tion. 6hoose a hih-"uality brand of activated charcoal ca!sules. ?or the
most accurate results, the ca!sules are inested Gust after a bowel movement. The
ideal dosae is based on body weiht *see Table 1.2-.
1.0.1.2. Observations
The first ste! is to note and record the time at which the charcoal is taken. This
marks the beinnin of the transit-time test. #atients are encouraed to observe the
consistency of their stool and note anythin unusual or different about the "uality,
texture, color, or com!osition of bowel movements.
Transit time test inter!retation
Twelve to eihteen hours is considered a healthy transit time. 8nfortunately,
many .mericans have a 9DM277 h transit time or loner. Slow transit time allows the
!roduc-tion and absor!tion of various toxins !roduced within the body M xenotoxins
that are absorbed from the chyme and stool directly into the bloodstream.
The loner the transit time, the reater the !ossibility that !utrefaction can occur
*with the overrowth of either commensal bacteria or !athoenic s!ecies-, leadin to
unhealthy waste !roducts that are too often reabsorbed and interfere with !ro!er
metab-olism. The result is !redis!osition toward chronic intestinal or systemic illness,
or the am!lification of existin conditions.
On the other hand, very short transit times may not !rovide ade"uate time to
diest and assimilate the food consumed. )t is recommended that the transit time be
rechecked twice a month until a healthy transit time is achieved.
2.+.'. Interventions
)nitial interventions for malada!ted transit time are relatively basic and can be
im!lement-ted by !atients throuh sim!le chanes in lifestyle.
1.0.9.2. 5ietary fiber
The Standard .merican 5iet is fiber deficient, ty!ically includin less than <
day
-2
. Low dietary fiber intake re"uires the body to work harder to !ush waste alon.
One of the best ways to su!!ort an o!timal transit time of 21M2C h is to increase fiber
content in the diet. Hood fiber intake also !rovides considerable benefit to ut health
and con-tributes to a healthy microflora !o!ulation. .dditional benefits of fiber
include
!revention of the develo!ment of !athoens in the intestine and their
adherence to the ut wallE
im!roved blood cholesterol levelsE
im!roved vitamin activationE
better absor!tion and elimination of toxins such as heavy metalsE
enhanced mental clarity and reduced brain foE
lower carbohydrate content and therefore healthier induction of lucose into
the bloodstreamE
reduced body weiht and lower body mass index *$urakami et al., 144<-.
?iber !rovides a cleansin function to swee! !athoens away from the intestinal tract
M fewer !athoens means that fewer immune defenses are re"uired, resultin in lower
levels of inflammation in the body.
On the most basic level, fiber !romotes ood elimination. The ideal oal is intake
of 74M244 of total soluble and insoluble fiber throuhout the day, with a balance of
C4R soluble fiber and 14R insoluble fiber to su!!ort healthy diestion. ?or exam!le,
on a diet that !rovides about 94 fiber daily, <M27 of additional fiber is indicated.
Su!!lemen-tation with 20M94 from multi!le forms of un!rocessed fiber is
recommended, selectin a source that contains no stimulants, artificial sweeteners, or
flavors.
1.0.9.1. Exercise and !hysical activity
6ore body strenth is a function of breath and stretch. ;ith the !ractice of
abdominal breath-in, stress is reduced and core body strenth is enhanced. Twenty
minutes a day of entle stretchin com!lements the breath in maintainin visceral,
core connective tissue, and mus-culoskeletal health. ;alkin as a source of
s!ontaneous, !leasant irreular movement can be enhanced by the !ractice of Traer
movement education, ?eldenkrais techni"ue, .lexander work, or such classic
a!!roaches as hatha yoa. )n the system !referred by the individual, a entle
a!!reciation for im!roved flexibility, resilience, comfort, and tolerance is suested
B. SYSTEMIC INFL*ENCES ON GI HEALTH
Seventy !ercent of the body%s immune system lies alon the diestive tract *the
#eyers !atches housed here are also known as Hut .ssociated Lym!hoid Tissue
*H.LT--. )n a healthy !erson, the food is broken down com!letely and is never
immunoenic. 'owever, malabsor!tion, takes a toll on the immune system. . !oorly
functionin diestive system has lost ability to turn food that is consumed into a form
the body can use. #oor diestion creates the same !redicament as !oor nutrition M a
lack of nutrients to su!!ort immune res!onse and !hysioloic function.
Today, loss of tolerance and homeostasis accounts for an estimated one third of all
chronic disease. =et, research and clinical ex!erience have shown that healin can be
stim-ulated and re!air induced with the !rotocol described here+ identifyin and then
avoidin offendin substances, followin an alkalini/in diet, and individuali/in
su!!lementation. This conce!t has been extensively tested in controlled outcome
studies on insulin resistance and diabetes, in cases of )BS and chronic fatiue
syndrome. 6linical outcome studies su-est that autoimmune conditions res!ond to
this com!rehensive clinical a!!roach over C4R of the time throuh a!!lication of
lower risk, lower cost, safer, and yet more effective !ersonali/ed interative thera!ies
known as The .lkaline ;ay.
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;u, O.W., Shu, L.'., Sun, $., ;an, '., Hao, '., 144D. Effect of lutamine on
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=amauchi, &., Suita, (., $iki, .., et al., 144D. Hastrointestinal candida
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RELEVANT WEBSITES
htt!+,,www.'ealthstudiescolleium.or
htt!+,,www.EL)S..6T.com
htt!+,,www.#E(W8E.com
htt!+,,www.#E(W8E;heyHuard.com
htt!+,,www.ncbi.nlm.nih.ov,!ubmed
CHAPTER B
A$ti!Hi/a$ts i$ I$'a..at!ry B!0(
Dis(as(I *&(rati+( C!itisI a$/ Cr!h$
Dis(as(
H. Asa,#ra
*
I T. Kitah!ra

*
>oukann 6linics, >awasaki, >anaawa, :a!an

)nternational 8niversity of 'ealth and ;elfare, .tami, Shi/uoka, :a!an


.> THE PATHOGENESIS OF *LCERATIVE COLITIS AND
CROHN DISEASE
)ntestinal mucosa has a sinle cell layer of e!ithelial cells that se!arates the ut
lumen harborin the commensal flora and foodborne !athoenic antiens from the
body. &ormal intestinal mucosa has no hy!ersensitivity aainst the commensal flora
because of oral tolerance. Hut-associated lym!hoid tissue !rotects the intestinal
mucosa from the intestinal antiens by !roducin secretory )., transformin rowth
factor *TH?--a, and interleukin *)L--24, which are immuno-su!!ressive cytokines.
8lcerative colitis *86- and 6rohn disease *65- are chronic nons!ecific
inflammatory bowel diseases *)B5s-. The number of !atients sufferin from these
diseases has been in-creasin all over the world, es!ecially in &orth .merica and
;est Euro!e. 86 is a disease mainly involvin the colon and rectum and 65 is a
disease mainly involvin the terminal ileum and,or the colon. #athoenesis of )B5 is
not fully clarified, but the combined association of environmental factors, disease
susce!tibility enes, and dysreulated immune reaction is very im!ortant for the
!athoenesis of )B5.
>.>. Dysr("#at(/ I..#$( R(a&ti!$
'uman and murine studies on 65 have shown an increased ex!ression of T-
hel!er 2 *Th2- cytokines by intestinal lamina !ro!ria lym!hocytes characteri/ed by
excessive !roduction of )L-21,)L-19, interferon *)?&--b and tumor necrosis factor
*T&?--c. T-hel!er cells are thouht to differentiate into Th2 and Th1, and recently
Th2< cells !roducin )L-2<. 86 is an aty!ical Th1 cell involvin Th2< cells. )n these
diseases, !rimary dysreulation of the mucosal immune system leads to excessive
immunoloic res!onses to the normal microflora or chanes in the com!osition of
intestinal micro-flora, and deraned e!ithelial barrier function may elicit !atholoic
res!onses from the normal mucosal immune system *Strober et al., 144<-.
)ntestinal dendritic cells extend their transe!ithelial dendrites into the intestinal
lumen and sam!le intestinal contents for sins of intestinal forein substances.
$acro!haes, the maGor !o!ulation of tissue-resident mononuclear !haocytes, !lay
roles in bacterial rec-onition and elimination as well as in the !olari/ation of innate
and ada!tive immunities. )ntestinal macro!haes !roduce several anti-inflammatory
cytokines like )L-24 and TH?-a and also !roinflammatory cytokines such as T&?-c,
)?&-b, )L-D, and reactive oxyen s!ecies *(OS-. ;hile a small number of 6527
B
cells
are !resent in normal human intestine, these cells are sinificantly increased in the
intestinal mucosa of !atients with 65 and 86. 6527
B
and 6599
B
cells *6599 is a
marker of intestinal macro!hae- !roduced lare amounts of !roinflammatory
cytokines such as )L-21,)L-19!74, )L-19, T&?-c, and )L-D in res!onse to commensal
bacteria stimuli *>amada et al., 1440-. These macro!haes in 65 are derived from
monocytes oriinated from bone marrow. )?&-b in the mucosa of 65 led to abnormal
macro!hae differentiation, resultin in hy!er!ro-duction of )L-19. Hranuloma
consistin of e!ithelioid macro!haes and lym!hocytes in the diestive tract was
found in D4M<4R of !atients with 65.
>.?. E$+ir!$.($ta Fa&t!rs
5iet, intestinal microbes or their com!onents, a!!endectomy, breast feedin,
!ublic sanitation, and early domestic hyiene includin runnin hot water su!!lies
and fixed bath or shower are listed as environmental factors *Timmer, 1449-. 'ih
!revalence of )B5 occurred first in the 8S., 6anada, and ;est Euro!e, and then in
East Euro!e and ?ar Eastern .sia with economical develo!ment. 5ietary animal
meats and fats, and milk !roducts are characteristic of ;esterni/ed foods *.sakura et
al., 144C-. :owett e t al.*1447- re!orted that dietary factors, such as a hih meat or an
alcoholic beverae intake, were identified with an increased likelihood of rela!se for
86 !atients. One of the rea-sons is that hydroen sulfide, which is a bacterially
derived cell !oison and harmful to the colon, is !roduced in the lare intestine from
dietary animal meats and milk.
?ish oils are rich in the lon-chain n-9 !olyunsaturated fatty acids, that is,
eicosa!en-taenoic *E#.- and docosahexaenoic acids *5'.-. Linseed and reen !lant
tissues are rich in the !recursor fatty acid, c-linolenic acid. $ost of veetable oils are
rich in the n-D !olyunsaturated fatty acids linoleic acid, the !recursor of arachidonic
acid. .rachidonic acid-derived eicosanoids *!rostalandin E1- are !roinflammatory.
Therefore, fish oils are considered anti-inflammatory. #roinflammatory cytokine
secretion such as T&?-c, )L-C, and )L-2a by human macro!haes was induced by
!almitic acid, stearic acid, but not by the shorter chain saturated fatty acids. (atio of
d9,dD is im!ortant for !athoenesis of )B5. . Euro!ean !ros!ective cohort study
showed that the hihest "uartile of intake of linoleic acid was associated with an
increased risk of 86 *O(e1.73- *TGonneland et al., 1443-. . statistically sinificant
!rotective odds ratio for the trend across tertiles of !atients with 86 was found for
5'. *O(, 4.79, 30R 6), 4.11M4.CD- and borderline sinificant differences for trends
for total n-9 !olyunsaturated fatty acids *O(, 4.0D, 30R 6), 4.1CM2.29- and E#. *O(,
4.09, 30R 6), 4.1<M2.49-.
5ietary lutamine su!!lement had !ro!hylactic effects on )L-C and T&?-c
!roduc-tion in trinitroben/ene sulfonic acid-induced colitis. 5ietary lycine !revented
chemi-cal-induced ex!erimental colitis in the rats. 'istidine-added normal diets, but
not alanine and lutamine, su!!ressed intestinal inflammation of )L-24-,- cell transfer
colitis models in a dose-de!endent manner *.ndou et al., 1443-. One of the
mechanisms to su!!ress an inflammatory res!onse by histidine will be that dietary
histidine showed anti-inflammatory effects on the !roduction of T&?-c by
macro!haes and monocytes. 'istidine has a ca!acity as a scavener of the hydroxyl
radicals *O'
-
- and inhibited the !roduction of )L-C by intestinal e!ithelial cell lines
treated by T&?-c or oxidative stress. Thus, foods includin amino acids may
influence the !athoenesis of )B5.
The human intestinal microflora is estimated to contain 044M1444 s!ecies and
their !o!ulation is estimated to be 24
29
-24
20
. ;hen mice or rats develo!in
nons!ecific enterocolitis similar to 86 and 65 in the conventional conditions were
raised in the erm-free conditions, their enterocolitis was not induced or reduced in
clinical severity, suestin that intestinal microbes are very im!ortant for the
!athoenesis of )B5 *Schult/ et al., 2333-.
>.B. Dis(as( S#s&(3tibiity G($(s
)B5 is thouht to be com!lex enetic disorders. Henome-wide scans study
showed many susce!tibility enes for 65 includin &O51, )L19(, .TH2DL2,
)(H$, T&?S?20, and so on. 6aucasian 65 !atients had mutations in
6.(520,&O51 *nucleotide-bindin oliomeri/ation domain 1-. 6.(520,&O51
function is thouht to be an antibacterial factor in human intestinal e!ithelial cells and
the intracellular !e!tidolycan rece!tors as a link between intracellular bacterial
sensin and the induction of auto!hay *'isamatsu et al., 1449-. Sinle nucleotide
!olymor!hism of &O51 that activates nuclear factor &?-fB is one of the candidates
for the susce!tibility enes of 65. 6.(520,&O51 is ex!ressed in intestinal
e!ithelial cells and triers human beta-defensin *'B5--1 transcri!tion. )n 65
!atients with a mutation in the &O51 ene, the ileal #aneth cell defensins, '5-1 and
'5-9, which are antimicrobial !e!tide, were diminished.
.uto!hay is an intracellular catabolic !rocess that destroys a cell%s own damaed
!ro-teins and oranelles via the lysosome. )m!aired auto!hay can lead to diseased
states such as ranuloma formation that is fre"uently found in 65. Several sinle
nucleotide !oly-mor!hisms of auto!hae-related enes are found in 65. The
auto!hay-related 2D like 2 *.TH2DL2- !rotein recruited by &O51 to the !lasma
membrane !lays an im!ortant role in the elimination of bacterial invasion *Travassos
et al., 1424-. 5efects of auto!hay reulated by &O51 and .TH2DL2 are thouht to
be im!ortant factors for the !atho-enesis of 65.
?. ROS IN *C AND CD
?.>. R(a&ti+( OHy"($ a$/ Nitr!"($ S3(&i(s
(eactive oxyen and nitroen s!ecies have been im!licated in the !athoenesis of
a variety of acute and chronic inflammatory diseases. Oxyen is converted to reactive
metabolites by reduction in the followin !rocesses+ the res!iratory chain reaction
such as cytochrome c oxidase, the activated !haocytic cells throuh nicotinamide
adenine dinucleotide !hos!hate *&.5#'- oxidase in the membranes of neutro!hils
and macro!haes, and ischemic-re!erfusion !rocesses by which adenosine
tri!hos!hate is converted to hy!oxanthine and xanthine dehydroenase by xanthine
oxidase throuh which the toxic metabolites are !roduced in the !resence of oxyen.
The first oxyen metabolite !roduced is su!eroxide anion *O
-
1
-, by a one-electron
reduction of oxyen. 'ydroen !eroxide *'
1
O
1
-, O'
-
, hy!ochlorous acid *'O6l-,
and water *'
1
O- are subse"uently formed by a chain of reactions. ?ormation of
su!eroxide or '
1
O
1
may be inGurious to tissue directly. )t is also thouht that the
!rimary mediators of tissue dam-ae are the secondarily derived oxidants such as O'
-
and 'O6l. Sources of (OS in the astrointestinal tract include mucosal oxidases such
as xanthine oxidase, amine oxidase, and aldehyde oxidase as well as the &.5#'
oxidase found in the resident !haocytic leukocytes *macro!haes- of the mucosal
lamina !ro!ria. The final mediator of toxicity seems to be the O'
-
derived from the
iron-cataly/ed interaction between su!eroxide and '
1
O
1
*Hrisham and Hraner, 23CC-
*?iure 9.2-.
Su!eroxide dismutase *SO5- converts O
-
1
to '
1
O
1
that is decom!osed to water
either by catalase or lutathione *HS'- !eroxidase. $yelo!eroxidase from the
!haoly-somes in the !resence of 6l
-
ions and '
1
O
1
!roduces the reactive
antimicrobial 'O6l.
Fi"#r( B.> $etabolic !rocess of reactive oxyen s!ecies. SO5, su!eroxide dismutaseE
HS', lutathione.
The most reactive oxyen radical, O'
-
, is formed either s!ontaneously or cataly/ed
by metal ions from the combination of O
1
-
and '
1
O
1
*Babbs, 2331-.
(OS are im!ortant in the normal function of cells. The res!iratory chain kee!s the
cell active, and !haocytic cells use (OS to destroy inested microoranisms.
.rachido-nic acid metabolism, cyclooxyenase, and li!oxyenase systems throuh
oxyen consum!tion are very im!ortant in cellular homeostasis, cell membrane
!rotection, and the immune res!onse. 'owever, excessive !roduction of (OS is
sometimes inGu-rious to cells. The oxyen radicals O
1
-
and O'
-
can induce li!id
!eroxidation and mem-brane destabili/ation, 5&. damae and inactivation of
!roteolytic en/ymes and !rotease inhibitors, resultin in cell death and tissue damae.
?.?. ROS i$ IBD
)n 86, res!iratory burst activities of both !olymor!honuclear leukocytes *#$&L-
and monocytes in the !eri!heral blood were found to be hihly associated with the
disease activities *Suematsu et al., 23C<a-. The sinificant increase in
chemiluminescence activity of !atients with 65 was found only in the monocyte
fraction, but not in the #$&L fraction when com!ared with that of controls. The
chemiluminescence value of mono-cytes in 65 was es!ecially hiher in !atients who
had anal fistula *>itahora et al., 23CC-. 'owever, there was no sinificant chane in
the chemiluminescence values of #$&L from 65 !atients between with and without
anal fistula. #rimin neutro!hils with bacterial li!o!rotein or li!o!olysaccharide
*L#S- dose-de!endently increased the su!er-oxide !roduction in both 86 and
controls. &o differences were found in the su!eroxide !roduction between 86 and
controls. 'owever, the whole blood luminal-enhanced chemiluminescence was
correlated with the 6rohn%s disease activity index *5.)- and 6-reactive !rotein.
.ddition of a/ide, SO5, deferoxamine, and dimethylthiourea resulted in a decrease of
chemiluminescence values.
)nterleukin-C *)L-C- is a !e!tide that induces not only chemotaxis of neutro!hils
but also the release of (OS from the neutro!hils. Levels of )L-C and myelo!eroxidase
*$#O- in oran culture media of bio!sy s!ecimens of colonic mucosa obtained from
!atients with active 86 were sinificantly hiher than those from !atients with
inactive 86 and controls *.ne/aki et al., 233C- *?iure 9.1-. (OS of bio!sy
s!ecimens in active 86 measured by luminol-de!endent chemiluminescence were
markedly increased when com!ared with those in inactive 86 and controls *?iure
9.9-. The levels of )L-C were closely correlated to luminol-de!endent
chemiluminescence and $#O levels.
The !ro!ortion of activated !latelets in the !eri!heral blood was sinificantly in-
creased in )B5 !atients. )n the absence of a !lateletM#$&L interaction, the levels of
(OS !roduction by #$&L did not sinificantly differ between 86 !atients and
normal controls *&6-. .ctivated !latelets from 86 !atients enhanced the amount of
(OS !roduction by indicator #$&L more than those from &6 *Su/uki et al., 1442-.
Fi"#r( B.? Levels of myelo!eroxidase *$#O- and interleukin *)L--C in the colonic
mucosa oran culture media from !atients with ulcerative colitis and controls. &6, normal
controlsE 86, ulcerative colitis.
Fi"#r( B.B Levels of luminol-de!endent chemiluminescence *L-6l- in the colonic mucosa
from !atients with ulcerative colitis and controls. 6ont., controlsE 86 inact, ulcerative colitis
inactive staeE 86 active, ulcerative colitis active stae.
The !eak levels of both !horbol myristate acetate- and o!soni/ed-induced
/ymosan-induced chemiluminescence activities in monocytes were sinificantly
hiher in !atients with 65 than in the controls *>itahora et al., 23CC-. 'owever,
chemiluminescence of #$&L showed no sinificant difference between 65 and the
controls. These data suested that excessive active oxyen s!ecies released by
monocytes and !erha!s macro!haes may !lay an im!ortant role in formation of the
intestinal lesions in 65.
$acro!haes isolated from the intestine of )B5 !atients had been shown to be
more res!onsive to stimulation for O
-
1
!roduction than !eri!heral blood monocytes.
)ntestinal macro!haes from )B5 !atients had an increased s!ontaneous and
stimulated (OS !ro-duction. The inability to efficiently reulate the activation of
tissue macro!haes as well as the recruitment and activation of !haocytic leukocytes
such as neutro!hils, eosino!hils, and monocytes will result in a remarkable increase in
the !roduction of (OS within the tissue throuh activation of the !haocyte-
associated, (OS-!roducin &.5#' oxidase. )n normal conditions, most tissues
!ossess sufficient amounts of !rotective en/ymatic sub-stances such as SO5, catalase,
HS' !eroxidase, and none/ymatic antioxidants such as thiols, ascorbate, and -
toco!herol. These substances will decom!osemost of the inGurious oxidants. 'owever,
levels of these substances were decreased in 86 *>awakami et al.144<-. The
over!roduction of (OS could easily overwhelm the !rotective mechanisms, resultin
in oxidative damae to cells and tissues. The !haocytic leukocytes !lay a role as the
most im!ortant source of inGurious (OS.
)nteraction of !roinflammatory aents such as cytokines, leukotriene B7, !latelet-
activatin factor, com!lement com!onents, and bacterial !roducts on the !haocyte
!lasma membrane results in the activation of the latent membrane-associated &.5#'
oxidase.
There was an increased ex!ression of the oxidase in the intestinal macro!haes
obtained from !atients with 65 when com!ared with healthy control tissues. The
neutro!hils from !atients with 86 could enerate increased levels of su!eroxide when
com!ared with those from healthy controls. Thus, increased su!eroxide !roduction
can attribute to monocytes invadin into the mucosa and the vascular endothelium
!ermeability in !atients with 86.
.ctivation of #$&L and monocytes induces enhanced formation of the !otent
oxidant, 'O6l from $#O-cataly/ed oxidation of 6l
-
by '
1
O
1
. 'O6l !ossesses the
oxidi/in e"uivalents of '
1
O
1
and is more toxic than either O
1
-
or '
1
O
1
. 'O6l is a
chlorinatin and an oxidi/in aent that nons!ecifically reacts with sulfhydryls,
!olyun-saturated fatty acids, 5&., !yridine, nucleotides, and aromatic amino acids.
(OS may mediate e!ithelial and mucosal inGury indirectly by alterin a balance
between the !rotease and anti!rotease that exists within the intestinal tissues.
&eutro!hil-derived oxidants such as 'O6l inactivate !rotease inhibitors. 'uman
neutro!hils use the $#O-'
1
O
1
Mchloride system to enerate chlorinated oxidants that
activate collaenolytic metallo!roteinase. Oxidative inactivation of !rotease inhibitors
with the oxidant-mediated activation of metallo!roteinase induces the intestinal
environment favorable for !roteinase-mediated deradation of the mucosal interstitial
matrix and e!ithelial cells.
)n the inflamed mucosa of colitis, there are lare number of #$&L, monocytes,
and lym!hocytes. The !rimary role of immune res!onse is !rotective and this
!rotective immune system involves not only activation of Th2 cells and the
subse"uent release of their !roinflammatory cytokines, but also activation of intestinal
macro!haes and other !haocytic leukocytes to release additional !roinflammatory
cytokines. These cy-tokines such as T&?-c, )L-2a, )L-D, )L-C, )L-21, )L-2<, )L-11,
and )L-19 as well as (OS and &O are !rimary to !rotect the body from invadin
microoranisms. 'owever, dys-reulatin immune system by which !roduction of
!roinflammatory is excessive and im-munosu!!ressive cytokines such as )L-24 and
TH?-a are deficient is thouht to be a key event involved in the !athoenesis of )B5.
The chronically inflamed intestine is subGected to substantial oxidative stress.
These in-creases in (OS were due to neutro!hil or monocyte-derived oxidants such as
su!eroxide, '
1
O
1
, O'
-
, and hy!ochlorite, because an administration of radical
scaveners *oxy!uri-nol-, antioxidant en/ymes *SO5, catalase-, and en/yme
inhibitors *sodium a/ide- decreased (OS-related chemiluminescence.To detoxify
li!id hydro!eroxides !revents chronic intes-tinal inflammation. The features of
!atients with 86 are a de!letion of endoenous oxidant defense substances such as
ascorbate, a-carotene, c-toco!herol, and HS' as well as the re-ulatory, su!!ressive
T cells *>awakami et al., 144<-.HS' is a naturally occurrin ubi"uitous tri!e!tide *b-
Hlu-6ys-Hly- !resented in hih levels within tissues. &ormal cellular HS' levels are
maintained by de novo synthesis from sulfur-containin !recursor amino acids
*cysteine, methionine- and reeneration via reduction of HS' disulfide.
&itric oxide *&O- is known to have o!!osin effects in inflamed tissue. )nducible
nitric oxide synthase *i&OS--derived &O directly or indirectly contributes to the initi-
ation and !roression of inflammation and tissue inGury. The interaction between &O
and O
1
-

!roduces the hihly cytotoxic oxidant O&OO *)schiro!oulos et al., 2331-. &O
can directly inhibit &.5#' oxidase and thereby limit su!eroxide eneration. Su!-
!ression of &O !roduction and the resultant enhancement of &.5#' oxidase
function may account for the more severe inflammation.
(ectal luminal levels of &O were measured with chemiluminescence techni"ue by
usin a tonometric balloon method and this study showed !atients with active )B5
had increased &O levels and healthy control had low levels of &O. (ectal &O levels
were correlated with disease activity in )B5 and decreased markedly in )B5 !atients
res!ond-in to anti-inflammatory treatment. i&OS ex!ression and &O !roduction in
the rectal mucosa of !atients with )B5 were sinificantly hiher than those of
controls.
B. O=IDANTS AND ANTIO=IDANTS IN THE
E=PERIMENTAL COLITIS
There are many studies on whether scavenin of O
1
-
by SO5 or intervention of
xan-thine oxidase by allo!urinol was effective in the !revention of ex!erimental
colitis. Sul-fasala/ine and 0-aminosalicylic acid *0-.S.- reduced sinificantly the
intestinal inflammatory reaction in the ex!erimental colitis as well as human )B5. 0-
.S. may exert its anti-inflammatory activity by inhibitin 0-li!oxyenase activities.
But hihly selective 0-li!oxyenase inhibitors were not effective in treatin !atients
with )B5. The effects of sulfasala/ine and its metabolites, 0-.S. and sulfa!yridine
on res!iratory burst of #$&L were investiated by luminol-de!endent
chemiluminescence for the de-tection of $#O-mediated active oxidants and by
lucienin-de!endent chemilumines-cence for the detection of su!eroxide anion
*Suematsu et al., 23C<b-. They attenuated (OS from #$&L at the concentrations
com!arable to clinical doses. They have a !otent antioxidant activity by scavenin a
variety of free radicals and the ability to su!!ress neutro!hil-derived hy!ochlorus
acid. Es!ecially, salicylates are remarkably effective as O'
-
scaveners.
There are three clinically succeeded trials for the treatment of human 86 with
antioxidantsE curcumin, SO5, and fish oils that are discussed in the next section.
. variety of ex!erimental colitis are em!loyed to clarify roles of (OS in the
!atho-enesis of )B5+ acetic acid-induced colitis, trinitroben/ene sulfonic acid
*T&BS- induced colitis, dextran sulfate sodium *5SS- induced colitis, enetically
deficient mice, trans-enic mice, and so on.
B.>. C#r&#.i$
6urcumin is a s!ice that ives the s!ecific flavor and yellow color to curry and the
maGor constituent of turmeric !owder extracted from the rhi/omes of the !lant
Curcuma longa L that is found in South and Southeastern .sia. 6urcumin has many
beneficial effects includin anti-inflammatory, antioxidant, anticarcinoenic,
antimicrobial, he!ato!ro-tective, and anti-anioenic actions. The food derivative
curcumin has been shown to inhibit &?-gB activity. The &?-gB !roty!e is com!osed
of the heterodimer (el. *!D0- and &?-gB2 *!04- subunits. This heterodimer is the
maGor &?-gB !rotein found in the nucleus of cytokine-stimulated intestinal e!ithelial
cells. &?-gB activation is closely reulated by its endoenous inhibitor )gB which
com!lexes with &?-gB in the cyto!lasma. .fter cytokine stimulation, Sartor%s rou!
showed that curcumin blocked intestinal e!ithelial cells ene ex!ression by inhibitin
the sinal leadin to )>> activation without directly interferin with &)> or )>>
*:obin et al., 2333-. Block-ade of )>> activation caused inhibition of )gBc
!hos!horylation and deradation and &?-fB activation and decreased )>> activity.
.nother mechanism of curcumin has anti-oxidative !ro!erties and function as a free
radical scavener. (OS reulate &?-gB activity in cells by modulatin &?-gB
activation.
.dministration of curcumin *244 m k
-2
, intra!eritoneally- for seven consecutive
days before 5SS challene to rats decreased the inGurious effects of 5SS and
ameliorated the inflammatory !arameters. #retreatment with curcumin before 5SS
administration resulted in a remarkable decrease in the concentration of serum T&?-c
by about 7DR and reduced the colonic $#O activity by about 0<R when com!ared
with rats treated with 5SS alone. )n addition, !rior administration of curcuma before
5SS increased colonic HS' content by 22<R and the colonic HS'-S-transferase
activity by about <3R, and reduced the colonic malondialdehyde *$5.- levels by
0DR when com!ared with only 5SS-treated rats. These data suest that curcumin
could have a !rotective role in colitis !robably via reulation of oxidant,antioxidant
balance and modulation of the release of T&?-c. #retreatment of mice with curcumin
sinificantly ameliorated the T&BS-induced colitis by a reduction in $#O activity
and $5. activity in the inflamed colon mucosa. )n addition, &?-gB activation in
colon mucosa was su!!ressed in the curcumin-treated mice. 6urcumin treatment
induced a marked su!!ression in )L-21 m(&. ex!ression by mucosal cells of T&BS-
treated mice, resultin in a reduced ability to induce )?&-b. The mechanism by which
curcumin inhibits )L-21 !roduction may be throuh the downreulation of &?-gB-
mediated activation and bindin to the !74-kB site. 6urcumin was shown to block
cytokine-mediated &?-gB activation and !roinflam-matory ene ex!ression by
inhibitin inhibitory factor )-gB kinase activity. The curative effect of curcumin
accom!anied by reduced levels of su!eroxide anions, &O, $5., and serine !rotease
may be the scavenin ca!ability of (OS and nitroen. 6urcumin induced
downreulation of 6OO-1 and i&OS ex!ression and a reduction in the activation of
!9C mitoen-activated !rotein kinases.
B.?. S#3(r!Hi/( Dis.#tas(
SO5 is one of the antioxidant !roteins. SO5 cataly/es the dismutation of
su!eroxide anion to '
1
O
1
, which is subse"uently detoxified to oxyen and water by
catalase or HS' !eroxidase. SO5 does not bind to cellular membranes and is ra!idly
excreted from the kidney. There are three isoforms of SO5 found in humansE
co!!er,/inc *6u,Nn--SO5, mananese *$n--SO5, and extracellular *E6--SO5. Since
6u,Nn-SO5 is a key antioxidant and is reduced in inflamed colonic tissues, this
en/yme as a treatment for )B5 had been investiated. .dministration of 6u,Nn-SO5
su!!ressed the develo!ment of ex!erimental colitis. These findin raised the
!ossibility that SO5 could be of thera!eutic benefit in the treatment of )B5.
'owever, clinical early trials of 6u,Nn-SO5 for !atients with )B5 had no remarkable
im!rovement, because of its low affinity in !lasma and short half-life of only a few
minutes. #6-SO5 is a lecithini/ed human 6u,Nn-SO5, in which four
!hos!hatidylcholin-derivative molecules are covalently bound to each SO5 dimmer.
Study to clarify the effects of #6-SO5 and unmodified SO5 was done in the
ex!erimental colitis induced by 7R 5SS *)shihara et al., 1443-. 6olon shortenin by
inflammation, cry!t loss, and infiltration of leukocytes were sinificantly ameliorated
in the #6-SO5-treated animals more than in the unmodified SO5-treated animals.
'owever, hiher doses of #6-SO5 had no sinificant effect on colon 5.). 5ecrease
in levels of su!eroxide anion released from activated neutro!hils was more
!ronounced in #6-SO5 than in unmodified SO5. )ncreased '
1
O
1
levels in 5SS-
treated animals were decreased by #6-SO5 administration only in the !resence of
simultaneous admin-istration of catalase. .n increased m(&. ex!ression of T&?-c
and )L-2a in 5SS-induced colitis was su!!ressed by administration of #6-SO5, but
that of )L-D and )L-19!23 was not su!!ressed. These data suested that #6-SO5
lowered the intestinal levels of (OS, resultin in su!!ressin the ex!ression of T&?-c
and )L-2a and amelioratin 5SS-induced colitis. )ncreased levels of &?-gB in 5SS-
induced colitis were also su!!ressed by administration of a low dose of #6-SO5 and
a hih dose of #6-SO5 with catalase.
.nother method to clarify role of SO5 is a SO5 ene transfer. E6-SO5, an
iso/yme of SO5s functions mainly in body fluids. The synenic fibroblasts
retrovirally transduced with the E6-SO5 ene transfer was inoculated subcutaneously
in the backs of 5SS-induced colitis mice. This treatment showed a sinificant
im!rovement in 5.) score, histoloic severity, and mucosal tissue levels of
inflammatory cytokines, C-hydroxydeoxy"uanosine, and$5.of colitis mice. )n
colitic interleukin-24 knockout mice, treatment of colitis mice with SO5 reduced an
increased ex!ression of endothelial vascular cell adhesion molecule-2 and mucosal
addressin cell adhesion molecule-2, and also colonic li!id hydro!eroxidation and
$#O activity.
Lactobacillus gasseri ex!ressin $n-SO5 was used to ameliorate colitis of the
)L-24 deficient mouse model. L# gasseri !roducin $n-SO5 sinificantly reduced
inflamma-tion in )L-24 deficient mice com!ared with untreated controls. The anti-
inflammatory effects of L# gasseri were associated with a reduction in the infiltration
of neutro!hils and macro!haes *6arroll et al., 144<-. Similar study usin L#
plantarum and Lactococcus lactis enineered to ex!ress SO5 was tried to ameliorate
T&BS-induced colitis in rats.
B.B. H(rba R(.(/i(s
Several herbal remedies were tried for the treatment of ex!erimental colitis
*Lanmead et al., 1441-.
!lippery elm bar& is derived from the sli!!ery elm or redtree native to &orth
.merica and has soothin !ro!erties in the case of inflammation of the
astrointestinal tract. )t is !o!ular amon )B5 !atients in the 8>.
'enugree& is an aromatic herb that !roduces methi seeds and is said to have a
beneficial effect on inflamed intestines. )t contains steroidal sa!onins that form the
basis for !hys-ioloical steroid !roduction.
Mexican yam is a tro!ical !erennial starch-rich tuberi/ed root, which is a sta!le
food in some areas and contains sa!onins in the form of dioscorea. )t is used for the
treatment of Goint and ut inflammation.
(evil)s cla* is a flowerin !lant native to southern .frica. The tuberi/ed
secondary roots are used for liver and stomach ailments. )t is said to have analesic
and anti-inflammatory !ro!erties. )t contains flavonoids that are !roven free radical
scaven-ers. #lant !henols are able to donate hydroen to oxyen radicals to form
stable inert com!ounds.
Tormentil is a member of the rose family that rows wild all over Euro!e. )t is
said to be effective in the treatment of diarrhea and bowel inflammation. )t contains a
hih concen-tration of tannins known to have a !otent su!eroxide-scavenin effect.
+ei tong ning, a traditional 6hinese medicine, is used for the treatment of !atients
with !e!tic ulcer and other intestinal inflammatory diseases.
These herbal remedies were assessed in an in vitro study on whether or not they
have scavener effects aainst oxyen radicals enerated by mucosal bio!sy
s!ecimens obtained from !atients with active 86 usin luminol-enhanced
chemiluminescence analysis. .ll herbs exce!t fenureek scavened su!eroxide dose-
de!endently like 0-.S.. .ll herbs scavened !eroxyl dose-de!endently. Oxyen
radical released from bio!sy s!ecimens was reduced after incubation in all herbs
exce!t $exican yam.
,cteoside, herbal !henylethanoid, is isolated from !lantain herb, Plantago
lanceolata L, and reduces the heat and !ain of inflammation, because it exhibits anti-
oxidative !otential and inhibits 0-hydroxy-D, C,22,27-eicosatetraenoic acid and
leukotriene B. )n addition, it inhibits the en/ymatic activity of i&OS ex!ressed in both
macro!haes and neutro!hils and 0-li!oxyenase *'ausmann et al., 144<-. )n 5SS-
induced colitis, acteoside sinificantly ameliorated colitis. ;hen stimulated
lym!hocytes from mesenteric lym!h nodes of colitis mice were treated with
acteoside, a sinificant reduction of )?&-b secretion was found. )nhibition of
oxidative burst activity with acteoside decreased mucosal tissue damae in 5SS-
induced colitis.
-reen tea is a well-known antioxidant and contains !oly!henols. This is discussed
in the next section.
B.D. Fr(( Ra/i&a S&a+($"(rs
Tempol is a membrane-!ermeable radical scavener and metal-inde!endent SO5-
mimetic. Treatment of dinitroben/ene sulfonic *5&BS- acid-induced colitis rats with
tem!ol sinificantly reduced the a!!earance of diarrhea and loss in body weiht. This
was associated with reduction in the deree of both neutro!hil infiltration and li!id
!er-oxidation in the inflamed colon. Tem!ol attenuated the colon shortenin and
damae score in 5SS-induced colitis mice.
%daravone, a free radical scavener, and tem!ol su!!ressed increased serum )L-D
levels and colonic $#O levels in 5SS-induced colitis mice
I,C, bis*2-hydroxy-1,1,D,D-tetramethyl-7-!i!eridinyl-decandioate, is a
innovative non-!e!tidyl, low-molecular-weiht radical scavener which is reactive
with most oxyen, nitroen, and carbon centered radicals. ).6 inhibited the reduction
in body weiht ain, decreased colonic damae and inflammation and T&?-c levels in
5&BS-induced colitis.
Prohibitin is a reulator of antioxidant and its levels were decreased durin colitis
and cultured intestinal e!ithelial cells overex!ressin #rohibitin were !rotected from
oxidative stress. )t is a reulator of nuclear factor erythroid 1-related factor 1
ex!ression *a transcri!t-tional reulator of oxidant res!onses- in intestinal e!ithelial
cells durin oxidative condi-tion. #rohibitin !revented inflammation-associated
oxidative stress and inGury throuh sustained activation of nuclear factor erythroid 1-
related factor 1 *Theiss et al., 1443-.
,llopurinol is used for the treatment of out and is a scavener of oxyen-derived
free rad-icals. Oanthine oxidase is ca!able of eneratin su!eroxide anionsby
convertin hy!oxanthine to xanthine and uric acid. )n ex!erimental colitis induced by
intracolonic administration of 0R acetic acid, s!ecific su!eroxide anion scavener
methoxy!olyethylene lycol+SO5, and reac-tive oxyenmetabolites scavener,
salfasala/ine sinificantly decreased the severity of inflame-mation. The xanthine
oxidase inhibitors, tunsten, and !terin aldehyde failed to im!rove inflammation, but
another xanthine oxidase inhibitor, allo!urinol, did limit the inflammation. )n this
colitis model, the xanthine oxidase !athway is not a maGor source of (OS.
B.E. G#tathi!$(
HS' is the most abundant cellular antioxidant synthesi/ed by animal cells and
!lays an essential role in cell bioloy and modulates cell res!onse to redox chanes
associated with (OS. !-adenosylmethionine, a HS' !recursor, is an obliatory
intermediate in the conversion of methionine to cysteine in the trans-sulfuration
!athway. !-.denosyl-methionine, 1*.$ !)-n-!ro!ylthia/olidine-7
h
-carboxylic acid,
and reen tea !oly!he-nols sinificantly im!roved colon lesions and increased body
weihts in 5SS-treated mice *O/ et al., 1440-. These antioxidants normali/ed the
!atholoical findins. The blood levels of reduced HS', and increased amyloid . and
T&?-c im!roved to normal when mice were treated with antioxidants.
/-.cetylcystein *&.6- as a HS' !recursor sinificantly reduced increased levels
of colonic $#O activity, (OS, T&?-c, and interleukin-2a. )n addition, it increased
#O&2 activity and HS' levels. 'owever, the other study re!orted that &.6 substan-
tially reduced the deree of colonic inGury !robably by reulatin free radical
!roduction and inhibitin inflammation, but its low doses had no !rotective effects.
&.6 alone and in combination with 0-.S. su!!ressed 6OO1 ene ex!ression and
!rostalandin E1 levels to control levels in T&BS-treated rats.
L-6arnitine administration im!roved histoloic scores and decreased $5. and
$#O levels in acetic acid-induced colitis. Su!!lementation of L-carnitine !revented
the de!le-tion of reduced HS' levels and increased SO5 levels without chane in
catalase activity.
B.G. iNOS I$hibit!r
i&OS is a reactive oxyen and nitroen metabolites-metaboli/in en/ymes.
.rinine in-creases &O levels while &.$E *a nonselective &OS inhibitor- lowers
oxidant levels. )n ex!erimental colitis induced by T&BS, the levels of &O and of
thiobarbituric acid-reactive substances *TB.(S, a marker of li!id !eroxidation- were
found to be sini-ficantly hiher in the arinine-administered rou! when com!ared
with lycine, and these levels were decreased on administration of &.$E to both
lycine- and L-arinine-administred rou!. HS' !eroxidase activity and HS' levels
were sinificantly hiher in arinine-administered rou! com!ared with lycine.
Sinificantly hiher 6u,Nn SO5 activity was found in the L-arinine B L-&.$E
rou! com!ared with arinine alone.
)n mice enetically deficient in i&OS or in the s!ecific i&OS inhibitor *2744 ;--
treated mice, the onset and severity of colitis induced by 5SS were sinificantly atten-
uated. The res!onses to 5SS did not differ between wild ty!e and !7< !hox , mice
while enhanced !rotection was noted in i&OS inhibitor-treated !7< !hox , *a !art of
&.5#'- mice. The combined blockade of i&OS and &.5#' oxidase was more
effective in !rotectin mice from 5SS-induced colitis than either intervention alone.
Therefore, diverent roles for su!eroxide enerated by membrane-associated &.5#'
oxidase in activated neutro!hils and i&OS-derived &O were found in intestinal
inflam-mation. &O may !lay a role in oxidant damae in ex!erimental colitis. On the
other hand, the healin of 5SS-induced colonic lesions was sinificantly im!aired by
admin-istration of L-&.$E or aminouanidine and the effect of L-&.$E was
sinificantly reverted by the co-administration of L-arinine. Therefore, &O !roduced
by i&OS contributed to the healin of 5SS-induced colonic lesions.
D. ANTIO=IDANTA IN H*MAN IBD
D.>. C#r&#.i$
'anai et al. !erformed a randomi/ed, multicenter, double-blind !lacebo-controlled
trial on the maintenance thera!y for 86. )n the trial, 70 !atients with "uiescent 86
received curcumin, 2 after breakfast and 2 after the evenin meal !lus
sulfasala/ine or 0-.S. and 77 !atients received !lacebo and sulfasala/ien or 0-.S..
Two of forty-three !atients receivin curcumin rela!sed durin D months of thera!y
while C of 93 !atients in the !lacebo rou! rela!sed. They concluded curcumin seems
to be a !romisin and safe medication for maintainin remission for !atients with
"uiescent 86 *'anai et al., 144D-.
D.?. S#3(r!Hi/( Dis.#tas(
#6-SO5 has a hih affinity to cellular membranes and scavenes su!eroxide
anion !roduced at affected tissues. #6-SO5 has a lon half-life in serum and
maintains en/y-matic activity for D h after inGection. #6-SO5 was intravenously
inGected once daily ini-tially for 27 days as in!atient treatment followed by twice
weekly for another 1 weeks as out!atient treatment. ?orty and eihty milliram
treatment rou!s at 7 weeks reduced 86-5.) total scores in 2< of 11 *C2R- and 22 of
14 *D2R-, res!ectively. $ean of 865.) total scores at 7 weeks was <.3B2.3 before
the treatment to 7.2B1.7 after that in 74 m rou! and C.0B2.7 to 0.DB2.7 in C4 m
rou!, res!ectively *Su/uki et al., 144C-. 'owever, unmodified SO5 trial usin
co!!er /inc SO5 for the treatment of 65 showed a !oor res!onse, because levels of
unmodified SO5 in the tissues seems to be not enouh for the treatment of 65 due to
short half-life of it.
D.B. Fish Oi
?ish oil su!!lement contains E#. and 5'.. There have been many re!orts about
effects of fish oil containin omea-9 fatty acids on the treatment of )B5. ?our
months of this su!!lement on the treatment with !redonisolone or sulfasala/ine in
!atients with 86 resulted in reduction in leukotriene B7 levels of rectal dialysates and
!redo-nisolone dose, and im!rovement in histoloic findins and weiht ain. Similar
o!en trial of ten !atients with mild to moderate 86 was !erformed by Solomon et al.
Seven of ten !atients had moderate to marked im!rovement and their steroid dose
could be reduced in four of five !atients on !redonisolone. 'owever, three !atients
had little or no im!rovement. The other study was done to clarify whether the
combination ther-a!y of sulfasala/ine and fish oil omea-9 fatty acid was effective for
!atients with 86. This study showed no sinificant chanes in any laboratory
indicator or in the simoi-dosc!y or histoloy scores. 'owever, oxidative stress was
decreased in total !lasma antioxidant ca!acity and microsomal li!id !eroxidation
inhibition assay *Barbosa et al., 1449-. )ncreased chemiluminescence induced by tert
butyl hydro!eroxide and microsomal li!id !eroxidation inhibition in !atients with 86
was reverted to baseline levels by treatment with combination thera!y. Levels of
!lasma $5., eryth-rocyte li!id !eroxidation and catalase in the combination rou!
were not different from those in the only sulfasala/ine rou!
6ochrane database systematic review showed a similar rela!se rate durin mainte-
nance of 86 remission between omea 9 treated !atients and controls *((+ 2.41, 30R
6)+ 4.02M1.49- *Turner et al., 144<-. On the other hand, it showed a marinal
sinificant benefit of omea 9 fatty acid thera!y for maintainin remission of 65
*((+ 4.<<, 30R 6)+ 4.D2M4.3C-.
D.D. A!3#ri$!
One hundred and eihty-four !atients who received !roctocolectomy and ileal
!ouch-anal anastomosis were randomi/ed to receive !osto!erative !ro!hylactic
allo!urinol 244 m twice daily or !lacebo control. The cumulative risk for a first
attack of !ouchitis was 94R in the allo!urinol rou! and 1DR in the !lacebo rou!
after 17 months. Therefore, allo!urinol did not reduce the risk of a first attack of
!ouchitis.
)n summary, reactive oxyen and nitric s!ecies !lay an im!ortant role in the
!atho-enesis of 86 and 65. 'owever, antioxidants are so weak in amelioratin the
severe inflammation of )B5 that they may be effective in addition to usual reimens
of )B5.
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abstract from 65 4403CD.
CHAPTER D
O.("a%G a$/ O.("a%B
P!y#$sat#rat(/ Fatty A&i/s a$/
I$'a..at!ry B!0( Dis(as(s
P.C. Ca/(r
8niversity of Southam!ton, Southam!ton, 8>
ABBREVIATIONS
ARA .rachidonic acid
CD 6rohn%s disease
DHA 5ocosahexaenoic acid
DSS 5extran sodium sulfate
EPA Eicosa!entaenoic acid
IBD )nflammatory bowel disease
IFN )nterferon
IL )nterleukin
IJB )nhibitory subunit of &?kB
LAT Linker of activated T cells
LT Leukotriene
NFJB &uclear factor ka!!a B
PG #rostalandin
PPAR #eroxisome !roliferator activated rece!tor
P*FA #olyunsaturated fatty acid
STAT Sinal transducers and activators of transcri!tion
TNF Tumor necrosis factor
*C 8lcerative colitis
.> INTROD*CTION
8lcerative colitis *86- and 6rohn%s disease *65- are the two main forms of
inflamma-tory bowel disease *)B5-. 65 can affect any !art of the astrointestinal
tract, while 86 !rimarily affects the colon *?arrell and #e!!ercorn, 1441E Shanahan,
1441-. )B5s are multifactorial conditions involvin both enetic and environmental
com!onents, with the outcome bein driven by an aberrant immune res!onse to
normal commensal micro-biota in individuals who have a weakened ut e!ithelial
barrier *$ac?arlane et al., 1447-. There is fairly stron evidence for a enetic link in
65+ a mutation in the &O51, 6.(5-20 *called )B5-2- ene has been found in 94R
of !atients with 65 *Ouraet al., 1442-. &O51 is a cyto!lasmic rece!tor for certain
!e!tides found in bacterial cell walls, which may result in 65 !atients with this
mutation havin a reduced ability to clear invasive bacteria. )ndeed, there is evidence
for microbial involvement in both forms of )B5, with a disturbed interaction between
the mucosal immune system and the com-mensal ut microbiota bein evident. )n
both forms of )B5, there are lare infiltrates of neutro!hils in the inflamed tissue. The
T-cell res!onse !rofiles associated with 86 and 65 are different, in that a Th2 !attern
of cytokine formation develo!s in 65 with increased !roduction of tumor necrosis
factor *T&?--c, interferon *)?&--b, and inter-leukin *)L--21, )L-D, and )L-2a, whereas
86 resembles more a modified Th1 !rofile, where cytokines includin )L-0 and )L-24
are u!reulated, but )L-7 is not. )n addition to this chane in cytokine !rofile,
intestinal B lym!hocytes !roduce lare amounts of )H. T&?-c is ex!ressed in the
intestinal mucosa of !atients with )B5 and triers in-flammation via a nuclear factor
ka!!a B *&?iB--de!endent sinalin cascade. $any of the cytokines involved act on
sinal transducers and activators of transcri!tion *ST.T- family *$usso et al., 1440-.
ST.T-9 sinalin has been found in 86 and 65, where it has been shown to be
confined to areas of active inflammation and infiltratin macro-!haes and T cells.
ST.T-9 induces transcri!tion of the !roinflammatory cytokine )L-D, which can
increase resistance of T cells to a!o!tosis, lenthenin the chronicity of 65, due to
the accumulation of reactive T cells. Other factors im!licated in 65 include
!roduction of matrix metallo!roteinases, which can derade extracellular matri-ces,
causin ulceration and tissue destruction.
The aim of this cha!ter is to review the evidence for involvement of lon-chain
!oly-unsaturated fatty acids *#8?.s- in )B5, either as !redis!osin environmental
factors or as !rotective or thera!eutic factors, and to !ro!ose mechanisms to ex!lain
these roles.
?. P*FAS) STR*CT*REI NOMENCLAT*REI SO*RCESI AND
INTERCONVERSION
This cha!ter considers two families of lon-chain #8?.s, the omea-D or n-D
family and the omea-9 or n-9 family. These two families have characteristic
structural features, se!arate dietary sources, and distinct bioloical !ro!erties. #8?.s
are fatty acids with two or more double bonds within the fatty acyl hydrocarbon
chain. #8?.s are rarer in the human diet than saturated or monounsaturated fatty
acids *British &utrition ?oun-dation, 2331-. The terms Sn-D% and Sn-9% refer to the
characteristic structural feature of the two families of fatty acid+ the number *i.e., D or
9- indicates the carbon atom in the hydrocarbon chain on which the first double bond
is found if the terminal methyl carbon is defined as carbon number one. The sim!lest
n-D #8?. is linoleic acid, an 2C-carbon fatty acid with two double bonds in the
hydrocarbon chain *?iure 7.2- and described by the shorthand term 2C+1n-D. The
sim!lest n-9 #8?. is a-linolenic acid, an 2C-carbon fatty acid with three double
bonds in the hydrocarbon chain *?iure 7.2-
Fi"#r( D.> The biosynthesis of !olyunsaturated fatty acids. 5'., docosahexaenoic acidE
E#., eicosa!entaenoic acid.
and described by the shorthand term 2C+9n-0. &either linoleic acid nor a-linolenic acid
can be synthesi/ed in animalsE these are readily synthesi/ed in !lants. ?urthermore, in
animals, n-D and n-0 #8?.s cannot be interconverted, althouh this does occur in
!lants. The !arent n-D and n-9 #8?.s may be converted to other fatty acids, with the
!osition of the methyl-terminal double bond retained, that is, linoleic acid ives rise to
other n-D #8?.s and a-linolenic acid to other n-0 #8?.s *?iure 7.2-. These
metabolic transfor-mations are achieved by the insertion of additional double bonds
into the hydrocarbon chain *termed unsaturation- and by elonation of the
hydrocarbon chain. Thus, linoleic acid can be converted via b-linolenic acid *2C+9n-D-
and dihomo-b-linolenic acid *14+9n-D- to arachidonic acid *14+7n-DE .(.- *?iure
7.2-. By an analoous set of reac-tions cataly/ed by the same en/ymes, c-linolenic
acid can be converted to eicosa!entae-noic acid *14+0n-9E E#.- *?iure 7.2-. Both
arachidonic acid and E#. can be further metaboli/ed, E#. ivin rise to
docosa!entaenoic acid *11+0n-9- and docosahexaenoic acid *11+Dn-9E 5'.- *?iure
7.2-.
Linoleic acid is found in sinificant "uantities in many veetable oils, includin
corn, sunflower, and soybean oils, and in !roducts made from such oils, such as
mararines *British &utrition ?oundation, 2331-. c-Linolenic acid is found in reen
!lant tissuesE in some common veetable oils, includin soybean and ra!eseed oilsE in
some nutsE and in flaxseed *also known as linseed- and flaxseed oil. Between them,
linoleic and c-linolenic acids contribute over 30R, and !erha!s as much as 3CR, of
dietary #8?. intake in most ;estern diets *British &utrition ?oundation, 2331-, with
linoleic acid intake bein in excess of that of c-linolenic acid. Ty!ical ;estern adult
intakes of linoleic acid and c-linolenic acid would be a!!roximately 24 and 2 day
-2
.
5ietary intakes of the lon-er-chain, more unsaturated #8?.s are much lower than
those of linoleic and c-linolenic acids. .(. is found in meat, and ty!ical
consum!tion is of the order of a few hundreds of millirams !er day. E#.,
docosa!entaenoic acid, and 5'. are found in fish, es!ecially the so-called Soily% fish
*tuna, salmon, mackerel, herrin, and sardine-. .n oily fish servin could !rovide 2M
9.0 of n-9 #8?.s, with lean fish !rovidin about one-tenth of this. Ty!ical intakes
of these marine n-9 #8?.s are in the tens to hundreds of millirams !er day *British
&utrition ?oundation, 2333E $eyer et al., 1449-. The com-mercial !roducts known as
fish oils also contain these lon-chain n-0 #8?.s, which ty!-ically will contribute to
about 94R of the fatty acids !resent.
B. INTAKE OF n,- AND n,' P*FAS AND RISK OF
DEVELOPING IBD
One study associated the hiher intake of n-D #8?.s as the :a!anese diet has
become more ;esterni/ed with the increase in 65 incidence in :a!an, suestin a
causal rela-tionshi! *Shoda et al., 233D-. $ore recently, usin data from the Euro!ean
#ros!ective 6ohort study *E#)6-, 'art et al. *144C- investiated the relationshi!
between fatty acid intakes in the diet of 293 subGects with incident 86 com!ared with
ae- and sex-matched controls. There was no relationshi! for intake of total saturated
fatty acids or total monounsaturated fatty acids, but there was a weak !ositive
relationshi! *i.e., in-creased risk for develo!in 86- for total #8?.s, with an odds
ratio of 2.0D for the hih-intake "uartile com!ared with the lowest "uartile of intake.
.s indicated above, n-D #8?.s make u! the bulk of dietary #8?.s. . further
analysis of the same data set revealed that linoleic acid intake was sinificantly
associated with the risk of develo!-in 86 *The )B5 in E#)6 Study )nvestiators,
1443-, with an adGusted odds ratio of 1.0 for the hihest "uartile of intake com!ared
with the lowest "uartile of intake. There was no association with intakes of c-linolenic
acid, E#., or 5'. *The )B5 in E#)6 Study )nvestiators, 1443-. .di!ose tissue
fatty acids reflect dietary intake of #8?.s *.rab, 1449-. .di!ose tissue fatty acids
were com!ared between !atients who went on to develo! 86 and ae- and sex-
matched controls *de Silva et al., 1424-. . hiher adi!ose tissue .(. concentration
was associated with sinificantly hiher risk of develo!in 86 within the E#)6-
5enmark #ros!ective 6ohort Study *adGusted risk in hihest "uartile 7 com!ared
with lowest "uartile-. .di!ose tissue E#. and 5'. were both associated with lower
risk of develo!in 86 but the variation in effect was wide, such that it was not
statistically sinificant. 'owever, within the E!ic-&orfolk 6ohort, dietary 5'.
!rotected aainst develo!ment of incident 86, with an adGusted odds ratio of 4.1 for
the hihest tertile of intake com!ared with the lowest tertile *:ohn et al., 1424-.
5ietary E#. was also associated with lower risk but the effect did not "uite reach
sinificance *:ohn et al., 1424-. Taken toether, these studies suest that a hiher
intake of either of the two n-D #8?.s *linoleic acid and .(.- is associated with
increased risk of develo!in )B5, es!ecially 86, while a hiher intake of 5'., and
!er-ha!s also E#., is associated with decreased risk. These effects on risk may relate
to the actions of these fatty acids on inflammatory !rocesses, driven in !art by their
effects on li!id mediators.
D. LIPID MEDIATORS) BIO SYNTHESISI ROLES IN IBDI
AND THE IMPACT OF $%B P*FAS
D.>. Ei&!sa$!i/s G($(rat(/ 'r!. Ara&hi/!$i& A&i/
Eicosanoids are key mediators and reulators of inflammation and immunity
*Lewis et al.,2334E Tilley et al., 1442- and are enerated from 14 carbon #8?.s.
Eicosanoids, whichinclude !rostalandins *#Hs-, thromboxanes, leukotrienes *LTs-,
and other oxidi/ed de-rivatives, are enerated from arachidonic acid by the metabolic
!rocesses summari/ed in ?iure 7.1. Eicosanoids are involved in modulatin the
intensity and duration of immune and inflammatory res!onses and they have cell- and
stimulus-s!ecific sources and fre-"uently have o!!osin effects. Thus, the overall
!hysioloical *or !atho!hysioloical- outcome will de!end on the cells !resent, the
nature of the stimulus, the timin of eicosanoid !roduction, the concentrations of
different eicosanoids enerated, and the sensitivity of taret cells and tissues to the
eicosanoids enerated. Because there is a rel-atively hih amount of .(. in immune
cell membrane !hos!holi!ids, this fatty acid is ty!ically the maGor !recursor for
eicosanoid mediators, which are !roduced in reatly increased amounts on cellular
stimulation. Thus, amon the mix of eicosanoids !ro-duced, those synthesi/ed from
arachidonic acid *e.., #HE
1
and LTB
7
- !redominate, althouh the exact eicosanoid
!rofile de!ends on the cell ty!e concerned *e.., neutro-!hils and mast cells !roduce a
lot of #H5
1
, whereas monocytes !roduce a lot of #HE
1
- and the nature of the
stimulusE the !rofile will also chane over time as the nature of the res!onse to the
stimulus alters.
D.B. Ara&hi/!$i&%A&i/%D(ri+(/ Ei&!sa$!i/s i$ IBD
)nduction of colitis in laboratory animals results in the !roduction of inflammatory
eicos-anoids such as #HE
1
and LTB
7
in the colonic mucosa *'irata et al., 1442E &ieto
et al.,1441-. )n human )B5, the intestinal mucosa contains elevated levels of
inflammatory ei-cosanoids such as LTB
7
*Sharon and Stenson, 23C7-. LTB
7
has
distinct !roinflammatory
Fi"#r( D.? 1utline of the path*ay of eicosanoid synthesis from arachidonic acid# C12$
cyclooxygenase3 "%T%$ hyroxyeicosatetraenoic acid3 "P%T%$ hydroperoxyeicosatetraenoic
acid3 L12$ lipoxygenase3 LT$ leu&otriene3 P-$ prostaglandin3 T2$ thromboxane# .eproduced
from Calder$ P#C#$ 4556# n-0 Polyunsaturated fatty acids$ inflammation$ and inflammatory
diseases# ,merican 7ournal of Clinical /utrition 80$ 9:5:!;9:9<!$ *ith permission#
actions such as inducin leukocyte infiltration and subse"uent release of inflammatory
mediators, and thus, it is hihly likely to be !layin a !atholoic role in )B5s. This is
su!!orted by the re!orted !rotective effect on mucosal inGury of the LT biosynthesis
in-hibitor $>-CCD in acetic-acid-induced rat colitis *Em!ey et al., 2332-. This effect
was associated with decreased colonic LTB
7
concentrations. 'owever, the role of
#HE
1
in )B5 is less certain, and there is evidence that it may be !rotective. ?or
exam!le, indo-methacin, an inhibitor of #H synthesis, worsened mucosal inGury in
acetic-acid-induced rat colitis *Em!ey et al., 2332-. ?urthermore, the LT synthesis
inhibitor not only de-creased colonic concentrations of LTB7 but also increased those
of #HE
1
*Em!ey et al., 2332-, suestin that a shift of .(. metabolism in favor of
#Hs is beneficial. (ecent studies have clearly demonstrated mechanisms by which
#HE
1
may act in an anti-inflammatory manner+ #HE
1
was shown to inhibit 0-
li!oxyenase, thus decreasin !roduction of the inflammatory 7-series LTs *Levy et
al., 1442-, and to induce 20-li!oxyenase, thus !romotin the formation of li!oxins
*Levy et al., 1442E Iachier et al., 1441- that have been found to have anti-
inflammatory effects. )ndeed, oral admin- stration of a li!oxin.7 analo attenuated
weiht loss and mortality in mice simultaneously subGected to oral treatment with
dextran sodium sulfate *5SS- to induce colitis *Hewirt/ et al., 1441-.
D.B. Fatty A&i/ M!/i'i&ati!$ !' Ei&!sa$!i/ Pr!'i(s
.nimal studies have shown a direct relationshi! between the .(. content of
immune cell !hos!holi!ids and the ability of those cells to !roduce #HE
1
*#eterson et
al., 233C-, such that #HE
1
!roduction is increased by .(. feedin *#eterson et al.,
233C- and decreased by E#. or 5'. feedin *6ha!kin et al., 2332E #eterson et al.,
233CE =a"oob and 6alder, 2330-E increased intake of E#. and 5'. reduces the .(.
content of im-mune cell !hos!holi!ids. >elley et al. *233C- re!orted increased
!roduction of #HE
1
and LTB
7
by blood mononuclear cells in human volunteers 21
weeks after oral administra-tion of 2.0 .(. !er day. )t is well documented that ex
vivo #HE
1
and 7-series-LT !roduction by human immune cells can be sinificantly
decreased by fish oil su!!lemen-tation of the diet for a !eriod of weeks to months
*6auhey et al., 233DE Endres et al., 23C3E Lee et al., 23C0E $eydani et al., 2332E
S!erlin et al., 2339E von Schacky et al., 2339-. Studies in humans demonstratin a
diminution of .(.-derived eicosanoid !roduction by oral n-9 fatty acids have
ty!ically used fairly hih intakes of the latter, for exam!le, between 1.7 and 27.9
E#.B5'. !er day. Little is known about the doseMres!onse relationshi! between n-9
#8?. intake and eicosanoid !roduction by human immune cells. (ecently, the effect
of increased oral intake of E#. *2.90, 1.<, 7.40 day
-2
for 21 weeks- on ex vivo
!roduction of #HE
1
by human mononuclear cells was examined in a !lacebo-
controlled, randomi/ed, double-blind study *(ees et al.,144D-. #HE
1
!roduction was
decreased by E#. intake in a dose-de!endent manner. 8nder the conditions of this
ex!eriment, an E#. intake of 2.90 day
-2
was not suffi-cient to influence ex vivo
#HE
1
!roduction, whereas an E#. intake of 1.< day
-2
si-nificantly decreased #HE
1
!roduction. This suests that chanes in the fatty acid com!osition induced with 2.90
E#. !er day were not sufficient to modify arachidonic acid metabolism leadin to
#HE
1
!roduction, whereas the chanes induced with 1.< E#. !er day were
sufficient to im!act on this metabolism. #HE
1
!roduction was sinif-icantly !ositively
related to .(. content of the cells and to the ratio of .(. to E#. in the cells as well
as sinificantly inversely related to the E#. content of the cells, showin the
im!ortance of the absolute and relative amounts of substrate *i.e., .(.-.
E#. is also a substrate for the cyclooxyenase and li!oxyenase en/ymes that
!roduce eicosanoids, but the mediators !roduced have a different structure from the
arachidonic-acid-derived mediators, and this influences their !otency. )ncreased
synthesis of 0-series LTs has been demonstrated usin macro!haes from fish-oil-fed
mice *6ha!kin et al., 2332- and neutro!hils from humans su!!lemented with oral fish
oil for several weeks *Endres et al., 23C3E Lee et al., 23C0E S!erlin et al., 2339-.
Trans-enic *Sfat-9)- mice bearin the C# elegans =n-0 desaturase% ene and so able to
convert n-D to n-0 #8?.s resultin in reatly elevated n-9 #8?. content in their
tissues were shown to !roduce lare amounts of #HE9 within colonic tissue after
induction of colonic in-flammation by 5SS *'udert et al., 144D-. The functional
sinificance of the !roduction of eicosanoids from E#. is that E#.-derived
mediators are often much less bioloically active than those !roduced from
arachidonic acid *Baa et al., 1449E Holdman et al.,23C9E Lee et al., 23C7-.
D.D. R(s!+i$s) N!+( A$ti%I$'a..at!ry a$/ I$'a..ati!$%
R(s!+i$" M(/iat!rs Pr!/#&(/ 'r!. EPA a$/ DHA
E#. and 5'. also !roduce resolvins throuh !athways involvin
cyclooxyenase and li!oxyenase en/ymes *Serhan et al., 1444a,b-. These mediators
have been demonstrated in cell culture and animal feedin studies to be anti-
inflammatory, inflammation resolv-in, and immunomodulatory *Serhan et al., 1441-.
?or exam!le, resolvin E2, derived from E#., was shown to !rotect aainst
chemically induced colitis in mice and this was associated with decreased infiltration
of ranulocytes into colonic tissue and decreased ex!ression of several inflammatory
enes *T&?-c, )L-21!74, cyclooxyen-ase-1, and inducible nitric oxide synthase-
within that tissue *.rita et al., 1440-. (esolvin synthesis is increased by feedin fish-
oil-rich diets to laboratory rodents *'on et al.,1449- and was shown to occur in fat-2
mice in which colitis had been induced *'udert et al., 144D-.
E. INFL*ENCE OF MARINE $%B FATTY ACIDS ON
INFLAMMATORY CYTOKINES
E.>. Tra$s&ri3ti!$ Fa&t!rs I$+!+(/ i$ R("#ati$"
I$'a..at!ry G($( EH3r(ssi!$
)n addition to effects on inflammation mediated by chanes in the !attern of
eicosanoids and other li!id mediators !roduced, marine n-0 #8?.s have also been
shown to alter the !roduction of inflammatory cytokines *see below-. This effect may
be mediated by altered activation of key transcri!tion factors involved in reulatin
inflammatory ene ex!ression. Two transcri!tion factors that are likely to !lay a role
in inflammation are &?iB and !eroxisome !roliferator activated rece!tor *##.(--b.
&?iB is the !rin-ci!al transcri!tion factor involved in u!reulation of inflammatory
cytokine, adhesion molecule, and cyclooxyenase-1 enes *>umar et al., 1447E Sial,
144D-. &?iB is acti-vated as a result of a sinalin cascade triered by extracellular
inflammatory stimuli and involvin !hos!horylation of an inhibitory subunit
*inhibitory subunit of &?iB *)iB--, which then allows translocation of the remainin
&?iB dimer to the nucleus *#erkins, 144<-. Thus, ex!ression of inflammatory enes
is u!reulated. &?iB is a reconi/ed taret for controllin intestinal inflammation
*:obin and Sartor, 1444E Schottelius and Baldwin, 2333E Nhan et al., 144D-. The
second transcri!tion factor, ##.(-b, is also ex!ressed in intestinal tissue *$ansen et
al., 233D- and is believed to act in an anti-inflammatory manner *S/anto and &ay,
144C-. 6olonic bio!sies of !atients with 86 show lowered ##.(-b ex!ression
*5esreumaux et al., 2333-, ##.(-b knockdown mice show enhanced susce!tibility to
chemically induced colitis *5esreumaux et al., 1442-, and ##.(-b aonists reduce
colitis in murine models *5esreumaux et al., 1442E Su et al., 2333-. Thus,
u!reulation of ##.(-b is also a reconi/ed taret for controllin intestinal
inflammation *5ubu"uoy et al., 144D-. ;hile ##.(-b directly reulates inflammatory
ene ex!ression, it also interferes with the activation of &?iB creatin an intriuin
interaction between these two transcri!tion factors *van den Berhe et al., 1449-. Both
&?iB and ##.(-b may be reulated by n-0 #8?.s *Bensiner and Tontono/, 144CE
Hrimaldi, 144<E Lo et al.,2333E $arion-Letellier et al., 1443E &ovak et al., 1449E Nhao
et al., 1447-.
E.?. Fatty A&i/ M!/#ati!$ !' Tra$s&ri3ti!$ Fa&t!r
A&ti+ati!$ a$/ I$'a..at!ry Cyt!,i$( Pr!/#&ti!$
Both E#. and 5'. inhibited endotoxin-stimulated !roduction of )L-D and )L-C
by cul-turedhuman endothelial cells *de 6aterina et al., 2337E >halfoun et al., 233<-,
and E#. or fish oil inhibited endotoxin-induced T&?-c !roduction by cultured
monocytes *Babcock et al., 1441E Lo et al., 2333E &ovak et al., 1449E Nhao et al.,
1447-. E#. or fish oil decreased endotoxin-induced activation of &?kB in human
monocytes *Lo et al., 2333E &ovak et al., 1449E Nhao et al., 1447-, and this was
associated with decreased )iB !hos!horylation *&ovak et al., 1449E Nhao et al.,
1447-, !erha!s due to decreased activation of mitoen-activated !rotein kinases *Lo et
al., 1444-. These observations su-est direct effects of marine n-0 #8?.s on
inflammatory ene ex!ression via inhibition of activation of the transcri!tion factor
&?iB. .nimal feedin studies with fish oil su!-!ort the observations made in cell
culture with res!ect to the effects of marine n-0 #8?.s on &?iB activation and
inflammatory cytokine !roduction. ?or exam!le, com!ared with feedin corn oil, fish
oil lowered &?iB activation in endotoxin-activated murine s!leen lym!hocytes *Oi et
al., 1442-. ?eedin fish oil to mice decreased ex vivo !roduction of T&?-c, )L-2a, and
)L-D by endotoxin-stimulated macro!haes *Billiar et al., 23CCE (enier et al., 2339E
=a"oob and 6alder, 2330- and decreased circulatin T&?-c, )L-2a, and )L-D
concentrations in mice inGected with endotoxin *Sadehi et al., 2333-. Several studies
in healthy human volunteers involvin su!!lementation of the diet with fish oil have
demonstrated decreased !roduction of T&?-c, )L-2a, and )L-D by endotoxin-
stimulated monocytes or mononuclear cells *a mixture of lym!hocytes and
monocytes- *6auhey et al., 233DE Endres et al., 23C3E $eydani et al., 2332E Trebble
et al., 1449E ;allace et al., 1449-, althouh not all studies confirm this effect *6alder,
144D-.
G. INFL*ENCE OF MARINE $%B FATTY ACIDS ON T CELLS
6ell culture studies have clearly shown the effects of various fatty acids, includin
n-0 #8?.s, on T-cell functional res!onses and sinalin, that these effects are dose
de!en-dent *6alder et al., 2332- and that they relate to chanes in the fatty acid
com!osition of lym!hocyte !hos!holi!ids *6alder et al., 2337-. E#. and 5'. have
both been shown to inhibit T-cell !roliferation *6alder and &ewsholme, 2331a,bE
6alder et al., 2332, 2337- and the !roduction of )L-1 *6alder and &ewsholme,
2331a,bE ;allace et al., 1442- in vitro. .nimal feedin studies involvin fairly hih
amounts of fish oil, or of E#. or 5'., have also re!orted reduced T-cell
!roliferative res!onses *:olly et al., 233<E >ew et al., 1449E ;allace et al., 1442E
=a"oob et al., 2337-. $echanisms believed to be involved include alterations in the
!hysical state of the !lasma membrane *6alder et al., 2337-E modification of the
!rofile of eicosanoid mediators, which in turn influence T-cell function *6alder et al.,
2331E $iles et al., 1449-E and direct effects on transcri!tion factor activation *$iles
and 6alder, 233C-. )n recent years, there has been some focus on whether marine n-0
#8?.s influence T-cell functions via effects on li!id rafts *Stulni and Neyda, 1447E
Stulni et al., 233C, 1442E Neyda and Stulni, 144D-, since li!id rafts a!!ear to be
intimately involved in T-lym!hocyte res!onses to activation *'arder, 1447E >atairi
et al., 1442E (a//a" et al., 1447-. ?eedin mice a diet rich in n-0 #8?. resulted in
incor!oration of E#. and 5'. into the li!ids of the rafts of s!leen T cells and this
was associated with a decreased s!hinomyelin content *?an et al., 1449-. 'ence,
inco-r!oration of n-0 #8?.s into membrane li!ids is a likely mechanism for !rotein
dis!lacement from rafts. #hos!horylation of the sinalin !rotein linker of activated T
cells *L.T- is the most u!stream ste! that is inhibited by marine n-0 #8?. treatment
of T cells *Neyda et al., 1449-, and it a!!ears that L.T dis!lacement from rafts is a
molecular mechanism mediatin inhibition of T-cell res!onses by n-0 #8?.s, at least
in vitro *Neyda et al., 1441-. )m!ortantly, animal studies have shown that dietary fish
oil affects early sinalin events in T cells, such as !hos!horylation of !hos!holi!ase
6-b2 *Sanderson and 6alder, 233C-, and have linked alterations of T-cell rafts by
dietary n-0 #8?.s with functional chanes such as decreased !roliferation and )L-1
!roduction *?an et al., 1447-.
5es!ite the consistent !icture of the effect of n-0 #8?.s on T cells that emeres
from in vitro and animal studies and the increasin understandin of the !ossible
mechanism involved, human data are inconsistent+ althouh some studies show that
increased intake of n-0 #8?.s from fish oil decreases human T-cell !roliferation
*$eydani et al., 2332E Thies et al., 1442- and )L-1 !roduction *$eydani et al., 2332-,
several other studies have re!orted no effect. One reason for this may be an
insufficient dose of n-9 #8?.s !ro-videdin some studies, but that does not ex!lain all
the inconsistency.
K. EFFICACY OF n,' P*FAS IN ANIMAL MODELS OF IBD
The reconition that n-0 #8?.s !ossess a rane of anti-inflammatory and
immunomod-ulatory !ro!erties *Table 7.2- that are of relevance to )B5 has !rom!ted
a series of studies investiatin their efficacy in animal models *.ndoh et al., 1449E
6ha!kin et al., 144<E Em!ey et al., 2331E 'udert et al., 144DE &ieto et al., 1441E Shoda
et al., 2330E Iilaseca et al., 2334E =uceyar et al., 2333-. These have !rimarily
involved chemically induced colitis. The outcomes of these studies are summari/ed in
Table 7.1. Two studies re!ort that diets rich in c-linolenic acid decrease colonic
damae and inflammation when com-!ared diets rich in n-D #8?. *Shoda et al.,
2330E =uceyar et al., 2333-. ;hether these effects are due to c-linolenic acid itself or
are related to conversion of c-linolenic acid to its loner chain, more unsaturated
derivatives, such as E#., is not clear from these studies. ?our studies re!ort that
dietary fish oil decreases chemically induced colonic damae and inflammation
com!ared with an n-D #8?.-rich diet *Em!ey et al., 2331E &ieto et al., 1441E
Iilaseca et al., 2334E =uceyar et al., 2333-. These effects on disease severity were, in
all cases, associated with a reduction in !roduction of .(.-derived eicosanoids *see
Table 7.1 for details-. . more recent study investiated 5SS-induced colitis in fat-9
mice *'udert et al., 144D-. These mice are able to !roduce n-0 #8?.s from n-D
#8?.s due to
Tab( D.> Summary of the (ane of .nti-)nflammatory and
)mmunomodulatory Effects of $arine n-0 #8?.s
A$ti%i$'a..at!ry !r
i..#$!.!/#at!ry (''(&t
M(&ha$is.@sA i,(y t! b( i$+!+(/
5ecreased synthesis of arachidonic-acid-
derived eicosanoids *many with
!roinflammatory actions-
5ecreased arachidonic acid in cell
membrane !hos!holi!ids
)nhibition of arachidonic acid metabolism
)ncreased !roduction of E#.-derived
eicosanoids *many with less
inflammatory and some with anti-
inflammatory actions-
)ncreased cell membrane !hos!holi!id
content of E#.
)ncreased !roduction of E#.- and 5'.-
derived resolvins *with anti-inflammatory
and inflammation resolvin actions-
)ncreased cell membrane !hos!holi!id
content of E#. and 5'. cou!led with
inhibition of arachidonic acid metabolism
5ecreased synthesis of !roinflammatory
cytokines *T&?-c, )L-2a, )L-D, )L-C-
5ecreased activation of &?gB *via
decreased !hos!horylation of )gB-
.ctivation of ##.(-b
.ltered activity of other transcri!tion
factors 5ifferential effects of arachidonic
acid-derived versus E#.-derived
eicosanoids
5ecreased T-cell reactivity 5ecreased !roduction of intracellular
sinals due to modified membrane rafts
!ource+ $odified from 6alder, #.6., 144D# n-0 #olyunsaturated fatty acids,
inflammation, and inflammatory diseases. .merican :ournal of 6linical &utrition C9,
2040SM2023S, with !ermission.
5'., docosahexaenoic acidE E#., eicosa!entaenoic acidE )iB, inhibitory subunit
of &?iBE )L, interleukinE &?iB, nuclear factor ka!!a BE ##.(, !eroxisome
!roliferator activated rece!torE T&?, tumor necrosis factor.
Tab( D.? Summary of Studies )nvestiatin the Effect of 5ietary n-9
#8?.s on .nimal $odels of 6olitis
R('(r($&( A$i.a M!/( Di(tary 'ats
&!.3ar(/ a$/
/#rati!$
Fi$/i$"s
Iilaseca et al.
*2334-
(at T&BS Sunflower oil
versus cod liver
oilE 7 weeks
!rior to T&BSE
u! to 04 days
!ost-T&BS
6olonic
damae,
ulceration, and
inflammation
were lower in
the cod liver
oil rou!
es!ecially at
04 days Time-
de!endent
elevation in
colonic
luminal
thromboxane
B
1
was blunted
in the cod liver
oil rou!
Em!ey et al.
*2331-
(at .cetic acid Saturated fat
versus n-D
#8?. versus
fish oilE D
weeks !rior to
acetic acid
)n the absence
of miso!rostol
!retreatment,
ileal fluid
absor!tion was
normal in the
fish oil rou!
but not in the
other two
rou!s
;ith
miso!rotol
!retreatment,
colonic fluid
loss was
reversed *to
absor!tion- in
the fish oil
rou! but not
in the other
two rou!s and
colonic inGury
was lower in
the fish oil
rou! than in
the other two
rou!s
Shoda et al.
*2330-
(at T&BS Safflower oil
versus !erilla
oil *rich in a-
linolenic acid-
6olonic
ulceration was
lower and
colonic weiht
was hiher in
the !erilla oil
rou!
#lasma LTB
7

