Chapterreference 12

See discussions, stats, and author profiles for this publication at: https://www.researchgate.
net/publication/286025873
Riboﬂavin (Vitamin B2)
Chapter · July 2013

DOI: 10.1201/b15413-7
CITATIONS READS
32 13,618
2 authors, including:
John T Pinto
New York Medical College
198 PUBLICATIONS 6,566 CITATIONS
SEE PROFILE
Some of the authors of this publication are also working on these related projects:
Aging and hypertension View project
Impact of Organoselenium Compounds on Prostate Cancer View project
All content following this page was uploaded by John T Pinto on 25 January 2016.
The user has requested enhancement of the downloaded file.

6 Riboflavin (Vitamin B2)
John Thomas Pinto and Richard Saul Rivlin
Contents
6.1 Introduction........................................................................................................................... 192
6.2 History................................................................................................................................... 193
6.3 Chemistry.............................................................................................................................. 194
6.3.1 Molecular Characteristics of Flavins......................................................................... 194
6.3.2 Flavin Photochemistry............................................................................................... 194
6.3.3 Coenzymic Derivatives of Riboflavin....................................................................... 194
6.3.4 Covalently Bound Flavins......................................................................................... 194
6.3.5 Reactivity of Isoalloxazine Ring............................................................................... 196
6.4 Dietary and Supplemental Sources of Riboflavin................................................................. 197
6.4.1 Food Sources............................................................................................................. 197
6.4.2 Effects of Food Preparation and Processing............................................................. 197
6.4.3 Forms of Dietary Riboflavin and Luminal Metabolism............................................ 199
6.5 Physiology.............................................................................................................................. 199
6.5.1 Intestinal Absorption................................................................................................. 199
6.5.2 Riboflavin Transport, Plasma Binding, and Carrier Proteins....................................200
6.5.2.1 Transmembrane Transport Proteins............................................................200
6.5.2.2 Other Specialized Riboflavin Transport Proteins....................................... 201
6.5.2.3 Transmembrane Riboflavin Efflux Protein.................................................202
6.5.2.4 Riboflavin-Binding Proteins in Plasma...................................................... 203
6.5.2.5 Specific Riboflavin Carrier Proteins...........................................................204
6.5.3 Riboflavin Storage and Turnover of Flavin Coenzymes............................................206
6.5.3.1 Flavokinase.................................................................................................207
6.5.3.2 Cytosolic FAD Synthetase..........................................................................208
6.5.3.3 Mitochondrial FAD Synthetase..................................................................208
6.5.3.4 FAD Pyrophosphatase................................................................................ 210
6.5.3.5 FMN Phosphatase....................................................................................... 211
6.5.4 Riboflavin Excretion.................................................................................................. 212
6.5.4.1 Riboflavin Tubular Secretion and Reabsorption......................................... 212
6.5.4.2 Drugs and Riboflavin Excretion................................................................. 213
6.5.4.3 Urinary Metabolites of Riboflavin.............................................................. 213
6.6 Riboflavin Deficiency............................................................................................................ 214
6.6.1 Metabolic Adaptations to Riboflavin Deficiency....................................................... 216
6.6.2 Organelle Adaptations to Riboflavin Deficiency....................................................... 217
6.6.3 Clinical Signs of Riboflavin Deficiency.................................................................... 218
6.6.3.1 Dermal Abnormalities................................................................................ 218
6.6.3.2 Hematological Abnormalities..................................................................... 219
6.6.3.3 Neuropathic Abnormalities......................................................................... 219
6.7 Major Metabolic Processes Utilizing Flavin Coenzymes..................................................... 221
6.7.1 Flavoenzyme Complexes: Electron Transport Chain and Citric Acid Cycle............ 221
6.7.1.1 Complex I.................................................................................................... 222
191
K15043_C006.indd 191 4/10/2013 4:56:58 PM

192 Handbook of Vitamins
6.7.1.2 Complex II and the Citric Acid Cycle......................................................... 223

6.7.1.3 Other Flavoenzymes Involved in Electron Shuttles.................................... 223
6.7.2 Flavoenzymes Involved in Lipid Metabolism...........................................................224
6.7.2.1 Acyl-CoA Dehydrogenase and β-Oxidation...............................................224
6.7.2.2 Cytochrome b5 Reductase and Fatty Acid Desaturation............................ 226
6.7.2.3 Flavoenzymes, Cholesterogenesis, and Steroidogenesis............................. 226
6.7.2.4 Flavoenzymes and Formation of Ether Lipids and Sphingolipids.............. 230
6.8 Antioxidant Activities............................................................................................................ 231
6.9 The Unfolded Protein Response............................................................................................ 233
6.10 Malaria................................................................................................................................... 235
6.11 Homocysteine Metabolism.................................................................................................... 237
6.12 Riboflavin and Cell Signaling Factors................................................................................... 239
6.13 Extended Use and Medical Applications for Riboflavin.......................................................240
6.13.1 Riboflavin Photosensitization....................................................................................240
6.13.1.1 Mechanisms of Riboflavin Photosensitization............................................240
6.13.1.2 Photosensitization of Cells.......................................................................... 242
6.13.1.3 Photosensitization for Neonatal Jaundice................................................... 242
6.13.1.4 Photosensitization and Blood-Borne Pathogens......................................... 242
6.13.1.5 Photosensitization and Treatment of Corneal Disorders (Keratoconus)..... 243
6.13.2 Treatment of Migraine and Other Uses with Riboflavin...........................................244
6.14 Requirements and Assessment..............................................................................................244
References.......................................................................................................................................246
6.1 Introduction
Although riboflavin can be synthesized de novo by plant, yeast, and prokaryotic cells, mammals
need to obtain riboflavin by consuming plant-based foods or, if necessary, supplemental sources.
Riboflavin is the precursor to the coenzymes, flavin mononucleotide (FMN) and flavin adenine
dinucleotide (FAD). Historically, both FMN and FAD are referred to as nucleotides. Since the
phosphate group of FMN is not esterified to a ribose moiety but rather to the N-10 ribitol side chain
of riboflavin, a more appropriate term is riboflavin-5′-monophosphate. In similar fashion, FAD has
its adenosine monophosphate bound in a pyrophosphate arrangement with riboflavin-5′-phosphate
and thus could be viewed as adenosine riboflavin pyrophosphate. For convenience, this chapter will
provide the more widely utilized and familiar terms FMN and FAD as the standard abbreviations
for the flavin coenzymes flavin mononucleotide and flavin adenine dinucleotide, respectively.
Because of its distinctive and flexible chemical properties, the isoalloxazine ring of the flavin
coenzymes (Figure 6.1) is the focal point for electron transfers, thus making flavin nucleotides
capable of participating in a wide variety of reduction/oxidation (redox) reactions. Despite the
electron-accepting capabilities of the isoalloxazine ring, about 10% of flavin-dependent enzymes
catalyze nonoxidation–reduction reactions. In view of the highly conjugated heterocyclic nature
of the isoalloxazine ring, both two-electron and one-electron reactions cause the flavin moiety to
exist in three different redox states: fully oxidized (quinone), one-electron reduced (semiquinone),
and two-electron reduced (hydroquinone) forms. The conversion to its physiologically active forms,
FMN and FAD, is regulated by nutritional status in particular, protein calorie malnutrition, meta-
bolic rate, hormones, and drugs. Interaction of flavin coenzymes with their respective apoflavopro-
tein involves both noncovalent and covalent associations. Of the several hundred flavoenzymes in
nature, only 25 have been identified as being covalently linked. Flavoenzymes function within an
eclectic array of cellular processes that involve electron transport; metabolism of lipids, drugs, and
xenobiotic substances; cell signaling; and protein folding.
K15043_C006.indd 192 4/10/2013 4:56:58 PM

Riboflavin (Vitamin B2) 193
Flavin adenine dinucleotide (FAD)
NH2
Riboflavin 5’-phosphate (FMN)
N
OH OH O O N
Riboflavin 3’ 5’ O O O
1’ 2’ 4’ P P CH2 N
OH O N
O_ O_
H3C N N O
10 1
8α 8 9 2
7α 7 4a 3NH
6 5 4
H3C N HO OH
Isoalloxazine ring O
FIGURE 6.1 Flavin structures. Structural formulas of isoalloxazine derivatives are shown: riboflavin and
the two coenzymes derived from riboflavin, riboflavin-5′-phosphate (FMN) and FAD.
Understanding the relationship of riboflavin and flavoenzymes to the other B vitamins and
their respective enzymes has helped to establish its importance in several categories of disease
and disease intervention. Within the past several years, novel uses of riboflavin and approaches
to supplementation on the basis of the photoreactive properties of the isoalloxazine ring have
evolved. In the presence of ultraviolet (UV) light, riboflavin generates reactive oxygen species
(ROS). Advantage has been taken of this feature of riboflavin for inactivation of pathogens in
blood products and other bodily fluids, management of progressive keratoconus, or induction
of melanin formation in the skin. A number of reviews on riboflavin and flavoproteins have
emphasized the role of riboflavin in health (Powers 2003), the regulation of riboflavin metabo-
lism (Rivlin and Pinto 2001), and the inborn errors of riboflavin metabolism with neurological
sequelae (Baxter 2003).
6.2 History
Isolation of riboflavin may represent one of the earliest discoveries of a food-derived chromophore.
The British chemist Alexander W. Blyth in 1879 isolated from milk whey a water-soluble, yellow
fluorescent compound he called lactochrome, appropriately named for its color and origin. The
importance of lactochrome was not fully realized until later investigational studies by McCollum
and Kennedy (1916), Emmett and Luros, and Smith and Hendrick that showed the preventive capa-
bilities of water-soluble food extracts against beriberi, pellagra, and pellagra-like dermatitis.
These water-soluble B fractions were termed B1 for thiamine, B2 for the lactochrome, subse-
quently named riboflavin, B3 for niacin, and B6 for pyridoxine (Emmett and Luros 1920). Several
years later, the physiological role of the yellow growth factor was shown by Warburg and Christian
(1932) to be a component of a yeast “Zwischenferment,” which was designated the “old yellow
enzyme.” This enzyme is an example of a large family of flavoenzymes isolated from bacterial
sources with reductase capabilities (Williams and Bruce 2002). Stern and Holiday (1934) found that
after its dissociation from the enzyme, the chromophore contains an isoalloxazine ring; Theorell
(1934) demonstrated that its ribityl side chain was phosphorylated at the 5′-OH moiety.
Kuhn et al. (1935) and Karrer et al. (1935) eventually succeeded in synthesizing the vitamin, now
termed riboflavin, and Theorell (1937) identified the isoalloxazine derivative from the old yellow
enzyme as riboflavin-5′-phosphate, also called FMN. The structure of a second coenzymic form
was established by Warburg and Christian (1938) as FAD and was shown to participate as the coen-
zyme of d-amino acid oxidase. Thus, both coenzymic forms are synthesized from dietary sources
K15043_C006.indd 193 4/10/2013 4:56:59 PM

of riboflavin, which is intracellularly phosphorylated first to FMN and then pyrophosphorylated

with an AMP moiety to produce FAD.
6.3 Chemistry
6.3.1 Molecular Characteristics of Flavins
Riboflavin (vitamin B2) chemically is 7,8-Dimethyl-10-[(2S,3S,4R)-2,3,4,5-tetrahydroxypentyl]
benzo[g]pteridine-2,4-dione, also 7,8-dimethyl-(N-10-ribityl) isoalloxazine and has a molecular
weight of 376.36 with a solubility in water at room temperature of approximately 12 mg/dL. In
older literature, riboflavin is referred to as vitamin G because of its green fluorescent properties in
UV light. The water solubility of riboflavin can be increased by addition of phenolic compounds;
extraction from aqueous solutions can be accomplished using benzyl alcohol or 2-phenyl ethanol
as solvents.
Solutions of riboflavin exhibit four characteristic UV/visible spectral absorption bands centered
around 220, 266, 375, and 447 nm. Riboflavin can be conveniently assayed using its natural yellow-
green fluorescence spectra. Distinct advantage is taken of the high molar absorptivity (12.2 × 103 cm2 M−1)
and the more solvent-stable 447 nm absorption (excitation) band, which results in an intense emission
maxima at 530 nm (Weimar and Neims 1975).
6.3.2 Flavin Photochemistry
If a solution of riboflavin is exposed to UV light, singlet oxygen is generated, which photooxidizes
the ribityl side chain to yield lumiflavin (7,8,10-trimethylisoalloxazine), under alkaline conditions
(pH > 8) or lumichrome (7,8-dimethylalloxazine) at neutral to acidic conditions (Huang et al. 2006).
In both instances, the ribityl side chain is cleaved, preventing phosphorylation and subsequent ade-
nylation of riboflavin to its metabolically active derivatives, FMN and FAD. The products of ribo-
flavin photodegradation are both antagonists to riboflavin action and may be significantly effective
when foods are stored improperly or exposed to light. Also, phototherapy of neonatal jaundice and
of certain skin disorders has the potential to promote systemic riboflavin losses (Lucas and Bates
1984). The structure–function relationships of the various biologically active flavins have been com-
prehensively reviewed elsewhere (Merrill et al. 1981).
6.3.3 Coenzymic Derivatives of Riboflavin

FMN and FAD serve as coenzymes for flavoproteins involved in a wide variety of oxidation–reduction
reactions in intermediary metabolism. Although ~90% of the flavoproteins are considered redox
active, about 10% of them do not display intrinsic redox activity but participate in transferase, lyase,
isomerase, and ligase reactions (Henriques et al. 2010). In most cases, flavoenzymes are composed
of noncovalent complexes of the flavin moiety bound to the apoprotein via hydrophobic, electro-
static, charge transfer or van der Waals interactions. The dissociation constants of the flavin nucleo-
tides are within the nanomolar range or lower (Janin and Chothia 1978). Thus, unlike the pyridine
nucleotide coenzymes, flavin coenzymes remain an integral part of the enzyme under physiological
conditions.
6.3.4 Covalently Bound Flavins

There are additional cellular forms of FAD that are covalently linked from the 8-α (methyl) or
the C6-position of the isoalloxazine ring, and sometimes at both sites. Flavinylation of specific
proteins usually occurs at the 8-alpha position via N(1) or N(3) of histidyl, the thiol of cysteinyl,
K15043_C006.indd 194 4/10/2013 4:56:59 PM

or the hydroxyl group of tyrosyl or threonyl residues. Under most circumstances, especially for
those covalently bound flavoproteins that anchor the flavin moiety via dual linkage (6-S-cysteinyl-
8alpha-N1-histidyl FAD), flavinylation increases the redox potential of the flavin moiety and thus
its oxidation potential (Mewies et al. 1998). Prokaryotic cells that contain all 25 members of the
known covalently bound flavin family (23 attached at C8α) possess two flavoenzymes (tri- and
dimethylamine dehydrogenases) attached at the C6-position through a thioether linkage (Heuts et
al. 2009). Mammalian enzymes with covalently bound flavins include mitochondrial enzymes, sar-
cosine dehydrogenase, N,N-dimethylglycine dehydrogenase, succinate dehydrogenase, and mono-
amine oxidase, as well as the peroxisomal enzyme, l-pipecolate oxidase (Hassan-Abdallah et al.
2005; Yagi et al. 1976). Those mammals capable of synthesizing ascorbic acid from its precursors
also contain l-gulono-γ-lactone oxidase, a nonfunctional and often missing flavoenzyme in pri-
mates, guinea pigs, and certain orders of fish and birds (Kenney et al. 1976).
Sequential analysis of amino acids around the active site of covalently linked flavoproteins has
revealed that flavinylation of the apoprotein may be performed posttranslationally by the apoen-
zyme itself (Leferink et al. 2008). Thus, after secondary protein folding, the flavoprotein presents
“catalytically” active amino acid residues at the active site, which mediate formation of covalent
linkage to the isoalloxazine ring. As an example, in monoamine oxidase, formation of the 8α-S-
cysteinyl linkage to the isoalloxazine ring is thought to occur via a Michael addition reaction
between a cysteine sulfhydryl group and the methylene moiety generated at the 8α-methyl group
by proton extraction, yielding an iminoquinone methide form of the isoalloxazine ring (Heuts et
al. 2009).
In addition to flavinylation with FAD, Vibrio cholerae contains FMN covalently bound to threo-
nine residues within a family of enzymes that couples oxidation–reduction reactions to translo-
cation of Na+ ion across the cell membrane (Backiel et al. 2008). As mentioned earlier, covalent
linkages with either FMN or FAD via a dual site attachment raise the oxidation potential of the
flavin moieties respectively to levels greater than that of their noncovalently bound forms.
Figure 6.2 illustrates the enzymic conversion of riboflavin to its coenzymic forms. The ini-
tial reaction involves phosphorylation of riboflavin to riboflavin-5′-phosphate (FMN) followed by
attachment of adenosine monophosphate to FMN with formation of FAD. Both reactions require
ATP. Flavinylation of specific proteins occurs posttranslationally subsequent to FMN or FAD
formation.
Flavokinase
Riboflavin + ATP Riboflavin-5’-phosphate + ADP
Zn++
FAD synthetase-1, -2
(aka FAD pyrophosphorylase )
Riboflavin-5’-phosphate + ATP FAD + PPi
Mg++
Nonenzymatic
posttranslational
FAD (prokaryotes and eukaryotes) flavinylation Covalently
FMN (prokaryotes) bound
flavoproteins
Apoflavoproteins
FIGURE 6.2 Conversion of riboflavin to riboflavin-5′-phosphate, FAD, and covalent attachment. Flavokinase
catalyzes phosphorylation of riboflavin to form riboflavin-5′-phosphate (FMN). FAD is formed from the action
of FAD synthetase (FAD pyrophosphorylase) on FMN after combination with a second molecule of adenos-
ine triphosphate. Flavinylation of specific client apoenzymes occurs posttranslationally through the covalent
attachment of either FAD or FMN.
K15043_C006.indd 195 4/10/2013 4:56:59 PM

6.3.5 Reactivity of Isoalloxazine Ring

As shown in Figure 6.1, the isoalloxazine ring forms the basic functional structure for riboflavin,
FMN, and FAD, and it is the heterocyclic nature of this conjugated ring system that can sustain three
stably configured oxidation states: fully oxidized, semiquinone (one-electron reduction), and hydro-
quinone (two-electron reduction). Fully oxidized flavins have a planar configuration although the
association with some apoenzymes can distort the ring to accommodate a single nucleophilic attack
at the N5 or C4a position with formation of a semiquinone (see Figure 6.3). Semiquinone flavins can
exist either in a neutral radical (FAD•) or as an anionic radical (FAD•−) state. Although thermody-
namically unstable within aqueous environments, the flavosemiquinones are stabilized within the
confines of their apoenzymes, thus allowing the unpaired electron to delocalize throughout the con-
jugated heterocyclic ring system. In redox transfer reactions, flavoproteins that can geometrically
restrict the isoalloxazine structure between a planar (fully oxidized) or bent (fully reduced) configu-
ration can influence the residence time of electrons that enter and leave the ring. Thus, depending
upon the constraints imposed upon the isoalloxazine ring by flavoproteins, a configuration bowed
through the N5–N10 axis favors an ultrafast electron flux (usually in the picosecond to femtosecond
range) while a more planar configuration extends the residence time of electrons to the nanosecond
range (Kao et al. 2008).
Stabilization of single electron transfers is critical to allow flavins to shuttle electrons appro-
priately down the electron transport chain within mitochondria. Reactions of flavin semiquinones
can result from a single electron transfer to the isoalloxazine ring (reduction) or a single electron
transfer from the isoalloxazine ring (oxidation). Shifting the conformational equilibrium of a fla-
voprotein from an electron-accepting conformation (flavin-shielded mode) toward an electron-
donating conformation (flavin-deshielded mode) may increase electron transfer toward the proper
electron acceptors along the electron transport chain or directly toward molecular oxygen, resulting
in the formation of superoxide anion (O− 2 ). A full two-electron reduction can result in a neutral
flavohydroquinone (FADH2) or an anionic flavohydroquinone (FADH−) state displaying a bowed
conformation along the N5–N10 axis (Beinert et al. 1985). Again, apoprotein interactions with fla-
vohydroquinones can minimize the extent of ring bending along the axis and thus retrain the rate Should retrain
here be changed
of deactivation. to retain instead?
Please confirm.
N N
1 1
N5 nucleophilic
5 attack 5
N N
X– X
N
1 N
1
C4α nucleophilic
C4α attack C4α
N
N
Y
Y– H
FIGURE 6.3 Nucleophilic attack on flavoquinones. Nucleophilic attack of the isoalloxazine ring at the N5
or C4a position with formation of a semiquinone. Semiquinone flavins exist either in a neutral radical (FAD•)
or in an anionic radical (FAD•−) state.
K15043_C006.indd 196 4/10/2013 4:57:00 PM

6.4 Dietary and Supplemental Sources of Riboflavin

6.4.1 Food Sources
Significant dietary sources of riboflavin in the United States include plant foods as well as ani-
mal sources, namely, meat, poultry, fish, and dairy products, particularly eggs, milk, and cheese.
In developing countries, plant sources contribute nearly all of the dietary riboflavin intake. Mean
intake of riboflavin and other B vitamins such as folate and thiamine, as well as minerals, calcium,
and magnesium are higher in most vegetarians than in nonvegetarians (Farmer et al. 2011). Green
vegetables, such as broccoli, collard greens, and turnips, are reasonably good sources of riboflavin.
Natural grain products tend to be relatively low in riboflavin, but fortification and enrichment of
cereals and grains have led to a considerable increase in riboflavin intake from these food items.
High-quality, protein-rich foods are excellent sources of riboflavin as well as of other B vitamins.
At the molecular level, the metabolic role of riboflavin as a coenzyme within plant and animal
food sources is intimately associated with those of the other B vitamins (Depeint et al. 2006).
Flavoenzymes catalyze a diverse number of reactions and interact with pathways of other enzymes.
Thus, it is not surprising that if a given individual’s diet has inadequate amounts of riboflavin, it is
very likely to be inadequate in other vitamins as well. A primary deficiency of dietary riboflavin
has wide implications for other vitamins, as flavin coenzymes are involved in the metabolism of five
vitamins: folic acid, pyridoxine, vitamin K, niacin, and vitamin D (Rivlin 1994).
6.4.2 Effects of Food Preparation and Processing

In general, food preparation and processing can make foods safer and healthier as well as extend shelf
life. Despite some of these benefits, processing can also adversely influence the amount of riboflavin
that is bioavailable from dietary sources. Blanching, milling, fermenting, and extruding can result in
physical removal of the vitamin (Reddy and Love 1999). Since riboflavin is relatively stable to thermal
processing, microwaving of foods or exposure to infrared radiation would not have an impact as much as
stovetop cooking, since heating is performed more evenly and for shorter periods. Exposure to UV light,
as noted earlier, would result in appreciable losses of riboflavin, particularly during cooking and process-
ing. Prolonged storage of milk in clear bottles or containers may result in flavin degradation (Wanner
1960). Thus, in view of milk being an excellent source of riboflavin for children, milk is generally no lon-
ger sold in clear bottles. Opaque plastic containers that are coated with UV-blocking agents may provide
greater protection than cartons, particularly when milk is stored on a grocery shelf exposed to continuous
fluorescent lighting (Cladman et al. 1998). Milk and milk products must be perfectly protected against
light; otherwise, significant amounts of riboflavin and vitamin A (retinol), which is also susceptible to
UV light, will be lost and food quality will deteriorate (Mestdagh et al. 2005).
Large amounts of riboflavin are lost during the sun-drying of fruits and vegetables. The precise
magnitude of the loss varies with the duration and intensity of exposure to UV light and the extent of
inter- and intramolecular forces surrounding the isoalloxazine ring (Cardoso et al. 2012). Aqueous solu-
tions of riboflavin exposed to varying intensities of UV light exhibit approximately 70% loss of ribo-
flavin within 4 h. Rates of formation of the photodegradation products of riboflavin are also a function
of pH and temperature (Koaio 1966). By contrast to flavin degradation in aqueous solutions, the extent
of flavin degradation in irradiated foods varies considerably (Furuya et al. 1984). As a coenzyme, the
flavin component is complexed within a protein matrix and the isoalloxazine ring is usually confined
within intramolecular complexes with purines (particularly adenine), pyrimidines, or aromatic amino
acid moieties (tryptophan, phenylalanine, tyrosine, and histidine). Thus, internal quenching through
intramolecule interactions stabilizes the isoalloxazine nucleus and makes it less susceptible to photoly-
sis (Tsibris et al. 1965). Foods cooked at elevated temperatures or under slightly acidic conditions expe-
rience increased dissociation of these complexes. In addition, the practice of adding sodium bicarbonate
as baking soda to accentuate the green color of vegetables can result in accelerated photodegradation of
riboflavin. The riboflavin content of common food items with the highest amounts is shown in Table 6.1.
K15043_C006.indd 197 4/10/2013 4:57:00 PM

Table 6.1
Top Sources of Riboflavin and Their Caloric Content
Top Food Sources Riboflavin (mg/100 g) Energy (kcal/100 g)
Yeast, Baker’s dry (active) 5.41 282
Liver, lamb, broiled 5.11 261
Yeast, Torula 5.06 277
Kidneys, beef, braised 4.58 252
Liver, hog, fried in margarine 4.36 241
Yeast, Brewer’s, debittered 4.28 283
Liver, beef or calf, fried 4.18 242
Cheese, pasteurized, process American 3.53 375
Turkey, giblets, cooked 2.72 233
Kidneys, lamb, raw 2.42 105
Kidneys, calf, raw 2.40 113
Eggs, chicken, dried, white powder 2.32 372
Whey, sweet, dry 2.21 354
Eggs, chicken, dried, white flakes 2.16 351
Liver, turkey, simmered 2.09 174
Whey, acid dry 2.06 339
Heart, hog, braised 1.89 195
Milk, cow’s dry, skim, solids, instant 1.78 353
Liver, chicken, simmered 1.75 165
Liver, beef or calf, fried 4.18 242
Cheese, pasteurized, process American 3.53 375
Liver, chicken, simmered 1.75 165
Corn flakes, with added nutrients 1.40 380
Almonds, shelled 0.93 598
Cheese, natural, Roquefort 0.59 369
Eggs, chicken, fried 0.54 210
Beef, tenderloin steak, broiled 0.46 224
Mushrooms, raw 0.46 28
Cheese, natural Swiss (American) 0.40 372
Wheat flour, all-purpose, enriched 0.40 365
Turnip greens, raw 0.39 28
Cheese, natural Cheddar 0.38 402
Wheat bran 0.35 353
Soybean flour 0.35 333
Bacon, cured, cooked, sliced medium 0.34 575
Pork, loin, lean, broiled 0.33 391
Lamb, leg, good or choice, lean roasted 0.30 186
Corn meal, de-germed, enriched 0.26 362
Chicken, dark meat without skin, fried 0.25 220
Bread, white, enriched 0.24 270
Milk, cow’s, whole, 3.7% 0.17 66
Source: From Ensminger, A.M. et al., Food and Nutrition Encyclopedia, CRC Press, Boca Raton, FL, 1994. With permission.
Figures are given in terms of the riboflavin and calorie amounts in 100 g (approximately 3.5 oz.) of the items as usually
consumed. Portion size and moisture content differ among food items.
K15043_C006.indd 198 4/10/2013 4:57:00 PM

6.4.3 Forms of Dietary Riboflavin and Luminal Metabolism

Most flavin-rich foods contain vitamin B2 as phosphorylated derivatives, FMN, FAD, and their cova-
lently bound forms. Very little dietary riboflavin is found as free riboflavin from sources in nature,
although some dairy products will contain free riboflavin. Dairy products are major contributors to
overall riboflavin nutriture for all age groups (Ranganathan et al. 2005). Under ordinary circumstances,
the main sources of free riboflavin are commercial multivitamin preparations, which are consumed
increasingly by the general public. Also, ready-to-eat and vitamin-fortified cereals contribute to the
quantity of dietary riboflavin for children, adolescents, and young adults (Galvin et al. 2003; van den
Boom et al. 2006; Ziegler et al. 2006). The phosphorylated forms of riboflavin in food must be con-
verted, for the most part, to free riboflavin by digestive enzymes prior to transport into the enterocyte.
Luminal alkaline phosphatase exhibits both phosphatase and pyrophosphatase activity required to
hydrolyze FMN and FAD, respectively, back to riboflavin. In addition to food items, the microbial flora
in humans and animals can play a significant role in supplying flavins (Hill 1997). Rumen microbial
flora are rich sources of flavins to the ruminant and subsequently to their milk production. Fecal bac-
terial flora can account for a major source of riboflavin in coprophagous rodents. In humans, dietary
studies have shown that gut bacterial flora contribute significantly to the stores of B vitamins as well as
menaquinone (vitamin K2). To illustrate further the influence of gut bacterial to riboflavin metabolism,
studies have also shown that excess flavins that escape absorption in the duodenum and jejunum can be
metabolized or degraded by the gut flora of the large bowel. Intestinal microbial action on dietary ribo-
flavin yields isoalloxazine catabolites (e.g., 7,8-dimethyl-10-[2′-hydroxyethyl]isoalloxazine), which are
absorbed, enter the bloodstream, and subsequently diffuse into cells. These metabolites are devoid of
the ribityl side chain and thus have no nutritional value and are promptly excreted in the urine (Chastain
and McCormick 1987). Unfortunately, covalently bound flavins, which can account for approximately
10%–15% of the total dietary flavin pool particularly from plant sources, are not utilized and are subse-
quently degraded and excreted in human urine (Decker 1993). Significant amounts of 7 alpha-hydroxy-,
8 alpha-hydroxy-, and 8 alpha-sulfonylflavin metabolites are excreted and reflect the action of both
tissue microsomes and microbial oxidations. Lumiflavin, the photodecomposition product of riboflavin
under neutral and alkaline conditions, also contributes to the total pool of urinary flavin analogs.
6.5 Physiology
6.5.1 Intestinal Absorption
Contrary to earlier studies suggesting that riboflavin was absorbed by processes involving simple
diffusion, recent studies have identified the presence of specialized saturable transporters, not only
for riboflavin but also for each of the other B vitamins. Coupled with these site-specific transporters,
the absorption of riboflavin is generally enhanced in the presence of food owing to a combination
of variations in gastric and intestinal motility, bile salt secretion (Jusko et al. 1971; Mayersohn et al.
1969), and changes in splanchnic blood flow. Thus, factors that (a) permit more prolonged contact
of dietary riboflavin with the apical surface of the intestinal mucosal cell, (b) increase its solubility
within the unstirred water layer, and (c) enhance its translocation and removal from the enterocyte
would be expected to increase overall intestinal absorption of riboflavin. In view of these lumi-
nal factors in digestion, studies measuring urinary riboflavin excretion after single doses between
5 and 100 mg have affirmed that the maximum absorption of riboflavin occurs with doses between
20 and 50 mg (Ramanujam et al. 2011). In healthy adult humans, pharmacokinetic studies using
orally and intravenously administered riboflavin and incorporating a two-compartment open model
system determined that the maximal amount of riboflavin that can be absorbed from a single dose
is 27 mg (Zempleni et al. 1996). This amount represents approximately 16 times the recommended
dietary allowance (RDA). Therefore, the common practice of some megavitamin enthusiasts to
consume massive doses of multivitamins may have marginal benefits with respect to riboflavin,
as the unabsorbed amounts would be catabolized by the microbial flora or eliminated in the stool.
K15043_C006.indd 199 4/10/2013 4:57:00 PM

A number of other luminal factors influence the rate of intestinal absorption of riboflavin (Rivlin
1991). Diets high in psyllium fiber decrease the rate of riboflavin absorption, whereas wheat bran has
no detectable effect (Roe et al. 1988). The time from oral administration to peak urinary excretion of
riboflavin is prolonged by the antacids aluminum hydroxide and magnesium hydroxide. Total urinary
excretion is unchanged by these drugs, however, and their major effects appear to be delaying the
rate of intestinal absorption rather than inhibiting net absorption. Alcohol interferes with both the
digestion of food flavins into riboflavin and the direct intestinal absorption of the vitamin (Pinto et
al. 1987). This observation suggests that the initial rehabilitation of malnourished alcoholic patients
may be accomplished more rapidly and efficiently with vitamin supplements containing riboflavin
rather than solely with food sources comprising predominantly phosphorylated flavin derivatives.
The practicality of this hypothesis needs to be clinically tested since individuals recovering from
chronic alcohol consumption have multiple dietary requirements in addition to riboflavin.
Transition metals, particularly copper, zinc, iron, molybdenum, and chromium, which them-
selves play important roles in biological oxidation processes, have been noted to form complexes
with riboflavin that may affect the bioavailability not only of riboflavin but also of the metal as
well (Clarke et al. 1980; McCormick 1990; Spence and Tocatlian 1961; Sugiyama 1991). Thallium
toxicity has been related to direct interactions with riboflavin, FMN, and FAD (Mulkey and Oehme
1993). However, nephrotoxicity of thallium appears to be more closely associated with an affinity
for sulfhydryl moieties rather than an interaction with flavin moieties. Accordingly, toxicant chan-
neling may account for thallium-induced nephrotoxicity as a thallium–S-glutathione conjugate can
undergo a β-lyase-generated activation to Tl-SH and react with macromolecules at thiophilic centers
(Cooper and Pinto 2006). This is further supported by the finding that buthionine sulfoximine, an
inhibitor of glutathione synthesis, diminishes thallium nephrotoxicity by decreasing thallium con-
centration in renal medulla (Appenroth and Winnefeld 1999). When complexes form, some metals,
such as zinc, exhibit greater bioavailability (Agte et al. 1992). The semiquinone state of riboflavin,
in particular, forms complexes with metals such as Cu+ and Fe++ that are in their lower valence state
(Favaudon and Lhoste 1975). Metal–riboflavin complexes are usually in the form of charge-transfer
complexes between the 3-d orbital of the metal ion and the pi electron ring system of the flavin.
Moreover, the complexed flavin accepts electrons in one-electron rather than two-electron steps in
complex arrangements at the N(5) and O(4) centers (Yu and Fritchie 1975). Whether these interac-
tions bear clinical significance is not known with certainty and requires further study.
In addition to metals, a variety of drugs and dietary constituents can also complex with riboflavin
and FMN. Among the substances in this category are dietary constituents (caffeine and nicotin-
amide) (Ahmad et al. 2009; Sevrioukova 2009), drugs (theophylline, resorcinol, timolol, pindo-
lol, and cloxacillin) (Criado and García 2004; Meisel et al. 1980; Roy et al. 2006), and amino
acids (tryptophan, histidine, and tyrosine) (Spector 1980). The binding of riboflavin to most of
these agents involves fundamental mechanisms of excitation energy transfer that include singlet
oxygen states, intramolecular charge-transfer states, and intramolecular proton transfer. Because
these complexes represent specific and perhaps nonspecific interactions between the ring systems of
each compound and riboflavin, the nutritional, pharmaceutical, and clinical implications need to be
examined individually. For example, the combination of riboflavin with theophylline or aminophyl-
line can cause protein degradation and hemolysis or can have implications regarding phototherapy
of neonatal jaundice. In addition, pi-stacking interactions between coplanar ring systems can help
stabilize the vitamin or drug as in the case of caffeine or nicotinamide binding.
6.5.2 Riboflavin Transport, Plasma Binding, and Carrier Proteins