levels were
lower in the
!erilla oil
rou!
=uceyar et al.
*2333-
(at T&BS 6otton and
sunflower oils
versus fish oilE
D weeks before
T&BSE 27 days
!ost-T&BS
6olonic
inflammation
and damae
were lower in
the fish oil
rou!
6olonic tissues
LTB
7
and
LT6
7
were
lower in the
fish oil rou!
&ieto et al.
*1441-
(at T&BS $ix of olive
oil, soybean oil,
and coconut oil
*D1.0+22.2+1D.7-
versus
6olonic
damae was
lower in the
fish oil rou!
than in the
other two
rou!s
Tab( D.? Summary of Studies )nvestiatin the Effect of 5ietary n-9
#8?.s on .nimal $odels of 6olitisYcontZd
R('(r($&( A$i.a M!/( Di(tary 'ats
&!.3ar(/ a$/
/#rati!$
Fi$/i$"s
mix of olive oil
and fish oil
<4+94- versus
mix of olive oil
and !i brain
!hos!holi!ids
*<4+94-E 2 or 1
weeks !ost-
T&BS
6olonic
mucosa #HE1
was lower in
the olive oil
and fish oil
rou!s than in
the brain
!hos!holi!ids
rou! at both
time !oints
6olonic
mucosa LTB7
was lower in
the fish oil
rou! than in
the other two
rou!s at both
time !oints
.ndoh et al.
*1449-
(at T&BS 'ih linoleic
acid li"uid diet
*77 linoleic
acid+2 c-
linolenic acid-
versus c-
linolenic-acid-
rich li"uid diet
*9 linoleic
acid+2 c-
linolenic acid-E
21 daysE T&BS
iven
intraileally
after 1 days
starvationE
)leal
inflammation,
ulceration, and
damae were
lower in the c-
linolenic acid
rou! )leal
inflammatory
cell infiltrate
and bleedin
were lower in
the c-linolenic
acid rou!
Serum )L-D
concentrations
were lower in
sacrifice 17 h
!ost-T&BS
the c-linolenic
acid rou! C h
!ost-T&BS
'udert et al.
*144D-
;ild-ty!e and
fat-9
transenic
mouse
5SS Safflower oilE
24 weeks !rior
to 5SSE C days
!ostfirst 5SS
ex!osure
Body weiht
loss, colon
shortenin, and
colonic
damae and
inflammation
were lower in
fat-9 mice
6olonic tissue
from fat-9
mice *but not
from wild
ty!e- contained
LTB
0
, #HE
9
,
resolvin E2,
resolvin 59,
and
neuro!rotectin
5) 6olonic
tissue tumor
necrosis factor-
c, )L-2a, and
inducible nitric
oxide synthase
m(&. levels
and &?iB
activation were
lower in fat-9
mice 6olonic
tissue Toll-
interactin
!rotein and
trefoil factor 9
m(&.
Tab( D.? Summary of Studies )nvestiatin the Effect of 5ietary n-9
#8?.s on .nimal $odels of 6olitisYcontZd
R('(r($&( A$i.a M!/( Di(tary 'ats
&!.3ar(/ a$/
/#rati!$
Fi$/i$"s
levels were
hiher in fat-9
mice
6ha!kin et al.
*144<-
$ouse )L-24
knockout
6orn oil versus
fish oilE u! to
24 weeks
6olonic
inflammation
was lower in
the fish oil
rou!
5SS, dextran sulfate sodiumE )L, interleukinE LT, leukotrieneE #H, !rostalandinE
T&BS, trinitroben/ene sulfonic acid.
the !resence of an Sn-0 #8?. desaturase% ene. They show much less colonic damae
and inflammation than wild-ty!e mice, and this is associated with a marked chane in
the !attern of inflammatory mediators !resent in colonic tissue. . study in )L-24
knockout mice, which s!ontaneously develo! colitis, demonstrated sinificantly
reduced colonic inflammation of the mice when fed fish oil com!ared to n-D #8?.-
rich corn oil *6ha!kin et al., 144<-.
L. H*MAN ST*DIES OF MARINE $%B P*FAS IN IBD
L.>O+(r+i(0 a$/ S#..ary !' H#.a$ St#/i(s
Lon-chain n-0 #8?.s are incor!orated into ut mucosal tissue of !atients with
)B5 who su!!lement their diet with fish oil *'awthorne et al., 2331E 'illier et al.,
2332E Loren/ et al., 23C3-. There are a number of re!orts that fish oil induces anti-
inflammatory effects in )B5 !atients, such as decreased LTB
7
!roduction by
neutro!hils *'awthorne et al., 2331E $c6all et al., 23C3E Shimi/u et al., 1449- and
colonic mucosa *Shimi/u et al., 1449E Stenson et al., 2331-, decreased #HE
1
and
thromboxane B1 !roduction by colonic mucosa *$c6all et al., 23C3-, and decreased
!roduction of #HE
1
and )?&-b by blood mononuclear cells *Trebble et al., 1447-.
Small o!en-label or !ilot studies re!orted clinical benefit of fish oil
su!!lementation in 86 *Salomon et al., 2334-. . number of randomi/ed, !lacebo-
controlled, double-blind studies of fish oil in )B5 have been re!orted *.lmallah et al.,
233C, 1444E .slan and Triadafilo!oulos, 2331E Bellu//i et al., 233DE ?eaan et al.,
144CE Hreenfield et al., 2339E 'awthorne et al., 2331E Loeschke et al., 233DE Loren/ et
al., 23C3E Loren/-$eyer et al., 233DE $ant/aris et al., 233DE Stenson et al., 2331E
Trebble et al., 1440E Iarhese and 6oomansinh, 1444E see Table 7.9-. Ty!ically,
these studies have not assessed the effect of n-9 #8?.s on the cellular as!ects of
inflammation or immune function but have focused on clinical outcomes. The dose of
lon-chain n-0 #8?.s used in these trials was between 1.< and 0.D day
-2
and
averaed about 7.0 day
-2
. Some of these trials indicate benefits of fish oil, which
include im!roved clinical score *three
Tab( D.B Summary of the (esults of #lacebo-6ontrolled Studies 8sin
5ietary $arine n-9 #8?.s *in the ?orm of ?ish Oil- in #atients with
)nflammatory Bowel 5iseases
R('(r($&( Dis(as( D!s( !'
EPA
MDHA @"
/ay
N>
A
D#rati!$
@0((,sA
Pa&(b! E''(&t !'
.ari$( $%B
P*FAs !$
&i$i&a
!#t&!.(s
Loren/ et al.
*23C3-
86 and
65
2.CB2.9 21 Olive oil 5ecreased
simoidosco!e
score
5ecreased
disease
activity in 86
*n.s.-
&o effect on
disease
activity in 65
.slan and 86 1.<B2.C 21 $ixed oils 5ecreased
Triadafilo!oulos
*2331-
disease
activity
5ecreased use
of
corticosteroids
&o effect on
simoidosco!e
score
&o effect on
ut mucosal
histoloy
score
'awthorne et al.
*2331-
86 7.0B2.2 01 Olive oil 5ecreased use
of
corticosteroids
in rela!sin
!atients
)nduction of
remission in
rela!sin
!atients &o
effect on
rela!se for
!atients in
remission
Stenson et al.
*2331-
86 9.1B1.1 2D Linoleicacid-
rich
veetable oil
5ecreased ut
mucosal
histoloy
score
5ecreased use
of
corticosteroids
)ncreased
weiht Hain
Hreenfield et al.
*2339-
86 1.1B2.0
for 7
weeksE
then
2.2B4.<0
for 14
weeks
17 Olive oil &o effect on
simoidosco!e
score
&o effect on
ut
Tab( D.B Summary of the (esults of #lacebo-6ontrolled Studies 8sin
5ietary $arine n-9 #8?.s *in the ?orm of ?ish Oil- in #atients with
)nflammatory Bowel 5iseases
R('(r($&( Dis(as( D!s( !'
EPA
MDHA @"
/ay
%>
A
D#rati!$
@0((,sA
Pa&(b! E''(&t !'
.ari$( $%B
P*FAs !$
&i$i&a
!#t&!.(s
$ucosal
histoloy
score &o
effect on
rela!se
&o effect on
rectal bleedin
Bellu//i et al.
*233D-
65 2.CB4.3 01 Short-chain
fatty acids
)ncreased
maintenance
in remission
5ecreased
rela!se
Loeschke et
al. *233D-
86 Total 0.2 247 6orn oil &o effect on
ut mucosal
histoloy
score &o
effect on
disease
activity &o
effect on
rela!se
Loren/-
$eyer et al.
*233D-
65 1.CB2.0 01 6orn oil &o effect on
rela!se
$ant/aris et
al. *233D-
86 9.1B1.2 01 Olive oil &o effect on
rela!se
.lmallah et
al. *233C,
1444-
86 9.1B1.7 17 Sunflower
oil
5ecreased
simoidosco!e
score
5ecreased ut
mucosal
histoloy
score
5ecreased
disease
activity
Iarhese and
6oomansinh
*1444-
86 Total 0.D 17 Sunflower
oil
5ecreased
disease
activity
5ecreased
simoidosco!e
score
Trebble et al.
*1440-
65 2.DB2.2 17 Olive oil &o effect on
disease
activity &o
effect on body
com!osition
Tab( D.B Summary of the (esults of #lacebo-6ontrolled Studies 8sin
5ietary $arine n-9 #8?.s *in the ?orm of ?ish Oil- in #atients with
)nflammatory Bowel 5iseasesYcontZd
R('(r($&( Dis(as( D!s( !'
EPA
MDHA @"
/ay
N>
A
D#rati!$
@0((,sA
Pa&(b! E''(&t !'
.ari$( $%B
P*FAs !$
&i$i&a
!#t&!.(s
?eaan et
al. *144C-
65
1B4.C
0C $edium-
chain
trilycerides
&o effect on
rela!se
&o effect on
disease
activity
*two
se!arate
studies-
!ource+ $odified from 6alder, #.6., 144D. n-9 #olyunsaturated fatty acids,
inflammation, and inflammatory diseases. .merican :ournal of 6linical &utrition C9,
2040SM2023S, with !ermission.
65, 6rohn%s diseaseE 5'., docosahexaenoic acidE E#., eicosa!entaenoic acidE
86, ulcerative colitis.
studies-, im!roved ut mucosal histoloy *two studies-, im!roved simoidosco!ic
score *three studies-, lower rate of rela!se *one study-, and decreased use of
corticosteroids *three studies- *Table 7.9-. 'owever, a number of trials do not re!ort
such benefits. .lthouh one study with an enterically coated fish oil showed a
sinificantly lower rate of rela!se over 21 months in !atients with 65 *Bellu//i et al.,
233D-, two recent trials with a similar desin and fish oil !re!aration and that used a
similar dose of n-9 #8?.s could not re!licate this findin *?eaan et al., 144C-.
L.?. M(ta%a$ays(s
.lthouh reviews of trials of fish oil in )B5 have fre"uently concluded that there
is some benefit from fish oil *Bellu//i, 1444, 1441E $ills et al., 1440E (oders, 233CE
Teitelbaum and ;alker, 1442-, meta-analyses do not reach this conclusion. . meta-
analysis identi-fied 29 studies of fish oil su!!lementation in )B5 *i.e., both 86 and
65- re!ortin outcomes related to clinical score, simoidosco!e score, ut mucosal
histoloy score, and induced remission and rela!se but concluded that there were
sufficient data to !er-form meta-analysis only for rela!se and only for 86 *$acLean
et al., 1447-. The !ooled risk of rela!se with lon-chain n-9 #8?.s relative to
!lacebo from these studies was 2.29 *30R 6)+ 4.32, 2.0<-, and it was concluded that
Sn-9 fatty acids have no effect on relative risk of rela!se in 86% and Sthere was a
statistically nonsinificant reduction in re"uire-ment for corticosteroids for n-9 fatty
acids relative to !lacebo in two studies.% )n the last few years, a series of meta-
analyses on this subGect have been !ublished *5e Ley et al., 144<E Turner et al., 144<,
1443-. One of these considered maintenance of remission in 65 and included six
studies, includin one !ublished in abstract form only and one on !ediatric !atients
*Turner et al., 1443-. The authors identified a Smarinal sinif-icant benefit of n-9
#8?.s for maintainin remission% *relative risk 4.<<E 30R 6)+ 4.D2, 4.3C-, but
because of heteroeneity amon studies and a lack of effect in the two larest studies
included, the overall conclusion was that n-9 #8?.s are S!robably ineffective for
maintenance of remission in 65.% The other two meta-analyses considered
maintenance of remission in 86 *5e Ley et al., 144<E Turner et al., 144<-. The first of
these included three studies and concluded that Sn-9 #8?.s are not effective in
maintainin remission in 86% *Turner et al., 144<-E relative risk was 2.41 *30R 6)+
4.02, 1.49-. The second of these included six studies includin two !ublished only as
abstracts and concluded that Sthere is insufficient information to reach of conclusion%
*5e Ley et al., 144<-. Thus, des!ite some favorable studies *Table 7.9-, the !resent
overall view is that there is only weak evidence that lon-chain-9 #8?.s have
clinical benefits in human )B5.
O. CONCL*SIONS
?atty acids may influence inflammation and immunity throuh a variety of
mechanismsE many of these are mediated by, or at least associated with, chanes in
fatty acid com!o-sition of immune cell membranes. 6hanes in these com!ositions
can modify membrane fluidity, li!id raft formation, !hos!holi!id-based cell sinalin
leadin to altered ene ex-!ression, and the !attern of li!id mediator !roduction.
'uman immune cells are ty!ically rich in the n-D fatty acid .(., but the contents of
.(. and of the n-9 fatty acids E#. and 5'. can be altered throuh oral
administration of E#. and 5'.. Eicosanoids !ro-duced from .(. have roles in
inflammation and reulation of immune functions. E#. also ives rise to eicosanoids,
and these may have !ro!erties different from those of .(.-derived eicosanoids. E#.
and 5'. ive rise to newly discovered resolvins that are anti-inflammatory and
inflammation resolvin. )ncreased membrane content of E#. and 5'. *and
decreased .(. content- results in a chaned !attern of !roduction of eicos-anoids
and !robably also of resolvins, althouh the latter are not well examined in the human
context. 6hanin the fatty acid com!osition of immune cells also affects T-cell
sinalin and T-cell reactivity. Thus, the fatty acid com!osition of human immune
cells influences their functionE the contents of .(., E#., and 5'. a!!ear to be
es!ecially im!ortant. $ost studies investiatin the influence of fatty acids on human
immune were on cells from !eri!heral bloodE there has been little attention !aid to
cells of the ut-associated lym!hoid tissue. 'owever, there has been considerable
interest in fatty acids and ut inflammation. 5ietary fish oil, a source of E#. and
5'., has been shown to have beneficial effects on animal models of colitis induced
by chemicals or by knockout of the )L-24 ene. E#. and 5'. are incor!orated into
ut mucosal tissue of !atients with )B5 who su!!lement their diet with fish oil, and
there are re!orts that this is asso-ciated with anti-inflammatory effects. . number of
clinical trials of E#. and 5'. in both 86 and 65 have been re!orted. .lthouh
some of these trials indicate benefits of fish oil, which include im!roved clinical
score, im!roved ut mucosal histoloy, im-!roved simoidosco!ic score, lower rate
of rela!se, and decreased use of corticosteroids, a number of trials do not re!ort such
benefits. $eta-analyses concluded that at !resent, there is no clear evidence of
efficacy of n-9 fatty acids in human )B5. )t is not clear why the anti-inflammatory
effects observed after fish oil treatment do not translate into clinical im!rovements,
but there is a need to understand this issue further.
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mani!ulation on rat lym!ho-cyte subsets and !roliferation. )mmunoloy C1, D49MD24.
=uceyar, '., O/utemi/, O., 'useyinov, .., et al., 2333. .dministration of n-9
fatty acids by mucosal enema !rotective aainst trinitroben/ene-induced colitis in
rats. #rostalandins, Leukotrienes, and Essential ?atty .cids D2, 993M970.
Neyda, $., Staffler, H., 'oreGsi, I., ;aldhausl, ;., 1441. L.T dis!lacement
from li!id rafts as a molecular mechanism for the inhibition of T cell sinallin by
!olyunsaturated fatty acids. :ournal of Bioloical 6hemistry 1<<, 1C72CM1C719.
Neyda, $., Stulnii, T.$., 144D. Li!id (afts A 6o.+ an interated model of
membrane orani/ation in T cell activation. #roress in Li!id (esearch 70, 2C<M141.
Neyda, $., S/ekeres, ..B., Saemann, $.5., et al., 1449. Su!!ression of T cell
sinalin by !olyunsaturated fatty acids+ selectivity in inhibition of mitoen-activated
!rotein kinase and nuclear factor activation. :ournal of )mmunoloy 2<4, D499MD493.
Nhan, S.N., Nhao, O.'., Nhan, 5.6., 144D. 6ellular and molecular
immuno!athoenesis of ulcerative colitis. 6ellular and $olecular )mmunoloy 9, 90M
74.
Nhao, =., :oshi-Barve, S., Barve, S., 6hen, L.'., 1447. Eicosa!entaenoic acid
!revents L#S-induced T&?-al!ha ex!ression by !reventin &?-ka!!aB activation.
:ournal of the .merican 6ollee of &utrition 19, <2M<C.
CHAPTER E
A&!h! a$/ Gastr!i$t(sti$a Tra&t
F#$&ti!$
S.B. Bhar/0a1
#unGab 8niversity, 6handiarh, )ndia
.lcoholic beveraes are enerally divided into three ty!es de!endin u!on their
alcohol content+ beers *7R-, wines *24R-, and li"uors *74R-. .lcoholic beveraes
like wines and beers have shown less damain effect on the ut mucosa than other
hih-rade li"uors *>noll et al., 233C-. .lthouh alcohol is a common inredient in
all alcoholic beveraes, these also contain many other substances that exert additional
effects u!on the astroin-estinal *H)- a!!aratus, thereby differentiatin various
alcoholic beveraes in terms of both com!osition and bioloical action u!on the
diestive tube *BuGanda, 1444-.
.lcohol or ethanol consum!tion *excess- has been associated with multi!le
!athol-oies at all levels. )n the diestive a!!aratus, alcohol has enerally been related
to its toxic effects u!on the liver *.chord, 2339- and !ancreas *Schenker and
$ontalvo, 233C-, but relatiively little attention has been centered on its actions within
the H) tract *BuGanda, 1444-. The mechanisms and !atho!hysioloy underlyin the
effects of ethanol on the orans of H) tract are not yet com!letely understood
*Siemundi et al., 1449-. )n addition to the causation of liver cirrhosis, chronic
!ancreatitisi, or neuroloical disorders, acute and chronic ethanol consum!tion may
ive rise to functional abnormalities of the dies-tive tract. Ethanol consum!tion is
now considered to be an im!ortant risk factor for the develo!ment of H) discomfort
and com!lications *LaheiG et al., 1447-. 5urin ethanol consum!tion, the H) tract is
ex!osed to hih concentrations of alcohol com!ared to other tissues. Therefore,
chronic alcoholics may !resent with hiher !revalence of H) com!laints such as
heartburn, chest and abdominal !ain, nausea, vomitin, flatulence, or diarrhea
com!ared to nonalcoholics *?ields et al., 2337E LaheiG et al., 1447-.
.lcohol is the cause of 7R of the lobal burden of disease when measured as
disability adGusted life years *5.L=s- and resulted in 9.1R of deaths in the year 1444
*(ehm et al., 1449a,bE;orld 'ealthOrani/ation *;'O-, 1441-. 5.L=s is a measure
that combines years of life lost because of early mortality, that is, death before life
ex!ectancy, with the hihest life ex!ectancy worldwide *currently :a!an-, with years
of life lost to im!erfect health *$urray et al., 1444-. The relationshi! between alcohol
consum!tion and 5.L=s demonstrates that a substantial burden of disease is
attributable to alcohol consum!tion. )n 2334, this was estimated lobally to be hiher
than the burden of disease attributable to the consum!tion of tobacco *$urray and
Lo!es, 233<-.
)nternational com!arisons of drinkin rates in adults based on eneral !o!ulation
surveys have been conducted in the last decade. )n 1441, Leiffman observed that the
hihest mean annual alcohol consum!tion *number of drinkin occasions !er bottles
consumed within a year- was in the 8nited >indom and the lowest consum!tion was
in Sweden. )n 1442, $akela et al. re!orted that amon the Scandinavian countries,
5enmark had the hihest and &orway had the lowest annual alcohol consum!tion. To
conduct valid com!arisons of diverse countries, a !roGect called HE&.6)S *Hender,
.lcohol and 6ulture+ .n international Study-, a multinational study that includes the
8S &ational )nstitute on .lcohol .buse and .lcoholism, Euro!ean commission,
and;'O *2333-, was undertaken. The study currently involves 1D diverse countries
includin $exico, .rentina, Sri Lanka, >a/akhstan, ?rance, )srael, 'unary, :a!an,
.ustria, and the 8nited States, and is investiatin ender differences in alcohol use
and conse-"uences on a cross-cultural level. The effects of alcohol-drinkin !atterns
are currently under study in different develo!in countries *5emers et al., 1442-. .
theme that has emered from a !ilot study from )ndia on social construction of
meanins attached to alcohol use is+ .lcohol use is a symbol of economic status,
caste, a !erson%s karma, a !eriod of turmoil, and ender !rivilee *&immaadda,
2333-. )n a community-based study from &orth )ndia on alcohol users, alcohol has
been said to be a social !roblem on a survey of 730 males *#al et al., 1449-.
.> AC*TE AND CHRONIC INGESTION OF ALCOHOL
.lcohol intake is of two ty!es+ acute and chronic inestion. The acute intake is the
short-term use of hih concentrations of ethanol *`D4R v,v- or !ure ethanol *model of
acute inGury-. The acute ex!osure of the intestinal mucosa to ethanol may result in
structural damae and im!aired absor!tion of nutrients and vitamins. This is
normali/ed within a day after cessation of alcohol inestion *#ersson, 2332-. .cute
intake of alcohol is related to acute diseases such as accidents and inGuries *(ossow et
al., 1442-.
The chronic intake of alcohol is defined as the consum!tion of alcohol of more
than D4 day
-2
in males and of more than 74 day
-2
in females for at least 9 months
accord-in to the latest criteria *;'O, 1444-. $any chronic diseases are related
overall to lon ex!osure of alcohol on tissue, leadin to cancer, liver cirrhosis, etc.
The reularity of drinkin and heavy drinkin also leads to coronary heart disease
*6'5E (ehm et al.,1449a,b-. .lcoholic beveraes in amounts enerally acce!ted are
associated with health risks when mean daily alcohol intake in males is of more than
74 day
-2
and in females, more than 14 day
-2
*'ullinhorst, 2333-. ?or consum!tion
which results in a hih risk of adverse conse"uences, the thresholds should be more
than 244 of alcohol !er day for men and more than D4 of alcohol !er day for
women. )t has been claimed by various workers that the effects of acute and chronic
inestion of alcohol vary considerably and de!end on the concentration and dose of
the alcoholic beverae toether with various factors such as nutritional status, ender,
and ethnicity but it is very difficult to acce!t these statements. This difficulty is
further intensified by the attem!t to extra!olate the findins obtained in ex!erimental
studies in animals and in vitro to the effect of alcohol on humans.
The use of different consum!tion thresholds for men and women, which are based
on ender differences in alcohol metabolism, is still controversial. .ccordin to a
study by Hraham et al. *233C-, different thresholds are less im!ortant for the study of
ender behavior than differences in the effects of alcohol *e.., alcohol-related
inGuries-. These are moderated by the slower drinkin !ace fre"uently found in
women than the study of biomedical ender differences *e.., liver disease-.
6onversely, a recent caseMcontrol study found marked ender differences in the risk
of alcohol-related inGury at different levels of alcohol consum!tion. )n this, women at
all levels of alcohol consum!tion had a substantially reater risk of inGury than men
*Stockwell et al., 1441-. .lcohol is more he!atotoxic in women *$c6ullouh and
O%6omnor, 233C-E it also influences the sex steroid metabolism and therefore may
influence women differently from men, as evidenced by the observation that relatively
low body weiht in alcohol drinkers, as com!ared to nondrinkers, is es!ecially
evident in women *;illiamson et al., 23C<-.
.? ABSORPTION OF ALCOHOL
Ethanol is absorbed throuh themucosa of the entire H) tract by sim!le diffusion.
The rate of diffusion of ethanol across the intestinal mucosa is determined by the
concentration radient between the intestinal lumen and the sube!ithelial ca!illaries,
reional blood flow, and the !ermeability of mucosa. The rate of ethanol absor!tion is
decreased by delayed astric em!tyin and by the !resence of food in the stomach.
These findins had already been described about 04 years ao *Bode et al., 23C4-.One
of the ex!lanations is that there is first-!ass metabolism *?#$- of alcohol in the
stomach, which has recently been demonstrated *6rabb et al., 1447-. ?#$of ethanol is
observed only with the intake of small doses of ethanol. )ts "uantitative im!ortance
for the overall metabolism of alcohol, as re!orted by different authors, varies from
24R or less *.mmon et al., 233D- to 74M<4R *Hentry et al., 2337-.;hether ?#$of
alcohol occursmainly or exclusively in the stomach or not is still a matter of debate
*Levitt and Levitt, 2337-.
.fter oral inestion of ethanol by man, a !eak ethanol concentration is reached in
the duodenum and the !roximal GeGunum durin the first 70 min. There is a moderate
increase in the ileum ethanol concentration !arallel to the serum concentration
*'alsted et al., 23<9-. )n a study in which alcohol was inested with a meal, the
absor!tion in the stomach was <0R and 10R in the duodenum *6ortot et al., 23CD-. )n
other studies, the main absor!tion of ethanol has been throuh the intestinal wall
*'alsted et al., 23<9-. )n the u!!er duodenum, alcohol concentrations u! to 24R can
be found, de!endin on factors such as ty!e of beverae, content of the stomach, and
velocity of stomach em!tyin. The maGor !art of an inested drink is absorbed in the
u!!er small intestine by !assive diffusion. )n earlier studies, it has been shown that
acute alcohol inestion causes mucosal damae in the u!!er H) tract, es!ecially in the
duodenum *Bode and Bode, 2331E 5inda et al., 2339-. .fter oral administration of
ethanol in a dose of 4.C k
-2
, levels of ethanol reater than 4.7R *w,v- *the !eak
level in duodenum and !roximal GeGunum is between 2R and 0R- are maintained for
u! to D4 min. )n the same study in humans, the !eak ethanol concentration after 70MD4
ethanol in 14R solution varied between D.0R and 3.7R in the duodenum and
between 0.<R and D.7R in the GeGunum *5inda and Beck, 23C2-. 'ih concentrations
of ethanol, similar to those of the inested alcoholic beveraes, are reached only in the
u!!er small intestine, that is, the duodenum and GeGunum *Bode, 23C4E $e/ey, 23C0-.
Even after the oral adm-inistration of a sinle lare dose of alcohol, the concentration
of ethanol !resent in the ileum is not sinificantly different to that of levels in the
blood, suestin that ethanol enters the ileum from the vascular s!ace. The damain
effects of alcoholic beveraes on the mucosa as a result of hih luminal ethanol
concentrations are therefore to be ex!ected !redominantly in the duodenum and u!!er
GeGunum.
$any hos!itali/ed alcoholic !atients have fat, vitamin B
21
, or xylose
malabsor!tion, and in one study, as many as 39R had at least one abnormal !arameter
of absor!tion *(oin et al., 23D3-. 'owever, the commonly used absor!tion tests
mainly reflect chanes in carrier-mediated !rocesses at the intestinal brush border or
the test substances are affected by luminal diestive !rocesses.
.B ALCOHOL CONS*MPTION AND GI TRACT
(elatively little attention has been centered on alcohol actions within the H) tract.
B.>. E''(&t !$ th( Es!3ha"#s
The most known effects of excess alcohol inestion at eso!haeal level are the
develo!-ment of eso!haeal varicosities secondary to liver damae caused by alcohol,
and the $alloryM;eiss syndrome *or tearin of distal third of the eso!haus- due to
vomitin. .lcohol not only causes direct damae to the eso!haeal mucosa but also
has noxious effects when it is no loner in contact with the mucosa. The
!atho!hysioloical mechanisms by which alcohol damaes the e!ithelium include
alterations in e!ithelial trans!ort, intercellular Gunction disorders, and im!airment of
the mucosal barrier, facilitatin hydroen ion !enetration into the mucosa *Bor et al.,
2333-.
.cute ethanol a!!lication exerts effects on the u!!er and lower s!hincters as well
as on the !eristalsis of the human eso!haus that may be attributed to a disturbance in
the function of the autonomous nervous system *Teysson and Siner, 1449-. .cute
ethanol consum!tion leads to a transient decrease in the lower eso!haeal s!hincter
!ressure and inhibition of the !rimary !eristaltic movement of the distal eso!haus
body causin im!aired eso!haeal clearance *$incis et al., 2330-. 6hronic ethanol
consum!tion induces secondary motility disorders in the distal eso!haus with
!roloned contractions of hiher am!litude than normal as well as simultaneous and
double-!eaked contractions. The effect on the lower eso!haeal s!hincter is o!!osite
to that of acute ethanol admin-istration *Teysson and Siner, 1449-. 6hronic alcohol
abuse with a normal meal facilitates acid reuritation by reducin the !ressure of the
lower eso!haeal s!hincter and slowin both eso!haeal motility and astric
em!tyin *Stermer, 1441-. .lcohol also tends to facilitate or cause astroeso!haeal
reflux and eso!haeal mucosal damae, reardless of ty!e of alcoholic beverae
involved *$incis et al., 2330-.
B.?. E''(&t !$ th( St!.a&h
)n 2339, 6hari et al. reviewed the effects of alcohol and different alcoholic
beveraes u!on astric acid and astric secretion. .lcohol at low concentrations *u!
to 0R alcohol- stimulated acid secretion, whereas hiher doses either exerted no
effect or showed inhib-itory action. .lcoholic beveraes !roduced by fermentation,
such as beer and wine, are !otent stimuli of astric acid out!ut as well as release of
astrin. On the other hand, beveraes with a hiher alcohol content that are !roduced
by distillation such as whisky *74R v,v- do not stimulate astric acid out!ut or the
release of astrin *Siner et al.,23C<-. The mechanisms !ro!osed to ex!lain the
stimulatory effect of low-dose alcohol u!on the astric mucosa are mediation via the
cholineric system, to!ical stimulation of !arietal cells with an increase in cyclic
.$# !roduction, and histamine release.
The administration of low-dose alcohol accelerates astric em!tyin whereas hih
doses delay em!tyin and reduce bowel motility *Bor et al., 2333-. Ethanol concentra-
tions of C !er 244 ml or more resulted in delayed astric em!tyin when com!ared
with water or low ethanol concentrations *6ooke, 23<4-. )n 2331, #feiffer et al.
investiated astric em!tyin and bowel transit followin different beveraes and
found wine and beer to increase astric em!tyin and intestinal motility versus
!hysioloical saline or alcohol. On the other hand, ethanol has also been seen to cause
!yloric relaxation, which may facilitate astric em!tyin, thouh it could also favor
duodenoastric reflux *#haosawasdi et al., 23<3-. $any studies have been carried out
to analy/e the im!act of ethanol on astric motility M with inconsistent results
de!endin on dose and ty!e of beverae studied and on the administration of a li"uid
or solid test meal *Teysson and Siner, 1449-. . study in mice has shown that chronic
astric administration of 74R ethanol *v,v- damaes the intramural neurons of the
stomach, which may im!air astric motility *:edr/eGewska and Nielinska, 2334-.
B.B. Gastri& M(tab!is.
The liver is the !rinci!al site for ethanol oxidation in the mammalian body.
.lthouh H) metabolism is "uantitatively much lower *14R- com!ared to the liver, it
is of im!ortance as it affects the systemic availability of alcohol and leads to the local
!roduction of toxic acetaldehyde, which is !ossibly involved in the !athoenesis of
tissue inGury *6rabb et al., 1447-. 'owever, it has been found that alcohol can also be
oxidi/ed in the H) tract and the so-called ?#$ of ethanol has been demonstrated
*5i!adova et al., 23C<-.
?#$ is the difference between the amount of alcohol administered orally and the
amount reachin the systemic circulation and conce!tually is a result of metabolism
of alcohol by the ut or liver durin the absor!tion !hase. ?#$ is im!ortant because it
reduces the amount of alcohol reachin taret orans and may also !redis!ose ut
tissues to inGury from alcohol metabolism.
.lthouh liver is the !rimary site of alcohol metabolism, ethanol can also be
oxidi/ed in the H) tract. .lcohol dehydroenase *.5'- isoen/ymes have been
identified in the stomach and intestine. Hastric .5' has been im!licated in astric
?#$ of ethanolE by oxidi/in ethanol directly in the stomach, this en/yme may limit
the amount of inested ethanol that is delivered to the !ortal circulation.
Hastric .5'activity is lower in women than inmen *?re//a et al., 2334E Seit/ et
al., 2339- and can also be inhibited by certain drus, such as as!irin and histamine '
1
rece!tor blockers *6abelleria et al., 2332-. ;hen astric .5'activity is reduced,
blood ethanol levels after ?#$ are reduced. The astric mucosa contains several
.5', for exam!le, b-.5', -.5', and -.5', that could be involved in the
metabolism of ethanol *6rabb et al., 1447-. -.5', a maGor astric .5' isoen/yme,
was absent in the stomach bio!sies of about 94R of .sians and those lackin in this
en/yme had lower ?#$ of ethanol *5ohmen et al., 233D-, suestin that -.5' is
im!ortant in astric oxidation of ethanol. The relationshi! between ?#$ and the rate
of astric em!tyin suests that !roloned contact of the inested alcohol solution
with the stomach favors absor!tion of the alcohol transastrically, where it could be
subGect to oxidation in stomach mucosal cells.
)n 233D, .mmon et al. ave ethanol intravenously and
1
'-labeled ethanol by
mouth or administered into the duodenum. They found that ?#$ was CM3R of the
oral dose and estimated that the astric contribution to ?#$ was about DR of the oral
dose. Hender differences in ?#$ are !robably not a maGor factor *.mmon et al.,
233D-. The overall im!ortance of ?#$ miht lie in the !otential for astric ?#$ to
!rotect the liver and other orans from low doses of ethanol and for certain drus to
block ?#$ *6abelleria et al., 2332-, resultin in intoxication from smaller than
ex!ected doses of ethanol. ?urthermore, this !rocess is an obvious !oint of
intersection of diet and timin of meals with ethanol consum!tion.
;hen astric .5' activity is reduced, blood ethanol levels after ethanol inestion
are increased. Some investiators have arued that individuals with reduced astric
.5' activity are more susce!tible to liver diseases, because of increased delivery of
ethanol to the liverE however, in liht of the many factors involved in ethanol
absor!tion and eliminations, this hy!othesis has not been !roved. Overall, the
im!ortance of astric .5' and astric ?#$ in the !atho!hysioloic mechanisms of
alcoholic liver disease *.L5- is unsettled *Smith et al., 2331-.
#ro!erties of .5' in man+
G($( !&#s S#b#$it ty3( K
.
@(tha$!A V
.aH
Tiss#(
/istrib#ti!$
6lass )
.5'2 c 7 07 Liver
.5'1 a 4.40M97.4 M Liver, lun
.5'9 b 4.DM2.4 M Liver, stomach
6lass ))
.5'7 j 97 74 Liver, cornea
6lass )))
.5'0