6.5.2.1 Transmembrane Transport Proteins
After dephosphorylation of dietary flavins, the direct transport of riboflavin through cell membranes
is controlled by highly specific riboflavin transporters (RFTs). Real-time polymerase chain reaction
(PCR) methodologies have revealed several RFTs expressed within the intestine, designated RFT-1,
K15043_C006.indd 200 4/10/2013 4:57:00 PM

-2, and -3 (Subramanian et al. 2011). Translocation of riboflavin across the mucosal barrier requires
coordination of RFTs localized within domains in the brush border (apical surface), the basolateral
surface, and intracellular vesicular structures within the enterocyte. Human riboflavin transporter
(hRFT)-1 is expressed at the basolateral surface; hRFT-2, which is more abundantly expressed than
hRFT-1, is exclusively located at the apical membrane, and hRFT-3 is largely confined inside intra-
cellular vesicles (Subramanian et al. 2011).
Members of the hRFT family of transport proteins are differentially expressed in other polarized
epithelia. In addition to the small intestine, hRFT-1, which has been identified as an RFT G-protein-
coupled receptor 172B (GPR172B), is strongly expressed in placenta and in human embryonic kid-
ney (HEK-293) and Caco-2 cells (Yonezawa et al. 2008). Messenger RNA for the apical membrane
RFT, hRFT-2, is abundantly expressed in testis and shows strong expression within prostatic cells
(Yamamoto et al. 2009). The uptake of riboflavin by RFT-1 and RFT-2 is independent of extracellular
Na+ and Cl− but does exhibit differences in pH sensitivities, in that RFT-2 more efficiently transports
riboflavin under acidic conditions (pH 6) and less efficiently under neutral and basic conditions (pH
8.4) where RFT-1 is not affected within either pH range. The apparent Michaelis–Menten constants
of hRFT-1 and hRFT-2 for riboflavin are 1.38 and 0.98 μM, respectively. Transport of riboflavin by
both carriers is strongly inhibited by riboflavin analogs, lumiflavin, FMN, and FAD.
Two missense sequence variations have been observed in RFT-1 and are associated with Q70R
and V296M substitutions within the transport protein. However, in vitro analysis of both variations
shows that riboflavin transport is unaffected by these amino acid variations. By contrast, studies in
a child presenting with elevated plasma acylcarnitines and organic aciduria (suggestive of a mild
multiple acyl-CoA dehydrogenase deficiency [MADD]) revealed a deletion in RFT-1 that spanned
exons 2 and 3 in one allele from the mother (Ho et al. 2011). Thus, studies of maternal riboflavin
deficiency have focused and yielded insight on possible genetic defects in RFTs.
In a similar fashion, mutations in the gene C20orf54 that encodes the hRFT-2 have been impli-
cated in the Brown–Vialetto–Van Laere syndrome (Bosch et al. 2011). This syndrome manifests
as insidious neurological defects characterized by sensorineural deficits that progress to overall
paralysis, culminating in respiratory failure. Studies show that a variety of mutations in hRFT-2 can
affect the transporter functionality by altering membrane targeting (mutants P28T, E36K, E71K, and
R132W were retained within the endoplasmic reticulum [ER]) and its transporter activity (mutants
W17R and L350M were expressed at the cell membrane but were nonfunctional) (Nabokina et al.
2012). The biochemical profile observed in this syndrome is similar to that displayed in MADD,
which is characterized by elevations in serum levels of ethylmalonic/adipic acid. The clinical profile
includes progressive muscle weakness and paralysis of the diaphragm. Without intervention with
high doses of riboflavin, neurological deterioration, hypotonia, respiratory insufficiency, and early
death will ensue (Anand et al. 2012). Defects in these transporters coupled with marginal ribofla-
vin deficiency during pregnancy can contribute significantly to the development of the riboflavin-
responsive disorders occurring in newborn infants.
hRFT-3, which localizes to intracellular vesicles within the enterocyte, has been isolated and
characterized in cells within the brain. hRFT-3 exhibits 86.7% and 44.1% amino acid homology to
that of hRFT-1 and hRFT-2, respectively (Yao et al. 2010). After rapid endocytosis of riboflavin,
hRFT-3 may assist in the intracellular trafficking of riboflavin into acidic vesicular compartments
(Huang et al. 2003; Huang and Swaan 2000). Since hRFT-3 is highly expressed in the brain, it may
regulate vesicular sorting of riboflavin as well as control riboflavin homeostasis within the central
nervous system. The apparent Michaelis–Menten constant of hRFT-3 for riboflavin is 0.33 μM,
whereas those for hRFT-1 and -2 are approximately four times higher. The lower Km of hRFT-3 may
be important for this protein to effectively compartmentalize and regulate intracellular riboflavin.
6.5.2.2 Other Specialized Riboflavin Transport Proteins

In addition to hRFT-1, -2, and -3, specialized carrier-mediated proteins for riboflavin uptake have been
isolated in a variety of other cells. It is unknown whether these proteins bear any homology to the
K15043_C006.indd 201 4/10/2013 4:57:00 PM

hRFTs or represent a separate family of riboflavin transport proteins. As riboflavin is an essential nutri-
ent for development and maintenance of the central nervous system, specialized transporters may have
evolved to transfer riboflavin from retinal or choroidal blood supplies. Although the uptake mechanism
and cellular translocation of riboflavin within this system are poorly understood, this protein system
appears to be regulated by protein kinase A, protein kinase C, or Ca2+/calmodulin pathways.
Studies using retinal pigment epithelial (RPE) cells (Said et al. 2005) and the retinoblastoma cell
line (Y-79) (Kansara et al. 2005) show that riboflavin uptake is not dependent on Na+ but is depen-
dent on energy and temperature. Although these proteins differ in their pH dependency, riboflavin
transport is saturable and inhibited by flavin analogs. The apparent Km for each is approximately
80 and 19 nM, respectively. The uptake of riboflavin by Hep2 (Said et al. 1998) and HK-2 (Kumar
et al. 1998) cells was found to be similar to that in RPE and Y-29 cells, each having an apparent
Michaelis–Menten constant for riboflavin uptake of 0.41 and 0.67 μM, respectively. In each of these
cell lines, the process of riboflavin uptake is adaptively regulated in riboflavin deficiency. Thus, the
cellular uptake and transport pathways of riboflavin exhibit facilitative mechanisms and relative
selectivity at physiological concentrations.
6.5.2.3 Transmembrane Riboflavin Efflux Protein

Is this correct as The protection of cells from excessive accumulation of riboflavin, as well as the facilitation of an
modified?
efflux of riboflavin into extracellular fluids such as blood plasma, cerebrospinal fluid, bile, semen,
and breast milk, is controlled by an ATP-binding cassette (ABC) transporter (ABCG2), located
on the apical surface of endothelial cells (Ifergan et al. 2005; van Herwaarden and Schinkel 2006;
van Herwaarden et al. 2007). This efflux protein, initially identified as the breast cancer resistance
protein, belongs to the multidrug resistance (MDR) transporter family of proteins that include over
40 members, mostly represented by P-glycoprotein (ABCB1) and MDR proteins (ABCC). These
proteins extrude a wide variety of endogenous and exogenous xenobiotic compounds from endothe-
lial cells after their uptake from the intestinal lumen and bloodstream (Ifergan et al. 2009). Thus,
ABCG2 and other MDR proteins protect cells from potential toxicity by transporting drugs and
toxins from the hepatocyte into bile, from intestinal epithelia back to the lumen, and from brain
or retinal endothelium to the capillary lumen. This transporter system restricts cellular access by
potential toxins and creates a major obstacle to successful cancer chemotherapy (Jonker et al. 2005).
In addition to its direct role in restricting cellular exposure to xenobiotics, a seemingly para-
doxical function of this transport protein is the secretion of riboflavin into breast milk and other
extracellular fluids such as semen, cerebrospinal fluid, and bile. After internalization by one of the
riboflavin transport proteins (see Section 6.5.2.1), riboflavin can conversely undergo exocytosis.
This process can be enhanced by increasing the extracellular concentrations of riboflavin to polar-
ized endothelial cells or by disrupting the endosomal and lysosomal trafficking patterns of proteins
within cells (Bareford et al. 2008).
In particular reference to the secretory epithelia (lactocytes) within mammary tissue, expres-
sion of the ABCG2 protein is strongly induced during lactation and expression is abated during
weaning periods. In addition, nuclear factors, such as hypoxia-inducible factor (HIF)-1, peroxisome
proliferator-activated receptor gamma, and the aryl hydrocarbon receptor, have also been shown to
be involved in the regulation of ABCG2 expression (Iqbal et al. 2012). Dietary riboflavin provides the
main source of riboflavin necessary for exporting riboflavin into breast milk, and lactating women
who restrict consumption of dairy products and key dietary constituents compromise the protein and
nutrient quality of breast milk (Mannion et al. 2007; Ortega et al. 1999). Thus, as a consequence of
this protein, not only is dietary riboflavin (and perhaps other vitamins) actively pumped into milk
but dietary toxins and a variety of drugs are also eventually secreted into the alveolus. The exposure
of suckling infants to xenobiotics coupled with the need to transfer specific vitamins from mother to
infants via milk presents significant pharmacological and toxicological issues for both dairy produc-
ers and consumers. Several intriguing hypotheses have been proposed elsewhere to reconcile the
presence of and the functional need for ABCG2 in mammary tissue (Mannion et al. 2007).
K15043_C006.indd 202 4/10/2013 4:57:00 PM

In studies using ABCG2 knockout mice (Abcg2−/−), steady-state secretion of riboflavin into milk
was reduced 60-fold compared to that in wild-type mice (van Herwaarden et al. 2007). Similarly,
FMN concentrations were reduced by approximately 6-fold, suggesting that FMN may also be
pumped by the ABCG2 transporter. By contrast, FAD levels were not compromised, suggesting
that other mechanisms independent of ABCG2 are functioning to sustain milk flavin status. Pups
fed milk from Abcg2−/− mothers are asymptomatic with regard to riboflavin deficiency as milk FAD
can be hydrolyzed to riboflavin within the newborn intestinal lumen (Vlaming et al. 2009). Thus,
while riboflavin transport into breast milk appears to be dependent on ABCG2 expression within
lactating mammary gland, FAD secretion into the alveolus is independent of this efflux protein
(Foraker et al. 2003).
The absence of riboflavin deficiency in Abcg2−/− mice may be partially explained by the transport
of immunoglobulins into breast milk. As discussed in Section 6.5.2.4, most human immunoglobu-
lins contain two high-affinity binding sites for both riboflavin and FAD; both ligands exhibit dis-
sociation constants within the picomole to nanomole range (Watson and Ford 1988). The presence
of secretory IgA in milk is provided by T and B lymphocytes (IgA-secreting cells) that migrate
into lactating mammary gland (Van de Perre 2003). Studies in humans have focused on both pla-
cental transfer of IgG and milk transfer of IgA immunoglobulins in newborn disease prevention.
Secretory IgA (dimeric form) as opposed to serum IgA (monomeric form) is the major immuno-
globulin in breast milk. Together with secretory IgM and IgG, these immunoglobulins can provide
a fully breastfed infant with as much as 0.5 to 1 g of protein per day. These proteins coupled with
the flavin binding capacity of the six major milk proteins (α-casein, β-casein, κ-casein, lactofer-
rin, α-lactalbumin, and β-lactoglobulin) can undergo transcytosis particularly when associated with
riboflavin (Holladay et al. 1999). Thus, several mechanisms may be operative and mammary tissue
may express redundant pathways to assure adequate supply of this vital nutrient into breast milk.
6.5.2.4 Riboflavin-Binding Proteins in Plasma

Complete absorption of riboflavin from the gastrointestinal tract into the bloodstream is facili-
tated by the presence of specialized RFTs. However, complete mucosal-to-serosal transport can
be attenuated by sequestering dietary riboflavin to meet the metabolic demands of flavoenzymes
inherent within the enterocytes. Additional “barriers” encountered before riboflavin reaches the
general circulation entail uptake and metabolism by red and white cells and hepatobiliary extraction
as the portal blood flows through the liver prior to reaching other organ systems. This latter effect of
hepatic riboflavin extrusion, as discussed in Section 6.5.2.3, is carried out primarily by the ABCG2
efflux protein from the hepatocyte into bile.
The major binding of riboflavin and its phosphorylated derivatives to proteins in plasma is to
albumin and several classes of immunoglobulins, IgA, IgG, and IgM (Innis et al. 1985). Purified
albumin exhibits weak and nonspecific binding affinity for riboflavin (Kd = 3.8 to 10.4 mM). By
contrast, FMN and the flavin-derived photodegradation products, lumiflavin and lumichrome, bind
more tightly to albumin. The affinity of albumin for flavins follows in the order at pH 5.8: lumiflavin
and lumichrome, FMN, riboflavin, and FAD (Sawada et al. 2001). Binding of riboflavin to immuno-
globulins is attributed primarily to subfraction, IgG and IgA, both of which exhibit marked varia-
tions in their capacity to bind riboflavin. Thus, differences in riboflavin binding capacity among
these proteins within human plasma may reflect marked variations in binding affinities of immu-
noglobulins for flavins within the human circulation. In general, binding of low-molecular-weight
compounds such as riboflavin to various plasma proteins with different affinities may influence
rates of transfer into cells.
Protein binding of riboflavin delays its renal clearance compared to no protein binding or may
increase residence time of the total plasma concentration (both free unbound and protein-bound
fractions) within the general circulation. The availability of both binding and transport proteins can
have marked influences on translocation of the flavin ligand as well as its binding protein within
the cell. Riboflavin binding to proteins, particularly albumin, also affects transcytosis of the protein.
K15043_C006.indd 203 4/10/2013 4:57:00 PM

Association of riboflavin with albumin facilitates its entry into cells, a process commonly observed
with epithelial cells within capillary beds (Holladay et al. 1999). The distribution of riboflavin within Is this correct as
modified?
tissues involves dynamic processing that includes protein binding, metabolism, membrane permeabil-
ity, and excretion, all of which may be interactive with, competing with, or reinforcing of one another.
In addition to albumin, most human plasma immunoglobulins contain two high-affinity binding
sites for both riboflavin and FAD. The estimated dissociation constants for the two separate binding
sites for riboflavin are 2.43 and 0.068 nM, and for FAD, the values are 1.73 and 0.078 nM (Watson
and Ford 1988). In view of the influence that riboflavin binding has on albumin transcytosis, it
would be of interest to determine whether binding of riboflavin to these high-affinity sites facilitates
transcytosis of important immunoglobulins. This knowledge would provide an impetus to examine
riboflavin status for modifying or improving immune surveillance (Fabian 1991).
Coupled with this observation is the ability of riboflavin to function as an endogenous photosen-
sitizer through its ability to generate ROS when photoexcited. Studies show that regardless of the
specificity of an immunoglobulin to bind a particular antigen, all antibodies can intercept ribofla-
vin-generated reactive oxygen. This process has been termed the antibody-catalyzed water oxida-
tion pathway, which can occur at physiologically relevant concentrations of riboflavin (<50 nM)
(Nieva et al. 2006). Generation of ROS by the riboflavin-induced photooxidation pathway acceler-
ates hemolysis of sheep red blood cells. It is unknown whether this process is operative in vivo
through surface capillary photoirradiation or exhibits pathophysiological effects.
In view of their high binding affinities for riboflavin and their potential transport capacity for this
vitamin, plasma immunoglobulins from patients with a variety of malignant diseases were tested for
abnormalities in riboflavin binding (Innis et al. 1986). These studies found elevated levels of a sub-
class of immunoglobulins from patients with breast cancer and melanoma and suggested that they
may contribute to limited urinary excretion and clearance of riboflavin in some patients with cancer.
It is unknown whether these proteins have potential as prognostic indicators for the extent of malig-
nancy. Binding of riboflavin to these immunoglobulins usually does not produce any overt clinical
signs. However, two unusual cases of riboflavin binding have been observed in women with multiple
myeloma who developed yellow skin and hair as a result of formation of a monoclonal IgG lambda and
IgG lambdaDOT (Farhangi and Osserman 1976; Merlini et al. 1990). Both these immunoglobulins
exhibited the highest affinity for riboflavin (binding constant 108/M) followed by FMN and FAD; bind-
ing of this magnitude by these immunoglobulins was not detected for nonflavin compounds as controls.
6.5.2.5 Specific Riboflavin Carrier Proteins

Studies in birds and in other oviparous species have identified a riboflavin carrier protein (RCP),
which is obligatory for the transport and deposition of riboflavin into the yolks of the developing
oocytes. This 37 kDa protein is synthesized in the livers of laying hens under control of estradiol
(Murthy and Adiga 1978). After synthesis of the 238-amino-acid protein, RCP is posttranslationally
cleaved of 19 amino acids at the carboxy-terminus and is cotransported with vitellogenin, a glyco-
lipoprotein, into the yolk. There, it undergoes a further intracellular cleavage reaction to remove
additional 11–13 residues at the carboxy-terminus (MacLachlan et al. 1994). The liver RCP trans-
ported in serum undergoes other posttranslational modifications that include N-linked oligosaccha-
ride attachment and phosphorylation of eight serine residues in the carboxyl-terminal portion of the
protein (MacLachlan et al. 1993). The oviduct in birds also synthesizes an RCP, which is delivered
to the egg white compartment. The egg white form of RCP is expressed by a gene similar to that in
liver but displays a different pattern of glycosylation (Clagett 1971).
Hormones other than estrogen also influence synthesis of RCP. Hyperthyroid status in birds
requires greater levels of estrogen for RCP induction by the liver. Progesterone stimulates RCP for-
mation by the oviduct but not by the liver (DurgaKumari and Adiga 1986). Birds genetically unable
to produce RCP have normal maternal riboflavin metabolism but cannot produce eggs capable of
sustaining embryogenesis beyond 13 to 14 days of incubation (White 1996). These eggs can be
rescued by exogenous injection of riboflavin through the shells. Riboflavinuria in these animals
K15043_C006.indd 204 4/10/2013 4:57:00 PM

appears to result from lack of functional RCP, which normally is required for the transport of ribo-
flavin into the yolk and albumen compartments. These studies illustrate the importance of riboflavin
as a critical nutrient for embryonic growth and overall gestational needs.
RCP is also present in sera of pregnant cows, monkeys, and humans (Visweswariah and Adiga
1987). The characteristic function of RCP has been evolutionarily conserved in terms of its physi-
cochemical and immunological properties. RCP is the prime mediator of riboflavin supply for the
maintenance of pregnancy and for the development of the fetus in mammals, including primates.
Pregnancy in rodents can be terminated after treatment with antiserum to chicken RCP. The immu-
nological cross-reactivity of chicken RCP antibodies with RCPs from different species illustrates
the remarkable degree of conservation within the linear antigenic regions of RCP.
In addition to females, males also produce RCP. Leydig cells within the testis secrete RCP in
response to luteinizing hormone. Stimulation of RCP synthesis by Leydig cells in vitro can also be
initiated by both 8-bromo cyclic AMP and cholera toxin (Subramanian and Adiga 1996b). These
results indicate that secretion of RCP is coupled to stimulation of a membrane-bound heterotrimeric
G-protein that controls production of cAMP. Immature, testosterone-treated rat Sertoli cells also
secrete RCP in response to follicle-stimulating hormone (Subramanian and Adiga 1996a).
Thus, in addition to providing riboflavin for embryogenesis, RCP assists in transporting riboflavin
across the blood–testis barrier. RCP localizes in the sperm head region and has been implicated in play-
ing an important role in sperm–egg interactions in hamsters, a model used for studies involving mamma-
lian sperm structure and function. Immunization of male rats and monkeys with denatured RCP impairs
the fertilizing potential of sperm cells (Adiga et al. 1997). Thus, in light of the role that RCP plays in the
development and maturation of spermatozoa and oocytes, RCP may have potential as a target to promote
contraceptive methods that are nonhormonal, safe, and acceptable to both males and females.
Studies in humans show that serum levels of RCP in pregnant women increase about 4 months
after conception and remain elevated for up to 8 months (Natraj et al. 1988). Despite the apparent
low, basal maintenance levels of RCP (<1 ng/mL) in the maternal circulation prior to 4 months
of pregnancy, RCP levels in amniotic fluid are elevated two- to threefold as compared to that in
maternal serum. These findings suggest that the developing fetal compartment is able to sequester
riboflavin (Dancis et al. 1988; Prasad et al. 1992). Mammalian placental trophoblasts are capable
of synthesizing RCP. Riboflavin bound to RCP is internalized via a Ca2+-dependent RCP receptor
located within clathrin-coated pits on the cell membrane (Mason et al. 2006). This lattice-shaped
region on the cytosolic surface facilitates receptor-mediated endocytosis by invagination of the “pit”
area into vesicles. After endocytosis and release of riboflavin, RCP is degraded within endosomes,
and the RCP receptors, in a manner similar to that of the transferrin receptor, are recycled back to
the membrane surface for recapture of additional ligand, riboflavin–RCP complexes.
Because of its role in growth and development and estrogen sensitivity, RCP has been examined
as a potential marker in hormone-sensitive cancers. RCP is overexpressed in patients with clinically
confirmed breast cancer. Serum RCP levels in premenopausal women (14.75 ± 5.25 ng/mL) and in
postmenopausal women (17.0 ± 4.6 ng/mL) are four- to sevenfold higher than in normal control sub-
jects of the same age. Cancer-free, cycling, and postmenopausal women exhibit serum RCP values
of 2.1 ± 1.0 ng/mL (Rao et al. 1999). The rise in serum levels of RCP in breast cancer patients cor-
relates with progression of the disease and differs between estrogen-responsive and -nonresponsive
disease. Immunohistochemical analyses of metastatic cells within lobular and ductal breast car-
cinomas reveal that invading cells are the origin of the circulating RCP (Karande et al. 2001). It
is currently unknown whether other estrogen-responsive cancers, such as those of the ovary and
cervix, overexpress RCP. Such a finding would complicate interpretation of serum RCP as a specific
biomarker and prognostic indicator for early stages of breast cancer.
Increased expression of RCP has also been shown in transrectal prostate biopsy specimens irre-
spective of their stage and Gleason score. Studies using highly metastatic human PC3 cells in cul-
ture reveal that antibodies to RCP added to the medium not only block riboflavin uptake but also
inhibit cell proliferation (Johnson et al. 2009). Further investigations using androgen-responsive and
K15043_C006.indd 205 4/10/2013 4:57:00 PM

-nonresponsive prostate cancer cells may possibly reveal the potential suitability of measuring RCP as
a marker for metastatic potential of human prostate cancer. Patients with liver diseases ranging from
hepatitis to cirrhosis to hepatocellular carcinoma exhibit varying amounts of elevated serum RCP.
Patients presenting with hepatocellular carcinoma express the highest serum values (Rao et al. 2006).
In view of the various types of tumors overexpressing RCP, the utility of RCP as a specific
biomarker for a given tumor may not be practical. Nevertheless, one consistent finding in clinical
evaluations of cancer thus far is that all biopsied adenocarcinomas of breast and prostate as well as
sera from patients with hepatocellular carcinoma overexpress RCP. Thus, measurement of serum
RCP levels may be helpful for differential diagnosis of cancers if coupled to a panel of other protein
biomarkers of proliferative diseases such as human epidermal growth factor receptor 2, prostate-
specific membrane antigen (Pinto and Heston 1999), or serum alpha-fetoprotein.
In addition to its potential as a biomarker for cancers, RCP may be helpful in blunting tastes of
certain foods. Both the apo- and holoprotein forms of RCP are unique among a variety of proteins
for their ability not only to suppress sweet flavors but also, more potently, to suppress bitter tastes
(Maehashi et al. 2008). RCP exhibits wide-ranging capabilities to inhibit structurally diverse bitter
substances, which suggests that it may function by a direct interaction with bitter receptor proteins
or bitter receptor cells. The blunting effect of RBP on bitter taste may be useful in the development
of oral pharmaceutical preparations to mask certain tastes. Lastly, because of its ability to bind
with various isoalloxazine structures, RCP may be useful for creating artificial flavoenzymes by
coupling it with flavin analogs other than riboflavin (de Gonzalo et al. 2011).
6.5.3 Riboflavin Storage and Turnover of Flavin Coenzymes

As noted above, riboflavin represents the parent isoalloxazine congener from which the flavin coen-
zymes FMN and FAD are synthesized. Thyroid hormones, in particular triiodothyronine (T3),
regulate the activities of the flavin biosynthetic enzymes (Lee and McCormick 1985), the synthesis
of the apoflavoproteins (Rivlin 1970), and the formation of covalently bound flavins (Pinto and
Rivlin 1979) in all mammalian tissues except adult brain. Studies in adrenalectomized rats show
that the mineralocorticoids aldosterone and dihydroaldosterone but not glucocorticoids increase
flavokinase activity in the adrenals and in the renal cortex and medulla (Trachewsky et al. 1985).
Mineralocorticoid enhancement of renal FMN and FAD synthesis may be a causative factor in the
increased reabsorption of Na+ (Trachewsky 1978). The effect of aldosterone on flavokinase activa-
tion is not observed in other tissues such as liver and heart. Hormonal and other factors that control
biosynthesis of flavin coenzymes are shown in Figure 6.4.
Thyroid hormones (T4, T3)

Aldosterone (adrenal gland/kidney)
+ Infection + Riboflavin +
deficiency
− − ± −
FAD
Flavokinase synthetase Covalently
Riboflavin FMN FAD bound
FMN FAD
flavoenzymes
phosphatase pyrophosphatase
FIGURE 6.4 Factors controlling flavin and covalently bound flavin biosynthesis. Control by thyroid hor-
mones and aldosterone on the metabolic pathway of the conversion of riboflavin into FMN, FAD, and cova-
lently bound flavin is shown. Flavin synthetic pathways are also modulated during infection and riboflavin
deficiency. Pathways up- and downregulated are indicated by (+) and (–), respectively.
K15043_C006.indd 206 4/10/2013 4:57:00 PM

6.5.3.1 Flavokinase
The sequence of events in the synthesis of the flavin coenzymes from riboflavin and its control by
thyroid hormones are shown in Figure 6.2. ATP:riboflavin-5-phosphotransferase (riboflavin kinase,
flavokinase; EC 2.7.1.26) is the first biosynthetic enzyme in the riboflavin-to-FAD pathway (Rivlin
1969). Flavokinase catalyzes the initial phosphorylation of riboflavin from ATP to form FMN.
Flavokinases from rat liver (Merrill and McCormick 1980) and rat intestine (Kasai et al. 1990) have
been isolated and purified and have molecular weights of ~28 and 13 kDa, respectively. Flavokinase
can use both ATP and dATP as phosphate donors and exhibits a preference for Zn++ as a metal
ion activator, although Mg++ can substitute producing lower reaction rates even at the pH optimum
of 9.3. The Km values of flavokinase for riboflavin and ATP are 11 and 3.7 μM, respectively. The
requirements for Zn++ and the presence of reactive sulfhydryl residues at the active site are demon-
strated by the ability of cadmium ion and sulfhydryl reagents (N-ethylmaleimde) to inhibit FMN
formation.
Glutathione, dithiothreitol, and other sulfhydryl-reducing agents protect flavokinase from inhibi-
tion (Bandyopadhyay et al. 1997). A critical connection in maintaining sulfhydryl domains within
flavokinase is further illustrated by the observation that enhanced activity of flavokinase renders
prostate cancer cells resistant to cisplatin and hydrogen peroxide. Overexpression of flavokinase not
only increases flavin coenzyme formation but also increases expression of glutathione reductase
and glutathione S-transferase π necessary for minimizing oxidative stress within cells (Hirano et
al. 2011). These findings suggest that flavokinase activity has implications not only for eliminating
oxidative stresses within cells but also for regulating the malignant progression of prostate cancer.
The activity of flavokinase is markedly reduced when the diet is deficient in riboflavin
(<0.4 mg/1000 kcal) (Prentice and Bates 1981). In addition to marginal restrictions of dietary
riboflavin, respiratory infection in rodents (Brijlal et al. 1996) also decreases activity of flavoki-
nase as observed by diminished formation of FMN and increased urinary excretion of riboflavin.
Interestingly, the activity of FAD synthetase is not altered, nor is the activity of FMN phosphatase
(EC 3.1.3.2), the second enzyme in the back conversion of FAD to riboflavin, changed. By contrast,
FAD synthetase (EC 3.6.1.9) activity is actually slightly increased under these conditions (Prentice
and Bates 1981). Thus, with dietary riboflavin deficiency, tissue FAD levels appear to be conserved
at the expense of levels of riboflavin and FMN.
The pattern of activity expressed by the flavin synthetic and dephosphorylating enzymes dur-
ing infections is similar to that during consumption of riboflavin-restricted diets. Since thyroid
hormones regulate activity of the flavin biosynthetic enzymes, studies have focused on the low
circulating levels of T3 commonly observed during respiratory infections as a major contributor
to decreases in flavokinase activity (Hashimoto et al. 1994). Decreased serum thyroid hormone
levels and changes in thyroid function are known to occur during starvation and in severely ill
patients and are collectively referred to as the “nonthyroidal illness syndrome” (Boelen et al. 2011).
The decreased activity of flavokinase during infection may reflect either decreases in de novo
biosynthesis or diminished activity of the enzyme. Other possible explanations for decreased fla-
vokinase levels during inflammation may reflect overexpression of tumor necrosis factor (TNF),
which causes flavokinase to be sequestered within the NADPH oxidase membrane complex
(Yazdanpanah et al. 2009). Flavokinase tethers directly to a TNF-receptor-1 binding protein that
couples with NADPH oxidase. Thus, flavokinase binds to both the TNF-receptor-1-death domain
and p22(phox), a subunit of NADPH oxidase assembly, and links death receptors 4/5 with Nox1. In
human HeLa and Jurkat tumor cells, death receptors 4 and 5 have been shown to activate Nox1 by
recruiting flavokinase, resulting in ROS-mediated apoptotic cell death in these tumor cells (Park
et al. 2012). These results suggest that flavokinase binding is critical for activation of NADPH oxi-
dase. It is unknown whether the recruitment of flavokinase to the six-member, membrane-bound
complex represents a loss of FMN biosynthetic function for the entire cell or gain of function
expressly for the activated complex.
K15043_C006.indd 207 4/10/2013 4:57:00 PM

After phosphorylation, a fraction of FMN becomes bound to FMN-binding domains within a

select panel of obligatory FMN-requiring apoflavoproteins. Some of these monoflavin enzymes
include inducible nitric oxide synthase (NOS), mitochondrial NADH-ubiquinone oxidoreductase
(complex I), pyridoxine 5′-phosphate oxidase, and iodotyrosine deiodinase. In addition, some difla-
vin enzymes contain both FMN and FAD as coenzymes in equimolar ratios. The first mammalian
enzyme to be identified with this arrangement was microsomal NADPH-cytochrome P450 reduc-
tase (Dignam and Strobel 1975). Human methionine synthase reductase (MSR), which plays a vital
role in methionine and folate metabolism by sustaining methionine synthase (MS) activity, is also a
member of the diflavin oxidoreductase family (Finn et al. 2003; Paine et al. 2000).
6.5.3.2 Cytosolic FAD Synthetase

Most flavoproteins use FAD rather than FMN as a coenzyme for a wide variety of metabolic reac-
tions. After the flavokinase reaction, the major portion of FMN is pyrophosphorylated to AMP by
FAD synthetase (ATP:FMN adenylyl transferase, FADS or FMNAT, aka FAD pyrophosphorylase,
EC 2.7.7.2) (Oka and McCormick 1987). FAD synthetase is a dimeric enzyme with a molecular
weight of ~97 kDa. Maximum activity occurs at pH 7.1 and requires Mg++ ion. Conversion of FMN
to FAD by the enzyme is essentially irreversible; however, pyrophosphorolytic hydrolysis of FAD
can occur at a very low rate. The Km values for FMN and ATP are 9.6 and 53 μM, respectively.
FAD synthetase plays a regulatory role for FAD homeostasis within eukaryotic cells (Huerta et
al. 2009).
As mentioned above, thyroid hormones increase the activity of the flavin biosynthetic enzymes
but the effect is more pronounced for flavokinase than it is for FAD synthetase. Under hyperthyroid
conditions, regulation of overall synthesis of flavin coenzymes and subsequent activation of their
client flavoenzymes occurs at the level of biosynthesis rather than degradation as the activities of
FMN phosphatase and FAD pyrophosphatase are not influenced by thyroid hormones (Lee and
McCormick 1985). By contrast, hypothyroidism diminishes FAD synthetase activity but the effect
is more pronounced on flavokinase activity. In a rodent model of riboflavin restriction, even at
early stages of deficiency, flavokinase activity was markedly compromised but little to no effect
was observed on the activity of FAD synthetase even at later stages of deficiency (3–5 weeks on
0.3–1 mg riboflavin/kg diet) (Lee and McCormick 1983).
X-ray crystallographic analysis of the protein structure of FAD synthetase reveals a novel flavin-
binding mode and a unique enzyme-bound FAD conformation. After assembly of FMN and ATP
within the active site, FAD synthetase quickly releases pyrophosphate but binds FAD very tightly
although not covalently. Reaction kinetics performed in vitro reveal that mild chaotropic agents are
required to dissociate the flavin attachment from the enzyme (Torchetti et al. 2011). Release of FAD
represents the rate-limiting step in final delivery of FAD to client apoflavoproteins. This tightly
controlled process for release of FAD may provide an explanation for the decreased formation of
covalently bound flavoproteins despite relative increases in hepatic FAD synthesis that occur during
riboflavin deficiency (Pinto and Rivlin 1979).
6.5.3.3 Mitochondrial FAD Synthetase

Studies in eukaryotic cells have identified two isoforms of FAD synthetase that are distinct in size
and intracellular location (Brizio et al. 2006). FAD synthetase located within the cytosolic compart-
ment is referred to as FAD synthetase-2 whereas FAD synthetase-1, which displays an additional
97-amino-acid domain at the N-terminus portion, localizes to the mitochondrial matrix. Sequence
analysis of isoform-1 displays a 17-amino-acid region that represents a cleavable mitochondrial
targeting sequence, which suggests that the protein is synthesized via nuclear DNA (Torchetti et al.
2010).
The presence of the two FAD synthetase isoforms supports the previous finding of a riboflavin/
FAD cycle identified in rat liver mitochondria (Barile et al. 2000). The formation of intramitochondrial
K15043_C006.indd 208 4/10/2013 4:57:00 PM

FAD requires transport of riboflavin from the cytosolic compartment into mitochondria and subse-
quent conversion to FMN via a putative mitochondrial flavokinase. Newly synthesized FAD com-
bines with its imported client apoflavoproteins when inside the mitochondria. This process may also
explain why diminished formation of covalently linked flavoproteins, which are exclusively resi-
dential mitochondrial proteins, occurs despite increased FAD synthesis observed during riboflavin
deficiency. Mammalian mitochondria exclusively harbor covalently linked flavoenzymes whose syn-
thesis within mitochondria may be dependent on bioavailability of the client apoflavoproteins and
not solely on the availability of FAD. Unassociated FAD is exported from the mitochondria to the
cytosol via a flavin exchange protein, FLX1, which belongs to a family of mitochondrial transport
proteins (Tzagoloff et al. 1996). FLX1 plays a crucial role in maintaining normal levels of FAD-
binding enzyme activity within the mitochondria and may also serve as a key regulator for unbound
FAD within the mitochondria (Bafunno et al. 2004).
Flavins unaffiliated with their client proteins or not confined within their proper domains in
the electron transport chain may force unscheduled electron shunting within the chain and result
in inappropriate formation of ROS. Leakage of electrons from complexes I and III of the electron
transport chain is the major source of reactive oxygen in the mitochondria. Generation of superox-
ide occurs at both the matrix side of the inner mitochondrial membrane and the cytosolic side of
this membrane (Ghiasi et al. 2012). An intrinsic contributor of ROS is the overexposure of riboflavin
to exogenous sources of UV light. It is of interest in this regard that riboflavin, transported into
mitochondria, may require immediate transformation to FMN and FAD by the mitochondrial FMN
and FAD biosynthetic enzymes. In blood samples from patients with amyotrophic lateral sclero-
sis (ALS), high-throughput sequencing using quantitative reverse-transcriptase PCR has revealed
that mRNA expression of FAD synthetase, flavokinase, and other electron transport chain proteins
are decreased in blood samples from patients with ALS compared to expressed levels in controls
(Lin et al. 2009). Sporadic ALS is an age-associated disease with cytoskeletal abnormalities and
slow demise of motor neurons caused by oxidative stress. It is unknown whether the low mRNA
expression in this disorder represents mitochondrial or cytosolic isoforms of the flavin biosynthetic
enzymes. In any event, compromised reduction in the activities of the flavin biosynthetic enzymes
(flavokinase and FAD synthetase) in patients with ALS could result in accumulation of free ribofla-
vin that may contribute to formation of excessive ROS. FMN and FAD serve as coenzymes and are
stabilized against photoreactivity and electron transfer while buried within their protein domains
whereas riboflavin does not share the same extent of privileged binding. Human motor neurons
are particularly susceptible to injury if marked changes occur in components of the mitochondrial
respiratory chain enzymes (Crugnola et al. 2010). Unattended riboflavin present within the inner
compartments of mitochondrial membranes may short circuit the tightly controlled flow of elec-
trons through the electron transport chain.
Advances in development of novel chemotherapeutic agents have been made by recognizing the
diversity in reactivity of both flavokinase and FAD synthetase with riboflavin analogs. Flavokinase
and FAD synthetase can convert roseoflavin (RoF) and 8-demethyl-8-amino-riboflavin (AF) into
coenzyme analogs of FMN (RoFMN and AFMN) and FAD (RoFAD) (Pedrolli et al. 2011). In addi-
tion to targeting enzymes, the coenzyme moieties are major therapeutic targets and thus coenzymic
mimics can function as effective chemotherapeutic and anti-infective agents. mRNAs that bind Is this correct as
modified?
FMN directly (a riboswitch), which regulates riboflavin metabolism within the organism, have been
identified. FMN-specific riboswitches (aka RFN elements) direct the expression of bacterial genes
critical for biosynthesis and transport of riboflavin (Serganov et al. 2009). The RoFMN and AFMN
analogs can inactivate a variety of flavoenzymes and bacterial riboswitches and thus have potential
to serve as lead compounds for development of antimicrobial agents (Mansjö and Johansson 2011).
Targeting coenzymes within vital metabolic pathways of microorganisms may be a more effective
approach to combat the increased resistance developed by microorganisms to the current armamen-
tarium of existing antimicrobial agents.
K15043_C006.indd 209 4/10/2013 4:57:00 PM

6.5.3.4 FAD Pyrophosphatase
The dephosphorylation or back conversion of FAD to riboflavin requires FAD pyrophosphatase
(EC 3.6.1.18) and FMN phosphohydrolase (aka, FMN phosphatase, EC 3.1.3.2). The FAD and FMN
dephosphorylating enzymes convert FAD to FMN and FMN to riboflavin, respectively, and were
initially isolated from the intestinal brush border membrane of the rat (Akiyama et al. 1982). The
pH maxima for rat FAD pyrophosphatase and FMN phosphohydrolase are each around 7.0. Both
enzymes are inhibited by EDTA and inactivated by heating for 15 and 25 min at 56°C. Each enzyme
plays an important role in converting dietary flavins (FAD and FMN) to riboflavin, which is then
recognized by the RFT-2 on the intestinal mucosal brush border.
Human placental syncytiotrophoblast microvilli also contain flavin dephosphorylating
enzymes supporting the need for the specific RFT-1 within the placenta (Lee and Ford 1988). The
FAD pyrophosphatase isolated from syncytiotrophoblasts has “moonlighting” characteristics as
it also displays 5′-nucleotidase activity. This enzyme is a glycoprotein with a molecular weight
of 74 kDa.
Similar to the intestinal enzyme, EDTA treatment of the placental enzyme inhibits the FAD
hydrolyzing as well as its 5′-nucleotidase activity; however, Co++ restores the pyrophosphorylase but
not the 5′-nucleotidase activity to normal levels. The presence of both FAD pyrophosphatase and
FMN phosphatase within the syncytiotrophoblast microvilli supports the need for dephosphory-
lation of flavins to parent riboflavin within the maternal circulation prior to placental transport
into the fetal compartment. Unlike the phosphorylating enzymes flavokinase and FAD synthetase,
activities of FAD pyrophosphatase and FMN phosphohydrolase are apparently not influenced by
thyroid hormones or by dietary riboflavin status (Lee and McCormick 1983, 1985). The activities of
the dephosphorylating enzymes however decrease with age (Lee and McCormick 1985). Figure 6.5
illustrates the biosynthesis and degradation of flavin coenzymes and the subsequent effects of each
process on the stability of their client proteins.
Activation/stabilization of client
Biosynthesis flavoenzymes
Flavokinase FAD
synthetase
Riboflavin FMN FAD
FMN FAD
Degradation
Flavokinase FAD
synthetase
Riboflavin FMN FAD
FMN FAD
Inactivation/destabilization of
client flavoenzymes
FIGURE 6.5 Formation and degradation of flavin coenzymes. The upper diagram illustrates the biosynthe-
sis of FMN and FAD by flavokinase and FAD synthetase, respectively, and the subsequent activation of their
client apoflavoenyzmes. The lower diagram illustrates the degradation of FAD and FMN by FAD pyrophos-
phatase and FMN phosphatase, respectively, coupled with the eventual inactivation and destabilization of
their client flavoenzymes.
K15043_C006.indd 210 4/10/2013 4:57:01 PM

6.5.3.5 FMN Phosphatase
After FAD pyrophosphohydrolase reaction of FAD to FMN, complete hydrolysis of FMN to ribo-
flavin involves removal of the terminal 5′-phosphate moiety from the ribityl side chain. A low-
molecular-weight (~18 kDa) FMN phosphatase was isolated from bovine kidney and shown to
have properties similar to acid phosphatases (ACPs). The enzyme demonstrated high efficiency for
FMN, is activated by guanosine, and is strongly inhibited by copper and p-chloromercuribenzoate
(Granjeiro et al. 1997).
FMN phosphatase has been associated with other ACP1 gene products, namely, low-molecular-
weight phosphotyrosine protein phosphatase (LMW-PTP) (Fuchs et al. 1992). Thus, like the FAD
pyrophosphatase isolated from syncytiotrophoblasts, the LMW-PTP/FMN phosphatase appears to
exhibit moonlighting activity that includes dephosphorylation of both phosphotyrosine and FMN.
Characteristic features of the enzyme have been more closely examined in Chinese hamster ovary
cells and in erythrocytes (Apelt et al. 2009). Kinetic analyses of LMW-PTP using FMN and phos-
photyrosine as substrates have demonstrated kcat/Km of 7.3 × 103 s−1 M−1 and 1.7 × 10 −1 s−1 M−1,
respectively, indicating a greater efficiency of the enzyme for FMN. Thus, the physiological func-
tion of FMN phosphatase appears to be more closely aligned with a role for LMW-PTP in flavin
metabolism.
Investigations of the common genetic variants among the ACP1 gene products have revealed
a causal association with various aspects of flavin metabolism. Studies in humans with the com-
mon ACP1 genotypes (ACP1*A, ACP1*B, and ACP1*C) have observed a negative correlation
between the activities expressed by the LMW-PTP phenotypes and that of glutathione reductase,
an FAD-requiring enzyme. Low basal activity of erythrocyte glutathione reductase coupled with its
enhanced activity in the presence of exogenous FAD (activity coefficient) and low glutathione levels
are used as indicators of tissue flavin saturation and as a biomarker of oxidative stress.
Erythrocytes from individuals expressing the ACP*A allozyme have lower FMN phosphatase
activities than those with ACP1*B and ACP1*C phenotypes. Interestingly, comparative studies
among individuals with the various ACP1 alleles show that those expressing the genotype AA have
higher basal levels of glutathione reductase, a greater proportion of glutathione reductase present as
the holoenzyme, and higher endogenous levels of FAD (Mohrenweiser and Novotny 1982).
These findings suggest that a loss of FMN phosphatase activity as observed in the ACP1 geno-
type AA may provide individuals with greater resistance to diseases associated with oxidative stress
such as acute ischemic stroke (Gariballa and Ullegaddi 2007), hypertension (Chaves et al. 2007),
and diabetes (Dincer et al. 2002). Thus, genetic alterations or perhaps use of specific inhibitors,
such as uric acid (Granjeiro et al. 2002), that interfere with the dephosphorylating activity of FMN
phosphatase may possibly improve stability and reactivity of flavoenzymes. This hypothesis needs
to be tested experimentally.
By contrast, increased activity or gain of function of the flavin dephosphorylating enzymes would
be expected to adversely affect the endogenous levels of FAD and FMN. In support of this concept,
the activity of FAD pyrophosphatase is elevated (to ~270% of normal) in the muscle of an individual
diagnosed with subacute carnitine-deficient lipid storage myopathy (Vergani et al. 1999). Muscle
FAD and FMN concentrations were decreased to approximately 50% that of control subjects. In a
second patient presenting with lipid storage myopathy, FAD pyrophosphatase activity was within
the normal range but muscle flavin coenzyme concentrations were 22% of control levels. Although
it was not determined whether FMN phosphatase activity was also increased, in either or both cases,
riboflavin supplementation corrected the biochemical abnormalities of increased organic aciduria.
Mitochondria display a pair of flavin dephosphorylating enzymes that differ from their isoforms
in the cytosol. Mitochondrial FAD pyrophosphatase and FMN phosphohydrolase differ from the
extramitochondrial enzymes in their pH maxima and sensitivity to inhibitors (Barile et al. 1997).
Thus, in view of the complete complement of both flavin biosynthetic and dephosphorylating
enzymes, as well as the presence of a flavin exchange protein, FLX1, it has been postulated that a
K15043_C006.indd 211 4/10/2013 4:57:01 PM

riboflavin–FAD cycle occurs within mitochondria and further that it plays a major role in control-
ling mitochondrial flavoprotein turnover. Understanding the homeostatic control and turnover of
the flavin coenzymes within the mitochondrial compartment is crucial to clarifying the regulatory
functions of flavoenzymes in maintenance of normal cellular metabolism and bioenergetics.
6.5.4 Riboflavin Excretion
6.5.4.1 Riboflavin Tubular Secretion and Reabsorption
After release from flavoproteins, flavin elimination occurs primarily through renal excretion pre-
dominantly in the form of riboflavin; FMN and FAD are not found in urine in measurable amounts.
Studies on renal clearance using a rodent perfusion model showed that riboflavin is actively secreted
and reabsorbed through the proximal tubules (Kumar et al. 1998). Both processes are dependent on
plasma riboflavin concentrations in that net riboflavin secretion decreases at 0.01 μM and results in
increased riboflavinuria at 1 μM. Renal tubular reuptake and secretion of riboflavin are saturable
and temperature dependent in both directions. Although Na+ concentration has no known effect on
tubular uptake and excretion of riboflavin, a Ca2+/calmodulin-mediated pathway appears to play a
role in the regulation of tubular uptake of riboflavin (Yanagawa et al. 2000). Pharmacokinetic stud-
ies of riboflavin clearance after its oral or intravenous administration indicate that urinary excretion
accounts for approximately half the overall riboflavin removed from plasma (Zempleni et al. 1996).
Urinary excretion of riboflavin can be a useful indicator of food intake in free-living individuals
(Tsuji et al. 2010). In a similar manner, consumption of dietary supplements that contain riboflavin
reveals a dose-dependent cumulative increase in urinary riboflavin excretion up to approximately
25 mg/day (Itokawa et al. 1992). Positive correlations occur among levels of dietary riboflavin,
protein, and energy intake. Thus, riboflavinuria also exhibits a positive correlation with intakes
of riboflavin, protein, and energy. Negative correlations are observed between riboflavin excretion
and elevated erythrocyte glutathione reductase activity coefficient, an assay indicative of riboflavin
deficiency (Fogelholm et al. 1993).
Fasting in humans affects urinary excretion of riboflavin. A 1-day fast increases by threefold
the urinary content of riboflavin (Fukuwatari et al. 2010). By contrast, extended periods of fasting
in animals result in decreased urinary riboflavin excretion. Hepatic riboflavin stores are mobilized
during early periods of food deprivation and can transiently maintain plasma levels to meet the
nutritional needs of peripheral tissues. It is important to remember that accurate interpretation of
high urinary riboflavin levels should be made in consideration of recent periods of fasting or con-
sumption of riboflavin-rich food items.
Increased demands for riboflavin by peripheral tissues especially during periods of exercise
result in decreases in urinary excretion of riboflavin (Soares et al. 1993). Assessment of physical
performance in women 50–67 years of age showed that urinary riboflavin levels decrease in asso-
ciation with added physical demands of exercise training but that riboflavin supplementation does
not enhance physical endurance (Winters et al. 1992). Despite the effects of estrogen on intracellular
riboflavin-binding proteins and metabolism, gender differences do not appear to affect riboflavin
excretion, although studies have shown that oral contraceptive agents can exacerbate a preexistent
riboflavin deficiency (Anderson et al. 1976; Thorp 1980). Hormones regulate food intake, which, in
turn, influences the levels of riboflavin excretion (Martini et al. 1994).
Mobilization of flavins from tissues to plasma results in increased urinary loss of riboflavin.
Studies in humans and animals show that the metabolic response to inflammatory stresses such as
infection with pneumococcal pneumonia involves release of FAD from hepatic stores. An increased
secretion of FAD into plasma results in a concomitant increase in urinary riboflavin excretion
(Brijlal et al. 1999). Flavin release from the liver may be part of the evolutionarily conserved acute
phase response. Hepatocytes play a pivotal role in innate immunity and the control of systemic
inflammatory responses to infectious and noxious stress factors (Bode et al. 2012; Quinton et al.
K15043_C006.indd 212 4/10/2013 4:57:01 PM

2012). By contrast, mobilization of flavins has not been observed in muscle, small intestine, and
brain (Brijlal and Lakshmi 1999), further indicating the importance of hepatocytes as a major con-
tributor to the acute phase response. Thus, continued mobilization of riboflavin from liver during
inflammatory stress coupled with an increase in riboflavinuria may result in rapid deterioration of
riboflavin status and further compromise antioxidant balance within the cell. Such an effect would
explain the inability of natural host defenses to regenerate reduced glutathione (GSH) during recov-
ery from acute respiratory distress syndrome (Kozar et al. 2000). Thus, accurate interpretation of
urinary riboflavin levels must be viewed with an understanding of not only the dietary content of
riboflavin but also nondietary factors such as physiological stress.
6.5.4.2 Drugs and Riboflavin Excretion

Drugs such as probenecid that inhibit the organic anion transporter diminish urinary tubular secre-
tion of riboflavin and elevate its plasma levels (Jusko and Levy 1967). Carrier-mediated transport
mechanism may be involved since tubular secretion of riboflavin is also decreased by para-
aminohippuric acid, an organic anion, and by lumichrome, an analog of the isoalloxazine ring
of riboflavin. Phenothiazine drugs, which bear a structural resemblance to the isoalloxazine ring,
inhibit both riboflavin secretion and reabsorption in the proximal tubules (Pelliccione et al. 1983).
However, despite the concomitant inhibition of these transport processes, riboflavinuria is the net
result because riboflavin filtration appears to exceed the decrease in urinary riboflavin secretion
under these circumstances (Pinto et al. 1985; Pinto and Rivlin 1987). Increased plasma levels of
unbound riboflavin would be expected to undergo rapid glomerular filtration and enhanced renal
clearance. Thus, malutilization of riboflavin characterized by reduced tissue uptake, diminished
apoflavoprotein binding, decreased FAD formation, or increased dephosphorylation of FMN and
FAD to riboflavin would result in elevated levels of plasma riboflavin. Psychotropic drugs and tri-
cyclic antidepressants that bear analogous structures to riboflavin impair conversion of riboflavin to
FMN and FAD and as a result cause increased riboflavinuria (Pinto et al. 1981).
In addition to interactions with tricyclic drugs, riboflavin metabolism is also affected by tet-
racyclic drugs. Both in vitro and in vivo studies show that Adriamycin and tetracycline bind
competitively to sites similar to those that bind flavin coenzymes and have a common cytotoxic
effect through inhibition of FAD biosynthesis, particularly in skeletal and cardiac muscle (Dutta
et al. 1988; Pinto et al. 1986; Raiczyk and Pinto 1988). Intraperitoneal injections of Adriamycin in
rats for six consecutive days cause increased urinary riboflavin excretion that begins 2 days after
Adriamycin administration (Ogura et al. 1991).
Ingestion of boric acid greatly increases urinary excretion of riboflavin. When consumed, borates
react with polyhydroxyl moieties on the ribityl side chain of riboflavin as well as on other molecules
that have polyhydroxyl groups, such as glucose and ascorbic acid. In both rats and guinea pigs,
excretion of a dose of radiolabeled riboflavin is greater in boric acid–fed animals than in controls
(Roe et al. 1972). Repletion with riboflavin greatly ameliorates the toxicity of administered boric
acid.
Studies in humans after accidental ingestion of boric acid, for example, boraxo powder or borate,
reveal that most patients excrete riboflavin at levels that exceed those in the 100th percentile of
the Ten-State Nutrition Survey. Urinary excretion of riboflavin expressed per gram of creatinine
increases within the first 24 h after borate ingestion (Pinto et al. 1978). As demonstrated in animal
models, administration of riboflavin should be effective in humans after accidental ingestion of
boric acid, although in practice it may be difficult to provide adequate amounts of riboflavin because
of its low solubility and limited absorptive capacity from the intestinal tract.
6.5.4.3 Urinary Metabolites of Riboflavin

Several derivatives and metabolites of riboflavin have been identified (Ohkawa et al. 1983a; West and
Owen 1969). In addition to the 60% to 70% of urinary flavins contributed by riboflavin itself, other
K15043_C006.indd 213 4/10/2013 4:57:01 PM

major derivatives include 7-α-hydroxymethylriboflavin (10%–15%), 8α-sulfonylriboflavin (5%–10%),

8-α-hydroxymethylriboflavin (4%–7%), riboflavinyl peptide ester (5%), and 10-hydroxyethylflavin
(1%–3%). These flavin derivatives represent largely metabolites from covalently bound flavoproteins
as well as intestinal riboflavin degradation by microorganisms. Traces of lumiflavin and other deriva-
tives have also been found in urine. Radiolabeled riboflavin administered to rats revealed urinary
metabolites of lumichrome, namely, 7-carboxy lumichrome and 8-carboxy lumichrome (Ohkawa et
al. 1983b) as well as a glucosylated derivative of riboflavin, riboflavinyl glucoside (Ohkawa et al.
1983c). It is unknown to what extent these urinary metabolites represent metabolism by mammalian
cytochrome P450–dependent monooxygenase system or absorption products from gastrointestinal
microbial flora.
In rats fed a riboflavin-deficient diet for 25 days and then administered a rescue dose of radiola-
beled riboflavin, a variety of conjugated metabolites of riboflavin were identified that were capable of
supporting growth of a standardized test organism for riboflavin, Lactobacillus casei (Christensen
1971; Tillotson and Karcz 1977). In addition, non-growth-supportive metabolites were found in
addition to radiolabeled urea. These studies suggest that, at least in rodent models, the metabolic
fate of riboflavin involves modification of its ring components, conjugation as glucosyl derivatives,
and extensive degradation to urea. Laboratory investigations have not determined whether a ribo-
flavin metabolite or the urinary profile of metabolites has any diagnostic potential in evaluating
diseases.
Urinary monitoring techniques for riboflavin have proven to be very effective tools in determin-
ing bioavailability of riboflavin from foods and for use as an objective biomarker of compliance
in pharmaceutical and clinical research studies. By contrast, use of plasma riboflavin appearance
methods underestimates riboflavin bioavailability because a major portion of recently absorbed
constituents from foods including riboflavin are removed by the hepatic “first pass” effect (Dainty
et al. 2007). The ingestion of riboflavin with foods and medications coupled with urinary measure-
ment is an accepted method of compliance detection (Anton 1996). Ingestion of wheat bran cereal
containing varying tracer amounts of riboflavin correlates with the level of the tracer dose and can
be used as a biomarker to validate fiber supplement intake (Switzer et al. 1997). Thus, urinary ribo-
flavin determinations provide a valid marker for adherence assessment of drugs, food, and supple-
ment compliance in large-scale clinical evaluation trials.
6.6 Riboflavin Deficiency
Animals cannot synthesize riboflavin and must obtain the vitamin from exogenous sources.
Riboflavin is essential for normal cellular function, growth, and development; deficiency leads to a
variety of fundamental and clinical abnormalities that range from mitochondrial dysfunction and
hemolytic anemia to growth retardation and neurological disorders. In addition to maintaining
adequate levels through dietary intake, homeostasis of riboflavin metabolism depends on normal
intestinal absorptive processes. Riboflavin has an impact on its own absorption and transport by
directly affecting gastrointestinal cell morphology. Depletion of dietary riboflavin impairs the size
and cellularity of the duodenal crypts by altering mitotic potential and increasing aneuploidy of
cells within the microvilli (Nakano et al. 2011).
Mechanisms involving riboflavin absorption and transport have been discussed earlier in this
chapter. Interference with these absorptive processes includes digestive and absorptive disorders,
intestinal diseases including resection, drug interactions, and chronic alcohol use, all leading to the
development of frank deficiency or suboptimal status and eventual clinical abnormalities. In addi-
tion, congenital defects of riboflavin transport such as Brown–Vialetto–Van Laere and Fazio–Londe
syndromes (C20orf54) and haploinsufficiency of GPR172B cause persistent riboflavin deficiency
(Bosch et al. 2011; Ho et al. 2011).
K15043_C006.indd 214 4/10/2013 4:57:01 PM

Aside from these rare genetic defects, isolated deficiencies of riboflavin are not widely prevalent
in the general population. Several of the physical and clinical symptoms that appear to be exclusive
features of riboflavin deficiency are in reality not pathognomonic or unique to riboflavin. Thus,
the classical glossitis, angular stomatitis, cheilosis, and dermatitis observed in advanced cases of
diminished riboflavin intake may be due to other vitamin deficiencies as well. In fact, when defi-
ciency of riboflavin does occur, it is almost invariably in association with multiple nutrient deficits
(Baker et al. 1980; Rivlin 1994). Clinical aspects of riboflavin deficiency (Hoppel and Tandler 1990)
and its interference with normal cell cycle progression and regulation of gene expression have been
reviewed elsewhere (Powers et al. 2012).
Dietary inadequacy and diminished intestinal transport are not the only causes of riboflavin defi-
ciency. In approaching riboflavin deficiency, as well as other nutrient deficiencies, it may be useful
to think in terms of risk factors. That is to say, the consequences of a poor diet may be intensified if
the patient is also experiencing hormonal imbalances, is using certain drugs for prolonged periods,
is abusing alcohol, is elderly, or has malabsorptive or other underlying illnesses affecting vitamin
metabolism (Bjarnason et al. 1996; Buckman and Heise 2010; O’Morain 1990).
Certain endocrine abnormalities, such as adrenal and thyroid hormone insufficiency, specific
drugs, and diseases may interfere significantly with vitamin utilization (Cimino et al. 1987; Rivlin
1991). Psychotropic agents, such as chlorpromazine; antidepressants, including imipramine and
amitriptyline (Pinto et al. 1981); cancer chemotherapeutic drugs, namely, Adriamycin; and some
antimalarial agents, for example, quinacrine (Dutta et al. 1985, 1988), impair riboflavin utilization
by inhibiting the conversion of this vitamin into its active coenzyme derivatives. Figure 6.6 shows
the structural similarities among riboflavin; the tricyclic compounds chlorpromazine, imipramine,
and amitriptyline (top); and the tetracyclic drugs Adriamycin and tetracycline (bottom). There is
evidence that alcohol causes riboflavin deficiency by inhibiting both its digestion from dietary
sources and its intestinal absorption (Dutta et al. 1995; Pinto et al. 1987).
N Cl N
CH2-(CH2)2-N(CH3)2 CH2-(CH2)2-N(CH3)2 CH-(CH2)2-N(CH3)2
Chlorpromazine Imipramine Amitriptyline
O
H3C N
NH
H3C N N O
CH2-(CHOH)3-CH2-OH
Riboflavin
O OH O OH O OH O O
OH
CCH2OH CNH2
OH
H H OH
H H3C OH OH
OCH3 O OH O O CH3 N(CH3)2
Adriamycin Tetracycline
OH
NH2
FIGURE 6.6 Structural formulas of riboflavin (middle); chlorpromazine, imipramine, and amitriptyline
(top); and Adriamycin and tetracycline (bottom) showing ring similarities and the potential for ring stacking
through π–π interactions.
K15043_C006.indd 215 4/10/2013 4:57:01 PM

6.6.1 Metabolic Adaptations to Riboflavin Deficiency

With the onset of riboflavin deficiency, one adaptation that occurs is a fall in the hepatic free ribo-
flavin pool to nearly undetectable levels, with relative sparing of the pools of FMN and FAD that
are needed to fulfill critical metabolic functions (Fass and Rivlin 1969). Another adaptation to
riboflavin deficiency in its early stages is an increase in hepatic de novo synthesis of GSH from
its amino acid precursors, in response to the diminished back conversion of oxidized glutathione
to GSH (Rivlin 1994). The transient increase in GSH synthesis observed during riboflavin defi-
ciency is associated with activation of the nuclear factor erythroid 2–related factor 2 (Nrf2)–anti-
oxidant responsive element pathway (McMahon et al. 2001). This stress–response pathway involves
enhanced transcription of gamma-glutamylcysteine synthetase (EC 6.3.2.2), glutathione synthetase
(EC 6.3.2.3), and other phase 2 enzymes, glutathione transferases, NAD(P)H:quinone reductase,
UDP-glucuronosyltransferase, and epoxide hydrolase (Lee et al. 2003).
Since riboflavin deficiency is accompanied by increased levels of ROS, cells become challenged
by excessive oxygen-free radicals and more susceptible to oxidative stress. In response, cells initiate
a well-orchestrated antioxidative defense manifested through Nrf2 gene transcription. Cellular GSH
plays an important role in the defense against oxidative stress and induction of gamma-glutamylcysteine
synthetase is effective for protecting endothelial cells.
Increased de novo biosynthesis of GSH may also be mediated by prostaglandins (PGs). Treatment
of L-1210 cells with PGA2 results in a persistent rise in GSH owing to a sustained increase in the
activity of gamma-glutamylcysteine synthetase (Ohno and Hirata 1990). Kidneys from riboflavin-
deficient animals exhibit marked increases in PG biosynthesis (Pelliccione et al. 1985) and thus may
also trigger increased de novo synthesis of GSH biosynthetic enzymes.
These data in their entirety strongly suggest that induction of GSH synthesis during riboflavin
deficiency may be provoked by increased oxidative stress and promoted by Nrf2 or PG-mediated
pathways. The compensatory reactions resulting from depressed activity of glutathione reductase,
a key FAD-requiring enzyme, and the compensatory induction of gamma glutamylcysteine/GSH
synthetase reactions are shown in Figure 6.7.
Riboflavin
Riboflavin sufficiency FMN
FAD
Reduced Oxidized
Glycine
Glutamate γ-Glutamyl
γ-Glutamyl- Glutathione Glutathione
+ cysteine glutathione Glutathione
cysteine synthetase reductase
Cysteine synthetase
(GSH) (GSSG)
FAD
R i b o f l a v i n d e f i c i e n c y, N r f 2, P G’ s
FMN
Riboflavin
FIGURE 6.7 Regeneration of GSH under normal and riboflavin-deficient conditions. The diagram represents two
major pathways for the formation of reduced GSH in erythrocytes, namely, (a) diminished formation of oxidized
glutathione (GSSG) via the FAD-dependent, glutathione reductase pathway and (b) increased de novo biosynthesis
via gamma-glutamylcysteine synthetase and glutathione synthetase. Bold arrows are used to emphasize the pre-
dominant pathways, thin arrows represent pathways that are operating below maximal levels, and the dotted arrow
indicates diminished enzymatic activity. De novo biosynthesis of GSH may also be driven by induction of the Nrf2
antioxidant transcription pathway (not fully illustrated) and by PG induction of gamma-glutamylcysteine synthetase.
K15043_C006.indd 216 4/10/2013 4:57:02 PM

Prolonged riboflavin deficiency in rats results in decreases in the number of villi within the small
intestine; villus length increases, as does the rate of transit of developing enterocytes along the villus
(Williams et al. 1996). These structural abnormalities, together with the accelerated rate of intestinal
cell turnover, may help explain why dietary riboflavin deficiency leads to both decreased iron absorp-
tion and increased iron loss from the intestine (Powers et al. 1993). They also suggest that changes in
cell cycle may result directly from riboflavin deficiency as riboflavin repletion does not reverse this
process and clonogenicity is lost (Yates et al. 2003). Thus, a clear link has been made between mater-
nal riboflavin deprivation in rats and intestinal development not only during the fetal period but also
extending into neonatal life and even into young adulthood (Williams et al. 1996; Yates et al. 2001).
In studies of duodenal biopsies from a cross section of gastroscopy patients, crypts were shorter
and hypocellular and exhibited fewer cell divisions from subjects in the lowest quartile than from
those in the highest quartile of riboflavin status (Nakano et al. 2011). Low riboflavin status also
resulted in irreversible loss of proliferative capacity of intestinal cells, an effect that may be associ-
ated with the unfolded protein stress response observed during riboflavin deficiency (see Section
6.9). These studies in humans coupled with those from rat models suggest that riboflavin deprivation
at a critical stage in crypt development results in irreversible loss in their proliferative potential and
may translate into later detrimental consequences of gastrointestinal function.
The role of riboflavin in iron transport and metabolism is described later in this chapter (see
Sections 6.6.3.2 and 6.7.2.3.4). Ultrastructural studies of marrow erythroblasts from rats made ribo-
flavin deficient reveal morphological abnormalities within their nuclear compartments that involve
asynchrony of nuclear–cytoplasmic maturation (Norton et al. 1979). During the latter stages of
erythroblast development, cells of deficient animals retain a larger portion of their ribosomes com-
pared to those from control animals. These studies appear to reflect morphological changes similar
to those observed in intestinal cells during riboflavin deficiency in that improperly folded proteins
result in discontinued nuclear development.
Critical effects of riboflavin deficiency as detected in liver, intestine, and other tissues have also
been found in lens as revealed by electron microscopic analysis (Hasegawa and Yagi 1975). Riboflavin-
deficient diets cause lens epithelia to swell, their mitochondria to degenerate, and their cytoplas-
mic compartment to undergo vacuolization, a process associated with necrosis and necroapoptosis
(Degterev et al. 2005). These changes are similar to those observed during initial periods of cataracto-
genesis. Thus, the potential role of riboflavin in the prevention of cataractogenesis has been suggested
(Jacques 1997). Riboflavin intakes ranging from the recommended daily allowance (1.2–1.6 mg) to
multivitamin preparations containing 10–30 mg riboflavin have been associated with reduced cataract
formation. The use of riboflavin for a 5-year period in the Nurses’ Health Study was associated with
a decreasing rate of development of lens opacification (Jacques et al. 2005). Early stages of cataract
formation within the general population were not found to correlate with dietary riboflavin deficiency;
however, a relationship between riboflavin deficiency and late-stage cataract formation was identified
particularly in association with human aging (Skalka and Prchal 1981). During cataractogenesis, lens
proteins unfold and thiols that are buried become exposed and react with GSH, cysteine, and protein
sulfhydryl groups to form disulfide-cross-linked aggregates (Craghill et al. 2004).
The activity of glutathione reductase in the lens is markedly reduced by UV-A light owing to sen-
sitivity of its FAD moiety (Linetsky et al. 2003). This effect results in a decreased GSH-to-oxidized
glutathione ratio compared with that of clear lenses. Loss of endogenous lens GSH is the result of
both reversible and irreversible glutathionylation. Irreversible attachment of GSH to lens proteins
results in the formation of nonreducible thioether linkages (lanthionine) as opposed to reducible,
mixed disulfide GSH attachments (reviewed in Cooper et al. 2011).
6.6.2 Organelle Adaptations to Riboflavin Deficiency