2444 M $ost tissues
6lass )I
.5'< k,u 14 2024 Stomach,
eso!haus,
other mucosa
6lass I
.5'D M 94 [ Liver, stomach
6lass I)
.5'C M M M &ot detected in
man, found in
deer muscles,
rat liver
Of the seven .5' enes in humans, .5'1 and .5'9 are known to be
!olymor!hic with three and two alleles. .lleles associated with somewhat hiher
.5'activity *.5'1
l
1 and .5'9
l
1- may enhance the adverse effect of alcohol,
while those with slower activity *.5'1
l
2 and.5'9
l
1- may increase alcohol
tolerance and !romote alcoholism *(oberts, 1447-. . mutation in the !romoter reion
*6
1
allele- of cytochrome #
704
en/yme, 6=#1E2, results in enhanced en/yme activity
and a!!ears to be associated with some increase in risk for cirrhosis in both :a!anese
and 6aucasian !o!ulations *(oberts, 1447-. One recent study suested that the
ethanol concentrations of the consumed beveraes, and not interindividual variations
in the activities of first-!ass alcohol metaboli/in en/ymes, are associated with H)
sym!toms in alcoholics *LaheiG et al., 1447-.
B.D. E''(&t !$ th( S.a I$t(sti$(
B.D.>. Abs!r3ti!$ !' .a&r!% a$/ .i&r!$#tri($ts
2. $onosaccharides+ 5ecreased absor!tion of 5-xylose has been re!orted
in alcohol-abusin subGects without clinically overt liver disease, but
the !revalence of disturbed absor!tion of 5-xylose varies markedly. )n
five studies, 2CM<DR !atients of alcohol abuse had altered 5-xylose
absor!tion. This variation may reflect differences in the mean daily
alcohol intake of the subGects studied and,or in their nutritional status
*Bode et al., 2334-. The absor!tion of lucose does not seem to be
affected by chronic alcohol administration *Bode et al., 2334-.
1. .mino acids+ 6hronic ethanol intake did not affect the absor!tion of
L
-
leucine from he small intestine of rats fed on a nutritious diet *$artines
et al., 23C2-. Thus, it seems that the inhibition of amino acid absor!tion
induced by acute alcohol administration *Beck et al., 23C2- is not
maintained when alcohol is consumed chronically.
'.4.2. .rotein/ "at/ and comple car%ohydrates
Steatorrhea and increased fecal nitroen excretion are well-known features of
alcohol-abusin !atients with exocrine !ancreatic insufficiency and,or advanced
.L5. 'owever, increased fecal fat excretion has also been re!orted in alcoholics
without cirrhosis or chronic !ancreatitis *Bode and Bode, 2334-. )ncreased fecal
nitroen excretion was also observed in about 04R of alcoholics who had neither
advanced liver disease nor chronic !ancreatitis *(oin et al., 23D3-. The
!athomechanism of the disturbed diestion and, or absor!tion of li!ids and !rotein in
alcoholics has not been clarified. The findin that ethanol interferes with the intestinal
hydroli!ids of !e!tides *5inda et al., 23C7- miht at least !artially ex!lain the
Smalabsor!tion% of !rotein.
.n im!ortant contribution to this field came from a study on alcoholics without
con-foundin disorders such as cirrhosis or !ancreatic insufficiency *#feiffer et al.,
2331-. ;hen the duodenal and GeGunal absor!tion of a nutrient solution containin a
mixture of !roteins, li!ids, and carbohydrates *34R dextrin maltose- was determined
usin an intestinal !erfusion techni"ue, the duodenal absor!tion of all these nutrients
was lower in alcoholics com!ared to ae-matched controls, but GeGunal absor!tion
rates were not decreased when the data for the duodenal and the GeGunal sements
were combinedE no statistically sinificant differences between the two rou!s were
detectable for any of the nutrients *#feiffer et al., 2331-. The authors ex!lained that
the different absor!tion rates in the duodenum versus the GeGunum of the alcoholics
were due to the more !ronounced alcohol-induced mucosal damae in the duodenum.
'.4.'. 0ater and electrolytes
Both in healthy subGects and in alcoholics, chronic alcohol consum!tion leads to
mark-edly reduced water and sodium absor!tion in the GeGunum and ileum *#feiffer et
al.,2331-. The reduced water absor!tion may contribute to diarrhea in alcoholics.
'.4.4. 1itamins
2. ?olic acid+ ?olate deficiency, documented by low serum levels, has been
described in about D4M<4R of bine drinkers *Lindenbaum, 23C4-.
(educed absor!tion is one of the causative factors. #ure nutrition with
inade"uate dietary folate intake a!!ears to be of reater im!ortance. .
more !ronounced folate deficiency may further aravate GeGunal u!take of
nutrients *Lindenbaum, 23C4-.
1. Iitamin B21+ 6hronic alcohol abuse may cause malabsor!tion of vitamin
B21 *Lindenbaum, 23C4-. 'owever, des!ite the fact that alcohol may
interfere with vitamin B21 absor!tion, few alcoholics have clinical
evidence of vitamin B21 deficiency *'alsted et al., 2334-.
9. Thiamine+ 6onflictin results have been !ublished reardin the effect of
chronic ethanol inestion on the intestinal absor!tion of thiamine in man.
)n one study, a reduced absor!tion was observed, while in other studies, no
effect on thiamine absor!tion was seen *Thomson, 1444-. ?rom the results
of studies !erformed in rats, it has been suested that alcohol
administration inhibits only the active trans!ort of thiamine at low
concentrations of the vitamin *'oyum!a et al., 23<0-. .lcohol abuse in
cirrhotics was found to be associated with low !lasma thiamine
concentrations and reduced thiamine !hos!horylation *Tallaksen et al.,
2331-. )nade"uate dietary thiamine intake is assumed to be an im!ortant
cause for thiamine deficiency in alcohol-abusin subGects.
7. Iitamin BD+ Iitamin BD *!yridoxal-0 !hos!hate- deficiency is observed in
a hih !ro!ortion of alcohol abusers *'alsted, 2334-. The deficiency of
this vitamin a!!ears to be caused by factors other than malabsor!tion
*'alsted, 2334-.
0. Iitamin 6+ Low blood levels of ascorbic acid are !rimarily found in
im!overished alcoholics. Leadin causes of vitamin 6 deficiency in
alcoholics are inade"uate intake and increased urinary excretion, but
alcohol has also been re!orted to im!air absor!-tion of this vitamin
*Lieber, 23CC-.
D. Iitamins ., 5, E, and >+ #lasma concentrations of the fat-soluble
vitamins are !artially below normal levels in alcoholic individuals. The
!revalence of low levels of these vitamins varies markedly de!endin on
the nutritional status and the !res-ence of more advanced staes of liver
disease and chronic !ancreatitis. )ntestinal absor!tion of these vitamins is
im!aired in the !resence of !ancreatic insufficiency or cholestasis but they
a!!ear to be absorbed normally in alcoholics without end-stae oran
diseases.
Calcium and magnesium+ The absor!tion of both minerals is not sinificantly
affected by acute or chronic alcohol inestion. 'y!omanesemia is a fre"uent findin
in alcoholics but it is caused mainly by increased manesium excretion in the urine or
as a result of diarrhea *BuGanda et al., 1444-
?inc+ .lcohol abuse is fre"uently associated with /inc deficiency *$c6lain et
al.,2334-. $aGor reasons for /inc deficiency noted in alcoholics with or without liver
disease are inade"uate dietary /inc intake and increased urinary loss of /inc. )m!aired
/inc absor!tion is another likely cause of /inc deficiency in alcoholics *$c6lain et al.,
2334- althouh in some studies no abnormalities of /inc absor!tion in alcoholics were
found
Iron+ )ncreased iron stores are common in alcoholics *.dams, 233C-. The effect of
chronic alcohol consum!tion on iron absor!tion in man has, however, not been clearly
defined *#ersson et al., 2332-. ?rom the results of a more recent study, it was
concluded that unreulated increased iron absor!tion via the non-carrier-mediated
!aracellular route contributes to the iron overload in chronic alcoholics *5uane et al.,
2331-.
!elenium@ 5iminished contents of selenium in serum and erythrocytes have
re!eatedly been observed in alcoholics with and without liver disease *Thuluvath and
Trier, 2331-. ;hether or not loss of this trace element is due to malabsor!tion
contributin to sele-nium deficiency in alcoholics remains an unanswered "uestion.
'.4.+. Increased gut permea%ility
)ncreased intestinal !ermeability to macromolecules followin acute alcohol
inestion was re!orted in earlier animal ex!eriments for hemolobin, horseradish
!eroxidase, and !olyethylene lycol *#EH- 2044 $r *Bode et al., 23C4-. The
increased !ermeability was shown to be connected to destabili/ation of intercellular
tiht Gunctions of entero-cytes, thus facilitatin an extracellular shunt !athway for the
u!take of macromolecular absor!tion.
Evidence of an increased intestinal !ermeability caused by alcohol consum!tion in
human subGects was first re!orted when smaller molecules *$r 044- were used as a
!ermeability !robe *BGarnason et al., 23C7-. 6hronic ethanol reversibly affects the
inte-rity of small intestinal villi without sinificantly affectin H) !ermeability. )n
contrast, a sinle oral dose of ethanol increases astroduodenal !ermeability but has
no effect on lactulose or mannitol !ermeability of the small intestine. These reional
chanes in ut !ermeability may contribute to alcohol-induced H) sym!toms
*>eshavar/ian et al., 2337-. (ecently, sinificantly increased intestinal !ermeability
in actively drinkin alcoholics has been confirmed for macromolecules such as #EH
7444 and 24444 $r *#arlesak et al., 1444-. The enhanced intestinal !ermeability for
these macromolecules had already been found in alcoholics without advanced staes
of liver disease M a findin that stronly su!!orts the assum!tion that the increased
!ermeability of the ut mucosa is caused by ethanol itself and is not a conse"uence of
advanced *(ao et al., 1447-. Hastroduodenal !ermeability is also increased in !rimary
biliary cirrhosis. Hut !ermeabil-ity in *#EH $r 24 444- !atients at different staes of
.L5 is shown below+
Pati($ts # PEG i$ #ri$(M+( @PA
6ontrols 97
?atty liver 2< <*72-
l
.lcoholic he!atitis 2C <*93-
ll
.lcoholic cirrhosis 23 D*92.0-
l
#arlesak et al. *1444-.
l
#V4.40 versus controls
ll
PV4.442 versus controls.
The hy!othesis that the acute and chronic inestion of lare "uantities of alcohol
enhances the translocation of macromolecules throuh the intestinal mucosa is
su!!orted by the observation of a transient endotoxemia followin acute alcohol
consum!tion in healthy volunteers and in alcoholics with fatty liver *Bode et al.,
233C-. 5irect evidence of enhanced translocation of endotoxin from the ut lumen
into the !ortal blood has been achieved in ex!eriments usin TsukamotoM?rench
model in rats *$athurim et al., 1444-. The intermittent endotoxemia stimulates
>u!ffer cells and other macro!haes in the liver, thereby enhancin the release of
reactive oxyen s!ecies and !roinflammatory mediators such as tumor necrosis factor
al!ha *T&?-c-, interleukin 2 *)L-2-, and inter-leukin D *)L-D- *Bode et al., 233C-.
6hronic over!roduction of such mediators may induce the influx and activation of
neutro!hil leukocytes, endothelial lesions, increased !ermeability of sinusoids,
disturbed microcirculation, and other inGurious events that finally lead to severe forms
of liver inGury and oran damae *Bode et al., 233CE 5emeo et al., 1441E $c6lain et
al., 2339-.
B.E. E''(&t !$ M#&!sa M!r3h!!"y
)n ex!erimental animals *rodents, dos-, acute administration of alcoholic
solutions at concentrations corres!ondin to those of commonly available alcoholic
beveraes leads to mucosal damae in the small intestine that extends to loss of
e!ithelium at the ti!s of the villi, hemorrhaic erosions, and hemorrhae in the lamina
!ro!ria *5inda et al., 23C2-. )n accordance with these findins are the results obtained
in a lare, !ros!ective caseMcontrol study that !rovided evidence that consum!tion of
alcoholic beveraes sinificantly increases the risk of maGor duodenal bleedin in
non-!redis!osed individuals *>elly et al., 2330-. On the basis of extensive
ex!erimental studies, other authors have !ro!osed an indirect effect of alcohol on the
mucosal microcirculation that leads to an enhanced transca!illary fluid filtration and
subse"uent ru!ture of the e!ithelial linin *5inda et al., 23C2-. )n additional studies on
animals, the same rou! !rovided evidence that alcohol induces villus contraction,
which leads to villus ti! bleb formation, lym!hatic obstruction, and, eventually,
exfoliation of the ti!s of the villi when blebs ru!ture *Beck et al., 23C3-. ?rom the
results of more recent ex!eriments, these authors have suested that the initial event
in res!onse to alcohol is an increased influx of leukocytes, leadin to an enhanced
release of noxious mediators, such as reactive oxyen s!ecies *5inda et al., 233D-,
leukotrienes *Beck et al., 23CC-, and histamine by mast cells.
8nlike the effects of acute inestion, whether or not chronic alcohol inestion
damaes, the mucosa of the small intestine remains controversial *Harcia et al., 2330-.
)n ex!erimental rats, sinificant small intestinal histoloic alterations have been
re!orted as shortenin of villi and increased microvascular !ermeability *Estrada et
al., 233D-, as well as com!ensatory hy!erenerative !rocesses and !ossibly
carcinoenesis *#ronko et al., 1441-. $oreover, the number of mononuclear cells was
hiher in chronic ethanol-fed ex!erimental rats *Ia"uera et al., 1441-. These studies
describe usin liht microsco!y, and both normal histoloy and sinificant
histoloical alterations includin inflammatory infiltrates and blebs at the a!ex of the
villus.
)n man, chronic alcohol administration has iven conflictin results. The close
asso-ciation between hih alcohol intake and the risk of acute bleedin in both the
stomach and the duodenum re!orted recently *>elly et al., 2330- su!!orts the
assum!tion that the ty!e of mucosal inGury in acute alcohol ex!osure discussed above
does also occur after heavy drinkin in subGects chronically abusin alcohol. The
results of a recent study suest that chronic alcohol abuse induces alterations of the
matrix network and increases the number of myofibroblast-like cells in the duodenal
mucosa M findins com!atible with the develo!ment of fibrosis in the intestinal
mucosa *6asini et al., 2333-. )n one study, the chanes in distribution and function of
intestinal )ELs in relation to intestinal barrier dysfunction in ex!erimental cirrhosis
show !roliferative res!onse of )ELs, which was sinificantly increased in the cirrhotic
rou! as com!ared to controls *)namura et al., 1449-.
)t has also been found that ethanol sinificantly reduces cell rowth in cultured rat
intestinal e!ithelial cells *&ano et al., 2334-. )t has been s!eculated that continuous
villus cell loss throuh normal !rocesses accom!anied by im!aired mitotic activity in
the cry!t e!ithelium will eventually lead to reduced villus heiht and ultimately to
subtotal villus atro!hy *&ano et al., 2334-.
The most characteristic feature of the normal mucosal ultrastructure of absor!tive
e!ithelium is the brush border, which is situated at the luminal !ole of the enterocyte.
Brush border is com!osed of two different substructures+ the surface *microvillus-
membrane and the underlyin cytoskeleton.
The microvillus membrane consists of a li!id bilayer, the luminal surface of which
is lined with a fine filamentous coat, rich in acid muco!olysaccharides, known as
lycol-calyx. The membrane !ossesses a lare com!onent of interal !roteins, many
of which are lyco!roteins and have been identified with s!ecific brush border
en/ymes.
The cytoskeleton is com!osed of actin filaments com!risin the microvillus core
and continues as terminal web into the u!!er !art of the cellular cyto!lasm. ;ithin
the cyto!lasm, there is abundant endo!lasmic reticulum and numerous mitochondria,
Holi com!lex, and lysosomes. The nucleus situated in the lower half of the cell is
elli!tic or irreularly rounded with shallow indentation *;an and .ndersson, 2337-.
'.+.1. !ther cell types with distinct ultrastructural "eatures
8ndifferentiated cry!t cells+ These are round and lare, often havin a centrally
situated nucleus with s!arse endo!lasmic reticulum, few mitochondria, and an
abundance of unattached ribosomes.
5ifferentiated cry!t cells+ They are elonated with a more basally situated nucleus
and a more discernible brush border with an abundance of rouh endo!lasmic
reticulum, more numerous mitochondria, and an active Holi com!lex.
#aneth cells+ They can be distinuished by lare membrane-bound electron-dense
secretory dro!lets *04M144- that are often surrounded by a lihter halo.
Hoblet cells+ They have microvilli at their luminal end and a basally situated
nucleus with an abundant, rouh endo!lasmic reticulum. The cells contain dro!lets
like !aneth cells which contain a homoeneous flocculent material that is less
electron-dense than !aneth cells.
Enterochramaffin cells+ They contain intracyto!lasmic electron-dense ranules of
varyin si/e but smaller than exocrine secretory cells *'aubrich et al., 2330E 'olmes
and Lobley, 23C3-.
)n ex!erimental alcohol-fed rats, ultrastructural evaluation of the intestinal
e!ithelium revealed loss of contact between enterocytes and abnormal dilatation of
the cisternae of the Holi a!!aratus. ?urther increased lysosome-like vesiculation and
enhanced lyso-somal beta-alactosidase activity was observed. The total number of
absor!tive entero-cytes in the e!ithelium was reduced by 94R in ethanol-ex!osed
newborn rats as com!ared to controls *Estrada et al., 233D-. )t has also been shown
that ethanol alone and !erha!s a hih level of acetaldehyde may be res!onsible for the
inhibition of intestinal !rotein synthesis and related !atholoical deranements
*$arway and #reedy, 2330-. )n a sinle small study on alcoholics by #ersson et al. in
2334, ultrastructural evaluation of intestinal e!ithelium showed bacterial adhesion to
the mucosal surface in addition to deeneration of villus cells.
B.G. E''(&t !$ M#&!sa E$-y.(s
.lcohol can interfere with the activity of many en/ymes located in the brush
border or other com!artments of the enterocytes.
'.-.1. 2rush %order en3ymes
5isaccharidases+ They are en/ymes that remove sinle lucose residues from
c*2m7-- linked disaccharides. )n ex!eriments !erformed in rodents for the activity of
disacc hari-dases *lactase, sucrase, maltase, and trehalase-, both reduced and
unchaned values have been re!orted followin acute or chronic alcohol
administration *#ersson et al., 2331-. 'owever, an increase in disaccharidases was
found in cultured rat cells after ex!osure to ethanol *&ano et al., 2334-. )n humans,
#erlow et al. had re!orted in 23<< that chronic alcohol inestion decreased GeGunal
disaccharide activities even in the absence of overt malnutrition. The decrease in
en/yme activity !roduced by alcohol is associated with increased morbidity after
lactose administration. Lactase activity in GeGunal bio!sies was found to be below
normal in only a small !ercentae of white alcoholics but was a reular findin in
.merican blacks, in whom lactase deficiency is more fre"uently found than in whites
*Bode, 23C1-. 5ecreased lactase activity can be a contributin factor to milk
intolerance with diarrhea commonly observed in alcoholics after a !eriod of heavy
drinkin *>eshavar/ian et al., 23CD-. 6onflictin data have been re!orted concernin
the effect of chronic alcohol abuse on the activity of sucrase and maltase *Bode,
23C1-. Some studies have re!orted no chane in activity of mucosal disaccharidases
*Nucoloto et al., 233D- whereas others have shown a decrease in lactase level with no
chane in maltase and sucrase level *Lhatoo et al., 1444-.
9.D.2.2. Hamma lutamyl transferase
HHT is an en/yme of amino acid metabolism that cataly/es the transfer of amma
lutamyl residues to amino acids or small !e!tides. HHT is involved in the synthesis
and metabolism of lutathione throuh the amma lutamyl cycle *Holdber et al.,
23D9-. There is ood evidence that this !lays a role in the absor!tion of amino acids
from the lomerular filtrate and from the intestinal lumen throuh a translocation
mech-ani sm.)t a!!ears to be a villus-s!ecific en/yme at least in rat mucosa *6ornell
et al., 23<D-. 6hronic ethanol feedin to rats resulted in a sinificant increase in serum
HT# activity M an indication of hih intestinal HT# activity. The histochemical
stainin of HT# in the mucosa of the small intestine of these animals demonstrated
enhanced HT# activity at the microvilli of the brush border membrane, lamina !ro!ria
of the mucosa, and endo!lasmic reticulum of intestinal e!ithelial cell *)shii et al.,
23CC-. 6hronic alcohol administration increases the activity of this en/yme in the
intestinal mucosa in animal ex!eriments and in man *Bode and Bode, 1449E #ersson
et al., 2332-. )t has been suested that intestinal HHT may contribute to the increased
serum HHT values commonly seen after hih alcohol intake in man. )n a small study
in humans by Harcia et al. in 2330, sinificant increases were observed in the HHT in
the intestinal mucosa of alcoholics with active alcohol intake as com!ared to the
control rou!. The chanes were ra!idly reversible followin abstinence and
correlated with the chanes observed in serum, thus suestin an en/ymatic
induction mechanism. HHT is still used as a traditional marker of excessive alcohol
use *6onirave et al., 1449-.
9.D.2.1. .lkaline !hos!hatase
#hos!hatase removes !hos!hate from certain oranic !hos!hates such as
hexose!ho-s!hate, lycero!hos!hate, and nucleotides derived from the diet and
diestion of nucleic acids by nucleases. )ts workin !' is C.D. )ntestinal alkaline
!hos!hatase *.#- differs from nonintestinal .# by substrate s!ecificity *>eidin,
23DD- and electro!horetic mobility *Baroana et al., 23<7-. .# is a hydrolytic en/yme
actin o!timally at alkaline !', exists in blood in numerous distinct forms which
oriinate mainly from bone and liver, but also from other tissues such as kidney,
!lacenta, intestine, testes, thymus, lun, and tumors *$oss and 'enderson, 2333-.
There are conflictin results with reard to the effect of acute *Baroana et al., 23<7E
&ano et al., 2334- and chronic *'aGGar et al., 23C2- ethanol administration on this
en/yme. The level of intestinal .# has shown to be increased in man *'aue et al.,
233C- whereas in ex!erimental rats it is shown to be decreased *5eoleiviera et al.,
2330- as well as in rabbits *>eslam et al., 23C0-. .nother study has shown increased
.# activity in rats *>aur et al., 2337- an rat intestinal cell line *&ano et al., 2334-. )t
has been observed that ethanol feedin induced hy!erli!idemia *>aur et al., 2337- and
.# levels increased under hy!erli!emic conditions *Ellakim et al., 2333-. But the
exact status is not known in chronic alcoholics.
9.D.2.9. Leucine amino !e!tidase
Leucine amino !e!tidase *L.#- cataly/es the hydrolysis of di- and tri!e!tides. )t
is a marker of !e!tide diestion and leucine release, and a re!resentative of amino
acid absor!tion in the small intestine *'eifer et al., 23<1-. Little is known about the
function of this exo!e!tidase beyond the fact that it is secreted by the mucosa of the
small intestine
L.# activity has not been shown to be altered by chronic ethanol administration
in rats *>aur et al., 2337-. )n a small study on humans, activity of L.# was lower in
chronic alcoholics, #V4.40 *Bode et al., 23C1a,b-. . study by Bhalla et al. in 1447
showed that !renatal ex!osure of ethanol to rat !u!s leads to a decrease in their body
weiht and lenth of intestine, and reduces the u!take ca!acity of enery-de!endent
lycine and
L
-leucine trans!orters as com!ared to controls.
9.D.2.7. Lactate dehydroenase
Lactate dehydroenase *L5'- is an en/yme of anaerobic lycolysisE it converts
!yruvate to lactate usin &.5', thus re!resentin anaerobic metabolism of lucose.
)t is also known as an en/yme re!resentin the viability of the cell+
#yruvate B &.5' B ' -` Lactate B &.5
B
?ive isoen/ymes of L5' have been described. $easurement of the combination
of ascetic fluid, total !rotein, lucose, and L5' has been found to be valuable in
distinuishin small bowel !eritonitis *SB#- due to ut !erforation from ascites
*.kriviadis and (unyon, 2334-. The !revalence of SB# in cirrhotic !atients with
ascites who are hos!itali/ed ranes from 24R to 94R with a!!roximately half the
e!isodes !resent at the time of hos!itali-/ation *Harcia, 2331-. The ascitic fluid
concentration ofL5'is usually less than half of the serum level in uncom!licated
cirrhotic ascites. )n SB#, the ascitic fluid L5' level rises because of neutro!hil
release of L5', such that the ascitic fluid level is reater than that of serum. )n
secondary !eritonitis, the L5' level rises even hiher than SB# and may be several
times hiher than in serum *.kriviadis and (unyon, 2334-. Ethanol was oxidi/ed to
acetate by an en/yme system com!risin L5'-recyclin&.5in two model duodenal
fluids and in canine duodenal as!irate in vitro *;hitmire et al., 2332-.
'.-.2. 4em%rane en3ymes
&a
B
M>
B
.T#ase, adenylate cyclase, and uanylate cyclase
$embrane &a
B
M>
B
.T#ase is im!ortant for the maintenance of the ionic e"uilib-
rium of intestinal cells and the !roduction of .T#, a hih-enery molecule, in the case
of excess !otential difference. )t is involved in the active trans!ort mechanisms, for
lucose and amino acids, across the membrane *;ilson and 'oyum!a, 23<3-. )n
eneral, cells maintain a low intracellular &a
B
concentration and hih intracellular >
B
concentration alon with a net neative electrical !otential inside. The !um! that
maintains these radients is .T#ase, which is activated by &a
B
and >
B
.T#ase both in
the brush border and in the basolateral membrane *Oneil et al., 23CD-. Thus, it inhibits
enery-de!endent trans!ort systems *5-lucose and amino acid absor!tion-. The
effect on acute and chronic administration of ethanol on GeGunal and ileal water and
electrolyte trans!ort was studied in healthy volunteers by tri!le lumen intestinal
!erfusion techni"ue *$ekhiGan and $ay, 23<<-. )t a!!eared that the diarrhea seen in
chronic alcoholics could be ex!lained in !art by the effect of ethanol in sodium
trans!ort, without any accom!anyin chanes in serum folate levels.
)ntestinal adenylate cyclase is involved in the formation of c.$#. )t has been
shown that ethanol increases the activity of adenylate cyclase and therebyc.$#*Bode
et al., 23C4-.This may !lay a role in enhanced intestinal secretion of sodium and
water. )n the colonicmucosa of rat, enhanced !roduction of colonic cyclic .$# has
been observed *Siet/ et al., 23C9-.
B.G.B. C(#ar ($-y.(s
9.D.9.2. Hlucose D !hos!hatase
The conversion of lucose D !hos!hate to lucose for lycoen synthesis is
cataly/ed by the s!ecific !hos!hatase, lucose D !hos!hatase. )n a small study by
Bode et al. in 23C1 on GeGunal bio!sies of chronic alcoholics, the activity of lycolytic
en/ymes was not affected.
9.D.9.1. Hlucose D !hos!hate dehydroenase
Hlucose D !hos!hate dehydroenase *H-D#5'- is the first en/yme in the !entose
!hos-!hate !athway, a re!resentative of an alternative route for the metabolism of
lucose, for the !roduction of &.5#' re"uired for fatty acid synthesis, and ribose
residue for nucleotide, nucleic acid biosynthesis, etc. 5ehydroenation of lucose D
!hos!hate to D !hos!holuconate occurs via formation of D !hos!holuconolactone
cataly/ed by H-D#5', an &.5#-de!endent en/yme. (eaction occurs in cytosol.
Three to four !ercent of cellular &.5#B H-D-# 5' activity is associated with
!eroxisomal fractions *Ta!!ia et al., 233C-. . sinificant decrease in the enterocyte
level of antioxidant en/ymes includin H-D#5' in Aibrio cholerae 4293-mediated
intestinal infection has been found in rabbit ileum *Horawara et al., 233C-..decline in
the s!ecific activity of H-D#5'was seen in the -iardia lamblia-infected mice *&ain
et al., 2332-. $itochondrial en/ymes in rat liver or intestine were investiated in
ex!erimental models with ethanol-fed rats. Smooth endo!lasmic reticulum was
increased in content and !entose !hos!hate shunt was inhib-ited by chronic ethanol
treatment, estimated from activities of &.5' ferricyanide and decreased activity of
H-D#5'. The chanes in he!atic en/ymes with ethanol treatment were !aralleled
with those of intestinal ones, indicatin that metabolic chanes in the intestine
contribute in some way to the formation of alcoholic fatty liver *Ohtani, 23C4-.
9.D.9.9. )socitrate dehydroenase
)socitrate dehydroenase *)65'- is a citric acid or tricarboxylic acid cycle
en/yme. )socitrate underoes dehydroenation in the !resence of )65' to form
oxalosuccinate. )n mammalian tissues, there are two &.5#-s!ecific )65'
isoen/ymes located in the mitochondria and cyto!lasm res!ectively. The isoen/ymes
of these two cell com!art-ments are under inde!endent enetic control. )n mammalian
cells, the heart and skeletal muscle contain !redominantly the mitochondrial &.5#-
s!ecific en/yme *$c?arlane et al., 23<<-. ?ive isoen/ymes have been re!orted in
various human tissues after electro-!horetic se!aration of )65' *5ror et al., 23<4-.
)n 2332, #reedy et al. studied the metabolic conse"uences of chronic ethanol
feedin in male ;istar rats and concluded that there was a decrease in citric acid
cycle activity found by !roton nuclear manetic resonance s!ectrosco!ic analysis,
which also showed reduced urinary excretion of citrate and 1-oxolutarate.
B.K. Ba&t(ria F!ra
The bacterial flora of the H) tract !lays an essential role in human !hysioloy. )n
healthy adults, the constant interaction between many microoranisms !roduces a
stable colonic ecosystem which maintains the interity of the enterocyte, modulates
metabolic and immunoloic !rocesses, and !rotects aainst coloni/ation by invasive
!athoens *5onaldson, 23D7-.
.s many as 744 different aerobic and anaerobic microoranisms have been
isolated. The nature and number of microoranisms differ from one !art of the H)
tract to the other. )n addition, the manitude of the flora and its com!osition vary
between !o!ulations and !atient rou!s. $icroflora of the stomach !redominantly
consists of Hram-!ositive aerobic microoranisms, the most commonly isolated
s!ecies bein !treptococci$ !taphylo-cocci$ Lactobacilli, and various funi. The
bacterial concentration in the stomach is usually less than 24
9
cfu ml
-2
. Oral anaerobes
such as Peptostreptococcus and 'usobacterium s!ecies may be !resent in small
numbers but 6oliforms, 6lostridia, and acteroide are distinctly uncommon
*5onaldson, 23D7-.
$icroflora of the !roximal small intestine is similar to that of the stomach. The
bacterial concentration is 24
9
M24
7
cfu ml
-2
. The !redominant s!ecies are aerobic and
Hram !ositive, althouh 6oliforms and anaerobic bacteria can be fre"uently isolated
in low concentration. )n the distal ileum, the concentration of oranisms is 24
0
M24
3
oranisms ml
-2
. 6oliforms are consistently !resent and anaerobic bacteria such as
bacteroides, bifidobacteria, fusobacteria, and clostridia are found in substantial
concentration. Bacterial concentration in the colon is 24
22
M24
21
cfu ml
-2
. .naerobic
bacteria outnumber aerobes by factors of 24
1
M24
7
. #redominant isolates are
bacteroides, bifidobacteria, and eubacteria. .naerobic Hram-!ositive 6lostridia,
Enterococci, and various Enterobacteriacea are common *Simon and Horbach, 23C7-.
The intestinal habitat of an individual contains 944M044 different s!ecies of
bacteria. The stomach and small intestine contain only a few s!ecies of bacteria
adherin to the e!ithelia. The scarcity of the bacteria in the u!!er tract seems to be
because of the com!osition of the luminal medium *acid, bile, !ancreatic secretion-
which kills most inested microoranisms, and because of the !hasic !ro!ulsive
motor activity toward the ileal end, which im!edes the stable coloni/ation of bacteria
in the lumen. By contrast, the lare intestine contains a com!lex and dynamic
microbial ecosystem with hih densities of livin bacteria *Huarner and $alaaleda,
1449E Simon and Horbach, 23C7-. S!arsely !o!ulated in its !roximal !osition, the
distal small intestine constitutes a /one of transition from the minimal flora of the
stomach to the lush and diverse bacterial flora of the colon. The bacteria ordinarily
encountered in the duodenum, GeGunum, and initial !ortions of the ileum are similar to
those encountered in the stomach. .s the ileocecal valve is a!!roached, however, the
number and variety of Hram-neative bacteria bein to increase *Horbach, 23<2-. )n
fact, the diversity in the H) tract a!!ears to be the result of stron host selection and
coevolution *Backhed et al., 1440-.
)n only one study usin a s!ecially devised tube, marked "ualitative and
"uantitative alterations of the GeGunal microflora have been documented in recent
alcoholics *Bode et al., 23C7-. &early 04R of the alcoholics had a marked increase in
the total number of bacteria *reater than 24
0
colony-formin units !er ml of GeGunal
as!irate- with a !redominant increase in bacteria from fecal flora.
#revalence of bacterial overrowth in alcoholics was determined usin the '
1
breath test after inestion of lucose or lactulose *Bode et al., 233C-. Bacterial
overrowth in the u!!er small intestine miht contribute to mucosal damae and
disturbed absor!tion in subGects chronically abusin alcohol. ?urthermore, it miht
increase the !roduction of bacterial toxins, es!ecially endotoxins from Hram-neative
bacteria, which, in combina-tion with mucosal inGury induced by alcohol, miht
contribute to an enhanced endotoxin *Bode et al., 233CE =amashina et al., 1449-
translocation from the luminal side into the !ortal blood. The e!ithelial monolayer
selects the luminal substances to be trans!orted from the lumen. This barrier normally
excludes endotoxin, a com!onent of the outer wall of Hram-neative bacteria, !resent
in the bacterial flora in the intestine. 'owever, this !otentially harmful substance
triers inflammation in case it crosses the ut barrier *(aet/ et al., 2332E Schafer et
al., 1441-. . maGor cause of alcohol-induced endotoxemia is now thouht to be an
overrowth of enteric bacteria *.sai et al., 1449-. 6ontinuous bacterial translocation
in !atients can cause s!ontaneous bacterial !eritonitis, an im!or-tant com!lication of
advanced disease. )n this settin, overrowth of bacteria within the small bowel has a
bier role than do colonic sources *Huarner et al., 233<-. %scherichia coli is the most
fre"uently im!licated oranism in bacteremic e!isodes of cirrhosis *Levy et al., 1449-.
'owever, in a recent study on ex!erimental rats, reduced rates of bacterial
translocation were observed after chronic ethanol intake. This miht have occurred
because of a cellular dysfunction or inefficient adhesion of bacteria to intestinal
e!ithelium *Sou/a et al., 1449-.
)n 23CD, Baroana et al. investiated the role of intestinal bacterial overrowth in
the !roduction and metabolism of ethanol, where rats with a GeGunal self-fillin
diverticulum *blind loo!- were com!ared to controls with a self-em!tyin
diverticulum. Both in rats with a blind loo! and controls, the areas under the curve of
blood ethanol concentrations were smaller after intraastric than after intravenous
ethanol administration *2 ethanol !er k body weiht-. This difference was
exaerated in rats with a blind loo!. Thus, a considerable amount of ethanol is
oxidi/ed in the H) lumen of rats with a blind loo!. The bacterial count was hiher in
the blind loo!, leadin to acetaldehyde !roduction. The resultin hih concentrations
of acetaldehyde, both in the intestinal lumen and !ortal blood, may have deleterious
effects on the H) mucosa and the liver. They concluded that in the H) tract, bacterial
overrowth could aravate the direct he!atotoxicity of alcohol because of its im!act
on acetaldehyde !roduction.
.cetaldehyde, a hihly reactive and toxic metabolite of ethanol, has been linked to
several toxic effects of ethanol on orans *=okoyoma et al., 233C-. )n addition, acetal-
dehyde !roduced via microbial ethanol oxidation has been !ro!osed to be a
!athoenic factor in ethanol-associated H) inGury *Salas!uro et al., 2333E Seit/ et al.,
2334-. Bacterial analysis has revealed that mainly Hram-!ositive aerobic bacteria and
yeasts are associated with hiher acetaldehyde !roduction *'omann et al., 233<-.
'.5.1. 2acteriocolonic meta%olism o" alcohol
?rom the results of recent ex!erimental research and of studies !erformed in man,
there is increasin evidence that the ut flora forms an im!ortant oran for ethanol
metabolism in the lare bowel *#ronko et al., 1441-. $any facultative anaerobic and
aerobic H) bacterial strains have substantial .5' activity in vitro and,
corres!ondinly, are ca!able of oxidi/in ethanol to acetaldehyde in vitro and in vivo
*:okelainen et al., 233D-. The .L5's of intestinal bacteria, such as members of the
Enterobacteriacea, however, have a limited ca!acity to metaboli/e acetaldehyde
!roduced by their .5's *&osova et al., 1444-. This may ex!lain the accumulation of
acetaldehyde in the lare intestine durin ethanol oxidation in vitro *:okelainen et al.,
233D-. .lcohol is trans!orted to the lumen of the colon via the blood stream and
converted to acetaldehyde by bacterial .5'. .s the ca!acity of the lare number of
bacteria in the colon for the first ste! of alcohol oxidation by .5' is much hiher
than that for the second ste! from acetaldehyde to acetate, the concentration of the
former increases distinctly. ;ith enhanced concentrations of acetaldehydes, a toxic
com!ound may contribute to the damae of the colonic mucosa and after bein
!artially absorbed into the blood may lead to liver inGury. .nother im!lication of the
bacterioloic !athway of alcohol breakdown miht be the local carcinoenic action of
acetaldehyde.
$oreover, it has been demonstrated that durin ethanol challene, acetaldehyde
level is reulated not only by intracolonic microbes, but also by colonic mucosal cells
*Iisa!aa et al., 1441-. The enetic inactivation of colonic.L5'amon .L5'1-
deficient !ersons may thus ex!lain the three- to fourfold increased risk of colorectal
cancer in that !o!ulation.
D. CONSE;*ENCES OF ALCOHOL AB*SE
Lon-term alcohol abuse is known to exert harmful effects on a number of the body%s
oran systems, mostly affected amon those include liver, H) tract, !ancreas, and the
im-mune, cardiovascular, and skeletal systems.
D.>. A&!h!i& Li+(r Dis(as(
The liver is a vital oran in !rocessin fats, suars, !roteins, and vitamins and in
reulatin blood clottin. Lon-term heavy alcohol use is the leadin cause of illness
and death from liver disease.
'owever, because malnutrition is also common in alcoholics, the early findins
were used to bolster the arument that malnutrition, rather than alcohol !er se, could
ex!lain the !athoenesis of alcohol-induced liver inGury. Over the !ast four decades, a
more balanced view has evolved. Based on studies in humans, subhuman !rimates,
and rodents, it is now established that alcohol can cause liver damae in the absence
of dietary deficits. )n both ?rance and Hermany, a close correlation exists between !er
ca!ita alcohol consum!tion and the likelihood of cirrhosis. ?urthermore, althouh the
incidence of malnutrition amon alcoholics has been decreasin, the number of deaths
attributable to alcoholic liver inGury has been risin. Lastly, no relationshi! has been
documented between nutritional status and severity of alcohol-induced liver inGury as
defined histoloically. )t is now clear that nutrition and toxic effects of alcohol are
often intertwined at the biochemical level by inducin microsomal cytochromes, that
chronic ethanol consum!tion is known to result in enery wastae and to !romote the
breakdown of nutrients *$ann and Truswell, 1441E Shils et al., 2337-.
4.1.1. )epatic meta%olism
The liver is the !rimary site of ethanol metabolism. ;ithin the liver, ethanol can
be oxidi/ed by three en/yme systems+
The alcohol dehydroenases
6ytochrome #
704
6atalases
.5's are cyto!lasmic en/ymes with numerous isoforms in human liver *Bosron
et al., 2339-. The class ) en/ymes, which have the lowest >m values and hihest
substrate s!ec-ificity for ethanol, are encoded by three se!arate enes .5'2, .5'1,
and .5'9. ?or .5'1 and .5'9, !olymor!hisms have been identified. Iariations
in.5'isoforms can account for sinificant differences in ethanol elimination rates
amon ethnic rou!s. .5' is the en/yme res!onsible when blood and tissue ethanol
concentrations are low.
;hen tissue levels exceed 24 m$ *under chronic ethanol consum!tion-, it induces
the activity of 6=#1E2 in the liver by as much as five- to tenfold. Ethanol-related
induction of 6=#1E2 is likely to account for more ra!id elimination of ethanol
observed in alcoholics. Both .5' and 6y#1E2 convert ethanol to acetaldehyde. The
en/yme system of catalase which is !resent in !eroxisomes and mitochondria is the
least im!ortant in these !athways.
The chanes in the liver of !ersons with .L5 rane from fatty liver to end-stae
cirrhosis. )t is conservatively estimated that there are more than 1 million cases of
.L5 worldwide *$edical 6onse"uences of .lcohol .buse, 1444-.
(ecent studies from the 8nited States *Ion and Bell, 1447- and the 8nited >in-
dom *?isher et al., 1441- have found .L5 to be the main cause of death from chronic
liver disease. This hih !revalence of .L5 is not in the least !artially due to the risin
!revalence of he!atitis 6 but the increased consum!tion of distilled s!irits and beer
amon youner subGects and women as com!ared to wine *;orld 5rink Trends,
1441-. )f this trend is to be maintained, it is likely that there will be a marked increase
in .L5 in future years.
There are three ty!es of .L5+
2. ?atty liver+ 8sually reversible with abstinence.
1. .lcoholic he!atitis+ 6haracteri/ed by !ersistent liver inflammation.
9. 6irrhosis+ 6haracteri/ed by !roressive scarrin of liver tissue.
2. 'e!atic steatosis+ ?atty liver, the most common he!atic lesion associated
with alcoholism, is found in <4M244R of !atients takin excessive
amounts of alcohol *'all, 2330-. The deree of steatosis can rane from
fatty chane in only a few cells to involvement of almost every he!atocyte,
and clinically, there is little or no Gaundice, ascitis, sinificant !ortal
hy!ertension, or liver failure. Oxidation of ethanol to acet-aldehyde by
.5' and oxidation of the acetaldehyde results in increased eneration of
&.5'. The reaction yields two molecules of reduced &.5 for each
molecule of ethanol oxidi/ed. The increased &.5',&.5 ratio enhances
li!oenesis either directly by the mitochondrial elonation !athway or
indirectly by increasin the relative concentration of &.5#' throuh the
transhydroenation mechanism. )ncreased concentration of &.5#' also
results from oxidation of ethanol by 6=#1E2. The increased
&.5#',&.5# ratio !resumably drives the induction of li!id metabolism
*Lieber, 233<-.
1. .lcoholic he!atitis+ The classical clinical syndrome of alcoholic he!atitis
consists of Gaundice, varyin derees of he!atic failure, abdominal distress,
fever, and leucocytosis. The syndrome a!!ears in !atients who have been
consumin excess amounts of alcohol for a !eriod of 0 years or more.
Takeuchi and Takada *23<0- have su-ested that circumstances which
enhance ethanol oxidation by the$EOS could subGect the he!atocyte to
intense toxicity for that metabolite. .dditionally, the !roduction of
acetaldehyde by the cytochrome #704 system *the $EOS-, which is also
concentrated in /one 9, offers an alternative ex!lanation for the /one-9
location of the brunt of al-cohol- induced he!atic inGury
9. 6irrhosis+ The clinical !icture of alcoholic cirrhosis is the resultant of three
vectors+ !ortal hy!ertension, loss of !arenchymal function, and
extrahe!atic inGury !roduced by alcoholism. )n .L5, activatin factors
include acetaldehyde and li!id aldehydes, as well as cytokines released
from >u!ffer cells, endothelial cells, and he!atocytes. The activation of
stellate cells is a!!arently a two-!hase !henomenon. The first is initiation
by cytokines released from >u!ffer and endothelial cells, which is
!rovoked by intestinal u!take of endotoxin. Endotoxin u!take is enhanced
by alcohol as wellas by factors released from he!atocytes in res!onse to
inGury induced by acetaldehyde li!id aldehydes and TH?7 *5en et al.,
233<-. The second-!hase S!er!etuation% involves stimulation by li!id
aldehyde and acetaldehyde and by cytokines *#5H?, TH?B, endothelin,
and $6#)E $aher and ?redman, 2330-.
.cute liver inGury+ $ay cause fastin hy!olycemia, attributed to de!leted liver
lycoen reserves and retardation of luconeoenesis. .ssociated with anorexia,
nausea and vomitin may be exacerbated by concomitant alcoholic astritis, and
nutritional conse"uences are minimal.
6hronic liver inGury+ &utritional com!lications are fre"uent and extensive when
liver function becomes im!aired in chronic liver inGury, !articularly cirrhosis, and
result in one of the followin factors+
)nade"uate dietary intake *usually !rotein-
$aldiestion and malabsor!tion *defective fat assimilation- resultin in
steatorrhea and deficiencies of fat-soluble vitamins with clinical
manifestations such as niht blindness, osteo!orosis, and hemorrhae.
5efective metabolism *es!ecially !rotein M decreased he!atic synthesis of
ex!ort !roteins M albumin, coaulation factors, decreased urea synthesis- and
decreased metabolism of aromatic amino acids. The effect of advanced liver
disease on !rotein catabolism is, however, controversial. Hlucose tolerance is
fre"uently abnormal in the cirrhotic !atient and has been linked to insulin
resistance. .bnormalities of water- and fat-soluble vitamins are common in
cirrhosisE as a result of these derane-ments, vitamin re!letion strateies
re"uire modification in !atients with liver failure *Shils et al., 2337-.
'e!atic ence!halo!athy *'E- is a neuro!sychiatric syndrome, which can develo!
in acute or chronic liver disease *'aussiner et al., 1444, 1449-. )t is functional in
nature, !otentially reversible, and !reci!itated by rather heteroeneous factors such as
bleedin and hih !rotein diet, hy!onatremia, sedatives, and inflammation. (ecent
data suest that 'E in liver cirrhosis is the clinical manifestation of a low-rade
chronic cerebral edema, which can transiently exacerbate under the influence of
!reci!itatin factors *'aussiner et al., 2337, 1444-. )n this context, astrocytes !lay
an im!ortant role, and are a maGor site of ammonia detoxification in 'E
In vivo '-$(S studies on the brain from cirrhotic !atients with 'E consistently
show a de!letion of myoinositol accom!anied by an increase in the
lutamine,lutamate sinal, which indicates astrocyte swellin as an early
!athoenetic event in 'E *'aussiner et al., 2337, 1444-. )t was suested that the
increase in brain water does not solely result from ammonia-induced astrolial
lutamine accumulation, but is also induced by other 'E-relevant factors such as
hy!onatremia *6ordoba et al., 2333-, T&?c *6han et al., 1442-, extracellular
lutamate *Bender et al, 233C-, and aonists of the !eri!heral-ty!e ben/odia/e!ine
rece!tor *Bender and &orenber, 233C-. Such triers are hallmarks of the so-called
!reci!itatin factors of 'E, which may syneris-tically increase astrocyte swellin
induced by ammonia. .strocyte swellin in the sense of a cytotoxic brain edema
occurs in the absence of a clinically overt increase of intra-cranial !ressure already in
the subclinical stae of 'E in cirrhotic !atients *'aussiner et al., 2337E $oran,
233C-. 'owever, even small chanes in astrocyte hydration may influence astrocyte
function, lioneuronal communication, and finally trier the clinical !icture of 'E.
'y!o-osmotic swellin of cultured rat astrocytes results in an almost immediate
oxidative stress res!onse and increased tyrosine nitration of distinct !roteins *Schliess
et al., 1447-. 'y!o-osmotic astrocyte swellin *Schliess et al., 1447-, ammonia
*Schliess et al., 1441-, ben/odia/e!ines *Hor et al., 1449-, and inflammatory
cytokines such as T&?c, interferon-c, and increased !rotein tyrosine nitration *#T&-
were observed in rat astrocytes. Thus, many factors known to !reci!itate 'E in
cirrhotic, !atients, such as ammonia, hy!onatremia, ben/odia/e!ines, and
inflammatory cytokines, induce astrolial #T&.
4.1.2. &iver "unction tests
The term liver function tests is assined for biochemical tests that assess the
functional he!atic reserve and include tests containin bilirubin, as!artate
transaminase *.ST-, alanine transaminase *.LT-, .#, HHT, and L5'.
.lthouh most cases of liver disease can be dianosed by other seroloical
markers, dianosis could not be made in C2 of 2217 !atients *<R- with abnormal liver
tests *5aniel and $arshal, 1444-. This suests that the maGority of asym!tomatic
individuals with serum .LT abnormalities may not have demonstrable liver disease.
Elevated transaminases M .LT is found in the cytosol of liver, whereas two .ST
isoen/ymes are located in the cytosol and mitochondria. Both en/ymes are released
into the blood in increasin amounts when the liver cell membrane is damaed. The
most common causes of elevated transaminase levels are chronic he!atitis B and 6,
alcohol-related liver inGury, nonalcoholic steatohe!atitis, hemochromatosis, ;ilson%s
disease, al!ha-antitry!sin deficiency, and a recently reconi/ed cause, celiac s!rue.
2. $inor elevation *V1 times- of u!!er limit of normal *8L&-. This is a common
condition.
1. $ild elevation *1 to V0 times- of 8L&.
.lcohol use is a common cause of mild elevation of transaminases. The "uantity
of alcohol and lenth of time that alcohol has been consumed account for the
develo!ment of disease *6arey, 1444-. .lcoholic he!atitis is commonly associated
with an .ST,.LT ratio of a!!roximately 1+2 and the .ST rarely exceeds 944 )u dl
-2
.
Other causes are drus, he!atitis 6, nonalcoholic steatohe!atitis, etc
2. $oderate elevation of .ST,.LT *0M20 times- of 8L& M entire s!ectrum of
he!atic diseases.
1. Severe .ST,.LT elevation *`20 times- of 8L& M severely ill !atient with
liver disease.
7.2.1.2. Serum bilirubin
Bilirubin is an endoenous oranic anion which binds reversibly to albumin and is
trans!orted to the liver, where it is conGuated to lucuronic acid and excreted in the
bile. 'e!atobiliary disease is indicated when the conGuated fraction of total bilirubin
exceeds the 8L&, even if the total serum bilirubin concentration is normal or near
normal *(osen and >eeffe, 233C-.
7.2.1.1. .lbumin
$ost circulatin !roteins in !lasma are synthesi/ed in the liver. .lbumin accounts
for D0R of serum !rotein and has a half-life of about 9 weeks. Ex!anded !lasma
volume or decreased albumin synthesis can result in hy!oalbuminemia. )n eneral,
albumin levels are affected by severe chronic liver disease, urinary !rotein losses,
hy!ercatabolic states, and H) losses *?riedman et al., 233DE Noli et al., 2332-.
7.2.1.9. )nternational normali/ed ratio
6oaulation factors reflect synthetic liver function. $ost of them, includin
fibrinoen, are vitamin k-de!endent factors *!rothrombin and factors I)), )O, and O-
and factor I *?riedman et al., 233D-. Severity and !ronosis of liver disease can be
assessed usin international normali/ed ratio. 'owever, international normali/ed ratio
is not absolutely sensitive, and can be normal in cirrhosis *$oseley, 233D-.
7.2.1.7. .lkaline !hos!hatase
Sources+ .# oriinates !redominantly from two sources+ liver and bone *5aniel et
al., 2333-. )t is also !resent in kidneys, small bowel, and !lacenta.
Serum 0 -nucleotidase or HHT is usually elevated in !arallel with the elevation in
.# in !atients with liver disorders, but they are not increased in !atients with bone
disorders. )f the increase in .# is less than 04R above the normal level, the results of
all the other liver en/yme tests are normal, and the !atient has no sym!toms and
observation alone is suested *Tukey and Strassbur, 1444-.
7.2.1.0. Hamma lutamyl transferase
Source+ HHT is a membrane en/yme found in he!atocytes and biliary e!ithelial
cells. Serum HHT !rovides a very sensitive indicator of the !resence or absence of
he!ato-biliary disease. Elevated levels of HHT have been re!orted in a wide variety of
clinical conditions, includin !ancreatic disease, myocardial infarction, renal failure,
diabetes, and alcoholism. 'ih serum HHT levels are also found in !atients takin
medications such as !henytoin and barbiturates *Tiribelli and Ostrow, 233D-.
Serum HHT can be used to confirm the he!atic oriin of elevated .L# or to
su!!ort the dianosis of alcohol abuse. 5ue to lack of s!ecificity and the hihly
inducible !ro!erty of this en/yme, an extensive evaluation of an isolated HHT
elevation in an otherwise asym!tomatic individual is not warranted *Nobair, 233C-.
D.?. A&!h!i& Pa$&r(atitis
.n association of alcohol abuse and !ancreatic inGury was re!orted as early as
2C<C *?riedreich, 2C<C-. .lthouh the !revalence of alcoholic !ancreatitis at lare is
unknown, clinicians usually aree that both acute and chronic alcoholic !ancreatitis
are res!onsible for sinificant numbers of illness and death. Sym!toms shared by
acute and chronic !ancreatitis are abdominal !ain and interference with normal
!ancreatic functions. .!!roximately 14R of !atients with alcoholic !ancreatitis die
within 14 years of disease *.!te et al., 1447-. 6hronic calcifyin !ancreatitis is rare in
countries with low alcohol consum!tion exce!t in some tro!ical countries where
malnutrition is wides!read as in southwest )ndia *.ubry et al., 23CC-.
#ancreatitis is a !otentially fatal inflammation of the !ancreas often associated
with lon-term alcohol consum!tion. )nitial sym!toms include vomitin as well as
acute abdominal !ain, which may be locali/ed to the back and u!!er abdomen. )n
mild cases, the !ain may last 1M9 days. )n severe cases, however, the !ain may !ersist
for several weeks and the risk of death rises to about 94R.
Less commonly, !ancreatitis can be com!letely !ainless and is only dianosed
from sym!toms of insufficient !ancreatic function, such as diabetes and steatorrhea.
.!!rox-imately 0MD years after the onset of the disease, evidence of chronic
!ancreatic disease develo!s as a result of !roressive destruction of !ancreatic tissue
resultin in !ersistent !ain, weiht loss, diabetes, and maldiestion of food. . lare
!ros!ective study has re!orted that chanes in the !ancreas related to chronic
!ancreatitis were more likely to occur in alcoholics who had recurrent acute
inflammation of the !ancreas *.mmann and $uellhau!t, 2337-. . !ostmortem study
of 17< !atients with fatal alcoholic !ancre-atitis demonstrated that in 09R of !atients,
no evidence existed of chronic chanes in the !ancreas. Ex!eriments show that
re!eated e!isodes of acute !ancreatitis in rats !roduce chronic chanes in the
!ancreas, includin fat de!osits, atro!hy, and fibrosis *Elsasser et al., 2331-.
Effect of alcohol on !ancreatic en/ymes M Oral or intraduodenal ethanol causes a
moderate stimulation of !ancreatic bicarbonate and en/yme out!ut, and intravenous
ethanol inhibits basal and hormonally stimulated !ancreatic exocrine secretion in
humans, dos, cats, !is, rabbits, and rats *&ieberall et al., 233C-. 5urin chronic
alcoholism, the ethanol-induced inhibition is re!laced by an enhanced en/yme out!ut
that causes intraductal !rotein !reci!itation. The occurrence of !rotein !reci!itates is
considered to be a crucial ste! in the develo!ment of chronic alcoholic !ancreatitis in
humans. Other ethanol-induced secretory alterations that may contribute to the
develo!ment of alcoholic !ancreatitis are *2- a decreased secretion of try!sin
inhibitor, *1- an increased cholineric tone, and *9- chanes in the concentration of
lithostathine *Siemund et al., 1449-. Lon-term alcohol consum!tion may lead to
!remature activa-tion of diestive en/ymes in the acinar cell. .lcohol increases the
synthesis of diestive en/ymes in the !ancreas *.!te et al., 2330- and also increases
the fraility of /ymoen ranules *'aber et al., 2337-, !otentially allowin /ymoens
to leak into the cell. The increase in lysosomal fraility a!!ears to be mediated by two
com!ounds known to accumulate in the !ancreas after chronic alcohol consum!tion+
cholesteryl esters and fatty acid ethyl esters *?.EEsE 'aber et al., 2337-. (ecent
evidence demonstrates that cytochrome #
704
1E2 also is !resent in the !ancreas and,
moreover, is induced by chronic alcohol administration *&orton et al., 233D-. This has
led to the conce!t of Sdrinkers% !ancreas% in which the effects of alcohol and its
metabolic by-!roducts lead to excessive accumulation of diestive and lysosomal
en/ymes in the acinar cell.
Elevated ?.EE concentrations have been detected in !ostmortem orans from
alcoholics and !atients acutely intoxicated by alcohol, and ?.EEs have been
im!licated as a mediator of ethanol-induced oran damae *5ic/falusy et al., 1442-.
?.EEs have been shown to uncou!le oxidative !hos!horylation in rabbit heart
mitochondria *Lane and Sobel, 23C9-, to inhibit !rotein synthesis and cell
!roliferation in human he!atoblastoma *'e!H1- *S/c/orkowski et al., 2330- cells, and
to increase fraility of !ancreatic lysosomes *'aber et al., 2339-. .fter acute alcohol
intoxication, the hihest concentrations were found in adi!ose tissue, liver, !ancreas,
and heart, whereas the concentrations in chronic alcoholics were hihest in adi!ose
tissue and !ancreas *La!osata and Lane, 23CD-. Liver, duodenal mucosa, and
!ancreas were found to have hihest ca!acities to synthesi/e ?.EEs. ?.EE
hydroly/in activity was hihest in liver and !ancreas, but hardly detectable in
adi!ose tissue or heart. .s orans like the !ancreas, heart, and brain have limited
ca!acity to oxidi/e ethanol but are also inGured in alcoholics, it is suested that
factors other than oxidation may be res!onsible for ethanol-induced damaes in these
orans.
. number of factors that may distinuish alcoholics who develo! !ancreatitis
from those who do not have been investiated. These factors include diet, amount of
alcohol consum!tion, hereditary factors, fat intolerance, and smokin *'aber et al.,
2330-.
$any studies have !rovided conflictin results, !robably because they com!ared
subGects with alcoholic !ancreatitis *i.e., the ex!erimental rou!- with subGects from
the eneral !o!ulation *i.e., the control rou!-. Thus, the control and ex!erimental
rou!s differed from each other with res!ect to two factors+ alcoholism and
!ancreatitis. The essential com!arison in such studies must be between alcoholics
with the disease and alcoholics without the disease, which we have included in our
study.
5es!ite numerous studies !erformed in animal ex!eriments and man, the
!athoen-esis of alcoholic !ancreatitis has not been classified until now *Bode and
Bode, 1444-.
E. EFFECT OF ALCOHOL ON IMM*NEI
CARDIOVASC*LARI AND SKELETAL SYSTEM
Both acute and chronic alcohol ex!osure su!!ress all branches of the immune
system, in-cludin early res!onses to infection and the tumor surveillance system.
?irst, alcohol im-!airs the ability of;B6s*neutro!hils- to mirate to sites of inGury
and infection M a !rocess called chemotaxis *>ovacs and $essinham, 1447-. )n
addition, removin ;B6s and !roteins that act as messeners between immune cells
from an animal that has not been iven alcohol and culturin them in the !resence of
alcohol, or isolatin these cells from humans or animals after alcohol administration
has been shown to alter !roduction of these macro!haes and cytokines *S/abo,
2333-.
6hronic heavy drinkin is a leadin cause of cardiovascular illness such as
deener-ative disease of the heart muscle *cardiomyo!athy-, disorders associated with
decreased blood su!!ly to the heart muscle *6'5-, hih blood !ressure, heart rhythm
disorders *arrhythmias-, and stroke *6orrao et al., 1444-.
.lcohol also affects the skeletal system, there is increased risk of accidental
inGury, and alcoholics also may suffer from a enerali/ed decrease in bone mass.
'eavy drinkin may lead to osteo!orosis, characteri/ed by severe back !ain, s!inal
deformity, and increased risk of wrist and hi! fractures *6lark, 23CC-.
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.merican :ournal of 6linical &utrition 94, 2CC4M2CCD.
.chord, :.L., 2339. (eview of alcoholic he!atitis and its treatment. .merican
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CHAPTER G
Da$"(r!#s H(rba W(i"ht%L!ss
S#33(.($ts
A. G!$-6(-%St#art
8niversity of Texas at El #aso, El #aso, TO, 8S.
>. INTROD*CTION
Obesity is usually defined as a body mass index of 94 or reater. This affliction is
associated with a myriad of health !roblems since it is a maGor risk factor for
cardiovascular disease, certain ty!es of cancer, and ty!e 1 diabetes *656, 1424-. )n
144<M144C, more than one third of the adults in the 8nited States were obese *?leal
et al., 1424-.
The Orani/ation for Economic 6oo!eration and 5evelo!ment *OE65- has stated
that the 8nited States *8S- and $exico are amon the most obese nations in the
world, es!ecially com!ared to certain .sian countries, such as >orea and :a!an, for
exam!le *OE65, 1421-.
Both in $exico and the 8S, more than two thirds of adults are both overweiht
and obese, in contrast to a!!roximately 13R of adults in 6hina. )n the !ast 14 years,
the !rev-alence of overweiht and obesity have occurred in all three countries. )n
$exico, as well as 6hina, the economic and health system costs of obesity-related
diseases *cardiovascu-lar, cancer, ty!e 1 diabetes, etc.- are the !rimary concerns
*#o!kin, 1422-.
5ue to the reat increase in obesity rates, many !eo!le in various countries are
seek-in a solution to their weiht !roblem, sometimes fallin victims to Snatural% or
Smiracle% !roduct marketin scams, which claim efficacy and safety for weiht loss,
but without any clinical !roof. This article focuses on two !otentially toxic herbal
!roducts sold as weiht-loss su!!lements+ yellow oleander and candle nut tree seeds,
known in S!anish s!eakin countries as Codo de 'raile and /ueB de la India,
res!ectively.
?. THE S*RGE OF HERBAL PROD*CT *SE WITHIN
COMPLEMENTARY AND ALTERNATIVE MEDICINE
)n the !ast two decades, there has been a dramatic increase in the use of herbal
!roducts in the 8S. Studies suest that herbal !roduct use is even more common
alon the 8SM$exico border, com!ared to the rest of the country *Hon/Kle/-Stuart et
al., 144DE (ivera et al., 144<-.
Since the 8S 6onress !assed the 5S'E. .ct of 2337, an herbal !roduct can be
classified as a nutritional su!!lementE so, for this reason, many of the !roducts bouht
as nutritional su!!lements may contain herbs in some form or another *herbal teas or
infusions, extracts, essential oils, syru!s, etc.-.
B. HERBAL S*PPLEMENT IDENTITYI EFFICACYI AND
SAFETY) CHAOS IN THE CYBER MARKETPLACE
Some !eo!le are still under the misuided im!ression that since herbal !roducts
are Snatural,% they must all be safe. .nother misconce!tion is that herbal !roducts are
devoid of side effects.
?actual information is often lackin reardin certain herbal su!!lements
em!loyed for weiht loss. .dded to this is the !ractice of mislabelin some herbal
su!!lements, makin the use of these !roducts confusin, risky, or sim!ly ineffective.
The lack of ade"uate "uality control !resent in certain herbal !roduct com!anies
around the world leads to variability in the "uantity and "uality of the !roducts%
content. . !ortion of the information available about many certain herbal weiht-loss
su!!le-ments currently in the market may be exaerated or misleadin. This is
es!ecially true of some individuals and orani/ations that are mainly interested in
marketin their !roducts only for !rofit, with little reard for the !ersonal welfare of
the consumer.
Serious intoxications have occurred around the lobe due to mislabelin of herbal
!roducts or erroneous identification of some !lant s!ecies. . sim!le mistake made in
the correct identification of an herb or the incorrect use of a certain !art of a !lant can
have serious or even fatal results.
This !roblem is exem!lified in the so-called S6hinese 'erb &e!hro!athy,% in
which the inclusion of the wron s!ecies of !lant containin aristolochic acid in a
weiht-loss !re!aration im!orted from 6hina, had disastrous results amon the !eo!le
who inested it *6heun et al., 144D-.
Some !lants have not been ade"uately studied with res!ect to their use in s!ecial
!o!ulations, includin !renant or lactatin women, small children, and the elderly,
for exam!le.
This situation is further com!licated by the fact that interactions between herbal
!roducts and certain !rescri!tion and over-the-counter medications can sometimes be
serious *'u et al., 1440-.
Iarious herbal su!!lements which are new to the western market, oriinatin in
.sia, .frica, or South .merica, for exam!le, have not been ade"uately tested for
!urity, safety, or efficacy, !rior to their introduction into the 8S market.
8nfortunately, some of the information currently available about many medicinal
!lants *includin herbal weiht-loss su!!lements- is either misleadin or sim!ly
lackin. This is es!ecially true of the medicinal !lants from $exico, which, for the
most !art, have not been studied in de!th for their !otential thera!eutic a!!lications.
.necdotal infor-mation by itself is not a reliable source of evidence for the safe use of
any herbal !roduct.
Some of the manufacturers or distributors of certain weiht-loss su!!lements tend
to encourae self-dianosis as well as self-medication, both of which can be
!otentially danerous to the consumer.
D. IDENTITY OF HERBAL PROD*CTS
)n order to !rotect the consumer%s health, it is of reat im!ortance to know exactly
what !lant or combination of !lants is bein inested. There aremany documented
cases of mis-labelin and adulteration in certain herbal su!!lements *(u!arel and
Lockwood, 1422-.
8nfortunately, some of the !lants and funi that !ossess medicinal or nutritional
value may be confused with !oisonous s!ecies by the inex!erienced lay!erson.
Because of this, cases of intoxication from certain herbal su!!lements, wild !lants,
and mushrooms occur each year in many countries around the world *#frohne and
#fander, 1440-.
E. THE INTERNET AS A SO*RCE OF INFORMATION
ABO*T HERBAL WEIGHT%LOSS S*PPLEMENTS
:ordan and 'aywood *144<- evaluated both the "uality and "uantity of dru
information available to consumers in the 8S on 91 )nternet websites, which sell
herbal weiht-loss dietary su!!lements. The selected websites were evaluated for the
!resence of active or inactive inredient names, dose, and other ?ood and 5ru
.dministration *?5.- labelin re"uirements. The information !resented in the
website reardin dru,su!!le-ment safety for the most common herbal inredients in
the !roducts evaluated was com!ared to standard herbal dru information references.
The authors concluded that the !otential risks associated with certain herbal weiht-
loss su!!lements may not be accurately mentioned on )nternet websites which sell
them.
G. YELLOW OLEANDER OR QCODO DE FRAILE4
Other common names+ =J yotl, =oyote *literally Srattle%-, .yoyote, .lcanfura,
6ame, 6abalona de la 'uasteca, 'ueso de ?raile, Solimann, Ienenillo, Semilla del
Brasil, and .lmendra "uema-rasa *Sfat-burnin almond%-.
This attractive ornamental shrub or medium-si/ed *9M3 m- tree *Thevetia s!!. M
.!ocynaceae-, with briht yellow-orane flowers and milky latex, is oriinally for
tro!-ical .merica but is now found in various tro!ical and subtro!ical areas around
the world. The !rinci!al s!ecies include+ Thevetia peruviana$ Thevetia thevetioides,
and Thevetia ovata# These !lants belon to the .!ocynaceae botanical family, lon
known to include various !oisonous s!ecies, includin oleander */erium oleander-,
amon many others *;astaff, 144C-.
.lthouh all !arts of the !lant are toxic, yellow oleander has been used in
$exican traditional medicine for various health com!laints, includin its use *seed- as
a charm aainst the Sevil eye% and fallen fontanel in babies. . !oultice made from the
leaves has been a!!lied externally to cure hemorrhoids, and the milky latex has been
a!!lied ty!ically as a skin antise!tic *$artine/, 23C3-.
(ecently, the seed has been !ro!osed as a cure for obesity, taken Sin minute "uan-
tities,% accordin to some commercial !roduct labels and ads on the )nternet. )n
various !arts of $exico, there have been numerous cases of !oisonin with this
!roduct sold as ca!sules under various commercial names. The !atients com!lained of
vomitin, diar-rhea, numbness of the extremities, intense abdominal !ain, headaches,
and dehydration. This situation !rom!ted the federal medical authorities to ban its use
and confiscate any !roduct containin the seed sold in !harmacies or convenience
stores but not with com-!lete success, since the !roduct is still available under various
commercial names on the )nternet *LJ!e/-Iillafranco and 6amacho-Lacroix, 1441-.
K. TO=ICITY OF THEVETIA SPP.
=ellow oleander *Thevetia neriifolia- !roduces seeds which are hihly !oisonous
and con-tain three lycosides M thevetin, thevetoxin, and !eruvoside. 6linical
manifestations of !oisonin rane from mild to !otentially fatal. The toxic lycosides
have sinificant car-diovascular effects with varyin rhythm abnormalities *Bose et
al., 2333-.
.ll !arts of these !lants are toxic *es!ecially the seeds- and contain a variety of
cardiac lycosides includin thevetins . and B. )nestion of yellow oleander results
in nausea, vomitin, abdominal !ain, diarrhea, cardiac dysrhythmias, and
hy!erkalemia *Bandara et al., 1424-.
The yellow oleander tree contains !hytochemicals, known as cardenolides, which
are ca!able of exertin !ositive inotro!ic effects on the heart. The cardiotonic
!ro!erties of this !lant have been used both thera!eutically, as well as in !oisonin,
since ancient times. The !hysioloical action of the cardenolides is similar to that of
the diitalis ((igitalis purpurea and (igitalis lanata- lycosides and includes en/yme
inhibition of !lasmalemma &a
B
, >
B
.T#ase.
'owever, there are differences in toxicity and extra cardiac effects between the
oleander and diitalis cardenolides. 8nlike dioxin toxicity, serum manesium
concen-trations are less likely to be affected in yellow oleander !oisonin. The effect
of mane-sium concentrations on toxicity and outcome is not known *Lanford and
Boor, 233DE (aGa!akse, 1443-.
)nestion of yellow oleander seeds *T# peruviana- has become a !o!ular method
of suicide in Sri Lanka and other countries in .sia. The seeds contain very toxic
cardiac lycosides that cause vomitin, di//iness, electrolyte disturbances, and
cardiac dysrh-ythmias, such as conduction block affectin the sinus and .I nodes.
There are thousands of cases of intoxication re!orted each year, some of which have a
fatal outcome *Eddleston et al., 2333, 1444a,bE ?onseka et al, 1441-.
Iarious herbal or herbal,mineral combinations used in traditional medicine are
cur-rently bein used around the world as thera!eutic remedies for common ailments.
6om-monly known cardiotoxic herbs, such as T# peruviana *yellow oleander-, for
exam!le, may be included in diverse thera!eutic !re!arations. These herbs may
!roduce cardiac ar-rhythmias, mainly ventricular ecto!ics, ventricular tachycardia, as
well as various derees of arterioventricular *.I- blocks. #eo!le affected by these
!re!arations commonly de-velo! related sym!toms, such as feelins of discomfort,
di//iness, chest discomfort, and ventricular arrhythmias. )n these situations, it is
im!erative that !eo!le seek !ro!er med-ical attention and discontinue the use of the
herbal !re!aration. 8nfortunately, some !eo!le believe that any herbal remedy is
always safe and are !rone to be intoxicated due to self-medication *&a/eri et al.,
1443E 5wivedi et al., 1422-.
L. SAFETY ISS*ES
There are no clinical studies to substantiate the claims that yellow oleander seed
can hel! reduce weiht. 'owever, there are a !lethora of !ublications confirmin its
hih toxicity to both humans and animals. ?or this reason, no !art of the !lant should
ever be inested, in any "uantity.
O. CANDLE N*T TREE @QN*EC DE LA INDIA4A
Other common names+ )ndian walnut, >ukui, Tuitui
The tree *,leurites moluccana M Eu!horbiaceae- is oriinally from )ndonesia but
is now cultivated in South .merica, es!ecially in Bra/il. Some ads a!!earin on the
)nternet mention that this tree is native to the .ma/on reion, which is not correct.
>R.PARTS OF THE PLANT *SED IN TRADITIONAL
MEDICINE
The fruits and leaves, but not the seeds, of this !lant are used in traditional .sian
medicine for the treatment of headache, mornin sickness, fever, inflammation,
onorrhea, and to lower cholesterol *Ostraff et al., 1444E #edrosa et al., 1441-. ,#
moluccana extracts showed antibacterial activity aainst !taphylococcus aureus and
Pseudomonas aeruginosa *Locher et al., 2330-.
>>.WEIGHT LOSS AND OTHER HEALTH CLAIMS MADE
ON THE INTERNET FOR CANDLE N*T TREE SEEDS
6andle nut tree seeds have recently been marketed aressively, es!ecially on the
)nter-net. The seeds are touted as havin Sfat burnin% and other weiht loss
!ro!erties, but no clinical trials have ever been undertaken in humans to evaluate their
!otential beneficial or toxicoloical effects.
Some commercial ads claim that the seeds of the !lant can hel! in weiht loss, as
well as to lower cholesterol. Some sites also mention the seed is useful for a wide
array of con-ditions includin arthritis, baldness, cellulite, consti!ation, hemorrhoids,
to im!rove skin conditions, as an a!!etite su!!ressant, and as an aid to sto! cravins
for tobacco *smokincessation-.
6om!anies and individuals marketin the seeds mention that a very small !ortion
*a!-!roximately 2,C to 2,7- of the seed should be boiled in water !rior to takin it
before bedtime. 'owever, since no exact dose has been s!ecified, it may be difficult
to measure exactly how much of the seed will actually be inested. .lso, the method
of takin the seed may vary accordin to the com!anies% )nternet site, which adds to
the confusion and misinformation.
Some com!anies marketin candle nut seed mention it is !ossible to lose u! to 24
k *11 lbs- in Gust a few weeks after takin this !roduct. The seed acts a stron
cathartic and may cause diarrhea, dehydration, and loss of electrolytes. #roloned use
of the seeds can affect the astrointestinal system, causin intestinal muscle atony.
There are also re!orts of !eo!le sufferin from cram!in, diarrhea, and vomitin.
.lterations in heart rate due to the inestion of the seeds have also been re!orted
*(amPre/, 1443-
>?.SAFETY ISS*ES
. woman was hos!itali/ed in &avarra *S!ain- after inestin a candle nut seed
!roduct for weiht loss. The !atient showed hy!otension, bradycardia, and was
semiconscious. .ct-in on advice from a friend who had used the !roduct for weiht
loss, she inested Sone fourth% of a seed for many days !rior to bein admitted to the
emerency room. The herbal !roduct label used by the intoxicated !atient stated that
it is a Snatural, nontoxic, and 244R effective% !roduct *#am!lona, 144<-.
There are no known clinical studies in humans to verify the various claims
reardin weiht loss *or any other health benefits- made by some commercial
com!anies which market candle nut tree seeds. Since the seeds can have a drastic
!urative action *'ockin, 233<E &elson et al., 144<E ;astaff, 144C-, they should
not be inested in any "uantity, es!ecially by !atients with colitis or irritable bowel
syndrome.
.dditionally, no toxicoloical studies have been made in order to establish the
!os-sible side effects of takin candle nut seeds for !roloned !eriods of time.
#atients suf-ferin from any form of liver, heart, or kidney disease should best avoid
takin this !roduct.
The seeds should es!ecially be avoided durin !renancy and lactation, as well as
in small children and the elderly. Some !eo!le may be alleric to this !lant.
>B.CONCL*SION
Obesity is ra!idly becomin a worldwide e!idemic, alon with the various health
comor-bidities associated with this condition. Even thouh many !eo!le seek a
Snatural% solution for weiht loss, they should be aware that not all herbal !roducts
available for sale on the )nternet are safe and that self-medication with these !roducts
can be danerous. This is es!ecially true of both candle nut tree and yellow oleander
seeds, which, due to their !otential toxicity, should not be inested in any "uantity.
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*9-, 1<9M1C2.
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Cha3t(r K
Mi, Ba&t(ria) R!( i$ Tr(ati$"
Gastr!i$t(sti$a A(r"i(s
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Takanashi $ilk #roducts 6o., Ltd., =okohama, >anaawa, :a!an

)nner $onolia $enniu 5airy *Hrou!- 6o. Ltd., 'uhhot, 6hina


ABBREVIATION
I"E )mmunolobulin E
IL )nterleukin
OVA Ovalbumin
>. INTROD*CTION
Hastrointestinal *H)- allery is an immediate hy!ersensitivity reaction of the
diestive sys-tem after the inestion of certain foods or drus. .lthouh H) allery
differs from food allery, which can affect other oran systems, H) allery is also
considered as immuno-lobulin E *)E--mediated, immune-mediated !atholoical
reaction which is same to food and alleric disorders, at least !artly. 6haracteristic
sym!toms include itchin andswellin of the mouth and oral !assaes, nausea,
vomitin, diarrhea *sometimes contain-in blood-, severe abdominal !ain, and, in
severe cases, ana!hylactic shock. Treatment includes identification and removal of the
alleren. )n an acute attack, e!ine!hrine may be administered as a stimulant, and
muscle relaxants may be iven to reduce intestinal s!asms that cause abdominal !ain.
)n childhood, H) allery is most often caused by hy!ersensitivity to cow%s milk and is
characteri/ed by diarrhea and colicky !ain, some-times with vomitin, ec/ema,
res!iratory distress, and thrombocyto!enia.
H) allery and other alleric diseases are becomin the serious !roblems in items
of !ublic health in :a!an as in many industrial countries. These diseases can im!air
the "uality of life of both !atients and their family, which neatively affects work
!roductivity, school !erformance, and social activities. .lthouh the s!ecific
mechanisms behind the e!idemic-oloical increase in the incidence of allery such as
H) allery still remains unclear, a hyienic environment with low microbial ex!osure
and less diverse fecal and oral micro-biota may !rovide less stimulation to the ut
immune cells, favorin allery-!rone immunity and influencin the overall mucosal
barrier. The intestinal microbiota may be amon the !ros!ective tarets in
themanaement of alleric disorders includin H) allery.
#robiotic bacteria can offer a safe and !ractical means of modulatin the function
and metabolic activity of the human intestinal microbiota and there has been much
interest in the !otential of usin !robiotic bacteria for treatin alleric diseases.
(ecent clinical and animal studies have su!!orted the hy!othesis that lactobacilli,
!articularly some selected strains with immunomodulatory !ro!erties, can modify the
res!onses of the host, thereby inducin beneficial effects aainst various alleric
disorders.
?. COLONICATION AND S*CCESSION OF H*MAN
INTESTINAL MICROBIOTA WITH AGE
The H) tract serves as one of the biest interfaces between the body and the
external environment. This H) tract is hihly s!eciali/ed oran system that allows one
to consume food in discrete meals as well as very diverse array foodstuffs to meet
nutrient needs. The orans of the H) tract include the mouth, eso!haus, stomach,
small intestine, and lare intestineE in addition, the !ancreas and liver secrete into the
small intestine. The system is connected to the vascular, lym!hatic, and nervous
systems to facilitate reulation of the d iestive res!onse, delivery of absorbed
com!ounds to oran of body, and reulation of the food intake.
.s one of the characteri/ed res!ects of H) tract, there are numerous endoenous
mi-crobes that coloni/e the surface of H) tract throuh whole life of the host, which
consist a com!lex community inside of the host known as intestinalmicroflora. )n the
healthy adults, microbial cells have estimated to outnumber somatic and erm cell by
a ratio of 24+2. The develo!ment of this utmicroflora is initiated durin the birth
!rocess. The fetus exists in a sterile environment until birth. .fter birth, the infant is
!roressively coloni/ed by bacteria from the mother%s vaina and feces and from the
environment. ;hen the nutrition and s!ace are not limited, the commensals with hih
division rates !redominateE e.. entero-bacteria *%scherichia coli- and enterococcus
a!!ear. The succession of microbes in infant%s intestinal tract also de!ends on the
feedin mode. The flora of breast-fed babies has been found to be relatively sim!le,
usually exclusively dominated by ifidobacterium. 'owever, the recent com!arative
studies showed that bifidobacteria were the !redominant fecal bac-teria in both rou!s
of infants. )n the bottle-fed infants, the count and fre"uencies of occurrence of
bacteroides, enterobacteriaceae, and stre!tococci were sinificantly hiher than those
in the breast-fed infants..fterweanin,when an adult diet is consumed, the stools of
infants bein to shift to adult bacterial flora+ bifidobacteria decrease remarkably and
constitute only 0M20R of total flora. The number of Bacteroidaceae, eubacteria,
!e!tococ-cace ae, and usually clostridia outnumber bifidobacteria, while aerobic
bacteria such as %# coli and stre!tococci, which had been rearded as the !redominant
s!ecies, are always detected, but account for less than2Rof the total bacterial count.
Lactobacilli,meas!haerae, and veil-lonellae are often found in adult feces, but the
counts are usually less than 24
<

-2
of feces. By the end of the secondary years, the
microflora becomes more stable and looks like that of adult. .s the !o!ulation
increase and nutrient are de!leted, the intestinal niches become occu!ied with more
s!eciali/ed s!ecies with an advanced symbiotic relationshi! between the host and
microbiota. Once the climax microflora has become established, the maGor bacterial
rou!s in the intestine of adult usually remain relatively constant over time.
The habitats of the intestinal microflora vary in different !arts of the human H)
tract in the healthy !ersonsE acid stomach contents are usually sterile. )mmediately
after a meal, counts of around 240 bacteria !er milliliter of astric Guice can be
recordedE bacteria in-cludin stre!tococci, enterobacteriaceae, Bacteroides, and
bifidobacteria are derived from the mouth and the meal. The flora of the small
intestine is relatively sim!le and lare numbers of oranisms are not found. Total
counts are enerally 24
7
or less !er milliliter, exce!t for the distal ileum, where the
total counts are usually about 24
D
ml
-2
. )n the duodenum and GeGunum, stre!tococci,
lactobacilli, and veillonellae are mainly found. Toward the ileum, %# coli and
anaerobic bacteria increase in number. )n the cecum, the com!osition suddenly
chanes and is similar to that found in feces, and the concen-tration may reach 24
22

-2
of content.
$ore than 744 s!ecies have been estimated to oriinally reside in the colon of the
healthy adults, which may attain !o!ulation levels nearly as hih as 24
21

-2
in the
colon and almost weiht 2,1 content. This bacterial community is dominated by the
strict anaerobes, and contains less facultative anaerobes with a rate of anaerobes and
aerobes as 2444+2. )n accordance with the metabolic activity, the maGor bacteria
!resent in the intestinal flora of the healthy adult can be divided rouhly into three
rou!s. Hrou! 2 is lactic acid bacteria *L.B- includin ifidobacterium$
Lactobacillus$ and !treptococcus *in-cludin %nterococcus-, which may !ose a
symbiotic relationshi! with host. Hrou! 1 includes !utrefactive bacteria such as
Clostridium perfringens$ Clostridium s!!., Bacteroides, #e!tococcaceae, Aeillonella$
%# coli$ !taphylococcus$ and Pseudomonas aeruginosa. Others are like %ubacterium$
.uminococcus$ Megasphaera$ Mitsuo&ello$ C# butyricum, and Candida. &ormally,
near stability exists in these habitats and each !erson has an individually fixed
microflora as far as "ualitative structure is concerned.
The intestinal microbiota !lay an im!ortant role in normal bowel function and
main-tenance of host health, throuh the formation of short chain fatty acids,
modulation of immune res!onses, and develo!ment of coloni/ation resistance. These
functions of the intestinal microbiota are the conse"uences of the activities of the
nemouse intestinal bac-teria, as a whole community, with a well-orani/ed structure
built on the balance amon the various bacterial members. Therefore, the functions of
the intestinal microbiota are very sensitive to the factors that can alter the structures of
the intestinal microflora "ual-itatively and !unitively, such as ain, !hysioloical
state, disease, diet, and stresses.
)ncreasin evidence indicates that the intestinal microbiota !lays a critical role in
the homeostasis of immune function in humans and that an abnormal intestinal
microbiota miht be the im!ortant as!ects of the !atholoy of various alleric disease
and other autoimmune diseases *'e et al., 1442, 1441E Ouwehand et al., 1442-.
B. PROBIOTICS AS A PRACTICAL WAY IN THE
MANAGEMENT OF ALLERGY
#robiotics have been suested to be a live microbial feed su!!lement that
beneficially affects the host by im!rovin its intestinal microbial balance *?uller,
23C3-. The definition of !robiotic has been further elaborated with the ex!ansion of
knowlede on the symbiotic interactions between intestinal microbes and the host
s!ecies *'e and Benno, 1422-. . recent definition, !ublished followin ex!ert
consultation at a 1442?.O,;'Omeetin, is T!robiotics are live microoranisms,
which when administered in ade"uate amounts confer a health benefit on the host.U
This newly !ro!osed definition em!hasi/es the im-!ortance of the clinically !roven
health benefits of !robiotics to the host, as well as their action in the host%s ut tract
and effects on intestinal microbiota. The ability of microbes, even if transient, to reach
and coloni/e the host intestine followin oral administration, is enerally considered
one of the key !ro!erties of !robiotics. On the other hand, for se-lection of new
microbes for !robiotic usae, the effects of microbes on the !hysioloical functions,
!articularly on immunity, of the host should be evaluated.
#resently, most !robiotics consist of L.B, !articularly lactobacilli and
bifidobacteria, which !ossess transient adhesive and coloni/in !ro!erties to the
human H) tract. Evi-dence from recent clinical and animal studies has su!!orted the
idea that lactobacilli and bifidobacteria, !articularly some selected strains, can modify
the host immune res!onses and !rotect them from !hysioloical disorders and
infective diseases, as well as these mi-crobes can normali/e intestinal microbiota and
reduce the fecal toxicity *?iure <.2EBenno et al., 233DE 'e et al., 1444, 1440E 'osoda
et al., 233D, 233CE Sawada et al., 144<-.
)n a recent study, more direct evidence was obtained for the !ossibility of
microbes to alleviate )E-mediated immune res!onses of allery *'arata et al., 1424-.
Sixteen heat-killed bifidobacteria isolated from human intestine and a !robiotic strain
Lactobacillus HH *LHH- was tested for their ability to influence )E-mediated
deranulation of rat baso-!hilic leukemia *(BL-1'9- cells in vitro. The bifidobacteria
su!!ressed )E-mediated deranulation of (BL-1'9 cells by 2.DM0D.7R in a strain-
de!endent manner. Bifidobac-teria from healthy infants ex!ressed hih inhibitory
effects on )E-mediated deranulation *72M00R-, while those from alleric infants
varied reatly in their effects aainst deranulation. Bifidobacteria taxonomically
identified as ifidobacterium bifidum exhibited much stroner inhibitory effects
aainst )E-mediated deranulation than those taxo-nomically identified as #
adolescentis *pV4.40-. These results indicate that the intestinal bifidobacteria miht be
one of the factors influencin )E-mediated alleric res!onses.
D. SELECTION AND EVAL*ATION OF PROBIOTIC FOR
POSSIBILITY IN ALLERGIC MANAGEMENT
(ecently, more and more scientists have focused their academic interest on
evaluation and selection of new !robiotic for their !otent health !romotin effects
aainst various
Fi"#r( K.> #ro!osed activation of !robiotics in astrointestinal tract of host animal
alleric disorders. .mon the !ros!ective, microbes for manaement of allery is
Lactobacillus gasseri T$6490D. This bacterium was oriinally isolated from a
healthy :a!anese adult *'osoda et al., 233C-. This bacterium was taxonomically
identified well with the !henoty!e and enoty!e methods *H6 content, 2Ds5&.-full
se"uence analysis, 5&.,5&. hybridi/ation-. T$6490D was tolerant to astric
acidity and bile toxicity, and adhered to ut e!ithelial cell and mucus in a strain-
de!endent manner *Hueimonde et al., 144DE :alonen et al., 1440E $orita et al.,
1441a,b-. T$6490D did not induce the local inflammation of e!ithelium
characteri/ed by interleukin *)L--C !roduction, but exhibited the a!!ro!riate !ro!erty
as an immunoreulator *'arata et al., 1443E 'e et al., 144DE )liev et al., 1440E $orita
et al., 1441a-. .natomical analysis also indicated that this bacterium can be taken u!
by ## tissue after oral admin-istration in mice *?iure <.1E 'arata et al., 1443-.
D.>. Sti.#ati!$ !' Cyt!,i$( Pr!/#&ti!$ by TMCRBEG a$/
E$ha$&(.($t !' T%h(3(r > Ty3( R(s3!$s(
T$6490D with other lactobacilli, oriinally from food and human intestine, were
tested for their ability to induce cytokine secretion by the murine macro!hae-like cell
line, :<<7.2 *$orita et al., 1441b-. The tested bacteria induced a!!arent )L-D, )L-24,
)L-21, and T&?- !roduction by :<<7.2 cells in a strain-de!endent fashion. 'owever,
Fi"#r( K.? 6ryosections of #eyerZs !atch usin confocal laser scannin microsco!e.
no )L-2 was detected in the s!ent media after ex!osure to any of the tested
lactobacilli. T$6490D sinificantly induced more )L-D, )L-24, )L-21, and T&?-
!roduction by :<<7.2 cells com!ared to the other tested bacteria *?iure <.9-. These
results suest that T$6490D can stimulate human immune res!onses, throuh the
activation of macro-!haes, characteri/ed by an elevation of both inflammatory and
anti-inflammatory cyto-kine secretion
D.?. I.3i&ati!$ by TMCRBEG t! I"E%M(/iat(/ I..#$(
R(s3!$s(s
T$6490D exhibited the !otential to modify )E !roduction in vitro by
s!lenocytes from ovalbumin *OI.--sensiti/ed B.LB,c mice *>awase et al., 144<b-.
?urthermore, T$6490D su!!ressed )E !roduction more than did other tested
lactobacilli. )n the !ar-allel, T$6490D sinificantly !romoted interferon *)?&--
!roduction and su!!ressed )L-7, )L-0 !roduction in the culture of the tested murine
s!lenocytes. The combination of LHH and T$6490D induced )?&- !roduction more
com!ared to either of them. Oral administration of cell !re!aration of T$6490D
sinificantly inhibited the increase of serum )E in OI.-sensiti/ed B.LB,c mice.
The combination of heat-killed LHH and T$6490D sinificantly decreased serum
)E !roduction even in a very low dosae.
The fermented milk !re!ared with T$6490D was administered at 144 ml day
-2
for
7 weeks to 20 volunteers with hih serum )E levels and !erennial alleric rhinitis
*$orita et al., 144D-. The serum total )E concentration was sinificantly reduced
after 1C days% ex!osure to the fermented milk *?iure <.7-. The serum )E s!ecific to
.cari and those to :a!anese cedar !ollen *:6#- was also sinificantly declined. T-
hel!er 2 *Th2- cells in the com!osition of #B$6s were sinificantly increased after
27 and 1C days *?iure <.0-. These results suest that T$6490D can downreulate
serum )E synthesis by enhancement of Th2 immune res!onses of the alleric
disorders in subGects with hih concentration of serum )E.
Fi"#r( K.B 6ytokine !roduction by :<<7.2 cells after ex!osure to Lactobacilli.
>.B. A(+iati!$ !' A(r"i& Sy.3t!.s a$/ I.3r!+(.($t !'
R(at(/ I$'a..ati!$ by TMCRBEG
&asal vascular !ermeability of OI.-sensiti/ed Brown &orway rats was evaluated
by an-aly/in brilliant blue concentration in the !erfusate from their nose after the
ex!osure of the nasal mucus to OI.. The oral administration of LHH and T$6490D
sinificantly inhibited the increase in OI. stimulated nasal vascular !ermeability
*>awase et al.144D-. The serum total )E of the rats fed with !robiotic bacteria was
also decreased, althouh such chanes were not statistically sinificant. These results
indicate that LHH and T$6490D may alleviate the nose alleric sym!toms by
su!!ressin the increase in the nasal vascular !ermeability caused by local
inflammation associated with alleric
Fi"#r( K.D Serum total )E of the subGects before and after the administration of the
fermented milk !re!ared with L# gasseri T$6490D.
Fi"#r( K.E Th2,Th1 in #B$6s of the subGects before and after the administration of the
fermented milk !re!ared with L# gasseri T$6490D.
rhinitis. S!ecific !robiotics L.B may influence systemic and local immune res!onses
of the host, while they !ersist in the ut of the host.
?urthermore, T$6490D and LHH were investiated for their ability to alleviate
nasal blockae associated with alleric rhinitis usin a uinea !i model *>awase et
al.144<a,b-. The increases in s(aw at 24 min and 0 h after the ex!osure of the nasal
mucosa to OI. were sinificantly alleviated in the uinea !is, orally administrated
with LHH and T$6490D com!ared with those of the control. The total numbers of
leukocytes,!articularly eosino!hils and neutro!hils from the nasal cavity lavae fluid,
and the OI.-s!ecific )E concentration in the serum were also decreased in the
uinea !is, orally administrated with LHH and T$6490D, althouh the decreases
were not statistically sinificant. These results suest that LHH and T$6490D can
alleviate antien-induced nasal blockae in early-!hase and late-!hase inflammatory
res!onses as-sociated with alleric rhinitis. On the other hand, the administration of
the combination of LHH and T$6490D su!!ressed inflation of eosino!hil
sinificantly in rats with OI.-induced alleric conGunctivitis *'ata et al., 144C-.
T$6490D and other L.B !roliferated human #B$6s from healthy subGects in a
dif-ferent manner com!ared to %# coli and su!!ressed )L-7 and )L-0 !roduction from
#B$6s isolated from alleric !atients sufferin from cedar !ollen after stimulation
with 6ryG-2, the key antien of :6# *>awase et al., 1443a-. The fermented milk
!re!ared with LHH and T$6490D showed its !otential to alleviate the sym!toms
such as nasal blockae of :6# allery and im!licate the related immune res!onses in a
randomi/ed double-blind, !lacebo-controlled trail. ?urthermore, the cell !re!aration
of LHH and T$6490D also ex!ressed the !otential to im!rove !erennial alleric
rhinitis in same randomi/ed double-blind, !lacebo-controlled way *>awase et al.,
1443b-.
D.D. C!!$i-ati!$ !' TMCRBEG i$ H#.a$ I$t(sti$( a$/
Stabii-(/ I$t(sti$a Mi&r!bi!ta i$ A(r"i& S#b1(&ts
The !resent study aimed to develo! an innovative, strain-s!ecific means of
identify-in !robiotic L# gasseri T$6490D and to determine whether orally
administered T$6490D could be recovered from the human intestine *>awase et al.,
1422-. 'ih molecular weiht enomic 5&. was isolated from T$6490D and 27
reference strains of L. gasseri includin the ty!e strain. The 5&. sam!les were
diested with the selected rare-cuttin restriction endonucleases !ma), !ac)), and
,pa), and resultin framents were se!arated by !ulsed-field el electro!horesis
*#?HE- in a si/e rane between 14 and 134 kb. T$6490D could be distinuished
from the other L# gasseri strains on the basis of the !maI and !acII macrorestriction
!atterns. ?urthermore, L# gasseri strains isolated from the feces of subGects inested
T$6490D were identical to T$6490D in !maI, !acII, and ,paI macrorestriction
framents of diested 5&.. These results su-est that #?HE of enomic 5&.
diested with !maI and !acII could be a !ractical means of identification of
T$6490D. ?urthermore, these results indicate that inested T$6490D can survive in
and coloni/e the human intestine.
. randomi/ed double-blind, !lacebo-controlled trial was conducted to ascertain
the intestinal microbiota alterin !ro!erties of Lactobacillus rhamnosus HH and L#
gasseri T$6490D *T$6490D- in :a!anese cedar Cryptomeria Caponica !ollinosis
*:6#sis- !atients. ?ecal bacteria communities were examined before and after
fermented milk administra-tion usin culture, fluorescence in situ hybridi/ation
*?)S'-, and terminal restriction frament lenth !olymor!hism *T-(?L#- methods
*>ubota et al., 1443-. Test rou! subGects showed the !resence of LHH and T$6490D
alon with a sinificant increase in fecal lactobacilli *pV4.442- after administration of
LHH and T$6490D fermented milk. 6ulture and ?)S' analysis revealed no
sinificant chanes in other intestinal bacterial rou!s. Each subGect exhibited a
characteristic T-(?L# !rofile !attern that varied "uan-titatively and "ualitatively with
:6# sheddin. #rofile chanes were observed in 01R of !lacebo rou! subGects and in
1<R of test rou! subGect%s !ostadministration, indicatin that LHH and T$6490D
su!!ressed intestinal microbiota chanes in :#6sis !atients. The results suest that
intestinal microbiota miht be more sensitive to ex!osure to environ-mental allerens
than that ex!ected from the results of eneral culture method studies. Stabili/ation of
intestinal microbiota by selected !robiotic strains such as LHH and T$6490D could
be beneficial to homeostasis of the intestinal microbiota and useful in manaement of
:6#sis. The fecal bifidobacteria community was examined before and after the
intervention with "uantitative real-time #6( *"(T-#6(- *>ubota et al., 1422-. There
were no sinificant differences in the !revalence of the ifidobacterium s!ecies, both
before and after the administration in each rou! and between the two rou!s. "(T-
#6( analysis revealed no sinificant chanes in other !redominate intes-tinal
ifidobacterium s!ecies. These results suest that :6#sis develo!ment of sym!toms
shows that a bi chane does not take !lace to intestinal ifidobacterium.
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lactobacilli. ?ood (esearch )nternational 3, 7D<M7<2.
'arata, H., 'e, ?., Takahashi, >.,i et al., 1443. 5ifferentiated im!lication of
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'e, ?., $orita, '., >ubota, .., et al., 1440. Effect of orally administered non-
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'e, ?., $orita, '., Ouwehand, .., 144D. Bifidobacteria and lactobacilli exhibited
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'e, ?., Ouwehand, ..6., )solauri, E., 'ashimoto, '., Benno, =., Salminen, S.,
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)liev, ).5., >ita/awa, '., Shimosato, T., et al., 1440. Stron immunostimulation in
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:alonen, L., Hueimonde, $., 'e, ?., Salminen, S., 1440. 5is!lacement of adhered
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6linical &utrition 27, S214.
>awase, $., 'e, ?., >ubota, .., 'arata, H., 'iramatsu, $., 144<a. Orally
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13M9C.
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)nhibitory effect of Lactobacillus gasseri T$6490D and Lactobacillus HH on
enhanced vascular !ermeability of nasal mucosa in ex!erimental alleric rhinitis of
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Lactobacillus gasseri T$6490D from human intes-tine. $icrobioloy and
)mmunoloy 00, 0C3M037.
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intestinal microbiota in :a!anese cedar !ollinosis !atients. $icrobioloy and
)mmunoloy 09, 23CM140.
>ubota, .., 'e, ?., >awase, $., 'arata, H., 'iramatsu, $., )ino, '., 1422.
5iversity of intestinal Bifidobacteria in !atients with :a!anese cedar !ollinosis and
!ossible influence of !robiotic intervention. 6urrent $icrobioloy D1, <2M<<.
$orita, '., 'e, ?., ?use, T., et al., 1441a. .dhesion of lactic acid bacteria to
6aco-1 cells and their effect on cytokine secretion. $icrobioloy and )mmunoloy
7D, 139M13<.
$orita, '., 'e, ?., ?use, T., et al., 1441b. 6ytokine !roduction by the murine
macro!hae cell line :<<7.2 after ex!osure to lactobacilli. Bioscience, Biotechnoloy,
and Biochemistry DD, 23D9M23DD.
$orita, '., 'e, ?., >awase, $., et al., 144Da. #reliminary human study for
!ossible alteration of serum 22. )mmunolobulin E !roduction in !erennial alleric
rhinitis with fermented milk !re!ared with Lactobacillus
gasseri T$6490D. $icrobioloy and )mmunoloy 04, <42M<4D.
Ouwehand, ..6., )solauri, E., 'e, ?., et al., 1442. 5ifferences in ifidobacterium
flora com!osition in alleric and healthy infants. The :ournal of .llery and 6linical
)mmunoloy 24C, 277M270.
Sawada, :., $orita, '., Tanaka, .., Salminen, S., 'e, ?., $atsuda, '., 144<.
)nestion of heat-treated Lactobacillus rhamnosus HH !revents develo!ment of
ato!ic dermatitis in &6,&a mice. 6linical and Ex!erimental .llery 9<, 13DM949.
CHAPTER L
N#triti!$a F#$&ti!$s !'
P!ysa&&hari/(s 'r!. S!y Sa#&( i$
th( Gastr!i$t(sti$a Tra&t
M. K!bayashi
'iashimaru Shoyu 6o., Ltd., 'yoo, :a!an
>. INTROD*CTION
Soy sauce, traditionally used in :a!an and several oriental countries, is a li"uid
seasonin currently used in cookin worldwide. The daily consum!tion of soy sauce
in :a!an is estimated at about 94 ml !er !erson accordin to the data from the :a!an
Soy Sauce Brewers .ssociation. Studies suest that soy sauce contains certain
bioactive com!onents in addition to taste and aroma com!ounds and has various
bioloical functions, includin anticarcinoenic, antimicrobial, antioxidative, and
anti!latelet activities, and the inhibi-tion of an aniotensin )-convertin en/yme
*$urooka and =amashita, 144C-. Therefore, soy sauce is considered to be not only a
traditional seasonin but also a functional food. ?urther study is needed to elucidate
the bioloical functions of soy sauce inredients. )n this cha!ter, nutritional functions
of !olysaccharides from soy sauce are described with es!ect to the reulation of iron
and li!id absor!tion in the astrointestinal tract.
?. BREWING OF 5APANESE SOY SA*CE
!hoyu is the :a!anese name for soy sauce. $any varieties of shoyu are !roduced
in :a!an and other oriental countries. Their characteristics de!end on the various ty!es
and dif-ferent ratios of raw materials used, the ty!es of microoranisms em!loyed,
and the con-ditions of fermentation *>obayashi, 1440E $urooka and =amashita,
144CE =okotsuka, 23CD-. )n :a!an, naturally brewed shoyu is !roduced as shown in
?iure C.2. $oistened soybeans are cooked at hih !ressure and tem!erature, while
wheat is roasted and crushed. These two materials are mixed with a small amount of
seed mold, ,spergillus oryBae or ,# soCae. This mixture is !laced on a lare !orous
!late throuh which tem!erature- and moisture-controlled air is !assed to !rovide the
a!!ro!riate conditions for mold rowth and en/yme !roduction. The mold-cultured
material is termed &oCi, and the &oCi is mixed with a hih concentration of &a6l in
water to make a mash called moromi. The moromi, containin about 20R &a6l, is
stored in lare tanks for several
Fi"#r( L.> Brewin of soy sauce from raw materials to the final !roduct Sshoyu#%
months at room or elevated tem!erature. The moromi is fermented with lactobacilli
and yeasts and then well aed. The aed moromi is !ressed, and the li"uid !art *raw
soy sauce- is !asteuri/ed to obtain the final !roduct, soy sauce *shoyu-.
B. POLYSACCHARIDES FROM SOY SA*CE
)n soy sauce, !roteins of the raw materials are com!letely deraded into !e!tides
and amino acids by microbial !roteolytic en/ymes after fermentation, and so no
allerens such as soybeans and wheat are !resent *>obayashi, 1440E >obayashi et al.,
1447a-. )n contrast, !olysaccharides oriinatin from the cell wall of soybeans are
resistant to en/ymatic hy-drolysis and are !resent even after fermentation *>obayashi
et al., 1447c-. )n 23<1, >ikuchi and =okotsuka !urified !olysaccharides from soy
sauce and investiated their !ro!erties in detail *>ikuchi and =okotsuka, 23<1-. The
cell wall !olysaccharides of soy-beans, one of the main raw materials of soy sauce,
contain a lare amount of alacturonic acid and are only slihtly hydroly/ed by mold
en/ymes durin the &oCi and moromi staes. These !olysaccharides make u!
a!!roximately 2R of the soy sauce and are termed shoyu !olysaccharides *S#S-
*>obayashi et al., 1447c-. .lthouh it has been more than 94 years since
!olysaccharides such as !ectic substances were first re!orted to be !resent in soy
sauce, their bioloical activities have still not been investiated. (ecently, >obayashi
et al. demonstrated that S#S have !otent antialleric effects in vitro and in vivo, and
oral su!!lementation of S#S was an effective intervention for !atients with alleric
rhinitis in two double-blind, !lacebo-controlled clinical studies *>obayashi, 1440,
1424E >obayashi et al., 1447b,c, 1440-. ?urthermore, $atsushita et al. found that S#S
have reulatory ef-fects on both the balance of Th2,Th1 cell res!onses and the
intestinal immune system *>obayashi, 1424E $atsushita et al., 144D, 144C-. The
nutritional functions of S#S were further examined in the astrointestinal tract, and
S#S were found to enhance the absor!-tion of iron and reduce elevated levels of
triacyllycerol in animal and human studies *>obayashi et al., 144D, 144C-.
D. IRON ABSORPTION
D.>. Ir!$ D('i&i($&y a$/ F!rti'i&ati!$
)ron deficiency is a maGor health !roblem in develo!in countries and affects
about 9M0 billion !eo!le worldwide, mainly women of re!roductive ae, infants, and
youn children *;an et al., 144C-. )ron-deficiency anemia is the most severe form of
iron deficiency. Low iron bioavailability could be due to low solubility in the
intestinal tract and the !resence of inhibitors such as !hytic acid from cereal-based
diets *;an et al.,144C-. The fortification of foods is often rearded as the most cost-
effective lon-term a!!roach to reducin the !revalence of iron deficiency. Soy sauce
is a !o!ular traditional seasonin in 6hina, Thailand, and Iietnam, makin it a
candidate for use as an iron carrier *Thuy et al., 1440E ;alc/yk et al., 1440E ;an et
al., 144C-. )n Southeast .sia, where iron deficiency is wides!read, and a maGor !ublic
health !roblem, soy sauce would be a !referred food condiment for iron fortification
with rice meals because of its consum!-tion and cost. Baynes et al. were the first to
re!ort a !romotive effect of soy sauce on iron absor!tion in clinical ex!eriments
*Baynes et al., 2334-. 6om!ared with commonly used ?e salt fortificants, &a?eE5T.
has a hiher rate of absor!tion in the human body, fewer adverse effects, and less
influence on the bioavailability of other minerals *;an et al.,144C-. The successful
use of &a?eE5T.-fortified soy sauce and fish sauce in anemic women and children
has since been re!orted in 6hina, Thailand, and Iietnam *Thuy et al., 1440E ;alc/yk
et al., 1440E ;an et al., 144C-. )nterestinly, Baynes et al. also re!orted that soy
sauce added to a rice meal a!!eared to enhance iron absor!tion what-ever its
mechanism of action *Baynes et al., 2334-. .lthouh these findins may have some
relevance to iron nutrition in those .sian countries in which rice forms a substantial
!art of the sta!le diet, the mechanism !romotin the effect of soy sauce on iron
absor!-tion remains unclear.
D.?. Ir!$ F!rti'i&ati!$ 0ith S!y Sa#&(
#oor bioavailability of iron from soybeans alone and an inhibitory effect of
soybeans on the absor!tion of iron from a mixed diet have been re!orted *Baynes et
al., 2334-. .lthouh the inhibitory com!onent of soybeans has not yet been identified,
hih-molecular-weiht !rotein would inhibit the bioavailability of iron, !ossibly via
the formation of relatively stable !roteinMiron areates *Baynes et al., 2334-. Soy
sauce lacks hih-molecular-weiht !roteins because amino acids and small !e!tides
are diested by microbial hydrolyses durin lon-term fermentation *>obayashi,
1440E >obayashi et al., 1447a-. )n 2334, Baynes et al. re!orted a !romotive effect of
soy sauce on the ab-sor!tion of iron from a rice meal, since fermented soy sauce does
not have the inhibitors of iron bioavailability !resent in most soybean !roducts
*Baynes et al., 2334-. Similarly, in 2334, $acfarlane et al. found that a fermented
soybean !roduct, miso, also im!roved iron bioavailability *$acfarlane et al., 2334-.
Therefore, traditionally fermented soybean !roducts such as shoyu *soy sauce- and
miso would contribute to iron nutrition in com-munities in which rice is the sta!le
diet. .lthouh the mechanism res!onsible for the !romotive effect on iron absor!tion
remained unclear, fermented !roducts of soy sauce miht be involved. Therefore, we
examined the ca!acity of S#S to bind iron, as well as the effect of S#S on iron
absor!tion in animals and humans, in order to clarify the !ro-motin activities of S#S
as a functional dietary com!onent from soy sauce *>obayashi et al., 144D-.
D.B. Pr!.!ti+( E''(&t !' SPS !$ Ir!$ Abs!r3ti!$
)ron deficiency, the most common nutritional deficiency in the world, is more
often the result of !oor iron bioavailability from diets that a!!arently contain ade"uate
amounts than of inade"uate intake. The maGor source of iron in these diets is nonheme
iron, the bioavailability of which de!ends on the s!ecific form of iron in the diet and
its solubility in the intestinal tract *>im and .tallah, 2339-. 5ietary liands that form
soluble iron chelates at intestinal !' values are assumed to increase bioavailability,
whereas those that form low solubility chelates, or can !reci!itate iron, reduce its
bioavailability *>im and .tallah, 2339-. The effect of dietary fiber on iron
bioavailability has been the focus of many studies. Some studies have re!orted that
fiber-rich diets decreased iron absor!tion, whereas others have shown no effect *>im
and .tallah, 2339-. #ectin is an im!ortant com!onent of the water-soluble dietary
!olysaccharides and contains carboxylic rou!s ca!able of formin com!lexes with
mineral ions. Because the free carboxyl rou!s in the alacturonic acid units can form
com!lexes with !olyvalent cations such as 6a, $, Nn, and ?e, !ectin in diets could
reduce the bioavailability of minerals *>im and .tallah, 2339-. 'owever, studies in
vivo showed that !ectin !romoted iron absor!tion, suestin that differences in the
molecular weiht and deree of esterification of !ectins influence solubility and
strenth of bindin to minerals *>im and .tallah, 2339E >im et al., 233D-.
8nlike heme iron, inoranic iron needs to be solubili/ed in the astrointestinal
tract to be effectively absorbed. >obayashi et al. re!orted that S#S derived from soy
sauce en-hanced intestinal iron absor!tion by increasin the solubility of inoranic
iron *>obayashi et al., 144D-. ?errous and ferric irons are dissolved at low !', in the
stomach for exam!le, but !reci!itated at neutral !' in the intestinal tract. )n the
!resence of S#S, however, ferrous and ferric irons are dissolved at neutral !'. S#S
would enhance the solubility of iron at neutral !', by formin the com!lex ?eMS#S.
Therefore, S#S of soy sauce would function as an iron stabili/er at neutral !', for
exam!le, in the small intestine. ?urthermore, S#S enhanced the absor!tion and
!oolin of iron in orans such as the liver and s!leen, and thus decreased iron
excretion into the feces in rats made anemic by feed-in them ?e-deficient diets for 1
weeks *>obayashi et al., 144D-. )n a double-blind, !lacebo-controlled clinical study of
healthy women, serum iron, hematocrit, and hemo-lobin levels were sinificantly
*pV4.40- hiher in the S#S-administered rou! than in the !lacebo-administered
rou! after 7 weeks of treatment *>obayashi et al., 144D-. .fter 7 weeks of treatment,
no sinificant abnormality was detected in routine blood tests, includin he!atic and
renal function, and concentrations of !roteins and li!ids. )n con-clusion, S#S of soy
sauce act to enhance iron absor!tion by stabili/in astrointestinal conditions
*>obayashi et al., 144D-. Therefore, soy sauce is useful as an antianemic aent in
daily life, and the S#S from soy sauce would be safe and ex!ected to !romote the iron
fortification of food.
E. LIPID ABSORPTION
E.>. Tria&y"y&(r! Abs!r3ti!$
)n develo!ed countries, the incidence of lifestyle-related diseases such as diabetes,
hy!er-li!idemia, and hy!ertension has been steadily increasin. Obesity is one of the
most im!ortant risk factors for various diseases in modern society. Excess inestion
of li!idsis the most common cause of obesity. Hiven the affiliation between dietary
li!ids and obesity, and even heart disease, a reduction in li!id intake is a loical
nutritional recourse *'su et al., 144D-. Therefore, dietary thera!y is im!ortant and
could be considered the first choice in treatment, and an effective way to !revent
obesity is to inhibit fat absor!-tion from the intestine or increase the metabolic rate
and fat oxidation *&akai et al., 1440-. 5ietary li!ids contain triacyllycerol,
!hos!holi!ids *!hos!hatidylcholine-, and sterols *cholesterol-, which re!resent a
hihly concentrated form of enery. 'owever, dietary li!id consum!tion is hih in
many !o!ulations. 5iestion by !ancreatic li!ases is an essential ste! in the
absor!tion of dietary li!ids. Studies in mice have suested that inhibition of
diestive li!ase activity could sinificantly affect dietary li!id absor!tion and increase
the fecal excretion of li!ids *'su et al., 144D-. )n mammals, the diestion of dietary
triacyllycerol involves three maGor li!ases+ !reduodenal *linual or astric-, car-
boxylester, and !ancreatic li!ases *TsuGita et al., 1449-. 8nder acidic conditions in the
stomach, fat is hydroly/ed by !reduodenal li!ases, which leads to the hydrolysis of
dietary triacyllycerol, !roducin diacyllycerol and free fatty acids. #ancreatic
li!ases convert triacyllycerol to 1-monoacyllycerol and free fatty acids.
6arboxylester li!ases have a broad s!ecificity, actin on triacyllycerol,
diacyllycerol, monoacyllycerol, and choles-terol esters. These li!olytic !roducts are
dis!ersed as micelles and carried to sites of fat absor!tion. Li!id absor!tion takes
!lace in the a!ical !art of the !lasma membrane of e!ithelial cells or enterocytes
linin the ut.
#ancreatic li!ase is a key en/yme for li!id absor!tion. )t is well established that
dietary fat is not directly absorbed from the intestine unless it has been subGected to
the actions of !ancreatic li!ase *&akai et al., 1440-. Therefore, to su!!ress serum
triacyllycerol levels and weiht ain, it would be effective to reduce fat absor!tion
by inhibitin li!ases. Orlistat, a s!ecific !ancreatic li!ase inhibitor, is used clinically
to !revent obesity and hy!erli!idemia *&akai et al., 1440-. Li!ase inhibitory materials
derived from natural !roducts, such as chitosan, !ectin, chondroitin sulfate, and the
!oly!henolic constituents of ra!e seed extract and oolon tea, have been re!orted
*'an et al., 2333E >oseki et al., 23C3E &akai et al., 1440E TsuGita et al., 1449-. )n
animal studies, water-soluble dietary fibers, such as lucomannan and !ectin, stronly
affected li!id metabolism, which led to decreased serum cholesterol and
triacyllycerol levels *=amada et al., 2333-. ?urther-more, in human studies, some
dietary factors, -conlycinin from soybeans, mannooli-osaccharides from coffee
mannan, and !oly!henol-enriched oolon tea, su!!ressed serum li!id levels and
reduced fat storae via mechanisms such as the inhibition of fat absor!tion, excretion
of fat, or reulation of li!id metabolism *'su et al., 144DE >ohno et al., 144DE >umao
et al., 144D-.
E.?. R(/#&i$" E''(&t !' SPS !$ Tria&y"y&(r! Abs!r3ti!$
To clarify the hy!oli!idemic effect of S#S as a functional dietary com!onent, we
exam-ined the effects of S#S on !ancreatic li!ase activity in vitro and on fat
absor!tion induced by a hih-fat diet in vivo usin mice and rats *>obayashi et al.,
144C-. ?urthermore, we evaluated the efficacy of S#S in reducin human serum
triacyllycerol levels in two clinical studies *>obayashi et al., 144C-.
.n in vitro study by >obayashi et al. revealed that S#S inhibited !ancreatic
li!ases *>obayashi et al., 144C-E this suests that S#S su!!ress fat absor!tion by
inhibition of !ancreatic li!ases. ?urthermore, S#S decreased immediately and
radually serum triacyl-lycerol levels in animals on hih-fat diets both in the short
and lon term *>obayashi et al., 144C-. (ecently, Takano et al. have constructed an
ex!erimental system to monitor the !ortal concentrations of nutrients such as lucose
and calcium usin rats that have underone !ortal vein catheteri/ation *Takano et al.,
144<-. This method was used to investiate the effect of S#S on li!id absor!tion in
rats with enteral feedin, althouh the lym!hatic absor!tion of triacyllycerol was not
directly monitored. )n catheteri/ed rats, S#S continuously lowered serum
triacyllycerol and nonesterified fatty acid levels raised by a hih-fat diet *>obayashi
et al., 144C-. Since S#S are !ectin !olysaccharides deraded from soybeans durin the
brewin !rocess, they would function to excrete fat as dietary fibers *=amada et al.,
2333-, and also to reduce the absor!tion of fat by inhi-bitin li!ases as chitosan *'an
et al., 2333-. 'owever, the !recise mechanisms underlyin the decreased
triacyllycerol synthesis and accumulation caused by S#S remain unknown.
)n a clinical study of healthy men, >obayashi et al. found that elevated serum
triacyl-lycerol levels were sinificantly *pV4.40- lower in the S#S-administered
rou! than in the !lacebo-administered rou! D h after a hih-fat meal *>obayashi et
al., 144C-. )n a clinical study of hy!ertrilyceridemic men, serum triacyllycerol
levels in the S#S-administered rou! were sinificantly *pV4.40- lower than the
baseline *4 week- after 7 weeks of treatment *>obayashi et al., 144C-. )n conclusion,
S#S of soy sauce would be effective in su!!ressin increases in serum triacyllycerol
levels when a hih-li!id diet is iven and reducin hih-serum triacyllycerol levels
in !atients with hy!ertrilycer-idemia as a dietary treatment. Therefore, soy sauce is
useful as a hy!oli!idemic seasonin and the S#S would be a safe and im!ortant
com!onent for the !revention and,or ame-lioratio nof visceral fat syndrome, or the so-
called metabolic syndrome.
G. CONCL*SION
)n the brewin of soy sauce, !roteins of the raw materials are com!letely deraded
into !e!tides and amino acids by microbial !roteolytic en/ymes after fermentation,
and no allerens remain *>obayashi, 1440E >obayashi et al., 1447a-. )n contrast,
deraded !oly-saccharides from the cell wall of soybeans, termed S#S, are resistant to
en/ymatic hydrolysis and are found in soy sauce even after fermentation *>obayashi
et al.,1447c-. ;e have demonstrated the enhanced absor!tion of iron and reduced
elevated levels of triacyllycerol by S#S in animal and human studies *>obayashi et
al., 144D, 144C-. (ecently, we have also demonstrated that S#S have both antialleric
activities and reulatory effects on the immune system in vitro$ in vivo, and in clinical
studies *>obayashi, 1440, 1424-. ?rom these results, immunoloical and nutritional
functions of S#S are summari/ed in ?iure C.1. Therefore, soy sauce is a !otentially
!romisin functional food.
Fi"#r( L.? )mmunoloical and nutritional functions of S#S.
Ma,i! K!bayashiI PhD
A''iiati!$) 5irector, (esearch Laboratory, 'iashimaru Shoyu 6o., Ltd.
Dat( !' birth+ 5ecember 3, 23D0
A&a/(.i& A33!i$t.($ts)
1442 Iisitin (esearcher, 'yoo #refectural )nstitute of Technoloy
1441 Iisitin Lecturer, School of Bioscience and Biotechnoloy, Tokyo )nstitute
of Technoloy
1440M!resent Iisitin .ssociate #rofessor, School of 'uman Science and
Environment, 8niversity of 'yoo
E/#&ati!$)
23C7M23CC 5e!artment of ?ermentation Technoloy, ?aculty of Enineerin,
'iroshima 8niversity, B.S. $arch 23CC
23CCM2334 Laboratory of ?ermentation Technoloy, 5e!artment of )ndustrial
6hem-istry, Hraduate School of Enineerin, 'iroshima 8niversity, $.S. $arch
2334
2334M2339 Laboratory of ?ermentation Technoloy, 5e!artment of )ndustrial
6hem-istry, Hraduate School of Enineerin, 'iroshima 8niversity, #h5 $arch 2339
A0ar/s)
1440 Technoloy .ward of the :a!an Soy Sauce Technoloy 6enterE S.lleren
deradation of wheat and soybeans in soy sauce brewin%
144D Technoloy .ward of the Brewin Society of :a!anE S;heat alleren
deradation in soy sauce brewin%
144< Encouraement .ward of the Society for Biotechnoloy, :a!an *Eda
.ward-E S?unctional research and develo!ment of soy sauce%
144C Technoloy .ward of the :a!an Soy Sauce Technoloy 6enterE S?unctions
of shoyu !olysaccharides%
R(s(ar&h T!3i&s)
(esearch and develo!ment of soy sauce brewin and its functions
P#bi&ati!$s+ CC includin 74 reviews
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Baynes, (.5., $acfarlane, B.:., Bothwell, T.'., et al., 2334. The !romotive effect
of soy sauce on iron absor!tion in human subGects. Euro!ean :ournal of 6linical
&utrition 77, 723M717.
'an, L.>., >imura, =., Okuda, '., 2333. (eduction in fat storae durin chitin-
chitosan treatment in mice fed a hih-fat diet. )nternational :ournal of Obesity 19,
2<7M2<3.
'su, T.?., >usumoto, .., .be, >., et al., 144D. #oly!henol-enriched oolon tea
increases fecal li!id excre-tion. Euro!ean :ournal of 6linical &utrition D4, 2994M
299D.
>ikuchi, T., =okotsuka, T., 23<1. Studies on the !olysaccharides from soy sauce
#art ). #urification and !ro!erties of two acidic !olysaccharides. .ricultural and
Bioloical 6hemistry 9D, 077M004.
>im, $., .tallah, $.T., 2339. )ntestinal solubility and absor!tion of ferrous iron
in rowin rats are affected by different dietary !ectins. :ournal of &utrition 219,
22<M217.
>im, $., .tallah, $.T., .marasiriwardena, 6., Barnes, (., 233D. #ectin with low
molecular weiht and hih deree of esterification increases absor!tion of
0C
?e in
rowin rats. :ournal of &utrition 21D, 2CC9M2C34.
>obayashi, $., 1440. )mmunoloical functions of soy sauce+ hy!oallerenicity
and antialleric activity of soy sauce. :ournal of Bioscience and Bioenineerin 244,
277M202.
>obayashi, $., 1424. )mmunoloical functions of !olysaccharides from soy
sauce. )n+ ;atson, (.(., Nibadi, S., #reedy, I.(. *Eds.-, 5ietary 6om!onents and
)mmune ?unction. 'umana #ress, Breinisville, #., !!. 072M001.
>obayashi, $., 'ashimoto, =., Taniuchi, S., Tanabe, S., 1447a. 5eradation of
wheat alleren in :a!anese soy sauce. )nternational :ournal of $olecular $edicine 29,
C12MC1<.
>obayashi, $., $aishi, &., $atsushita, '., et al., 144C. 'y!oli!idemic effect of
!hoyu !olysaccharides from soy sauce in animals and humans. )nternational :ournal
of $olecular $edicine 11, 0D0M0<4.
>obayashi, $., $atsushita, '., Shioya, )., et al., 1447b. Wuality of life
im!rovement with soy sauce inre-dients, Shoyu !olysaccharides, in !erennial
alleric rhinitis+ a double-blind !lacebo-controlled clinical study. )nternational :ournal
of $olecular $edicine 27, CC0MCC3.
>obayashi, $., $atsushita, '., Tsukiyama, (., Saito, $., Suita, T., 1440. !hoyu
!olysaccharides from soy sauce im!rove "uality of life for !atients with seasonal
alleric rhinitis+ a double-blind !lacebo-controlled clinical study. )nternational :ournal
of $olecular $edicine 20, 7D9M7D<.
>obayashi, $., $atsushita, '., =oshida, >., et al., 1447c. In vitro and in vivo
anti-alleric activity of soy sauce. )nternational :ournal of $olecular $edicine 27,
C<3MCC7.
>obayashi, $., &aatani, =.,$aishi, &., et al., 144D. #romotive effect of !hoyu
!olysaccharides fromsoy sauce on iron absor!tion in animals and humans.
)nternational :ournal of $olecular $edicine 2C, 2203M22D9.
>ohno, $., 'irotsuka, $., >ito, $., $atsu/awa, =., 144D. 5ecreases in serum
triacyllycerol and visceral fat mediated by dietary soybean a-conlycinin. :ournal of
.therosclerosis and Thrombosis 29, 17<M100.
>oseki, $., TsuGi, >., &akaawa, =., et al., 23C3. Effects of um arabic and
!ectin on the emulsification, the li!ase reaction, and the !lasma cholesterol level in
rats. .ricultural and Bioloical 6hemistry 09, 921<M9291.
>umao, T., ?uGii, S., .sakawa, .., Takehara, )., ?ukuhara, )., 144D. Effect of
coffee drink containin man-nooliosaccharides on total amount of excreted fat in
healthy adults. :ournal of 'ealth Science 01, 7C1M7C0.
$acfarlane, B.:., van der (iet, ;.B., Bothwell, T.'., et al., 2334. The effect of
traditional oriental soy !rod-ucts on iron absor!tion. .merican :ournal of 6linical
&utrition 02, C<9MCC4.
$atsushita, '., >obayashi, $., Tsukiyama, (., =amamoto, >., 144D# In vitro and
in vivo immunomodulatin activities of !hoyu !olysaccharides from soy sauce.
)nternational :ournal of $olecular $edicine 2<, 340M343.
$atsushita, '., >obayashi, $., Tsukiyama, (., et al., 144C. Stimulatory effect of
!hoyu !olysaccharides from soy sauce on the intestinal immune system. )nternational
:ournal of $olecular $edicine 11, 179M17<.
$urooka, =., =amashita, $., 144C. Traditional healthful fermented !roducts of
:a!an. :ournal of )ndustrial $icrobioloy and Biotechnoloy 90, <32M<3C.
&akai, $., ?ukui, =., .sami, S., et al., 1440. )nhibitory effects of oolon tea
!oly!henols on !ancreatic li!ase in vitro. :ournal of .ricultural and ?ood 6hemistry
09, 7039M703C.
Takano, =., $atsuura, T., =oshikawa, =., et al., 144<. $odulation of the
intestinal 6a
1B
u!take by a cheese whey !rotein diest. Bioscience, Biotechnoloy,
and Biochemistry <2, 27C<M2739.
Thuy, #.I., Berer, :., &akanishi, =., et al., 1440. The use of &a?eE5T.-
fortified fish sauce is an effective tool for controllin iron deficiency in women of
childbearin ae in rural Iietnam. :ournal of &utrition 290, 103DM1D42.
TsuGita, T., Sumiyoshi, $., 'an, L.>., et al., 1449. )nhibition of li!ase activities
by citrus !ectin. :ournal of &utritional Science and Iitaminoloy 73, 974M970.
;alc/yk, T., Tunti!o!i!at, S., Neder, 6., et al., 1440. )ron absor!tion by human
subGects from different iron fortification com!ounds added to Thai fish sauce.
Euro!ean :ournal of 6linical &utrition 03, DDCMD<7.
;an, B., Nhan, S., Sun, :., Lee, L., 144C. Social mobili/ation and social
marketin to !romote &a?eE5T.-fortified soya sauce in an iron-deficient !o!ulation
throuh a !ublic-!rivate !artnershi!. #ublic 'ealth &utrition 21, 2<02M2<03.
=amada, >., Tokunaa, =., )keda, .., et al., 2333. 5ietary effect of uar um and
its !artially hydroly/ed !roduct on the li!id metabolism and immune function of
S!raueM5awley rats. Bioscience, Biotech-noloy, and Biochemistry D9, 12D9M12D<.
=okotsuka, T., 23CD. Soy sauce biochemistry. .dvances in ?ood (esearch 94,
230M913.
CHAPTER O
N#triti!$ T Di(tary Fib(rsI a$/
Ch!(ithiasis) Ch!(ithiasis a$/ Li3i/
L!0(ri$"
R. Shar.a
*
,