In experimental animals, hepatic architecture is markedly disrupted in riboflavin deficiency. Mito
chondria in riboflavin-deficient mice progress through a series of morphological changes ranging
K15043_C006.indd 217 4/10/2013 4:57:02 PM

from elongation of cristae to development of cristae clusters that result in cup-shaped mitochondria
that tend to nest within each other. With cristae structures increasing in length and number, mito-
chondria, which normally range in size from 0.5 to 10 μm, expand to diameters greater than 8 μm
(Hoppel and Tandler 1975; Tandler et al. 1969; Tandler and Hoppel 1980).
Although these structural abnormalities occur, changes in oxidative metabolism appear mar-
ginal, as minor losses are observed in the activity of the flavin-dependent components of the elec-
tron transport chain, namely, complexes I and II (Veitch et al. 1989). Despite the relatively normal
appearance of oxidative metabolism by these mitochondria in vivo, unscheduled shunting of elec-
trons, particularly within complexes I and II, may also prevail, thereby increasing formation of
ROS. In contrast to activities of flavoproteins associated with complexes I and II, activities of the
three FAD-dependent acyl-CoA dehydrogenases in lipid metabolism are severely depressed as well
as the mitochondrial acyl-CoA dehydrogenases involved in the metabolism of the branched-chain
amino acids. Morphological changes in mitochondria induced during riboflavin deficiency also
compromise the activity of the mitochondrial lipid transporter, carnitine palmitoyltransferase.
Other critical organelles markedly impaired during riboflavin deficiency are peroxisomes. These
organelles contribute to several crucial metabolic processes such as β-oxidation of fatty acids, bio-
synthesis of ether phospholipids, and metabolism of ROS (Van Veldhoven 2010). Because of their
dynamic ability to rapidly assemble, multiply, and degrade in response to metabolic needs, peroxi-
somes, like mitochondria, are indispensable to human health and development. FAD-dependent
enzymes are critical for lipid metabolism within peroxisomes. Lipid-metabolizing pathways
involving flavoenzymes include biosynthesis of ether phospholipids and degradation of long-chain
fatty acids, methyl branched fatty acids, PGs, bile-acid precursors, and xenobiotics by either α- or
β-oxidation pathways, the latter of which also metabolizes ω-oxidized fatty acids (Poirier et al.
2006).
By contrast to effects on mitochondria, riboflavin deficiency results in increases in both mRNAs
and activities of peroxisomal FAD-dependent fatty acyl-CoA oxidase and peroxisomal carnitine
palmitoyltransferase (Veitch et al. 1989). Effects similar to these have also been observed during
starvation (Brady et al. 1989). Despite the relatively normal appearance of riboflavin metabolism
within peroxisomes during starvation, the activities of other peroxisomal flavoproteins, d-amino acid
oxidase and glycolate oxidase, are markedly reduced. Riboflavin deficiency also reduces activity of
pipecolate oxidase, an exclusive peroxisomal covalently linked (cysteinyl) flavoprotein, resulting in
decreased oxidation of l-pipecolic acid, a metabolite of lysine (Wanders et al. 1989). Abnormalities
in peroxisomal function induced during riboflavin deficiency resemble those observed in Zellweger
syndrome (Bennett et al. 1991), an autosomal recessive disorder characterized by dysfunctional
peroxisomes. Apparently compensatory mechanisms prevail during riboflavin deficiencies that
involve coordination of oxidative metabolism among mitochondria and peroxisomes. It is likely that
metabolic coordination among organelles is critical to maintain vital cellular function. Part of this
cooperative effort may entail translocating flavin coenzymes for their most beneficial use among
flavoenzymes within the mitochondrial and peroxisomal compartments.
6.6.3 Clinical Signs of Riboflavin Deficiency

6.6.3.1 Dermal Abnormalities
Dermal abnormalities constitute one of the more variable and multisystemic clinical manifesta-
tions of riboflavin deficiency. Although some signs may be closely aligned with onset of ribofla-
vin deficiency, marked overlap of skin manifestations is observed in multiple vitamin deficiency
states (Barthelemy et al. 1986). Mucocutaneous lesions appear during acute and chronic riboflavin-
deficient conditions (Roe 1991) and consist of scaliness and incrustation of red-brown material
consistent with changes in lipid metabolism, particularly those of essential fatty acid deficiencies
(Miller 1989). A disproportion of soluble to insoluble collagen in skin occurs in riboflavin- as well
K15043_C006.indd 218 4/10/2013 4:57:02 PM

as pyridoxine-deficient rats, which suggests that collagen maturation requires participation of fla-
vin- and pyridoxal-5′-phosphate-dependent enzymes (Prasad et al. 1983).
Other clinical signs of riboflavin deficiency include scrotal dermatitis, angular stomatitis, chei-
losis, and magenta tongue, which is often accompanied by deterioration of filiform papillae (Lo
1984). These cutaneous and mucous membrane changes associated with riboflavin deficiency may
likewise be difficult to distinguish from those of other fat- and water-soluble vitamin deficiencies.
Animals fed riboflavin-deficient diets develop corneal vascularization, which is usually preceded
by leukocytic infiltration beneath the corneal stroma (Tisdall et al. 1943). Corneal vascularization is
also associated with congenital malformations in the eye of spring of maternal riboflavin-deficient
rats. Penetration of leukocytes within the stromal layer and subsequent inflammation result in capil-
lary formation. The conjunctivae become inflamed and the lens is opaque with cataract formation
(Goldsmith 1975). Neovascularization of the cornea remains long after the inflammatory insult
subsides (Fromer and Klintworth 1975).
6.6.3.2 Hematological Abnormalities
Later stages of riboflavin deficiency are associated with anemias (Goebel and Goebel 1972).
Riboflavin plays a significant role in erythropoiesis, at least in part by impairing iron metabo-
lism. Flavohemoproteins, such as NAD(P)H oxidoreductase, reduce ferric ion to facilitate transport
across the cell membrane and to assist mobilization of bound Fe+3 from liver ferritin (Zhu et al.
1999). Riboflavin supplementation of human adult and pediatric populations with anemia has con-
firmed this role (Buzina et al. 1979). Riboflavin-deficient children administered riboflavin demon-
strate a decrease in serum iron and an increase in hemoglobin, supporting the relationship between
riboflavin and iron utilization.
Unlike other clinical symptoms, such as dermatitis, stomatitis, and cheilosis, the anemia of ribo-
flavin deficiency is clearly distinct from the anemias associated with other B vitamin deficiencies
(Lane and Alfrey 1965). Deficiencies of vitamin B12 and folic acid are associated with megaloblastic
anemia; pyridoxine-responsive anemia is hypochromic and microcytic and the marrow exhibits
erythroid hyperplasia coupled with the presence of sideroblasts. A deficiency of pantothenic acid in
man has not been shown to result in anemia and induced niacin deficiency in man does not appear
to result in anemia as well.
On the basis of the requirement of FMN-dependent pyridoxine phosphate oxidase for the con-
version of pyridoxine phosphate to the active coenzyme, pyridoxal phosphate, one might anticipate
that a deficiency of riboflavin might result in a deficiency of pyridoxal phosphate. The anemia
manifested by riboflavin-deficient patients does not resemble the hypochromic microcytic anemia
that responds to pyridoxine administration nor does administration of pyridoxal, which bypasses
the need of pyridoxal phosphate oxidase, improve the anemia caused by riboflavin deficiency (Lane
and Alfrey 1965).
Additionally, riboflavin deficiency decreases activity of erythrocyte glutathione reductase criti-
cal for supplying GSH necessary for glutathione peroxidase that effectively regulates cellular redox
potential (Beutler 1969a,b). Under conditions of normal metabolic stress, decreased activity of glu-
tathione reductase alone is not generally associated with increased sensitivity of erythrocyte mem-
branes to oxidant-induced injury (Beutler and Srivastava 1970). By contrast, riboflavin deficiency
accompanied by increased oxidative stress renders red cell membranes vulnerable to oxidative
injury, resulting in hemolytic anemia.
6.6.3.3 Neuropathic Abnormalities
A relationship between nutritional status and peripheral neuropathies is well established with tobacco
and ethanol abuse, protein-energy malnutrition, and deficiencies in thiamine, niacin, pyridoxine,
cobalamin, and vitamins A, D, and E all being causative (Kumar 2007; Lanska 2010). Several of
the most notoriously publicized neuropathic conditions include dry beriberi, Wernicke–Korsakoff
K15043_C006.indd 219 4/10/2013 4:57:02 PM

syndrome, pellagra, neural tube defects, subacute combined degeneration of the spinal cord, and
spinocerebellar ataxia.
Although not widely appreciated, demyelinating peripheral neuropathy is a feature of late-stage
riboflavin deficiency. Ultrastructural studies in riboflavin-deficient rats demonstrate marked degen-
eration of the structural integrity of myelin lamellae. By contrast, ultrastructural integrity of non-
myelinated nerve fibers found in gray matter is not affected by the deficiency. Cellular organelles
of both myelinated and nonmyelinated nerve fibers remain intact and are presumably functional
(Norton et al. 1976). Alterations in myelin formation may be explained by direct influences of
flavoenzymes in sphingo- and phospholipid formation as well as in one-carbon and antioxidant
metabolism. Studies in chickens have shown that ultrastructural changes occur within Schwann
cells, which are hypertrophic and exhibit marked lipid accumulation and concentric interdigitations
of their cytoplasmic processes (onion bulb features) (Cai et al. 2007, 2009; Jortner et al. 1987).
Other characteristic changes occurring in riboflavin deficiency include the presence of redundant
myelin folds (paranodal tomacula). Redundancy within myelin folds is thought to involve a remy-
elination process; however, overlap in this process occurs because of premature formation of the
tomaculous (sausage-like) features during the demyelination phase (Cai et al. 2006). Other studies
in riboflavin-deficient chickens have shown that sciatic nerves are enlarged and are accompanied
by segmented demyelinated layers and infiltration of leukocytes. ACP activity is elevated within
Schwann cells of affected nerves (Johnson and Storts 1988). This enzyme, as noted earlier in the
chapter, has a high affinity for hydrolyzing FMN to riboflavin, which may exacerbate further losses
of endogenous flavin during riboflavin deficiency.
Central nervous system responses to damage caused by riboflavin deficiency also occur in astro-
cytes and microglia, manifested as an increase in the number of cells positive for glial fibrillary
acidic protein (GFAP), a protein associated with central nervous system repair as a result of injury.
Laboratory studies using animal models of traumatic brain injury demonstrated that administration
of riboflavin (7.2 mg/kg body weight) after trauma reduces the size of brain lesions and decreases
the number of astrocytes that stain positive for GFAP (Hoane et al. 2005). Upregulation of GFAP
is associated with posttraumatic glial scarring that occurs during the brain’s attempt to reestablish
glial borders around the site of injury.
Supplementation of riboflavin also significantly reduces edema and lesion volume and markedly
improves behavioral and sensorimotor responses. In view of these beneficial responses to ribofla-
vin supplementation under conditions of riboflavin deficiency, ultrastructural changes after brain
trauma may be exacerbated and myelin damage may be promoted particularly within lamellar bod-
ies that originate from lipids of intracellular organelles. Riboflavin deficiency can avert lipid reori-
entation during myelin regeneration by promoting edema and causing a disordered arrangement of
tubuli (Schmitz and Muller 1991). Thus, riboflavin supplementation may have therapeutic potential
for treatment of peripheral neuropathy and traumatic brain injury.
In addition to a role in maintaining the structural integrity of myelin lamellar bodies, riboflavin
availability also controls neuronal excitation by regulating NMDA receptor–mediated neurotrans-
mission indirectly as a coenzyme for d-amino acid oxidase (Kawazoe et al. 2007). d-serine is
synthesized from l-serine in the brain by a vitamin B6 –dependent serine racemase and functions as
a potent agonist for NMDA-mediated excitatory neurotransmission (Horio et al. 2011). d-serine is
released by both neurons and astrocytes to regulate NMDA receptors.
Human FAD-dependent d-amino acid oxidase (hDAAO) degrades d-serine and blocks NMDA-
mediated neuroexcitation (Pollegioni et al. 2007). pLG72 is a novel primate-specific protein that
regulates d-amino acid oxidase. Recent studies demonstrate that overexpression of hDAAO in glio-
blastoma cells decreases levels of d-serine, which is nullified when pLG72 is expressed, suggesting
a negative effector role for pLG72 on regulation of hDAAO. Increases in hDAAO activity and loss
of pLG72 expression were recently proposed to be among the molecular mechanisms leading to
the onset of schizophrenia susceptibility (Sacchi et al. 2008). If this observation is confirmed and
extended, it would suggest a potential role for riboflavin in prevention and treatment of mental illness.
K15043_C006.indd 220 4/10/2013 4:57:02 PM

In terms of riboflavin bioavailability, the weak binding of FAD to hDAAO (Kd = 7 ± 2 μM)
coupled with the remarkable stability of the apo-homodimer form in the absence of its flavin coen-
zyme may represent a mechanism of controlling the activity of hDAAO relative to riboflavin acces-
sibility (Caldinelli et al. 2009, 2010). It is interesting to speculate that the competitive inhibition
of hDAAO by quinacrine and several tricyclic antipsychotic agents (Hellerman et al. 1946), which
also impair bioactivation of riboflavin (Pelliccione et al. 1983; Pinto et al. 1981, 1985), may possibly
manifest their behavioral effects through regulation of d-amino acid oxidase. Such interactions may
reinforce the importance of adherence to medication among patients with schizophrenia (McCabe
et al. 2012). By enhancing formation of d-serine or avoiding its excessive degradation, maintaining
proper excitatory status, riboflavin may play a major role in the pathophysiology of schizophrenia
(Habl et al. 2009). Thus, in view of the control of B vitamins over neuronal excitation, it is intrigu-
ing to speculate that both pyridoxal phosphate, the coenzyme of serine racemase (which synthesizes
d-serine), and FAD, the coenzyme of hDAAO, which degrades d-serine, may be potential target
sites for control of schizophrenia.
In summary, the clinical features of human riboflavin deficiency generally do not have absolute
specificity. In all species studied, riboflavin deficiency is often accompanied by deficiencies of other
lipid- and water-soluble vitamins. Because of the ubiquitous nature of flavoenzymes, riboflavin
deficiency causes profound biochemical, physiological, structural, and functional changes in an
apparently ordered fashion. Riboflavin depletion causes alterations predominately in energy and
lipid metabolism within organelles, which subsequently result in changes in substrate trafficking
and adjustments in oxidation reduction capacity of cells. Oxidative stresses induced by formation of
ROS are often accompanied by inflammatory responses within tissues and eventual organ dysfunc-
tion. Early changes due to riboflavin deficiency are readily reversible. Later anatomical changes,
such as formation of cataract, are largely irreversible despite treatment with riboflavin.
6.7 Major Metabolic Processes Utilizing Flavin Coenzymes

Aside from the exploitation of riboflavin for its unique photodynamic properties, the major func-
tion of riboflavin, as noted earlier, is to serve as the precursor of the flavin coenzymes, FMN and
FAD, and of covalently bound flavins. These coenzymes are widely distributed throughout cellular
metabolism and catalyze numerous oxidation–reduction reactions involved primarily in energy,
carbohydrate, lipid, and amino acid metabolism. Flavoenzymes influence other major metabolic
pathways such as phase 1 metabolism of drugs and xenobiotic substances as well as phase 2 con-
jugation reactions. The interdependency among the vitamins is further exemplified by the need
for flavoenzymes to activate other vitamins, namely, pyridoxine, folate, vitamin K, and vitamin
D. Flavins are coenzymes for an eclectic array of proteins that function as electron transferases,
dehydrogenases, oxidoreductases, monooxygenases, hydroxylases, and oxidases. The multiplicity of
electronic signaling, proton exchange reactions, and electron discharge within the isoalloxazine ring
of reduced flavin coenzymes have been previously reviewed (Macheroux et al. 2005).
Although the majority of flavoproteins catalyze redox processes, a small percentage catalyze
reactions that do not involve a net oxidation–reduction reaction. A detailed review of these reactions
has been previously published (Bornemann 2002). Flavoenzymes are widely utilized in cellular
signaling, repair, and maintenance functions. These processes and the role of flavoproteins as sens-
ing molecules in plant phototropism and nitrogen fixation have been comprehensively reviewed (De
Colibus and Mattevi 2006; Henriques et al. 2010; Massey 2000).
6.7.1 Flavoenzyme Complexes: Electron Transport Chain and Citric Acid Cycle

As both FMN and FAD are part of the respiratory chain, riboflavin plays a central role in control-
ling the flow of electrons along the electron transport chain to the ultimate electron acceptor, molec-
ular oxygen. Using the unique conjugated features of the isoalloxazine ring, flavoproteins function
K15043_C006.indd 221 4/10/2013 4:57:02 PM

as mediators between two-electron acceptor/donors and one-electron acceptor/donors within the

electron transport chain. The major components of the chain have been traditionally organized into
four complexes embedded with the inner membrane of the mitochondria (Rich and Maréchal 2010).
These complexes consist of a number of subunits containing several different electron carriers. The
first two complexes, complex I and complex II, contain flavoprotein reductases (dehydrogenases)
and electron-transferring flavoproteins (ETFs) while complexes III and IV are composed of a ubi-
quinone (coenzyme Q10 [CoQ]) component and a variety of cytochromes, respectively.
6.7.1.1 Complex I
Complex I (NADH-ubiquinone oxidoreductase, EC 1.6.5.3) is a critical link among aerobic metabo-
lism of the mitochondria, glycolysis, the citric acid cycle, and fatty acid metabolism. The major
components of complex I are FMN and several iron–sulfur centers. Electron transport through this
complex begins with a two-electron (hydride, H:) transfer from one of the NADH moieties from
the citric acid cycle to the FMN moiety. The fully reduced flavohydroquinone (FMNH2) displays
a bowed configuration through the N5–N10 axis, which favors an ultrafast sequential transfer, one
electron at a time, to the iron–sulfur clusters (Hirst 2009). Two FMN moieties within this complex
serve as single and dual electron transferring agents (Albracht et al. 2003). The final electron trans-
fer involves another sequential single electron shift to CoQ with formation of CoQH2 hydroquinone.
An unscheduled release of an electron from the reduced FMN component within mitochondrial
complex I is regarded as a major contributor to oxidative stress. When reoxidation of FMN hydro- or
semiquinone is impaired, the rate of formation of ROS markedly increases. Under normal condi-
tions of electron transport, NADH and FMN assume a spatial conformation with a stable stacking
arrangement between their ring structures. This arrangement greatly facilitates hydride transfer and
subsequent formation of CoQH2 (Berrisford and Sazanov 2009).
Studies on mitochondrial oxygen consumption in isolated mitochondria show that increasing the
availability of FMN or FAD enhances the rate of reaction of complex I but not that of complex II or
III (Mazzio and Soliman 2004). This finding suggests that by increasing flavin availability within
complex I, greater hydrophobic ring stacking is enabled, the distance between reactive rings is
shortened, and the entire complex is stabilized to facilitate electron transfer. By contrast, increased
reactivity of complex II would not be anticipated owing to the highly specific arrangement of the
covalently linked FAD within the enzyme active site and the marked differences in the redox poten-
tials between covalently and noncovalently bound flavin prosthetic groups.
Biochemical and genetic abnormalities within complex I correlate highly with mitochondrial
disorders manifesting predominantly as human neurodegenerative diseases. In an exploratory study
of mitochondria from children with autism, the majority of cases exhibited complex I activities
below control levels (Giulivi et al. 2010). Isolated complex I deficiency has been observed in patients
with mutations in the Acad9 gene, which encodes for the FAD-dependent mitochondrial acyl-CoA
dehydrogenase family member 9 (Zhang et al. 2002). Patients presenting with this defect are easily
fatigued, exhibit exercise intolerance, and have lactic acidemia. Supplementation of these patients
with riboflavin improves exercise intolerance and the activity of complex I within the subsarcolem-
mal mitochondria (Gerards et al. 2011; Scholte et al. 1995). However, it is important to consider that
early treatment with riboflavin may block disease progression but that supplementation may not
necessarily reverse established cellular or tissue damage (Horvath 2012).
Studies using combinations of anticancer agents to enhance chemotherapy found that estra-
diol and tamoxifen particularly affected mitochondrial function by targeting the FMN site within
complex I. Since inhibition of complex I favors formation of ROS, a combination of estradiol and
tamoxifen can be used to accelerate oxidative stress within cancer cells (Moreira et al. 2006). These
results raise the possibility that targeting the FMN site of complex I may prove important for cancer
therapy. Complex I has key importance to cancer therapy because mitochondrial failure of neoplas-
tic cells may rapidly contribute to disease arrest and apoptosis.
K15043_C006.indd 222 4/10/2013 4:57:02 PM

6.7.1.2 Complex II and the Citric Acid Cycle

Complex II includes the covalently attached flavoenzyme, succinate dehydrogenase (SDH,
succinate:ubiquinone oxidoreductase, E.C. 1.3.99.1), which directly links the major energy-producing
pathways, the electron transport chain, and the citric acid cycle. Of the enzymes that function within
the citric acid cycle, SDH is the only one that is an integral component of the inner mitochondrial
membrane. This enzyme complex is a heteroligomer composed of four subunits, namely, SDHA,
SDHB, SDHC, and SDHD. Complex II, like complex I, contains iron–sulfur clusters linked with
the flavin component. But rather than using FMN, complex II uses FAD covalently linked to a his-
tidyl residue on SDH as its electron acceptor. After interaction with succinate, the enzyme abstracts
a hydride ion yielding FAD hydroquinone and fumarate. The final transfer of electrons is similar
to that of complex I in that CoQH2 is formed. The ease of electron transfer through complex II is
facilitated by the linear arrangements and proximities of the flavin and iron–sulfur centers to the
quinone and heme components within the membrane domain (Cecchini 2003).
Mutations in the genes encoding mitochondrial SDH or the different subunits associated with
it are involved in the development of the paraganglioma syndrome (Chetty 2010). Paragangliomas
are rare (2–8 cases/million/year) neuroendocrine tumors that arise from the neural crest cells in an
extraadrenal location. The clinical phenotype is determined by whether the tumors are catechol-
amine secreting (functional sympathetic) or nonsecreting (nonfunctional parasympathetic). A more
complete description of the clinical manifestations in patients affected by these mutations has been
published (Waguespack et al. 2010).
In one mutation identified in humans, an alteration in the Sdha gene that causes a substitution of a
cysteine moiety for arginine-408 results in loss of covalent attachment of FAD to the flavoprotein (Birch-
Machin et al. 2000). Loss of covalent attachment of FAD to SDH leads to a dysfunctional enzyme unable
to oxidize succinate. It is also important to consider that marked changes in the activities of any of the
components within the hierarchical organization of electrons through complex II that result in increased
electron transfer to the FAD moiety could have deleterious consequences. Redistributing electrons
toward FAD coupled with diminished oxidation of the FAD hydroquinone can result in overproduction
of ROS, superoxide and hydrogen peroxide, causing oxidative stress. Thus, preservation of mitochondrial
function at the level of complexes I and II has an added benefit in reducing formation of ROS.
Defects in any of the aforementioned SDH subunits of complex II or sequential presentation of
electrons through complex I could mimic features of chronic hypoxia, termed pseudohypoxia, with
concomitant induction of HIF-1 (Agani et al. 2000). Loss of activity of SDH through riboflavin
deprivation or decreased FAD attachment will result in an accumulation of succinate. A buildup
of succinate prevents degradation of HIF, which occurs normally under normal oxygen conditions
(Selak et al. 2005). Stabilization of HIF-1 during hypoxic conditions upregulates several genes that
promote cell survival under low-oxygen conditions. These include activation of an anaerobic switch
that favors glycolysis and attenuates mitochondrial activity by inhibiting complex I activity.
The overall effect is a critical adaptive mechanism to reprogram the mitochondria to prevent
apoptosis (Tello et al. 2011). Hypoxia-induced HIF-1 increases expression of a variety of factors in
response to malutilization of molecular oxygen that results in recruitment of ERK and AKT kinases
that promote antiapoptotic programming and confer protection against oxygen deprivation. Thus,
in the absence of mitochondrial reprogramming, persistent energy deficits would lead to further
mitochondrial lesions and dysfunction prior to mitochondrial release of cytochrome c and initiation
of apoptosis. Specific citric acid cycle intermediates, namely, alpha-ketoglutarate and l-aspartate
can prevent mitochondrial lesions by aerobically maintaining electron flux and proton extrusion at
complex I with subsequent substrate-level ATP production.
6.7.1.3 Other Flavoenzymes Involved in Electron Shuttles

In addition to complexes I and II, two other flavoenzymes supply electrons to the electron trans-
port chain via interacting with the ubiquinone pool, sn-glycerophosphate dehydrogenase, an inner
K15043_C006.indd 223 4/10/2013 4:57:02 PM

membrane–bound shuttle enzyme, and the flavoprotein ubiquinone oxidoreductase (EC 1.5.5.1),
which accepts electrons from the ETFs.
6.7.1.3.1 Mitochondrial sn-Glycerophosphate Dehydrogenase

Mitochondrial sn-glycerophosphate dehydrogenase (sn-glycerol 3-phosphate:quinone oxidoreduc-
tase, EC 1.1.5.3) is a flavin-dependent dehydrogenase essential for shuttling NADH reducing equiva-
lents between glycolysis and the electron transport chain. This enzyme in mammals is tightly bound
to the outer surface of the inner mitochondrial membrane and together with the cytosolic enzyme,
glycerol-3-phosphate dehydrogenase (NAD+) (EC 1.1.1.8), forms the glycerol-3-phosphate shuttle.
This pathway yields NADH produced primarily from glycolysis in the cytosol and is reoxidized
to NAD+ by the electron transport chain. This shuttle plays a critical role in transferring reducing
equivalents from cytosolic NADH to the mitochondrial matrix (MacDonald and Brown 1996).
6.7.1.3.2 ETF Dehydrogenase

The ETF dehydrogenase (electron transfer flavoprotein-ubiquinone oxidoreductase, EC 1.5.5.1)
accepts electrons from the ETF and reduces CoQ. Like complex II, this enzyme complex contains
one iron–sulfur cluster and uses FAD as the electron acceptor/donor. Site-directed mutagenesis of
the enzyme has revealed that the dehydrogenase FAD moiety accepts electrons from the iron–sulfur
cluster and transfers those electrons to CoQ (Swanson et al. 2008). The iron–sulfur cluster accepts
electrons from the ETF. Thus, the iron–sulfur cluster is the immediate donor of electrons to the
FAD and the immediate electron acceptor from the ETF.
6.7.1.3.3 Electron-Transferring Flavoprotein

ETFs are soluble heterodimeric FAD-containing proteins within the mitochondrial matrix that
function as electron acceptor/donor intermediates between the iron–sulfur cluster of the ETF dehy-
drogenase and the family of acyl-CoA dehydrogenases responsible for metabolism of fatty acids
and branched-chain amino acids (Gregersen 1985). The ETF has a highly mobile redox-active FAD
domain that serves as the key metabolic branch point for acceptance and delivery of electrons
(Toogood et al. 2007). The conformational flexibility of the flavin domain enables ETF to recognize
structurally distinct acyl-CoA dehydrogenase while maintaining a degree of specificity.
Defects either in the ETF or in the electron transfer dehydrogenase are the major causes of
MADDs (Schiff et al. 2006). With regard to the dehydrogenase, two amino acid mutations located
within the FAD-binding domain are thought to contribute indirectly to the binding instability (Er et
al. 2011). Defects in FAD binding, diminished protein biosynthesis, and rapid degradation of ETF
are also responsible for the defects associated with MADD (Yamaguchi et al. 1991). Alteration in
FAD binding most likely contributes to the responsiveness of certain forms of MADD to ribofla-
vin supplementation. Although the underlying biochemical variations associated with MADD are
not fully defined, riboflavin-responsive, nonresponsive, and myopathic forms of this disorder have
been identified. In the latter two forms, CoQ supplementation and riboflavin together with carnitine
supplementation, respectively, have improved patients with MADD (Liang et al. 2009).
6.7.2 Flavoenzymes Involved in Lipid Metabolism

6.7.2.1 Acyl-CoA Dehydrogenase and β-Oxidation
Mitochondrial β-oxidation of fatty acyl-CoA esters involves replicate cycles of four sequential reac-
tions for the oxidation of fatty acids. Acyl-CoA dehydrogenases are a class of flavoenzymes that
catalyze the initial step in each reaction cycle. Several of these dehydrogenases exhibit overlapping
chain length specificities but have been conveniently categorized into three distinct groups on the
basis of their specificity for short-, medium-, or long-chain fatty acid acyl-CoA substrates. Disorders
of fatty acid β-oxidation exhibit autosomal recessive inheritance characterized by symptoms often
K15043_C006.indd 224 4/10/2013 4:57:02 PM

precipitated by catabolic stress that may occur at any time during life. Affected patients may be
asymptomatic or manifest a variety of symptoms, such as hepatic encephalopathy with hypoketotic
hypoglycemia and coma similar to that of Reye syndrome, cardiomyopathy, arrhythmias, progres-
sive myopathy, fulminant hepatic failure, or sudden infant death. A comprehensive review of mito-
chondrial β-oxidation disorders has been published elsewhere (Sim et al. 2002). Of the enzymes
involved in β-oxidation of fatty acid, the majority of defects occur within the acyl-CoA dehydroge-
nase family; the medium-chain acyl-CoA dehydrogenase is the most commonly deficient enzyme
within the class that leads to metabolic disorders. Disorders within this flavoprotein class present
with overlapping phenotypes and the same defective enzyme may be associated with considerable
clinical heterogeneity. Table 6.2 illustrates the types of acyl-CoA dehydrogenases and the approxi-
mate chain length of fatty acids they oxidize.
Figure 6.8 summarizes the pathway of electrons transferred after oxidation of fatty acids to their
ultimate delivery site, the electron transport chain, and prior to formation of ATP.
Table 6.2
Types of Acyl-CoA Dehydrogenases
Acyl-CoA Dehydrogenases Chain-Length Oxidized
Very long-chain acyl-CoA dehydrogenase C14–C20
Long-chain 3-hydroxy acyl-CoA dehydrogenase C12–C16
Medium-chain acyl-CoA dehydrogenase C6–C10
Short-chain acyl-CoA dehydrogenase <C6
See review by Ghisla and Thorpe (2004).
Mitochondrial e-transport chain

CoQH2
CoQ
Mitochondrial
ETF matrix
ETF oxidoreductasered
oxidoreductaseox
ETFred
ETFox
FADH2
H H O H O
FAD
R-CH2- Cβ Cα - C-S-CoA R-CH2- Cβ Cα - C-S-CoA
H H Acyl CoA
H
Dehydrogenase
Fatty Acyl CoA Trans-Δ2–Enoyl CoA
FIGURE 6.8 Acyl-CoA dehydrogenase, the first reaction in β-oxidation of fatty acids. A proton is removed
from the Cα carbon by the flavoenzymes, acyl-CoA dehydrogenase, followed by hydride (H:) transfer from
the Cβ carbon to the FAD moiety. The two electrons removed are sequentially transferred to the ETF and then
to the ETF oxidoreductase. The reduced ubiquinone (CoQH2) eventually delivers the electron cargo to the
electron transport chain.
K15043_C006.indd 225 4/10/2013 4:57:02 PM

6.7.2.2 Cytochrome b5 Reductase and Fatty Acid Desaturation

The flavoprotein, cytochrome b5 reductase, plays a pivotal role in catalyzing double bond forma-
tion in fatty acids after the acyl chain length exceeds 16 carbons. Dehydrogenation of stearoyl-
CoA is initiated by stearoyl-CoA desaturase in coordination with a heme protein, cytochrome b5
(Nakamura and Nara 2004). All three proteins (cytochrome b5 reductase, stearoyl-CoA desaturase,
and cytochrome b5) are associated with the ER membrane. Cytochrome b5 reductase transfers a pair
of electrons from NADH produced by the desaturase through FAD to cytochrome b5. Oxidation of
reduced cytochrome b5 is coupled to reduction of nonheme Fe3+ to Fe2+ in the desaturase. Mammalian
cells possess four desaturases: Δ9 desaturase, Δ6 desaturase, Δ5 desaturase, and Δ4 desaturase. The
Δ9 desaturase synthesizes oleic acid, a monounsaturated, ubiquitous component of all cells in the
human body. Δ9 desaturase produces oleic acid by desaturating stearic acid, a saturated fatty acid
either synthesized in the body from palmitic acid or ingested directly. Desaturation occurs in the
middle of the aliphatic chain without a subsequent hydration step similar to that in β-oxidation:
CH3(CH2)16CO-SCoA → CH3(CH2)7CH = CH(CH2)7CO-SCoA

Stearoyl CoA Oleoyl CoA
Δ6 and Δ5 desaturases are required for synthesis of the key polyunsaturated fatty acids, eicos-
apentaenoic and docosahexaenoic acids, both of which are synthesized from α-linolenic acid.
Arachidonic acid is synthesized from linoleic acid. Completion of their formation requires suc-
cessive action by elongases as well as by desaturases. The long-chain polyunsaturated fatty acids
possess distinct biological properties that impart beneficial effects on cells and tissues that underlie
their capacity for preventing disease.
Each of the aforementioned desaturases interacts directly with the cytochrome b5 reductase
within the membrane of the ER, which suggests a critical role for this flavoenzyme in controlling
fatty acid desaturation. Support for this postulated regulatory function is provided by the finding
that the 14-carbon fatty acid, myristic acid, regulates the activity of mammalian desaturases by acti-
vation of the membrane-bound cytochrome b5 reductase (Rioux et al. 2011). Prior to the activation
and association of this flavoprotein within the membrane of the ER, it must undergo a posttransla-
tional N-terminal myristoylation. It is intriguing to speculate whether the requirement to commence
fatty acid desaturation after the fatty acid length exceeds 16 carbons bears any relationship to acti-
vation of cytochrome b5 reductase by a 14-carbon length myristic acid.
6.7.2.3 Flavoenzymes, Cholesterogenesis, and Steroidogenesis

6.7.2.3.1 NADPH-Cytochrome P450 Oxidoreductase
NADPH-cytochrome P450 oxidoreductase (EC 1.6.2.4) (CPR) functions as a key regulatory flavo-
enzyme that donates electrons to a number of acceptor proteins. Some of these proteins include the
superfamily of microsomal cytochrome P450 heme proteins, cytochrome b5, heme oxygenase, and
squalene monooxygenase, which are involved primarily in oxidative metabolism of drugs, xenobi-
otic substances, lipids, and steroids (Murataliev et al. 2004). Studies using mouse knockout mod-
els for CPR indicate that the flavoenzyme is indispensable for the biosynthesis of cholesterol and
metabolism of drugs as no other electron donor is capable of providing reducing equivalents to the
microsomal cytochrome P450 heme proteins (Gu et al. 2003).
The enzyme CPR is an 82 kDa, membrane-bound protein that possesses a diflavin binding
domain similar to that of NOS (EC 1.14.13.39), MSR (EC 1.16.1.8), NADPH sulfite reductase (EC
1.8.1.2), and cytoplasmic NR1 protein (Aigrain et al. 2011). Enzymic activity catalyzed by CPR
begins with an electron flow from NADPH to the flavin electron acceptors prior to reacting with its
target acceptor proteins. The electron transfer usually follows the pathway:
NAD(P)H → FAD → FMN → protein acceptor
K15043_C006.indd 226 4/10/2013 4:57:03 PM

The flavin moieties are located in domains distinct from each other and separated by a flexible
hinge region. After transfer of an electron pair from NADPH, FAD aligns its isoalloxazine ring with
that of FMN and discharges its cargo electrons. The redox environment of FMN allows sequential
electron flow to a variety of partner proteins that include both heme and flavoproteins.
With regard to cholesterol biosynthesis, CPR plays a pivotal role in serving as the electron donor
to primarily two other flavoenzymes in the pathway, squalene monooxygenase and 3-β-hydroxysterol
delta-24-reductase (DHCR24). The transfer of electrons between CPR and a flavin acceptor of a
partner reductase is illustrated in Figure 6.9.
6.7.2.3.2 Squalene Monooxygenase

Squalene monooxygenase (EC 1.14.99.7) is a key FAD-dependent enzyme and potential regulatory
site in the cholesterol biosynthetic pathway that relies on CPR for reducing equivalents. The enzyme
catalyzes oxidation of squalene to (3S)-2,3-oxidosqualene, which undergoes cyclization catalyzed
by the lanosterol cyclase to form the first C30 steroidal intermediate, lanosterol (Laden et al. 2000).
Squalene monooxygenase may play a central role in the overall regulation of cholesterol biosyn-
thesis in that the addition of cholesterol to hepatocytes in culture decreases squalene monooxygen-
ase mRNA levels, suppresses squalene monooxygenase activity, and results in the accumulation of
squalene (Bogh et al. 2010).
In this reaction, a series of 1,2-methyl groups and hydride transfers occur along the squalene
chain to form the four rings. This process of cyclizing the squalene chain is considered one of the
most complex single enzymatic reactions thus far identified with an enzyme that is 90 kDa in size
(Hamdane et al. 2009). In the ER, sterol carrier protein 1 (also supernatant protein factor [SPF])
(Mokashi et al. 2004) binds squalene in the presence of phosphatidylserine, FAD, NADPH, and
oxygen and promotes squalene epoxidation (Lin 1980). Purified SPF stimulates squalene monooxy-
genase and squalene-2,3-oxide lanosterol cyclase. The enzyme also conducts a side reaction with
2,3-oxidosqualene to produce a potentially harmful 2,3;22,23-squalene dioxide, a reaction that has
an absolute requirement for exogenous FAD (Bai et al. 1992; Nagumo et al. 1995).
B
H
Box
O H
O O O
O O
H3C N H3C N H C N
NH NH – H2O 3 NH
H3C N N O H3C N N O H3C N N O
R R R
NADPH
e–
NADPH cytochrome P450 reductase
H+
NADP+
O−
O H O2 O H O
O
H3C N H3C N H3C N
NH H+ NH O2 NH
R R R
FIGURE 6.9 Flavoprotein-mediated interaction with NADPH-cytochrome P450 reductase. B represents a

recipient substrate that is oxidized.
K15043_C006.indd 227 4/10/2013 4:57:03 PM

Squalene monooxygenase is an attractive potential target for drugs to lower serum cholesterol
(Belter et al. 2011). In view of its flavin requirement, the apparent Km of 0.3 μM is consistent with
a freely dissociable FAD moiety; the Km for CPR, a requisite reductant flavoprotein, is in the low
nanomolar range (Laden et al. 2000). The expression of CPR is lower than that compared to most
of its other partner proteins (even heme partner enzymes). In view of the high affinity of protein–
protein interactions, the targeting of CPR may have potential for limiting monooxygenase activity
(Feidt et al. 2009). Inhibitors of the lower cholesterol biosynthetic pathway limit the production of
key metabolites (namely, dolichol, farnesyl, and geranylgeranyl) essential for protein signaling and
cell proliferation. By contrast, postsqualene blockade at the site of squalene monooxygenase would
result in the accumulation of squalene, which is considered nontoxic and thus spares needed isopre-
nyl units for other vital cell metabolic processes.
Several dietary constituents have been shown to target squalene monooxygenase, which may
explain their marginal effects on lowering serum cholesterol and improving cardiovascular function.
Of interest are the green tea polyphenolics (epigallocatechin-3-O-gallates), red wine stilbenoids (res-
veratrol, trans-3,4′,5-trihydroxystilbene), and allylsulfides from garlic (allylcysteines). These deriva-
tives act as reversible, noncompetitive inhibitors by scavenging the ROS formed at the active site of
the flavoenzyme, namely, the flavin 4a hydroperoxide (Abe et al. 2000; Gupta and Porter 2001).
6.7.2.3.3 3-β-Hydroxysterol Delta-24-Reductase and 7-Dehydrocholesterol Reductase

Desmosterol and 7-dehydrocholesterol are the last intermediates before the formation of cholesterol,
depending on the order of enzymatic reactions. Desmosterol differs from cholesterol in having an
unsaturated Δ24,25-double bond, whereas 7-dehydrocholesterol differs in having the unsaturated
Δ7,8-double bond. Several enzymes are involved in postsqualene cholesterol synthesis that leads to
the formation of desmosterol and 7-dehydrocholesterol. Two flavoenzymes that complete the conver-
sion to cholesterol are DHCR24 (E.C. 1.3.1.72) and 7-dehydrocholesterol reductase (DHCR7) (EC
1.3.1.21). Although both enzymes are reductases and utilize NADPH for donor electrons, DHCR7
does not form a partner complex with CPR (Zou et al. 2011).
DHCR24 is a member of the category of FAD-dependent oxidoreductases. DHCR24 catalyzes
the reduction of the Δ24 double bond to convert desmosterol to cholesterol. The protein contains a
leader sequence that directs it to the ER membrane. A missense mutation in this flavoprotein causes
desmosterolosis, which presents biochemically with elevated desmosterol and phenotypically with
spasticity, microcephaly, and micrognathia in affected patients (Waterham et al. 2001; Zolotushko
et al. 2011).
DHCR7 is the terminal reaction in cholesterol biosynthesis that catalyzes reduction of
7-dehydrocholesterol to cholesterol. The functional DHCR7 enzyme is a 55.5 kDa NADPH-requiring
protein localized to the microsomal membrane. Deficiency in DHCR7 (caused by gene mutations)
results in the disorder called Smith–Lemli–Opitz syndrome (DeBarber et al. 2011). This syndrome
is characterized by increased levels of 7-dehydrocholesterol and reduced levels of cholesterol result-
ing in multiple developmental malformations and behavioral problems (Porter and Herman 2011).
It is important to consider here that 7-dehydrocholesterol is the immediate precursor of both choles-
terol and vitamin D3 (cholecalciferol) (Bogh et al. 2010; Hafner et al. 2011). Cholesterol cannot be oxi-
dized back to 7-dehydrocholesterol, and therefore, cholesterol is not a precursor of vitamin D3. Rather,
both vitamin D3 and cholesterol share the common precursor, 7-dehydrocholesterol. In view of the reg-
ulatory function of several flavoenzymes within the cholesterol biosynthetic pathway, riboflavin status
potentially would have an impact on controlling vitamin D3 formation from 7-dehydrocholesterol.
This derivative is converted in skin to vitamin D3 in the presence of UV irradiation.
In addition, the intracellular levels of cholesterol per se may influence vitamin D3 status at two
sites controlled by flavoenzymes (Glossmann 2010), namely, at the squalene monooxygenase and
the cytochrome P450 27A1. At the first site, both expression of mRNA and activity of squalene
monooxygenase are suppressed by addition of cholesterol in media of cell culture models (Gill et
al. 2011; Li and Porter 2007). This inhibition at the squalene level would decrease formation of
K15043_C006.indd 228 4/10/2013 4:57:03 PM

7-dehydrocholesterol and limit available substrate for photosynthesis of vitamin D3. At the second
site, both cholesterol and cholecalciferol share the same mitochondrial heme protein, cytochrome
P450 27A1, for hydroxylation at their respective 25- and 26-carbon sites. Although the Km of vita-
min D3 is similar to that of cholesterol, the kcat is ~4-fold lower, suggesting lower turnover rates
(Gupta et al. 2007). These effects of cholesterol on diminishing vitamin D3 synthesis are compatible
with the observations that vitamin D3 insufficiency is associated with increased blood cholesterol
concentrations (Forrest and Stuhldreher 2011) and that obese children and adults usually express
low circulating levels of 25-hydroxyvitamin D3 (Gagnon et al. 2012; Kumar et al. 2009; Sacheck et
al. 2011).
6.7.2.3.4 Flavoproteins and Cytochrome P450 Heme Proteins in

Cholesterogenesis, Steroidogenesis, and Drug Metabolism
Like the cytochrome b5 involved in fatty acid desaturation as well as squalene monooxygenase
and DHCR24 for cholesterol biosynthesis, cytochrome P450 heme proteins also depend upon
NADPH-dependent flavoproteins for reducing equivalents. These heme proteins are members of
the superfamily of cytochrome P450 heme enzymes involved in converting lanosterol to cholesterol,
cholesterol to bile acids, and cholesterol to steroid hormones (Hafner et al. 2011). The cytochrome
P450 enzymes catalyze various hydroxylation reactions on the cholesterol ring by functioning as
monooxygenases that require O2 as well as the reductant function of CPR. One oxygen atom is
incorporated into a substrate and the other oxygen atom is reduced to water.
Cytochrome P450 heme proteins are classified as two types of enzymes on the basis of their asso-
ciation with two related NADPH-donor flavoproteins. Type I proteins are found in mitochondria,
where they receive electrons from the flavoprotein ferredoxin reductase (also called adrenodoxin
reductase) coupled with an iron–sulfur protein, ferredoxin (or adrenodoxin). Type II heme enzymes
are found in the ER and receive their electrons from CPR or sometimes assisted by cytochrome b5.
A combination of 57 type I and type II enzymes that play key roles in steroidogenesis, eicosanoid
metabolism, metabolism of drugs, and xenobiotic compounds has been identified. The specific func- Is this correct as
modified?
tion of ~25% of these proteins, which are considered orphan P450 enzymes, is unclear (Miller 2011).
Although the sequence of electron transfer from the two flavoenzyme complexes to their recipi-
ent heme proteins bears some similarities, the donor flavoproteins exhibit two major differences
in the number and form of the flavin moiety. In type I transfers, ferredoxin reductase has one
FAD domain, while in type II transfers, CPR uses both FAD and FMN to transfer electrons. In
the FAD-dependent ferredoxin reductase type I transfers, the pair of electrons is transmitted to
a 14 kDa iron–sulfur protein, ferredoxin, localized between the mitochondrial matrix and inner
membrane. Thus, the pattern of electron shuttle involves NADPH to FAD-ferredoxin reductase,
FAD-ferredoxin reductase to ferredoxin, and ferredoxin to heme protein–associated exchanges.
Both cytochrome 27A1 and 27B1 are type I enzymes that activate vitamin D3 to its major circulat-
ing and hormonally active forms, respectively. These flavin-dependent heme proteins align further
the association between riboflavin and vitamin D status (Miller 2008).
Type II transfers have been described previously in this chapter (see NADPH-cytochrome P450 You may want
to indicate the
reductase) and differ from type I in that the ferredoxin has been replaced using an FMN moiety exact section
number referred
that, after receiving two electrons from FAD, sequentially donates them to the heme proteins. Some to here.
type II reactions will recruit cytochrome b5 for this transfer but only after the first electron has been
delivered by CPR.
Although several polymorphisms and missense mutations have been identified in the CPR gene,
two missense mutations, in particular V492E and R457H, result in instability and folding of CPR
that involve FAD binding (Kranendonk et al. 2008). The binding affinity of V492E is lower than
that of R457H for FAD; however, its activity as an electron donor is more easily reconstituted
with addition of exogenous FAD. By contrast, R457H displays a more tightly bound FAD but rees-
tablishment of activity is somewhat more resistant to FAD reconstitution. Regions of polypeptide
unfolding within CPR differ among these CPR variants. V492E is less stable because of its low
K15043_C006.indd 229 4/10/2013 4:57:03 PM

binding affinity for FAD and demonstrates a gradual, localized unfolding in the absence of FAD as
demonstrated by partial trypsin digestion (Xia et al. 2011). R457H, on the other hand, binds FAD
more tightly but unfolding is observed more globally throughout the CPR variant. Both mutants are
stable to partial trypsinization in the presence of FAD (Xia et al. 2011). Thus, the presence of FAD
markedly stabilizes CPR function and suggests a preventive role for riboflavin supplementation in
patients identified with these mutations.
The majority of cytochrome P450 heme enzymes are associated with metabolism of drugs and
xenobiotic substances. These enzymes are located within the microsomal enzyme system and have
an obligatory requirement for CPR as their electron donor partner protein. Thus, the importance
of riboflavin in drug metabolism is recognized by the fact that six cytochrome P450 heme proteins
have obligate requirements for CPR and metabolize 90% of drugs and xenobiotic agents passing
through the small intestine, liver, lung, kidney and placenta (Guengerich 2003).
Studies in riboflavin-deficient animals show marked decreases in the levels of CPR and cytochrome
b5 proteins and complete recovery after repletion with riboflavin (Patel and Pawar 1974; Yang 1974).
In addition, induction of microsomal drug-metabolizing enzymes by phenobarbital during riboflavin
deficiency requires the presence of added riboflavin (Shargel and Mazel 1973); thus, reversal of activi-
ties of drug-metabolizing enzymes occurs when riboflavin is administered to deficient animals.
Cytochrome P450 proteins are key enzymes associated with catalyzing both toxication and detox-
ication of drugs and environmental pollutants. When considering the influence of riboflavin status on
drug metabolism, it is important to consider whether the metabolic product of a reaction leads to a
less active or inactive metabolite than the parent compound or enhances activity to a more toxic agent.
Riboflavin deficiency may be beneficial under conditions of exposure to a drug or potential toxicant if
the hepatic metabolite is more toxic than the parent compound. Thus, biotransformation by the cyto-
chrome P450 heme system may include both detoxication and toxication processes. Environmental
and genetic factors cause interindividual and intraindividual differences in drug metabolism and may
significantly influence the balance between toxication and detoxication reactions. Riboflavin status of
an individual coupled with genetic polymorphisms may leave patients with decreased, absent, or even
increased capacity to metabolize drugs and toxins (Meyer 1996). Thus, a number of dietary and life-
style factors can induce or inhibit drug-metabolizing enzymes and cause intraindividual variation.
6.7.2.4 Flavoenzymes and Formation of Ether Lipids and Sphingolipids

Other areas of lipid metabolism influenced by riboflavin involve the formation of ether lipids and
sphingosine. Biosynthesis of ether lipids resides within peroxisomes and requires two enzymes,
dihydroxyacetone phosphate acyltransferase and alkyl dihydroxyacetone phosphate synthase, a fla-
voenzyme that catalyzes formation of the ether bond in the sn-1 position of the hydroxyacetone
derivative. Dihydroxyacetone phosphate is the immediate precursor for synthesis of glycerol ether
phospholipids (Razeto et al. 2007) and C16:0, C18:0, or C18:1 fatty alcohol moieties are the source
of the alkyl group at the sn-1 site (Figure 6.10). This peroxisomal flavoenzyme does not display the
intrinsic redox activity characteristic of approximately 90% of other flavoenzymes in that the non-
redox reaction converts an ester to an ether linkage (de Vet et al. 2000).
Two flavoenzymes are involved in the pathway leading to the formation of sphingosine and sphin-
golipids, namely, 3-ketosphinganine reductase and dihydroceramide desaturase. After a condensation
reaction between serine and palmityl-CoA, the resultant 3-keto-sphinganine is reduced to sphinga-
nine by the action of 3-ketosphinganine reductase (Hannun and Obeid 2008). Ceramide is formed by
the desaturation of dihydroceramide by dihydroceramide desaturase. This enzyme plays a vital role
in regulating the balance between sphingolipids and dihydrosphingolipids and, as described earlier
regarding the function of desaturases, has a requirement for molecular oxygen and NADPH (Fabrias
et al. 2012). These enzymes are responsible for the first steps in sphingosine biosynthesis and are
localized to the ER where they have access to reducing equivalents from electron donor flavoproteins
(Mandon et al. 1992). An overview for the role of riboflavin in lipid metabolism is shown in Table 6.3.
K15043_C006.indd 230 4/10/2013 4:57:03 PM

O O
HO C R1
CH2 O C R1 CH2 O CH2 R2
HO CH2 R2
C=O C=O
–2 Alkyl-dihydroxyacetone
–2
CH2 O PO3 phosphate synthase CH2 O PO3
1-acyl hydroxyacetone (FAD dependent) 1-O-alkyl-hydroxyacetone
phosphate phosphate
FIGURE 6.10 Catalysis by alkyl dihydroxyacetone phosphate (DHAP) synthase involves exchange at the
sn-1 position of an acyl chain in acyl-DHAP for a long-chain fatty alcohol, yielding an ether-linked intermedi-
ate, alkyl-DHAP. Alkyl moieties at the sn-1 position can be derived from C16:0, C18:0, or C18:1 fatty alcohols.
DHAP serves as the glycerol precursor for the synthesis of the glycerol ether phospholipids. Examples of
glycerol ether lipids are plasmalogens characterized by a vinyl ether linkage at the sn-1 position and an ester
linkage at the sn-2 position and platelet-activating factor that contains an ether linkage in the sn-1 position and
an acetyl moiety in the sn-2 position.
Table 6.3
Role of Riboflavin in Lipid Metabolism
β-Oxidation Acyl-CoA Dehydrogenase
Fatty acid desaturation Desaturase
Cholesterol metabolism Squalene monooxygenase
Sphingosine formation Dihydroceramide reductase
Synthesis of ether lipids Plasmalogen, PAF
1-Carbon metabolism Formation of phospholipids
6.8 Antioxidant Activities
In the wake of contemporary interest in dietary antioxidants, one vitamin that is often not appre-
ciated sufficiently as a member of the armamentarium of antioxidants is riboflavin. One reason,
perhaps, is that riboflavin when exposed to UV light particularly in the presence of molecular
oxygen, tryptophan, lipids, and ascorbic acid generates ROS (Isenberg and Szent-Györgyi 1958).
Light-exposed total parenteral nutrition (TPN) solutions with multivitamin supplements contribute
to peroxide loads when used in preterm infants (Laborie et al. 1998). Peroxide generation in TPN
solutions is dependent on the extent of light exposure and the content of riboflavin and ascorbate
(Zaniolo et al. 2010). Thus, riboflavin has little significant antioxidant action per se. However, when
converted to FMN and FAD and subsequently incorporated into select antioxidant proteins, ribofla-
vin becomes a vital contributor to the antioxidant capacity of cells.
A major protective role against lipid peroxidation and general oxidative stress within cells is
provided by the glutathione/glutathione disulfide (GSH/GSSG) redox cycle (Deneke and Fanburg
1989). This redox couple is critical for maintaining intracellular redox potential by adjusting
the GSH/GSSG ratio to meet cellular demands. The intracellular ratio of GSH/GSSG is gener-
ally ≥100:1 (Schafer and Buettner 2001), which justifies the need for a steady supply of reducing
equivalents (NADPH) or of sulfur-containing amino acids (cysteine) to maintain adequate intra-
cellular levels of GSH. As the master antioxidant in the cell, GSH is present in various cell types
in concentrations that range from 0.1 to 12 mM (Meister and Anderson 1983). When exposed to
an oxidant, GSH is subsequently oxidized to GSSG. Examples of the antioxidant properties of
GSH include its participation in the reduction of hydrogen peroxide (H2O2) to water and organic
K15043_C006.indd 231 4/10/2013 4:57:03 PM

peroxides (ROOH) to a corresponding alcohol (ROH) by a variety of antioxidant enzymes that

include peroxiredoxins (Hofmann et al. 2002; Poole et al. 2011) and a family of glutathione per-
oxidases (Lubos et al. 2011; Tappel 1974). These enzymes require GSH, which is regenerated,
in turn, from its oxidized form (GSSG) by the FAD-containing enzyme, glutathione reductase,
and NADPH. Approximately 10% of the glucose within erythrocytes is utilized by the hexose
monophosphate pathway to produce the reducing equivalents provided by NADPH, which is gen-
erated by glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Because
NADPH is a stronger reductant than GSH, GSSG formed as a result of oxidative processes is
reduced back to GSH (see reaction below) by glutathione reductase, a process favored by the
relatively high intracellular ratio of NADPH/NADP+ compared to that of NADH/NAD+ (Bulger
and Brandt 1971; Månsson et al. 1976). The following equation illustrates the stoichiometry of
1 mol of GSSG being converted to 2 mol of GSH by the reducing equivalence of NADPH from
the hexose monophosphate pathway:
GSSG + NADPH + H+ → 2GSH + NADP+
Diminished glutathione reductase activity would be expected to lead to diminished concen-

trations of GSH that serve as substrate for glutathione peroxidase and glutathione S-transferase.
Decreased activities of these enzymes would limit the rate of degradation of lipid peroxides and
xenobiotic substances (Deneke and Fanburg 1989). In view of its critical role in maintaining GSH,
riboflavin nutritional status plays an important role in maintaining redox balance and metaboliz-
ing drugs and xenobiotic substances. Studies in riboflavin-deficient animals have found that the
activity of glucose-6-phosphate dehydrogenase is significantly compromised within erythrocytes
(Dutta et al. 1995; Taniguchi and Hara 1983). This observation provides an additional mechanism
to explain the diminished glutathione reductase activity in vivo during riboflavin deficiency and
the eventual decrease in antioxidant activity that develops. Riboflavin deficiency is associated with
increased hepatic lipid peroxidation and riboflavin supplementation limits this process. Feeding a
riboflavin-deficient diet to rats increases basal as well as stimulated lipid peroxidation (Rivlin and
Dutta 1995).
There have been numerous reports that riboflavin deficiency is associated with compromised
oxidant defense and furthermore that restoration of riboflavin status should be part of an overall
therapeutic strategy for patients whose conditions induce an oxidative stress (Allard et al. 2006;
Depeint et al. 2006; Gariballa and Ullegaddi 2007; Wacker et al. 2000). An overview of the impor-
tance of GSH and its role in enzymatic and nonenzymatic reactions throughout cell function is
shown in Table 6.4.
Table 6.4
Metabolic Processes Involving Glutathione (GSH)
Detoxification reactions (Phase I and Phase II)
Protein synthesis
Nucleic acid synthesis
DNA repair
Protein sulfhydryl protection
Immune function
Cellular differentiation and aging
Maintenance of intracellular redox potential
K15043_C006.indd 232 4/10/2013 4:57:03 PM

6.9 The Unfolded Protein Response

All cells contain low but measurable concentrations of ROS and free radicals. In view of the impor-
tance of FAD-dependent glutathione reductase in maintaining high levels of GSH, normal cells
with an adequate supply of riboflavin maintain an equilibrium between free-radical production
and clearance (Droge 2002). In addition, a cadre of enzymatic and nonenzymatic processes orches-
trated directly and indirectly by the GSH/GSSG redox cycle (see Figure 6.11) function to maintain
homeostatic control of various oxidants within the cell. The cytosolic ratio of GSH/GSSG for most
cells falls within a range from 30:1 to 100:1, indicating an antioxidant environment. If a continuous
imbalance occurs with the GSH/GSSG ratio falling below a certain nadir, the aberrant level of oxi-
dants can trigger transcriptional induction of a cell stress survival signal or initiate a programmed
cell death response (apoptosis) (Banhegyi et al. 2007).
In addition to oxidation of GSH to GSSG under conditions of oxidative stress, thiol groups in cys-
teinyl domains of proteins (P-SH) can undergo sequential and often reversible oxidations to sulfenic
acid (P-SOH) and sulfinic acid (P-SO2H). Subsequent oxidation to sulfonic acid (P-SO3H) is essen-
tially irreversible (Lei et al. 2008). Formation of protein cysteine sulfenic moieties in unfolded
proteins can assist in the formation of correct intramolecular disulfide bonds (Rehder and Borges
2010). Excessive accumulation of unfolded proteins resulting through extensive oxidation of cys-
teinyl moieties can trigger increased expression of genes involved in growth arrest and apoptosis
(Boot-Handford and Briggs 2010). Reaction of P-SOH with GSH will generate a glutathionylated
protein (PSSG) following the equation:
P-SOH + GSH → PSSG + H2O2.
Glutathione S-
Glutamate
Glycine Electrophiles conjugates
+ Cysteine
Glutathione
de novo S-transferase
biosynthesis
GSH Glutathione
peroxidase
Glucose-6-phosphate Mutagens
Glutathione dehydrogenase
NADPH NADP+
reductase 6-Phosphogluconate
FAD dehydrogenase Free
radicals
Toxins
GSSG
FIGURE 6.11 The GSH/GSSG redox cycle. The left side of the cycle illustrates reactions that maintain the
levels of GSH, namely, reduction of GSSG by glutathione reductase and de novo biosynthesis from precursor
amino acids via the biosynthetic enzymes, gamma-glutamylcysteine synthetase and glutathione synthetase,
respectively. The right side of the cycle illustrates reactions that consume GSH, namely, conjugation with reac-
tive electrophiles by glutathione S-transferases and direct oxidation to GSSG by peroxide degradation with
glutathione peroxidases or interactions with mutagens, free radicals, and toxins. As illustrated in the center,
the cycle is kept supplied with NADPH-reducing equivalents provided by the hexose monophosphate shunt
enzymes, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase.
K15043_C006.indd 233 4/10/2013 4:57:04 PM

Attachment of glutathione forming a mixed disulfide will protect the protein against poten-
tial loss of function resulting from further oxidation of the P-SOH moiety to P-SO2H or P-SO3H
(Townsend 2007). Glutathionylated proteins can be readily reduced back to P-SH, thus conserving
the integrity of a protein and preempting expenditure of cellular energy necessary for de novo pro-
tein biosynthesis.
Critical for cell differentiation, growth, and survival is the conversion of several signaling mol-
ecules and transcription factors that are regulated by reversible S-glutathionylation (Cooper et al.
2011; Dalle-Donne et al. 2009). Regulation of protein function by glutathionylation/deglutathio-
nylation is analogous to the “on–off” regulation of proteins by phosphorylation/dephosphorylation
(Fratelli et al. 2005), thus illustrating the importance of this process in redox control of metabolic
pathways.
In line with sulfhydryl exchange processes through glutathionylation and deglutathionylation of
proteins, the ER regulates secondary protein folding through similar sulfhydryl–disulfide exchange
reactions catalyzed by two distinct flavin-linked sulfhydryl oxidase families, namely, ER oxidore-
ductin 1 (Ero1) and quiescin-sulfhydryl oxidases (QSO) (Kodali and Thorpe 2010). These flavopro-
tein oxidases catalyze a direct, but not error-free, formation of disulfide arrangements into nascent
polypeptides with concomitant generation of hydrogen peroxide from molecular oxygen. Thus, in
contrast to the cytosolic compartment, the redox environment within the confines of the ER is
considerably more oxidative, exhibiting a GSH/GSSG ratio range of 3:1 to 1:1 (Hwang et al. 1992).
Considering the extant low GSH/GSSG ratio, this highly favored oxidizing environment of the
ER is aptly suited for disulfide bond formation. The flavoproteins (Ero1 and QSOs) are responsible
for the generation of intramolecular disulfide bridges during protein folding and processing within
the ER. Newly synthesized unfolded proteins have designated sulfhydryl moieties linked via disul-
fide arrangements so that the nascent polypeptides can fulfill their functional conformation assign-
ments. Only properly folded proteins proceed from the ER to the cell surface for secretion (Naidoo
2009). Thus, in addition to its role in controlling protein biosynthesis, the ER is a critical center
for protein processing and maintenance of protein quality control. Newly synthesized proteins that
fail to assemble properly, along with damaged and oxidized mature proteins, provoke an oxidative
burden within the ER (ER stress) and are targeted for degradation by several intracellular signal
transduction pathways, termed collectively as the “unfolded protein response” (UPR) (Ron and
Walter 2007).
The catalytic proficiency of most sulfhydryl oxidases is low since mispairings of sulfhydryl moi-
eties result in improperly folded proteins in the majority of cases. To improve efficiency of protein
folding, cells with a large secretory load rely on a partnership between the sulfhydryl oxidases and
another key flavoprotein family, protein disulfide isomerases (PDIs). These proteins are responsible
for disassemblance of incorrect disulfide bond pairing (reduction) and rearrangements (isomeriza-
tion) to properly folded proteins. These latter processes rather than the direct disulfide oxidations
by the sulfhydryl oxidases have been considered to be rate limiting during folding and refolding
of disulfide bonds (Ellgaard and Ruddock 2005). The disulfide isomerization reactions can occur
via both a series reduction and reoxidation cycles and a redox neutral shuffling of vicinal disulfide
moieties.
As mentioned earlier, the flavoprotein-Ero1 family of enzymes is important for direct disulfide
bond formation and is considered to be the primary oxidizing agent (electron acceptor) (Higa and
Chevet 2012). Two glycosylated isoforms of Ero1 (Ero1α and Ero1β) that assist with initiating oxi-
dative folding have been identified in humans. The pattern of electron transfer involves the accep-
tance of electrons by the Ero1-flavin moiety from sulfhydryl moieties on PDI. The disulfide bond
of PDI subsequently cross-reacts with sulfhydryl moieties on nascent polypeptides creating folded
proteins. The oxidized form of PDI can also react with GSH and the reduced form of PDI can react
with GSSG, forming a mixed disulfide. Such rapid interplay among sulfhydryl moieties is postu-
lated to enable functional cross-talk among PDIs, Ero1, nascent polypeptides, and the glutathione
cycle (Kim et al. 2012).
K15043_C006.indd 234 4/10/2013 4:57:04 PM

The overall influence of riboflavin on the UPR is evident at several different levels critical for cell
metabolism (Manthey et al. 2006). First, oxidative folding within the ER is highly sensitive to levels
of free cellular FAD. Within the ER, riboflavin deficiency would compromise activities of the key
flavoenzymes involved in the initial folding and activation of proteins, namely, Ero1, QSO, and PDI.
In addition, the activity of glutathione reductase would decrease, resulting in lower ratios of GSH/
GSSG. A fall in GSH, the major reducing agent, in turn, would affect the hydrogen peroxide and
alkyl hydroperoxide degradating capacities of several glutathione peroxidases, thioredoxin reduc-
tases, and the peroxiredoxin family of proteins. Lastly, riboflavin deficiency creates an oxidative
stress response in mitochondria and peroxisomes, leading to alterations in lipid, protein, and DNA
metabolism (Yoshida 2009).
The effects of riboflavin on the UPR are particularly magnified in cells burdened with high lev-
els of secretory proteins. Hepatocarcinoma cells (HepG2) secrete a variety of plasma proteins, for
example, albumin, transferrin, apolipoprotein B-100, and a series of acute phase proteins, fibrino-
gen, alpha 2-macroglobulin, alpha 1-antitrypsin, and plasminogen. HepG2 cells grown in media
containing varying concentrations of riboflavin indicate that moderate deficiency can result in early
signs of the UPR. Thus, consistent with ER stress, increases were observed in expression of genes
encoding ubiquitin-activating enzyme E1 and X box–binding protein coupled with decreased secre-
tion of apolipoprotein B-100 and other specific proteins (Manthey et al. 2005; Werner et al. 2005).
Since hydrogen peroxide is the product of the flavin-linked disulfide-generating enzymes, cells with
a heavy load of secretory proteins such as the liver would develop significant levels of oxidative
stress and exhibit increased needs for GSH.
By contrast, in immortalized T lymphocytes (Jurkat cells) that have a low secretory burden,
secretion of interleukin-2 is impaired only in severely riboflavin-deficient cells but not in mod-
erately deficient cells (Camporeale and Zempleni 2003). These findings are consistent with the
hypothesis that ER stress observed during riboflavin deficiency may be influenced by cellular pro-
tein secretory capacity and that cells with minimal secretory function are less compromised during
riboflavin deficiency.
6.10 Malaria
It is not commonly recognized that riboflavin deficiency has a protective role against malaria both
in experimental animals and in humans (Das et al. 1988; Kaikai and Thurnham 1983). In regions
endemic for malaria over the centuries, riboflavin-deficient erythrocytes served as important pro-
tective factors for the inhabitants of these regions (Anderson et al. 1994). With dietary riboflavin
deficiency, parasitemia is decreased dramatically, and symptomatology of infection is diminished.
In a study with human infants suffering from malaria, normal riboflavin nutritional status was asso-
ciated with high levels of parasitemia. In a similar fashion, supplementation with iron and vitamins
that included riboflavin results in increased malaria parasitemia (Oppenheimer et al. 1983).
Further evidence for a beneficial role of riboflavin deficiency in malaria is provided by stud-
ies using specific antagonists of riboflavin, for example, galactoflavin and 10-(4′-chlorophenyl)-3-
methylflavin (Becker et al. 1990; Schönleben-Janas et al. 1996). The antimalarial efficacy of these
flavin analogs as well as other isoalloxazine derivatives (Geary et al. 1985; Haynes et al. 2011) may
possibly be related to their ability to penetrate erythrocytes and function as glutathione reductase
inhibitors.
The exact mechanism by which riboflavin deficiency appears to inhibit malarial parasitemia is not
known with certainty. One possibility relates to effects on the redox status of erythrocytes, which is
an important determinant of growth of malaria parasites. Thus, riboflavin deficiency decreases the
activity of FAD-dependent glutathione reductase. Decreased activity of this enzyme coupled with
oxidative stress associated with the increased parasitic load within erythrocytes reduces the GSH/
GSSG ratio, which, in turn, can increase the magnitude of lipid peroxidation. Increased peroxide
formation may account for antiparasitic effects in host cells during riboflavin deficiency (Rockett
K15043_C006.indd 235 4/10/2013 4:57:04 PM