I R.K. Ta$/!$
*
,

#ush!awati Sinhania )nstitute of Liver, and Biliary 5iseases, &ew 5elhi, )ndia

.mity 8niversity, &oida, )ndia

#ush!awati Sinhania (esearch )nstitute of Liver, Hall Bladder 5iseases, &ew


5elhi, )ndia
>. INTROD*CTION
Cholilithiasis *ko-le-li-T')-ah-sis- was defined for the first time in fourteenth
century as a condition where a !erson has allstones *stones in the allbladder- by
Wuincke and 'o!!e-Seyler *2349-. 6holelithiasis is a common health !roblem
worldwide, affectin a!!roximately 2 out of 2444 !eo!le. )n the 8nited States,
cholelithiasis condition affects about 14R of the !o!ulation above 74 years of ae and
is more !revalent in women, &ative .mericans, and !ersons with cirrhosis of the
liver. .!!roximately 14R of !eo!le above D0 years of ae have allstones but most
have no sym!toms re!orted by &)55> *1443- and .fdhal *144<-. ?ive million
.mericans suffer from dianosed sym!tomatic cholesterol saturation disorder while
an even larer number are believed to be sufferin from an undianosed
asym!tomatic cholelithiasis-related disease. The cholesterol satura-tion in bile and
blood is a common chronic disease as hy!ercholesterolemia and it leads to allstones
mainly com!licated with atherosclerosis, obesity, and fatty liver sooner or later.
$ostly allstones in &orth .merica are com!osed !rimarily of cholesterol *<0R-.
Other 10R of allstones are com!osed of !iment *D4R in :a!an- as re!orted by
Nubler et al. *233C-. This cha!ter introduces the basic conce!ts of cholelithiasis
includin oriin, sym!toms, dianosis, !atho!hysioloy, role of diet thera!y, nutrition
su!!lements, nutrition manaement, diet testin by en/yme assay, dietary fibers, and
nutrition treat-ment with future !ros!ective on success of cholelithiasis !revention
and treatment in the followin sections.
?. HOW CHOLELITHIASIS IS ORIGINATED AND
COMPLICATEDU
Hallbladder is a !ouch-like oran located Gust below the liver and it stores bile
from liver. Bile is an emulsion made of three main li!ids includin cholesterol,
!hos!holi!ids, and li!o!roteins. Bile is a balanced mixture of water, cholesterol *fat-,
bile salts lecithin, and bilirubin *yellow !iment-. Bile !asses throuh tubes called
bile ducts and is released into the intestines after eatin. Bile hel!s in diestin fats
and other substances. 6holesterol !lays a maGor role in bile salt formation and kee!in
biliary salts in balance *(oda et al., 2331-. Cholelithiasis is referred to as the !resence
of stones in the allbladder. Cho-lecystitis is defined as an acute or a chronic
inflammation of the allbladder. Choledocho-lithiasis refers to the !resence of stones
in the common bile duct. Hallstones result from su!ersaturation of cholesterol in the
bile. .ccelerated nucleation of cholesterol is a !heno-menon which is not well
understood. )n hy!ercholesterolemic stae, bile acts as an irritant, !roducin
inflammation in the allbladder, and !reci!itates out of bile, makin stones.
6om!lications of allbladder disease also include cholanitisE necrosis, em!yema,
and !erforation of allbladderE biliary fistula throuh duodenumE allstone ileusE and
ad-enocarcinoma of the allbladder. 'owever, several risk factors !lay their role in
chole-lithiasis such as ender *women four times as likely to develo! cholesterol
stones as men-, ae *above 74 years of ae-, multi!le !arity, obesity, use of estroen
and cholesterol low-erin drus, bile acid malabsor!tion with H) disease, enetic
!redis!osition, ra!id weiht loss, ethnic and hereditary factors, diabetes, liver
cirrhosis, lon-term intravenous nutri-tion *total !arenteral nutrition *#&--, and certain
o!erations for !e!tic ulcers *(oda et al., 2331E ?iure 3.2-.
B. SYMPTOMS OF CHOLELITHIASIS
The most common sym!tom of cholelithiasis is a severe, steady !ain felt in the
riht u!-!er abdomen. )t is usually Gust below the riht rib cae. The !ain may also be
felt in the riht shoulder and between the shoulder blades. There may also be nausea
*feelin sick to the stomach- and vomitin *throwin u!-. These sym!toms may o
away on their own, only to come back later, es!ecially after a fatty meal. Other sins
and sym!toms may include
enetic factors
some ethnic rou!s are more susce!tible, such as &ative .mericans and
'is!anics
obesity
excess alcohol consum!tion
oral contrace!tives
hih-fat, low-fiber diet
ra!id weiht loss
women who have had many children *multi!arity-
hemolytic disorders such as sickle cell anemia and hereditary s!herocytosis
liver cirrhosis
diabetes
female ender
inflammatory bowel diseases such as crohns
Fi"#r( O.> Left+ Biochemical basis of cholelithiasis and bile metabolism in he!atobiliary
systems showin beneficial effects of low-fat, hih-fiber-bran diets containin !lant sterols
and stenols. (iht+ Three maGor !hysioloical !athways of !lasma li!o!rotein metabolism are
shown+ *2- trans!ort of dietary *exoenous- fat *left-, *1- trans!ort of he!atic *endoenous- fat
*center-, and *9- reverse cholesterol trans!ort *bottom-. Sites of action of the six maGor li!id-
alterin drus on exoenous and endoenous !athways of li!o!rotein metabolism are *2-
inhibition of '$H-6o. reductase by statins and de!endence on '$H-6o.(+ 6holesterol
hydroxylaseE *1- bindin of bile acids by se"uestrants, interferin with their reabsor!tion by
the ileal bile acid trans!orter *)B.T-E *9- bindin of a cholesterol absor!tion inhibitor to the
&iemann #ick 62L2, decreasin the absor!tion of dietary and biliary cholesterolE *7-
decreased mobili/ation of free fatty acids *??.- by niacin, leadin to decreased u!take of
??. by liver and reduced IL5L, )5L, and L5L !roductionE *0- inhibition of TH synthesis by
dM9 fatty acidsE *D- u!-reulation of li!o!rotein li!ase *L#L- and decreased !roduction of
a!o6-))), an inhibitor of L#L, by a fibric acid derivative, leadin to decreased IL5L-TH.
The he!atic cholesterol !ool is decreased by the aents at ste!s 2, 1, and 9, each leadin to an
u!-reulation of the L5L(. )n tissues, the li!id synthesis and oxidation at different cellular
oranelle !lay role shown with dots. L6.T, Lecithin cholesterol acyl transferaseE .B6.-),
.T#-bindin cassette !rotein .-)E .B6H, .T#-bindin cassette !rotein HE B., bile acidsE
6E, cholesteryl estersE 6$, chylomicronsE 6$(, chylomicron remnantsE S(-., class .
scavener rece!torE S(B2, class B scavener rece!tor.
Gaundice *yellowin of the skin or whites of the eyes-
feelin bloated or havin too much as in the stomach
!ale colored stools *bowel movements-
dark colored urine.
D. DIAGNOSIS OF CHOLELITHIASIS
)nitially bile analysis and all bladder scans are indicative of any chane in
cholesterol con-tents in bile or alteration in all bladder sha!e. Some basic !rocedures
are described in the followin sections.
ile and blood tests+ Bile acid content analysis for cholesterol, !hos!holi!ids, and
bile salts ive the first indication of all bladder disease or cholesterol bile saturation.
)n ad-vanced stae of bile saturation, dianostic scannin is done by endosco!ic
retrorade cho-lanio !ancreatora!hy *E(6#- to find stones, tumors, or other
!roblems. 5ye is !ut into the endosco!y tube to show u! better on O-rays and stones
may be removed durin E(6#. $oreover, other advanced scan tests are liver and
gallbladder "I(, scan and oral cholecystography usin radioactive dye in veins
*Haby, 1443-. #ictures are then taken by a s!ecial O-ray scanner that can Ssee% the dye
in the body. Other !romisin techni"ue is abdominal ultrasound to see inside the
abdomen. Sound waves are used to show !ictures of the abdomen on a TI-like
screen. Our rou! re!orted ultrasound mor!holoy of cholesterol calculi in allstones
!revalent in &orth )ndia usin ultrasonora!hy *Nhurikhin et al., 1424-. Hallstones
showed a cholesterol content of more than <4 m R labeled as cholesterol calculi.
?ive sonora!hic !atterns were documented *full moon 10.3R, half moon 10.3R,
crescent 11.1R, comet tail 22.2R, no distinct !attern 27.CR-, may have a bearin on
manaement as shown in ?iure 3.1.
PATHOPHYSIOLOGY
Hallstone disease may be thouht of as havin the followin four staes+ *2- the
lithoenic state, in which conditions favor allstone formationE *1- asym!tomatic
allstonesE *9- sym!tomatic allstones, characteri/ed by e!isodes of biliary colicE and
*7- com!licated cholelithiasis.
Hallstone formation occurs because certain substances in bile are !resent in
concen-trations that a!!roach the limits of their solubility. ;hen bile is concentrated
in the all-bladder, it can become su!ersaturated with these substances, which then
!reci!itate from the solution as microsco!ic crystals. These crystals are tra!!ed in
allbladder mucus, !roducin allbladder slude. Over time, the crystals row,
areate, and fuse to form macrosco!ic stones. Occlusion of the ducts by slude and
stones !roduces the com!li-cations of allstone disease. The two main substances
involved in allstone formation are cholesterol and calcium bilirubinate.
Fi"#r( O.? Hallstone sha!es identified by ultrasonora!hy in allstone !atients.
.eproduced *ith permission from Paul$ !##$ erry$ M#$ Tandon$ .#D#$ 9<<<# !onographic
sub-types of cholesterol gallstones among Indian patients@ ,n initial experience# Indian
7ournal of .adiology and Imaging Eserial onlineF < (9)$ <;99#
E.>. Ch!(st(r! Gast!$(s
$ore than C4R of allstones in the 8nited States contain cholesterol as their
maGor com-!onent. Liver cells secrete cholesterol into bile alon with !hos!holi!ids
*lecithin- in the form of small s!herical membranous bubbles, termed unilamellar
vesicles. Liver cells also secrete bile salts, which are !owerful deterents re"uired for
diestion and absor!tion of dietary fats. Bile salts in bile dissolve the unilamellar
vesicles to form soluble areates called mixed micelles. This ha!!ens mainly in the
allbladder, where bile is concentrated by reabsor!tion of electrolytes and water.
6om!ared with vesicles *which can hold u! to one molecule of cholesterol for every
molecule of lecithin-, mixed micelles have a lower carryin ca!acity for cholesterol
*about one molecule of cholesterol for every three mol-ecules of lecithin-. )f bile
contains a relatively hih !ro!ortion of cholesterol to bein with, then as bile is
concentrated, !roressive dissolution of vesicles may lead to a state in which the
cholesterol carryin ca!acity of the micelles and residual vesicles is exceeded. .t this
!oint, bile is su!ersaturated with cholesterol, and cholesterol monohydrate crystals
may form. Thus, the main factors that determine whether cholesterol allstones will
form or not are *2- the amount of cholesterol secreted by liver cells, relative to lecithin
and bile salts, and *1- the deree of concentration and extent of stasis of bile in the
allbladder.
E.?. Ca&i#.I Biir#bi$I a$/ Pi".($t Gast!$(s
Bilirubin, a yellow !iment derived from the breakdown of heme, is actively
secreted into bile by liver cells. $ost of the bilirubin in bile is in the form of
lucuronide con-Guates, which are "uite water soluble and stable, but a small
!ro!ortion consists of unconGuated bilirubin. 8nconGuated bilirubin, such as fatty
acids, !hos!hate, carbon-ate, and other anions, tends to form insoluble !reci!itates
with calcium. 6alcium enters bile !assively alon with other electrolytes.
)n situations of hih heme turnover, such as chronic hemolysis or cirrhosis, uncon-
Guated bilirubin may !resent in bile at hiher concentrations. 6alcium bilirubinate
may then crystalli/e from the solution and eventually form stones. Over time, various
oxida-tions cause the bilirubin !reci!itates to take on a Get black color, and stones
formed in this manner are termed blac& pigment stones. Black !iment stones
re!resent 24M14R of allstones in the 8nited States.
Bile is normally sterile, but, in some unusual circumstances *e.., above a biliary
stric-ture-, it may become coloni/ed with bacteria. The bacteria hydroly/e conGuated
biliru-bin, and the resultin increase in unconGuated bilirubin may lead to
!reci!itation of calcium bilirubinate crystals. Bacterial hydrolysis of lecithin leads to
the release of fatty acids, which com!lex with calcium and !reci!itate from the
solution. The resultin concretions have a clay-like consistency and are termed bro*n
pigment stones. 8nlike cholesterol or black !iment stones, which are formed almost
exclusively in the allblad-der, brown !iment stones often form de novo in the bile
ducts. Brown !iment stones are unusual in the 8nited States but are fairly common
in some !arts of Southeast .sia, !ossibly related to liver flukes.
E.B. MiH(/ Gast!$(s
6holesterol allstones may become coloni/ed with bacteria and can elicit
allbladder mucosal inflammation. Lytic en/ymes from bacteria and leukocytes
hydroly/e bilirubin conGuates and fatty acids. .s a result, over time, cholesterol
stones may accumulate a substantial !ro!ortion of calcium bilirubinate and other
calcium salts, !roducin mixed allstones. Lare stones may develo! a surface rim of
calcium resemblin an eshell that may be visible on !lain O-ray films.
G. ROLE OF DIET THERAPY AND CHALLENGES IN
CHOLELITHIASIS TREATMENT
&aturally occurrin synthetic bile acids *ursodeoxycholic acid or
chenodeoxycholic acid *656.-- is a choice to reduce cholesterol and kee! a balance
of li!ids with bile salts in bile or to !romote radual dissolution of all stones. )n
advanced !ersistent allstones, cholecystectomy is the choice under su!ervision.
'owever, recurrences are seen in u! to 04R of !atients after any treatment is
discontinued. (ecent re!orts indicate the recurrence risks of #&, !ure bile salts, and
cholecystectomy *5ella 6orte et al., 144CE 5ray et al., 144<E 'artl et al., 1443E >elly,
1424E Stroohle et al., 144DE Teitelbaum et al., 1440E Nhurikhin et al., 1424-. )n fact,
#& is not associated with increased mortality, overall fre"uency of com!lications, or
loner lenth of hos!ital stay *LOS-. 'owever, risks of #& com!li-cations *e..,
refeedin-syndrome, hy!erlycemia, bone deminerali/ation, catheter infec-tions- can
be minimi/ed by carefully monitorin the !atients and use of nutrition su!!ort teams
!articularly durin lon-term #&. 6om!lications may be due to refeedin syn-drome
in !atients sufferin from severe malnutrition with the initiation of refeedin or
metabolic, hy!ertrilyceridemia, hy!erlycemia, osteomalacia and osteo!orosis, and
he!atic com!lications includin fatty liver, nonalcoholic fatty liver disease,
cholestasis, cholecystitis, and cholelithiasis. Efficient monitorin in all ty!es of #&
can result in reduced #&-associated com!lications at reduced costs. &ew findins
suest water and electrolyte balance, blood suar, and cardiovascular function
should reularly be moni-tored durin #&. (eular checks of serum electrolytes and
trilycerides as well as addi-tional monitorin measures are necessary in !atients with
altered renal function, electrolyte-free substrate intake, li!id infusions, and in
intensive care !atients in ho!e of better outcome. The metabolic monitorin of
!atients under lon-term #& should be carried out accordin to standardi/ed
!rocedures. $onitorin metabolic determinants of bone metabolism is !articularly
im!ortant in !atients receivin lon-term #&. $arkers of intermediary, electrolyte,
and trace element metabolism re"uire reular checks. These re!orts leave us in
dismay to search other ways of reducin cholelithiasis such as nutrition su!!lements
and dietary thera!y or other factors *'eshka et al., 233DE 'oy et al., 2337E :ayanthi et
al., 233CE >amrath et al., 2331E >lawansky and 6halmers, 2331E $Qnde/- SKnche/ et
al., 1442E $orKn et al., 233<E #ausawasdi et al., 233DE (oslyn et al., 23C9E Serrano
#a/ et al., 2330-. )n the next section, the focus is on different diet and nutri-tion
a!!roaches which are initially beneficial in asym!tomatic !atients to hihliht that
nutrition su!!lementation and diet thera!y is im!ortant and dru treatment is not
needed always.
G.>. Di(t A/+i&(
s 5urin an cholelithiasis attack, si! water occasionally, but don%t eat.
s .t other times, eat low-fat diet. ?atty meals may brin on mild attacks.
s )f !erson is overweiht, bein a weiht reduction !roram.
)n the next section, different nutrient su!!lements influencin cholelithiasis
are introduced.
G.?. N#triti!$ S#33(.($ts i$ Ch!(ithiasis
Henerally, it is considered that cholelithiasis !atients in advanced staes of
allstone sur-ical removal undero !ostcholecystectomy-related nutritional
deficiencies includin severe !rotein-calorie malnutrition, fat malabsor!tion,
micronutrient deficiencies of vitamin B21, thiamine, folate, and the fat-soluble
vitamin deficiency of vitamins 5, >, E, 6, and microelements iron, calcium,
aluminum.
. brief descri!tion of different su!!lements and usae in cholelithiasis or their
deficiency as risk after cholecystectomy is iven below.
Iitamin . is considered he!atotoxic and !oor in reducin cholesterol, bile salts,
and !hos!holi!ids in bile *6ardenas et al., 233DE Hranados and 6anrdenas, 2337-. Still
the role of retinol remains controversial in cholelithiasis.
Iitamin 5 malabsor!tion is considered sinificant in bone mineral density
disorders es!ecially in cholelithiasis !atients with !ostcholecystectomic syndrome
who show biliary insufficiency *?isher et al., 1443E >oricheva et al., 1424E
$alinowski, 144D-.
$icronutrient antioxidant vitamin E without sulfur-containin amino acid is
associ-ated with disturbed lutathione homeostasis. Theoretical considerations and
ex!erimen-tal studies suest a causal connection between micronutrient antioxidant
insufficiency and the develo!ment of human allstones *$arcinowska-Suchowierska
et al., 2330E ;orthinton et al., 233<, 1447-.
Little is known about the role of fat-soluble vitamins > and 5 in liver function
and bone metabolism in biliary and !ancreatic diseases associated with cholestasis
and,or fat malabsor!tion *Leblanc et al., 23C3E Lin et al., 233DE Saito, 23C<E Nhou et
al., 1444-.
-.2.1. 1itamin C
Iitamin 6 miht hel! in !revention of allstones. Huinea !is develo!ed
allstones when fed with a diet hih in cholesterol and low in vitamin 6, but not when
fed the same diet with an ade"uate amount of vitamin 6 *'olloway and (ivers, 23C2E
:enkins 23<C-. Iitamin 6 is a cofactor for the en/yme < c-hydroxylase, which is the
rate-limitin en/yme in the conversion of cholesterol to bile acids *?iure 3.2-.
Iitamin 6 !revents allstone formation by !romotin the conversion of
cholesterol to bile salts, thereby decreasin the lithoenicity of bile.*Hinter et al.,
23<3E 'orni and ;eiser, 23<D-. Iitamin 6 su!!lementation also inhibited
cholelithiasis and accelerated the conversion of cholesterol to bile salts in hamsters
*Hinter and $ikus, 23<<-.
)n a cross-sectional study of <471 women !artici!atin in the Third &ational
'ealth and &utrition Examination Survey, 23CCM2337, a sinificant inverse
association was observed between serum vitamin 6 levels and !revalence of
allbladder disease *Simon and 'udes, 1444-. 'owever, no such association was
found in men !artici!atin in the same survey. )n a study of !atients with allstones,
daily su!!lementation with 1 vitamin 6 for 1 weeks decreased the lithoenicity of
bile. Sixteen !atients with allstones scheduled for cholecystectomy received 044 m
vitamin 6 four times daily for 1 weeks before sureryE another 2D !atients scheduled
for cholecystectomy did not receive vitamin 6 *control rou!-. 5urin surery, bile
was taken from the allbladder of each !atient. 6om!ared with the control !atients,
vitamin 6-treated !atients had sinificantly hiher concentrations of !hos!holi!ids in
bile. The mean nucleation time of bile *the time re"uired for the formation of
cholesterol crystals, the first ste! in stone formation- was < days in the vitamin 6
rou! and 1 days in the control rou! *!V4.42- *Hustafsson et al., 233<-. These
findins suest increasin vitamin 6 intake decreases the risk of de-velo!in
allstones. 'owever, additional research is needed to confirm the !ossibility and
determine the o!timal dosae.
Essential fatty acids, es!ecially 6-CM6-21 chain fatty acids, !rovide enery
balance in !hos!holi!id, cholesterol, and bile salts. .rachidonic acid and linoleic acid
showed !roven effect in allstone !revention *$iyake et al., 23D7-. Later
!olyunsaturated li!ids were re!orted to !lay a mechanistic role in
hy!ocholesterolemic effect *#aul et al., 23C4-. 5iets containin casein, rice flour,
lucose, and fiber with *minimal essential fatty acids without cholesterol- !romoted
!iment stones showed normal li!o!rotein !rofile, whereas low-fat diet limited in
essential fatty acids containin cholesterol and lactose !romoted cholesterol
allstones associated with an ex!anded IL5L !ool *'ayes et al., 2331-.
-.2.2. &ecithin
#hos!holi!ids, mainly lecithin, increase the solubility of biliary cholesterol and
lower the !hos!holi!id concentrations in !atients with allstones than in those
without allstones. 'owever, status of !hos!holi!id content of lithoenic and normal
bile is not established yet *.nderson and Bouchier, 23D3-. Lecithin contains hih
concentrations of !hos!ho-li!ids but decreases the lithoenicity of bile by increasin
biliary !hos!holi!id concen-trations. )n an uncontrolled trial, su!!lementation of
eiht allstone !atients with a relatively low dose of lecithin *244 m three times
daily- for 2CM17 months showed sinificant increase in biliary !hos!holi!id content
and sinificant decrease in biliary cholesterol levels *Tu/hilin et al., 23<D-. )n another
study, daily administration of 7.0 soybean lecithin for 9 weeks resulted in a
nonsinificant CR im!rovement in the cholesterol saturation index of bile *'olan et
al., 23<3-. Still the role of lecithin is not well defined and confirmed.
G.B. Physi!&h(.i&a Basis !' Ch!(ithiasis
Hallstones, mainly cholesterol allstone formation, result from a number of
!hysioloical events such as he!atic secretion of bile saturated with cholesterol,
nucleation of choles-terol monohydrate crystals in all bladder, and im!aired
em!tyin of all bladder contents. Bile saturation and nucleation show conse"uences
of cholesterol all stones.
Bile is an a"ueous solution, enriched with water-insoluble hydro!hobic li!ids such
as cholesterol and !hos!holi!ids, otherwise sus!ended in deterent bile acids. Such
dissolved solids make u! about 9R by weiht he!atic bile. Bile salts are the
!rominent solute, averain 14M94 mmol l
-2
in he!atic bile. #hos!holi!id
concentrations averae < mmol l
-2
and cholesterol averaes 1M9 mmol l
-2
in soluble
form. Bilirubin is !resent in concentrations of about 4.1R by weiht. . fraction of
these !roteins !lay an im!ortant role in cholesterol crystal nucleation and allstone
formation.
The !rinci!al li!id com!onents *!hos!holi!id and cholesterol- are solubili/ed by
bile salt micelles. Bile salts are extremely effective solubili/ers of !hos!holi!ids. )n
addition, !resence of !hos!holi!id markedly increases the extent to which bile salts
can incor!o-rate cholesterol into micelles. The relative amounts of cholesterol,
!hos!holi!id, and bile salt, which may coexist in micellar solution, have been
determined em!irically by 6arey and Small *6arey and Small, 23<C- as shown in
?iure 3.9. )n the followin descri!tion, the bile homeostasis at ene level is
described for interested readers.
.t enetic level, both bile acids 656. and cholic acid *6.- can reulate the
ex!res-sion of enes involved in their synthesis creatin a feedback loo!. The
elucidation of this reulatory !athway came about as a conse"uence of the isolation of
a class of rece!tors called the farnesoid O rece!tors *?O(s-. The ?O(s belon to the
su!erfamily of nuclear rece!tors that includes the steroid,thyroid hormone rece!tor
family as well as the liver O rece!tors *LO(s-, retinoid O rece!tors *(O(s-, and the
!eroxisome !roliferator-activated rece!tors *##.(s-.
There are two enes encodin ?O(s identified as ?O(c and ?O(a. )n humans, at
least four ?O( isoforms have been identified as bein derived from the ?O(c ene as
a result of activation from different !romoters and the use of alternative s!licin+
?O(c2, ?O(c1, ?O(c9, and ?O(c7. The ?O( ene is also known as the &(2'7
ene *for nuclear rece!tor subfamily 2, rou! ', member 7-. The ?O( enes are
ex!ressed at hihest levels in the intestine and liver.
Similar to all rece!tors of this su!erfamily, liand binds the rece!tor in the
cyto!lasm and then the com!lex mirates to the nucleus and forms a heterodimer with
other
Fi"#r( O.B Left *a- Bile li!id analysis to evaluate the bile com!ositionE (iht+ *b-
Tricordinate !hase diaram showin intersectin !oint for the relative concentrations of
cholesterol, !hos!holi!ids, and bile salts in bile accordin to 6arey and Small *23<C-. &otice
left lower end !resents micelle. Bile su!ersaturated with cholesterol !reci!itates out of
solution.
members of the family. ?O( forms a heterodimer with members of the (O( family.
?ollowin heterodimer formation, the com!lex binds to s!ecific se"uences in taret
enes called ?O( res!onse elements *?O(Es- resultin in reulated ex!ression. One
maGor taret of ?O( is the small heterodimer !artner *S'#- ene. .ctivation of S'#
ex!ression by ?O( results in inhibition of transcri!tion of S'# taret enes. Of
sinificance to bile acid synthesis, S'# re!resses the ex!ression of the cholesterol-
<c-hydroxylase ene *6=#<.2-. 6=#<.2 is the rate-limitin en/yme in the synthesis
of bile acids from cholesterol throuh the classic !athway.
)n the .yurvedic tradition of medicine, any resin collected by ta!!in the trunk of
a tree is called guggul. The cholesterol-lowerin action of the uul from the $ukul
myrrh tree *Commiphora mu&ul- of )ndia is that a li!id com!onent of this extract
called guggulsterone *also called uul li!id- is an antaonist of ?O(. 'owever, in
addition to its effects on ?O( function, uulsterone has been shown to activate the
!renane Orece!tor *#O(-, which is another member of the nuclear rece!tor
su!erfamily. #O( is a reconi/ed rece!tor for lithocholic acid and other bile acid
!recursors. #O( activation leads to re!ression of bile acid synthesis because of its
!hysical association with he!atocyte nuclear factor 7c *'&?-7c- causin this
transcri!tion factor to no loner be able to associate with the transcri!tional
coactivator #H6-2c *##.(b coactivator 2c- which ultimately leads to loss of
transcri!tion factor activation of 6=#<.2.
The ex!ression of other enes involved in bile acid synthesis is also reulated by
?O( action. The action of ?O( can be to either induce or re!ress the ex!ression of
these enes. Henes that are re!ressed in addition to 6=#<.2 include S(EB#-2c,
sterol 21c-hydroxylase *ene symbole6=#CB2-, and solute carrier family 24
*sodium,bile acid cotrans!orter family-, member 2 *ene symboleSL624.2-. This
latter ene is iden-tified as the &a
B
-taurocholate cotrans!ortin !oly!e!tide *&T6#-.
&T6# is involved in he!atic u!take of bile acids throuh the sinusoidal,basolateral
membrane. Thus, bile acid-mediated re!ression of &T6# ene ex!ression would
reduce u!take of bile acids and !rotect the liver from the toxic effects of excess bile
acid accumulation. Bile acids re!ress the transcri!tion of another bile acid trans!orter
that is ex!ressed in the sinusoid-dal, basolateral membrane. This trans!orter is &a
B
-inde!endent and is called the oranic anion trans!ortin !oly!e!tide 2B2
*O.T#2B2, ene symboleSL6O2B2-. O.T#2B2 was formerly identified as O.T#-
6. The effect of bile acids on O.T#2B2 ex!ression is indirect and involves S'#-
mediated reduction in '&?-7c activity, which in turn reduces the ex!ression of
another liver-enriched transcri!tion factor '&?-2c which is the maGor activator of the
O.T#2B2 ene.
Henes induced by ?O( include liver bile salt ex!ort !um! *BSE#-, multidru
resis-tance !rotein 9 *$5(9-, and multidru resistance associated !rotein 1 *$(#1-.
The latter two enes are involved in ex!ort of oranic com!ounds and were identified
based on their ability to trans!ort drus out of cells thereby, allowin the cells to resist
the intended effects of the administered dru. The normal function of $5(9, which is
a member of the .T#-bindin cassette *.B6- family of trans!orters *$5(9 is also
iden-tified as .B6B7-, is the translocation of !hos!holi!ids throuh the canalicular
membrane of he!atocytes. Thus, it is inferred that the bile acid-mediated increase in
$5(9 ex!res-sion is necessary to allow he!atocytes to res!ond to bile acid toxicity
throuh the formation of cholesterol, !hos!holi!id, and bile acid containin micelles.
BSE# is also a member of the .B6 family of trans!orters *BSE# is also identified as
.B6B22- and it is involved in the !rocess of ex!ortin bile acids out of he!atocytes
thus reducin their toxicity to these cells. .lthouh uulsterones antaoni/e the
actions of ?O(, which would lead to a reduction in bile acid ex!ort, these li!ids have
been shown to activate the ex!ression of BSE# throuh an ?O(-inde!endent
mechanism. This latter effect !robably ex!lains the cholesterol-lowerin benefits
attributed to these com!ounds.
Similar with cholelithiasis, once cholesterol concentrations exceed their maximum
e"uilibrium solubility, multilamellar cholesterol vesicles fuse and areate into a
cluster that serves as a nidus for crystal formation *nucleation-. &ucleation may be
either homo-eneous or heteroeneous. 'omoeneous nucleation occurs if
crystalli/ation occurs without forein material. 'eteroeneous nucleation occurs
without forein material. 'eteroeneous nucleation occurs if crystalli/ation takes
!lace on a forein surface such as e!ithelial cells, !rotein, calcium salts, or a forein
body. Because nucleation occurs ra!idly at low levels of cholesterol su!ersaturation,
it occurs throuh heteroeneous !athway.
(ecently, !romoters and inhibitors of cholesterol crystal formation and rowth in-
fluence the viscosity of human bile. These !romotin and inhibitin factors may
directly influence the Snucleatin time% of bile. Biliary !roteins of molecular weiht
294 k5a have been !ro!osed as !otential !ronucleators. )n contrast, there are !roteins
in normal bile that inhibit nucleation. These antinucleatin factors may stabili/e
cholesterolM !hos!holi!id vesicles in Snormal% bile and retard crystalli/ation. #otential
candidate anti-nucleatin !roteins include a!oli!o!rotein .-) and .-)) *Hudheti et al.,
1440-.
6urrent conce!ts on allstone formation invoke the notion that nucleation of cho-
lesterol crystals occurs in a mucous el throuh !roteinMli!id interactions. The rate of
cholesterol crystal nucleation may be influenced by a balance between !ro- and
antinu-cleatin factors. )n allbladder, mucin also may !romote the stone formation.
$ucin causes a time- and concentration-de!endent acceleration of cholesterol
crystalli/ation. >uver et al. *1447- demonstrated that mucus hy!ersecretion occurs
before allstone formation in animals fed on allstone !romotin diet. 'owever,
inhibition of mucus secretion by usin hy!ocholestemic oriental !rinci!les may
!revent allstone formation but does not alter the develo!ment of diet-induced bile
cholesterol su!ersaturation.
Hallbladder stasis facilitates the rowth of microsco!ic crystals into macrosco!ic
stones. .nimal studies suest that a allbladder motility defect may antedate
allstone formation *;an et al., 1443-. )n addition, allbladder motility worsens as
stones develo!. Biliary stasis com!licates the cholelithiasis treatment with total
!arenteral nutri-tion and oral contrace!tives and !renancy *'onoren, 23C4-.
Bile saturation with cholesterol may result from the deficiency of secreted bile
salts or !hos!holi!ids or a dis!ro!ortionately increased secretion of cholesterol.
6holesterol stones are characteristically more common in women beyond !uberty.
'owever, differ-ences in !revalence of cholelithiasis between sexes decline after
meno!ause. #renancy associated with biliary cholesterol saturation, estroens,
contrace!tives, and allbladder em!tyin both factors contribute to increased
fre"uency of stones in women of child-bearin ae *>arayalctin et al., 1424-.
Limited information is available reardin biliary li!id com!osition and
cholesterol saturation in normal infants and children *5avit-S!raul et al., 1424E
>oivusalo et al., 1424-. Bile is relatively undersaturated with cholesterol in infants
and children com!ared with adults. This fact may be ex!lained by the observation that
bile salt !ools ex!and ra!idly after birth, and body si/e matched !ools in infancy and
childhood exceed those observed in youn adults with result of hih biliary bile saltM
cholesterol secretion ratio, with less saturated bile *Sichieri et al., 2332-.
'owever, !o!ulations at low risk of cholesterol cholelithiasis and obesity show no
biliary cholesterol saturation amon males in !uberty, whereas females before !uberty
show undersaturated bile and after !uberty, the same females show su!ersaturated bile
*Sichieri et al., 2332-. Enhanced cholesterol secretion and saturation may be observed
with several other related conditions of obesity, ileal resection, GeGunoileal by!ass,
6rohn%s disease, cystic fibrosis, and !renancy.
G.D. N#triti!$a Ma$a"(.($t !' Ch!(ithiasis
6holelithiasis+ 'y!erli!idemia is a conse"uence of im!aired bile flow, im!aired
li!o!ro-teins and li!ids. )ncreased he!atic cholesterol synthesis, li!id trans!ort, low
cholesterol-lecithin acyltransferase activity causes hy!ercholesterolemia. To treat it,
increased conversion of cholesterol to bile acids and enhanced elimination of bile
acids and cho-lesterol is a choice. Low cholesterol and low saturated fat diets, fat
restriction, low diet calorie content are some re!orted means to combat cholelithiasis
*6han et al., 1443E >atsinelos et al., 144D-.
&utrition assessment is used to monitor effects of nutritional rehabilitation.
;eiht for ae, heiht for ae, and weiht for heiht, skin fold thickness, mid arm
circumference, and !lasma !roteins are some tests to assess cholelithiasis.
&utritional Thera!y+ .fter nutrition assessment, !atients are advised for
customi/ed diet intake to receive calculated calories. To accom!lish the
recommended enery intake, artificial diets containin !olycose, olio!e!tides,
manesium, and mananese are su!!lemented in diet.
Enery+ 6alorie intake should be 210R of recommended diet allowance *(5.-
with ood !ractice lucose !olymer *!olycose !owder or medium chain trilyceride-
oil can be su!!lemented with diet *>amrath et al., 2331-.
G.D.>. Iron @FONT SICEA
5os fed with an iron-deficient diet had a hiher incidence of cholesterol crystals
in their bile than animals fed with a control diet *C4R vs. 14RE !V4.40-. The activity
of he!atic <c-hydroxylase *?iure 3.2-, was insinificantly less by D7R in iron-
deficient dos than in controls *!e4.4<-. These findins raise the !ossibility that iron
deficiency !lays a role in the !athoenesis of allstone formation in humans *:ohnston
et al., 233<-.
$ananese is a micronutrient and it is needed in micro"uantities as recommended
as a cofactor in the body. #revious studies suested the role of $n in allstones in
&ier !o!ulation with no clue of reducin cholesterol and incidence of colelithiasis
*.latise et al., 1424-. )n other study, #& was associated with $n in allstone bearin
children *>elly, 233C-. $anesium is a hydro!hilic element and is re!orted as
nutrient in !reven-tion of allstone formation *>o 144C-.
K. CHOLELITHIASIS ENCYME ASSAY DEVELOPMENT TO
TEST DIETS
)n our lab, cholesterol lowerin lithoenic diets includin a!!le and casein were
com-!ared by measurin microsomal 6ytochrome #704 !rotein and cholesterol <c-
hydroxylase *6'- activities in lithoenic diet fed hamsters. Lithoenic diet, casein,
and a!!le fiber diets were fed to hamsters for 9M0 weeks. ?or control rou!, animals
were fed on normal #urina chow without any su!!lement. The cholesterol lowerin
effect of lithoenic diet, casein, and a!!le diets were com!ared. The isolated liver
microsomes were se!arated from animals and tested for the 6' en/yme activity
measurement in all three rou!s. The lithoenic diet showed sinificantly enhanced
6' en/yme activ-ities, whereas animals fed on casein and a!!le diet reimen showed
moderate increase in microsomal 6' en/yme activity indicated cholesterol lowerin
in liver. 6' may be a biomarker of cholesterol status in the body and microsomal 6'
en/yme may be lowered down after treatment of casein and a!!le diets. )n !revious
ex!eriments, diet su!!lemen-ted bile acids and salts in combination showed
cholesterol desaturation and reduced all-stone formation in both animals and human
*.!rikian et al., 1441E 5onowski and Ehwald, 2333E )keami et al., 2334E Oakenfull
and ?enwick, 23<CE Sembries et al., 1447, 144DE Shimi/u et al., 233D-. The maGor
factor of cholesterol desaturation was the !hysical !hase transformation of cholesterol
into bile cholesterol esters and metabolic desaturation of cholesterol because of
cholesterol conversion into different li!o!roteins *Boll et al., 2333E >inowaki et al.,
1441E $aeda et al., 2330E &orlin et al., 1444E Souidi et al., 233CE Souidi et al., 1444E
Ialhouny et al., 23C0E Iidyashankar et al., 1424-. )n the followin section, the
methods that are develo!ed at our lab for interested readers to mea-sure he!atic 6' in
cholesterol lowerin and its reulation are described. (educed '$H-6o.+ 6' ratio
was ex!lored by a!!le diet su!!lementation to hamsters.
)n brief, hamsters weihin 204M110 were locally raised at animal laboratory at
.ll )ndia )nstitute of $edical Sciences, and !laced in !lastic caes fed on cholesterol-
rich diet for the !eriod of 2M7 weeks under constant su!ervision for any chane in
weiht as !er )nstitutional .nimal 6are and 8se 6ommittee. .nimals were rou!ed
in four rou!s+ control rou!, lithoenic or hih-cholesterol rou!, a!!le
su!!lemented rou!, and casein su!!lemented rou! fed on normal #urina *(odent
(alston #urina-, hih cho-lesterol diet, a!!le su!!lemented diet, and casein
su!!lemented diet, res!ectively, with ad libitum water $atheson et al. *2330-. The
lithoenic hih-cholesterol dietary additive su!!lements
2
were used to fed animals
and to develo! cholesterol all stones in animals as !ositive control animal rou!.
Other two ex!erimental animal rou!s included a!!le diet
1
and casein diet
9
rou!s.
The #urina diet contained sucrose *004 k
-2
wt- and vitamin mix *74 k
-2
wt-.
.fter different intervals, cytochrome #704 !rotein and 6' en/yme were estimated as
biomarkers of time-de!endent cholesterol chanes and cholesterol reulatin 6'
en/yme in liver.
K.>. Is!ati!$ !' H(3ati& Mi&r!s!.(s
The he!atic microsomal !re!arations from hamster livers were done by liver cell
isola-tion, density radient ultrafast centrifue at u24
0
centrifual force to isolate the
micro-somal fraction as source of cytochrome #704 and 6' en/yme activity. The
microsomal isolation was done by the authors% modified method as described
elsewhere *Einarsson et al., 23C3-.
K.?. Ch!(st(r! KV Hy/r!Hyas( E$-y.ati& Esti.ati!$ a$/
E$-y.( A&ti+ity
6holesterol was delivered in Tween C4 as !reviously described and modified by
our rou! *'onda et al., 2330-. Briefly, in a total volume of 044 Xl, each assay tube
contained <0 mmol l
-2
!hos!hate buffer, !' <.7, 2 mmol l
-2
E5T., 1.0 mmol l
-2
5TT,
0 mmol l
-2
$6l
1
, 9 mmol l
-2
&.5#', v7-
27
6wcholesterol *2.2u24
D
d!m, 244444
!mole4.1 mmol l-, 4.<C m Tween C4, and 044 X of liver microsomal !ro-tein. The
!reincubation time durin which &.5#' was omitted was 1 min at 9<
o
6. The
incubation time was 14 min.
.n im!roved assay for 6' activity in hamster liver microsomes was !erformed as
followin *Boll et al., 2333-. )n a total volume of 044 Xl, each assay tube contained <0
mmol l
-2
!hos!hate buffer, !' <.7, 2 mmol l
-2
E5T., 4.0 mmol l
-2
5TT, 0 mmol l
-2
$6l
1
, 2 mmol l
-2
&.5#', v
27
6wcholesterol *2.2u24
D
, 1D444 !mol, 01 Xmol l
-2
-, 7.0
m '#B65, 24 mmol l
-2
lucose-D-!hos!hate, 1 8 lucose-D-!hos!hate
dehydroenase, and 104 X of liver microsomal !rotein. .ssay tubes were incubated
at 9<
o
6 in a shakin water bath *$axi-shake, 'eto, OS), ?rance-. The !re-incubation
time durin which only &.5#' was omitted was 0 min and the incubation time
followin initiation of the assay with &.5#' was D min. The reaction was termi-
nated by addin 74 Xl of 0 mol,l &aO'. Nero-controls were always run in !arallel by
the addition of 0 mol,l &aO' at the beinnin of the !reincubation. The sterols were
extracted *after neutrali/ation- with 7.9 ml of dichloromethaneMethanol *0+2, v,v, !lus
2.1 ml of '
1
O-. . mixture of unlabeled <c- and <a-hydroxycholesterol was added and
v
27
6w<c-hydroxycholesterol was se!arated from <a-hydroxycholesterol by thin-layer
chromatora!hy *TL6- on silica el H usin a double miration techni"ue with ethyl
acetateMhexane *2+2, v,v-. )n all cases, activity was linear with res!ect to the amount
of microsomal !rotein added and the incubation time. The amount of endoenous
cholesterol !resent in assays raned from 7.<0 to 0.0 X for 104 X of microsomal
!rotein.
K.B. E$-y.( Ch!(st(r! KV Hy/r!Hyas( R(a&ti!$ a$/ E''(&t
!' Cyt!&hr!.( PDER a$/ Ch!(st(r! A//iti+(s
)n control and a!!le diet su!!lemented animal liver microsomal !re!arations, 6'
en-/yme activity was measured at different concentrations of cytochrome #704
!rotein. )n another set of ex!eriment, 6' activity was measured at different
cholesterol concen-trations added in the reaction mixture.
)n the followin section, the outcome of a!!le and casein in fiber diet fed
hamsters and the 6' en/yme activity chanes as indicator of cholelithiasis from
technical stand-!oint are described.
The weiht of animals varied in the rane of 14M74R after lithoenic diet. The
animals did not show any sym!toms of indiestion after dietary treatment of
casein and a!!le fiber.
The treatment of casein and a!!le fiber showed measurable difference in the
6' en/yme activity in microsomes. 'owever, control animal 6' activity
remained constant throuhout the study.
The 6' en/yme activity was linearly chaned with the hydroxylation of
cytochrome #704 !rotein. The cytochrome #704 hydroxylation was
!ro!ortional to the choles-terol added in the en/yme reaction medium *see
Table 3.2-.
The treatment of hih casein and a!!le fiber contents in hamsters did not
affect the en/yme activity in reaction medium and medium conditions.
K.D. Th( Ch!(st(r! KV Hy/r!Hyas( E$-y.( A&ti+ity a$/
Cyt!&hr!.( PDER C!$&($trati!$ i$ Li+(r C( Mi&r!s!.(s
The !urity of microsomes was 3DR based on microsco!y. )n isolated microsomes,
the indicator en/ymes showed the active state of microsomes without en/yme
deactivation throuhout the ex!eriments. The en/yme activity was substrate
Tab( O.> 6holesterol <c 'ydroxylase S!ecific .ctivities in .nimals ?ed
on 5ifferent 5iets
Mi&r!s!s!.a
Cyt!&hr!.( Cyt!&hr!.( PDERF."
3r!t(i$ @t!taA
Cyt!&hr!.( PDERF."
3r!t(i$ @'ra&ti!$ AA PDERF."
Di(t Hy/r!Hyas(
a
C!$t($t Ch!(st(r!
KV
C!$t($t KV
hy/r!Hyas(
6ontrol 1.14^4.10 2C.7^4.1 4.<4^4.2 2C.0^1.1 4.C0^4.20
Lithoenic 2.<3^4.14 24.1^4.3 4.D0^4.2 2D.0^1.4 4.<0^4.14
.!!le diet 9.70^4.04 17.D^1.4 2.1^4.20 14.0^1.0 2.C^4.20
6asein diet 1.C2^4.94 21.1^4.1 4.7C^4.0 - -
a
S!ecific activity of 6' en/yme
l
is ex!ressed as !moles cytochrome #704
cataly/ed !er minute.
concentration-de!endent and substrate-s!ecific. The end !roduct of cholesterol
conver-sion to hydroxycholesterol by 6' en/yme indicated the nature of en/yme
reaction as de!endent on cytochrome #704 and &.5# reduction at o!timum !' <.7
in Tris buffer me-dium. The addition of different cholesterol concentrations in
en/yme reaction medium containin control microsome showed the linear chane in
6' en/yme s!ecific activity. The cholesterol lowerin effect of diets in different
animal rou!s was in the increasin order of control rou! )`lithoenic diet ))`casein
rou! )))`a!!le diet rou! )I as shown in Table 3.2 and ?iure 3.2. The effect of
each individual diet was distinct and 6' activity was decreased in animals after diet
treatment while en/yme reaction medium did not show any chane in cholesterol and
cytochrome #704 contents. )t indicated further that en/yme activity chanes were due
to diet treatment.
.nimals on a!!le su!!lemented diets showed de!endence of 6' on cytochrome
#704 !rotein content and cholesterol in the reaction mixture. ;ith increasin cyto-
chrome #704 !rotein in microsomes, en/yme elevated and a!!le diet enhanced further
the en/yme activity. )t indicated the a!!le diet effect inde!endent from cytochrome
#704 !rotein. The cytochrome #704 !rotein stimulated en/yme activity behavior was
close to simoid !attern at constant cholesterol concentrations. En/yme activity
enhancement showed de!endence with both cytochrome #704 !rotein concentrations
and cholesterol concentrations. The cholesterol concentrations in reaction mixture and
en/yme activities showed linear relationshi! as shown in Table 3.1.
L. F*T*RE PROSPECTIVES ON CHOLELITHIASIS AND
N*TRITION
6holelithiasis and its com!lications in allstone formation are seen as a maGor
challene. $ain bottlenecks are lack of data available on reconition of biliary li!id
cut-off values as risk levels in children, adolescents, !renant,lactatin women in
different social rou!s, elderly in different sexes, ae rou!s amon different
ethnicities or countriesE challenes of #& and com!lications after cholecystectomyE
less documented benefits of nutrition
Tab( O.? Effect of 6ytochrome #704 and 6holesterol 6oncentration on
#urified 6holesterol <c 'ydroxylase System *$icrosomal-
C!$&($trati!$ !'
&yt!&hr!.( PDER
@i$ $a$!.!(s )
a
Ch!(st(r!
&!$&($trati!$
a//(/ i$ assay
.(/i#.
Ch!.KV
hy/r!Hyas(
a&ti+ity
*!icomoles,min- Di(t @"r!#3A
6ontrol 4.104 10 nmol 9D.0
.!!le diet 4.104 10 nmol 90.4
6ontrol 4.04 10 nmol 00.4
.!!le diet 4.04 10 nmol D4.9
6ontrol 2.4 10 nmol 0<.4
.!!le diet 2.4 10 nmol D0.0
6ontrolBcholesterol 4.0 04 X$
a
24.0
6ontrolBcholesterol 4.0 244 X$
a
1C.0
6ontrolBcholesterol 4.0 144 X$
a
97.1
6ontrolBcholesterol 4.0 744 X$
a
00.4
6ontrol+ Hrou! )E .!!le diet+ Hrou! )I.
a
Hrou! ) animal liver microsomes were added with different cholesterol
concentrations
and dietary su!!lementationE unconfirmed benefits of new synthetic diets from )ndus-
triesE lack of social, enetic, environmental, alleric, anthro!ometric, metabolic, and
ethnic risk factors contributin cholelithiasisE lack of overnment !olicy and
monitorin on un!rescribed nondru su!!lements available at o!en counters. )t is
!ossible that new diet reimens includin wild foods, veetarian life style may be a
new a!!roach to reduce cholesterol and biliary li!ids in bile and blood as !ro!osed
decades ao *Lei et al., 144CE #ixley et al., 23C0-. )t is ex!ected that nutrition
industries will make available new ready-made foods after ?5.,8S5. a!!roval to
reduce cholelithiasis and lithoenic effects in-cludin !roteins and !ectins. (ecent
review on nutritional thera!y of cholelithiasis suested immediate attention on
reconition and identification of cholelithiasis as risk in different taret rou!s with
active role of traditional dietary manaement to manae cholelithiasis and
dysli!idemia in s!ecial rou!s includin children, !renant women, and !atients after
cholecystectomy *#aul et al., 2333-. $anaement of cholelithiasis in children still
remains to be established and needs uidelines on cholelithiasis, identifica-tion of
allstone com!lications, and monitorin of !ostcholecystectomy *della 6orte et al.,
144CE 'artl et al., 1443-, new enetic risk factors, factors of affluence, and envi-
ronmental and metabolic factors *Boue et al., 1424-.
(ole of nonsterols, cholesterol, and bile salts is extensively investiated to rule out
the main mechanism of cholelithiasis, new risk factors, com!lications, and outcome of
allstones in children *;alcher et al., 1424-. &utrition deficiencies develo!ed after
all-stone removal !ose a challene if not !revented or treated in time. Still there is a
need to !revent or treat !ossible nutrition deficiencies develo!ed after the bariatric
surery, !ostcholecystectomy *Nieler et al., 1443-. )t is ex!ected that two maGor
achievements will chane the scenario of cholelithiasis and its !revention+ a!!roved
clinical and laboratory evaluation of li!id !rofile established to evaluate efficacy of
#& in cholecystitis *Haby, 1443E ;alcher et al., 1424-E new knowlede of dietary
surveys, benefits of diet reimens includin wild foods, veetarian life style in
different social ethnic rou!s *Nhurikhin et al., 1424-. )t is s!eculated that mother
Snature% has iven humans natural benefits of active and healthy life style *low fat,
low salt, no smokin,alcoholic, no !ills, vean habits- to kee! cholesterol low and
avoid cholelithiasis or allstone formation *Sharma et al., 1422-.
O. CONCL*SION
6holesterol, !hos!holi!ids, and bile salts remain in balance as emulsion in bile
stored in all bladder. #hysicochemical studies suest that cholelithiasis is due to
cholesterol saturation with com!lications of allstone formation due to nucleation and
de!osition of calcium, bilirubin, and !iments. 6holelithiasis is reconi/ed first by
biliary li!id !ro-file in clinical lab su!!orted with clinical evaluation and dianostic
!rocedures. )nitially cholelithiasis is !revented by dietary and nutrition
su!!lementation to kee! li!ids low but success rates or benefits are less understood or
unconfirmed in different taret rou!s or social ethnicities because of lack of data on
!hysical, environmental, enetic, and afflu-ence life styles. )n advanced staes of
cholelithiasis, allstones et develo!ed and bile salt thera!ies with modified diet
reimens are !rescribed to !revent nutrition deficiencies and com!lications. Still
today, #& com!lications after cholecystitis, cholecystectomy, and cholelithiasis risk
factors in children bearin allstones !ose a challene.
ACKNOWLEDGMENTS
.uthors acknowlede the assistance of $r >ishan Lal and senior research
fellowshi! from )ndian 6ouncil of $edical (esearch, &ew 5elhi. Senior author
#rofessor (akesh > Tandon is a consultant at #ush!awati Sinhania (esearch
)nstitute, Sheikh Sarai-), &ew 5elhi, )ndia.
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study of the !revention of allstone formation and !rotection from liver disorder in
hamsters. &i!!on Heka 'okan 0D *9-, 1D1M1<0.
Sembries, S., 5onowski, H., $ehrlLnder, >., ;ill, ?., 5ietrich, '., 1447. 5ietary
fiber-rich colloids from a!!le !omace extraction Guices do not affect food intake and
blood serum li!id levels, but enhance fecal excretion of steroids in rats. The :ournal of
&utritional Biochemistry 20 *0-, 13DM941.
Sembries, S., 5onowski, H., $ehrlLnder, >., ;ill, ?., 5ietrich, '., 144D.
#hysioloical effects of extraction Guices from a!!le, ra!e, and red beet !omaces in
rats. :ournal of .ricultural and ?ood 6hemistry 07 *1D-, 241D3M241C4.
Serrano #a/, #., Hon/Kle/ Bueno, I., 5ieo EstQve/, $., et al., 2330.
#osto!erative enteral nutrition with a hih content of mono- and !olyunsaturated fatty
acids. &utricion 'os!italaria 24 *7-, 119M11<.
Sharma, (., $offatt, (.:., 1422. 5iet and nutrition thera!y in dysli!idemia
manaement. )n+ 5ysli!idemia (oya > *Ed.-, )SB& 3<C-309-94<-<1D-4. )ntech
#ublishin 6om!any, 6roatia, !!. D<M<0.
Shimi/u, :., =amada, &., &akamura, >., Takita, T., )nnami, S., 233D. Effects of
different ty!es of dietary fiber !re!arations isolated from bamboo shoots, edible
burdock, a!!le and corn on fecal steroid !rofiles of rats. :ournal of &utritional
Science and Iitaminoloy *Tokyo- 71 *D-, 01<M093.
Sichieri, (., Everhart, :.E., (oth, '., 2332. . !ros!ective study of hos!itali/ation
with allstone disease amon women+ role of dietary factors, fastin !eriod, and
dietin. .merican :ournal of #ublic 'ealth C2 *<-, CC4MCC7.
Simon, :..., 'udes, E.S., 1444. Serum ascorbic acid and allbladder disease
!revalence amon 8S adults+ the Third &ational 'ealth and &utrition Examination
Survey *&'.&ES )))-. .rchives of )nternal $edicine 2D4, 392M39D.
Souidi, $., #ar"uet, $., Lutton, 6., 233C. )m!roved assay of he!atic microsomal
cholesterol < al!ha-hydroxylase activity by the use of hydroxy!ro!yl-beta-
cyclodextrin and an &.5#'-reeneratin system. 6linica 6himica .cta 1D3 *1-,
142M12<.
Souidi, $., #ar"uet, $., 5ubrac, S., .udas, O., BQcue, T., Lutton, 6., 1444. .ssay
of microsomal oxysterol <al!ha-hydroxylase activity in the hamster liver by a
sensitive method+ in vitro modulation by oxysterols. Biochimica et Bio!hysica .cta
27C< *2-, <7MC2.
Strqhle, .., ;aldmann, .., ;olters, $., 'ahn, .., 144D. Ieetarian nutrition+
#reventive !otential and !ossible risks. #art 2+ #lant foods. ;iener >linische
;ochenschrift 22C *23M14-, 0C4M039.
Teitelbaum, 5.'., Tracy :r., T.?., .outhmany, $.$., et al., 1440. 8se of
cholecystokinin-octa!e!tide for the !revention of !arenteral nutrition-associated
cholestasis. #ediatrics 220 *0-, 2991M2974.
Tu/hilin, S..., 5reilin, 5..., &arodetskaGa, (.I., Lukash, L.>., 23<D. The
treatment of !atients with allstones by lecithin. .merican :ournal of
Hastroenteroloy D0, 192M190.
Ialhouny, H.I., .damson, )., 6halcar/, ;., et al., 23C0. Effects of casein and soy
!rotein on he!atic and serum li!ids and li!o!rotein li!id distributions in the rat.
.therosclerosis 0D *1-, 21<M29<.
Iidyashankar, S., Sambaiah, >., Srinivasan, >., 1424. (eression of
!reestablished cholesterol allstones by dietary arlic and onion in ex!erimental mice.
$etabolism 03 *24-, 2741M2721.
;alcher, T., 'aenle, $.$., $ason, (..., >oeni, ;., )mhof, .., >rat/er, ;.,
1424. E$)L Study Hrou!. The effect of alcohol, tobacco and caffeine consum!tion
and veetarian diet on allstone !revalence. Euro!ean :ournal of Hastroenteroloy
and 'e!atoloy 11 *22-, 2970M2902.
;an, 5.W., 6ohen, 5.E., 6arey, $.6., 1443. Biliary li!ids and cholesterol
allstone disease. :ournal of Li!id (esearch 04 *Su!!l-, S74DMS722.
;orthinton, '.I., 'unt, L.#., $c6loy, (.?., 8bbink, :.B., Braan/a, :.$., 1447.
5ietary antioxidant lack, im!aired he!atic lutathione reserve, and cholesterol
allstones. 6linica 6himica .cta 973 *2M1-, 20<M2D0.
Nhou, :.?., 6hen, #., =an, :.L., Nhu, =.H., #en, 6.'., ;u, =.L., 1444.
Oxidative stress before and after o!eration in !atients with chronic cholecystitis
containin allstone. Biomedical and Environmental Sciences 29 *7-, 107M1D1.
Nhurikhin, ..I., >itiashvili, ).N., >utukov, I.I., 1424. 6linical and laboratory
evaluation of the efficiency of !arenteral nutrition in destructive forms of acute
calculous cholecystitis. >linicheskaia Laboratornaia 5ianostika 24, 1DM13.
Nieler, O., Sirveaux, $..., Brunaud, L., (eibel, &., Wuilliot, 5., 1443. $edical
follow u! after bariatric surery+ nutritional and dru issues. Heneral
recommendations for the !revention and treatment of nutritional deficiencies.
5iabetes A $etabolism 90 *D #t 1-, 077M00<.
Nubler, :., $arkowski, H., =ale, S., Hraham, (., (osenthal, T.6., 233C. &atural
history of asym!tomatic allstones in family !ractice office !ractices. .rchives of
?amily $edicine <, 194M199.
CHAPTER >R
I$/ia$ M(/i&i$a Pa$ts a$/ S3i&(s
i$ th( Pr(+($ti!$ a$/ Tr(at.($t
!' *&(rati+( C!itis
M.S. Bai"a
S
I 5. Na$/hi$i
S
I F. E..a

I M.V. V($,atara$"a$$a

I P.
V($,at(sh
W
I R. Faya/

l
?ather $uller $edical 6ollee, $analore, >arnataka, )ndia

8niversity of South 6arolina, 6olumbia, S6, 8S.

6onnexios Life Sciences, Banalore, >arnataka, )ndia

Toti!otentSc Scientific #roduct, Huraon, )ndia


ABBREVIATIONS
AKBA .cetyl-22-keto-beta-boswellic acid
CD 6rohn%s disease
CO= 6yclooxyenase
DNBS 5initroben/ene sul!honic acid
DSS 5extran sulfate sodium
GSH Hlutathione
IBD )nflammatory bowel disease
IFN%X )nterferon amma
IJB &uclear factor of ka!!a liht !oly!e!tide ene enhancer in B-cells inhibitor
IYBa &uclear factor of ka!!a liht !oly!e!tide ene enhancer in B-cells inhibitor,
al!ha
IKK )gBa kinase
IL )nterleukin
iNOS )nducible nitric oxide synthase
LO= Li!oxyenase
LPO Li!id !eroxidation
LPS Li!o!olysaccharide
MAPK $itoen-activated !rotein kinase
MPO $yelo!eroxidase
NF%ZB &uclear factor ka!!a B
NSAIDS &onsteroidal anti-inflammatory drus
PPARX #eroxisome !roliferator-activated rece!tor amma
SOD Su!eroxide dismutase
TGF%[> Transformin rowth factor beta 2
TNBS Trinitroben/ene sulfonic acid
TNF%V Tumor necrosis factor al!ha
*C 8lcerative colitis
>. INTROD*CTION
)nflammatory bowel disease *)B5-, clinically characteri/ed by bloody diarrhea,
cram!in, and abdominal !ain, is an immune-mediated chronic and rela!sin
inflammatory disease affectin the linin of the intestine *#odolskiy, 1441-. ?urther,
when com!ared with the averae !o!ulation, due to chronic inflammatory nature,
!atients with )B5 are at a hiher risk to develo! colorectal cancer *)t/kowit/ and =io,
1447-. 6rohn%s disease *65- and ulcerative colitis *86- re!resent the two most
common forms of the )B5 con-ditions *)t/kowit/ and =io, 1447E #odolskiy, 1441-.
These diseases closely resemble each other, but differ sufficiently to be considered as
inde!endent ailments *summari/ed in Table 24.2E #odolskiy, 1441-. 5es!ite much
effort and research, the etioloy and the !ro-cess of !athoenesis remain to be
deci!hered *)t/kowit/ and =io, 1447-.
'istorically, the )B5 incidence was hih in countries of &orth .merica and
&orth-ern and ;estern Euro!e, while it was low in Eastern Euro!e, .frica, South
.merica, .sia, and the #acific reion. 6onversely, recent studies suest that the
incidence fiures have stabili/ed or slihtly increased in the !reviously hih-!revalent
countries, while is on a rise in the !reviously low-incidence countries. 6ollectively,
these re!orts indicate that in the near future, )B5 could be a lobal health !roblem
and attention is warranted in understandin its etioloy, !athoenesis, and to develo!
affordable safe treatments *'unin et al., 1449-.
)n milder conditions, the anti-inflammatory com!ounds such as sulfasala/ine and
0-aminosalicylic acid are administered, whereas in the more severe and !ersistent
cases, the oral, rectal, and !arenteral administration of corticosteroids and
immunosu!!ressants is followed. 'owever, in extreme conditions, de!endin on the
need and !atient%s health, surery may be !erformed. )n most cases, the benefit
restricts to reduction of in-flammation and its com!lications *#odolskiy, 1441-.
6onversely, reular intake of these aents is unsafe as they !ossess lon-term
damae. The most common side effects are astric ulcerations with nonsteroidal anti-
inflammatory drusE 6ushin%s habitus, fraile skin, !ur!le striae, hy!erlycemia,
muscular weakness, flarin u! of latent infections, delayed healin of wounds, !e!tic
ulcerations, osteo!orosis, cataract, laucoma, and hy!othalamic !ituitary axis
su!!ression with corticosteroidsE and increased risk of bacterial, funal, viral, as well
as o!!ortunistic infections and develo!ment of lym!homas and related malinancies
with immunosu!!ressants *#odolskiy, 1441-. ?ur-ther, in some !atients, the condition
may become refractory leadin to severe morbidity and decrease in the "uality of life
*#odolskiy, 1441-.
(ecently, anti-tumor necrosis factor al!ha *T&?-c-, anti-al!ha four interin,
!erox-isome !roliferator-activated rece!tor amma liand, and !robiotic thera!y are
bein tried but their lon-term benefits are unknown *#odolskiy, 1441-. The
bioloical aents, in !articular, anti-T&? aents such as infliximab, adalimumab, and
certoli/umab have been tried but recent re!orts suest that lon-term use of these
T&?-c inhibitors miht
Tab( >R.> Etioloical ?actors )nvolved in )nflammatory Bowel 5isease
*&(rati+( &!itis Cr!h$\s /is(as(
Henetic backround Less !rofound $ore !rofound
Sex distribution $arinally more common in males $arinally more
common in females
6oncordance rate in
twins
Lower 'iher
Hene associations 5(B2
l
2041,5(B2
l
4249,5(B2
l
21 &O51 *6.(T20-
)B5 0 on ene 0 Less association $ore association
$5(2 ene $ore association Less association
$e!rin al!ha ene ?ive !olymor!hisms One !olymor!hism
TL( abnormalities Less association $ore association
)mmune !rofile Th1 res!onse Th2 res!onse
$ediators s!ecific to
each
)L-0, )L-29 )L-21, )L-2C, )L-19,
)L-1<, )L-12
5efect in a!o!tosis Less association $ore association
)nvolvement of
!latelets
Likely to be less Likely to be more
Environmental factors $ore !rofound Less !rofound
$icrooranisms Mycobacterium paratuberculosis$
Pseudomonas s!ecies, Listeria
s!ecies
acillus s!ecies,
adhesive %scherichia
coli$ 'usobacterium
varium
Trierin antiens E!ithelial antiens, functionally
altered aerobes
.naerobic bacteria,
cell wall bacterial
com!onents
Smokin #rotective (isk factor
'istory of !revious
a!!endicectomy
#rotective &o association
5iabetes )ncreased association &o sinificant
association
(heumatoid arthritis &o sinificant association )ncreased association
'ormone re!lacement
thera!y
&o sinificant risk )ncreased risk
5e!ression and anxiety $ore association Less association
increase the risk of infections and malinancies, es!ecially, non-'odkin%s lym!homa
*Lakatos and $iheller, 1424-.
Bioloicals that block leukocyte adhesion *natali/umab-, taret cytokines *such as
in-terleukin *)L--21,19 antibodies-, or inhibit T-cell sinalin *such as )L-D rece!tor
anti-bodies- are also bein investiated. 'owever, these drus also have a number of
contraindications and side effects, es!ecially, when used in combination with classical
im-munosu!!ressive aents or corticosteroids. The maGor areas of concern consist of
o!!ortunistic infections, malinancies, and diverse com!lications such as
inGection,infu-sion reactions and autoimmunity and contraindications, such as heart
failure and acute infectious diseases *Stallmach et al., 1424-.
The re!eated rea!!earance, fear of surery, severe morbidity, and derisory
res!onse to conventional drus, safety, and drawbacks of new drus entices the
!atient to use unconventional treatments, with a ho!e that it will decrease the
sym!toms of the dis-ease and concomitantly !erk u! the "uality of life *'ilsden et al.,
1449-. (ecent re!orts suest that at least 74R of )B5 !atients have used
com!lementary and alternative med-icines, and !redictions are that in the near future,
this number will increase *'ilsden et al., 1449-.
?. AY*RVEDIC PLANTS AND IBD
The traditional )ndian systemofmedicine, the .yurveda, whichmeans the science
of life, is one of the world%s oldest systems of medicines. The exact oriin of
.yurveda is unknown, but assum!tions are that the a!!roaches and conce!ts have
been refined and !erfected be-tween 1044 and 044 B6. .yurveda !redominantly uses
!lant-based formulations devel-o!ed throuh the ex!erimentation and ex!eriences of
!hysicians for centuries *:ata! et al., 1447-. Some of the )ndian !lants such as
Curcuma longa, Commiphora mu&ul, ,llium sativum, ,loe vera or ,loe barbadensis,
os*ellia serrata, -arcinia cambogia, Punica granatum, Tri-gonella foenum-
graecum, and ?ingiber officinale have been investiated for their beneficial use in
)B5. The scientific, Enlish, and the 'indi names are enlisted in Table 24.1. This
review addresses these observations.$ost of the studies are !reclinical and have
shown reat !rom-ise but warranties further investiations.
Tab( >R.? The Scientific &ame and 6ommon &ame, in Both Enlish
and 'indi, of the )ndian $edicinal #lants (e!orted to Be Effective in
#reventin,.melioration of )B5 in Ex!erimental Systems of Studies
S&i($ti'i& $a.( Fa.iy C!..!$ $a.( I$/ia$ $a.(
,llium sativum .lliaceae Harlic Lasuun
,loe vera or ,loe
barbadensis
.s!hodelaceae ,loe >umari
os*ellia serrata Burseraceae )ndian frankincense Salai
Commiphora
mu&ul
Burseraceae Huul,Hual,$ukul
myrrh tree
Huul
Curcuma longa Niniberaceae Turmeric 'aldi
-arcinia
cambogia
6lusiaceae $alabar tamarind Iriksahmala
Punica granatum Lythraceae #omeranate .nar
Trionella
foenum-raecum
?abaceae ?enureek $ethi
Niniber officinale Niniberaceae Hiner .drak
B. PHYTOCHEMICALS AND INDIAN MEDICINAL PLANTS
WITH ANTI%IBD EFFECTS
B.>. C#r&#.i$I th( A&ti+( Pri$&i3( !' T#r.(ri&
6urcumin *?iure 24.2-, the maGor constituent of the rhi/ome of C. longa
*turmeric-, a s!ice and colorin aent widely used in )ndian food, is one of the well-
studied !hytoche-mical for )B5 and other diseases. )t is a nontoxic aent with !otent
antioxidant, anti-inflammatory, and cyto!rotective effects. $yriad studies have shown
that curcumin is a formidable !ro!hylactic and thera!eutic aent aainst different
ulceroens *trinitroben/ene
Fi"#r( >R.> Structure of curcumin, boswellic acid, uulsterone, /erumbone, and
disoenin.
Fi"#r( >R.? $olecular tarets affected by curcumin for the !revention,amelioration of
ulcerative colitis. .rrow u!, increaseE down, decrease.
sulfonic acid, T&BSE dextran sulfate sodium, 5SSE dinitroben/ene sul!honic acid,
5&BSE and dinitrochloroben/ene- and that it also reduces the rela!se rate in human
86.
)n ex!erimental studies, it was observed that the curcumin treatment either when
!ro!hylactic or curative, or when administered throuh the oral or intra!eritoneal
routes im!roved the survival rate and decreased the ulceroen-induced wastin and
discomfort. .t the ross level, curcumin sinificantly decreased the macrosco!ic
scores of mucosal erosions and ulcerations. 6urcumin%s activities are associated with
its ability to scavene free radicals, influence multi!le sinalin !athways, es!ecially
the kinases *.>T, mitoen-activated !rotein kinase, extracellular sinalin kinase-,
inhibit cyclooxyenase *6OO--2, 6OO-1, li!oxyenase *LOO-, T&?-c, interferon
amma *)?&-b-, inducible nitric oxide synthase *i&OS-, inhibit the transcri!tion
factors &?-gB and activator !rotein-2, and modulate the cyto!rotective !athways
de!endent on nuclear factor erythroid-derived 1-related factor *'anai and Suimoto,
1443E ?iure 24.1-.
Studies have also shown that 9D4 m curcumin when administered three or four
times a day for 9 months also reduced the clinical rela!se in !atients with "uiescent
)B5 *'anai and Suimoto, 1443-. 6umulatively, all these results stronly suest that
curcumin is a !romisin medication for maintainin remission in !atients and that
randomi/ed con-trolled clinical investiations in lare cohorts of !atients are needed
to fully evaluate its clinical !otential in treatment of )B5.
B.?. G#""#st(r!$(I th( A&ti+( Pri$&i3( !' C. mukul
Huulsterone, *E- and N uulsterone chemically 7, 2<*14--!renadiene-9, 2D-
dione- *?iure 24.2-, is a !lant sterol !resent in the resin of C. mu&ul. Since anti"uity,
the resin has been used to treat a variety of ailments, includin obesity, bone fractures,
arthritis, inflammation, cardiovascular disease, diabetes, hy!erli!idemia,
atherosclerosis, and oste-oarthritis, in .yurveda *6heon et al., 144D-. Ex!erimental
studies have confirmed that the um uul and its a"ueous and steroid fractions
!ossess anti-inflammatory activities in various models of inflammation *6heon et al.,
144DE $encarelli et al., 1443-.
Huulsterone inhibited the 5SS-induced murine colitis as assessed by reduction
in the clinical disease activity score, colon lenth, and histoloy *6heon et al., 144D-.
$ech-anistic studies with both in vitro and animal models have shown that
uulsterone inhib-ited li!o!olysaccharide *L#S- or )L-2a-induced intercellular
adhesion molecule-2 ene ex!ression, nuclear factor ka!!a B *&?-Z]- transcri!tional
activity, )Z] !hos!horylation, deradation, and &?-Z] 5&. bindin activity in
intestinal e!ithelial cell. $oreover, uulsterone blocked the )ZBa kinase *)>>-
activity and attenuated the 5SS-induced u!reulation of )YB *nuclear factor of ka!!a
liht !oly!e!tide ene enhancer in B-cells inhibitor- and )>> !hos!horylation in
tissues *6heon et al., 144D-.
'owever, contradictin the above observations, studies have shown uulsterone
to be ineffective in !reventin the T&BS-induced colon inflammation in (ats,mice,
but had a !artial ameliorative effect on the oxa/olone-induced colitis. In vitro, studies
with 657B cells isolated from the intestinal lamina !ro!ria have demonstrated that
uul-sterone effectively reulates the function of effector T cells by modulatin cell
sinalin activation !athway caused by 659,651C. The net bioloical effects
resultin from ex-!osure to uulsterone includes attenuation of eneration of )L-1
and )L-7 and )?&-X as well as T cell !roliferation *$encarelli et al., 1443-. )n view of
the diverent observations, it is im!erative that detailed studies are warranted for
uulsterone to be of use in clinics.
D. INDIAN MEDICINAL PLANTS WITH ANTI%IBD EFFECTS
4.1. A. sativum
6ommonly known as arlic and indienous to .sia, the cloves are a reular in the
various cuisines and folk medicines. )n traditional medicines, the arlic cloves, both
raw and aed are used as a natural antiviral, antibacterial, and antifunal aent to treat
common couh, astrointestinal disorders, and as a cardio!rotective aent *'arisa et
al., 1443-. ?eedin rats with arlic *4.10 k
-2
body weiht-, orally for 7 consecutive
weeks and 9 days dur-in induction of colitis sinificantly reduced the acetic acid-
induced increase in the colon weiht and modulated the oxidant and antioxidant
!arameters. ;hen com!ared with the !lacebo-treated colitis rou!s, administerin
arlic restored the levels of lutathione *HS'- and the antioxidant en/ymes with a
concomitant decrease in li!id !eroxidation *L#O- levels. ?urther combinin arlic
*4.10 k
-2
body weiht- with amino acid L-arinine *D10 m k
-2
body weiht- for
the same !eriod further mitiated the chanes in both colon weiht and increased the
arlic effect on colon tissue contents of L#O and HS' *'arisa et al., 1443-.
4.2. A. vera or A. %ar%adensis
>nown as >umari in Sanskrit, this reen shrubby, !erennial, xero!hytic succulent
!lant is one of the most commonly used medicinal !lant in .yurveda *>orkina et al.,
1449-. In vitro studies have shown that ,# vera el inhibited the eneration of reactive
oxyen me-tabolite !roduction *su!eroxide and !eroxyl radical-, re!ressed the
!roduction of !ros-talandin in colorectal bio!sies, and decreased the levels of )L-C in
6a6O
1
cells *Lanmead et al., 1447a-. . double-blind randomi/ed !lacebo-controlled
trial also showed that the oral administration of nontoxic dose of ,loe el 244 ml
*diluted 1+2-, twice daily for 7 weeks, !roduced a clinical res!onse, decreased the
sim!le clinical colitis activity index, and im!roved the histoloical scores clearly
suestin that its beneficial effects may also extend to humans *Lanmead et al.,
1447b-.
>orkina et al. *1449- for the first time evaluated the !rotective,curative effects of
,# vera aainst the 5SS-induced 86 in rats. The authors observed that when
com!ared to the !lacebo-treated cohorts, the !retreatment with the ,loe !re!aration
*10 m k
-2
- was effective, as !arameters evaluatin the deree of inflammation,
electrical,mechanical im!airment in the ut, and oxidative stress were im!roved. The
simultaneous or !ost-treatment administration of the ,loe !re!aration was ineffective
suestin that ,loe !re!aration !ossess !reventive but not curative effects *>orkina
et al., 1449-.
4.'. 2. serrata
. serrata is a moderate to lare branchin tree aboriinal to the dry hilly areas of
)ndia *Hu!ta et al., 233<, 1442-. #re!arations from the oleo um resins of . serrata
*salai- have been used as a traditional remedy in .yurvedic medicine in )ndia for the
treatment of inflammatory diseases and for the chronic !ain associated with arthritis
*Hu!ta et al., 233<, 1442-. Boswellic acid *?iure 24.2-, the !entacyclic triter!enes,
are !otent anti-inflammatory aents and inhibit the leukotriene biosynthesis in
neutro!hilic ranulocytes by a nonredox, noncom!etitive inhibition of 0-LOO.
.dditionally, certain boswellic acids have been described to inhibit elastase in
leukocytes, inhibit !roliferation, induce a!o!tosis, and inhibit to!oisomerases
*.mmon, 144D-.
6linical studies have shown that administerin . serrata *904 m thrice daily for
D weeks- to !atients with rade )) and ))) 86 was effective and was associated with
min-imal side effects. The effect was com!arable to that of sulfasala/ine *2 thrice
daily- used as controls *Hu!ta et al., 233<-. .dministerin . serrata im!roved the
!atholoical con-ditions in maGority of the !atients as evaluated from the stool
!ro!erties, blood !arameters *'b, serum iron, calcium, !hos!horus, !roteins, total
leukocytes, and eosino!hils-, scan microsco!y, and histo!atholoy of rectal bio!sies
*Hu!ta et al., 233<, 1442-.
#reclinical studies validate the clinical observations and studies by Latella et al.
*144C- have shown that administerin os*ellia extracts *04 m k
-2
- to the T&BS-
induced colitis rats decreased the fibrosis associated with the chronic colonic
inflammation. . marked reduction in the disease activity index, colon weiht, lenth,
adhesions, strictures, dilatation, thickness, edema, ulcerations, and extension of the
damae was ob-served in the os*ellia extract-treated cohorts. (eduction in the
ex!ression of al!ha smooth muscle actin, collaen )M))), connective tissue rowth
factor, transformin rowth factor beta 2 *TH?-a2-, Smad9, and Smad< was also
observed in the colons. 'is-toloical studies substantiated the ross and biochemical
observations and the authors !ro!ose that the observed antifibrotic mechanism of
action seems to be mediated by the inhibition of TH?-a2,Smad9 !athway *Latella et
al., 144C-.
Studies have shown that acetyl-22-keto-beta-boswellic acid *.>B.-, an active
!rin-ci!le of . serrata abroated &?-B activation induced by T&?, )L-2a, okadaic
acid, doxorubicin, L#S, hydroen !eroxide, !olymethyl acrylate, and ciarette smoke.
.>B. did not directly affect the bindin of &?-B to the 5&. but inhibited
se"uentially the T&?-induced activation of )>>, )Ba *nuclear factor of ka!!a liht
!oly!e!tide ene enhancer in B-cells inhibitor, al!ha- !hos!horylation, )Ba
ubi"uitination, )Ba dera-dation, !D0 !hos!horylation, and !D0 nuclear translocation
*Takada et al., 144D-. . semi-synthetic form of .>B. also !revented the 5SS-
induced ex!erimental murine colitis as assessed by ross and histoloical
observations. The recruitment of adherent leukocytes and !latelets into inflamed
colonic venules was !rofoundly reduced indicatin that #-selectin-mediated
recruitment of inflammatory cells is a maGor site of action for this novel anti-
inflammatory aent *.nthoni et al., 144D-.
'owever, contradictory observations by >iela et al. *1440- suest that os*ellia
ex-tracts were ineffective in amelioratin 5SS or T&BS-induced colitis in mice.
?urther, the authors observed that the extract caused he!atomealy and steatosis in
mice, whereas the individual boswellic acids increased the basal and )L-2a-stimulated
&?-B activity in intestinal e!ithelial cells *>iela et al., 1440-.
4.4. G. cam%ogia
)s a !lant indienous to )ndia and !arts of Southeast .sia. The fruits, which
resemble a miniature !um!kin, are of use as both dietary and medicinal aent. The
decoction of the fruits is an im!ortant !re!aration in the various folk medicines to
treat ulcers and inflam-mations. )t is also a !o!ular weiht loss aent and has been
marketed in !arts of Southeast .sia *dos (eis et al., 1443-. Ex!erimental studies have
shown that the fruit extract !ossess hy!oli!idemic !ro!erties, antiadi!oenic, and
a!!etite su!!ressor effects *dos (eis et al., 1443-. (ecent re!orts suest that the
extract inhibited the T&BS-induced colitis in rats by !reventin the damae, and
decreasin the activity of myelo!eroxidase *$#O- and ex!ression of 6OO-1 and
i&OS to reduce the colonic levels of !rostalandin E1 *#HE1- and )L-2a and the
5&. damae in the colonocytes *dos (eis et al., 1443-.
4.+. .. granatum
6onsidered to be a mystical fruit, P# granatum is cultivated throuhout the
$editerranean reion, Euro!e, )ndia, #akistan, 6hina, Southeast .sia, and in
6alifornia and .ri/ona in the 8nited States is today an im!ortant !lant. .ll !arts of
the !lant have been of use in treatin a variety of ailments in the various traditional
and folk systems of medicine. Sci-entific studies have shown that !omeranate
!revents cancer, cardiovascular disease, di-abetes, dental conditions, .l/heimer%s
disease, arthritis, obesity, erectile dysfunction, bacterial infections and antibiotic
resistance, and ultraviolet radiation-induced skin dam-ae *Sinh et al., 1443-.
?eedin Swiss albino mice with hydromethanolic extract of the !omeranate flower
and its ellaic acid-rich fraction *244 m k
-2
and 144 m k
-2
, !.o.- decreased the
5SS-induced 86. The effect was com!arable to sul!hasala/ine *244 m k
-2
, !.o.-
and sodium cromolycate *74 m k
-2
, i.!.-. 'isto!atholoical studies showed that the
extracts reduced the deree of ulceration, levels of $#O, hista-mine, and li!id
!eroxides *Sinh et al., 1443-.
4.-. $. "oenum,graecum
6ommonly known as ?enureek, the seeds of the !lants are of use in traditional
med-icine. )t is used to induce labor, hel! diestion, and as a eneral tonic to im!rove
me-tabolism and health. Ex!erimental studies with laboratory animals and human
trials suest its !ossible hy!olycemic and antihy!erli!idemic !ro!erties *=amada et
al., 233<-. In vitro studies with cell-free systems have shown that fenureek was an
effective scavener of !eroxyl but not of su!eroxide radical in chemical assay and
also effective in scavenin the free radicals !roduced by the inflamed colorectal
mucosa *Lanmead et al., 1441-.
The steroidal sa!onin diosenin *?iure 24.2- !resent in fenureek has been
shown to su!!ress inflammation. #retreatment with diosenin sinificantly inhibited
the weiht loss and food intake. )t also !revented the indomethacin-induced intestinal
inflammation as confirmed by ross examinations and histoloical findins *=amada
et al., 233<-. 5ios-enin inhibited the ovalbumin-induced intestinal alleric res!onsesE
su!!ressed the intes-tinal inflammation, occurrence of diarrhea, infiltration and
deranulation of mast cells, and !resence of mucin-containin oblet cells in the
duodenum in mice. 5iosenin also reduced the cry!t de!th of the intestine and
su!!ressed the serum !roduction of ovalbumin-s!ecific )E and the total )E *'uan
et al., 1443-.
5iosenin su!!resses T&?-induced &?-B activation as determined by 5&.
bind-in, activation of )>>, )Ba !hos!horylation, )Ba deradation, !D0
!hos!horylation, and !D0 nuclear translocation throuh inhibition of .kt activation.
&?-B-de!endent re!orter ene ex!ression was also abroated by diosenin. T&?-
induced ex!ression of &?-B-reulated ene !roducts involved in cell !roliferation
*cyclin 52, 6OO-1, c-myc- and antia!o!tosis *inhibitor of a!o!tosis2, Bcl-1, Bcl-OL,
Bfl-2,.2, T&? rece!tor-associated factor 2, and cas!ase C homoloue ?L)6E
inhibitory !rotein- were also downreulated *Shishodia and .arwal, 144D-.
4.5. 6. o""icinale
The rhi/ome of ?. officinale commonly known as iner is an im!ortant s!ice and
an in-teral !art of several medicinal formulations in .yurveda, Siddha, 8nani,
Srilankan, Hreek, (oman, .sian, )ndian, $editerranean, and .rabic systems of
medicines. )t is used !rimarily as carminative, dia!horetic, antis!asmodic,
ex!ectorant, !eri!heral circulatory stimulant, astrinent, a!!etite stimulant, anti-
inflammatory aent, diuretic, and antifla-tulent *El-.bhar et al., 144C-.
#reclinical studies have shown that !retreatment with iner extract ameliorated
the acetic acid-induced edematous inflammation in the colon. 'isto!atholoical
studies showed that iner sinificantly attenuated the extent and severity of edema.
5es"uama-tion, necrosis, and inflammatory cell infiltration in the mucosa were also
observed. The levels and activity of colonic $#O, li!id !eroxides, !rotein carbonyl
content, T&?-c, and #HE1 were also decreased. .dministerin iner restored the
levels of HS', catalase, and su!eroxide dismutase. The !rotective effect of hih doses
of iner was com!arable to that of the standard sulfasala/ine *El-.bhar et al., 144C-.
Nerumbone *ses"uiter!enoidE ?iure 24.2-, a very minor constituent of ?.
officinale but a maGor com!onent of ?ingiber Berumbet, mitiated the 5SS-induced
acute colitis in )6( mice. Oral feedin of /erumbone reduced the inflammatory
biomarkers *)L-2c, )L-2a, T&?-c, #HE1, and #H?1c- in the colonic mucosa and
su!!ressed 5SS-induced colitis. &imesulide, a selective 6OO-1 inhibitor su!!ressed
the histoloical chanes induced by 5SS without affectin inflammatory biomarkers
but when combined with /erumbone enhanced the !rotective effects *$urakami et al.,
1449-.
E. AY*RVEDIC%BASED POLYHERBAL FORM*LATION
:ata! et al. *1447- studied the effect of a !olyherbal formulation, made as !er the
ancient authentic classical text of .yurveda and consistin of ,egle marmelos,
Coriandrum sativum, Cyperus rotundus, and Aetiveria BiBanioides. The authors
!re!ared a decoction of the herbal formulation and administered them for <
consecutive days before induction of colitis with acetic acid. )t was observed that the
herbal formulation was effective in !reventin acetic acid-induced colitis in mice. The
!rotective effects were e"uivalent to that of !red-nisolone that was used as the
standard dru. The herbal decoction decreased the levels of $#O and the
histo!atholoical observations showed lower score *:ata! et al., 1447-.
?eedin methanolic extract of ,# marmelos *144 m k
-2
- ameliorated 5&BS-
induced 86 in rats. Bael inhibited the 5&BS-induced decrease in food and water
intake, wastin, and restored the stool consistency. Bael reduced the ross chanes,
mucosal damae, and disease activity index. 'isto!atholoical study showed that bael
administration decreased the infiltrative neutro!hils and inflammation. The
biochemical assays showed a decrease in the levels of nitric oxide, L#O, and $#O.
6oncomitantly, the levels of antioxidant en-/yme in the bael-treated colitis cohort
were increased *#hoolsinh, 1440-.
G. CONCL*SION
)B5, which affects the considerable number of !eo!le in some !arts of the world,
is a chronic inflammatory condition of the astrointestinal tract. #harmacoloical
studies, with ex!erimental animals in the !ast decade, suest that herbal treatments
are effective in !reventin,amelioratin )B5 com!lications and these re!orts su!!ort
the ethnome-dicinal use in .yurveda. Of all the botanicals investiated, curcumin, the
active !rinci!le of turmeric, is observed to lead to disease "uiescence in humans and
is the most !rom-isin. 'owever, with other !lants, further ex!lorations are necessary
to elucidate their !harmacoloical activities and clinical utility in treatin of )B5.
Owin to their nontoxic nature and easy acce!tability, some of the
!lants,!hytochemicals studied may enter clinical trials and deserve detailed
!reclinical studies.
ACKNOWLEDGMENTS
The authors dedicate this article to Late 5r. Sumathi Bhide, ?ormer 'ead of
6arcino-enesis, 6ancer (esearch )nstitute, Bombay, )ndia, and the mentor of 5r.
$.S. Balia. 5r. $rs Bhide was a !ioneer scientist and one of the early workers on
the antimutaenic and chemo!reventive effects of curcumin and turmeric. The authors
5r. $.S. Balia and $s &andhini :ose!h are rateful to (ev. ?r. #atrick (odrius
*5irector-, (ev. ?r. 5enis 5%Sa *.dministrator-, and 5r. :ai #rakash .lva, *5ean- of
?ather $uller $edical 6ollee for !rovidin the necessary facilities and su!!ort.
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beta-boswellic acid !oten-tiates a!o!tosis inhibits invasion and abolishes
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CHAPTER >>
Gi$"(r @6ingi%er o""icinale R!s&!(A) A$
A$&i($t R(.(/y a$/ M!/(r$ Dr#" i$
Gastr!i$t(sti$a Dis!r/(rs
M.S. Bai"a
*
, A.R. Shi+asha$,ara
*
I R. Ha$ia/,a
*
I P.L. Paatty
*
I R. Ar!ra

I R.
Faya/

*
?ather $uller $edical 6ollee, $analore, >arnataka, )ndia

)nstitute of &uclear $edicine and .llied Sciences, 5elhi, )ndia

8niversity of South 6arolina, 6olumbia, S6, 8S.


ABBREVIATIONS
CAT 6atalase
GR Hlutathione reductase
H
?
O
?
'ydroen !eroxide
LPO Li!id !eroxidation
>. INTROD*CTION
The astrointestinal tract *H)T-, consistin of the mouth, eso!haus, stomach,
intestine, colon, and rectum, is the !rimary oran for diestion and absor!tion. The
synchronous !eristalsis and secretions hel!s in the diestion both !hysically and
chemically *en/ymat-ically-. This occurs with the com!lex inter!lay of .uerbach and
$eissner%s !lexuses and a host of neurotransmitters M noradrenaline, acetylcholine,
nonadreneric noncholineric, and serotonin. The H)T is an im!ortant link for
assimilation of external environment into the internal milieu. The H)T is the !ortal for
entry of nutrients and essential mol-ecules includin drus and, to a limited extent, the
site for elimination of endoenous waste !roducts, drus, etc. H)T disturbances vary
from dys!e!sia, distressin nausea, vomitin, inflammations, infections, and cancers
to !hysical obstruction of intestine, volvulus, etc. *(amakrishna (ao et al., 1449-.
)n view of these functions, it is im!erative that the o!timal health of the H)T is
main-tained and re!orts suest that the traditionally used herbs and s!ices like
curcumin, ca!-saicin, !i!erine, arlic, onion, iner, mint, coriander, cumin, caraway,
aGowan, fennel, fenureek, mustard, and asafetida are extremely effective *#latel and
Srinivasan, 233DE 1444E (amakrishna (ao et al., 1449-. Of these, iner is the most
a!!reciated and well studied for amelioratin the various astrointestinal
disturbances, ailments, and diseases, and is the focus of this cha!ter.
?. GINGER AND HEALTH OF GASTROINTESTINAL
SYSTEM
The rhi/ome of ?ingiber officinale.oscoe, commonly known as iner, is
considered an im-!ortant s!ice lobally *.li et al., 144CE6hrubasik et al., 1440-. )n
addition to its culinary uses, iner !ossesses awidearrayofmedicinal uses
andisobservedtobeeffectiveaainstunrelated ailments like arthritis, s!rains, muscular
aches, !ains, sore throats, hy!ertension, dementia, fever, infectious diseases, and
helminthiasis *.li et al., 144CE 6hrubasik et al., 1440-.
The myriad !harmacoloical effects are su!!osed to be due to the !resence of
volatile com!ounds like /iniberene, curcumene, farnesene, bisabolene, -
ses"ui!hellandrene, 2, C-cineole, linalool, borneol, neral, and eraniol and the
nonvolatile ones like inerols, shoaols, !aradols, and /inerone. The other
constituents include iner !rotease, ca!sa-icin, inediol, alanolactone,
inesulfonic acid, alactosyl lycerols, inerlycoli!ids, diarylhe!tanoids, neral,
and !hytosterols *.li et al., 144C-. Some of the !hytochemical are de!icted in ?iure
22.2.
Fi"#r( >>.> Structures of some !hytochemicals !resent in the iner rhi/ome.
The com!osition of the !hytochemicals varies and is de!endent on the
tem!erature, water, humidity, soil conditions, the harvestin time, and ae of the
!lant,rhi/ome. Hinerols are hihly thermolabile due to the !resence of -hydroxy
keto rou! and read-ily undero dehydration to form the shoaols. The extent of this
conversion is likely to have a sinificant im!act on the medicinal benefits of iner,
as the two classes of com-!ounds vary in their bioavailability, !harmacokinetics, and
!harmacoloical !ro!erties. Shoaols may be further converted to !aradols by
hydroenation and are similar to the corres!ondin inerol *.li et al., 144CE
6hrubasik et al., 1440-.
.s seen in the 6hinese, .yurvedic, .rabic, Tibetan, 8nani, and various folk
systems of medicine, iner has a lon tradition of bein very effective in alleviatin
sym!toms of astrointestinal !roblems like consti!ation, indiestion, nausea, and
vomitin *.li et al.,144C-. Hiner is used as a stimulant and carminative, and is used
fre"uently for treatin dys!e!sia and colic *.li et al., 144CE 6hrubasik et al., 1440-. )t
has a sialaoue action, stimulatin the !roduction of saliva and !romotin the release
of bile from the all blad-der *.li et al., 144CE 6hrubasik et al., 1440-. #reclinical
studies have also shown that it !ossesses carminative, astro!rotective, antiulcerative,
and antiemetic !ro!erties to !re-vent e!iastric discomfort, dys!e!sia, stomachache,
abdominal s!asm, and cancer of the astrointestinal system *.li et al., 144CE
6hrubasik et al., 1440- *?iure 22.1-. )n the followin sections, the beneficial effects
of iner on the various astrointestinal systems are addressed.
B. GINGER IN ORAL HEALTH
Hlobally, dental caries and !eriodontal diseases are one of the most common
infections and at times can affect the "uality of life. #ark et al. *144C- investiated the
antibacterial
Fi"#r( >>.? Effect of iner on the various astrointestinal tracts.
effect of iner and its constituents on the rowth of oral bacteria associated with
!eri-odontitis. )t was observed that the ethanol and n-hexane extracts of iner
exhibited anti-bacterial activities on Porphyromonas gingivalis .T66 093<C, P#
endodontalis .T66 9074D, and Prevotella intermedia .T66 10D22. .ctivity-uided
fractionated studies were also !erformed and it was observed that v24w-inerol and
v21w-inerol effectively inhib-ited the rowth of these oral !athoens at a minimum
inhibitory concentration rane of DM94 ml
-2
and a minimum bactericidal
concentration rane of 7M14 ml
r2
*#ark et al., 144C-.
D. GINGER PREVENTS EPIGASTRIC DISCOMFORT AND
DYSPEPSIA
)n the various systems of medicine, iner has been re!orted to !ossess carminative
effect, decrease the !ressure on lower eso!haus, lessen intestinal cram!in, !revent
dys!e!sia, and reduce flatulence and bloatin *.li et al., 144CE 6hrubasik et al.,
1440-. (ecently, Lohsiriwat et al. *1424- studied the effect of iner on the eso!haus
and lower eso!h-aeal s!hincter function in healthy male volunteers. The authors
observed that the con-sum!tion of iner *2 of dried !owder- did not affect the
lower eso!haeal s!hincter !ressure at rest or the eso!haeal contractile am!litude
and duration when swallowin. 'owever, iner administration enhanced relaxation
of the lower eso!haeal s!hincter and decreased the eso!haeal contraction velocity,
and this !ossibly was res!onsible for the antiflatulent effects *Lohsiriwat et al., 1424-.
E. GINGER IS EFFECTIVE AGAINST VARIO*S GASTRIC
*LCEROGENS
Hastrointestinal ulcers, characteri/ed by inflamed lesions or excavations of
astrointestinal mucosa, are one of the most common disorders affectin humans and
are associated with severe morbidity and mortality. The exact cause of !e!tic ulcer is
not known, but factors such as smoke, anti-inflammatory drus, alcohol, stress, fatty
foods, and "elicobacter pylori infections are known to initiate and aravate the
!roblem *5e-Sousa et al., 144C-. The conventionally used drus are associated with
serious side effects like arrhythmias, im!o-tence, ynecomastia, arthralia,
hy!erastrinemia, and hemo!oietic chanes, and may be ineffective at !reventin the
incidences of rela!ses. 'ence, the search for new and ideal antiulcer dru continues.
$ulti!le studies have clearly shownthatwhole iner and some of its
!hytochemicals are !otent astro!rotective aents in various standard models of
astric ulcers. Hiner showed !rotective effects aainst '6l,ethanol, C4R ethanol, 4.D
$ '6l, 4.1 $ &aO' and 10R &a6l, indomethacin, as!irin, reser!ine, hy!othermic
restraint induced, swim stress-,ethanol stress-induced ulcers and !ylorus liation-
induced astric ulcerations in rats *.l-=ahya et al., 23C3E &anGundaiah et al., 1422E
=amahara et al., 23CC-. The decoction *boilin the iner in water till the volume of
water reduces to half or less- !re!ared from the dry iner was better than the roasted
iner in !reventin astric ulcers in rats, clearly indicatin that roastin deactivates
the active com!ounds *;u et al., 2334-. (ecently, the oil of iner was also re!orted
to afford !rotection aainst the as!irin and !ylorus liation-induced ulcerations in rats
*>hushtar et al., 1443-. The !hytochemical /iniberene was effective in '6l,ethanol
*=amahara et al., 23CC-, inerol aainst '6l *'orie et al., 1447- and'6l,ethanol
*=oshikawa et al., 2337-, D-inesulfonic, D-inerol, and D-shoaol aainst
'6l,ethanol *=oshikawa et al., 2337- induced astric ulceroenesis in rats.
Studies by $ahady et al. *1449- have shown that the methanol extract of the
iner, the extract fractions, and the isolated constituents, D-,C-, and 24-inerol and
D-shooal inhibited the rowth of the different strains of "# pylori in vitro with a
minimum inhibitory concentration in the rane of D.10M04 ml
r2
*$ahady et al.,
1449-. ?ractions contain-in inerols were the most active and inhibited the rowth
of all '. !ylori strains *minimum inhibitory concentration rane of 4.<CM21.0 ml
-
2
-. There was sinificant activity even aainst the more virulent 6a.Bstrains
*$ahady et al., 1449-. The iner-free !henolic and iner-hydrolysed !henolic
fractions also !ossess inhibitory ef-fects on the rowth of "# pylori, which scavene
free radicals, !ossess reducin !ower abilities, !rotect 5&., and inhibit li!id
!eroxidation *SiddaraGu and 5harmesh, 144<-.
G. GINGER IS AN EFFECTIVE ANTIEMETIC AGENT
S&ausea% in Hreek means sea sickness and vomitin *emesis-. &ausea and
vomitin are the two common distressin sym!toms that indicate deranement in
health or adverse effect of drus. Hiner is efficacious in !reventin nausea and
vomitin in early !renancy *Ensiyeh and Sakineh, 1443E ?ischer-(asmussen et al.,
2332E Smith et al., 1447E Iutyavanich et al., 1442E ;illetts et al., 1449- without
increasin the !renancy-related com!lications, the !renancy outcome, and
conenital abnormalities *#ortnoi et al., 1449ESmith et al., 1447-. Hiner is also
re!orted to !revent !osto!erative nausea and vomit-in *.!ariman et al., 144DE
#onroG!aw and 6hiamchanya, 1449E Tavlan et al., 144D-.
;ith reard to motion-related sickness, studies in animals have shown that iner
Guice !roduces antimotion sickness action and that this action was !ossibly due to its
effect on the central and !eri!heral cholineric, histamineric, and serotoneric
!athways *Wian and Liu, 2331-. 'owever, studies in human have shown diverent
results *Lien et al., 1449E $owrey, 23C1E Stewart et al., 2332-. Hiner is observed to
!revent motion sickness in human volunteers *Lien et al., 1449E $owrey, 23C1-.
'owever, contradictory to these observations, Stewart et al. *2332- have observed that
!retreatment with iner was ineffective in !reventin motion sickness *Stewart et al.,
2332-. 'owever, iner has been re!orted to reduce the sea sickness in human
volunteers and naval cadets *Hrrntved et al., 23CCE Hroslashntvedab et al., 23CC-.
Ex!erimental studies have shown that iner was effective in !reventin
chemothera!y-induced nausea and radiation-induced nausea and emesis. Oral feedin
of iner extracts !revented cis!latin-induced emesis in healthy monrel dos. The
ac-etone extract was more valuable than ethanolic extract, but was less effective than
ran-isetron *Sharma et al., 233<-. The acetone extract, 04R ethanolic extract, and
fresh iner Guice were also effective in !reventin cis!latin-induced delayed astric
em!tyin in rats *Sharma and Hu!ta, 233C-. The reversal !roduced by the iner Guice
was better than that of the 0-'T9 rece!tor antaonist ondansetron, while that of the
acetone extract was sim-ilar to it *Sharma and Hu!ta, 233C-.
Hinerol, the active !rinci!le of raw iner, was also re!orted to !ossess
antiemetic !ro!erty aainst cis!latin and the effect was similar to that of ondansetron,
used as a !ositive control *Wian et al., 1424-. $echanistic studies showed that
inerol caused a dose-de!endent su!!ression in the levels of substance # and &>2
rece!tors in the !ostrema and ileum areas and suest its action to be similar to that of
a!re!itant *Wian et al., 1424-.
Sharma et al. *1440- have also observed that intra!eritoneal administration of the
hydroalcoholic extract of iner 2 h before ex!osure to 1 Hy of irradiation was
effective in blockin the saccharin avoidance res!onse for five !osttreatment
observational days. . concentration and time-de!endent !rotective effect was
observed and a dose of 144 m k
-2
body weiht was the most effective in males
*Sharma et al., 1440-, while a dose of 104 m k
-2
was effective in female rats,
suestin the existence of sex dichot-omy in the effect *'aksar et al., 144D-.
.lthouh iner was effective in !reventin chemothera!y-induced nausea in ani-
mals, similar observations were not observed in all the studies !erformed in humans.
The antiemetic effects of iner aainst chemothera!y-induced nausea and vomitin
have been mixed and contradictory. )n a double-blind crossover study,
$anusirivithaya et al. *1447- have observed that the addition of iner to standard
antiemetic reimen of ynecoloical oncoloy !atients receivin cis!latin offered no
advantae in reducin chemothera!y-induced nausea and vomitin in acute !hase of
cis!latin-induced emesis, while in the delayed !hase, its effect was com!arable to that
of metoclo!ramide *$anusirivithaya et al., 1447-.
Hiner was also effective in reducin chemothera!y-induced nausea and vomitin
stimulated by low-dose cyclo!hos!hamide in combination with other emetoenic
anti-cancer drus *Sontakke et al., 1449-. These results indicate that the antiemetic
effect of iner was e"ual to that of metoclo!ramide, but inferior to ondansetron
*Sontakke et al., 1449-. Studies have also shown that combinin hih-!rotein meals
with iner reduces the chemothera!y-induced delayed nausea and the use of standard
antiemetic medica-tions in cancer !atients *Levine et al., 144D-.
6ontradictin these observations, Nick et al. *1443- have observed that iner is of
no benefit in reducin the !revalence or severity of acute or delayed chemothera!y in-
duced nausea and vomitin when combined with 0-'T9 rece!tor antaonists and,or
a!re!itant. ?urther, the authors also observed that the !artici!ants who took both
iner and a!re!itant had more severe acute nausea than !artici!ants on a!re!itant,
only su-estin a !ossible antaonism *Nick et al., 1443-.
$echanistic studies suest that vDw-, vCw-, and v24w-inerol and vDw-shoaol exert
their antiemetic effect at least in !art by actin on the 0-'T9 rece!tor ion-channel
com-!lex, !robably by bindin to a modulatory site distinct from the serotonin-
bindin site. This may include indirect effects via rece!tors in the sinal cascade
behind the 0-'T9 rece!tor channel com!lex such as substance # rece!tors and
muscarinic rece!tors *.bdel-./i/ et al., 144D-. The antiemetic activity of iner has
been shown to be mediated throuh its antaonist, &>) antaonist, antihistaminic and
!rokinetic effects, and was devoid of any adverse effects.
K. GINGER ALTERS GASTROINTESTINAL MOTILITY
#reclinical studies have shown that iner extract enhanced the intestinal travel of
char-coal meal in mice *Hhayur and Hilani, 144D-. In vitro studies have also shown
that iner *4.42M2444 m ml
-2
- inhibited both !reGunctional and !ostGunctional
inhibitory effects on ileal contractility and that the !reGunctional inhibitory effect of
iner on enteric ex-citatory transmission could involve a ca!sa/e!ine-sensible site
*Borrelli et al., 1447-.
The !hytochemicals vDw-shoaol, vDw-, vCw-, and v24w-inerol were also observed to be
effective in enhancin the trans!ort of a charcoal meal and their effects were similar
to or slihtly weaker than those of metoclo!ramide and don!eridone *=amahara et al.,
2334-. Ninerone, the !unent !hytochemical, also inhibited the s!ontaneous
contractile movements in the isolated colonic sements, and the effects were
concentration de!en-dent. This inhibitory effect was not affected by !retreatment with
ca!sa/e!ine, an antaonist of transient rece!tor !otential vanilloid 2, or tetrodotoxin,
a blocker of volt-ae- de!endent sodium channels directly on neurons, suestin that
/inerone acts on the smooth muscles *)wami et al., 1424-.
Studies have also shown that /inerone assuaed colonic motility in rats without
affectin blood !ressure and heart rate and that this effect was reversible and
re!roducible *)wami et al., 1424-. 6linical studies have also shown that administerin
iner accelerates astric em!tyin and stimulates antral contractions in healthy
volunteers *;u et al., 144C-.
L. GINGER IS EFFECTIVE ON DIGESTIVE ENCYMES
)n the various traditional systems of medicine, consumin tea brewed from fresh
iner after lunch or dinner is rearded to enhance diestion, and scientific studies
have substan-tiated this hy!othesis. #reclinical studies have shown that feedin rats
with iner *04 mR- incor!orated diet for C weeks enhanced the intestinal li!ase,
sucrase, and malt-ase activities *#latel and Srinivasan, 233D-.
Hiner increased the activity of !ancreatic li!ase and amylase in vitro
*(amakrishna (ao et al., 1449- and this extended to studies in animals *#latel and
Srinivasan, 1444-. ?eedin iner *04 mR- to rats is also observed to stimulate the
synthesis of try!sin and chymotry!sin *#latel and Srinivasan, 1444-. (ecent re!orts
also suest that iner also stimulated the activity of the brush-border membrane
en/ymes *lycyl-lycine di-!e!tidase, leucine amino !e!tidase, and amma-lutamyl
trans!e!tidase- in the GeGunal mucosa *#rakash and Srinivasan, 1424a-.
O. GINGER INCREASES ANTIO=IDANT ENCYMES IN THE
GIT
Excess eneration of the reactive oxyen s!ecies *(OS- !roduced durin
!athoenesis and in res!onse to various toxicants induces oxidative stress. The
antioxidant en/ymes !rotect the cell aainst oxidative stress and mutaenic effects of
the (OS, and !reclinical studies have shown that iner *4.0R- increases su!eroxide
dismutase, catalase *6.T-, lutathione reductase *H(-, and lutathione S-transferase
in both astric and intestinal mucosa of rats *#rakash and Srinivasan, 1424a-.
Se!arate studies have also shown that ad-ministerin iner *4.0R- in diet alleviates
the diminished activities of antioxidant en-/ymes in astric and intestinal mucosa in
rats treated with ethanol *#rakash and Srinivasan, 1424b- and in rats with diabetes
*>hadem-.nsari et al., 144C-.
>R.GINGER ALTERS THE BR*SH%BORDER MEMBRANE
FL*IDITY AND INCREASES THE S*RFACE AREA OF
THE BR*SH%BORDER MEMBRANE
The absor!tion alon the D-m lon intestine de!ends on the absor!tive surface
area in !articular. Hiner has been shown to favorably alter membrane fluidity of the
brush border. ?eedin iner *4.40R- to ;istar rats for C weeks in diets is re!orted to
increase the fluidity of the brush-border membrane in the GeGunal and ileal reions by
decreasin the cholesterolM!hos!holi!id ratio. Scannin electronic microsco!y of the
intestinal villi showed an increase in the microvilli lenth and !erimeter, suestin
that the beneficial effects may !artly be due to the increase in the absor!tive surface
of the small intestine *#rakash and Srinivasan, 1424a-.
>>.EFFECT OF GINGER ON THE INTESTINAL PATHOGENS
Enteric infections by viruses, bacteria, !roto/oans, nematodes, and helminthes
affect intestinal absor!tion, nutrition, and develo!ment. #reclinical studies have
shown that iner !ossesses anthelmintic effects aainst human ,scaris lumbricoldes
*>alesaraG, 23<7-, .nisakis larvae *Hoto et al. 2334-, and "aemonchus contortusa, a
!athoenic nem-atode of ruminants *)"bal et al., 1442-. 'owever, it was ineffective in
!reventin the entry of rotavirus into the $.-247 cells and the tro!ho/oites of
-iardia lamblia in vitro *5aswani et al., 1424-.
$ulti!le studies have shown that iner and some of its !hytochemical
com!onents !ossess antibacterial effects on both sensitive and dru-resistant bacteria
*>han et al., 1424E $ascolo et al., 23C3, Thonson et al., 1440-. The essential oil of
iner is observed to be effective on C# CeCuni$ %# coli 19:G@"G$ L# monocytogenes$
and !almonella enterica *?riedman et al., 1441-. 'owever, contradictory re!orts also
exist and recent re!orts by 5aswani et al. *1424- suest iner to be ineffective as an
antibacterial aent on some strains of the entero!athoenic %# coli, A# cholerae, and !#
flexneri.
>?.GINGER IS EFFECTIVE IN INFLAMMATORY BOWEL
DISEASES
)nflammatory bowel diseases, mainly the ulcerative 6 colitis and 6rohn%s disease,
are im-!ortant immune-mediated diseases of the H)T. Ex!erimental studies have
shown that !retreatment with iner extract ameliorated the acetic-acid-induced
edematous inflam-mation in the colon. The histo!atholoical studies confirmed that
iner attenuated the extent and severity of edema, des"uamation, necrosis, and
inflammatory cell infiltration in the mucosa. The levels and activity of colonic
myelo!eroxidase, li!id !eroxides, !ro-tein carbonyl content, T&?-c, rand #HE1 were
also decreased. .dministerin iner restored the levels of H(, 6.T, and su!eroxide
dismutase. The !rotective effect of the hihest doses of iner was com!arable to that
of the standard sulfasala/ine *El-.bhar et al., 144C-
>B.GINGER PREVENTS DIARRHEA
Hlobally, diarrhea caused by intestinal !athoens is a maGor health concern and a
maGor cause for infant mortality in the develo!in countries. $aGority of diarrheal
incidences are due to the enteric bacteria, es!ecially the entero!athoenic %# coli$
Aibrio cholera$ campylo-bacter$ !almonella typhi, and !higella flexneri. #reclinical
studies have shown that iner blocked the bindin of heat-labile enterotoxin to cell-
surface rece!tor H $2, which resulted in the inhibition of fluid accumulation in the
closed ileal loo!s of mice *6hen et al., 144<-.
Bioloical-activity-uided studies indicated that /inerone was !ossibly
res!onsible for the iner%s antidiarrheal efficacy *6hen et al., 144<-. Hiner reduces
coloni/ation of the e!ithelial cells, 'E!-1 by entero!athoenic %# coli, enteroinvasive
%# coli, and !higella flexneri *5aswani et al., 1424-. In vitro studies also showed that
iner did not arrest the rowth of A# cholerae, but inhibited the !roduction of cholera
toxin thereby im!lyin its affect to be selectively on the metabolic !athway,s
res!onsible for the toxin !roduc-tion *5aswani et al., 1424-.
Ex!erimental studies have also shown that the administration of iner
sinificantly inhibited the 0-'T induced diarrhea *'uan et al., 2334-. The active
!rinci!les vDw-shooal, vDw-dehydroinerdione, and vCw- and v24w-inerol have been
re!orted to !ossess anticathartic action *'uan et al., 2334-. In vitro studies with the
uinea !i ileum, rat stomach fundus, and rabbit aortic stri!s have shown that
alanolactone, a diter-!enoid isolated from iner-inhibited contractile, res!onds to 0-
'T. The inhibitory effect of alanolactone on the 0-'T res!onse in the stomach
fundus and aortic stri!s was less than that in the ileum and that the effect was related
to antaonism of 0-'T9 rece!tors *'uan et al., 2334-.
>D.CONCL*SION
Hiner has been used since anti"uity to treat various astrointestinal ailments and
scien-tific studies, with both !reclinical and human studies substantiatin most of
these ethno-medicinal observations. 5ue to its abundance, low cost, and safety in
consum!tion, iner remains a s!ecies with tremendous !otential and countless
!ossibilities for further investiation. The various !harmacoloical activities of iner
a!!ear to be due to the !resence of various !hytochemicals. ;ith reard to certain
!harmacoloical effects *anti-bacterial and antiemetic effects aainst motion sickness
and chemothera!y-induced nau-sea in humans-, contradictory results are seen and this
may be !ossibly due to the variation in the !hytochemicals in the iner used. Studies
should also be aimed at understandin which bioactive com!ound is res!onsible for
the beneficial effect, as this will hel! in understandin the !ossible mechanism of
action res!onsible for the observed !harmaco-loical effects and a!!reciation of
iner on a reular basis and durin ailments.
ACKNOWLEDGMENTS
The authors are rateful to (ev. ?r. #atrick (odrius *5irector-, (ev. ?r. 5enis
5%Sa *.dministrator-, and 5r :aya #rakash .lva *5ean- of ?ather $uller $edical
6ollee for their unstinted su!!ort.
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Based 6om!lementary and .lternative $edicine 1422, 1737C<.
#ark, $., Bae, :., Lee, 5.S., 144C. .ntibacterial activity of v24w-inerol and v21w-
inerol isolated from iner rhi/ome aainst !eriodontal bacteria. #hytothera!y
(esearch 11, 277DM2773. #latel, >., Srinivasan, >., 233D. )nfluence of dietary s!ices
or their active !rinci!les on diestive en/ymes of small intestinal mucosa in rats.
)nternational :ournal of ?ood Sciences and &utrition 7<, 00M03.
#latel, >., Srinivasan, >., 1444. )nfluence of dietary s!ices and their active
!rinci!les on !ancreatic diestive en/ymes in albino rats. &ahrun 77, 71M7D.
#onroG!aw, 5., 6hiamchanya, 6., 1449. The efficacy of iner in !revention of
!ost-o!erative nausea and vomitin after out!atient ynecoloical la!arosco!y.
:ournal of the $edical .ssociation of Thailand CD, 177M104.
#ortnoi, H., 6hn, Lu.-.., >arimi-Tabesh, L., >oren, H., Tan, $.#., Einarson,
(.&., 1449. #ros!ective com!arative study of the safety and effectiveness of iner
for the treatment of nausea and vomitin in !renancy. .merican :ournal of
Obstetrics and Hynecoloy 2C3, 29<7M29<<.
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ultrastructure and fluidity of the intestinal brush border in rats. British :ournal of
&utrition 247, 92M93.
#rakash, 8.&., Srinivasan, >., 1424b. Hastrointestinal !rotective effect of dietary
s!ices durin ethanol-induced oxidant stress in ex!erimental rats. .!!lied
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of iner. Nhonuo Nhon Oi =i :ie 'e Na Nhi 21 *30MC-, <4.
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Effect of inerol on sub-stance # and &>2 rece!tor ex!ression in a vomitin model
of mink. 6hinese $edical :ournal 219, 7<CM7C7.
(amakrishna (ao, (., #latel, >., Srinivasan, >., 1449. In vitro influence of s!ices
and s!ice-active !rinci!les on diestive en/ymes of rat !ancreas and small intestine.
&ahrun 7<, 74CM721.
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73M00.
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modulates amma radiation-induced conditioned taste aversion. #harmacoloy,
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and "elicobacter pylori rowth by !henolic antioxidants of ?ingiber officinale.
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randomi/ed controlled trial of iner to treat nausea and vomitin in !renancy.
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Thonson, 6., 5avidson, #.$., $ahakarnchanakul, ;., Iibulsresth, #., 1440.
.ntimicrobial effect of Thai s!ices aainst Listeria monocytogenes and !almonella
typhimurium (T95H. :ournal of ?ood #rotection DC, 1407M140C.
Iutyavanich, T., >raisarin, T., (uansri, (., 1442. Hiner for nausea and
vomitin in !renancy+ random-i/ed, double-masked, !lacebo-controlled trial.
Obstetrics and Hynecoloy 3<, 0<<M0C1.
;illetts, >.E., Ekanaki, .., Eden, :..., 1449. Effect of a iner extract on
!renancy-induced nausea+ a randomised controlled trial. The .ustralian and &ew
Nealand :ournal of Obstetrics and Hynaecoloy 79, 293M277.
;u, >.L., (ayner, 6.>., 6huah, S.>., et al., 144C. Effects of iner on astric
em!tyin and motility in healthy humans. Euro!ean :ournal of Hastroenteroloy and
'e!atoloy 14, 79DM774.
;u, '., =e, 5., Bai, =., Nhao, =., 2334. Effect of dry iner and roasted iner
on ex!erimental astric ulcers in rats. Nhonuo Nhon =ao Na Nhi 20, 1<CM1C4.
=amahara, :., 'uan, W.(., Li, =.'., Ou, L., ?uGimura, '., 2334. Hastrointestinal
motility enhancin effect of iner and its active constituents. 6hemical and
#harmaceutical Bulletin 9C, 794M792.
=amahara, :., $ochi/uki, $., (on, '.W., $atsuda, '., ?uGimura, '., 23CC. The
anti-ulcer effect in rats of iner constituents. :ournal of Ethno!harmacoloy 19,
133M947.
=oshikawa, $., =amauchi, S., >unimi, >., et al., 2337. Stomachic !rinci!les in
iner. ))). .n anti-ulcer !rinci!le, D-inesulfonic acid, and three
monoacyldialactosyllycerols, inerlycoli!ids ., B, and 6, from Niniberis
(hi/oma oriinatin in Taiwan. 6hemical and #harmaceutical Bulletin 71*D-, 211DM
2194
Nick, S.$., (uffin, $.T., Lee, :., et al., 1443. #hase )) trial of enca!sulated iner
as a treatment for chemothera!y-induced nausea and vomitin. Su!!ortive 6are in
6ancer 2<, 0D9M0<1.
CHAPTER >?
Th( R!( !' Mi&r!bi!ta a$/ Pr!bi!ti&s
!$ th( Gastr!i$t(sti$a H(ath)
Pr(+($ti!$ !' Path!"($ I$'(&ti!$s
M.C. C!a/!
*,
I 7. Gr-(8,!0ia,