et al. 1988). On the basis of an oxidative stress hypothesis, protection from malaria is afforded by
several oxidant drugs, vitamin E deficiency, and specific genetic abnormalities in which oxidative
defense mechanisms are compromised (Dutta 1991).
Is this correct as
modified?
Another possible association with riboflavin metabolism may relate to identified differences in
binding domains for flavin coenzymes by flavoenzymes between malarial parasites and humans.
Binding of FMN within parasite NADPH-cytochrome P450 oxidoreductase is about fourfold lower
than that in the human flavoprotein, suggesting weaker association of flavin moieties and more rapid
exchange with flavin analogs (Lian et al. 2011). In addition, flavin analogs can function as mimics
of flavins that maintain redox homeostasis in the malaria parasite (Haynes et al. 2011). Other stud-
ies show that some flavoenzymes within Plasmodium falciparum exhibit poor catalytic efficiency
owing to slower hydride transfer and unstable formation of FAD semiquinones (Balconi et al. 2009).
These studies would suggest that altering riboflavin status and targeting specific flavoenzymes are
potential strategies for controlling malaria infection.
Further support for the therapeutic potential of targeting an oxidative stress response in control-
ling parasitemia is the observation that malaria parasites (Plasmodium berghei) are highly suscep-
tible to ROS. Parasites are relatively more susceptible than erythrocytes to the damaging effects
of lipid peroxidation (Dutta 1993). These results have led to the hypothesis that the requirement
of parasites for riboflavin is higher than that of the host cells. Thus, marginal riboflavin deficiency
would be selectively detrimental to parasites. Support for this hypothesis comes from the finding
that the uptake of riboflavin and its conversion to FMN and FAD are significantly higher in para-
sitized than in unparasitized erythrocytes and furthermore that the rate of uptake of riboflavin is
proportional to the degree of parasitemia (Dutta 1991).
In a report of malaria patients in Gabon (Traunmüller et al. 2003), plasma levels of FAD, FMN,
and riboflavin were normal, but the authors point out that because of the high degree of hemolysis,
dehydration, and liver and kidney problems in these patients, plasma flavin levels may not be com-
parable with values found in other population groups. In other studies of mildly riboflavin-deficient
school-age children, riboflavin levels did not predict hemoglobin levels or anemia but did predict
iron deficiency among children free of malaria (Rohner et al. 2007). Low dietary riboflavin intake
per se may not be the critical factor contributing to the flavin deficiency in red blood cells.
Studies show that resistance to malaria is also observed in individuals with certain genetic
defects (Anderson et al. 1993; López et al. 2010). Such defects have resulted in deficiencies of
flavin-dependent enzymes, namely, FAD-dependent glutathione reductase, FMN-dependent pyri-
doxine phosphate oxidase, and glucose-6-phosphate dehydrogenase, which is the prime generator
of NADPH.
These studies suggest that the influence of riboflavin deficiency alone is complex and that defi-
ciency states may affect more adversely on metabolism in parasites than in host cells. However,
it is important to consider that effects on parasite growth are not limited to riboflavin deficiency
and that riboflavin-deficient conditions do not generally occur as isolated entities. Other nutrient
deficiencies can diminish parasitic growth (Shankar 2000) but can also be detrimental to the host.
Undernutrition, in general, can exacerbate diarrhea and respiratory infections but can exacerbate,
can alleviate, or have marginal effects upon malaria outcomes (Caufield et al. 2004). Improvements
of nutritional status reduce severity of malaria episodes and result in fewer deaths.
In view of the importance of maintaining adequate nutritional status during malaria infection,
in deference to riboflavin deficiency, supplementation of riboflavin may also adversely affect devel-
opment and function of P. falciparum. Malaria parasites ingest over 50% of their host’s hemoglo-
bin and thus retain high levels of Fe+++ methemoglobin within their food vacuoles (Mansouri and
Winterhalter 1973). A model has been developed whereby administration of riboflavin at concen-
trations normally used to treat patients with congenital methemoglobinemia activates NADPH-
cytochrome b5/cytochrome b5 reductase, the flavoenzyme that reduces Fe+++ methemoglobin to Fe++
hemoglobin (Akompong et al. 2000). A continued reduction of methemoglobin by the flavoenzyme
creates a futile redox cycle within parasites, so that ingestion of hemoglobin by the parasite is
K15043_C006.indd 236 4/10/2013 4:57:04 PM

continually oxidized and then reduced. The model predicts a localized competition for available
NADPH in the parasite between the NADPH-cytochrome b5 reductase and glutathione reductase
resulting in a decreased GSH/GSSG ratio. This shift in coenzyme use favors a more oxidative envi-
ronment within the parasite. Thus, the net effect of riboflavin supplementation under these condi-
tions would culminate in the arrest of parasite maturation and differentiation.
6.11 Homocysteine Metabolism
Of the vitamins demonstrated to play a direct role in the regulation of homocysteine metabolism,
riboflavin can be considered a significant “silent partner.” Methionine and folate are the key com-
ponents of one-carbon metabolism that provides the methyl groups for numerous methyl transferase
reactions via the ubiquitous methyl donor, S-adenosyl methionine (SAM). After discharge of its
methyl group, SAM is converted to S-adenosyl homocysteine (SAH) and then hydrolyzed to homo-
cysteine. Homocysteine is involved in the pathogenesis of vascular disease, including cardiovascu-
lar, cerebrovascular, and peripheral vascular disorders (Graham et al. 1997).
The metabolism of methionine and other sulfur amino acids is responsive to nutrient intake, is
regulated by hormones (growth hormone, insulin, and thyrotropin-releasing hormone), and requires TRH was
expanded as
a number of B vitamins (cobalamin, folate, pyridoxine, and riboflavin) as coenzymes (Métayer et thyrotropin-re-
leasing hormone.
al. 2008). The production of methionine involves the remethylation of homocysteine by MS (EC Is this correct?
2.1.1.13) using the coenzyme 5-methyltetrahydrofolate (CH3THF). Cobalamin serves as an inter-

mediary in the methyl transferase reaction, which causes its cobalt moiety to cycle after several
hundred enzymic iterations between its reactive state, cob(I)alamin, and its less reactive forms,
cob(III)alamin and cob(II)alamin. Cob(I)alamin is a strong nucleophile that is capable of extracting
the 5-methyl group from CH3THF. After catalyzing several hundred reactions, the reactive nature
of cob(I)alamin makes it vulnerable to oxidation as it is converted to cob(II)alamin, a form inca-
pable of capturing the methyl moiety from CH3THF (Fujii et al. 1977). In a manner similar to that
of other NADPH-cytochrome P450 reductases, mammalian MSR (EC 1.16.1.8) restores the activity
of MS through the FAD/FMN-directed reductive methylation of the cob(II)alamin bound to the
enzyme (Olteanu and Banerjee 2001). MSR is also capable of catalyzing conversion of exogenous
aquacob(III)alamin to cob(II)alamin for uptake by apo-MS (Yamada et al. 2006).
Remethylation of homocysteine to methionine is necessary for subsequent formation of SAM,
which is critical for methylation processes particularly during initial periods of embryogenesis
(Kalhan and Marczewski 2012). After transmethylation, SAM is converted to SAH, the major
source of homocysteine. In addition to reconversion to methionine, homocysteine is coupled to a
transsulfuration pathway that produces cysteine and glutathione that requires a variety of pyridoxal
phosphate–dependent enzymes. Metabolic regulation of the remethylation and transsulfuration
pathways is controlled by the availability of SAM. SAM is an allosteric regulator of the FAD-
dependent N5,N10-methylenetetrahydrofolate reductase (MTHFR) (EC 1.5.1.20). This flavoenzyme
is responsible for the reductive conversion of N5,N10-methyleneTHF into CH3THF (Fowler 2001).
N5,N10-methyleneTHF is the product of several flavoenzyme reactions within the one-carbon
cycle. Hence, choline oxidase (EC 1.1.99.1), dimethylglycine dehydrogenase (EC 1.5.99.2), and
sarcosine dehydrogenase (EC 1.5.99.1) catalyze the oxidative demethylation of choline, dimeth-
ylglycine, and sarcosine, respectively, to yield active one-carbon units at the level of -CH2-, which
is transferred to THF to form N5,N10-methyleneTHF (Gadda 2012; Porter et al. 1985; Slow and
Garrow 2006). The reaction of N5,N10-methyleneTHF by MTHFR to form CH3THF is irreversible
in vivo and is the only methyl source for the cob(I)alamin-dependent conversion of homocysteine
to methionine (Selhub 1999). Experimental riboflavin deficiency is associated with low MTHFR
activity and reduced levels of CH3THF. In most tissues, this reaction provides the sole pathway for
homocysteine remethylation. Concentrations of homocysteine in tissues and blood are also depen-
dent upon its degradation through vitamin B6 –dependent transsulfuration in the liver and kidneys
(Lamarre et al. 2012).
K15043_C006.indd 237 4/10/2013 4:57:04 PM

In general, riboflavin has received less consideration for its importance in homocysteine metabo-
lism than have other B vitamins, folate, cobalamin, and pyridoxal. Thus, riboflavin not only is a
key link in the utilization of dietary folates and cobalamins but also controls homocysteine remeth-
ylation and transsulfuration in association with methyl donor flavoenzymes within the one-carbon
cycle as well as the FAD/FMN diflavin enzyme, MSR, the FAD-dependent N5,N10-MTHFR neces-
sary for methionine formation.
In addition to depending on the availability of the four B vitamin coenzymes, homocysteine
levels are also altered because of polymorphisms in genes encoding for the three major enzymes
in the methionine-synthesizing pathway. In particular, mutations in MS (A2756G), MSR (three
polymorphisms, A66G, A129T, and C524T), and N5,N10-MTHFR (two polymorphisms C677T and
C1298T) contribute to risk factors for several major pathologies that include cardiovascular disease,
birth defects, osteoporosis, Alzheimer’s disease, and renal failure (Chen et al. 2012; Hultdin et al.
2011; Yang et al. 2012). Although still controversial, elevated levels of homocysteine are believed to
be an independent risk factor for several of these pathologies. However, it is important to consider
that a lack of available methionine or a change in the SAH/SAM ratio may also contribute to these
diseases (Smulders and Blom 2011).
Of the three major enzymes and several polymorphisms associated with genes encoding for
these enzymes, mutations in MSR and N5,N10-MTHFR have particular relevance to riboflavin bind-
ing. Amino acid sequence studies on MSR have identified mutations at the conserved tryptophan
moiety at position 697 (W697) (Meints et al. 2011). These investigations demonstrated that W697
is juxtaposed to the isoalloxazine ring and significantly contributes to binding of FAD to MSR.
Site-directed mutagenesis analysis of W697 indicates that the presence of tryptophan is critical
for the attenuation of hydride transfer from NADPH and the assurance of coenzyme selectivity.
Tryptophan also accelerates transfer of electrons from FAD to FMN. As in other members of the
NADPH-cytochrome P450 reductase family of enzymes, conserved FAD stacking with tryptophan
promotes efficient interflavin electron transfer (Meints et al. 2011).
In addition to substitutions at W697, dysfunction of MSR also involves substitution of an alanine
(A) for a threonine (T) at position 129. The A-to-T transfer causes alterations in the FMN binding
domain. Thus, FMN binding is destabilized, which would affect efficient transfer of electrons from
NADPH to FAD and then to FMN. This mutation does not affect the NADPH- or FAD-binding
domains and is confined to the FMN binding motif (Gherasim et al. 2008). The mutant enzyme
is deplete of FMN and has ~8% efficiency of the wild-type MSR to reactivate MS. In a manner
similar to other diflavin reductase mutations, this mutation may be responsive to riboflavin therapy
(Gherasim et al. 2008).
The C677T polymorphism in the gene encoding for N5,N10-MTHFR results in a thermolabile
form of the protein. Accordingly, an alanine-to-valine mutation decreases the rate of FAD asso-
ciation and the diminished FAD binding is linked to marked changes in quaternary structure
(Guenther et al. 1999; Pejchal et al. 2006; Yamada et al. 2001). The release of FAD can be prevented
by increasing the availability of folate (Martínez-Frías 2008). Thus, the mutant enzyme has lower
affinity for its flavin cofactor than the wild-type enzyme. The diminished binding coupled with the
requirement for folate shows that plasma homocysteine is inversely related to riboflavin, particularly
in individuals with the homozygous genotype, MTHFR C677T TT, compared with CC individu-
als. The requirement for folate to stabilize the mutant MTHFR has been especially observed in
cancer patients with this mutation who show increased sensitivity to methotrexate toxicity. Patients
with MTHFR C677T polymorphism treated with methotrexate show increased risk of liver toxic-
ity, myelosuppression, oral mucositis, gastrointestinal toxicity, and skin toxicity (Yang et al. 2012).
In studies of the MTHFR C677T TT homozygous, heterozygous (CT), and wild-type (CC) geno-
types, riboflavin significantly lowered blood pressure in patients homozygous for the 677C→T poly-
morphism in MTHFR. Riboflavin intervention (2 mg) reduced mean blood pressure specifically
in patients with TT genotype (144/87 to 131/80 mmHg; p < 0.05 systolic; p < 0.05 diastolic). By
contrast, no response was observed in the other genotype groups (Horigan et al. 2010).
K15043_C006.indd 238 4/10/2013 4:57:04 PM

Thus, mutations affecting flavin binding within the two methionine-metabolizing enzymes MSR
and MTHFR have been identified. In MSR, two different mutations affect the flavin binding sites,
one interfering with FAD and the other with FMN. A mutation leading to the heat-sensitive form of
MTHFR has been identified and exhibits a genetic variation in ~10% to 15% of the population of
Europe and North America (Jacques et al. 1996). Homozygous individuals are especially sensitive
to folate, and those with folate levels in the lower part of the so-called normal range tend to have
elevated serum levels of homocysteine. Riboflavin shares the property of stabilizing this enzyme
variation (McNulty et al. 2002). Furthermore, studies (Rozen 2002; Yamada et al. 2001) support
that the state of riboflavin nutrition governs homocysteine metabolism in patients who are homozy-
gous for this genetic variation. It seems likely that patients with this genotype would respond more
rapidly and effectively to riboflavin supplementation than those individuals without this genetic
variation (Frosst et al. 1995). Furthermore, dietary intake of riboflavin in food is inversely related
to serum homocysteine concentrations in the United States (Ganji and Kafai 2004). Consistent with
this finding is the observation that riboflavin improves the genomic instability of the genetic vari-
ant of MTHFR (Hustad et al. 2000; Kimura et al. 2004; Stern et al. 2003; Stuart et al. 2003). Other
investigators have suggested that folate and riboflavin together lower plasma homocysteine concen-
trations regardless of genotype (Ganji and Kafai 2004).
6.12 Riboflavin and Cell Signaling Factors

Riboflavin participates in a wide variety of metabolic processes in microorganisms and plants that
involve growth regulation, priming of defense responses against pathogens, phototropisms, and
regulation of circadian or biological rhythms. All of these processes rely on the strategic location of
flavoenzymes capable of reacting with light, molecular oxygen, and numerous substrates to catalyze
one- and two-electron transfers. By functioning in this manner, several flavoenzymes are able to
participate significantly in many signal transduction pathways (Dong and Beer 2000; Green and
Paget 2004; Macheroux et al. 2011; Massey 2000; Tokutomi et al. 2008). Several flavoproteins that
play critical roles in mammalian redox signaling and programmed cell death have been identified
and reviewed. These include nitric oxide synthetase (Mowat et al. 2010), apoptosis-inducing factor
(AIF), proline dehydrogenase, and NADPH oxidase (Natarajan and Becker 2012). These flavoen-
zymes exhibit a high degree of homology within their carboxyl-terminal domain with that of other
oxidoreductases and can form homodimers.
The family of NOSs is composed of constitutive and inducible diflavin isoforms. In addition to
the major cytosolic (NOS I, NOS II) and membrane-bound (NOS III) forms, NOSs also exist as
splice variants and multiple isoforms. NOSs form dimers in their catalytically active states which,
when assembled, receive electrons from NADPH at their reductase domain (FAD and FMN) and
transfer them to the oxygenase domain that contains heme iron and tetrahydrobiopterin (Alderton
et al. 2001; Groves and Wang 2000). These enzymes catalyze a five-electron oxidation reaction
that converts l-arginine to l-citrulline with concomitant formation of nitric oxide. Nitric oxide
plays a critical role in signal transduction pathways in the cardiovascular and nervous systems and
is of major importance for cytostatic and cytotoxic activities in the immune system and cancer
progression.
Of particular note in mammalian cell signaling pathways is the role of a mitochondrial FAD-
dependent oxidoreductase, AIF, which, when activated, becomes a critical component in a
caspase-independent cell death pathway (Natarajan and Becker 2012). AIF is an inner mitochon-
drial membrane-bound enzyme that shares protein homology with a variety of other oxidoreductase
family proteins and is marginally linked to the oxidative phosphorylation complexes I and III.
After activation of a number of proapoptotic signaling proteins such Bax, calpain, and poly(ADP-
ribose) polymerase 1, AIF is proteolytically dissociated from the inner mitochondrial membrane and
the truncated form (tAIF) translocates to the nucleus where it binds with cyclophilin A (Candé et al.
2004) and a phosphorylated form of histone H2AX (γH2AX) (Artus et al. 2010). The former enzyme
K15043_C006.indd 239 4/10/2013 4:57:04 PM

is a peptidyl–prolyl cis–trans isomerase but loses its isomerase activity when bound to tAIF to con-
duct its apoptotic effect. γH2AX is a histone nucleosome core protein that usually marks double-
stranded DNA breaks and acts as a scaffolding protein to facilitate docking of DNA repair enzymes.
When complexed with tAIF and cyclophilin A, γH2AX apparently cannot accommodate DNA repair
enzymes (Artus et al. 2010). Thus, a synchronized collaboration among the three proteins, tAIF,
cyclophilin A, and γH2AX, forms a DNA-degradation complex necessary for chromatinolysis. It is
also important to consider that, as demonstrated under cell-free conditions, once AIF is activated,
subsequent loss of the flavin moiety or a mutation in the FAD-binding domain does not affect its
capacity to function with the cyclophilin A/γH2AX complex (Loeffler et al. 2001; Susin et al. 2000).
In its capacity as an oxidoreductase, AIF resides in a dimeric form after reduction with its
preferred pyridine nucleotide, NADH, to form FADH− (Miramar et al. 2001). Once reduced, the
FADH− forms a charge-transfer complex with NAD+, which demonstrates remarkable stability in
air. The reduced form of AIF exhibits high binding affinity for NAD+ (Churbanova and Sevrioukova
2008). These qualities coupled with its slow reactivity with molecular oxygen and its refractory
nature in the presence of low redox potential quinones (Misevičienė et al. 2011) argue against AIF
having a significant role in mitochondrial electron transfer reactions.
Control of the NADH/NAD+ redox ratio particularly at the interface of the inner mitochondrial
membrane may alter the equilibrium of AIF between its monomeric and dimeric forms. The reduced
form of FAD favors formation of the dimeric form, which has a high binding affinity for NAD+
and is refractory to N-terminal proteolysis at the membrane binding domain (Sevrioukova 2011).
Depletion of NAD+ particularly when excessive poly(ADP-ribose) polymerase is activated during
DNA strand breakage would shift the equilibrium to the monomeric form, allowing proteolytic
cleavage and escape of the monomeric AIF from the mitochondrial to nuclear compartment. Thus, it
is intriguing to consider that the level of reduced flavin in AIF may serve as a regulator or sensor for
available NAD+. High-affinity binding between the flavin–NAD moieties would favor membrane
attachment and retention of AIF within mitochondria. Conversely, loss of available NAD+ would
allow AIF to be cleaved from the membrane more readily and translocate to the nucleus.
Thus, utilizing the terminology from other proteosomes that reference “Janus kinases” and
“Janus chaperone” proteins, AIF is a “Janus oxidoreductase” that faces in opposite life/death direc-
tions. AIF’s “life” direction is toward the mitochondria where it may function as a NADH oxidase
or “sensor” of available NAD+ in mitochondria (Delavallée et al. 2011; Miramar et al. 2001). After
exiting the mitochondria, its “death” direction is toward the nucleus where it associates with its
nuclear partners to elicit caspase-independent apoptosis.
6.13 Extended Use and Medical Applications for Riboflavin

6.13.1 Riboflavin Photosensitization
The photoreactive properties of riboflavin and its derivatives that enable the isoalloxazine ring to
become a strong oxidizer have been exploited for clinical and therapeutic situations other than their role
as a vitamin or coenzyme. Flavins are used in phototherapy of neonatal jaundice to reduce the presence
of pathogens in blood products, to treat progression of corneal disorders, and to accelerate sun tanning.
6.13.1.1 Mechanisms of Riboflavin Photosensitization

The mechanisms by which riboflavin exerts its photodynamic activities is through its reactivity
with a wide range of electron-donating substrates through Type I, Type II, or mixed Type I–Type
II photochemical mechanisms (Silva and Edwards 2006). After photoexcitation of riboflavin to a
reactive triplet state, a Type I reaction, which is favored at low oxygen concentrations, requires a
substrate (e.g., an amino acid, protein, or lipid) to react directly with the excited flavin moiety to
generate radicals or radical ions (Martin et al. 2002). These molecules, in turn, react with molecular
oxygen to form superoxide, which may subsequently become reduced to H2O2 and hydroxyl radical.
K15043_C006.indd 240 4/10/2013 4:57:04 PM

A Type I reaction may underlie the antimutagenic activity of riboflavin against food-borne hetero-
cyclic amines (Edenharder et al. 1999) and effect efficacy of pharmaceutical formulations when
riboflavin and certain drugs are coingredients (Sue-Chu et al. 2009). In a Type II mechanism, the
excited triplet of riboflavin reacts directly with oxygen to form singlet molecular oxygen. Singlet
oxygen can add across double bonds or other electron-dense centers within a variety of substrates.
By contrast to Type II mechanisms, Type I reactions, under hypoxic conditions, protect riboflavin
against oxidative degradation by singlet oxygen to form lumichrome or lumiflavin. Type I and Type
II photochemical reactions with riboflavin are depicted in Figures 6.12 and 6.13, respectively.
O O O
•
H3C N N N
NH hν H3C NH Intersystem
H3C
NH
crossing
•
R R R
1Riboflavin 1Riboflavin* 3Riboflavin**
ground state Singlet-excited state Triplet-excited state

τ = 5 ηs τ = 15 μs
ISC 3Riboflavin
Riboflavin + hν
3Riboflavin + AH: Riboflavin • + A– •
Riboflavin • + O2 Riboflavin + O2– •
2H+ + A– • + O2– • H2O2 + A
hν
AH: + O2 H2O2 + A
Riboflavin
FIGURE 6.12 Generation of hydrogen peroxide (H2O2) in the presence of riboflavin, UV light (UV-A),
reducing agent (AH:), and molecular oxygen (O2). Exposure of riboflavin to UV-A light results in electron spin–
allowed transitions to highly fluorescent short-lived (5 ns) singlet-excited states (1Riboflavin*). Intersystem
crossing (ISC) with a high quantum yield generates the triplet-excited state of riboflavin (3Riboflavin**).
3Riboflavin is relatively long lived (~15 μs in water at ambient temperature) and as a bi-radical is a very power-
ful oxidant compared to ground-state riboflavin. This represents a Type I mechanism.
1Riboflavin
Riboflavin + hν
1Riboflavin
ISC 3Riboflavin
3Riboflavin + O
2 Riboflavin + 1O2
1O
hν + O2 2
FIGURE 6.13 Generation of singlet-excited state (1O2) in the presence of riboflavin and UV light (UV-A)
and molecular oxygen (O2). Exposure of riboflavin to UV-A light results in electron spin–allowed transitions
to a highly fluorescent short-lived (5 ns) singlet-excited state (1Riboflavin*). Intersystem crossing (ISC) gener-
ates the triplet-excited state of riboflavin (3Riboflavin). 3Riboflavin reacts directly with oxygen to form singlet
molecular oxygen. As riboflavin itself eventually becomes the reducing agent and is cleaved to lumichome
or lumiflavin (pH dependent), the reaction does not represent a true photosensitizer role for riboflavin. This
represents a Type II mechanism.
K15043_C006.indd 241 4/10/2013 4:57:05 PM

Type I and Type II reactions with xenobiotic substances may also explain why riboflavin defi-
ciency enhances carcinogenesis by increasing activation of carcinogens, particularly nitrosamines.
Riboflavin may provide protection against damage to DNA caused by certain carcinogens through
its action as a coenzyme with a variety of cytochrome P450 enzymes as well as by a direct interac-
tion with its photoirradiated products. It is important to recognize the role of riboflavin as a dietary
factor capable of reducing risk of carcinogenesis while determining the full implications of the
photosensitizing actions of riboflavin on mutagenesis and carcinogenesis.
6.13.1.2 Photosensitization of Cells
Photosensitization of cells in the presence of riboflavin was first observed using cultures of Ehrlich
ascites tumors (Warburg et al. 1968). Hydrogen peroxide and nonperoxide photoproducts were iden-
tified as the ultimate effectors and were shown to be generated from riboflavin and tryptophan or
tyrosine via oxygen intermediates (Wang and Nixon 1978). The riboflavin-mediated photoreactions
are triggered by visible light of wavelengths below 500 nm. These and similar studies have in com-
mon that riboflavin-mediated photoreactions can potentially lead to injury or death of cells.
Investigations using photoirradiation of riboflavin have been conducted in cancer treatment mod-
els of leukemia and solid tumor formation. Irradiated riboflavin was shown to induce signaling
events specific to leukemic cells that culminated in cell death. Upregulation of the Fas–Fas ligand
cascade was suggested to underlie riboflavin-mediated effect (Santos de Souza et al. 2006). Further
studies, using androgen-independent human prostate cancer cells, have demonstrated similar find-
ings in that the photoproducts of riboflavin resulted in FasL–Fas-dependent upregulation followed
by inhibition of matrix-degrading proteases and vascular growth factors suggesting antimetastatic
potential (de Souza Queiroz et al. 2007). The exact mechanism by which tumor cells are affected
by riboflavin photoirradiation must be interpreted with caution particularly when considering both
under cell culture conditions and in intact animal models. Accordingly, the level of molecular oxy-
gen and the availability of reduced substrates (amino acids, proteins) may lead to either Type I or
Type II photochemical reactions or a combination of both.
Highly lipid-soluble derivatives of riboflavin may also have potential as photosensitizers in can-
cer treatment. Riboflavin-2′,3′,4′,5′ tetrabutyrate either alone or in combination with tryptophan
demonstrates distinct cytotoxicity in human promyelocytic leukemia cell line (HL-60) and in the
human epithelial cervical cancer cell line (HeLa) (Muñoz et al. 2011). Both caspase-dependent
and -independent mechanisms are involved in tumor cell death and illustrates the need for further
research to identify selectivity of Type I and Type II photochemical mechanisms on normal and
mitotic cells.
6.13.1.3 Photosensitization for Neonatal Jaundice

Light-induced injury involving riboflavin has been noted explicitly during treatment of neonatal
jaundice (hyperbilirubinema) using phototherapy at wavelengths below 500 nm (Sisson 1987).
Since riboflavin and bilirubin have similar absorption maxima, “blue” light irradiation of capillary
beds will cause photoisomerization of bilirubin accompanied by photodegradation of riboflavin
(Knobloch and Hodr 1989; Knobloch et al. 1988). Loss of riboflavin diminishes erythrocyte gluta-
thione reductase activity, which, in association with increased hydrogen peroxide generation, causes
red blood cell lysis. Supplementation with riboflavin is considered beneficial during treatment, but
dosage, time course, and mode of administration to infants undergoing phototherapy should be
carefully monitored to avoid unwanted side effects (Tontisirin et al. 1989).
6.13.1.4 Photosensitization and Blood-Borne Pathogens

UV light–activated riboflavin reduces replication of pathogens (viruses, bacteria, and protozoa) in blood
products (Goodrich 2000). Clinical trials have been completed on methods that selectively damage
DNA and RNA and thus inactivate pathogen-contaminated blood components without damaging cells
K15043_C006.indd 242 4/10/2013 4:57:05 PM

or plasma protein components of blood. A Mirasol pathogen reduction technology (PRT) system for
platelets and plasma harnesses the photoreactive properties of riboflavin to induce damage of nucleic
acid–containing pathogens (Marschner and Goodrich 2011). This system has been shown to be effec-
tive against clinically relevant pathogens and inactivates leukocytes (Marschner et al. 2010) without
significantly compromising the amount or efficacy of the blood product or resulting in product loss.
Not all pathogens appear susceptible to the direct photosensitizing effects of riboflavin.
Irradiation of a corneal infection induced by the amoeboid eukaryote, Acanthamoeba, did not
demonstrate antitrophozoite activity in the presence of riboflavin (Kashiwabuchi et al. 2011). By
contrast, direct treatment of fungal keratitis using photoirradiation standardized for corneal cross-
linking procedures (see Section 6.13.1.5) is effective in decreasing the intensity and severity of
infectious keratitis by Fusarium solani (Galperin et al. 2012). Because of safety considerations for
direct photoirradiation in the eye, higher and less energetic wavelengths are used compared to those
required during PRT for blood products. These studies illustrate further the urgent need to assess
the specific conditions under which phototherapy with riboflavin is efficacious in association with
safety issues for its use.
6.13.1.5 Photosensitization and Treatment of Corneal Disorders (Keratoconus)

Riboflavin administration followed by UV-A treatment slows or stops the progression of the corneal
disorder keratoconus. The treatment procedure is referred to as corneal collagen cross-linking. In
corneal cross-linking, a solution of riboflavin is applied to the patient’s corneal surface. After pen-
etration of riboflavin through the cornea, UV-A light therapy is applied with variable duration and
intensity. This process induces collagen cross-linking, which increases the tensile strength of the
cornea. The treatment has been shown in several studies to stabilize keratoconus (Wittig-Silva et
al. 2008). In another study of patients who already had keratoconus, riboflavin administered in eye
drops delayed its progression (Wollensak 2006; Wollensak et al. 2003). Treatment of keratoconus
with riboflavin and UV light increases the stiffness of the cornea, increases the cross-linking of
collagen, and in this manner may inhibit progression of the disorder.
A detailed analysis of the photochemical kinetics of riboflavin-assisted corneal cross-linking
has been recently published (Kamaev et al. 2012). This study carefully measured oxygen concentra-
tions, controlled temperature, and varied energy input to determine that the initial mechanism of
surface protein cross-linking supports a brisk Type I photosensitization reaction. As the concen-
tration of oxygen increases within the corneal field of operation, a Type II mechanism begins to
contribute to the cross-linking.
Exposure of riboflavin to visible or UV light produces potent intermediate photosensitizers.
Controlling the formation of excited states of riboflavin and channeling the reactive species toward
cancer cells, microorganisms and viruses show promise in disease prevention. Interactions of pho-
toirradiated riboflavin with tryptophan and other amino acid residues in proteins have proven ben-
eficial in the treatment of certain corneal defects but may be detrimental in skin in that accelerated
free-radical formation will promote increased wrinkling and risk for skin cancer.
Topical administration of riboflavin to the skin may increase melanin synthesis by stimulation
of free-radical formation (Joshi et al. 1987). The photoreactive properties of riboflavin in foods and
various food products, particularly meats and dairy, have been examined for practical importance
involving maintenance of food quality, flavor, and freshness, as well as packaging requirements,
storage, and human consumption. The photosensitizing properties of riboflavin also raise the pos-
sibility of some potential risks. Phototherapy in vitro leads to degradation of DNA and increase in
lipid peroxidation, which may have implications for carcinogenesis, mutagenesis, and other disor-
ders. Further research is needed to explore the full implications of the photosensitizing capabilities
of riboflavin and its phosphorylated derivatives. These and other considerations involving ribofla-
vin, light, and food safety have been recently reviewed (Cardoso et al. 2012) and remain a subject
needing further investigation.
K15043_C006.indd 243 4/10/2013 4:57:05 PM

6.13.2 Treatment of Migraine and Other Uses with Riboflavin

Studies using high doses of riboflavin either alone or in combination with β-blockers, CoQ, and
magnesium while accompanied with an appropriate diet have demonstrated efficacy in the preven-
tion of migraine (Sandor et al. 2000; Taylor 2011; Zencirci 2010). Investigations on the use of ribo-
flavin were based on treatment outcomes in patients who had rare mitochondrial diseases. Patients
with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) expe-
rienced a symptom complex similar to that of patients with migraine, namely, recurrent headaches,
pain, muscle fatigue, loss of appetite, vomiting, and seizures. In subjects with MELAS and another
mitochondrial disease, Leber’s hereditary optic neuropathy, riboflavin treatment alleviates symp-
toms (Arts et al. 1983). Several studies show that riboflavin is a promising, safe, and well-tolerated
alternative in migraine prophylaxis in adults (Schoenen et al. 1994, 1998) and children (Condò
et al. 2009) but some results in children have been equivocal. A high-dose (200 mg riboflavin),
double-blind, placebo-controlled study (MacLennan et al. 2008) showed a high placebo responder
rate and concluded that riboflavin is not an effective therapy for preventing migraine in children. In
another study, doses of 50 mg of riboflavin had no prophylactic effect on the severity of migraine
attacks but did provide evidence that riboflavin use may be effective on interval headaches (Bruijn
et al. 2010).
6.14 Requirements and Assessment

There are a variety of methods available for analysis of riboflavin and its derivatives in biological
samples. Bioassays (Baker and Frank 1975) measure the growth effect of vitamins but lack the pre-
cision of more sensitive analytical procedures. Fluorometric procedures take advantage of the inher-
ent fluorescent properties of flavins (Bessey et al. 1949). Some degree of purification of the urine
or tissues may be required before analysis is performed as there is often significant interference by
other natural substances that lead to quenching of fluorescence and methodological artifacts.
A procedure has been developed for measuring riboflavin by competitive protein binding, which
is applicable to studies in human urine (Fazekas et al. 1974). Riboflavin binds specifically to the
avian egg white riboflavin-binding protein and thereby provides the basis for quantitative analysis
(Kim et al. 1995). Other procedures based on binding to specific apoenzymes, such as d-amino
acid oxidase, are also in use. Currently, procedures using high-pressure liquid chromatography
(HPLC) are widely applied as they have great precision and can be used for analysis of riboflavin in
pure form as well as in biological fluids and tissues (Chastain and McCormick 1987). HPLC is the
method most widely employed at this time for determination of flavins in blood and other tissues.
In clinical studies that involve individual patients as well as population groups, the status of ribo-
flavin nutrition is generally evaluated by determining urinary excretion of riboflavin (Sauberlich et
al. 1972) and the erythrocyte glutathione reductase activity coefficient (EGRAC). Urinary riboflavin
determinations are made in the basal state, in random samples, in 24 h collections, or after a ribofla-
vin load test. Normal urinary excretion of riboflavin is approximately 120 mg/g creatinine/24 h or
higher. It is useful to express urinary excretion in terms of creatinine to verify the completeness of
the collection and to relate riboflavin excretion to this biological parameter. Expressed in terms of
the total amount of urinary flavins in the normal adult (not taking supplements), excretion is about
1.5–2.5 mg/day, which is very close to the RDA of the National Academy of Sciences. Riboflavin
excretion per se is only about 1.0–1.5 mg/day. Flavin metabolites account for an appreciable portion
of total urinary flavins, as noted above.
In riboflavin-deficient adult individuals, urinary riboflavin excretion is reduced to about 40 mg/g
creatinine/24 h. Thus, deficient individuals have reduced urinary excretion, reflecting diminished
dietary intake and depleted body stores. Normal urinary excretion is reduced with age, may be
reduced by physical activity (as discussed later), and is stimulated by elevated body temperature,
treatment with certain drugs, and various stressful conditions associated with negative nitrogen
K15043_C006.indd 244 4/10/2013 4:57:05 PM

balance (Chastain and McCormick 1987). Interpretation of urinary riboflavin excretion must be
made with these factors in mind.
Another potential drawback to using urinary riboflavin excretion as an assessment of nutritional
status of this vitamin is that the amount excreted reflects recent intake very sensitively. Thus, if an
individual has been depleted of riboflavin for a long time but consumes a food item high in ribofla-
vin, urinary excretion determined a few hours later may not be in the deficient range, but is likely to
be normal or even elevated. It is for this reason that attention has been directed to the development
of assessment techniques that more accurately reflect long-term riboflavin status. The method most
widely employed that largely meets these needs is the EGRAC assay, as noted earlier. The principle
of the method is that the degree of saturation of the apoenzyme with its coenzyme, FAD, should
reflect the body stores of FAD. In deficient individuals, relative unsaturation of the apoenzyme with
FAD leads to decreased basal activity of the enzyme. Therefore, the addition of FAD to the enzyme
contained in a fresh erythrocyte hemolysate from deficient individuals will increase enzyme activ-
ity in vitro to a greater extent than that observed in a preparation from well-nourished individuals
in whom the apoenzyme is more fully saturated with FAD.
The EGRAC is expressed as the ratio of in vitro enzyme activity with the addition of FAD to that
without. In general, most studies propose that an activity coefficient of 1.2 or less indicates adequate
riboflavin status, 1.2–1.4 denotes borderline-to-low status, and greater than 1.4 indicates a clear
riboflavin deficiency (Rivlin 1966; Sauberlich et al. 1972).
It must be kept in mind that a number of physiological variables influence the results of this
determination. In the inherited disorder of glucose-6-phosphate dehydrogenase deficiency, associ-
ated with hemolytic anemia, the apoenzyme has a higher affinity for FAD than that of the normal
erythrocyte and will affect the measured EGRAC. Thyroid function affects glutathione reduc-
tase activity, with the coefficient elevated in hypothyroidism and decreased in hyperthyroidism
(Menendez et al. 1974), reflecting that hypothyroidism has many biochemical features in common
with those of riboflavin deficiency (Rivlin et al. 1968).
The RDAs issued by the Food and Nutrition Board (2000) call for adult males aged 19–50 years
to consume about 1.3 mg/day. Adult females from 19 to 50 years of age should consume 1.1 mg/day.
It is recommended that intake be increased to 1.4 mg/day during pregnancy and to 1.6 mg/day in
lactation. There has been some concern as to whether these figures are applicable to all population
groups around the world. Chinese tend to excrete very little riboflavin, and their requirement may
be lower than that of Americans. Using reference values of <1.4 mg for 4 h urinary excretion ribo-
flavin after a 5 mg load, more than 70% of the subjects examined in rural China exhibited low levels
usually associated with high risk of riboflavin deficiency (Brun et al. 1990). Adults in Guatemala
have similar requirements in individuals older than 60 compared with those 51 years or younger
(Boisvert et al. 1993). This finding may not necessarily be relevant to populations of other countries.
The requirements of various national groups require further study. Environmental factors, protein-
calorie intake, physical activity, and other factors may have an impact on riboflavin requirements.
More research is needed on the requirements of the extremely old, who form an increasingly
large proportion of the population. They are also the population group that consumes the larg-
est number of prescribed and over-the-counter medications. As mentioned earlier in this chapter
(Section 6.5.4.1), women aged 50–67 who exercise vigorously for 20–25 min/day, 6 days a week
exhibit decreases in riboflavin excretion and slight elevations in their EGRAC (Winters et al. 1992).
Supplementation with riboflavin does not improve exercise performance. Comparable findings have
been observed in young women exercising vigorously (Belko et al. 1985). Similar observations of
reduced urinary riboflavin excretion and elevated EGRAC were made in young Indian males who
exercise vigorously (Soares et al. 1993). Thus, exercise studies suggest that an increased demand
for riboflavin occurs for selective biochemical functions during exercise but additional riboflavin
supplementation does not enhance performance.
To determine whether the status of riboflavin nutrition influences metabolic responses to exer-
cise, blood lactate levels were determined in a group of physically active college students from
K15043_C006.indd 245 4/10/2013 4:57:05 PM

Finland before and after an exercise period. A number of the students were initially in a state of
marginal riboflavin deficiency. After supplementation with vitamins, including riboflavin, which
produced improvement in the elevated EGRAC, the blood lactate levels were unaffected and were
related only to the degree of exercise (Fogelholm et al. 1993). Thus, to date, while exercise clearly
produces biochemical abnormalities in riboflavin metabolism, it has not been shown that these
abnormalities lead to impaired performance, nor has it been shown that riboflavin supplementation
under these conditions leads to improved endurance.
References
Abe, I., Seki, T., Umehara, K., Miyase, T., Noguchi, H., Sakakibara, J. and Ono, T. 2000. Green tea polyphe-
nols: novel and potent inhibitors of squalene epoxidase. Biochem Biophys Res Commun. 268: 767–71.
Adiga, P.R., Subramanian, S., Rao, J. and Kumar, M. 1997. Prospects of riboflavin carrier protein (RCP) as an
antifertility vaccine in male and female mammals. Hum Reprod Update. 3: 325–34.
Agani, F.H., Pichiule, P., Chavez, J.C. and LaManna, J.C. 2000. The role of mitochondria in the regulation of
hypoxia-inducible factor 1 expression during hypoxia. J Biol Chem. 275: 35863–7.
Agte, V.V., Chiplonkar, S.A. and Gokhale, M.K. 1992. Interaction of riboflavin with zinc bioavailability. Ann
N Y Acad Sci. 669: 314–6.
Ahmad, I., Ahmed, S., Sheraz, M.A., Aminuddin, M. and Vaid, F.H. 2009. Effect of caffeine complexation on
the photolysis of riboflavin in aqueous solution: a kinetic study. Chem Pharm Bull (Tokyo). 57: 1363–70.
Aigrain, L., Pompon, D. and Truan, G. 2011. Role of the interface between the FMN and FAD domains in the control
of redox potential and electronic transfer of NADPH-cytochrome P450 reductase. Biochem J. 435: 197–206.
Akiyama, T., Selhub, J. and Rosenberg, I.H. 1982. FMN phosphatase and FAD pyrophosphatase in rat intestinal
brush borders: role in intestinal absorption of dietary riboflavin. J Nutr. 112: 263–8.
Akompong, T., Ghori, N. and Haldar, K. 2000. In vitro activity of riboflavin against the human malaria parasite
Plasmodium falciparum. Antimicrob Agents Chemother. 44: 88–96.
Albracht, S.P., van der Linden, E. and Faber, B.W. 2003. Quantitative amino acid analysis of bovine
NADH:ubiquinone oxidoreductase (Complex I) and related enzymes. Consequences for the number of
prosthetic groups. Biochim Biophys Acta. 1557: 41–9.
Alderton, W.K., Cooper, C.E. and Knowles, R.G. 2001. Nitric oxide synthases: structure, function, and inhibi-
tion. Biochem J. 357: 593–615.
Allard, M.L., Jeejeebhoy, K.N. and Sole, M.J. 2006. The management of conditioned nutritional requirements
in heart failure. Heart Fail Rev. 11: 75–82.
Anand, G., Hasan, N., Jayapal, S., Huma, Z., Ali, T., Hull, J., Blair, E., McShane, T. and Jayawant, S. 2012.
Early use of high-dose riboflavin in a case of Brown-Vialetto-Van Laere syndrome. Dev Med Child
Neurol. 54: 187–9.
Anderson, B.B., Giuberti, M., Perry, G.M., Salsini, G., Casadio, I. and Vullo, C. 1993. Low red blood cell glu-
tathione reductase and pyridoxine phosphate oxidase activities not related to dietary riboflavin: selection
by malaria? Am J Clin Nutr. 57: 666–72.
Anderson, B.B., Scattoni, M., Perry, G.M., Galvan, P., Giuberti, M., Buonocore, G. and Vullo, C. 1994. Is the
flavin-deficient red blood cell common in Maremma, Italy, an important defense against malaria in this
area? Am J Hum Genet. 55: 975–80.
Anderson, K.E., Bodansky, O. and Kappas, A. 1976. Effects of oral contraceptives on vitamin metabolism. Adv
Clin Chem. 18: 247–87.
Anton, R.F. 1996. New methodologies for pharmacological treatment trials for alcohol dependence. Alcohol
Clin Exp Res. 20(7 Suppl): 3A–9A.
Apelt, N., da Silva, A.P., Ferreira, J., Alho, I., Monteiro, C., Marinho, C., Teixeira, P., Sardinha, L., Laires, M.J.,
Mascarenhas, M.R. and Bicho, M.P. 2009. ACP1 genotype, glutathione reductase activity, and riboflavin
uptake affect cardiovascular risk in the obese. Metabolism. 58: 1415–23.
Appenroth, D. and Winnefeld, K. 1999. Is thallium-induced nephrotoxicity in rats connected with riboflavin and/
or GSH?—reconsideration of hypotheses on the mechanism of thallium toxicity. J Appl Toxicol. 19: 61–6.
Arts, W.F.M., Scholte, H.R., Boggard, J.M., Kerrebijn, K.F. and Luyt-Houwen, I.E.M. 1983. NADH-CoQ
reductase deficient myopathy: successful treatment with riboflavin. Lancet 2: 581–2.
Artus, C., Boujrad, H., Bouharrour, A., Brunelle, M.N., Hoos, S., Yuste, V.J., Lenormand P., Rousselle, J.C.,
Namane, A., England, P., Lorenzo, H.K. and Susin, S.A. 2010. AIF promotes chromatinolysis and
caspase-independent programmed necrosis by interacting with histone H2AX. EMBO J. 29: 1585–99.
K15043_C006.indd 246 4/10/2013 4:57:05 PM

Backiel, J., Juárez, O., Zagorevski, D.V., Wang, Z., Nilges, M.J. and Barquera, B. 2008. Covalent binding of
flavins to RnfG and RnfD in the Rnf complex from Vibrio cholerae. Biochemistry. 47: 11273–84.
Bafunno, V., Giancaspero, T.A., Brizio, C., Bufano, D., Passarella, S., Boles, E. and Barile, M. 2004. Riboflavin
uptake and FAD synthesis in Saccharomyces cerevisiae mitochondria: involvement of the Flx1p carrier
in FAD export. J Biol Chem. 279: 95–102.
Bai, M., Xiao, X.Y. and Prestwich, G.D. 1992. Epoxidation of 2,3-oxidosqualene to 2,3;22,23-squalene dioxide
by squalene epoxidase. Biochem Biophys Res Commun. 185: 323–9.
Baker, H. and Frank, O. 1975.Analysis of riboflavin and its derivatives in biologic fluids and tissues. In
Riboflavin, ed. R.S. Rivlin, 49–79. New York: Plenum Press.
Baker, H., Frank, O. and Jaslow, S.P. 1980. Oral versus intramuscular vitamin supplementation for hypovita-
minosis in the elderly. J Am Geriatr Soc. 28: 42–5.
Balconi, E., Pennati, A., Crobu, D., Pandini, V., Cerutti, R., Zanetti, G. and Aliverti, A. 2009. The ferredoxin-
NADP+ reductase/ferredoxin electron transfer system of Plasmodium falciparum. FEBS J. 276: 3825–36.
Bandyopadhyay, D., Chatterjee, A.K. and Datta, A.G. 1997. Effect of cadmium on purified hepatic flavokinase:
involvement of reactive -SH group(s) in the inactivation of flavokinase by cadmium. Life Sci. 60: 1891–903.
Banhegyi, G., Benedetti, A., Csala, M. and Mandl, J. 2007. Stress on redox. FEBS Lett. 581: 3634–640.
Bareford, L.M., Phelps, M.A., Foraker, A.B. and Swaan, P.W. 2008. Intracellular processing of riboflavin in
human breast cancer cells. Mol Pharm. 5: 839–48.
Barile, M., Brizio, C., De Virgilio, C., Delfine, S., Quagliariello, E. and Passarella, S. 1997. Flavin adenine
dinucleotide and flavin mononucleotide metabolism in rat liver—the occurrence of FAD pyrophospha-
tase and FMN phosphohydrolase in isolated mitochondria. Eur J Biochem. 249: 777–85.
Barile, M., Brizio, C., Valenti, D., De Virgilio, C. and Passarella, S. 2000. The riboflavin/FAD cycle in rat liver
mitochondria. Eur J Biochem. 267: 4888–900.
Barthelemy, H., Chouvet, B. and Cambazard, F. 1986. Skin and mucosal manifestations in vitamin deficiency.
J Am Acad Dermatol. 15: 1263–74.
Baxter, P. 2003. Vitamin responsive conditions in paediatric neurology. In International Review of Child
Neurology Series, ed. P. Baxter, 47–53. London: MacKeith Press.
Becker, K., Christopherson, R.I., Cowden, W.B., Hunt, N.H. and Schirmer, R.H. 1990. Flavin analogs with
antimalarial activity as glutathione reductase inhibitors. Biochem Pharmacol. 39: 59–65.
Beinert, W.D., Rüterjans, H., Müller, F. and Bacher, A. 1985. Nuclear magnetic resonance studies of the old
yellow enzyme. 2. 13C NMR of the enzyme recombined with 13C-labeled flavin mononucleotides. Eur
J Biochem. 152: 581–7.
Belko, A.Z., Meredith, M.P., Kalkwarf, H.J., Obarzanek, E., Weinberg, S., Roach, R., McKeon, G. and Roe,
D.A. 1985. Effects of exercise on riboflavin requirements: biological validation in weight reducing
women. Am J Clin Nutr. 41: 270–7.
Belter, A., Skupinska, M., Giel-Pietraszuk, M., Grabarkiewicz, T., Rychlewski, L. and Barciszewski, J. 2011.
Squalene monooxygenase – a target for hypercholesterolemic therapy. Biol Chem. 392: 1053–75.
Bennett, M.J., Pollitt, R.J., Goodman, S.I., Hale, D.E. and Vamecq, J. 1991. Atypical riboflavin-responsive
glutaric aciduria, and deficient peroxisomal glutaryl-CoA oxidase activity: a new peroxisomal disorder.
J Inherit Metab Dis. 14: 165–73.
Berrisford, J.M. and Sazanov, L.A. 2009. Structural basis for the mechanism of respiratory complex I. J Biol
Chem. 284: 29773–83.
Bessey, O.A., Lowry, O.H. and Love, R.H. 1949. Fluorometric measure of the nucleotides of riboflavin and
their concentration in tissues. J Biol Chem. 180: 755–69.
Beutler, E. 1969a. Effect of flavin compounds on glutathione reductase activity: in vivo and in vitro studies.
J Clin Invest. 48: 1957–66.
Beutler, E. 1969b. Glutathione reductase: stimulation in normal subjects by riboflavin supplementation.
Science. 165: 613–5.
Beutler, E. and Srivastava, S.K. 1970. Relationship between glutathione reductase activity and drug-induced
haemolytic anaemia. Nature. 226: 759–60.
Birch-Machin, M.A., Taylor, R.W., Cochran, B., Ackrell, B.A. and Turnbull, D.M. 2000. Late-onset optic atro-
phy, ataxia, and myopathy associated with a mutation of a complex II gene. Ann Neurol. 48: 330–5.
Bjarnason, I., Sharpstone, D.R., Francis, N., Marker, A., Taylor, C., Barrett, M., Macpherson, A., Baldwin, C.,
Menzies, I.S., Crane, R.C., Smith, T., Pozniak, A. and Gazzard, B.G. 1996. Intestinal inflammation, ileal
structure and function in HIV. AIDS. 10: 1385–91.
Bode, J.G., Albrecht, U., Häussinger, D., Heinrich, P.C. and Schaper, F. 2012. Hepatic acute phase proteins –
regulation by IL-6- and IL-1-type cytokines involving STAT3 and its crosstalk with NF-κB-dependent
signaling. Eur J Cell Biol. 91: 496–505.
K15043_C006.indd 247 4/10/2013 4:57:05 PM

Boelen, A., Kwakkel, J. and Fliers, E. 2011. Beyond low plasma T3: local thyroid hormone metabolism during
inflammation and infection. Endocr Rev. 32: 670–93.
Bogh, M.K., Schmedes, A.V., Philipsen, P.A., Thieden, E. and Wulf, H.C. 2010. Vitamin D production after
UVB exposure depends on baseline vitamin D and total cholesterol but not on skin pigmentation. J Invest
Dermatol. 130: 46–53.
Boisvert, W.A., Castañeda, C., Mendoza, I., Langeloh, G., Solomons, N.W., Gershoff, S.N. and Russell, R.M.
1993. Prevalence of riboflavin deficiency among Guatemalan elderly people and its relationship to milk
intake. Am J Clin Nutr. 58: 85–90.
Boot-Handford, R.P. and Briggs, M.D. 2010. The unfolded protein response and its relevance to connective
tissue diseases. Cell Tissue Res. 339: 197–211.
Bornemann, S. 2002. Flavoenzymes that catalyze reactions with no net redox change. Nat Prod Rep. 19: 761–72.
Bosch, A.M., Abeling, N.G., Ijlst, L., Knoester, H., van der Pol, W.L., Stroomer, A.E., Wanders, R.J., Visser,
G., Wijburg, F.A., Duran, M. and Waterham, H.R. 2011. Brown-Vialetto-Van Laere and Fazio Londe
syndrome is associated with a riboflavin transporter defect mimicking mild MADD: a new inborn error
of metabolism with potential treatment. J Inherit Metab Dis. 34: 159–64.
Brady, P.S., Marine, K.A., Brady, L.J. and Ramsay, R.R. 1989. Co-ordinate induction of hepatic mitochondrial
and peroxisomal carnitine acyltransferase synthesis by diet and drugs. Biochem J. 260: 93–100.
Brijlal, S. and Lakshmi, A.V. 1999. Tissue distribution and turnover of [3H] riboflavin during respiratory infec-
tion in mice. Metabolism. 48: 1608–11.
Brijlal, S., Lakshmi, A.V. and Bamji, M.S. 1999. Mitochondrial oxidative metabolism during respiratory infec-
tion in riboflavin deficient mice. J Nutr Biochem. 10: 728–32.
Brijlal, S., Lakshmi, A.V., Bamji, M.S. and Suresh, P. 1996. Flavin metabolism during respiratory infection in
mice. Br J Nutr. 763: 453–62.
Brizio, C., Galluccio, M., Wait, R., Torchetti, E.M., Bafunno, V., Accardi, R., Gianazza, E., Indiveri, C. and
Barile, M. 2006. Over-expression in Escherichia coli and characterization of two recombinant isoforms
of human FAD synthetase. Biochem Biophys Res Commun. 344: 1008–16.
Bruijn, J., Duivenvoorden, H., Passchier, J., Locher, H., Dijkstra, N. and Arts, W.F. 2010. Medium-dose ribo-
flavin as a prophylactic agent in children with migraine: a preliminary placebo-controlled, randomised,
double-blind, cross-over trial. Cephalalgia. 30: 1426–34.
Brun, T.A., Chen, J., Campbell, T.C., Boreham, J., Feng, Z., Parpia, B., Shen, T.F. and Li, M. 1990. Urinary
riboflavin excretion after a load test in rural China as a measure of possible riboflavin deficiency. Eur J
Clin Nutr. 44: 195–206.
Buckman, S.A. and Heise, C.P. 2010. Nutrition considerations surrounding restorative proctocolectomy. Nutr
Clin Pract. 25: 250–6.
Bulger, J.E. and Brandt, K.G. 1971. Yeast glutathione reductase. II. Interaction of oxidized and 2-electron
reduced enzyme with reduced and oxidized nicotinamide adenine dinucleotide phosphate. J Biol Chem.
246: 5578–87.
Buzina, R., Jusic, M., Milanovic, N., Sapunar, J. and Brubacher, G. 1979. The effects of riboflavin administra-
tion on iron metabolism parameters in a school going population. Int J Vitaminol Nutr Res. 49: 136–43.
Cai, Z., Blumbergs, P.C., Finnie, J.W., Manavis, J. and Thompson, P.D. 2007. Novel fibroblastic onion bulbs in
a demyelinating avian peripheral neuropathy produced by riboflavin deficiency. Acta Neuropathol. 114:
187–94.
Cai, Z., Blumbergs, P.C., Finnie, J.W., Manavis, J. and Thompson, P.D. 2009. Selective vulnerability of periph-
eral nerves in avian riboflavin deficiency demyelinating polyneuropathy. Vet Pathol. 46: 88–96.
Cai, Z., Finnie, J.W., Blumbergs, P.C., Manavis, J., Ghabriel, M.N. and Thompson, P.D. 2006. Early paranodal
myelin swellings (tomacula) in an avian riboflavin deficiency model of demyelinating neuropathy. Exp
Neurol. 198: 65–71.
Caldinelli, L., Molla, G., Bracci, L., Lelli, B., Pileri, S., Cappelletti, P., Sacchi, S. and Pollegioni, L. 2010.
Effect of ligand binding on human D-amino acid oxidase: implications for the development of new drugs
for schizophrenia treatment. Protein Sci. 19: 1500–12.
Caldinelli, L., Molla, G., Sacchi, S., Pilone, M.S. and Pollegioni, L. 2009. Relevance of weak flavin binding in
human D-amino acid oxidase. Protein Sci. 18: 801–10.
Camporeale, G. and Zempleni, J. 2003. Oxidative folding of interleukin-2 is impaired in flavin-deficient jurkat
cells, causing intracellular accumulation of interleukin-2 and increased expression of stress response
genes. J Nutr. 133: 668–72.
Candé, C., Vahsen, N., Kouranti, I., Schmitt, E., Daugas, E., Spahr, C., Luban, J., Kroemer, R.T., Giordanetto,
F., Garrido, C., Penninger, J.M. and Kroemer, G. 2004. AIF and cyclophilin A cooperate in apoptosis-
associated chromatinolysis. Oncogene. 23: 1514–21.
K15043_C006.indd 248 4/10/2013 4:57:05 PM

Cardoso, D.R., Libardi, S.H. and Skibsted, L.H. 2012. Riboflavin as a photosensitizer. Effects on human health
and food quality. Food Funct. 3: 487–502.
Caufield, L.E., Richards, S.A. and Black, R.E. 2004. Undernutrition as an underlying cause of malaria morbid-
ity and mortality in children less than five years old. Am J Trop Med Hyg. 71: 2 suppl, 55–63.
Cecchini, G. 2003. Function and structure of complex II of the respiratory chain. Annu Rev Biochem. 72: 77–109.
Chastain, J.L. and McCormick, D.B. 1987. Flavin catabolites: identification and quantitation in human urine.
Am J Clin Nutr. 46: 830–4.
Chaves, F.J., Mansego, M.L., Blesa, S., Gonzalez-Albert, V., Jiménez, J., Tormos, M.C., Espinosa, O., Giner,
V., Iradi, A., Saez, G. and Redon, J. 2007. Inadequate cytoplasmic antioxidant enzymes response contrib-
utes to the oxidative stress in human hypertension. Am J Hypertens. 20: 62–9.
Chen, L., Liu, L., Hong, K., Hu, J. and Cheng, X. 2012. Three genetic polymorphisms of homocysteine-
metabolizing enzymes and risk of coronary heart disease: a meta-analysis based on 23 case-control stud-
ies. DNA Cell Biol. 31: 238–49.
Chetty, R. 2010. Familial paraganglioma syndromes. J Clin Pathol. 63: 488–91.
Christensen, S. 1971. Studies on riboflavin metabolism in the rat. VI. Properties of radioactive metabolites
excreted after administration of riboflavin-2- 14 C. Acta Pharmacol Toxicol (Copenh). 30: 177–84.
Churbanova, I.Y. and Sevrioukova, I.F. 2008. Redox-dependent changes in molecular properties of mitochon-
drial apoptosis-inducing factor. J Biol Chem. 283: 5622–31.
Cimino, J.A., Jhangiani, S., Schwartz, E. and Cooperman, J.M. 1987. Riboflavin metabolism in the hypothyroid
human adult. Proc Soc Exp Biol Med. l84: 15l–3.
Cladman, W., Scheffer, S., Goodrich, N. and Griffiths, M.W. 1998. Shelf-life of milk packaged in plastic con-
tainers with and without treatment to reduce light transmission. Int Dairy J. 8: 629–36.
Clagett, C.O. 1971. Genetic control of the riboflavin carrier protein. Fed. Proc. 30: 127–9.
Clarke, M.J., Dowling, M.G., Garafalo, A.R. and Brennan, T.F. 1980. Structural and electronic effects resulting
from metal-flavin ligation. J Biol Chem. 255: 3472–81.
Condò, M., Posar, A., Arbizzani, A. and Parmeggiani, A. 2009. Riboflavin prophylaxis in pediatric and adoles-
cent migraine. J Headache Pain. 10: 361–5.
Cooper, A.J.L. and Pinto, J.T. 2006. Cysteine S-conjugate β-lyases. Amino Acids. 30: 1–15.
Cooper, A.J.L., Pinto, J.T. and Callery, P.S. 2011. Reversible and irreversible protein glutathionylation: biologi-
cal and clinical aspects. Expert Opin Drug Metab Toxicol. 7: 891–910.
Craghill, J., Cronshaw, A.D. and Harding, J.J. 2004. The identification of a reaction site of glutathione mixed-
disulphide formation on gammaS-crystallin in human lens. Biochem J. 379: 595–600.
Criado, S. and García, N.A. 2004. Vitamin B2-sensitized photooxidation of the ophthalmic drugs Timolol and
Pindolol: kinetics and mechanism. Redox Rep. 9: 291–7.
Crugnola, V., Lamperti, C., Lucchini, V., Ronchi, D., Peverelli, L., Prelle, A., Sciacco, M., Bordoni, A., Fassone,
E., Fortunato, F., Corti, S., Silani, V., Bresolin, N., Di Mauro, S., Comi, G.P. and Moggio, M. 2010.
Mitochondrial respiratory chain dysfunction in muscle from patients with amyotrophic lateral sclerosis.
Arch Neurol. 67: 849–54.
Dainty, J.R., Bullock, N.R., Hart, D.J., Hewson, A.T., Turner, R., Finglas, P.M. and Powers, H.J. 2007.
Quantification of the bioavailability of riboflavin from foods by use of stable-isotope labels and kinetic
modeling. Am J Clin Nutr. 85: 1557–64.
Dalle-Donne, I., Rossi, R., Colombo, G., Giustarini, D. and Milzani, A. 2009. Protein S-glutathionylation: a
regulatory device from bacteria to humans. Trends Biochem Sci. 34: 85–96.
Dancis, J., Lehanka, J. and Levitz, M. 1988. Placental transport of riboflavin: differential rates of uptake at the
maternal and fetal surfaces of the perfused human placenta. J Obstet Gynecol. 158: 204–10.
Das, B.S., Das, D.B., Satpathy, R.N., Patnaik, J.K. and Bose, T.K. 1988. Riboflavin deficiency and severity of
malaria. Eur J Clin Nutr. 42: 277–83.
De Colibus, L. and Mattevi, A. 2006. New frontiers in structural flavoenzymology. Curr Opin Struct Biol. 16:
722–8.
de Gonzalo, G., Smit, C., Jin, J., Minnaard, A.J. and Fraaije, M.W. 2011. Turning a riboflavin-binding protein
into a self-sufficient monooxygenase by cofactor redesign. Chem Commun (Camb). 47: 11050–2.
de Souza Queiroz, K.C., Zambuzzi, W.F., Santos de Souza, A.C., da Silva, R.A., Machado, D., Justo, G.Z.,
Carvalho, H.F., Peppelenbosch, M.P. and Ferreira, C.V. 2007. A possible anti-proliferative and anti-
metastatic effect of irradiated riboflavin in solid tumours. Cancer Lett. 258: 126–34.
de Vet, E.C., Hilkes, Y.H., Fraaije, M.W. and van den Bosch, H. 2000. Alkyl-dihydroxyacetonephosphate syn-
thase. Presence and role of flavin adenine dinucleotide. J Biol Chem. 275: 6276–83.
DeBarber, A.E., Eroglu, Y., Merkens, L.S., Pappu, A.S. and Steiner, R.D. 2011. Smith-Lemli-Opitz syndrome.
Expert Rev Mol Med. 13: e24.
K15043_C006.indd 249 4/10/2013 4:57:05 PM

Decker, K.F. 1993. Biosynthesis and function of enzymes with covalently bound flavin. Annu Rev Nutr. 13: 17–41.
Degterev, A., Huang, Z., Boyce, M., Li, Y., Jagtap, P., Mizushima, N., Cuny, G.D., Mitchison, T.J., Moskowitz,
M.A. and Yuan, J. 2005. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for
ischemic brain injury. Nat Chem Biol. 1: 112–9.
Delavallée, L., Cabon, L., Galán-Malo, P., Lorenzo, H.K. and Susin, S.A. 2011. AIF-mediated caspase-
independent necroptosis: a new chance for targeted therapeutics. IUBMB Life. 63: 221–32.
Deneke, S.M. and Fanburg, B.L. 1989. Regulation of cellular glutathione. Am J Physiol. 257: L163–73.
Depeint, F., Bruce, W.R., Shangari, N., Mehta, R. and O’Brien, P.J. 2006. Mitochondrial function and toxicity:
role of the B vitamin family on mitochondrial energy metabolism. Chem Biol Interact. 163: 94–112.
Dignam, J. and Strobel, H. 1975. Preparation of homogeneous NADPH-cytochrome P-450 reductase from rat
liver. Biochim Biophys Res Commun. 63: 845–52.
Dincer, Y., Akcay, T., Alademir, Z. and Ilkova, H. 2002. Effect of oxidative stress on glutathione pathway in red
blood cells from patients with insulin-dependent diabetes mellitus. Metabolism. 51: 1360–2.
Dong, H. and Beer, S.V. 2000. Riboflavin induces disease resistance in plants by activating a novel signal trans-
duction pathway. Phytopathology. 90: 801–11.
Droge, W. 2002. Free radicals in the physiological control of cell function. Physiol Rev. 82: 47–95.
DurgaKumari, B. and Adiga, P.R. 1986. Hormonal induction of riboflavin carrier protein in the chicken oviduct
and liver: a comparison of kinetics and modulation. Mol Cell Endocrinol. 44: 285–92.
Dutta, P. 1991. Enhanced uptake and metabolism of riboflavin in erythrocytes infected with Plasmodium falci-
parum. J Protozool. 38: 479–83.
Dutta, P. 1993. Disturbances in glutathione metabolism and resistance to malaria: current understanding and
new concepts. J Soc Pharm Chem. 23: 11–48.
Dutta, P., Pinto, J.T. and Rivlin, R.S. 1985. Antimalarial effects of riboflavin deficiency. Lancet 2(8463):
1040–3.
Dutta, P., Raiczyk, G.B. and Pinto, J. 1988. Inhibition of riboflavin metabolism in cardiac and skeletal muscle
of rats by quinacrine and tetracycline. J Clin Biochem Nutr. 4: 203–8.
Dutta, P., Seirafi, J., Halpin, D., Pinto, J. and Rivlin, R. 1995. Acute ethanol exposure alters hepatic glutathione
metabolism in riboflavin deficiency. Alcohol. 12: 43–7.
Edenharder, R., Worf-Wandelburg, A., Decker, M. and Platt, K.L. 1999. Antimutagenic effects and possible
mechanisms of action of vitamins and related compounds against genotoxic heterocyclic amines from
cooked food. Mutat Res. 444: 235–48.
Ellgaard, L. and Ruddock, L.W. 2005. The human protein disulphide isomerase family: substrate interactions
and functional properties. EMBO Rep. 6: 28–32.
Emmett, A.D. and Luros, G.O. 1920. Water soluble vitamins. I. Are the antineuritic and the growth promoting
water-soluble B vitamins the same? J Biol Chem. 43: 265–9.
Ensminger, A.M., Ensminger, M.E., Konlande, J.E. and Robson, J.R.K. 1994. In Food and Nutrition
Encyclopedia, 1927. Boca Raton, FL: CRC Press.
Er, T.K., Chen, C.C., Liu, Y.Y., Chang, H.C., Chien, Y.H., Chang, J.G., Hwang, J.K. and Jong, Y.J. 2011.
Computational analysis of a novel mutation in ETFDH gene highlights its long-range effects on the FAD-
binding motif. BMC Struct Biol. 11: 43.
Fabian, R.H. 1991. Retrograde axonal transport and transcytosis of immunoglobulins: Implications for the
pathogenesis of autoimmune motor neuron disease. Adv Neurol. 56: 433–44.
Fabrias, G., Muñoz-Olaya, J., Cingolani, F., Signorelli, P., Casas, J., Gagliostro, V. and Ghidoni, R. 2012.
Dihydroceramide desaturase and dihydrosphingolipids: debutant players in the sphingolipid arena. Prog
Lipid Res. 51: 82–94.
Farhangi, M. and Osserman, E. F. 1976. Myeloma with xanthoderma due to an IgG lambdamonoclonal anti-
flavin antibody. N Engl J Med. 294: 177–83.
Farmer, B., Larson, B.T., Fulgoni, V.L. 3rd, Rainville, A.J. and Liepa, G.U. 2011. A vegetarian dietary pattern
as a nutrient-dense approach to weight management: an analysis of the national health and nutrition
examination survey 1999–2004. J Am Diet Assoc. 111: 819–27.
Fass, S. and Rivlin, R.S. 1969. Regulation of riboflavin-metabolizing enzymes in riboflavin deficiency. Am J
Physiol. 217: 988–91.
Favaudon, V. and Lhoste, J.M. 1975. The kinetics of flavin oxidation-reduction. II. Metal ion interactions.
Biochemistry. 14: 4739–44.
Fazekas, A.G., Menendez, C.E. and Rivlin, R.S. 1974. A competitive protein-binding assay for urinary ribofla-
vin. Biochem Med. 9: 167–76.
Feidt, D.M., Klein, K., Nüssler, A. and Zanger, U.M. 2009. RNA-interference approach to study functions of
NADPH: cytochrome P450 oxidoreductase in human hepatocytes. Chem Biodivers. 6: 2084–91.
K15043_C006.indd 250 4/10/2013 4:57:05 PM