I S. Sa.i$($

*
)nstitute of .rochemistry and ?ood Science *).T.-6S)6-, Ialencia, S!ain

8niversity of Turku, Turku, ?inland


> GASTROINTESTINAL TRACT AND G*T MICROBIOTA
The human astrointestinal tract is one of the main body surfaces in constant
contact with exoenous aents such as viruses, bacteria, and allerens. )t has a
!rimary role in the host defense aainst external aressions by means of the
intestinal mucosa, the immune sys-tem, and the interactions with the microbiota. The
intestinal tract is covered by a mucous membrane that forms a barrier between the
external and internal environment of the human body. $ucin lyco!roteins *mucins-
form the maGor macromolecular constitu-ents of e!ithelial mucus and have lon been
im!licated in health and disease. The inter-actions between mucus and bacteria
!resent in the astrointestinal tract are !ivotal for the ut health.
The establishment of ut microbiota starts at delivery. The microbiota of a
newborn develo!s ra!idly and is initially stronly de!endent on the maternal
microbiota, the mode of delivery, environment at birth, and hyiene conditions.
Subse"uently, it is influenced by feedin !ractices and the livin environment of the
infant. The human intestinal microbiota is a!!roximately com!osed of 24
29
M24
27
microoranisms whose collective enome *Smicrobiome%- contains at least 244 times
as many enes as human enome *Hill et al., 144D-. The human intestinal microbiota
!lays an im!ortant and com!lex role in the maintenance of host health and is involved
in diestion of dietary inredients, re-ulation of enery balance, immune system
!rimin and modulation, and !rotection from !athoens. The ut microbiota has
beun to receive rowin attention in recent years, since a remarkable relationshi!
between astrointestinal disorders and nonintestinal diseases has been identified. .n
individual balance of the microbiota !lays a critical role in maintenance of the health
status of the host. The !resence of bacterial !athoens may alter the intestinal
bacterial homeostasis *microbiota com!osition and activity-, leadin to either
increased risk of disease or !resence of s!ecific diseases. The maintenance of micro-
biota e"uilibrium is im!ortant to !reserve and to !romote health. $icrobiota may be
divided into commensals, symbiotic microoranisms, and !athoenic
microoranismsE althouh other transient bacteria such as food-associated bacteria
and !robiotics may be a !art of the microbiota. The astrointestinal develo!ment and
function de!end on the !resence of com!lex microbiota that !erforms numerous
metabolic, rowth-!romotin, and !rotective roles.
? G*T MICROBIOTA AND HEALTH
)m!ortant advances have recently been made toward establishin the identity of
s!ecific microbes and microbial rou!s or microbial com!onents contributin to
collective microbiota and its im!act on host !hysioloy and health. 6oncurrently,
s!ecific host fac-tors have been identified, which are involved in the develo!ment and
maturation functions.
. balance amon microbial rou!s !resent in human ut is re"uired for
maintainin health and well-bein of the host. ;hen this balance is disturbed, the
hostMmicrobe in-teractions can !roress toward a disease state. .berrancies in the
microbiota have been associated with a hiher risk of allery, ut inflammatory
conditions, and even obesity *6ollado et al., 1443-. The im!ortance of a healthy
microbial balance is a!!reciated usu-ally only after disturbances have occurred, for
exam!le, followin antibiotic thera!y or travelers% diarrhea. .ntimicrobial treatment
alters the ecoloical balance of the healthy microbiota, which can result in diarrhea,
antibiotic-associated diarrhea *..5-, and other diseases. Several studies have shown
the adverse effects of different antibiotics on the host ut microbiota in human
subGects *Sekirov et al., 1424-.
)n addition, microbial e"uilibrium can be altered by the !resence of certain
bacteria such as disease-causin bacteria, !athoens, yeasts, funi, and !arasites. )t
has been dem-onstrated that the eradication of "elicobacter pylori from the stomach,
which causes most ulcers and many ty!es of chronic stomach inflammation, re"uires
a combination of ther-a!ies. .ntibiotics *e.., amoxicillin, clarithromycin, or
nitroimida/oles- are used toether with acid su!!ression drus *!roton !um!
inhibitors or '
1
-rece!tor antaonists-, in tri-!le or "uadru!le combinations. Treatment
aimed at "# pylori eradication has been re!orted to ive rise to ecoloical disturbances
with su!!ression of healthy microbiota and emerence of antibiotic-resistant
microbes. Other im!ortant com!lication of anti-biotic treatment can be the
establishment of s!ecific !athoens, such as Clostridium difficile, and their
overrowth resultin in !seudomembranous colitis.
)nflammatory bowel disease *)B5- is a chronic and recurrent inflammation
enerally affectin the colon or the small intestine and includes ulcerative colitis,
!ouchitis, and 6rohn%s disease. The etioloy of )B5 remains unclear, but enetic
!redis!osition and alterations in microbiota are involved. There is evidence that the
immune system reacts abnormally toward the endoenous microbiota. The microbiota
in subGects with )B5 have been shown to be less stable com!ared to that of healthy
adults and have hiher levels of !otentially !athoenic bacteria as acteroides and
%scherichia coli attached to e!ithelial cells than healthy !eo!le *Iander!loe et al.,
1424-. )n addition, an altered ut microbiota has also been re!orted in !atients with
irritable bowel syndrome *)BSE Salonen et al., 1424-. The )BS is characteri/ed by an
abdominal !ain, bloatin, and chane in bowel habit, with an absence of any overt
mucosal abnormality and flatulence. On the other hand, the association between
microbiota com!osition and risk of colon cancer has been a subGect of discussion for
many years and some re!orts suested that fecal and colonic mucosa-associated
microbiota may be altered in these !atients *Hueimonde et al., 144<-.
.ltered microbial e"uilibrium in other human sites is often associated with
different diseases such as the !eriodontal disease and dental caries *5ewhirst et al.,
1424-, urinary tract infections and bacterial vainosis infections *>irGavainen et al.,
1443-, res!iratory infections, and also skin infections *Hrice and Sere, 1422-.
?urthermore, microbiota de-viations have been associated with enhanced risk of
s!ecific diseases such as alleries, ato!ic diseases, diabetes, obesity, and even autism
*Sekirov et al., 1424-. $ental !rob-lems, includin schi/o!hrenia, anxiety, stress, or
de!ression, are fre"uently associated with other disorders in which microbiota
disturbances may !lay a role, such as ulcerative colitis, celiac disease, metabolic
syndrome, or )BS. )nterestinly, the microbiota has been associated with the
develo!ment of the !hysioloical system of stress res!onse and even brain
develo!ment *'eiGt/ et al., 1422E &eufeld and ?oster, 1443-.
B G*T MICROBIOTA AND THERAPE*TIC ACTION OF
PROBIOTICS
There is a rowin interest in beneficial microbes with s!ecific functions in the
human ut, which can increase health and !revent and treat diseases, and which can
be used in foods or su!!lements *(eid et al., 1422-. The !rotective role of s!ecific
bacteria as !ro-biotics aainst astrointestinal !athoens and diseases and the
underlyin mechanisms have been extensively studied. The develo!ment of adGuvant
or alternative thera!ies based on bacterial re!lacement is considered im!ortant
because of the ra!id emerence of antibiotic-resistant !athoenic strains and the
adverse conse"uences of antibiotic ther-a!ies on the !rotective microbiota.
. !robiotic has been defined as a Slive microoranism which when administered
in ade"uate amounts confers a health benefit on the host% *?.O,;'O, 1441-.
#robiotics are usually considered as transient bacteria !assin throuh human ut
althouh !robio-tics are usually also commensal bacteria, normally inhabitin the
human astrointestinal tract. The most numerous !robiotic bacteria are s!ecies
belonin to Lactobacillus$ %ntero-coccus$ ifidobacterium, and also some
Propionibacterium strains, amon others, and they have been introduced as !robiotics
in food !roducts because of their health-!romotin effects. The !rotective role of
!robiotic bacteria aainst astrointestinal !athoens and the underlyin mechanisms
have received s!ecial attention. The demonstrated beneficial health effects of
!robiotic consum!tion include reulation of microbiota and stimulation and
develo!ment of the intestinal barrier effect, includin the immune system, beneficial
im!act on the bioavailability of nutrientsE reduction or alleviation of sym!toms of
lactose intoleranceE and reduction in the risk of s!ecific microbiota-associated
diseases such as acute astroenteritis caused by viruses or bacteria, es!ecially in
infants and children.
D. PROBIOTICS AND GASTROINTESTINAL HEALTH
The clinical interest in the a!!lication of !robiotics has been in the !revention and
treat-ment of astrointestinal infections and also related to the manaement of
astrointestinal infections caused by !athoens such as "# pylori$ C# difficile$ %# coli$
and !almonella amon others. Some beneficial intestinal effects of certain !robiotic
strains can be considered as well established, which has led to the !ublication of
recommendations for their use in clinical !ractice *S/aGewska, 1424-. Therefore,
modulation of an unbalanced microbiota forms the rationale of !robiotic thera!y.
5urin the !ast few decades, a lare number of studies have been conducted to assess
!robiotics, usin different formula and with s!ecific !ur!oses of !reventin or
treatin diseases. .lternative thera!ies to re!lace antibiotics based on enhancin the
barrier or re!lacement of !athoens are considered im!ortant because of the ra!id
emerence of antibiotic-resistant !athoenic strains and the adverse conse"uences of
antibiotic thera!y on the !rotective healthy intestinal microbiota. S!e-cific !robiotics
have been demonstrated to exert !rotective effects aainst different dis-eases caused
by !athoenic bacteria and also they are able to modulate the microbial disbalance
caused by antibiotic treatments and reduce the side effects from antibiotics such as
as, cram!in, or diarrhea. Similarly, !robiotics can be used to ease sym!toms of
lactose intolerance. #athoens may alter the intestinal !ermeability, while !robiotic
strains could !revent damae and re!air and restore the mucosal interity, increase the
e!ithelial resistance aainst !athoens, and induce cell !roliferation.
5iarrhea is an obvious taret for !robiotics and their role in !revention and
treatment of infant%s diarrhea is the best studied !robiotic effect. )n fact, !robiotics are
considered amon the alternatives for manaement of acute astroenteritis in children.
)n addition, s!ecific !robiotic strains have been shown to exert a !rotective effect
aainst acute diarrhea, rotavirus diarrhea, and ..5 *.llen et al., 1424E #reidis et al.,
1422-. (ecently, the efficacy of a hih-!otency !robiotic !re!aration on !revention
and treatment of radiation-induced diarrhea in cancer !atients *Hiralt et al., 144C-,
beneficial effects on "# pylori infection *Lionetti et al., 1424-, and alleviation of
sym!toms of astrointestinal diseases such as )BS *#arkes et al., 1424- have been
described. The effect of different !ro-iotics on )B5 has been evaluated with variable
results. The reatest evidence for efficacy of !robiotics on )B5 has been shown in the
!revention of !ouchitis *Huandalini, 1424-. 5ifferent human intervention trials have
shown effectiveness of the !robiotic !re!aration ISL}9 in maintainin remission of
!ouchitis. #robiotic Lactobacillus rhamnosus HH was found to !rolon the rela!se-
free time, althouh no effect on rela!se rate was observed and some !ositive results
have also been re!orted for !accharomyces boulardii. )n contrast, trials evaluatin the
a!!lication of different !robiotics on 6rohn%s disease have not been successful and
only some !reliminary studies re!orted modest beneficial effects *)annitti and
#almieri, 1424-. Some reviews and meta-analysis studies focused on the use of !ro-
biotics in )B5 concluded that further research is still needed. 5ifferent studies in
adults showed that ifidobacterium infantis$ L# rhamnosus HH, and also the mixture
of different !robiotics such as L# rhamnosus HH, L# rhamnosus L6<40$ # breve
Bb33, and Propionibac-rium freudenreichii :S can be effective in alleviatin
sym!toms of )BS *#arkes et al., 1424-. ?urthermore, !robiotics have been suested
to relieve consti!ation, and s!ecific strains such as # lactis 5&-2<9 424, L# casei
Shirota, or L# Cohnsonii La2 have been re!orted to be beneficial. (ecent studies
suest that certain !robiotics can reduce both the severity and incidence of
necroti/in enterocolitis in !reterm neonates while no effect has been ob-served with
other !robiotic strains, underlinin the hih strain s!ecificity of !robiotics
*5esh!ande et al., 1424-.
The hyiene hy!othesis su!!orts the ra!id increase of allery related to lower
microbe ex!osure at early life and subse"uent lower number of infections durin
infancy. (ecent studies re!orted microbiota differences between alleric and healthy
infants in hih-income countries, and these chanes in microbiota may be
counterbalanced by !robiotic bacteria. #ositive clinical effects of !robiotics in the
!revention and treatment of ato!ic diseases have been re!orted *#an et al., 1424E Tan
et al., 1424-. On the contrary, a recent meta-analysis *Boyle et al., 1443-, includin 21
randomi/ed-controlled trials *<C2 !artic-i!ants-, concluded that !robiotics are not a
clinically effective treatment for ec/ema, althouh when iven durin early infancy
for the !revention rather than the treatment of ec/ema the results for some !robiotic
strains are !romisin. .nother recent study *Iliaoftis et al., 144C- evaluated the
clinical evidence for the use of !robiotics as a ther-a!eutic modality for alleric
rhinitis and asthma by reducin sym!tom severity and med-ication use, althouh
further ood-"uality studies are needed.
5isturbances on the microbiota have been identified in other intestinal disorders,
includin diverticulitis and extraintestinal conditions such as uroenital, vainal, and
skininfections and in different diseases such as obesity, diabetes, autism, mental
health, and so far !robiotics have not been tested in some of these settins, and these
studies indicate !otential tarets for the future develo!ment of !robiotic !roducts.
)t is ex!ected that combinations of different !robiotics strains may be more
effective than sinle strains of !robiotics de!endin on the taret. )t is clear that
combinations mayalso be needed for counteractin com!lex microbiota aberrancies in
adults and infants. (ecently, it has been demonstrated that a combination of !robiotic
strains may com!le-ment or im!rove health benefits iven by individual strains *Lyra
et al., 1424-. #revious studies on the combinations of well-known !robiotics strains,
such as L# rhamnosus HH and # lactis Bb21, and their effects on the !athoen
adhesion to intestinal mucus system have been re!orted. #robiotic strains and their
combinations were able to inhibit sinificantly the adhesion of acteroides vulgatus$
C# histolyticum$ C# difficile, and !taphylococcus aureus *6ollado et al., 144<-. These
results em!hasi/ed the !otential a!!li-cation of !robiotic combinations in the
inhibition of !athoen adhesion to intestinal mucus but further studies are needed to
clarify the mechanisms involved in !athoen in-hibition by combinations of
!robiotics. #robiotic combinations that inhibit and dis!lace !athoens may be ood
candidates in case of s!ecific microbiota aberrancies related to disease risk reduction.
'owever, it is im!ortant to take into account the hih s!ecificity of these !rocesses
bein necessary to characteri/e the !ro!erties of the strains in order to select the best
combination for a s!ecific a!!lication. The chane of one strain in a com-bination
maymodify all the !ro!erties aainst !athoen adhesion reardin inhibition, dis-
!lacement, and com!etition mechanisms. These results su!!ort the idea that the use of
!robiotic combinations selected for s!ecific tarets may have a maGor im!act on the
disease.
One of the limitations of !robiotics in clinical a!!lication is that the most effective
!robiotic strains often !rove to be fraile durin industrial and manufacturin
!rocessin, and this may have an im!act on their !ro!erties *Hr/e~kowiak et al.,
1422-. )m!rovin the stress tolerance of !robiotic strains is thus an im!ortant
bioloical and clinical oal. S#athobiotechnoloy% describes the ex!loitation of hihly
ada!ted !athoenic stress sur-vival and host evasion strateies for the develo!ment of
im!roved !robiotic strains *Sleator and 'ill, 144C-.
E. MECHANISMS OF ACTION OF PROBIOTIC
Studies on several !robiotics and their !ro!erties have !rovided evidence that
s!ecific *but not all- !robiotics may counteract or dam!en ex!erimental and human
astrointes-tinal inflammation by their im!act on e!ithelial cell function, includin
e!ithelial cell barrier function, e!ithelial cytokine secretion, and throuh antibacterial
effects relatin to coloni/ation of the e!ithelial layer.
The mechanisms of action of !robiotics are often not well understood, and they
are likely to be multifactorial !rocesses. 'owever, mechanisms must be clearly
associated with their observed effects. Thereby, s!ecific !robiotic actions can be
rou!ed into the cateories+ anti-infective actions *treatment or !revention of viral,
yeast, funi, or bacteria !athoenesis-, metabolic functions *lactose malabsor!tion and
lowerin blood cholesterol-, and modulation of the immune system *im!rovement of
immune res!onse, amelioration of inflammatory syndromes, and immune-related
!atholoies-. The anti-infective effects of !robiotic bacteria involve mechanisms that
im!ly antaonism with other bacteria and viruses, as secretion of antimicrobial
substances and com!ounds, com-!ete for nutrients necessary for !athoen survival
and antitoxin effects and also, as com-!etitive bindin to rece!tors, adhesion to
mucosal and e!ithelial cells, stimulation of mucus secretion and enhancin the
!roduction of defensive molecules such as mucins and defensins, increasin and
reinforcin barrier function, increasin and reinforcin innate immune function,
reducin the secretory and inflammatory conse"uences of bacterial infection, and
im!rovin ut motility *6orr et al., 1443E Ohland and $ac&auhton, 1424E Salminen
et al., 1424-.
E.>. A$ti.i&r!bia S#bsta$&(s
The Lactobacillus and ifidobacterium s!!. are ca!able of !roducin oranic
acids as end !roducts of metabolism, as well as other antimicrobial metabolites. The
antimicrobial metabolites !roduced by !robiotics can be divided into two rou!s+ *i-
low-molecular mass com!ounds *below 2.444 5a- such as oranic acids, which have
a broad s!ectrum of action and *ii- antimicrobial !roteins, termed bacteriocins
*`2.444 5a-, which have a relatively narrow s!ecificity of action aainst closely
related oranisms and other ram-!ositive bacteria.
The oranic acids secreted in the fermentative metabolism of carbohydrates by
!ro-biotics have been considered to be the main antimicrobial com!ounds res!onsible
for their inhibitory activity aainst !athoens. )n addition, in the !resence of oxyen,
some !robiotics are able to enerate hydroen !eroxide that has oxidi/in effect on
bacterial cell surface. 6arbon dioxide is !roduced by heterofermentative bacteria and
its antimi-crobial activity is due to inhibition of en/ymatic decarboxylation and its
accumulation in the membrane causin dysfunction in the !ermeability of the
membrane. Bacteriocins are com!ounds with !otential antimicrobial activity
synthesi/ed by many bacterial s!ecies, includin lactic acid bacteria *6hen and
'oover, 1449-. Bacteriocins have been subdi-vided into four classes+ class ), the
lantibiotics, com!risin small *V0 k5a- heat-stable !e!tides that contain
!osttranslationally modified amino acidsE class )), the nonlantibiotic !e!tides,
com!risin small *V24 k5a- heat-stable !roteinsE class ))), com!risin lare *`94
k5a- heat-labile !roteinsE and class )I, com!risin an undefined mixture of !ro-teins,
li!ids, and carbohydrates. )n addition, the term bacteriocin-like com!ound has been
coined to refer to antaonistic substances that are incom!letely defined or do not fit
the ty!ical criteria definin bacteriocins and tend to have a broader s!ectrum of
activity. Some bacteriocin-like com!ounds have also been described for !robiotics
*SKnche/ et al., 1424- and a uni"ue bacteriocin, bifidocin B, from ifidobacterium
bifidum &6?B 2707 has been !urified. #robiotics have exhibited antaonistic effects
aains different !athoenic s!ecies, includin !almonella$ Listeria, and "elicobacter
amon others. Owin to the !otential interest of these antimicrobial !roteins in novel
thera!eutic develo!ments, further studies should be carried out on their enetics,
biochemistry, and mechanisms of action.
E.?. A/h(si!$ a$/ C!.3(titi+( I$hibiti!$ !' Path!"($s t! th(
I$t(sti$(
)ntestinal mucus has several rolesE it !rotects the mucosa from invasive
!athoenic microoranisms while !rovidin an initial bindin site, nutrient source,
and a matrix on which commensal bacteria can !roliferate. ?urthermore, adherence of
bacteria to intestinal e!ithelium is re"uired for both tem!orary coloni/ation of the ut,
and also adherence or !enetration is a !rere"uisite for infection by many !athoensE it
is also im-!ortant for the modulation of the immune system. 6oloni/ation of the
intestinal mu-cosa by bacterial enteric !athoens results in the induction of a stron
inflammatory res!onse aimed at controllin the offendin !athoen. 'owever, this
inflammatory res!onse has also been shown to decrease the viability of the ut
microbiota, allowin the !athoen to occu!y the vacated niches *Sekirov et al., 1424-.
&owadays, other mucosas are bein analy/ed because of their interest for health as,
for exam!le, nasal, oral, and vainal mu-cosa. Thus, intestinal coloni/ation with
non!athoenic bacteria as transient or commen-sal microbiota is of !articular
im!ortance for the !rotection of the host aainst !athoenic strains by com!etitive
exclusion. S!ecific !robiotics can influence adhesion of !otential !athoenic bacteria
to the intestinal e!ithelium. 5ifferent bacterial strains of the same enus and s!ecies
may exert com!letely different effects on the host, and thus, interac-tions cannot be
easily !redicted. The adhesive !ro!erties of !robiotics widely vary de!endin on the
strain, and furthermore, hih in vitro adherence ability in one strain does not always
uarantee its in vivo !ersistence and !rotective effect. 6om!etitive ex-clusion by
intestinal bacteria is based on bacteria-to-bacteria interaction mediated by com!etition
for available nutrients and also for mucosal-adhesion sites. )n order to ain a
com!etitive advantae, bacteria can also modify their environment to make it less
suitable for their com!etitors. The !roduction of antimicrobial substances, such as
lactic and acetic acid, is one exam!le of this kind of environmental modification.
)t has been shown that some lactobacilli and bifidobacteria share carbohydrate-
bindin s!ecificities with some entero!athoens *?uGiwara et al., 1442E &esser et al.,
1444-. This allows the strains to com!ete for the rece!tor sites on the host cell with
s!e-cific !athoens. )n eneral, it is considered that !robiotic strains are able to inhibit
the attachment of !athoenic bacteria by means of steric hindrance at enterocyte
!athoen rece!tors. )n addition, !robiotic strains may reduce the !athoen
coloni/ation and !os-sible subse"uent invasion by the reduction of the viability of a
!athoen by !roducinrowth inhibitors. Several !robiotic strains are able to com!ete
with !athoenic bacteria, includin # vulgatus$ C# histolyticum$ C# difficile$
%nterobacter aerogenes$ Listeria monocyto-genes$ !# aureus$ !almonella enterica$
Cronobacter sa&aBa&ii, enterotoxienic %#coli, and other !athoens for intestinal
e!ithelial cell bindin, and they can inhibit and dis!lace !atho-enic bacteria even if
the !athoens have been !reviously attached to intestinal cells *6ollado et al., 144<-.
(ecently, it has been re!orted that s!ecific combinations of !ro-biotic strains may
com!lement or im!rove health benefits iven by individual strains.The well-known
!robiotic combination ISL}9, a mixture of eiht bacteria, includin Lactobacillus
and ifidobacterium strains, has been re!orted to be im!ortant in !revention and
treatment of s!ecific human diseases *Huandalini, 1424-. . few in vitro and in vivo
studies have assessed other combinations such as L# rhamnosus$ P# freudenreichii, and
# lactis. This combination has then been demonstrated effective in clinical studies on
)BS !atients *>aGander et al., 144CE Lyra et al., 1424-. 'owever, this same !robiotic
combi-nation did not have an im!act on allery !revention in a study involvin
infants, while L# rhamnosus HH was !reviously demonstrated to be effective
*>alliomLki et al., 142E $arschan et al., 144CE (ose et al., 1424-. Thus, !reclinical
studies are always warrantedon any combination to verify the effect before human
intervention studies.
E.B. @N*MBERING CONFIRMATIONAA""r("ati!$
0ith Path!"($s
#athoen inhibition by s!ecific !robiotics may !rovide sinificant human health
!rotec-tion aainst !athoen infection actin as a natural barrier aainst !athoen
ex!osure in the astrointestinal tract. Oranisms with the ability to coareate with
other bacteria such as !athoens may have an advantae over noncoareatin
oranisms that are more eas-ily removed from the intestine and,or other environments
such as human ecosystem *oral, uroenital tract, skin, etc.-, water, or foods, avoidin
the !athoen infection. 6ell adhesion is a com!lex !rocess involvin contact of the
bacterial cell membrane and inter-actin surfaces. The ability to adhere allows
bacteria to remain in the intestinal tract and com!ete for adhesion sites. .dherence of
bacterial cells is usually related to cell surface characteristics, and areation has
also been correlated with adhesion, whereas coare-ation has been related to the
ability to interact closely with !athoens. 6oareation with !otentially ut
!athoens could therefore contribute to the !robiotic !ro!erties as-cribed to lactic acid
bacteria. )t has been demonstrated that the cellular areation could be !ositive to
!romote the coloni/ation of beneficial microoranisms, as suested for lactobacilli
in the astrointestinal or vainal tract. $any re!orts have suested that the
coareation abilities of Lactobacillus s!ecies miht enable it to form a barrier that
!revents coloni/ation by !athoenic bacteria *6ollado et al., 144<E Twetman et al.,
1443-, because of the !roduction of a microenvironment around these !athoens, in
which the inhibitin substances were enerated by Lactobacillus s!ecies. 8sin two
iso-enic strains of Lactobacillus crispatus, one with areation !henoty!e and
showin hih adhesion and the other nonareative, it was shown that this
areation !henoty!e favors intestinal coloni/ation and modulates ex!ression of
immune rece!tors in the mucosa *Ioltan et al., 144<-. Then, conse"uently, the ability
of !robiotics to areateand coareate are desirable !ro!erties for use in
commercial food !re!arations. Such studies should be conducted as !reclinical
re"uisites before embarkin on human inter-vention studies.
E.D. T!Hi$s A/h(r($&( a$/ R(.!+a by Pr!bi!ti&s
#robiotics have been re!orted to interact directly with !athoens and toxin
!roducers, as well as to bind and remove toxins from food and !athoens such as
bacteria and funi, which influence the health of the host *Salminen et al., 1424-. The
bindin !rocessa!!ears to be s!ecies- and strain de!endent. . !otential chance to
reduce ex!osure todietary mycotoxins is the use of !robiotics. The L# rhamnosus
strain HH and L# rhamnosus strain L6-<40 have been extensively studied and are
shown to have the reatest aflatoxinremoval ca!acity described to date *Salminen et
al., 1424-. S!ecific !robiotics have !roved to be effective in removin aflatoxins and
mycotoxin and ochratoxin in different model systems althouh the !ro!erties were
strain and toxin s!ecific. Only a few human studies have been re!orted on !robiotic
administration to humans ex!osed to dietary aflatoxin inestion. )n one intervention
trial, it has been demonstrated that dietary su!-!lementation of s!ecific !robiotic
bacteria ex!osed to aflatoxin B2 via diet resulted inreduced urinary excretion of one
aflatoxin metabolite, known to be a biomarker for liver cancer risk *El-&e/ami et al.,
144D-.
)t has recently been shown that s!ecific strains of !robiotic bacteria are able to
elim-inate cyanobacterial toxins from a"ueous solutions *&ybom et al., 144C-. Toxic
cyano-bacteria have been found in natural and artificial freshwater reservoirs in
several countries, and their toxins, microcystins, are sinificant !ublic health !roblem
due to both their acute and chronic toxicities because they have a !otent
he!atotoxicity and tumor-!romotin activity. )n addition, !robiotics can be
molecularly enineered to ex!ress rece!tors for the toxins and absorb and eliminate
them from bindin to their natural re-ce!tors, thus reducin or eliminatin the
!atholoical im!act of bacterial infection *#atonet al., 1424-.
E.E. I..#$( R("#ati!$ a$/ M!/#ati!$
The ut microbiota and transient bacteria *!robiotics- are known to influence the
devel-o!ment and reulation of the host%s defenses of immune and nonimmune nature
*O%?laherty et al., 1424-. #robiotics are able to stimulate, as well as reulate natural
and ac"uired immune res!onses by interactin with the mucosa-associated lym!hoid
tis-sue and are considered to be im!ortant mediators of immune reulation in the ut.
This interaction has broad-reachin sinificance to human health and could im!act
infectious diseases, some forms of cancer, alleric disease, autoimmune disorders, and
also a rane of intestinal inflammatory diseases. #robiotics are able to reulate
mucosal im-mune res!onses throuh induction of anti-inflammatory cytokines such as
)L-24 and TH?-b, while decreasin ex!ression of !roinflammatory cytokines such as
T&? and )?&- *6orr et al., 1443-. There is a lare variation in the induced res!onse
amon strains and s!ecies, but accumulatin evidence suests that some !robiotics
can stimulate a !rotective im-mune res!onse sufficiently to enhance resistance to
microbial !athoens *O%?laherty et al., 1424E #atel and Lin, 1424-. This
immunomodulatory role of !robiotics is an im-!ortant factor reulatin the immune
res!onse aainst astrointestinal !athoens and !re-ventin inflammatory conditions
in the H) tract.
.G CONCL*SIONS
)mbalances and dysfunctions of the astrointestinal tract are associated with
microbiota aberrancies. S!ecific !robiotics may re!resent the best o!tion to balance
ut microbiota es!ecially when they have been selected and characteri/ed for s!ecific
sub!o!ulations or disease rou!s. Since !robiotic health benefits are strain s!ecific, it
is likely that strains can be selectively aimed at !articular rou!s of !atients.
#robiotics have been usually used to treat and !revent s!ecific astrointestinal
disturbances, althouh new data su!!ort the !o-tential use of !robiotics in the
!revention and treatment of a number of diseases includin ato!ic diseases, immune
disorders, obesity, and diabetes, as well as other nonintestinal diseases. 'owever, as
!robiotic mechanisms of action are hihly s!ecific, it is im!ortant to characteri/e the
s!ecific !ro!erties of !robiotics and their combinations in order to select the best
strains or strain combinations for the s!ecific taret. .dvances have !rom!ted to
increase the interest of researchers and industry, and new a!!lications and tarets are
bein discovered.
ACKNOWLEDGMENTS
.ll authors !artici!ated in !re!aration of the manuscri!t. &one of the authors has
conflict of interests. This work was su!!orted by 6onsolider ?un-6-?ood 6S5144<-
444D9 from the S!anish $inistry of Science and )nnovation. $.6.6. is the reci!ient
of a S(amJn y 6aGal% research contract *(=6-1424-40D27- from the S!anish $inistry
of Science and )nnovation, S!anish Hovernment.
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CHAPTER >B
Pr!bi!ti&s a$/ Irritab( B!0( Sy$/r!.(
I. ;#r(shiI 5.R. E$/r(s
I$& (esearch, Enumclaw, ;., 8S.
>. INTROD*CTION
The role of the astrointestinal microbial flora in diestive health has been of
reat interest to researchers. )t is now well established that bacterial inhabitants of the
H) tract influence immunoloical function to a reat deree and modulate
inflammatory reactions. 6om-mon constituents of normal, healthy H) flora, includin
ifidobacteria and Lactobacilli, !romote the release of anti-inflammatory cytokines
and inhibit the release of !roinflam-matory cytokines via interactions with dendritic
cells *'art et al., 1447-. .n alteration of flora due to reductions in these and other
beneficial bacterial s!ecies can lead to activation of the immune system and an
increased likelihood of inflammatory reactions and, ulti-mately, tissue and cellular
damae. ?urthermore, several of these beneficial bacterial strains !ossess direct
antibacterial and antiviral !ro!erties *Wuiley and ?lourie, 144<-, an additional
mechanism whereby they enhance the health of the host oranism. ifido-bacteria are
also involved in the !roduction of the short-chain fatty acids that are essential to
astric mucosal health, and facilitate the removal of toxins and other harmful
substances from the diestive tract. These strains also !artici!ate in essential nutrient
synthesis and absor!tion *Si et al., 1447-. Evidence of the im!ortance of a balanced
H) flora for health in irritable bowel syndrome *)BS- comes from the findin that
antibiotic-induced chanes may be associated with an increased risk of develo!in the
disorder *Wuiley, 144<-. .ntibiotics im!act both !athoenic and commensal strains
of bacteria. .ddin to the evidence that altered or imbalanced H) flora !lays an
im!ortant role in this con-dition is the findin that antibiotic thera!y im!roves several
common sym!toms of )BS *#imentel et al., 1422-.
?. PATHOPHYSIOLOGY OF IBS
)BS is a disorder of unknown !atho!hysioloy characteri/ed by a constellation of
sym!-toms, the more common of which include cram!in, abdominal !ain and
tenderness, bloatin, consti!ation, and diarrhea. .ccordin to the &ational )nstitutes
of 'ealth, )BS affects as many as 14R of the 8S adult !o!ulation. )BS tends to occur
more often in women than men, with the initial onset of sym!toms bein !rior to the
ae of 90 in rouhly one-half of cases *)rritable Bowel Syndrome, 1424-.
;hile individuals with )BS often have uni"ue sym!toms, they can enerally
be@E$t(r *s(/A
rou!ed into three distinct subty!es based on bowel habits and !atterns+ diarrhea-
!redominant, consti!ation-!redominant, or mixed subty!e *>assinen et al., 144<-. )BS
sym!toms are often chronic in nature and sinificantly im!act "uality of life *WoL-.
Studies com!arin )BS sufferers to those with other chronic conditions suest that it
has a similar adverse effect on WoL as diabetes and renal failure *#arkes et al., 144C-.
The definitive cause of )BS is unknown. 'owever, evidence suests that some of
the factors include altered astrointestinal *H)- motility, visceral hy!ersensitivity
*overly sen-sitive to !ain and !ressure in the bowels-, inflammation of the H) tract,
increased !sy-choloical stress, and bacterial astroenteritis *as )BS sym!toms are
often triered by a !recedin infection- *>assinen et al., 144<-. ?urthermore, the
inter!lay between the nervous system and H) tract M the SbrainMut axis% M has
arnered serious attention as a factor in health and disease. This model !rovides an
ex!lanation for how sym!toms of )BS, es!ecially those involvin stress, altered H)
motility and enhanced visceral sensitive-ity, may occur *#arkes et al., 144C-. The
extraordinary inter!lay between the immense H) immune system and microbial
inhabitants of the H) tract cannot be overlooked as research into the causes of )BS
continues.
B. EVIDENCE OF INTESTINAL FLORA ALTERATIONS IN
IBS
Several rou!s have examined )BS !atients for alterations in bowel flora. ;hile
some researchers have found no sinificant differences in small intestinal bowel
overrowth *S)BO- between )BS !atients and healthy controls *#osserud et al., 144<E
;alters and Ianner, 1440-, others have found a sinificantly increased !revalence in
)BS !atients *Lu!ascu et al., 1440-. The detection method em!loyed in these studies
involves breath hydroen analysis, which has been critici/ed for bein variable in its
ability to detect S)BO *(iordan and >im, 144D-. 'owever, studies usin more
advanced methods have shown sinificant alterations of H) flora between )BS !atients
and healthy controls, and within )BS subty!es. ?or exam!le, a study usin real-time
#6( found that Lactobacilli were sinificantly lower in diarrhea-!redominant )BS
!atients versus consti!ation-!redominant !atients. Aeillonella s!!. were sinificantly
increased in consti!ation-!redominant )BS !atients com!ared to healthy controls.
?urthermore, a trend toward decreased ifidobacterium s!!. levels in diarrhea-
!redominant )BS !atients has been ob-served *$alinen et al., 1440-. . study
conducted in the &etherlands also found a twofold decrease in ifidobacteria in )BS
!atients in com!arison to healthy controls when analy/in the fecal microbial
com!osition utili/in fluorescent in situ hybridi/ation *?)S'- tech-ni"ues *>erckhoffs
et al., 1443-, while .nna >assinen%s research team in ?inland found sinificant fecal
flora alterations in )BS !atients versus controls usin 2DS ribosomal (&. testin
*>assinen et al., 144<-. Overall, these studies are hihly suestive that altered H)
flora !lays a role in the !athoenesis of at least some )BS sins and sym!toms.
Fi"#r( >B.> 5efinition of !robiotics. *,dapted from ',1I+"1 (4559) and the
International !cientific ,ssociation for Probiotics and Prebiotics.-
Hiven the evidence for altered bowel flora in )BS !atients, restorin healthy bowel
flora throuh su!!lementation with !robiotic oranisms may benefit individuals with
this condition. . re!ort !re!ared by a Goint ex!ert committee of the ?ood and
.riculture Orani/ation of the 8nited &ations *?.O- and the ;orld 'ealth
Orani/ation *;'O- in 1442 defined !robiotics as follows+ S#robiotics are live
microoranisms, which when administered in ade"uate amounts confer a health
benefit on the host.% There are other im!ortant criteria, which also factor into definin
!robiotic oranisms *?iure 29.2-.
6linical trials of !robiotic su!!lementation for )BS have concluded mixed results.
Several trials have shown benefits with others showin no results. This cha!ter
hihlihts the results of selected clinical trials demonstratin !ositive results of
!robiotic su!!lemen-tation for )BS sym!toms in adults. $ore com!rehensive reviews
have been !ublished elsewhere, and some of these are !ointed out in the S?urther
(eadin% section at the end of the cha!ter. The studies summari/ed in this cha!ter
resulted from a search for dou-ble- blind, !lacebo-controlled clinical trials on #ub$ed
for probiotics and irritable bo*el syn-drome. ?urther limits included those trials
investiatin oral su!!lementation with !robiotic !re!arations for the !ur!ose of
treatin the maGor sym!toms of )BS meetin the (ome dianostic criteria, the third
and most current iteration of which is known as (ome ))) *?iure 29.1-. The (ome
!rocess is a set of criteria used to cateori/e func-tional astrointestinal disorders and
is currently considered somewhat of a Sold standard% for the dianosis of )BS based
on a cluster of sym!toms *Bouchoucha et al., 144D-.
D. PROBIOTIC ORGANISMS AND IBS
One of the initial controlled investiations into !robiotic su!!lementation for
sym!toms of )BS was !ublished in 1444. &obaek et al. conducted a double-blind,
!lacebo-controlled *5B#6- trial of D4 study subGects who were randomi/ed to receive
either a rosehi! drink containin 0u24
<
6?8 *colony-formin units- Lactobacillus
plantarum
Fi"#r( >B.? (ome ))) dianostic criteria for )BS. (.eproduced from
http@IIromecriteria#org.)
5S$ 3C79 or a !lain rosehi! drink for 7 weeks *&obaek et al., 1444-. The !atients
had to have a normal colonic examination within D months !rior to the study, no
malabsor!-tion, and a normal blood test includin hemolobin, 6-reactive !rotein,
thyroid-stimulatin hormone, and T
7
. The !atients were also re"uired to avoid all
other lactic acid-containin !roducts. The outcome measures were flatulence,
abdominal !ain, def-ecation *fre"uency and consistency-, and overall H) function.
The researchers concluded that L# plantarum 5S$ 3C79 may be effective in reducin
abdominal !ain and flatulence. These results were encourain for the develo!ment of
!robiotic oranisms for )BS treatment. 6learly, larer studies of loner duration were
needed to confirm these initial findins and to attem!t to overcome the hih !lacebo
res!onse rate seen.
)n 1449, >im et al. !ublished a 5B#6 !ilot study conducted at the $ayo 6linic in
(ochester,$&*>im et al., 1449-. The study com!rised 10 subGects who met the (ome
criteria for )BS with the diarrhea-!redominant subty!e. SubGects were randomi/ed to
receive !lacebo or 7.0u24
22
6?8 ISL}9 !er day for C weeks in two divided doses
after a 1-week run-in !eriod. ISL}9 is a com!osite !robiotic formulation that
contains the followin viable oranisms+ ifidobacterium *# longum$ # infantis$ #
breve-, Lactobacillus (L# acidophilus$ L# casei$ L# bulgaricus$ L# plantarum) and
!treptococcus thermophilus. #atients were re"uired to record a weekly res!onse on
the satisfactory relief from )BS sym!tom "uestionnaire, a daily bowel fre"uency, and
consistency diary based u!on the validated Bristol stool form scale, and a daily diary
to record abdominal !ain, bloatin, flatulence, and urency on a 244-mm visual
analo scale *I.S-. Hastrointestinal and colonic transit was measured usin
scintira!hic methods. The authors concluded that ISL}9 had no effect on transit
time and, com!ared with the !lacebo rou!, had no effect on abdominal !ain,
flatulence, or urency. 'owever, a reduction in bloatin when com!ared to the
control rou! was noted, which may have clinical sinificance because many !atients
with )BS claim that bloatin and flatulence, rather than !ain, are the most difficult
sym!toms to co!e with. .ccordin to the authors, a maGor limitation of the study was
that it was !owered to detect sinificant differences in bowel transit rather than the
common sym!toms of )BS.
)n a re!eat of the study described above, the same rou! increased the study !o!u-
lation from 10 to 7C !atients *>im et al., 1440-. This investiation revealed a
sinificant decrease in flatulence *pe.42- for the treatment rou!, but no sinificant
differences were seen with res!ect to bowel function, abdominal !ain, or bloatin.
By 1440, the thinkin reardin )BS !atho!hysioloy shifted slihtly, with
researchers theori/in that a dysreulation of the brainMut axismay act as a !rimary
factor in the chanes in utmotility and visceral hy!ersensitivity seen in )BS.
?urthermore,mucosal bio!sies seem to indicate that chronic low-rade inflammation
is !resent in at least some !atients, which may !lay a role in the alteredmotility. .
?innish study conducted by >aGander et al. *1440- investiated an intervention with a
combination of e"ual amounts of Lactobacillus HH, L# rhamnosus LCG5:$ # breve
b<<, and Propionibacterium freudenreichii ss!. !hermanii 7! for a total daily dose
of CM3u24
3
6?8 *>aGander et al., 1440-. The D-month 5B#6 study com!rised 249
!atients meetin the (ome criteria for )BS. 6onsum!tion of !roducts containin
!robiotic oranisms was forbidden durin the studyE however, because of the duration
of the study, !atients !reviously on )BS medications were allowed to continue M
althouh they were advised not to chane these treatments or lifestyle for the study
!eriod. Each month, the !atients maintained a diary for a 2-week !eriod. The !rimary
outcome measures were abdominal !ain, distention, flatulence, and borborymi. The
secondary out-come measures were urency, strainin, belchin, heartburn, feelin of
incom!lete evacu-ation, vomitin, !ost-meal fullness, and either blood ormucus in the
stool. The(.&5-9D *a 9D-item health survey- was utili/ed as the "uality-of-life
"uestionnaire. Lastly, a food fre-"uency "uestionnaire was used to assess the
inestion of !otentially sym!tom-exacerbatin and as-!roducin foods. .t the end of
the study, the combined sym!tom score for the !rimary outcome measures was
reduced by 71R com!ared with DR for the !lacebo rou! *pV.420-.;hen looked at
se!arately, there was a trend toward an im!rovement in all of the !rimary outcome
measures, but only borborymi achieved statistical sinificance *pV.44C-. )t is
im!ortant to note that while some sym!toms of )BS were im!roved, no increase in the
"uality of life as measured by the (.&5-9D survey was noted.
. second D-month 5B#6 trial conducted in 1440 by &iv et al. looked at the
efficacy of Lactobacillus reuteri .T66 00<94 dosed at 1u24
C
6?8 day
-2
for )BS *&iv
et al., 1440-. ;hile each of the 07 subGects that com!leted the study ex!erienced
im!rovement in all of the study outcome measures, there was no statistical
sinificance between the treatment and !lacebo rou!s. The authors suest that the
lare !lacebo res!onse rate may be res!onsible for the lack of sinificance for this
smaller study. Two of the outcome measures, consti!ation *pe.4<27- and flatulence
*pe.43<2-, were marinally sinificant and may have reached statistical sinificance
had the study been !owered with an ade-"uate number of subGects.
O%$ahony et al. !ublished an C-week 5B#6 study of << subGects in 1440 to
inves-tiate the res!onse to sym!toms of )BS and any association of cytokine ratios
with ad-ministration of Lactobacillus salivarius 8667992, ifidobacterium infantis
90D17, or !lacebo *O%$ahony et al., 1440-. The !atients included in the study met the
current (ome cri-teria for )BS havin had all other oranic causes ruled out. The
treatment rou!s re-ceived 2u24
24
live bacterial cells in a malted milk !re!aration,
whereas the !lacebo rou! re-ceived the malted milk alone. .fter a 7-week run-in
!eriod where the study !artici!ants recorded sym!toms, as well as stool fre"uency
and consistency, they were randomi/ed to receive the treatment or !lacebo.
6om!liance was assessed by fecal flora analysis. The subGects continued to record
their sym!toms for a 7-week washout !eriod. The !rimary outcome measures were
abdominal !ain, distention, and bowel movement difficulty. Each of the sym!toms
was measured usin a <-!oint Likert scale as well as a 24-cm I.S. The study
demonstrated # infantis to be better than both L# salivarius and !lacebo for all of the
chief sym!toms of )BS studied, with statistical sinificance havin been achieved
*pV.40- with both the Likert and I.S scores for abdominal !ain, bowel move-ment
difficulty *which assessed both the consti!ation and diarrhea subty!es-, and the
com!osite score. The !lacebo res!onse rate was found to be similar to what has been
observed with many other )BS trials. ?urthermore, the researchers found an im!ortant
association with the cytokine ratios, which su!!orts the hy!othesis of altered ut
immune function and low-rade inflammation as !layin a role in the etioloy or
!athoenesis of )BS. )L-24 tends to be lower and )L-21 hiher in !atients with )BS.
;hen the )BS !atients% )L-24 to )L-21 ratios were com!ared to those of healthy
volunteers, a sinificant difference was observed *pe.449-. )nterestinly, the ratio
returned to that observed in the healthy volunteer rou! followin treatment with #
infantis 90D17, but not with L# salivarius 8667992. This may be the first study
conducted that has demonstrated a benefit of one !robiotic oranism over another,
while at the same time sinificantly better than !lacebo.
;horwell et al. desined a 7-arm follow-u! study that was !ublished in 144D to
con-firm the results of the !revious study of # infantis 90D17 in a 5B#6 trial
consistin of 9D1 female !atients dianosed with )BS of any subty!e *;horwell et al.,
144D-. .fter a 1-week run-in !eriod, the !atients were randomi/ed to receive either #
infantis at a sin-le daily dose of 2u24
D
, 2u24
C
, or 2u24
24
live bacterial cells or a
!lacebo in an identical ca!sule for a 7-week !eriod. The !atients were followed for a
1-week washout !eriod at the end of the treatment !hase of the study. Throuhout the
duration of the study, the !artici!ants recorded sym!toms usin a tele!hone-based
interactive voice recordin system *)I(S-, for the !ur!ose of recordin scores on a D-
!oint scale for abdominal !ain, distention, flatulence, urency, feelin of incom!lete
evacuation at defecation, and mucus in stool. .t baseline and at the end of the study,
the subGects com!leted an )BS "uality-of-life "uestionnaire as well as the 'os!ital
.nxiety and 5e!ression *'.5- Scale. . baseline analysis revealed no sinificant
differences between the rou!s. The !rimary end!oint was defined a !riori to be
abdominal !ain. Only the 2u24
C
6?8 day
-2
dose was statistically im!roved when
com!ared to the !lacebo rou! at week 7. Of the secondary efficacy variables
measured for the diarrhea subty!e, incom!lete evacuation *pe.44C-, strainin
*pe.44<-, bowel habit satisfaction *pe.427-, overall assessment of )BS sym!toms
*pe.41C-, and the com!osite score *pe.41<- for all outcome measures were
sinificantly better when com!ared to the control rou!. ?or the consti!ation subty!e,
the 2u24
C
6?8 day
-2
dose was statistically im!roved when com!ared to the !lacebo
rou! for abdominal !ain *pJ.49D- and for bowel habit satisfaction *pJ.47<-. $ost
im!ortantly, the overall scores for abdominal !ain *pJ.419-, bloatin *pJ.47D-, and
com!osite scores *pe.429-, reardless of subty!e, were also sinificantly im!roved
when com!ared to !lacebo for the 2u24
C
6?8 day
-2
dose. &one of the doses resulted
in sinificant chanes in either the WoL or '.5 scores. ;hen consid-erin why the
2u24
24
6?8 day
-2
dose was not sinificantly better for the trial rou! than the control
rou!, the authors conGectured that due to the hydrosco!ic nature of the !robiotic
oranism, the test article may have coaulated and not dissolved u!on inestion. The
hy!othesis was tested, and indeed, the 2u24
24
6?8 day
-2
dose was found to be
resistant to acid and !roloned aitation. The authors conclude that it is confirmed
that # infantis 90D17 is an effective treatment for female !atients with mild to
moderate )BS.
)n 144C, >aGander et al. !ublished another 5B#6 study investiatin a
multis!ecies !robiotic for the relief of )BS sym!toms *>aGander et al., 144C-. The
s!ecies in the for-mulation was the same as the 1440 study >aGander%s rou!
investiated *described above- with the exce!tion of a substitution of # animalis s!!.
Lactis Bb21 for # breve Bb33. The test article was administered as a milk-based
!roduct and the !lacebo was the milk-based !roduct alone. Each oranism was
!resent at a concentration of 2u24
<
6?8 ml
-2
. The subGects received 2.1 dL of the
milk-based !roduct !er day, which su!!lied a total of 7.Cu24
3
6?8 day
-2
. This 144C
study consisted of CD )BS !atients who met the current (ome criteria and who were
randomi/ed to receive either the !robiotic mixture or !lacebo for 0 months. This study
was slihtly smaller and of a 2 month shorter duration than the 1440 study. The )BS
sym!toms and the bowel habits were recorded in a diary and the authors reference
their 1440 study for that descri!tion. The '(WoL was used to assess "uality-of-life
chanes at the mid!oint and at the end of the study. The com!osite )BS sym!tom
score *the sum of abdominal !ain, distention, flatulence, and borborymi scores- was
the !rimary outcome measure and was hihly sinificant for the treatment rou! when
com!ared with the control *pe.44C9-. .t the end of the study, distention was
sinificantly im!roved *pe.419-, and abdominal !ain alone almost reached statistical
sinificance *pe.401-. .n analysis of the data collected usin the "uality-of-life
"uestion-naire resulted in a sinificant difference for the rou! receivin the !robiotic
!re!aration *pe.470-. )t is concluded that this multi!le s!ecies !robiotic mixture is
useful clinically for the alleviation of many of the sym!toms of )BS.
acillus coagulans is a novel !robiotic oranism that is often incorrectly referred
to as Lactobacillus sporogenes. Like most other commonly used !robiotics, #
coagulans is a Hram-!ositive, lactic acid-!roducin bacteria. 'owever, this s!ecies is
also a s!ore former, which ives it the !ro!erty of bein unusually resistant to hih
tem!erature, low !', and bile acids when com!ared to other !robiotic oranisms. )n
1443, 'un !ublished a 5B#6 study of 77 subGects who met the current (ome criteria
and who were randomi/ed to receive either a !lacebo or # coagulans HB)-D4CD once
daily for C weeks at a dose of Cu24
C
6?8 day
-2
*'un, 1443-. The !artici!ants recorded
self-assessment for severity of )BS sym!toms *abdominal !ain and bloatin- on a 0-
!oint scale as well as !roduct use on a daily basis. The author did not mention
whether the diary used was a validated tool. 6om!liance was measured by countin
ca!lets at !atient visits on days 1C and 0D. The author re!orts that due to statistically
sinificant differences between the rou!s at baseline after randomi/ation, a within-
rou! chane from baseline was used to evaluate efficacy. Therefore, a direct
com!arison between the treatment and !lacebo rou! was not !ossible. The within-
rou! anal-ysis resulted in a sinificant chane from baseline in the treatment rou!
for both abdominal !ain and bloatin *pV.42-. .s miht be ex!ected, the !lacebo
res!onse was hih and also resulted in a sinificant reduction in !ain *pV.40-.
Therefore, as many !revious studies have found, im!rovements were observed in both
the treat-ment and !lacebo rou!s.
.n additional !ilot study evaluated the treatment of )BS sym!toms with #
coagulans HB)-D4CD in males and females between 2C and <0 years of ae *5olin,
1443-. The !atients were classified as )BS-5 accordin to the (ome ))) criteria. Sixty-
one !atients were enrolled in the study, of which 00 met the inclusion criteria and 01
were included in the final analysis. .n C-week treatment !eriod was initiated
followin a 1-week obser-vation !eriod, durin which the !atients inested a ca!sule
containin 1u24
3
6?8 . coagulans HB)-D4CD daily or a !lacebo. #atients were
re"uired to com!lete a daily diary durin the initial 1-week observation !eriod notin
daily bowel movement counts and consistency. #atients returned to the clinic and
were asked to com!lete a WoL "uestion-naire and were randomi/ed to receive either
the treatment or !lacebo, and iven daily diary cards to assess stool count and
consistency alon with medication consum!tion. The study subGects were also asked
to rate abdominal !ain, tihtness, flatulence, and,or urency sym!toms at the end of
each <-day !eriod on a I.S. #atients returned for follow-u! visits at 7 and C weeks
where com!liance was assessed and WoL "uestion-naires were re!eated. The results
demonstrated that the averae number of bowel movements !er day was sinificantly
reduced in the # coagulans HB)-D4CD treatment rou! versus !lacebo *pe.471-.
There was a lare variability in baseline scores in !atient-assessed measures, which
!recluded assessment of treatment differences in those !arameters. 'owever, the
study confirmed that the treatment was well tolerated and !rovided benefits to !atients
with )BS-5.
E. DISC*SSION
The treatment of )BS with !robiotic oranisms has yielded mixed results to date,
but it is a thera!y that certainly holds !romise and has a stron rational basis. Overall,
studies !ub-lished more recently have demonstrated !robiotic su!!lementation to be
efficacious in im!rovin several sym!toms of )BS in contrast to the earlier studies.
Several limitations of earlier studies include small sam!le si/e, and short duration, as
well as the lare !lacebo res!onse rate, and therefore, they may not have been
!owered sufficiently to detect a treatment effect. . lare !lacebo effect is commonly
seen in trials of thera!eutics for )BS and may stem from several of the
!atho!hysioloical associations common to this condition, includin the role
emotional status !lays with the syndrome. This has !roven to be a substantial
challene for researchers.
$oreover, since the dianosis of )BS is currently based on the !resence of
hallmark sym!toms *in the absence of other oranic causes-, the variability of the
!atient !o!ula-tion dianosed with this condition !resents enormous challenes in
itself. Since more than 14R of the !o!ulation in the 8nited States is thouht to have
)BS, it follows that there would be reat interindividual variability amon !atients and
that this may lead to reat differences in res!onse to various thera!ies.
.nother sinificant issue that has been identified !reviously is inade"uate "uality
con-trol in the manufacturin of !robiotics *Berman and S!icer, 1449-. ;hile this
may cer-tainly im!rove with advances in manufacturin techni"ues and adherence to
the relatively new ood manufacturin !ractices *H$#s- for dietary su!!lements,
decreased !otency of !robiotics su!!lements in terms of an insufficient number of
oranisms 6?8 dose
-2
- or oranisms that are not viable, mislabeled, or contain either
the wron oranisms or !athoenic oranisms is of concern and !otentially
danerous. Since many of the studies failed to ade"uately address such !roduct
"uality considerations, some of the identified issues may have been a factor in the
inconsistency of the observed results.
.nother consideration in several of the studies, and somethin that needs to be
addressed in future studies assessin the efficacy of !robiotics for )BS, is that of
!ro!er dosin. O!timal dosaes for the beneficial strains need to be established to
reach the de-sired treatment effect. The consensus amon !ractitioners and
researchers seems to be that larer doses of !robiotic bacteria are necessary to
effectively re!o!ulate or re!lenish bowel flora and crowd out any undesirable or
!athoenic strains.
?inally, the reat variability of outcome measures between the )BS studies
reviewed makes it difficult to make a direct com!arison of efficacy for the various
strains of !ro-biotic oranisms investiated. $ore recent studies seem to indicate that
certain enera of bacteria, and s!ecific s!ecies within a !articular enus, are likely to
have a reater benefit than others. .dditional research will continue to define the most
effective strains.
This review of clinical trials of !robiotic oranisms for the treatment of sym!toms
of )BS has revealed clinically relevant and im!ortant benefits. The most !romisin
!robiotic oranisms based on research to date include the strain # infantis 90D17 used
in ;horwell%s 144D study and the combination of Lactobacillus HH, L# rhamnosus
L6<40, # animalis s!!. Lactis Bb21, and P. freudenreichii ss!. !hermanii 7! used in
>aGander%s 144C study. acillus coagulans HB)-D4CD is another !robiotic oranism
that has shown !romise. ?urther research will continue to delineate additional
beneficial effects of these and other health-!romotin bacteria.
?uture studies need to consider the issues associated with !robiotic "uality,
!otency, identity, and dosae as well as ade"uately address the lare !lacebo res!onse
rate by !owerin studies a!!ro!riately. ?urthermore, reater attention must be !aid to
standard-i/in outcome measures, which will allow enhanced detection of !ositive
and neative res!onses. The issue of interindividual variability must also be
considered, and the con-clusions made about !otential benefits must reflect the !atient
!o!ulation bein studied. There is a stron basis for the thera!eutic use of !robiotic
oranisms as a com!onent of a com!rehensive a!!roach for treatin sym!toms
associated with )BS. ?avorably im!act-in and alterin bowel flora may !rovide
sinificant relief to the millions of individuals sufferin from this condition.
GLOSSARY
Cyt!,i$(s 6ytokines are any of a number of substances, such as interferon,
interleukin, and rowth factors that are secreted by certain cells of the immune system
and have an effect on other cells.
D($/riti& &(s 5endritic cells are antien-!resentin cells that are likely to be
!ivotal in the balance between tolerance and active immunity to commensal
microoranisms that is fundamental to inflammatory conditions, includin 6rohn%s
disease and ulcerative colitis *Sta et al., 1449-.
Irritab( b!0( sy$/r!.( )rritable bowel syndrome *)BS- is a !roblem that
affects the lare intestine. )t can cause abdominal cram!in, bloatin, and a chane in
bowel habits. Some !eo!le with the disorder have consti!ation. Some have diarrhea.
Some o back and forth between consti!ation and diarrhea. .lthouh )BS can cause a
reat deal of discomfort, it does not harm the intestines.
Pr!bi!ti&s #robiotics are live microoranisms *in most cases, bacteria- that are
similar to beneficial microoranisms found in the human ut. They are also called
Sfriendly bacteria% or Sood bacteria.% #robiotics are available to consumers mainly in
the form of dietary su!!lements and foods.
R!.( III /ia"$!sti& &rit(ria 6riteria fulfilled for the last 9 months with
sym!tom onset at least D months !rior to dianosis, a system for dianosin
functional astrointestinal disorders based on sym!toms, for )BS are as follows+
recurrent abdominal !ain or discomfort *Sdiscomfort% means an uncomfortable
sensation not described as !ain- at least 9 days !er month in the last 9 months
associated with 1 or more of the followin+ *2- im!rovement with defecation, *1-
onset associated with a chane in fre"uency of stool, and *9- onset associated with a
chane in form *a!!earance- of stool.
S.a i$t(sti$a b!0( !+(r"r!0th Small bowel bacterial overrowth is a
condition in which abnormally lare numbers of bacteria row in the small intestine.
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controlled D-month intervention. .limentary #harmacoloy A Thera!eutics 11 *0-,
9C<M937.
>aGander, >., $yllyluoma, E., (aGili-StoGanovi, $., et al., 144C. 6linical trial+
multis!ecies !robiotic su!-!lementation alleviates the sym!toms of irritable bowel
syndrome and stabili/es intestinal microbiota. .limentary #harmacoloy A
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>assinen, .., >roius->urikka, L., $Lkivuokko, '., et al., 144<. The fecal
microbiota of irritable bowel syndrome !atients differs sinificantly from that of
healthy subGects. Hastroenteroloy 299 *2-, 17M99.
>erckhoffs, ..#.$., Samsom, $., van der (est, $.E., et al., 1443. Lower
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>im, '.:., 6amilleri, $., $c>in/ie, S., et al., 1449. . randomi/ed controlled trial
of a !robiotic, ISL}9, on ut transit and sym!toms in diarrhoea-!redominant irritable
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&obaek, S., :ohansson, $.L., $olin, H., .hrnQ, S., :e!!sson, B., 1444. .lteration
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!atients with irritable bowel syndrome. The .merican :ournal of Hastroenteroloy 30
*0-, 2192M219C.
O%$ahony, L., $c6arthy, :., >elly, #., et al., 1440. Lactobacillus and
Bifidobacterium in irritable bowel syn-drome+ sym!tom res!onses and relationshi! to
cytokine !rofiles. Hastroenteroloy 21C *9-, 072M002.
#arkes, H.6., Brostoff, :., ;helan, >., Sanderson, :.5., 144C. Hastrointestinal
microbiota in irritable bowel syndrome+ their role in its !athoenesis and treatment.
The .merican :ournal of Hastroenteroloy 249 *D-, 200<M20D<.
#imentel, $., Lembo, .., 6hey, ;.5., et al., 1422. (ifaximin thera!y for !atients
with irritable bowel syndrome without consti!ation. The &ew Enland :ournal of
$edicine 9D7 *2-, 11M91.
#osserud, )., Stot/er, #., BGoornsson, E.S., .brahamsson, '., SimrQn, $., 144<.
Small intestinal bacterial overrowth in !atients with irritable bowel syndrome. Hut
0D *D-, C41MC4C.
Wuiley, E.$.$., 144<. Bacterial flora in irritable bowel syndrome+ role in
!atho!hysioloy, im!lications for manaement. :ournal of 5iestive 5iseases C *2-,
1M<.
Wuiley, E.$.$., ?lourie, B., 144<. #robiotics and irritable bowel syndrome+ a
rationale for their use and an assessment of the evidence to date.
&euroastroenteroloy and $otility 23 *9-, 2DDM2<1.
(iordan, S.$., >im, (., 144D. Bacterial overrowth as a cause of irritable bowel
syndrome. 6urrent O!in-ion in Hastroenteroloy 11 *D-, DD3MD<9.
Si, :., =u, =., ?an, =., 6hen, S., 1447. )ntestinal microecoloy and "uality of life
in irritable bowel syndrome !atients. ;orld :ournal of Hastroenteroloy 24 *21-,
2C41M2C40.
Sta, ..:., et al., 1449. The dendritic cell+ its role in intestinal inflammation and
relationshi! with ut bac-teria. Hut 01, 2011M2013.
;alters, B., Ianner, S.:., 1440. 5etection of bacterial overrowth in )BS usin
the lactulose '1 breath test+ com!arison with 276-5-xylose and healthy controls. The
.merican :ournal of Hastroenteroloy 244 *<-, 20DDM20<4.
;horwell, #.:., .ltriner, L., $orel, :., et al., 144D. Efficacy of an enca!sulated
!robiotic ifidobacterium infantis 90D17 in women with irritable bowel syndrome.
The .merican :ournal of Hastroenteroloy 242 *<-, 20C2M2034.
F*RTHER READING
Bix"uert, :.$., 1443. Treatment of irritable bowel syndrome with !robiotics. .n
etio!athoenic a!!roach at last[ (evista Es!a\ola de Enfermedades 5iestivas 242
*C-, 009M0D7.
Brenner, 5.$., 6hey, ;.5., 1443. ifidobacterium infantis 90D17+ a novel
!robiotic for the treatment of irritable bowel syndrome. (eviews in
Hastroenteroloical 5isorders 3 *2-, <M20.
6ollado, $.6., )solauri, E., Salminen, S., San/, =., 1443. The im!act of !robiotic
on ut health. 6urrent 5ru $etabolism 24 *2-, DCM<C.
>liler, B., 6ohrssen, .., 144C. #robiotics. .merican ?amily #hysician <C *3-,
24<9M24<C.
Lee, '.-(., #imentel, $., 144D. Bacteria and irritable bowel syndrome+ the
evidence for small intestinal bacterial overrowth. 6urrent Hastroenteroloy (e!orts
C *7-, 940M922.
Lin, '.6., 1447. Small intestinal bacterial overrowth+ a framework for
understandin irritable bowel syn-drome. :ournal of the .merican $edical
.ssociation 131 *<-, C01MC0C.
$c?arland, L.I., 5ublin, S., 144C. $eta-analysis of !robiotics for the treatment
of irritable bowel syndrome. ;orld :ournal of Hastroenteroloy 27 *2<-, 1D04M1DD2.
$oayyedi, #., ?ord, ..6., Talley, &.:., et al., 1424. The efficacy of !robiotics in
the treatment of irritable bowel syndrome+ a systematic review. Hut 03 *9-, 910M991.
Ohland, 6.L., $acnauhton, ;.>., 1424. #robiotic bacteria and intestinal
e!ithelial barrier function. .merican :ournal of #hysioloy. Hastrointestinal and
Liver #hysioloy 13C *D-, HC4<MHC23.
#arkes, H.6., Sanderson, :.5., ;helan, >., 1424. Treatin irritable bowel
syndrome with !robiotics+ the evidence. #roceedins of the &utrition Society D3 *1-,
2C<M237.
#reidis, H..., Iersalovic, :., 1443. Taretin the human microbiome with
antibiotics, !robiotics, and !rebiotics+ astroenteroloy enters the metaenomics era.
Hastroenteroloy 29D *D-, 1420M1492.
Wuiley, E.$.$., 1424. #rebiotics and !robioticsE modifyin and minin the
microbiota. #harmacoloical (esearch D2 *9-, 129M12C.
RELEVANT WEBSITES
htt!+,,www.f!notebook.com,i,Bowel,)rtblBwlSyndrm.htm.
htt!+,,www.mayoclinic.com,health,irritable-bowel-syndrome,5S4424D.
htt!+,,www.ncbi.nlm.nih.ov,!ubmedhealth,#$'4442131,.
htt!+,,www.nlm.nih.ov,medline!lus,irritablebowelsyndrome.html.
htt!+,,www.nlm.nih.ov,medline!lus,tutorials,irritablebowelsyndrome,htm,index.
html.
htt!+,,www.webmd.com,ibs,uide,ibs-triers-!revention-strateies.
htt!+,,www.webmd.com,ibs,features,su!!lements-for-ibs-what-works.
CHAPTER >D
A$ti!Hi/a$tI L#t(!i$ EHhibits
A$ti%i$'a..at!ry E''(&t i$ In 1itro G#t
I$'a..ati!$ M!/(
M. Mi-#$!I Y. Nishita$i
>obe 8niversity, >obe, :a!an
ABBREVIATIONS
CD 6rohn%s disease
IBD )nflammatory bowel diseases
IFN%X )nterferon-b
IL )nterleukin
LPS Li!o!olysaccharide
NF- ZB &uclear factor-ka!!aB
NO &itric oxide
TNF%V Tumor necrosis factor-c
*C 8lcerative colitis
>. INTROD*CTION
)nflammatory bowel diseases *)B5- includin ulcerative colitis *86- and 6rohn%s
disease *65- are inflammatory disorders of the astrointestinal tract in humans. )B5
has in-creased sinificantly in develo!ed countries over the last few decades *Loftus
and Sand-born, 1441-. .lthouh the !athoenesis of )B5 remains unclear, it is widely
believed to be due to dysreulation of host immune res!onse *.treya et al., 144C-. )t
has been re!orted that macro!haes, intestinal e!ithelial cells, and T cells !roduce
lare amounts of !roinflammatory cytokines such as tumor necrosis factor *T&?--c,
interleukins *)L--D, )L-C, )L-21, )L-2<, and )L-19, and interferon *)?&--b in the
inflamed intestine of )B5 !atients *.rai et al., 2333-. Treatment of )B5 de!ends on
drus such as anti-T&?-c antibody, 0-aminosalicylic acid, corticosteroids,
a/athio!rine, merca!to!urines, and cyclos!orine *'anauer, 233DE #odolsky, 1441-.
'owever, the use of these drus is some-times limited by dru-induced toxicity.
There is an increasin need for alternative aents that may be e"ually or more
effective, but toxicity free, as well as chea!er.
?lavonoids have a variety of bioloical effects such as antitumor, antioxidant,
antialler-ic, antimicrobial, and antianioenic !ro!erties *Shimada et al., 144D-.
Some flavonoids are known for their anti-inflammatory effects, which may be
attributed to their ability to inhibit the !roduction of !roinflammatory enes
andmediators *=uan et al., 144D-. Luteo-lin *9%,7%,0, <-tetrahydroxylflavone-
contained in celery, reen !e!!er, !erilla leaf, and chamomile exerts anti-
inflammatory !ro!erties. )t has been re!orted that luteolin can in-hibit the
li!o!olysaccharide *L#S--induced T&?-c, )L-D, and inducible nitric oxide *&O-
!roduction in vitro *6hen et al., 144<E Oaorari et al., 1442-, su!!ress the L#S-
induced T&?-c in the serum, and reduce the intercellular adhesionmolecule *)6.$--2
ex!ression and leukocyte infiltration in the liver tissues *>otanidou et al., 1441-.
?urthermore, it was demonstrated that luteolin su!!ressed nuclear factor *&?--kB
activation via inhibition of both myeloid differentiation marker *$y5- CC and
toll,interleukin-2 rece!tor domain-containin ada!ter !rotein *T()?--de!endent
sinalin !athways of toll-like rece!tors *TL(s- *6hen et al., 1447E Lee et al., 1443-.
)n a !revious study, we established a ut inflammation in vitro model in which an
in-testinal e!ithelial cell line, 6aco-1 cells, was !laced in a transwell at the a!ical side
and a macro!hae cell line, (.;1D7.< cells, was !laced at the basolateral side
*Tanoue et al., 144C-. ;hen the (.;1D7.< cells were stimulated with L#S, )L-C and
T&?-c secretion increased. This ut inflammation model was a!!licable in the search
for anti-inflammatory factors aainst intestinal inflammation *Tanoue et al., 144C-. )n
the !resent study, to examine whether luteolin exerts anti-inflammatory effects, we
evaluated its ef-fect by usin an in vitro ut inflammation model consistin of 6aco-
1,(.;1D7.< cells stimulated with L#S.
?. L*TEOLIN S*PPRESSES IL%L .RNA E=PRESSION OF
Ca&!%? CELLS IN I# 1I$R! G*T INFLAMMATION
MODEL
)L-C is a chemokine that is inordinately secreted by the intestinal e!ithelial cells of
!atients with )B5, inducin excessive recruitment and transmiration of neutro!hils
into in-flamed tissues followed by inGury of the e!ithelium *Struyf et al., 1440-. To
test whether luteolin exerts a su!!ressive effect on the chemokine ex!ression of
intestinal e!ithelial cells, we assessed the effect of luteolin treatment on the )L-C
m(&. ex!ression of 6aco-1 cells in in vitro ut inflammation model. Luteolin *244
X$- was added to the a!i-cal com!artment in this model for 9 h, and thereafter, L#S
was added to the basolateral side at a final concentration of 2 n ml
-2
, and the cells
were incubated for an additional 9 h. Budesonide, a thera!eutic dru used for65
!atients in the 8nited States and Euro!e *6han and Lichtenstein, 144D-, was also
added in the same way as a !ositive control that can inhibit )L-C m(&. ex!ression.
Treatment with 244 X$ luteolin resulted in the downreulation of )L-C m(&.
ex!ression in 6aco-1 cells, with the su!!ressive rate reachin almost the same level as
that of budesonide *?iure 27.2*a--. $oreover, it was demonstrated that luteolin *244
X$- inhibited )L-C m(&. ex!ression in 6aco-1 cells at a lower concentration
com!ared to curcumin *204 X$-.
Fi"#r( >D.> The su!!ressive effect of luteolin on )L-C m(&. ex!ression and T&?-c
!roduction in in vitro ut inflammation model. Luteolin *244 X$- and curcumin *204 X$-
were added into the a!ical com!artment of the in vitro ut inflammation model for 9 h.
Subse"uently, L#S was added to the basolateral com!artment at a final concentration of 2 n
ml
-2
, followed by incubation for an additional 9 h. )L-C m(&. ex!ression in 6aco-1 cells was
detected by (T-#6(. T&?-c !roduction in the basolateral com!artment was determined by
an L313 cytotoxicity assay. Ialues re!resent the meansSE *ne9-.
l
#V4.42 com!ared with
the controls stimulated with L#S alone.
B. L*TEOLIN S*PPRESSES TNF% SECRETION FROM
RAW?GD.K CELLS IN I# 1I$R! G*T INFLAMMATION
MODEL
Lamina !ro!ria mononuclear cells from !atients with 65 s!ontaneously secreted
T&?-c, and then the newly !roduced T&?-c caused e!ithelial barrier destruction
*Nareie et al., 1442-. To examine the effect of luteolin on the !roinflammatory
cytokine !roduc-tion of intestinal immune cells, which were se!arated from the ut
lumen by an intestinal e!ithelial monolayer, luteolin was added to the a!ical
com!artment. .s shown in ?iure 27.2*b-, luteolin decreased the T&?-c !roduction
from (.;1D7.< cells com-!ared with L#S treatment, indicatin that luteolin exerts a
su!!ressive effect on the !roinflammatory cytokine secretion of intestinal immune
cells. ;e !reviously demon-strated that T&?-c secretion is essential to u!reulate the
)L-C m(&. ex!ression in 6aco-1 cells in this ut inflammation model *Tanoue et al.,
144C-. $oreover, the addi-tion of luteolin *244 X$- to the a!ical com!artment of
6aco-1 sinle culture model did not su!!ress the increase in )L-C m(&. ex!ression
induced by stimulation of recom-binant T&?-c from the basolateral side. These
results suest that the su!!ressive effect of luteolin on the )L-Cm(&. ex!ression of
6aco-1 cells shown in ?iure 27.2*a- occurs mainly throuh inhibition of T&?-c
secretion from L#S-stimulated (.;1D7.< cells.
D. L*TEOLIN S*PPRESSES N*CLEAR FACTOR%KAPPAB
TRANSLOCATION INTO THE N*CLE*S OF RAW?GD.K
CELLS IN I# 1I$R! G*T INFLAMMATION MODEL
T&?-c ene ex!ression is mainly reulated by nuclear factor-ka!!aB *&?-gB-
*Brown et al., 144C-. 'ence, the effect of luteolin on &?-gB activation in (.;1D7.<
cells was examined in in vitro ut inflammation model. &?-gB is a com!lex
com!osed of !D0 and !04 *$ukaida et al., 2337.- and translocates to the nucleus in
the !resence of stimulation such as by L#S. )mmunofluorescence stainin was carried
out to observe &?-gB !D0 translocation into the nucleus. #ro!idium iodide *#)-
stainin a!!eared red, and &?-g B !D0 stainin a!!eared reen. .s shown in ?iure
27.1*a-, &?-gB !D0 was locali/ed mainly in the cyto!lasm in the absence of L#S
stimulation. )n the !resence of L#S *2 n ml
-2
- stimulation, &?-gB nuclear
translocation was observed in (.;1D7.< cells *?iure 27.1*b--, and the
nuclear,cyto!lasmic ratio was increased com!ared with that of
Fi"#r( >D.? The su!!ressive effect of luteolin on &?-gB nuclear translocation in
(.;1D7.< cells stimulated by L#S in in vitro ut inflammation model. $edium *a, b- or 244
X$ luteolin *c- was added into the a!ical com!artment of the in vitro ut inflammation model
for 9 h. Subse"uently, L#S *b, c- was added to the basolateral com!artment at a final
concentration of 2 n ml
-2
, followed by incubation for an additional 94 min. The ratio of
nuclear to cytosolic &?-gB was !erformed accordin to the method of &oursadehi et al.
*144C-. Ialues re!resent the meansSE *ne9-.
l
#V4.42 com!ared with control stimulated with
L#S alone.
cells without L#S stimulation. 'owever, the addition of luteolin to the a!ical
com!art-ment downreulated &?-gB translocation into the nucleus of (.;1D7.<
cells *?iure 27.1*c--. These results indicate that the su!!ressive effect of luteolin on
T&?-c m(&. ex!ression occurs throuh the inhibition of L#S-induced &?-gB
activation in (.;1D7.< cells.
E. L*TEOLIN IS TRANSPORTED ACROSS THE Ca&!%?
CELL MONOLAYER IN I# 1I$R! G*T INFLAMMATION
MODEL
The results shown in ?iure 27.2 indicate that a!ical treatment with luteolin
induced downreulation of ex!ression of inflammation markers in (.;1D7.< cells
located be-neath the 6aco-1 cell monolayer. To evaluate the effects of luteolin
treatment on the 6aco-1 cell monolayer, luteolin *244 X$- was added to the a!ical
com!artment of the 6aco-1 cell sinle culture transwell system for D h. '#L6
analysis of the basolateral su!ernatant revealed that luteolin alycone and its
metabolites *lucuronides,sulfates- were !resent in the solution at concentrations of7
and 24 X$, res!ectively. $oreover, direct treatment of luteolin *7 X$- to the L#S-
stimulated (.;1D7.< cells was sinify-cantly inhibited T&?-c !roduction, but its
lucuronide did not. These results suest that luteolin was trans!orted across the
intestinal e!ithelial monolayer and that the aly-cone released from the monolayer
affected directly intestinal immune cells to !revent &?-gB activation associated with
some cytokine !roductions.
G. DISC*SSION
)t has been re!orted that the antioxidant luteolin is a food factor that exhibits anti-
inflammatory effects *'ouee et al., 1440E >im and :obin, 1440E >otanidou et al.,
1441E Niyan et al., 144<-. .mon these effects, >arrasch et al. re!orted the
su!!ressive effect of luteolin on ut inflammation usin an )L-24
-,-
mouse colitis
model *>arrasch et al., 144<-. 'owever, this su!!ressive effect on ut inflammation
has not been studied sufficiently at the cellular level. )n the !resent study, we
examined the effect of luteolin in in vitro ut inflammation usin the model of 6aco-
1,(.;1D7.< cells stimulated with L#S established in our !revious investiation
*Tanoue et al., 144C-. )L-C is se-creted abundantly by the intestinal e!ithelial cells of
!atients with )B5 and causes ex-cessive recruitment and transmiration of neutro!hils
*Struyf et al., 1440-. Luteolin *244 X$- addition to the a!ical com!artment of this in
vitro ut inflammation model su!!ressed the )L-C m(&. ex!ression in 6aco-1 cells
*?iure 27.2*a--. )t has been re!orted that the intestinal immune cells of )B5 !atients
such as lamina !ro!ria macro!haes !roduce !roinflammatory cytokines includin
T&?-c and )L-D in abun-dance *>amada et al., 144C-. Luteolin *244 X$- addition to
the a!ical com!artment of this ut inflammation model su!!ressed the T&?-c
secretion from (.;1D7.< cells *?iure 27.2*b--, indicatin that luteolin also exerts
anti-inflammatory effects on intes-tinal immune cells. ;e !reviously demonstrated
that basolateral T&?-c is essential for the u!reulation of )L-C m(&. ex!ression in
6aco-1 cells *Tanoue et al., 144C-. )t was ascertained that the addition of luteolin to
the a!ical com!artment of 6aco-1 sinle culture model did not exert a su!!ressive
effect on the increase in )L-C m(&. ex!res-sion of 6aco-1 cells induced by
stimulation with recombinant T&?-c from basolateral side. These results suest that
the su!!ressive effect of luteolin on the )L-C m(&. ex!ression of 6aco-1 occurs
mainly throuh the inhibition of T&?-c secretion from L#S-stimulated (.;1D7.<
cells. )t has been re!orted that the transcri!tional activity of T&?-c and )L-D ene
were reulated by &?-gB *Brown et al., 144C-. ;e hy!oth-esi/ed that the ability of
luteolin to reulate &?-gB activation in (.;1D7.< cells ac-counts for the decrease in
T&?-c m(&. ex!ression. To test this hy!othesis, &?-gB !D0 translocation into the
nucleus of (.;1D7.< cells was observed by immunofluo-rescence stainin. )n the
!resence of L#S stimulation in the basolateral com!artment, &?-gB translocated into
the nucleus *?iure 27.1*b--. Treatment with luteolin inhibited &?-gB nuclear
translocation in (.;1D7.< cells stimulated with L#S *?iure 27.1*c--. These results
suest that the addition of luteolin to the a!ical com-!artment su!!ressed the
increase in T&?-c m(&. ex!ression in (.;1D7.< cells
Fi"#r( >D.B The su!!ression of &?-gB activation by luteolin in in vitro ut inflammation
model.
throuh inhibition of &?-gB nuclear translocation. ;hen 6aco-1 cells were treated
with 244 X$ luteolin from the a!ical side, its alycone was detected in the basolateral
solution *a!!roximately 7 X$-. $eanwhile, luteolin metabolites *lucuronides,
sulfates- were also detected *a!!roximately 24 X$-.$urota et al. also re!orted the
!er-meability of luteolin throuh the 6aco-1 monolayer *$urota et al., 1441-. .n
addition into the a!ical solution at an estimated concentration of luteolin *7 X$- was
sufficient to sinificantly su!!ress T&?-c !roduction from L#S-stimulated
(.;1D7.< cells. These results suest that luteolin !enetrated the intestinal e!ithelial
monolayer and that the released alycone acts directly on the intestinal immune cells.
6hen et al. also re!orted that luteolin reduced the 5&. bindin activity of &?-gB in
L#S-activated macro!haes *6hen et al., 144<-.
)n summary, a !utative mechanism for the su!!ressive effect of luteolin on )L-
Cm(&. ex!ression in 6aco-1 cells was suested+ *2- luteolin !enetrated the 6aco-1
monolayer as alycone and its metabolites, *1- its alycone inhibits &?-gB
translocation into the nucleus of (.;1D7.< cells, and *9- the increases in T&?-c
m(&. ex!ression and !roduction of (.;1D7.< cells are su!!ressed, resultin in
inhibition of the )L-C m(&. ex!ression of 6aco-1 cells *?iure 27.9-. These findins
suest that luteolin miht be an alternative aent to the chemical drus which
!ossess the dru-induced toxicity.
REFERENCES
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CHAPTER >E
H#.a$ Mi&r!bi!.( a$/ Dis(as(s)
A M(ta"($!.i& A33r!a&h
M.C. C!a/!
*
I G. D\A#ria

I A. Mira

I M.P. Fra$&i$!

*
)nstitute of .rochemistry and ?ood Technoloy, S!anish &ational (esearch
6ouncil *).T.-6S)6-, Ialencia, S!ain

:oint 8nit of (esearch in Henomics and 'ealth 6entre for #ublic 'ealth
(esearch *6S)S#-, Ialencia, S!ain

8niversity of 6alifornia, $erced, 6., 8S.


ABBREVIATIONS
AAD .ntibiotic associated diarrhea
CD 6rohn%s disease
GIT Hastrointestinal tract
IBD )nflammatory bowel disease
IBS )nflammatory bowel syndrome
NGS &ext-eneration se"uencin
>. H*MAN MICROBIOTA
>.>. Mi&r!bia Di+(rsity !' th( H#.a$ Mi&r!bi!ta
The human astrointestinal tract *H)T- is inhabited by a com!lex and dynamic
!o!ula-tion of around 044M2444 different microbial s!ecies, which exhibits lare
variation amon individuals, in relation to internal and external factors, such as
enetic factors, ae, diet, and health, and remains in a com!lex e"uilibrium. .lthouh
the exact com-!osition of the microbiota is not known, advances in enomic
technoloies have recently beun to unravel our microbial !artners. )t is estimated
that each individual houses at least 2D4 such s!ecies from a consortium of 2444M2204
!revalent bacterial s!ecies *Win et al., 1424- whose collective enome *microbiome-
contains at least 244 times as many enes as the human enome. )t is known that
about <0R of the ut microbiome is covered by already known and dominant !hyla
*.ctinobacteria, ?irmicutes, and Bacteroidetes-, while about 10R is still unknown or
very little investiated *Ley et al., 1440-. The micro-biota has beun to receive
rowin attention in recent years, because a remarkable relationshi! between
astrointestinal disorders and nonintestinal diseases has been iden-tified. .n ade"uate
balance of the microbiota !lays a critical role in maintainin the health status of the
host. The !resence of bacterial !athoens or antibiotic treatments may alter the
intestinal bacterial homeostasis *microbiota com!osition and activity-, leadin either
to increased risk of disease or to s!ecific diseases.
>.?. H#.a$ Mi&r!bi!.( A&^#isiti!$ a$/ D(+(!3.($t
The develo!ment of the human microbiome is a com!lex !rocess that has been
tradi-tionally assumed to start at birth. 'owever, microbial coloni/ation of the human
body may bein earlier throuh trans!ort of maternal bacteria via the bloodstream to
the !la-centa, from where they could reach the umbilical cord or the amniotic fluid,
which is constantly bein swallowed by the fetus *:imene/ et al., 144CE Ialles et al.,
1421-. .t birth, the neonate is oin to be ex!osed to an avalanche of new microbes.
2DS r(&. !yrose"uencin has revealed that the bacterial communities from newborn
babies shortly after delivery are "uite similar across body habitats. Iainally
delivered infants ac"uire bacterial communities resemblin their own mother%s
vainal microbiota, dom-inated by Lactobacillus$ Prevotella, or !neathia, whereas
6esarean section infants ac"uire bacterial communities similar to those found on the
skin, dominated by !taphylococcus, Corynebacterium, and Propionibacterium
*5ominue/-Bello et al., 1424-.
$icrobiome develo!ment in the H)T has been far more studied than in other body
habitats. Throuhout the first year of life, the H)T microbiota chanes continuously
and increases in diversity until reachin an adultlike com!osition. This !rocess will be
affected by feedin habits, health status, and sanitary conditions. The early H)T
microbiota is often dominated by one or a few enera, namely %scherichia$
Clostridium$ acteroides, or ifidobacterium. .mon breast-fed infants,
ifidobacterium-dominated microbiotae are more fre"uent than amon formula-fed
infants, but other com!ositions are also common. . lare shift in microbiota
com!osition accom!anies the introduction of solid foods into the diet, althouh the
s!ecific taxa !resent before and after this !oint may vary *>oeni et al., 1422E #almer
et al., 144<E #enders et al., 144DbE Iaisham!ayan et al., 1424E Ialles et al., 1421-.
?unctional characteri/ation of the infant microbiome las behind studies of
!hyloe-netic com!osition, but recent metaenomic analyses have initiated this task
for H)T microbiota develo!ment. )n s!ite of taxonomical dis!arities, the infant H)T
ra!idly ac"uires a functional ene re!ertoire that is dominated by carbohydrate
metabolism enes and is broadly analoous to that of the adult. S!eciali/ed functional
re!ertoires may exist at different staes, as the earliest microbiota can be enriched in
enes facilitatin lactate utili/ation, whereas solid foods may !romote enrichment in
enes enablin uti-li/ation of a larer variety of carbohydrates, vitamin biosynthesis,
and xenobiotic dera-dation. 'owever, enes involved in !lant !olysaccharide
metabolism are also detected in the microbiota before the introduction of solid foods
*>oeni et al., 1422E >urokawa et al., 144<E Iaisham!ayan et al., 1424E Ialls et al.,
1421-.
>.B. F#$&ti!$s !' th( H#.a$ Mi&r!bi!ta
The human intestinal microbiota !lays an im!ortant and com!lex role in the
mainte-nance of host health and can be identified as an active Soran,% which is
involved in different !rocesses such as the im!rovement of nutrient bioavailability
and deradation of nondiestible dietary com!oundsE the su!!ly of new nutrientsE and
the removal of harm-ful, toxic, and nonnutritional com!ounds from the metabolism
and diet. These metabolic functions have im!ortant im!lications for human health and
nutrition. The ut micro-biota is essential for !rocessin dietary !olysaccharides, thus
affectin enery harvest from the diet. )t !rovides additional enery in the form of
short-chain fatty acids, includin acetate, !ro!ionate, and butyrate. $oreover, the
microbiota is involved in fat storae in the host *BLckhed et al., 1447-. )n addition,
several members of the intestinal micro-biota can im!rove the absor!tion of calcium,
manesium, and !hos!horus and can !ro-duce vitamins, mainly vitamin > and also
some B vitamins, and !rovide them to the host. The microbiota is re!orted to
contribute to human !rotein homeostasis. The intestinal immune system constitutes
the !rimary immune oran of the human body and an essen-tial element of host
defense aainst !athoenic microoranismsE it !rovides an im!ortant stimulus for
develo!ment of the host immune system and reulates innate and ada!tive immunity
*)solauri et al., 144C-.
>.D. A$aysis !' H#.a$ Mi&r!bi!ta) M(ta"($!.i&s
The com!osition of the microbiota was initially ins!ected by means of
microsco!ic observations and culture techni"ues. &umerous more recent studies have
sam!led the human H) microbiota by means of 2DS r5&. analyses. These analyses
have confirmed that Bacteroidetes and ?irmicutes are often the most common !hyla in
the H)T, but they have unearthed that a!!roximately <0R of the !hyloty!es *i.e.,
uni"ue 2DS se"uences- recovered corres!ond to hitherto undescribed oranisms.
>nowlede about the diversity of oral bacteria is even more meaer. Studies of oral
microbial diversity by molecular techni"ues are relatively recent, and #6( and
subse"uent clonin of the 2DS r(&. ene have shown the !resence of bacterial
rou!s !reviously thouht to be exclusive to other environments such as marine
sediments *#aster et al., 1442-. )n addition, the analysis of oral microbial diversity in
healthy individuals and in !atients with different !atholoies has !ointed out some
s!ecies that may be associated with the develo!ment of disease. Similar a!!roaches
have been followed to unravel bacterial diversity in other human niches such as the
skin, the vaina, or the res!iratory tract. $ore recent a!!roaches have made use of
!yrose"uencin to sinificantly increase the number of 2DS r(&. se"uences obtained
in #6(-am!lification studies. This a!!roach obviates the need for clonin and
ty!ically !rovides thousands of reads of the 2DS r(&. ene !er sam!le, !rovidin an
un!recedented level of detail.
.lthouh 2DS r5&. analyses have !rovided a wider re!resentation of the human
microbiota than culture-based techni"ues, this a!!roach cannot o beyond a!!raisin
bacterial diversity as it !rovides no information on the codin ca!abilities of the
detected s!ecies. Beyond 2DS studies, metaenomic techni"ues can !rovide data
elucidatin, not only the !hyloenetic !ositions but also the codin ca!abilities of the
members of a iven community. Lare-scale shotun se"uencin !roGects aimin at
this bacterial assemblae have recently been undertaken at several institutions
*>urokawa et al., 144<- and have revealed the existence of common ene sets in
human metaenomes, establishin stron links between the detected ene re!ertoires
and the !hysioloical functions of the micro-biota. 5es!ite the clonin bias, the
clonin metaenomic a!!roach has the enormous advantae of allowin functional
analysis of the inserted 5&., such as screenins for s!e-cific functions. ?or instance,
screenins for antimicrobial resistance in metaenomic fos-mid libraries have
indicated that the ut and oral cavity microbiotae are an im!ortant reservoir of
antibiotic resistance enes *Ialls et al., 1421-. ;hole enomes have also been
obtained for some of the most im!ortant cultivable H), and oral inhabitants and
numerous others will be se"uenced as !art of the 'uman $icrobiome #roGect *&elson
et al., 1424-. The availability of these com!lete reference enomes will reatly
enhance our abilities to inter!ret the results of lare-scale metaenomic !roGects.
&ext eneration se"uencin *&HS- techni"ues, !articularly 707-!yrose"uencin
and )llumina, now allow direct se"uencin of the metaenomic 5&. without the need
for clonin. Thus, the #6( bias and clonin bias can be circumvented, and these
a!!roaches, with their relative sim!licity and affordability, can be used to !rocure
lare numbers of data sets !rovidin information on the !hyloenetic diversity,
codin ca!-abilities, and ene ex!ression !atterns of the microbiota under different
conditions. The latter deserves !articular attention, as the direct !yrose"uencin of
c5&. sam!les may !rovide information about the active com!onent of the microbial
community and the enes ex!ressed under iven conditions *Hosalbes et al., 1422-.
Hiven the absence of !oly-. tails in bacterial m(&., the enrichment of (&.
sam!les in m(&. remains a technical challene, but once it is solved, the
metatranscri!tomic a!!roach will likely be a !romisin stratey to study ene
function within such a com!lex community. 6ur-rently, different metaenomic and
metatranscri!tomic initiatives to study the human microbiome are under way,
includin shotun, fosmid libraries, and direct !yrose"uen-cin a!!roaches. This
information will be invaluable for understandin the am!le bio-loical roles of the
microbial communities in human health and for evaluatin the health risks associated
with disturbances of the ecoloical system.
?. MICROBIOME AND H*MAN HEALTH
)n recent years, the increase in microbiota-related research has made !ossible
im!ortant advances toward establishin the identity of s!ecific microbes and
microbial rou!s or microbial molecules contributin to various as!ects of host
!hysioloy and health. Studies on human microbiota should include microbial
ecoloy and analysis of the com-!lex metabolism of the microbial community, as well
as various hostMmicrobial interac-tions occurrin at the interface between microbes
and host intestinal e!ithelia. Such studies should lead to an understandin of the
im!act of the microbiota on human health and disease. 6oncurrently, host factors
involved in various as!ects of develo!ment and maturation tareted by the microbiota
have been identified.
. balance amon the microbial rou!s !resent in the human ut is crucial for
main-tainin health. ;hen this balance is disturbed, the hostMmicrobe relationshi! can
!roress toward a disease state. The im!ortance of the microbiome in relation to
human disease has been stressed by a recent &ational )nstitutes of 'ealth initiative,
the S'uman $icro-biome #roGect,% which aims at assessin how chanes in the
microbiome correlate with human disease usin enomic and metaenomic
se"uencin *Sekirov et al., 1424-.
?.>. Ora Ca+ity a$/ St!.a&h Dis(as(s
The oral microbiota is extremely diverse. Studies based on molecular a!!roaches
such as the clonin of #6(-am!lified 2DS r(&. enes have identified over 944
!hylorou!s, and the total number of s!ecies has been estimated to be over 044
*#aster et al., 1442-. $ost of these s!ecies are considered commensal, but an
imbalance in !hysicochemical conditions *e.., a shift in !'- can ive rise to an
ecoloical chane favorin the rowth of !athoenic s!ecies. The incidence of oral
diseases caused by bacteria *dental caries, inivitis, and !eriodontitis- amon the
Guvenile and adult !o!ulation is dramatic. The ;orld 'ealth Orani/ation estimates,
for instance, that dental caries affects D4M34R of children of school ae. )n addition, it
has to be considered that the oral cavity is the entry !oint for bacteria into the H) tract,
and bad oral health has im!lications for other diseases such as cancer or astric ulcer
*.bnet et al., 1442-.
Hiven the dramatic effects of oral diseases on human health, it may seem
sur!risin that no efficient treatments have yet been develo!ed. This is !robably
related to the fact that the study of oral diseases is com!licated because of several
factors, mainly their com-!lex etioloy *at least three s!ecies have been shown to be
involved in !eriodontal dis-ease-, the difficulty in culturin *over 04R of oral bacteria
cannot be cultured by conventional methods-, and the ecosystem%s com!lexity
*between 144 and 044 bacterial s!ecies-. ;ith the arrival of enomics, metaenomics,
and hih-throuh!ut 5&. se-"uencin, these im!ediments can be eliminated, and
new strateies with extraordinary !otential to combat oral disease can be develo!ed.
The a!!lication of &HS to the study of microbial diversity has allowed massive
se-"uencin of 2DS r(&. am!licons, ivin estimates of several thousand s!ecies !er
!er-son. 'owever, the shorter se"uence lenth of these techni"ues may be inflatin
estimates of diversity, as a recent lare-scale !roGect manaed to se"uence 2277<
nearly full-lenth 2DS r(&. am!licons by clonin and subse"uent Saner se"uencin
*Bik et al., 1424-, reducin the estimates of s!ecies richness to fewer than 944 s!ecies
for 24 individuals. )nterestinly, the a!!lication of hih-throuh!ut se"uencin to
bacterial sam!les from saliva and dental !la"ue su!!orts the idea of a Shealthy
microbiome% formed by microbial s!ecies that consistently a!!ear to be associated to
the absence of disease *Naura et al., 1443-. This has recently been corroborated by the
metaenomic analysis of eiht dental !la"ue sam!les of individuals varyin in health
status *.lcara/ et al., 1421-, which suested a list of microbial s!ecies with a
!robiotic !otential to !revent cavities and !romote oral health.
The hih-throuh!ut se"uencin a!!roach to 2DS r(&. #6( !roducts has also
been a!!lied to stomach sam!les *Bik et al., 144D-. Hiven the extreme environmental
conditions *e.., standard !' values around 2.0M1.4-, it had been assumed that
microbial diversity in the stomach was low, as traditional culturin techni"ues
suested. 'owever, se"uence analysis of 19 astric endosco!y bio!sy sam!les
revealed 21C !hyloty!es, which included !reviously uncharacteri/ed s!ecies. This
un!recedented level of diversity su-ests a !otential role of the stomach microbiota
in human health and disease still to be discovered.
?.?. I$t(sti$a Dis(as(s
Hastrointestinal microbial homeostasis is im!ortant to maintain healthy conditions
in the intestine. The normal microbiota contributes to avoidin coloni/ation by
!athoenic bacteria. . breach of the microbial e"uilibrium *dysbiosis- can ensue from
various factors such as mucosal inflammation releasin microbe-killin factors, loss
of key s!ecies, and nutrient imbalances *Stecher and 'ardt, 144C-. Several
astrointestinal !atholoies such as inflammatory bowel disease *)B5-, inflammatory
bowel syndrome *)BS-, obesity, var-ious forms of colitis, and even autism have been
linked to disru!tions in human-associated microbiota or alterations of the intimate
cross-talk between these microbes and human cells *?ava and 5anese, 1422-.
8lcerative colitis and 6rohn%s disease *65- are two exam!les of )B5s still stronly
associated to an abnormal functionin of host mu-cosal barriers and to a
malfunctionin of the immune system, althouh several rou!s of bacteria have been
!ro!osed as res!onsible. Both are characteri/ed by an ina!!ro!riate, exaerated, and
continuous activation of the mucosal immune system stimulated by the resident
astrointestinal microbiota. )n both cases, dysbiosis is observedE for exam!le, a
metaenomic a!!roach a!!lied on 6rohn%s disease !atients hihlihted a reduction in
?irmicutes *!articularly Clostridium leptum- and an increase in some Hram-neative
bac-teria *#or!hyromonadaceae- often res!onsible for inflammatory !rocesses
*$anichanh et al., 144DE Iander!loe et al., 1424-. )n the case of Mycobacterium
avium subs!. paratuberculosis, also thouht to be res!onsible for 65, the association
to disease de!ends on a co-occurrence of host enetic susce!tibility and certain
resistance mechanisms of these bacteria *Behr and >a!ur, 144C-. The causes of )BS
are still not clear, but it is acce!ted that this syndrome is related to an abnormal
distribution of ut microbiota *Ianner, 144CE =amini and #imentel, 1424-
accom!anied by an oversecretion of microbial oranic acids *Tana et al., 1424-.
.nother issue in intestinal diseases is re!resented by antibiotic associated diarrhea
*..5-. )n most cases, antibiotic treatments re!resent the standard for controllin
inflam-mations or infections, but they can be fre"uently followed by an overrowth of
commen-sal bacteria, which could be res!onsible for ..5. Clostridium difficile is
one of the most im!ortant nosocomial !athoens, and the most commonly dianosed
as res!onsible for an infectious hos!ital diarrhea known as C# difficile associated
diarrhea, which can rane from mild self-limitin diarrhea to severe !seudo-
membranous colitis *Sekirov et al., 1424-. .nother intestinal disease is colorectal
cancer, which is the third most com-mon cancer and seems to be stronly linked to
dietary habits. .n association between ut microbiota com!osition and risk of colon
cancer has been described *Hueimonde et al., 144<-.
?.B. A(r"i(s a$/ At!3i& Dis(as(
.to!ic diseases, includin ec/ema, food allery, hay fever, and asthma, are
chronic in-flammatory disorders that occur in susce!tible individuals because of
aberrant immune res!onses aainst common environmental antiens. Their
!revalence in ;estern socie-ties has risen dramatically in the !ast decades, reachin
about 94R of the adult !o!ulation *Huarner et al., 144D-. Environmental and lifestyle
chanes are !robably res!onsible for this rise, by forcin the immune system to
develo! within a context of ex!osure to mi-crobes distinct to the one in which it
evolved. This Shyiene hy!othesis% is su!!orted by the fact that infants who are more
ex!osed to microbes have a much lower incidence of ato!ic disease *Braun-
?ahrlander et al., 1441-. .lthouh the initial formulation of the hyiene hy!othesis
im!licated a lack of early infectious diseases in the inade"uate !rimin of the immune
system *Strachan, 23C3-, it is now thouht that !ro!er develo!ment of the H)T
microbiota may be the !rinci!al re"uirement for modulation of the immune system
and induction of tolerance *Huarner et al., 144DE >alliomLki et al., 1442E (autava et
al., 1447E (omanani, 1447-. This revision of the hyiene hy!othesis is sometimes
termed the Smicrobiota hy!othesis% of ato!ic disease *&overr and 'uffnale, 1440-.
&umerous studies have linked early ut microbiota to the develo!ment of ato!ic
dis-eases, but no s!ecific microbes have yet been identified with consistently harmful
or !ro-tective roles reardin ato!y *#enders et al., 144<-. Several studies indicate a
link between bifidobacteria deficiency and increased incidence of ato!y *>alliomLki
et al., 1442E SGoren et al., 1443-. 'owever, two lare !ros!ective caseMcontrol
studies have failed to confirm this association *$urray et al., 1440E #enders et al.,
144Da- and conflictin results have been obtained reardin the !rotective role of
different ifidobacterium s!ecies *SGoren et al., 1443-. The effects of other H)T
bacterial enera on ato!y develo!ment are even less clear. )n s!ite of their
immunoreulatory !ro!erties in vitro, lactobacilli have rarely been confirmed to exert
a !rotective role in children *BGorksten et al., 2333-. 6lostridia have been !ositively
associated with ato!ic disease in numerous studies but neatively in others
*>alliomLki et al., 1442E SGoren et al., 1443-. Some discre!ancies may be related to
reional differences, as ex!osure to microbes, microbiota develo!ment, and timin of
maturation of the immune system may differ amon countries.
.t the cellular and molecular level, the mechanisms by which the H)T microbiota
affects the develo!ment of immunotolerance are currently debated and !robably
include several different routes, involvin reconition of microbial antiens by
dendritic cells and other cells of the innate immune system, followed by the
stimulation or re!ression of different subsets of T cells and their com!lex web of
interactions *#enders et al., 144<E (autava et al., 1447E (omanani, 1447E SGoren et
al., 1443-. The resultin ratios of T-reulatory and T-hel!er cells of different ty!es
*Th-2, Th-1, and Th-2<- are likely to interate the effects of the H)T microbiota
interactions with different com!onents of the immune system to modulate the
am!litude and class of the immune res!onse. S!ecific alterations in the ratios of these
ada!tive immune cells can dereulate the immune balance in different directions,
!romotin either the onset of ato!ies or the develo!ment of autoimmune diseases
*#enders et al., 144<E (autava et al., 1447-.
?.D. Ob(sity a$/ O+(r0(i"ht
Obesity is viewed as one of the maGor !ublic health !roblems of today, and its
im!act is hihest in children. The develo!ment of metabolic com!lications associated
with obesity durin childhood tracks into adulthood and increases the risk for
autoimmune diseases as ty!e 1 diabetes and early metabolic and cardiovascular
diseases. )n addition to risk factors such as diet, lifestyle, decreased !hysical activity,
and ra!id weiht ain in infancy, some re!orts have suested that the ut microbiota
is an im!ortant factor affectin the re-ulation of host enery homeostasis and
adi!osity, as differences in microbial com!osition can ex!lain an increased ca!acity
of the obesity-associated microbiome to harvest enery from the diet *BLckhed et al.,
1447E Turnbauh and Hordon, 1443E Tsai and 6oyle, 1443-. . subse"uent
metaenomic study *Turnbauh and Hordon, 1443- with 207 individuals showed that
obesity was associated with a markedly reduced bacterial diver-sity, a relative
de!letion of Bacteroidetes, and a hiher !ro!ortion of .ctinobacteria com-!ared with
lean subGects. 'owever, lower ratios of ?irmicutes to Bacteroidetes in overweiht
human adults com!ared to lean controls have recently been re!orted *Schwiert/ et al.,
1424-. )n addition, diets based on a hih intake of !rotein and,or low intake of
carbohydrate or low fat consum!tion may alter microbial com!osition and activity in
the lare intestine and thus im!act ut health *6ani et al., 144<E 5uncan et al., 144C-.
Other studies have examined ut microbiota com!osition in human obesity and ty!e-1
diabetes and the im!act of weiht reduction on microbiota *Larsen et al., 1424E
Santacru/ et al., 1424-, some of which contest the link between the !ro!ortion of
Bacteroidetes and ?irmicutes and human obesity *5uncan et al., 144C-. )n addition, it
has been shown that infants with hih numbers of ifidobacterium and low numbers
of !taphylococcus in early life may be !rotected from weiht ain durin later life
*>alliomLki et al., 144C-. )nfants from women with normal weiht ain durin
!renancy showed hiher levels of bifidobacteria than those from women with
excessive weiht ain, suestin a !otential role for ifidobacterium on infant
microbiota and weiht de-velo!ment *6ollado et al., 1424-. . recent study re!orts
that hih numbers of bifidobac-teria may correlate !ositively with normali/ation of
inflammatory status and im!roved lucose tolerance, and lucose-induced insulin
secretion *6ani et al., 144<-. .ltoether, the com!osition of the obese microbiome is
still "uestionable and more scientific evi-dence is needed to elucidate the relationshi!
between the ut microbial com!osition and metabolic diseases includin the
develo!ment of obesity. $icrobiota modification by use of !robiotics may offer new
directions for !reventive and thera!eutic a!!lications in reducin the risk of
overweiht and obesity.
?.E. Diab(t(s
5iabetes mellitus is a rou! of metabolic diseases characteri/ed by hih blood
suar *lucose- levels that result from defects in insulin secretion, action, or both. .
link be-tween ut microbiota com!osition and activity and the manaement of
lycemia asso-ciated with overweiht and diabetes has been re!orted *5el/enne and
6ani, 1422-. )n ty!e-2 diabetes, a combination of multi!le factors leadin to the
develo!ment of autoimmunity a!!ears to include an aberrant intestinal microbiota, a
leaky intestinal mucosal barrier, and an altered intestinal immune res!onsiveness
*&eu et al., 1424-. Ty!e 1 diabetes is a metabolic disease, the !rimary cause of which
is obesity-linked in-sulin resistance. (ecent research *Larsen et al., 1424- has re!orted
that ty!e 1 diabetes is associated with com!ositional chanes in the intestinal
microbiota. The relative abun-dance of ?irmicutes was sinificantly lower, while the
!ro!ortion of Bacteroidetes and #roteobacteria was somewhat hiher in diabetic
!ersons com!ared to nondiabetic counter!arts. .ccordinly, the ratios of
Bacteroidetes to ?irmicutes !ositively correlated with reduced lucose tolerance.
These results, assumin that diabetes and im!aired lucose tolerance are linked to
obesity, are in areement with the recent evidence obtained for overweiht !ersons by
Schwiert/ et al. *1424-.
2.-. 7kin 8iseases
The skin is the human body%s larest oran, coloni/ed by a diverse milieu of
micro-oranisms, which de!ends on different factors such as eora!hical location,
host, and environment *Hrice and Sere, 1422-. 'uman skin can be considered as an
ecosystem with a delicate balance between host and microoranisms, and disru!tions
in the balance can result in skin disorders or infections. .s defined by 2DS r(&.
metaenomic se"uencin, skin microbiota falls into four !hyla+ .ctinobacteria,
?irmicutes, Bacteroi-detes, and #roteobacteria, which also constitute the microbiota of
the inner mucosal surfaces *the astrointestinal tract and oral cavity-, but are found in
different !ro!ortions. . common feature of ut and skin microbial communities
seems to be low diversity at the !hylum level but hih diversity at the s!ecies level
*Hrice and Sere, 1422-. Some skin disorders have been shown to be related to the
microbiota. ?or instance, Seborrhoeic dermatitis is associated with reduced levels of
MalasseBia, and Propionibacterium acnes is as-sociated with acne, an inflammatory
disorder of the !ilosebaceous unit. .to!ic disease, as mentioned above, is also related
to alterations in microbiota com!osition that affect the immune system. 5ysreulation
of the skin immune res!onse is a!!arent in several skin disorders such as !soriasis
and ato!ic dermatitis, but how this dysreulation affects and,or results from chanes
in the microbiota remains unclear. Other skin !roblems are chronic wounds that affect
diabetic, elderly, and immobile individuals. Burn wounds commonly become infected
with !treptococcus pyogenes$ %nterococcus, or Pseudomonas aeruginosa and can
also become infected with funi and,or viruses.
?.K. Oth(r Dis(as(s
The microbial balance in the uroenital tract and vaina can be disturbed by
-ardnerella, Peptostreptococcus$ Prevotella, or aerobic cocci, and numerous
oranisms can cause urinary tract infections and bacterial vainosis *>irGavainen et
al., 1443E &elson et al., 1424E Lamont et al., 1422-. )n addition, microbial com!osition
differences have been re!orted in ')I-infected subGects com!ared to ')I-
seroneative rou!s *S!ear et al., 1422-.
&ew evidence on ae-related diseases im!licates microbial dysbiosis as a !otential
risk factor *Tiihonen et al., 1424-. . weakened immune system has also been linked
to an increased risk for chronic diseases such as autoimmune diseases, atherosclerosis,
ty!e 1 diabetes, and even .l/heimer%s disease. On the other hand, common enetic
and immunoloic mechanisms underlie the coincidence of inflammation in the bowel
and Goints. . recent study re!orts the association between bacterial astroenteritis and
the risk of develo!in arthritic sym!toms *Har et al., 144C- and aberrancies in the
microbiota have been identified in arthritis *Toivanen, 1449- and s!ondylitis
*Stebbins et al., 1443-.
The ability of microbiota to communicate with the brain and modulate behavior is
emerin as a conce!t in health and disease *'eiGt/ et al., 1422-. E!idemioloical
studies have indicated an association between neurodevelo!mental disorders, such as
autism and schi/o!hrenia, and microbial infections durin the !erinatal !eriod, but
dysbiosis in the ut microbiome could also lead to dysreulation of immune res!onses
both in the ut and in distal effector immune sites such as the central nervous system.
(ecent findins suest that alterin certain bacterial !o!ulations !resent in the ut
can lead to a !roinflammatory condition that may result in the develo!ment of human
multi!le sclerosis *Ochoa-(e!Kra/ et al., 1422-. )n addition, chanes in the
microbiome have been associated with autism syndrome toether with other enetic
and environmental factors *Ochoa-(e!Kra/ et al., 1422E Sekirov et al., 1424-.
?urthermore, mental !roblems, includin schi/o!hrenia, anxiety, stress, or de!ression,
are fre"uently associated with other disorders in which microbiota disturbances may
!lay a role *&eufeld and ?oster, 1443-. 6hanes in the ut microbiome have also been
associated to maGor de!ressive disorder showin reduced relative !ercentaes of
s!ecific commensal !o!ulations such as Lactobacilli and ifidobacterium *5owlati et
al., 1424-. .lterations of intestinal microbiota also seem to !lay an im!ortant role in
induction and !romotion of liver damae !rores-sion *6esaro et al., 1422-. Bacterial
overrowth, immune dysfunction, alteration of luminal factors, and altered intestinal
!ermeability are all involved in the !athoenesis of com!lications of liver cirrhosis,
such as infections, he!atic ence!halo!athy, s!ontane-ous bacterial !eritonitis, and
renal failure.
?inally, we are beinnin to understand that ex!osures to microbes before conce!-
tion, durin estation, and in the neonatal !eriod have !rofound effects on the
develo!-in immune system. (ecent studies and evidence are su!!ortin the Searly
!rorammin hy!othesis% in which the microbeMhost interactions will be a critical
factor influencin future human health and the develo!ment of immune-mediated
disease.
B. MICROBIOME AND PROBIOTICS
There is a rowin interest in beneficial microbes with s!ecific functions in the
human microbiome, which can be used in foods or su!!lements that increase human
health and !revent and treat diseases. . !robiotic has been defined as a Slive micro-
oranism that when administered in ade"uate amounts confers a health benefit on the
host% *?.O,;'O, 1441-. The most common !robiotic bacteria are s!ecies belonin
to Lactobacillus$ %nterococcus$ ifidobacterium, and also some Propionibacterium
strains.
5urin the !ast few decades, a lare number of studies have been conducted to
assess !robiotics, usin different formulas and with s!ecific !ur!oses of !reventin or
treatin diseases. Some beneficial intestinal effects of certain !robiotic strains can be
considered well established, which has led to the !ublication of recommendations for
their use in clinical !ractice *?loch and ;alker, 144C-. Therefore, modulation of an
unbalanced indienous microbiota forms the rationale of !robiotic thera!y *6ollado et
al., 1443- and o!ens new !revention and treatment !ossibilities on diseases in which
dysbiosis of the microbiome !lays relevant roles. So far, the metaenomic data
available from human intervention studies with !robiotics and their im!act on the
microbiome is limited. &evertheless, some studies are already underway and an
ex!losion of such data is ex!ected in the near future. )ncor!oratin such data with
studies on host ene res!onse to chanes in the microbiota durin !robiotic
administration will allow us to understand microbeMmicrobe and hostMmicrobe
interactions, and therefore, the influence of the ut microbiota on our !hysioloy and
well-bein.
D. CONCL*SIONS
The develo!ment of several diseases can be related to microbiota aberrancies, and
!ro-biotics may re!resent the best o!tion to reestablish microbial balance and host
health. $onitorin of microbiota !ersistence and effect is a need in modern ut
microbioloy. $etaenomic and bioinformatic methodoloies will offer a com!lete
!icture of micro-bial diversity at an affordable cost. . not-so-far future is envisaed
in which assessin microbial diversity in the ut, oral cavity, vaina, or skin will be as
inex!ensive and fast as a standard blood or urine test. ;hen robust links between
disease and microbial com-!osition are ex!erimentally validated, metaenomic data
may likely be used to im!le-ment !ersonali/ed medicine a!!roaches, allowin for
better dianostics, !revention, and treatment of disease.
ACKNOWLEDGMENTS
.ll authors !artici!ated in !re!aration of the cha!ter. &one of the authors have
conflict of interests. $66 is the reci!ient of a S(amJn y 6aGal% research contract
*(=6-1424-40D27- from the S!anish $inistry of Science and )nnovation, S!anish
Hovernment. H5 is the reci!ient of a S$iuel Servet% research contract *6#43,44473-
from Instituto de !alud Carlos ))), S!ain. This work was su!!orted by ?un-6-?ood
6S5144<-444D9 and $icroen 6S51443-4444D from the 6onsolider-)nenio
!roram from the S!anish $inistry of Science and )nnovation.
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Ekstrom, E., 1443. .ltered early infant ut microbiota in children develo!in allery
u! to 0 years of ae. 6linical and Ex!erimental .llery 93, 02CM01D.
S!ear, H.T., Hilbert, 5., Landay, ..L., et al., 1422. #yrose"uencin of the enital
microbiotas of ')I-sero!ositive and -seroneative women reveals Lactobacillus
iners as the !redominant Lactobacillus S!ecies. .!!lied and Environmental
$icrobioloy << *2-, 9<CM9C2.
Stebbins, S.$., Taylor, 6., Tannock, H.;., Baird, $..., 'ihton, :., 1443. The
immune res!onse to au-toloous bacteroides in ankylosin s!ondylitis is
characteri/ed by reduced interleukin 24 !roduction. :ournal of (heumatoloy 9D *7-,
<3<MC44.
Stecher, B., 'ardt, ;.-5.5., 144C. The role of microbiota in infectious disease.
Trends in $icrobioloy 2D *9-, 24<M227.
Strachan, 5.#., 23C3. 'ay fever, hyiene, and household si/e. British $edical
:ournal 133 *D<24-, 2103M21D4.
Tana, 6., 8mesaki, =., )maoka, .., 'anda, T., >ana/awa, $., ?ukudo, S., 1424.
.ltered !rofiles of intes-tinal microbiota and oranic acids may be the oriin of
sym!toms in irritable bowel syndrome. &eu-roastroenteroloy and $otility 11 *0-,
021M023.
Tiihonen, >., Ouwehand, ..6., (autonen, &., 1424. 'uman intestinal microbiota
and healthy aein. .ein (esearch (eviews 3 *1-, 24<M22D.
Toivanen, #., 1449. &ormal intestinal microbiota in the aetio!athoenesis of
rheumatoid arthritis. .nnals of the (heumatic 5iseases D1 *3-, C4<MC22.
Tsai, ?., 6oyle, ;.:., 1443. The microbiome and obesity+ is obesity linked to our
ut flora[ 6urrent Has-troenteroloy (e!orts 22 *7-, 94<M929.
Turnbauh, #.:., Hordon, :.)., 1443. The core ut microbiome, enery balance and
obesity. The :ournal of #hysioloy 0C< *#t 2<-, 7209M720C.
Iaisham!ayan, #..., >uehl, :.I., ?roula, :.L., $oran, :.L., Ochman, '.,
?rancino, $.#., 1424. 6om!ar-ative metaenomics and !o!ulation dynamics of the
ut microbiota in mother and infant. Henome Bi-oloy and Evolution 1, 09MDD.
Ialls, =., Hosalbes, $.:., de Iries, L.E., .bellann, :.:., ?rancino, $.#., 1421.
$etaenomics and develo!-ment of the ut microbiota in infants. 6linical
$icrobioloy and )nfection 2C *7-, 12M1D. Iander!loe, (., #anaccione, (., Hhosh,
S., (ioux, >., 1424. )nfluences of intestinal bacteria in human inflammatory bowel
disease. )nfectious 5isease 6linics of &orth .merica 17 *7-, 3<<M339.
Ianner, S., 144C. The small intestinal bacterial overrowth irritable bowel
syndrome hy!othesis+ im!lica-tions for treatment. Hut 0< *3-, 2920M2912.
=amini, 5., #imentel, $., 1424. )rritable bowel syndrome and small intestinal
bacterial overrowth. :ournal of 6linical Hastroenteroloy 77 *24-, D<1MD<0.
Naura, E., >eiGser, B.:., 'use, S.$., 6rielaard, ;., 1443. 5efinin the healthy
Score microbiome% of oral microbial communities. B$6 $icrobioloy 3, 103.
CHAPTER >G
F!at( Pr!/#&ti!$ by La&ti& A&i/
Ba&t(ria
5.E. Lai9!
*
I G.S. /( Gi!ri
*,I
5.G. L(Ba$&
*
*
6entro de (eferencia !ara Lactobacilos *6E(EL. M 6O&)6ET-, TucumKn,
.rentina

8niversidad nacional de TucumKn, TucumKn, .rentina


ABBREVIATIONS
EI>R%F!r.i.i$!%THF 0,24-?ormiminotetrahydrofolate
E%F!r.y%THF 0-?ormyltetrahydrofolate
>R%F!r.y%THF 24-?ormyltetrahydrofolate
EI>R%M(th($y%THF 0,24-$ethenyltetrahydrofolate
EI>R%M(thy($(%THF 0,24-$ethylenetetrahydrofolate
E%MTHF 0-$ethyltetrahydrofolate
ALIMTA $ultitareted antifolate dru
B. Bifidobacterium
DHF 5ihydrofolate
DNA 5eoxyribonucleic acid
GIT Hastrointestinal tract
GM Henetically modified
GM%LAB Henetically modified lactic acid bacteria
GTP Huanidine tri!hos!hate
L. Lactobacillus
LAB Lactic acid bacteria
L&# Lactococcus
MA $icrobioloical assay
NICE &isin-controlled ex!ression
NTDs &eural tube defects
3ABA p-.minoben/oate
RDA (ecommended dietary allowance
RNA (ibonucleic acid
St. !treptococcus
s#bs3. Subs!ecies
THF TetrahydrofolateE 0,D,<,C-tetrahydrofolate
>. INTROD*CTION
?olate is an essential micronutrient that !artici!ates in the metabolism of all livin
thins. )t belons to the water-soluble B-rou! vitamins that also include thiamin,
riboflavin, niacin, !yridoxine, !anthotenic acid, biotin, and cobalamin. Each one of
these vitamins is chemically different and acts syneristically with other vitamins to
maintain homeo-stasis since they !ossess determinant functions in metabolic
!rocesses such as enery !ro-duction and erythrocyte synthesis. .lthouh this rou!
of vitamins !resent in many foods, they can be easily lost or destroyed durin cookin
and food !rocessin, reasons why their deficiency is !resent in many reions of the
world.
'uman life cannot exist without folate because this vitamin is involved in many
key functions of human metabolism. Tetrahydrofolate *T'?- and its derivates
*enerally rou!ed under the denomination folates- are vital cofactors to metabolic
en/ymes that !artici!ate in one-carbon transfer reactions *?iure 2D.2-. .s the maGor
carriers and donors of one-carbon units, folates are involved in a wide number of key
metabolic func-tions includin 5&. re!lication, re!air, and methylation and
biosynthesis of nucleic acids, some amino acids, !antothenate, and other vitamins.
Fi"#r( >G.> 6hemical structure of *a- folic acid *!teroyl-
L
- lutamic acid- and *b- native-
food folates *i.e., reduced and one-carbon-substituted forms of !olylutamates-.
?olate also has antioxidant !ro!erties, makin it im!ortant in the !rotection of
animal enomes by inhibitin the attack of free radicals on 5&., and it !lays a key
role in 5&. re!air and re!lication mechanisms.
5ue to the wide rane of re"uirements for folates by animals, low folate intake
has been associated with a number of health disorders such as cancer, cardiovascular
disease, and neural tube defects *&T5s-.
?olic acid is often confused with folates, bein the first molecule a synthetic form
of folate that does not exist in nature, but is commonly used in food fortification
*?iure 2D.2-. )n this cha!ter, the term Sfolate% is used to refer to the natural forms of
folates and Sfolic acid% to describe the chemical used in fortification !rotocols.
?. FOLATE METABOLISM AND BIOAVAILABILITY
?olates are three-!art molecules com!osed of a !terin rin, ap-aminoben/oate
*!.B.- core, and a lutamate tail. The one-carbon units can be found in different
oxidation levels and are areated to the &-0 of the !terin domain, &-24 of the
p.B. domain, or linked to both.
?olate biosynthesis in microoranisms *?iure 2D.1- im!lies the conversion of
HT# *uanidine tri!hos!hate-, in seven consecutive ste!s, to the bioloically active
cofactor T'?. Two condensation reactions take !lace in the T'? biosynthesis
!athway+ the liation of p.B. with 1-amino-7-hydroxy-D-hydroxymethyl-<,C-
dihydro!teridine to form dihydro!teroate and the reaction of lutamate with
dihydro!teroate to !roduce dihydrofolate *5'?-. p.B. is synthesi/ed usin the
!entose !hos!hate !athwayE in this way, 5-erythrose-7-!hos!hate is condensed with
!hos!hoenol!yruvate to finally !roduce chorismate. The chorismate serves as a
bride between aromatic aminoacid syn-thesis *try!to!han, !henylalanine, and
tyrosine- and p.B. !roduction. )n %scherichia coli, the chorismate is converted by
chorismate synthetase com!onents ) and )) *#abB and #ab., E6 D.9.0.C- into 7-
amino-7-deoxychorismate. #yruvate is then broken down by 7-amino-7-
deoxychorismate lyase *#ab6 E6 7.2.9.9C- to !roduce p.B.. ;ithout p.B., T'?
cannot be !roduced, and since T'? is essential as the donor and acce!torof one-
carbon rou!s *e.., methyl, formyl, methenyl, and methylene- in the biosynthe-sis of
!urines and !yrimidines, formylmethionyl t(&.
f$et
, and some amino acids, life
without this vitamin could not exist.
&atural folates include 0-methyltetrahydrofolate *0-$T'?-, 0-
formyltetrahydrofolate *0-formyl-T'?-, 24-formyltetrahydrofolate *24-formyl-T'?-,
0,24-methylenetetrahydro-folate *0,24-methylene-T'?-, 0,24-
methenyltetrahydrofolate *0,24-methenyl-T'?-, 0,24-formiminotetrahydro-folate
*0,24-formimino-T'?-, 0,D,<,C-tetrahydrofolate *T'?-, and 5'?. )n nature, folates
are found as !olylutamates with a !olylutamyl b-chain *normally containin 1M<
lutamate residues- linked to the T'? lutamateE however, !teroyl!olylu-
tamateswith u! to 22 lutamic acid residues can also exist in nature. #olylutamylated
folates
Fi"#r( >G.? Schematic re!resentation of folate biosynthetic !athway in lactococci and
lactobacilli.
are the !referred substrates for most of the folate-de!endent en/ymes *6ossins, 1444E
Scott et al., 1444-. On the other hand, folate trans!orters !resent in animals enerally
!refer the monolutamyl folate forms *Scott et al., 1444-. Themain intracellular
folates are !teroyl!en-talutamates, whereas !teroylmonolutamates aremore
common outside the cells. .ll folate forms are somewhat unstable in different
derees, mainly due to the liht-induced oxidative breakdown between !terin and
p.B. domain *Scott et al., 1444-.
?olic acid, obtained by chemical synthesis, differs from natural folate in many
as!ects+ *i- oxidation level *not reduced-, *ii- no substitutions are !resent in the !terin
and p.B. domain, and *iii- it can cause adverse side effects that currently !ut in
doubt their use in fortification !rorams.
;hen studyin vitamin and mineral u!takes, it is im!ortant to understand their
bio-availability, which is defined as the !ro!ortion of a consumed nutrient that
becames avail-able to metabolic or storae !rocesses in the body *?iure 2D.9-. )t has
been re!orted that dietary folate bioavailability could be affected by the lenth of its
!olylutamyl chain in natural folates. )n conse"uence, the !olylutamyl chain must be
removed, u! to the !roximal domain, by the b-lutamyl hydrolase en/yme or human
conGuase that is lo-cated in the brush border of the small intestine. This en/yme is
!resent in sufficient amounts and is not a limitin factor in folate absor!tion. Some
studies have suested that the !olylutamyl form is only D4MC4R bioavailable
com!ared to the monolutamyl folates, and this was confirmed in human trials
com!arin chemically synthesi/ed he!-talutamic folate with reular monolutamyl
folate *$else-Boonstra et al., 1447-.
Fi"#r( >G.B ?olate absor!tion. Schematic re!resentation of folate absor!tion throuh the
human intestine. The !olylutamyl native-food folates are first deconGuated by human
conGuase located in the brush border of the intestine. The monolutamyl forms are
trans!orted inside the enterocytes throuh the folate rece!tor and then absorbed throuh the
!ortal vein and delivered to the liver. Then the folates bind with c-lobulin and are
trans!orted to the tissues where they are used by cells.
'owever, different results were obtained in rats in which !olylutamyl folates
with lon chains *containin eiht lutamic acid residues- showed a!!arently hiher
bioavailability, com!ared with the folyl!olylutamates *LeBlanc et al., 1424b-. One
!ossible ex!lanation for these differences is that the en/yme carboxy!e!tidase )) from
rats, which is re"uired for the transformation of !olylutamyl folates in monolutamyl
folates, miht have more af-finity for lon !olylutamyl folates than for short
!olylutamyl folates. The monolutamyl folates are subse"uently absorbed into the
blood and trans!orted to the he!atic !ortal vein.
Bioavailability can also be affected by folate-bindin !roteins !resent in foods,
which miht increase the efficiency of folate absor!tion by !rotectin dietary folates
from ca!-ture by intestinal bacteria. Other interactions that can affect bioavailability
of folate in-clude the effects of food in the intestinal !' with !otential modification of
the conGuase activity, !resence of antaonists, intestinal chanes influenced by
dietary factors, "uela-tion, and factors that modify the astric em!tyin rate. 5es!ite
the reat "uantity of information that has been !ublished on folate bioavailability,
knowlede of this im!ortant !art of the folate metabolism is still at its beinnin and
more studies have to be !erformed to understand all the mechanisms involved.
B. FOLATE DEFICIENCY) IMPLICATIONS IN HEALTH
AND DISEASE
'uman beins cannot synthesi/e folate, so they have to obtain it from external
sources *?iure 2D.7-. This vitamin is !resent in a wide variety of foods such as
leumes *bean, nut, !ea, etc.-, reen leaves *s!inach-, some fruits *citrus-, veetables
*broccoli and cauliflower-, and liver and dairy !roducts *milk and fermented-.
.lthouh folate is omni!resent in the human diet, deficiencies are fre"uent even in
develo!ed countries. The recommended dietary allowance *(5.- of folate in adults is
144M744 X day
-2
*?.O,;'O, 1441-.
)n some !o!ulation rou!s, there is an increased risk of vitamin deficiency,
es!ecially in elderly !eo!le because their food intake is lower and in children who
sometimes consume a restricted variety of foods.
B.>. F!at( a$/ NTDs
. low folate intake can contribute to many !atholoies because it can lead to
folate deficiency that is a key factor to the develo!ment of many diseases such as
.l/heimer%s and a causal factor of others such as cardiac diseases, osteo!orosis,
increased risk of breast and colorectal cancer, and &T5s. &T5s are conenital
malformations of the brain and s!inal cord caused by a failure in the closin of the
neural tube in the fetus between days 12 and 1C after conce!tion. The defects can
rane from anence!haly to s!ina bifida, the latter can in turn have varyin severities.
&T5s are an im!ortant cause of morbidity and
Fi"#r( >G.D ?olate concentration in dairy !roducts. (esults are ex!ressed as meanB,-
standard deviation.
mortality with an estimated lobal incidence of `944444 new cases !er year, which
result in 72444 deaths *:eatheesan et al., 144D-. )t is estimated that &T5s re!resent
about a tenth of all conenital conditions and is the third most im!ortant after
conenital cardiac diseases and 5own syndrome. )n countries where habitants do not
consume folate-rich diets, the incidence of &T5s is elevated with an inverse relation
to the economic situation. 'owever, in countries with hih incomes, an elevated risk
of &T5s is associated with !oor maternal education.
B.?. F!at( a$/ A$(.ia
Besides the &T5s, another im!ortant and fre"uent manifestation of folate
deficiency is mealoblastic anemia. This !atholoy is the result of the lack of 5&.
synthesis as a con-se"uence of an insufficient amount of !lasmatic folate. This
decreased 5&. re!lication leads to a lower !roduction of hemolobin durin
erythro!oiesis, which is manifested by the !resence of abnormally enlared
erythrocytes *mealocytes- that have a lower con-centration of hemolobin and other
hematoloical alterations. $ealoblastic anemia is caused not only by folate
deficiency but also by the deficiency of vitamin B
21
, as both have similar clinical
manifestations.
B.B. F!at( a$/ Car/i!+as&#ar Dis(as(
?olate deficiency has also been im!licated in the increasin !lasma homocysteine
con-centrations that in turn can elevate the risk of cardiovascular diseases. 'owever,
fortifi-cation with folic acid has been shown to be ineffective in decreasin the risk of
cardiovascular diseases and mortality in healthy adults. The .merican 'eart
.ssociation does not recommend B-vitamin su!!lementationE it suests that folate
levels should be obtained from a balanced diet to reduce the incidence of heart disease
and stroke *>elly and .nne, 1424-.
B.D. F!at( a$/ Oth(r Dis(as(s
Besides insufficient intake, folate deficiency can also be due to other causes, such
as certain cancer treatments where antifolate drus *such as methotrexate- are used.
&ow-adays, another multitareted antifolate dru *.L)$T.- is used, which tarets
various folate-de!endent en/ymes such as thymidylate synthase and dihydrofolate
reductase. .s a !art of current treatment !rotocols, folic acid is coadministered with
the antifolate drus to !rolon the treatment.
.nother cause of folate deficiency is malabsor!tion. The mechanism of folate
absor!-tion in the GeGunum has been recently describedE certain !atholoies *such as
celiac disease and tro!ical s!rue- can affect absor!tion and thus cause folate
deficiency. Surical removal of the u!!er intestinal tract *such as !artial astrectomy
or GeGunal resection-, cer-tain inflammatory diseases *such as 6rohn%s disease-, the use
of sulfasala/ine *a noncom!etitive inhibitor of the reduced folate carrier-, or alcohol
abuse can also affect folate absor!tion.
B.E. F!at( a$/ Ca$&(r
Since folate !lays a key role in 5&. synthesis and re!air by actin as a !recursor
in !urine synthesis and maintains 5&. stability by donatin one-carbon rou!s, it is
not sur!risin that this vitamin is involved in certain ty!es of cancers. Several in vitro
studies from animals and humans showed that folate deficiency can result in the
demethylation of cytosine, lobal 5&. hy!omethylation, !roto-oncoene activation,
and chromosomal instability *5uthie, 1422-. )t was also demonstrated that a low
intake of dietary folates can cause an insufficient incor!oration of uracil, breaku! of
5&. strands with chromosomal breakae, and malinant transformations *5uthie,
1422-.
)n animal studies, it was shown that low-folate diets are associated with an
elevated risk of colorectal cancer and that folic acid su!!resses the rowth of the
cancer *Hiovannucci, 1441-.
Since folate levels can affect 5&. methylation, folate has the !otential to act in
the carcinoenesis of colon cells by affectin not only their severity and duration but
also their enes, tissues, and malinant transformations. This is the reason why an
increased intake of dietary folate and hih levels of folate in blood are enerally
associated with decreased risks of certain kinds of malinancies, althouh a com!lete
!rotector role for this vitamin aainst carcinoenesis has been "uestioned by many
researchers. Em!iric evidence does not seem to su!!ort the hy!othesis that the
incom!lete 5&. methylation of the entire enome as a direct conse"uence of low
folate levels can increase the risk of colon cancer in humans. Even with the evidence
that folate deficiency causes enomic instability by inducin 5&. damae, inhibitin
5&. re!air, and increasin malinant transformations, definitive evidence that shows
a causal relationshi! between the bio-markers of enomic stability and cancer risk
does not exist *5uthie, 1422-. Some ex!er-imental evidence has suested that folate
deficiency would !romote initial staes of carcinoenesis, while hih doses of folic
acid could increase the rowth of cancerous cells. .s a conse"uence of fortification
with folic acid in the 8nited States, an im!ortant increase in nonmetaboli/ed folic
acid and circulatin folate concentration has been ob-served. These results are
creatin concerns reardin the safety of folic acid fortification !rorams, es!ecially
with res!ect to the risk of develo!in cancer *Smith et al., 144C-.
Iarious ex!lanations have been elaborated to ex!lain how folic acid could
contribute to cancer develo!ment, such as the !ossible !resence of nonidentified
!reneo!lastic inGuries with the !otential to develo! cancer in hih-risk !o!ulations
and the inability of the body to metaboli/e increased doses of B vitamins *6arroll et
al., 1424-. .nother event that could also be related to the develo!ment of many kinds
of human cancers is the overex!ression of the e!idermal rowth factor rece!tor,
transferrin rece!tor, and folate rece!tor. The folate rece!tor has been widely used as a
liand to deliver thera!eutic aents to cancerous cells due to its hih-affinity union
*Nhan et al., 1424-.
On the other hand, lare discre!ancies exist between the results of studies related
to !o!ulations with a history of adenomas, in which some researchers demonstrated a
clear reduction of the risk of recurrence of adenomas, while others did not observe the
same effects *6arroll et al., 1424-. .ll these conflictin results have raised concerns
about the su!!lementation with folic acid and the risk of develo!in cancers,
es!ecially res!ect to the new fortification !olicies of many countries *8lrich, 144C-.
This demonstrates that it is im!ortant to establish the riskMbenefit relationshi! of
folate and folic acid in reard to cancer incidence and their chemo!reventive effect.
D. FOLIC ACID FORTIFICATION AND
S*PPLEMENTATION
The increased occurrence of folic acid deficiency in the world and the ravity of
the !atholoies it causes *such as neural tube malformations, cardiac diseases, and
mealoblastic anemia- have oblied many overnments to ado!t different folic acid
fortification !olicies. Besides the above-mentioned causes of folate deficiencies, some
!o!ulation rou!s re"uire additional vitamin u!takes. )t has been re!orted that at least
a third of !renant women and infants do not reach their folate re"uirements throuh
their conventional diet alone. ?or all these reasons, fortification of flours and other
foods with folic acid has now been im!le-mented in over 0< countries. Thus, in
countries such as 6anada and the 8nited States, flour fortification with folic acid is
obliatory since 233CE other countries followed their lead, such as .rentina in 1441.
'owever, the effectiveness of these fortification !rorams de!ends larely on the
eatin habits of the !ro!osed consumers. ?or exam!le, flour fortification would be
ineffective in some .sian and .frican countries where many fam-ilies, es!ecially in
the !oorest reions that are most at risk of develo!in vitamin deficien-cies, do not
reularly consume commercial foods !re!ared with flour. On the other hand, many
countries have not ado!ted a &ational ?ortification #roram with folic acid because of
its !otential undesirable side effects. The main concerns are based on the fact that
folic acid is areated at concentrations that allow !ersons with low folate intake to
reach the (5., to !revent !atholoies associated with folate deficiencies. .t these
levels of fortifi-cation, those with normal or elevated folate inestions would be
ex!osed to an excessive folic acid intake, which in turn can mask the early
hematoloical manifestations of vitamin B
21
deficiency. This is im!ortant since it has
been estimated that 24M94R of !eo!le older than 04 years have a reduced ability to
naturally absorb vitaminB
21
, and conse"uently,14R of the eneral !o!ulation in
industriali/ed countries is !otentially deficient in this vitamin *.srar and O%6onnor,
1440-.
Since folate fortification levels are based on the re"uirements of the eneral
!o!ula-tion, some rou!s could be ex!osed to extremely hih levels of folic acid,
such as chil-dren, whose vitamin re"uirements are lower than those of adults. )t has
even been suested that the fetus could be ex!osed to excessive amounts of folic
acid due to su!-!lementation of the mother durin !renancy, in addition to eatin
fortified foods, and this could favor the selection of methylentetrahydrofolate
!olymor!hism that is associated with a rou! of debilitatin diseases.
;ith res!ect to su!!lementation, several different alternatives to folic acid have
been studied, such as the administration of *DS- 0-$T'?, which is more effective to
increase the concentration of folate in erythrocytes in !otentially childbearin women.
Since 0-$T'? does not mask B
21
deficiency, this folate form would be a more
efficient and secure alternative than su!!lementation with folic acid *Lamers et al.,
144D-. .l-thouh milk has not been traditionally considered an im!ortant source of
folates com-!ared to folate-rich food !roducts, in vitro studies usin a dynamic
astrointestinal model demonstrated that 0-$T'? in milk was easily released from
the milk matrix and hihly available for absor!tion *D4M<4R- *Ierwei et al., 1449-. )t
was shown that synthetic folic acid is absorbed and trans!orted to the liver where it is
reduced and a !or-tion is methylated *;riht et al., 1449-. )n contrast, natural folates
*such as 0-$T'? !resent in foods and !roduced by microoranisms- are reduced and
methylated before bein absorbed, makin them more bioavailable than folic acid
*.srar and O%6onnor, 1440-.
Other foods that have been !ro!osed as vehicles for folic acid su!!lementation
include rice. Since rice is a !oor source of essential micronutrients, includin folates,
met-abolic enineerin strateies have been develo!ed to increase their
concentrations. By overex!ressin two ,rabidopsis thaliana enes of the !terin and
the p.B. branches of the folate biosynthetic !athway, folate concentrations were 244
times hiher com-!ared to the values of wild-ty!e rice. 6onsum!tion of 244 of
!olished raw rains is sufficient to meet a fourfold intake of the adult daily folate
re"uirement *Storo/henko et al., 144<-. 'owever, !eo!le livin in lower
socioeconomic conditions are less willin to acce!t enetically modified *H$- rice
com!ared to hiher-income !o!ulations, a clear setback in fortification !rorams that
are to be directed to these hiher-risk !o!ulations whose folate intake is inade"uate.
.nother way to increase folate consum!tion is to increase its levels in foods that
are consumed by lare !o!ulation rou!sE the use of folate-!roducin microoranisms
is thus an interestin alternative to fortification with folic acid by !roducin
fermented foods with elevated concentrations of natural forms of this essential
vitamin.
E. FOLATE BIOSYNTHESIS AND LACTIC ACID BACTERIA
Lactic acid bacteria *L.B- are a rou! of microoranisms that are normally used
as starter cultures in the manufacture of a lare variety of fermented foods. Besides
their im!ortant fermentative ca!acities, L.B can also increase the safety, shelf life,
nutritional value, flavor, and overall "uality of fermented !roducts. 6ertain strains of
L.B are able to !roduce, release, and,or increase s!ecific beneficial com!ounds in
foods. These func-tional inredients are sometimes referred to as nutraceuticals that
can be defined as any substance considered as food or a !art of food that can confer
medical or health ben-efits includin the !revention and,or treatment of disease.
These inredients can be mac-ronutrients *such as unsaturated fatty acids !resent in
some oils-, micronutrients *such as vitamins-, or nonnutritive com!ounds *such as
hydrolytic en/ymes and flavonoids- and can be naturally !resent in foods *such as
omea-9 fatty acids in fish or vitamin 6 in citrus fruits- or added *such as milks
fortified with calcium and vitamin 5 and cereals fortified with folic acid- *'uenholt/
et al., 1441-. The selection of strains deliverin health-!romotin com!ounds
*nutraceuticals- is now the main obGective of several research rou!s. $ost bacteria
are auxotro!hic for several vitamins, but it has been shown that certain strains have
the ca!acity to synthesi/e B-rou! vitamins as demonstrated by the fact that some
fermented foods contain elevated levels of B-rou! vitamins as a result of microbial
biosynthesis.
E.>. F!at( Bi!sy$th(sis 'r!. La&ti& A&i/ Ba&t(ria
&umerous studies have shown that industrial lactic acid bacteria such as
Lactococcus *Lc.- lactis and !treptococcus *!t.- thermophilus have the ability to
synthesi/e folate. This ex!lains why some fermented dairy !roducts, includin yourt,
contain hiher amounts of folate than nonfermented milk !roducts. 'owever, some
works have shown that the ability of microbial cultures to !roduce or utili/e folate
varies considerably and is a strain-de!endent trait. The amount of folic acid found in
cow%s milk ranes from 14 to D4 X l
-2
, whereas its concentration in yourt may be
increased, de!endin on the starter cultures used and on the storae conditions, to
values above 144 X l
-2
*;outers et al., 1441-. This level de!ends on the strain of !t#
thermophilus and Lactobacillus *L.- delbruec&ii subs!. bulgaricus used because the
latter oranism utili/es folates for its rowth. )t is now known that not only do yourt
starter cultures and Lc# lactis have the ability to !roduce folates but other L.Bs also
have this im!ortant !ro!erty. Lactoba-cillus acidophilus is re!orted as bein able to
!roduce folate in chemically defined medium as can L# plantarum *LeBlanc et al.,
1424a-. Other L.B such as Leuconostoc lactis and ifidobacterium *.- longum were
also re!orted as folate !roducers. Some Propionibacterium *#.- strains, well-known
!roducers of vitamin B
21
, can !roduce hih "uantities of folates, so these L.B could
!otentially increase folate levels in milk.
)t was observed that a combination of !t#
thermophilus,bifidobacteria,%nterococcus *%.- faecium increased folate levels, and a
combination of !t# thermophilus and ifidobacterium animalis could increase folate
levels by sixfold, re!resentin 20R of (5. *6rittenden et al., 1449-. )t is well
established that !t# thermophilus strains are dominant !roducers of folates in milk,
!rinci!ally !roducin 0-$T'?, ivin rise to yourts with more than six times 0-
$T'? content com!ared with the control after 21 h of fermentation *'olasova et al.,
1447-. )n the case of # longum, some strains were reconi/ed as mod-erate !roducers
with a maximum increase of <9R of 0-$T'? after 21 h of fermentation *'olasova et
al., 1447-. On the other hand, Propionibacterium freundenreichii subs!. !herma-nii
strains did not influence 0-$T'? levels durin fermentation. )n all cases, the max-
imum concentration of 0-$T'? was hihest between D and 21 h of fermentation, and
then a decrease in the 0-$T'? content was observed *'olasova et al., 1447-.
)n addition to obtainin fermented milk !roducts usin ade"uately selected starter
cultures that can increase vitamin concentrations, it is !ossible to increase the folate
level naturally throuh the addition of some fruit com!onent *'olasova et al., 1440-.
There-fore, a ood flavor of fermented melon Guice or melon concentrate that !ossess
hih levels of folate and vitamin B
21
could be the beinnin of a line of !roducts with
a lon shelf life that can be directed at !o!ulations with B vitamin deficiencies.
Besides fermented dairy !roducts, microoranisms are able to increase folate
content in a wide variety of other foods. ?or exam!le, fermentation of rye douh to
!roduce bread is fre"uently accom!anied by increase in folate content *>ariluoto et
al., 144D-. )n these studies, the increase of this vitamin durin fermentation was
mainly due to folate synthesis by yeasts, whereas L.B did not !roduce folate but
rather consumed it. The ade"uate selection of strains, for exam!le, by re!lacin folate
consumers with folate-!roducin L.B, could sinificantly increase folate content in
these breads.
.lso it has been re!orted that it is !ossible to select starter cultures of L.B that
!ro-duce sinificant amounts of 0-$T'? *almost twice the basal concentration-
durin veetable fermentation *:aoerstad et al., 1447-. To o!timi/e the entire !rocess,
it is im-!ortant to carefully check the folate concentration in raw veetables. ?olate
losses durin !rocessin must be limited as much as !ossible and o!timi/in the
conditions to favor the microbioloical biosynthesis of folates is essential to increase
folate levels in the final !roduct.
.nother exam!le of use of L.B to im!rove folate level in fermented !roducts is
in the fermentation of corn flour, where an increase of folate of almost threefold after
7 days of fermentation at 94 6 has been obtained *$urdock and ?ields, 23C7-.
Other studies !erformed with the aim to determine if the exoenous vitamin can
affect folate synthesis by bacteria have shown that !roduction is strain de!endentE
some bifidobacteria did not !roduce folate when this vitamin was already !resent,
whereas others !roduced it reardless of the vitamin concentration. )t has been
suested that in some strains, folate biosynthesis miht not be reulatedE this is
confirmed by the fact that the final concentration of this vitamin was at least 04-fold
hiher than the re"uire-ment of all strains *#om!ei et al., 144<-.
)t has been shown that different forms of folates are !roduced by L.B, some even
!roduce folates with more than 9 lutamyl residues. .n exam!le is Lc# lactis, where
u! to 34R of the total !roduced folate remained in the cell and was identified as bein
0,24-methenyl-T'? and !resumably 24-formyl-T'?, both with four, five, or six
lutamate residues *Sybesma et al., 1449b-. )n !t# thermophilus, much less of the total
!roduced folate remained in the cell and was identified as bein 0-formyl-T'? and
0,24-methenyl-T'?, both with three lutamate residues. These differences in distribu-
tion can !robably be ex!lained by the different lenth of the !olylutamyl tail of the
two microoranisms. One of the main functions of the !olylutamyl tail is thouht to
be the retention of folate within the cell. )t can be assumed that cell retention of folate
is mainly a result of the neative chare of the carboxyl rou!s of *!olylutamyl-
folate *!D
a
of 7.D-. $oreover, in !t# thermophilus, the intra- and extracellular folate
distribution was influ-enced by the !'. 6ells that were rown at low !' had a larer
extracellular folate frac-tion than cells that were cultured at hih !'. 6onse"uently, at
low intracellular !', a hiher concentration of the folate is !rotonated and electrically
neutral, enhancin trans!ort across the membrane. )n Lc# lactis, !' did not seem to
affect the intra- and extracellular folate distribution *Sybesma et al., 1449b-.
The a!!lication of biofortification of daily !roducts usin vitamin-!roducin
micro-oranisms is an interestin alternative to the use of synthetic folic acid in
fermented foods. The careful selection of folate-!roducin strains and the
o!timi/ation of their !roduction are essential and could lead to natural enrichment of
folate in different !roducts *'olasova et al., 1447E Sybesma et al., 1449b-.
E.?. F!at( a$/ Pr!bi!ti&s
Because of the numerous beneficial !ro!erties that have been attributed to L.B,
these are the most commonly used !robiotic microoranisms and can be defined as
Tlive micro-oranisms which when administered in ade"uate amounts confer a health
benefit on the hostU *?.O,;'O, 1442-. )t was shown that !t# thermophilus !ossesses
certain !ro-biotic characteristics such as !rovidin resistance to bioloical barriers
*astric Guice and bile salts-, im!rovin intestinal microflora and lactose diestion in
lactose-intolerant individuals, stimulatin the ut immune system, !roducin a hih
"uantity of folate ex-tracellularly, and alleviatin the risk of certain cancers, ulcer,
and inflammation. To clas-sify a bacterium as a !robiotic, it has to satisfy a series of
re"uirements that vary de!endin on the research rou!, but basically it should
!ossess a enerally reconi/ed as safe *H(.S- status to be able to survive throuh the
astrointestinal tract *H)T- and adhere to the human intestinal cells in addition to
exertin health benefits in the host. Since some L.B can !roduce sinificant amounts
of folate and certain strains are able to survive in the H)T, these beneficial
microoranisms could be used as efficient !robiotics to !roduce or liberate folate in
the H)T. Some research have shown that the amount of folate synthe-si/ed in the
human H)T is sinificant and could be clinically im!ortant if it is available *(on et
al., 2332-. There is direct evidence in vivo that folate synthesi/ed by bacteria could be
absorbed throuhout the intact lare intestine and incor!orated into tissues *(on et
al., 2332-.
(ecent re!orts have shown that some !robiotic microoranisms *such as
bifidobac-teria and !ro!ionibacteria- have the ability to synthesi/e folates *'olasova
et al., 1447E #om!ei et al., 144<-. The oral administration of folate-!roducin
!robiotic strains may confer a more efficient !rotection aainst inflammation and
cancer by exertin the beneficial effects of !rovidin folate and by deliverin it to
colonic rectal cells *#om!ei et al., 144<-. )n humans, folate is also !roduced by the
microbiota in the small intestine and is assimilated by the host *6amilo et al., 233D-.
.lthouh microbial folate synthesis is believed to su!!ly only a minor source of the
total absorbed folate in humans *Bates, 2339-, the contribution of the microbiota to
the folate re"uirements of the hih cell turnover intestinal e!ithelium is unknown. .
mechanism for luminal folate absor!-tion by cells in the human colon has been
re!orted *5udeGa et al., 233<-, which suests that folate !roduced in situ by the
colonic microbiota may be utili/ed by cells in the co-lonic e!ithelium. .srar and
O%6onnor *1440- also showed that bacterially synthesi/ed folate is absorbed across
the lare intestine and incor!orated into the liver and kidneys of !ilets. They
!redicted that a!!roximately 2CR of the dietary folate re"uirement in !ilets could be
met by folate absor!tion across the lare intestine. .nother im!ortant findin is that
increased intestinal bifidobacteria !o!ulations have been correlated with an enhanced
folate status in rats *>rause et al., 233D-. )t is therefore !ossible that !robiotic bacteria
active in the intestinal tract may be able to contribute to the folate re"uirement of
colonic e!ithelial cells. 'owever, further research is re"uired to determine if these
bacteria !roduce folate in the intestinal environment, the form in which this folate oc-
curs, the availability of this folate for trans!ort and utili/ation by colonocytes from the
lumen, and the contribution of the intestinal microbiota to the total folate re"uirement
of colonic e!ithelial cells *LeBlanc et al., 1424a-.
Other !robiotic oranisms includin %# faecium and !accharomyces cerevisiae
boulardii have !otential to be used in !robiotic !roducts. =ourt is the most im!ortant
delivery vehicle for !robiotic oranisms. 6heddar cheese, di!s, and s!reads are
becomin !o!ular as alternative !roducts for incor!oration of !robiotics.
E.B. F!at( Pr!/#&ti!$ *si$" G($(ti&ay M!/i'i(/ La&ti& A&i/
Ba&t(ria
The folate biosynthesis enes have been identified in Lc# lactis$ L# plantarum
*>leerebe/em et al., 1449-, and L# delbruec&ii subs!. bulgaricus *van de Huchte et al.,
144D-. This new information o!ened the doors to numerous studies and allowed the
develo!ment of many metabolic enineerin techni"ues that are necessary not only to
understand the com!lex metabolic !athways but also for the enetic modification of
L.B to !roduce bioloical com!ounds. Lc# lactis is by far the most extensively
studied lactic acid bacte-rium, and over the last decades, a number of eleant and
efficient enetic tools have been develo!ed for this starter bacterium. These tools are
of critical im!ortance in metabolic enineerin strateies that aim to inactivate
undesired enes and,or *control- the overex!ression of existin or novel ones. )n this
res!ect, es!ecially, the nisin-controlled ex!ression *&)6E- system for controlled
heteroloous and homoloous ene ex!ression in Lc# lactis has !roven to be valuable.
The desin of rational a!!roaches to metabolic enineerin re"uires a !ro!er
understandin of the !athways that are mani!-ulated and the enes involved,
!referably combined with knowlede about the fluxes and control factors. $etabolic
enineerin of more com!licated !athways involved in sec-ondary metabolism has
only recently beun with the enineerin of exo!olysaccharide !roduction in Lc#
lactis *Levander et al., 1441- and continued with other com!licated !athways such as
the biosynthesis of folate *Hreen et al., 233D-. This biosynthesis includes !arts of
lycolysis, the !entose !hos!hate !athway, and the shikimate !athway for the
!roduction of the folate buildin block p.B., while the biosynthesis of !urines is re-
"uired for the !roduction of the buildin block HT#. )n addition, a number of s!ecific
en/ymatic ste!s are involved in the final assembly of folate and for !roduction of the
var-ious folate derivatives *Sybesma et al., 1449a-.
)t is well known that some L.B cannot synthesi/e folate because some of the
enes involved in folate biosynthesis are not !resent in their enomeE this is the case
for L# gasseri *;ekam! et al., 1447-, L# salivarius *6laesson et al., 144D-, L#
acidophilus, and L. Cohnsonii *van de Huchte et al., 144D-.
)t has been shown that metabolic enineerin can be used to increase folate levels
*Table 2D.2- in Lc# lactis *Sybesma et al., 1449aE ;ekam! et al., 144<-, L# gasseri
*;ekam! et al., 1447-, and L# reuteri *Santos et al., 144C-.
)n cells, folate is !resent !redominantly in the !olylutamyl form because many
folate-de!endent en/ymes have increased affinity for !olylutamyl folates than for
mono-lutamyl folates. The en/yme res!onsible for !olylutamyl folate synthesis and
the cor-res!ondin elonation of the chain is !olylutamyl synthetase *E6 D.9.1.2<-,
which is encoded by the fol6 ene in Lc# lactis. 8ntil now, all se"uenced microbial
enomes !os-sess fol6 or a similar ene *Sybesma et al., 1449a-.
The controlled overex!ression of folD% enes in Lc# lactis that codes for D-
hydroxy-methyl- dihydro!terin !yro!hos!hokinase * fol&- and HT# cyclohydrolase *
fol%- !ro-duced a tenfold increased in the !roduction of extracellular folate and a
threefold increase in the !roduction of total folatesE meanwhile, overex!ression of
fol, that codes for dihydrofolate reductase decreased *by 04R- the !roduction of total
folates. .lso it was observed that the combined overex!ression of folD% and folC
favored the accumulation of intracellular folate *Sybesma et al., 1449a-. ?urthermore,
the overex!ression of the first en/yme of the biosynthetic !athway *HT#
cyclohydrolase )- showed a bi !otential as a stratey to increase the flux throuh the
folate biosynthesis !athway. This !resum!tion is based on the fact that this en/yme in
acillus subtilis has a low turnover and is not reulated by neative feedback *5e
Sai/ieu et al., 2330-.
Even thouh inducible systems are useful, in food fermentations, it is !referable to
use constitutive !romoters. 6lonin the folD% ene next to a constitutive !romoter
resulted
Tab( >G.> Total ?olate #roduced By $icrooranisms Hrown )n
6hemically 5efined ?olate-?ree $edium
Mi&r!bia s3(&i(s F!at( @_"
%>
A

R('(r($&(
Lactococcus
Lactococcus lactis subs!#
cremoris
31M22D Sybesma et al. *1449b-
21M29 Hanadharan et al. *1424-
Lactococcus lactis subs!.
lactis
0<M132 Sybesma et al. *1449b-
29M27 Hanadharan et al. *1424-
Lactococcus lactis subs!#
lactis biovar diacetylactis
<3M244 Sybesma et al. *1449b-
Lactobacillus
Lactobacillus plantarum 70 Sybesma et al. *1449b-
Lactobacillus helveticus 1MC3 Sybesma et al. *1449b-
Lactobacillus acidophilus 2 Sybesma et al. *1449b-
Lactobacillus casei 91 Sybesma et al. *1449b-
Lactobacillus casei subsp#
rhamnosus
97 Sybesma et al. *1449b-
Lactobacillus delbruec&ii
subs!# bulgaricus
07 Sybesma et al. *1449b-
Propionibacterium
Propionibacterium thoenii 9D 'uenholt/ and Smid
*1441-
Propionibacterium
acidipropionici
9D 'uenholt/ and Smid
*1441-
Propionibacterium
Censenii
74 'uenholt/ and Smid
*1441-
Propionibacterium
freudenreichii subs!#
shermanii
2<M<C 'uenholt/ and Smid
*1441-
Propionibacterium s!# 3M13 'uenholt/ and Smid
*1441-
ifidobacterium
ifidobacterium
adolescentis
<4M224 #om!ei et al. *144<-
ifidobacterium animalis 4M1D #om!ei et al. *144<-
ifidobacterium bifidum 4M2 #om!ei et al. *144<-
ifidobacterium breve 4M1 #om!ei et al. *144<-
ifidobacterium
catenulatum
9 #om!ei et al. *144<-
ifidobacterium cuniculi M #om!ei et al. *144<-
ifidobacterium dentium 13 #om!ei et al. *144<-
ifidobacterium globosum M #om!ei et al. *144<-
ifidobacterium infantis 4M1< #om!ei et al. *144<-
ifidobacterium lactis M #om!ei et al. *144<-
ifidobacterium longum 4M1 #om!ei et al. *144<-
ifidobacterium magnum M #om!ei et al. *144<-
ifidobacterium
pseudocatenulatum
<0M34 #om!ei et al. *144<-
ifidobacterium suis M #om!ei et al. *144<-
ifidobacterium
thermophilus
M #om!ei et al. *144<-
!treptococcus
thermophilus
13M141 Sybesma et al. *1449b-
Leuconostoc
Leuconostoc lactis 70 Sybesma et al. *1449b-
Leuconostoc
paramesenteroides
77 Sybesma et al. *1449b-
a
?olate concentration was measured by a microbioloical assay method.
in the same increase in folate !roduction that was observed usin the &)6E system.
6ombinin the overex!ression of folD% with the increased or decreased ex!ression of
other folate biosynthesis enes, folate !roduction could be sinificantly increased
*Sybesma et al., 1449a-. Other studies were !erformed on the overex!ression of the
p.B. ene cluster on three different vectors, two nisin-inducible vectors, and one
con-stitutive vector. The over!roduction of p.B. did not lead to elevated folate
!ools. 'ence, by overex!ressin the p.B. and the folate biosynthesis ene clusters
simulta-neously, hih folate levels were reached inde!endent of the p.B.
su!!lementation *;ekam! et al., 144<-.
The over!roduction of p.B. leads to relatively low intracellular folate !ools and
a relatively hih secretion of folate.
There exists a very tiht relation between folate and p.B. biosynthesis. *i- The
deletion of the p.B. enes in Lc# lactis eliminated its ability to synthesi/e folate,
causin a com!lete inability to row in the absence of !urine
nucleobases,nucleosides. )n the !resence of !urine nucleobases,nucleosides, folate is
not re"uired for rowth and *ii- the combined overex!ression of folate and p.B.
biosynthesis !athways led to a strain that !roduces a hih folate concentration and
that does not rely on the su!!lementation of !recursors in the medium *;ekam! et
al., 144<-. These studies were reali/ed in L.B that have the ability to !roduce folates.
)n other trials, L# gasseri *.T66 99919- was con-verted from bein a folate consumer
into a hihly efficient folate-!roducin strain. )n this bacterium, the folate
biosynthesis enes are not !resent, exce!t for fol, and folC, which are involved in the
reeneration and retention of reduced folates absorbed from the me-dium. L# gasseri
was transformed usin a !lasmid that contains the com!lete folate ene cluster *fol,$
fol$ folD%$ folP, and ylg- and folC- from Lc# lactis $H29D9 convertin it into a
folate-!roducin strain *;ekam! et al., 1447-.
)t was also demonstrated that enineered L# lactis was able to im!rove the folate
status in deficient rats *LeBlanc et al., 1424b-. Su!!lementation with Lc# lactis
overex!ressin the folC$ folD%$ or folCKfolD% enes sinificantly im!roved the folate
status in deficient rats. The biosafety assessment of these enetically modified L.B
*H$-L.B- was !er-formed, and it was shown that these were Gust as safe as the
native strains from which they were derived *LeBlanc et al., 1424c-.
G. CONCL*SIONS
)n this review, it has been shown that folate biosynthesis by L.B could increase
Snatural% folate concentrations in certain foods such as yourts and fermented milks
throuh careful and s!ecific selection of the microbial s!ecies and cultivation
conditions. These folates would not cause danerous side effects, such as maskin of
vitamin B
21
deficiency, as is the case with folic acid. On the basis of its ability to
!roduce B-rou! vitamins, L.B could be useful to desin new functional foods that
could !revent vitamin deficiencies by im!rovin the nutritional values of foods. The
food industry should use this information for selectin folate-!roducin strains as !art
of their starter cultures to !roduce fermented !roducts with elevated levels of this
essential vitamin. $any benefits will be obtained from the use of these strains, such as
economic advantaes to food man-ufacturers by !rovidin a value-added effect
without increasin !roduction costs. .lso consumers would increase their folate
intakes by includin these novel fermented foods as !art of their normal diet and
lifestyle. )n addition, selected s!ecific folate-!roducin strains could also !rovide
health benefits for consumers.
ACKNOWLEDGMENTS
The authors would like to thank the 6onseGo &acional de )nvestiaciones
6ientPficas y TQcnicas *6O&)-6ET-, .encia &acional de #romociJn 6ientPfica y
TecnolJica *.&#6yT-, and the 6onseGo de )nves-tiaciones de la 8niversidad
&acional de TucumKn *6)8&T- for their financial su!!ort.
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