Finn, R.D., Basran, J., Roitel, O., Wolf, C.R., Munro, A.W., Paine, M.J. and Scrutton, N.S. 2003. Determination
of the redox potentials and electron transfer properties of the FAD- and FMN-binding domains of the
human oxidoreductase NRI. Eur J Biochem. 270: 1164–75.
Fogelholm, M., Ruokonen, I., Laakso, J.T., Vuorimaa, T. and Himberg, J.J. 1993. Lack of association between
indices of vitamin B1, B2, and B6 status and exercise-induced blood lactate in young adults. Int J Sport
Nutr. 3: 165–76.
Food and Nutrition Board. 2000. Institute of Medicine. Riboflavin, Chapter 5. In Dietary Reference Intakes for
Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin and Choline, 87.
Washington, DC: National Academy Press.
Foraker, A.B., Khantwal, C.M. and Swaan, P.W. 2003. Current perspectives on the cellular uptake and traffick-
ing of riboflavin. Adv Drug Deliv Rev. 55: 1467–83.
Forrest, K.Y. and Stuhldreher, W.L. 2011. Prevalence and correlates of vitamin D deficiency in US adults. Nutr
Res. 31: 48–54.
Fowler, B. 2001. The folate cycle and disease in humans. Kidney Int Suppl. 78: S221–9.
Fratelli, M., Goodwin, L.O., Ørom, U.A., Lombardi, S., Tonelli, R., Mengozzi, M. and Ghezzi, P. 2005. Gene
expression profiling reveals a signaling role of glutathione in redox regulation. Proc Natl Acad Sci USA.
102: 13998–4003.
Fromer, C.H. and Klintworth, G.K. 1975. An evaluation of the role of leukocytes in the pathogenesis of experi-
mentally induced corneal vascularization. Am J Pathol. 79: 537–54.
Frosst, P., Blom, H.J., Milos, R., Goyette, P., Sheppard, C.A., Matthews, R.G., Boers, G.J.H., den Heijer M.,
Kluijtmans, L.A.J., van den Heuve, L.P. and Rozen R. 1995. A candidate genetic risk factor for vascular
disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet. 10: 111–3.
Fuchs, K.R., Shekels, L.L. and Bernlohr, D.A. 1992. Analysis of the ACP1 gene product: classification as an
FMN phosphatase. Biochem Biophys Res Commun. 189: 1598–605.
Fujii, K., Galivan, J.H. and Hennekens, F.M. 1977. Activation of methionine synthase: further characterization
of flavoprotein system. Arch Biochem Biophys. 178: 662–70.
Fukuwatari, T., Yoshida, E., Takahashi, K. and Shibata, K. 2010. Effect of fasting on the urinary excretion of
water-soluble vitamins in humans and rats. J Nutr Sci Vitaminol (Tokyo). 56: 19–26.
Furuya, E.M., Warthesen, J.J. and Labuza, T.P. 1984. Effects of water activity, light intensity and physical struc-
ture of food on the kinetics of riboflavin photodegradation. J Food Sci. 49: 526–8.
Gadda, G. 2012. Oxygen activation in flavoprotein oxidases: the importance of being positive. Biochemistry.
51: 2662–9.
Gagnon, C., Lu, Z.X., Magliano, D.J., Dunstan, D.W., Shaw, J.E., Zimmet, P.Z., Sikaris, K., Ebeling, P.R. and
Daly, R.M. 2012. Low serum 25-hydroxyvitamin D is associated with increased risk of the development
of the metabolic syndrome at five years: results from a national, population-based prospective study (The Please provide
Australian Diabetes, Obesity and Lifestyle Study: AusDiab). J Clin Endocrinol Metab. Mar 22. [Epub update on the
publication
ahead of print]). status.
Galperin, G., Berra, M., Tau, J., Boscaro, G., Zarate, J. and Berra, A. 2012. Treatment of fungal keratitis from
fusarium infection by corneal cross-linking. Cornea. 31: 176–80.
Galvin, M.A., Kiely, M. and Flynn, A. 2003. Impact of ready-to-eat breakfast cereal (RTEBC) consumption on
adequacy of micronutrient intakes and compliance with dietary recommendations in Irish adults. Public
Health Nutr. 6: 351–63.
Ganji, G. and Kafai, M.R. 2004. Frequent consumption of milk, yogurt, cold breakfast cereals, peppers and
cruciferous vegetables and intakes of dietary folate and riboflavin but not vitamins B12 and B6 are
inversely associated with serum total homocysteine concentrations in the US population. Am J Clin Nutr.
80: 1500–7.
Gariballa, S. and Ullegaddi, R. 2007. Riboflavin status in acute ischaemic stroke. Eur J Clin Nutr. 61: 1237–40.
Geary, T.G., Divo, A.A. and Jensen, J.B. 1985. Nutritional requirements of Plasmodium falciparum in culture.
II. Effects of antimetabolites in a semi-defined medium. J Protozool. 32: 65–9.
Gerards, M., van den Bosch, B.J., Danhauser, K., Serre, V., van Weeghel, M., Wanders, R.J., Nicolaes, G.A.,
Sluiter, W., Schoonderwoerd, K., Scholte, H.R., Prokisch, H., Rötig, A., de Coo, I.F. and Smeets,
H.J. 2011. Riboflavin-responsive oxidative phosphorylation complex I deficiency caused by defective
ACAD9: new function for an old gene. Brain. 134(Pt 1): 210–9.
Gherasim, C.G., Zaman, U., Raza, A. and Banerjee, R. 2008. Impeded electron transfer from a pathogenic
FMN domain mutant of methionine synthase reductase and its responsiveness to flavin supplementation.
Ghiasi, P., Hosseinkhani, S., Noori, A., Nafissi, S. and Khajeh, K. 2012. Mitochondrial complex I deficiency
and ATP/ADP ratio in lymphocytes of amyotrophic lateral sclerosis patients. Neurol Res. 34: 297–303.
K15043_C006.indd 251 4/10/2013 4:57:05 PM

Ghisla, S. and Thorpe, C. 2004. Acyl-CoA dehydrogenases. A mechanistic overview. Eur J Biochem. 271:
494–508.
Gill, S., Stevenson, J., Kristiana, I. and Brown, A.J. 2011. Cholesterol-dependent degradation of squalene
monooxygenase, a control point in cholesterol synthesis beyond HMG-CoA reductase. Cell Metab. 13:
260–73.
Giulivi, C., Zhang, Y.F., Omanska-Klusek, A., Ross-Inta, C., Wong, S., Hertz-Picciotto, I., Tassone, F. and
Pessah, I.N. 2010. Mitochondrial dysfunction in autism. JAMA. 304: 2389–96.
Glossmann, H.H. 2010. Origin of 7-dehydrocholesterol (provitamin D) in the skin. J Invest Dermatol. 130:
2139–41.
Goebel, K.M. and Goebel, F.D. 1972. Hemolytic anemia and pancytopenia in glutathione reductase deficiency:
further experience with riboflavin. Acta Haematol. 47: 292–6.
Goldsmith, G.A. 1975. Riboflavin deficiency. In Riboflavin, ed. R.S. Rivlin, 221–42. New York: Plenum Press.
Goodrich, R.P. 2000. The use of riboflavin for the inactivation of pathogens in blood products. Vox Sang. 78
Suppl 2: 211–5.
Graham, I.M., Daly, L.E., Refsum, H.M., Robinson, K., Brattström, L.E., Ueland, P.M., Palma-Reis, R.J.,
Boers, G.H.J., Sheahan, R.G., Israelsson, B., Uiterwaal, C.S., Meleady, R., McMaster, D., Verhoef, P.,
Witteman, J., Rubba, P., Bellet, H., Wautrecht, J.C., de Valk, H.W., Sales Lúis, A.C., Parrot-Rouland,
F.M., Tan, K.S., Higgins, I., Garcon, D., Medrano, M.J., Candito, M., Evans, A.E. and Andria, G. 1997.
Plasma homocysteine as a risk factor for vascular disease. The European Concerted Action Project.
JAMA. 277: 1775–81.
Granjeiro, J.M., Ferreira, C.V., Granjeiro, P.A., Da Silva, C.C., Taga, E.M., Volpe, P.L. and Aoyama, H. 2002.
Inhibition of bovine kidney low molecular mass phosphotyrosine protein phosphatase by uric acid.
J Enzyme Inhib Med Chem. 17: 345–50.
Granjeiro, J.M., Ferreira, C.V., Jucá, M.B., Taga, E.M. and Aoyama, H. 1997. Bovine kidney low molecular
weight acid phosphatase: FMN-dependent kinetics. Biochem Mol Biol Int. 41: 1201–8.
Green, J. and Paget, M.S. 2004. Bacterial redox sensors. Nat Rev Microbiol. 2: 954–66.
Gregersen, N. 1985. Riboflavin-responsive defects of beta-oxidation. J Inherit Metab Dis. 8 Suppl 1: 65–9.
Groves, J.T. and Wang, C.C.-Y. 2000. Nitric oxide synthase: models and mechanisms. Curr Opin Chem Biol.
4:687–95.
Gu, J., Weng, Y., Zhang, Q.Y., Cui, H., Behr, M., Wu, L., Yang, W., Zhang, L. and Ding, X. 2003. Liver-specific
deletion of the NADPH-cytochrome P450 reductase gene: impact on plasma cholesterol homeostasis and
the function and regulation of microsomal cytochrome P450 and heme oxygenase. J Biol Chem. 278:
25895–901.
Guengerich, F.P. 2003. Cytochromes P450, drugs, and diseases. Mol Interv. 3: 194–204.
Guenther, B.D., Sheppard, C.A., Tran, P., Rozen, R., Matthews, R.G. and Ludwig, M.L. 1999. The structure
and properties of methylenetetrahydrofolate reductase from Escherichia coli suggest how folate amelio-
rates human hyperhomocysteinemia. Nat Struct Biol. 6: 359–65.
Gupta, N. and Porter, T.D. 2001. Garlic and garlic-derived compounds inhibit human squalene monooxygen-
ase. J Nutr. 131:1662–67.
Gupta, R.P., Patrick, K. and Bell, N.H. 2007. Mutational analysis of CYP27A1: assessment of 27-hydroxyl-
ation of cholesterol and 25-hydroxylation of vitamin D. Metabolism. 56: 1248–55.
Habl, G., Zink, M., Petroianu, G., Bauer, M., Schneider-Axmann, T., von Wilmsdorff, M., Falkai, P., Henn, F.A.
and Schmitt, A. 2009. Increased D-amino acid oxidase expression in the bilateral hippocampal CA4 of
schizophrenic patients: a post-mortem study. J Neural Trans. 116: 1657–65.
Hafner, M., Rezen, T. and Rozman, D. 2011. Regulation of hepatic cytochromes p450 by lipids and cholesterol.
Curr Drug Metab. 12: 173–85.
Hamdane, D., Xia, C., Im, S.C., Zhang, H., Kim, J.J. and Waskell, L. 2009. Structure and function of an
NADPH-cytochrome P450 oxidoreductase in an open conformation capable of reducing cytochrome
P450. J Biol Chem. 284: 11374–84.
Hannun, Y.A. and Obeid, L.M. 2008. Principles of bioactive lipid signalling: lessons from sphingolipids. Nat
Rev Mol Cell Biol. 9: 139–50.
Hasegawa, Y. and Yagi, K. 1975. Electron microscopic study on the lens of riboflavin-deficient albino rat.
J Nutr Sci Vitaminol (Tokyo). 21: 395–401.
Hashimoto, H., Igarashi, N., Yachie, A., Miyawaki, T. and Sato, T. 1994. The relationship between serum lev-
els of interleukin-6 and thyroid hormone in children with acute respiratory infection. J Clin Endocrinol
Metab. 78: 288–91.
Hassan-Abdallah, A., Bruckner, R.C., Zhao, G. and Jorns, M.S. 2005. Biosynthesis of covalently bound flavin: iso-
lation and in vitro flavinylation of the monomeric sarcosine oxidase apoprotein. Biochemistry. 44: 6452–62.
K15043_C006.indd 252 4/10/2013 4:57:05 PM

Haynes, R.K., Cheu, K.W., Tang, M.M., Chen, M.J., Guo, Z.F., Guo, Z.H., Coghi, P. and Monti, D. 2011.
Reactions of antimalarial peroxides with each of leucomethylene blue and dihydroflavins: flavin reduc-
tase and the cofactor model exemplified. Chem Med Chem. 6: 279–91.
Hellerman, L., Lindsay, A. and Bovarnick, M.R. 1946. Flavoenzyme catalysis; inhibition of d-amino acid oxi-
dase by competition with flavin-adenine-dinucleotide of atabrine (quinacrine); quinine, and certain other
compounds. J Biol Chem. 163: 553–70.
Henriques, B.J., Olsen, R.K., Bross, P. and Gomes, C.M. 2010. Emerging roles for riboflavin in functional
rescue of mitochondrial β-oxidation flavoenzymes. Curr Med Chem. 17: 3842–54.
Heuts, D.P., Scrutton, N.S., McIntire, W.S. and Fraaije, M.W. 2009. What’s in a covalent bond? On the role and
formation of covalently bound flavin cofactors. FEBS J. 276: 3405–27.
Higa, A. and Chevet, E. 2012. Redox signaling loops in the unfolded protein response. Cell Signal. 24: 1548–55.
Hill, M.J. 1997. Intestinal flora and endogenous vitamin synthesis. Eur J Cancer Prev. 6 Suppl 1: S43–5.
Hirano, G., Izumi, H., Yasuniwa, Y., Shimajiri, S., Ke-Yong, W., Sasagiri, Y., Kusaba, H., Matsumoto, K.,
Hasegawa, T., Akimoto, M., Akashi, K. and Kohno, K. 2011. Involvement of riboflavin kinase expression
in cellular sensitivity against cisplatin. Int J Oncol. 38: 893–902.
Hirst, J. 2009. Towards the molecular mechanism of respiratory complex I. Biochem J. 425: 327–39.
Ho, G., Yonezawa, A., Masuda, S., Inui, K., Sim, K.G., Carpenter, K., Olsen, R.K., Mitchell, J.J., Rhead, W.J.,
Peters, G. and Christodoulou, J. 2011. Maternal riboflavin deficiency, resulting in transient neonatal-
onset glutaric aciduria Type 2, is caused by a microdeletion in the riboflavin transporter gene GPR172B.
Hum Mutat. 32: E1976–84.
Hoane, M.R., Wolyniak, J.G. and Akstulewicz, S.L. 2005. Administration of riboflavin improves behavioral
outcome and reduces edema formation and glial fibrillary acidic protein expression after traumatic brain
injury. J Neurotrauma. 22: 1112–22.
Hofmann, B., Hecht, H.J. and Flohé, L. 2002. Peroxiredoxins. Biol Chem. 383: 347–64.
Holladay, S.R., Yang, Z., Kennedy, M.D., Leamon, C.P., Lee, R.J., Jayamani, M., Mason, T. and Low, P.S.
1999. Riboflavin-mediated delivery of a macromolecule into cultured human cells. Biochim Biophys
Acta. 1426: 195–204.
Hoppel, C.L. and Tandler, B. 1975. Riboflavin and mouse hepatic cell structure and function. Mitochondrial
oxidative metabolism in severe deficiency states. J Nutr. 105: 562–70.
Hoppel, C.L. and Tandler, B. 1990. Riboflavin deficiency. Prog Clin Biol Res. 321: 233–48.
Horigan, G., McNulty, H., Ward, M., Strain, J.J., Purvis, J. and Scott, J.M. 2010. Riboflavin lowers blood
pressure in cardiovascular disease patients homozygous for the 677CàT polymorphism in MTHFR.
J Hypertens. 28:478–86.
Horio, M., Kohno, M., Fujita, Y., Ishima, T., Inoue, R., Mori, H. and Hashimoto, K. 2011. Levels of D-serine
in the brain and peripheral organs of serine racemase (Srr) knock-out mice. Neurochem Int. 59: 853–9.
Horvath, R. 2012. Update on clinical aspects and treatment of selected vitamin-responsive disorders II (ribofla- Please provide
vin and CoQ(10)). J Inherit Metab Dis. Jan 10. [Epub ahead of print]. update on the
publication
Huang, R., Kim, H.J. and Min, D.B. 2006. Photosensitizing effect of riboflavin, lumiflavin, and lumichrome on status.
the generation of volatiles in soy milk. J Agric Food Chem. 54: 2359–64.
Huang, S.N. and Swaan, P.W. 2000. Involvement of a receptor-mediated component in cellular translocation of
riboflavin. J Pharmacol Exp Ther. 294: 117–25.
Huang, S.N., Phelps, M.A. and Swaan, P.W. 2003. Involvement of endocytic organelles in the subcellular traf-
ficking and localization of riboflavin. J Pharmacol Exp Ther. 306: 681–7.
Huerta, C., Borek, D., Machius, M., Grishin, N.V. and Zhang, H. 2009. Structure and mechanism of a eukary-
otic FMN adenylyltransferase. J Mol Biol. 389: 388–400.
Hultdin, J., Van Guelpen, B., Winkvist, A., Hallmans, G., Weinehall, L., Stegmayr, B. and Nilsson, T.K. 2011.
Prospective study of first stroke in relation to plasma homocysteine and MTHFR 677C>T and 1298A>C
genotypes and haplotypes – evidence for an association with hemorrhagic stroke. Clin Chem Lab Med.
49: 1555–62.
Hustad, S., Ueland, P.M., Vollset, S.E., Zhang, Y., Bjorke-Monsen, A.L. and Schneede, J. 2000. Riboflavin as a
determinant of plasma total homocysteine: effect modification by the methylenetetrahydrofolate C677T
polymorphism. Clin Chem. 46: 1065–71.
Hwang, C., Sinskey, A.J. and Lodish, H.F. 1992. Oxidized redox state of glutathione in the endoplasmic reticu-
lum. Science. 257: 1496–502.
Ifergan, I., Goler-Baron, V. and Assaraf, Y.G. 2009. Riboflavin concentration within ABCG2-rich extracellular vesi-
cles is a novel marker for multidrug resistance in malignant cells. Biochem Biophys Res Commun. 380: 5–10.
Ifergan, I., Scheffer, G.L. and Assaraf, Y.G. 2005. Novel extracellular vesicles mediate an ABCG2-dependent
anticancer drug sequestration and resistance. Cancer Res. 65: 10952–58.
K15043_C006.indd 253 4/10/2013 4:57:05 PM

Innis, W.S., McCormick, D.B. and Merrill, A.H., Jr. 1985. Variations in riboflavin binding by human plasma:
identification of immunoglobulins as the major proteins responsible. Biochem Med. 34: 151–65.
Innis, W.S., Nixon, D.W., Murray, D.R., McCormick, D.B. and Merrill, A.H., Jr. 1986. Immunoglobulins asso-
ciated with elevated riboflavin binding by plasma from cancer patients. Proc Soc Exp Biol Med. 181:
237–41.
Iqbal, M., Audette, M.C., Petropoulos, S., Gibb, W. and Matthews, S.G. 2012. Placental drug transporters and
their role in fetal protection. Placenta. 33: 137–42.
Isenberg, I. and Szent-Györgyi, A. 1958. Free radical formation in riboflavin complexes. Proc Natl Acad Sci
USA. 44: 857–62.
Itokawa, Y., Kimura, M., Nishino, K., Mino, M., Kitagawa, M., Matsuoka, M. and Otsuka, H. 1992. Blood
levels and urinary excretion of thiamin and riboflavin during oral administration of multivitamin tablets
to healthy adults. J Nutr Sci Vitaminol (Tokyo). Spec No: 438–41.
Jacques, P.F. 1997. Nutritional antioxidants and prevention of age-related eye disease. In Antioxidants and
Disease Prevention, ed. H.S. Garewal, 149–77. Boca Raton, FL: CRC Press.
Jacques, P.F., Bostom, A.G., Williams, R.R., Ellison, R.C., Eckfeldt, J.H., Rosenberg, I.H., Selhub, J. and
Rozen, R. 1996. Relation between folate status, a common mutation in methylenetetrahydrofolate reduc-
tase and plasma homocysteine concentration. Circulation. 93: 7–9.
Jacques, P.F., Taylor, A., Moeller, S., Hankinson, S.E., Rogers, G., Tung, W., Ludovico, J., Willett, W.C. and
Chylack, L.T., Jr. 2005. Long-term nutrient intake and 5-year change in nuclear lens opacities. Arch
Ophthalmol. 123: 517–26.
Janin, J. and Chothia, C. 1978. Role of hydrophobicity in the binding of coenzymes. Biochemistry. 17: 2943–7.
Johnson, T., Ouhtit, A., Gaur, R., Fernando, A., Schwarzenberger, P., Su, J., Ismail, M.F., El-Sayyad, H.I.,
Karande, A., Elmageed, Z.A., Rao, P. and Raj, M. 2009. Biochemical characterization of riboflavin car-
rier protein (RCP) in prostate cancer. Front Biosci. 14: 3634–40.
Johnson, W.D. and Storts, R.W. 1988. Peripheral neuropathy associated with dietary riboflavin deficiency in the
chicken. I. Light microscopic study. Vet Pathol. 25: 9–16.
Jonker, J.W., Merino, G., Musters, S., van Herwaarden, A.E., Bolscher, E., Wagenaar, E., Mesman, E., Dale,
T.C. and Schinkel, A.H. 2005. The breast cancer resistance protein BCRP (ABCG2) concentrates drugs
and carcinogenic xenotoxins into milk. Nat Med. 11: 127–9.
Jortner, B.S., Cherry, J., Lidsky, T.I., Manetto, C. and Shell, L. 1987. Peripheral neuropathy of dietary riboflavin
deficiency in chickens. J Neuropathol Exp Neurol. 46: 544–55.
Joshi, P.C., Carraro, C. and Pathak, M.A. 1987. Involvement of reactive oxygen species in the oxidation of
tyrosine and dopa to melanin and in skin tanning. Biochem Biophys Res Commun. 142: 265–74.
Jusko, W.J. and Levy, G. 1967. Effect of probenecid on riboflavin absorption and excretion in man. J Pharm
Sci. 56: 1145–9.
Jusko, W.J., Levy, G., Yaffe, S.J. and Allen, J.E. 1971. Riboflavin absorption in children with biliary obstruc-
tion. Am J Dis Child. 121: 48–52.
Kaikai, P. and Thurnham, D.I. 1983. The influence of riboflavin deficiency on Plasmodium berghei infection in
rats. Trans R Soc Trop Med Hyg. 77: 680–6.
Kalhan, S.C. and Marczewski, S.E. 2012. Methionine, homocysteine, one carbon metabolism and fetal growth.
Rev Endocr Metab Disord. 13: 109–19.
Kamaev, P., Friedman, M.D., Sherr, E. and Muller, D. 2012. Photochemical kinetics of corneal cross-linking
with riboflavin. Invest Ophthalmol Vis Sci. 53: 2360–7.
Kansara, V., Pal, D., Jain, R. and Mitra, A.K. 2005. Identification and functional characterization of riboflavin
transporter in human-derived retinoblastoma cell line (Y-79): mechanisms of cellular uptake and translo-
cation. J Ocul Pharmacol Ther. 21: 275–87.
Kao, Y.-T., Saxena, C., He, T.-F., Guo, L., Wang, L., Sancar, A. and Zhong, D. 2008. Ultrafast dynamics of
flavins in five redox states. J Am Chem Soc. 130: 13132–9.
Karande, A.A., Sridhar, L., Gopinath, K.S. and Adiga, P.R. 2001. Riboflavin carrier protein: a serum and tissue
marker for breast carcinoma. Int J Cancer. 95: 277–81.
Karrer, P., Scopp, K. and Benz, F. 1935. Synthesis of flavins IV. Helv Chim Acta. 18: 426–9.
Kasai, S., Nakano, H., Maeda, K. and Matsui, K. 1990. Purification, properties, and function of flavokinase
from rat intestinal mucosa. J Biochem. 107: 298–303.
Kashiwabuchi, R.T., Carvalho, F.R., Khan, Y.A., de Freitas, D., Foronda, A.S., Hirai, F.E., Campos, M.S. and
McDonnell, P.J. 2011. Assessing efficacy of combined riboflavin and UV-A light (365 nm) treatment of
acanthamoeba trophozoites. Invest Ophthalmol Vis Sci. 52: 9333–8.
Kawazoe, T., Park, H.K., Iwana, S., Tsuge, H. and Fukui, K. 2007. Human D-amino acid oxidase: an update
and review. Chem Rec. 7: 305–15.
K15043_C006.indd 254 4/10/2013 4:57:05 PM

Kenney, W.C., Edmondson, D.E., Singer, T.P., Nakagawa, H., Asano, A. and Sato, R. 1976. Identification of
the covalently bound flavin of L-gulono-gamma-lactone oxidase. Biochem Biophys Res Commun. 71:
1194–200.
Kim, M.J., Kim, H.J., Kim, J.M., Kim, B., Han, S.H. and Cha, G.S. 1995. Homogeneous assays for riboflavin medi-
ated by the interaction between enzyme–biotin and avidin–riboflavin conjugates. Anal Biochem. 231: 400–6.
Kim, S., Sideris, D.P., Sevier, C.S. and Kaiser, C.A. 2012. Balanced Ero1 activation and inactivation establishes
ER redox homeostasis. J Cell Biol. 196: 713–25.
Kimura, M., Umegaki, K., Higuchi, M., Thomas, P. and Fenech, M. 2004. Methylene tetrahydrofolate reduc-
tase C677T polymorphism, folic acid and riboflavin are important determinants of genomic stability in
cultured human lymphocytes. J Nutr. 134: 48–56.
Knobloch, E. and Hodr, R. 1989. Metabolism of bilirubin and riboflavin in the course of phototherapy for
hyperbilirubinaemia in the newborns. Czech Med. 12: 134–44.
Knobloch, E., Mandys, F. and Hodr, R. 1988. Study of the mechanism of the photochemical oxidation of bili-
rubin by high-performance liquid chromatography. J Chromatogr. 428: 255–63.
Koaio, J. 1966. Studies on flavins in organic solvents—ii. photodecomposition of riboflavin in the presence of
oxygen. Photochem Photobiol. 5: 55–62.
Kodali, V.K. and Thorpe, C. 2010. Oxidative protein folding and the quiescin-sulfhydryl oxidase family of
flavoproteins. Antioxid Redox Signal. 13: 1217–30.
Kozar, R.A., Weibel, C.J., Cipolla, J., Klein, A.J., Haber, M.M., Abedin, M.Z. and Trooskin, S.Z. 2000.
Antioxidant enzymes are induced during recovery from acute lung injury. Crit Care Med. 28: 2486–91.
Kranendonk, M., Marohnic, C.C., Panda, S.P., Duarte, M.P., Oliveira, J.S., Masters, B.S. and Rueff, J. 2008.
Impairment of human CYP1A2-mediated xenobiotic metabolism by Antley-Bixler syndrome variants of
cytochrome P450 oxidoreductase. Arch Biochem Biophys. 475: 93–9.
Kuhn, R., Reinemund K., Kaltschmitt H., Strobele, R. and Trischmann, H. 1935. Synthetisches 6,7-dimethyl-
9-d-riboflavin. Naturwissenschaften. 23: 260.
Kumar, C.K., Yanagawa, N., Ortiz, A. and Said, H.M. 1998. Mechanism and regulation of riboflavin uptake by
human renal proximal tubule epithelial cell line HK-2. Am J Physiol. 274(1 Pt 2): F104–10.
Kumar, J., Muntner, P., Kaskel, F.J., Hailpern, S.M. and Melamed, M.L. 2009. Prevalence and associations of
25-hydroxyvitamin D deficiency in US children: NHANES 2001–2004. Pediatrics. 124: e362–70.
Kumar, N. 2007. Nutritional neuropathies. Neurol Clin. 25: 209–55.
Laborie, S., Lavoie, J.C. and Chessex, P. 1998. Paradoxical role of ascorbic acid and riboflavin in solutions
of total parenteral nutrition: implication in photoinduced peroxide generation. Pediatr Res. 43: 601–6.
Laden, B.P., Tang, Y. and Porter, T.D. 2000. Cloning, heterologous expression, and enzymological characteriza-
tion of human squalene monooxygenase. Arch Biochem Biophys. 374: 381–8.
Lamarre, S.G., Molloy, A.M., Reinke, S.N., Sykes, B.D., Brosnan, M.E. and Brosnan, J.T. 2012. Formate can
differentiate between hyperhomocysteinemia due to impaired remethylation and impaired transsulfura-
tion. Am J Physiol Endocrinol Metab. 302: E61–7.
Lane, M. and Alfrey, C.P., Jr. 1965. The anemia of human riboflavin deficiency. Blood. 25: 432–42.
Lanska, D.J. 2010. Chapter 30: historical aspects of the major neurological vitamin deficiency disorders: the
water-soluble B vitamins. Handb Clin Neurol. 95: 445–76.
Lee, J.M., Calkins, M.J., Chan, K., Kan, Y.W. and Johnson, J.A. 2003. Identification of the NF-E2-related
factor-2-dependent genes conferring protection against oxidative stress in primary cortical astrocytes
using oligonucleotide microarray analysis. J Biol Chem. 278: 12029–38.
Lee, R.S. and Ford, H.C. 1988. 5′-Nucleotidase of human placental trophoblastic microvilli possesses cobalt-
stimulated FAD pyrophosphatase activity. J Biol Chem. 263: 14878–83.
Lee, S.S. and McCormick, D.B. 1983. Effect of riboflavin status on hepatic activities of flavin-metabolizing
enzymes in rats. J Nutr. 113: 2274–9.
Lee, S.S. and McCormick, D.B. 1985. Thyroid hormone regulation of flavocoenzyme biosynthesis. Arch
Biochem Biophys. 237: 197–201.
Leferink, N.G., Heuts, D.P., Fraaije, M.W. and van Berkel, W.J. 2008. The growing VAO flavoprotein family.
Arch Biochem Biophys. 474: 292–301.
Lei, K., Townsend, D.M. and Tew, K.D. 2008. Protein cysteine sulfinic acid reductase (sulfiredoxin) as a regula-
tor of cell proliferation and drug response. Oncogene. 27: 4877–87.
Li, L. and Porter, T.D. 2007. Hepatic cytochrome P450 reductase-null mice reveal a second microsomal reduc-
tase for squalene monooxygenase. Arch Biochem Biophys. 461: 76–84.
Lian, L.Y., Widdowson, P., McLaughlin, L.A. and Paine, M.J. 2011. Biochemical comparison of Anopheles
gambiae and human NADPH P450 reductases reveals different 2′-5′-ADP and FMN binding traits. PLoS
One. 6: e20574.
K15043_C006.indd 255 4/10/2013 4:57:05 PM

Liang, W.C., Ohkuma, A., Hayashi, Y.K., López, L.C., Hirano, M., Nonaka, I., Noguchi, S., Chen, L.H., Jong,
Y.J. and Nishino, I. 2009. ETFDH mutations, CoQ10 levels, and respiratory chain activities in patients
with riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency. Neuromuscul Disord. 19:
212–6.
Lin, J., Diamanduros, A., Chowdhury, S.A., Scelsa, S., Latov, N. and Sadiq, S.A. 2009. Specific electron trans-
port chain abnormalities in amyotrophic lateral sclerosis. J Neurol. 256: 774–82.
Lin, L.F. 1980. Roles of phospholipid and detergent in soluble protein activation of squalene epoxidase.
Linetsky, M., Hill, J.M., Chemoganskiy, V.G., Hu, F. and Ortwerth, B.J. 2003. Studies on the mechanism of
the UVA light-dependent loss of glutathione reductase activity in human lenses. Invest Ophthalmol Vis
Sci. 44: 3920–6.
Lo, C.S. 1984. Riboflavin status of adolescents in southern China. Average intake of riboflavin and clinical
findings. Med J Aust. 141: 635–7.
Loeffler, M., Daugas, E., Susin, S.A., Zamzami, N., Metivier, D., Nieminen, A.L., Brothers, G., Penninger,
J.M. and Kroemer, G. 2001. Dominant cell death induction by extramitochondrially targeted apoptosis-
inducing factor. FASEB J. 15: 758–67.
López, C., Saravia, C., Gomez, A., Hoebeke, J. and Patarroyo, M.A. 2010. Mechanisms of genetically-based
resistance to malaria. Gene. 467: 1–12.
Lubos, E., Loscalzo, J. and Handy, D.E. 2011. Glutathione peroxidase-1 in health and disease: from molecular
mechanisms to therapeutic opportunities. Antioxid Redox Signal. 15: 1957–97.
Lucas, A. and Bates, C. 1984. Transient riboflavin depletion in preterm infants. Arch Dis Child. 59: 837–41.
MacDonald, M.J. and Brown, L.J. 1996. Calcium activation of mitochondrial glycerol phosphate dehydroge-
nase restudied. Arch Biochem Biophys. 326: 79–84.
Macheroux, P., Ghisla, S., Sanner, C., Rüterjans, H. and Müller, F. 2005. Reduced flavin: NMR investigation of
N(5)-H exchange mechanism, estimation of ionisation constants and assessment of properties as biologi-
cal catalyst. BMC Biochemistry. 6(26): 1–11.
Macheroux, P., Kappes, B. and Ealick, S.E. 2011. Flavogenomics—a genomic and structural view of flavin-
dependent proteins. FEBS J. 278: 2625–34.
MacLachlan, I., Nimpf, J. and Schneider, W.J. 1994. Avian riboflavin binding protein binds to lipoprotein
receptors in association with vitellogenin. J Biol Chem. 269: 24127–32.
MacLachlan, I., Nimpf, J., White, H.B., 3rd and Schneider, W.J. 1993. Riboflavinuria in the rd chicken. 5′-splice
site mutation in the gene for riboflavin-binding protein. J Biol Chem. 268: 23222–26.
MacLennan, S.C., Wade, F.M., Forrest, K.M., Ratanayake, P.D., Fagan, E. and Antony, J. 2008. High-dose
riboflavin for migraine prophylaxis in children: a double-blind, randomized, placebo-controlled trial.
J Child Neurol. 23: 1300–4.
Maehashi, K., Matano, M., Nonaka, M., Udaka, S. and Yamamoto, Y. 2008. Riboflavin-binding protein is a
novel bitter inhibitor. Chem Senses. 33: 57–63.
Mandon, E.C., Ehses, I., Rother, J., van Echten, G. and Sandhoff, K. 1992. Subcellular localization and
membrane topology of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine
N-acyltransferase in mouse liver. J Biol Chem. 267: 11144–8.
Mannion, C.A., Gray-Donald, K., Johnson-Down, L. and Koski, K.G. 2007. Lactating women restricting milk
are low on select nutrients. J Am Coll Nutr. 26: 149–55.
Mansjö, M. and Johansson, J. 2011. The riboflavin analog roseoflavin targets an FMN-riboswitch and blocks
Listeria monocytogenes growth, but also stimulates virulence gene-expression and infection. RNA Biol.
8: 674–80.
Mansouri, A. and Winterhalter, K. 1973. Nonequivalence of chains in hemoglobin oxidation. Biochemistry.
12:4946–49.
Månsson, M.O., Mattiasson, B., Gestrelius, S. and Mosbach, K. 1976. Continuous regeneration of NAD(P)+ by
flavins covalently bound to sepharose. Biotechnol Bioeng. 18: 1145–59.
Manthey, K.C., Chew, Y.C. and Zempleni, J. 2005. Riboflavin deficiency impairs oxidative folding and secre-
tion of apolipoprotein B-100 in HepG2 cells, triggering stress response systems. J Nutr. 135: 978–82.
Manthey, K.C., Rodriguez-Melendez, R., Hoi, J.T. and Zempleni, J. 2006. Riboflavin deficiency causes protein
and DNA damage in HepG2 cells, triggering arrest in G1 phase of the cell cycle. J Nutr Biochem. 17:
250–6.
Marschner, S. and Goodrich, R. 2011. Pathogen reduction technology treatment of platelets, plasma and whole
blood using riboflavin and UV light. Transfus Med Hemother. 38: 8–18.
Marschner, S., Fast, L.D., Baldwin, W.M., 3rd, Slichter, S.J. and Goodrich, R.P. 2010. White blood cell inacti-
vation after treatment with riboflavin and ultraviolet light. Transfusion. 50: 2489–98.
K15043_C006.indd 256 4/10/2013 4:57:05 PM

Martin, C.B., Tsao, M., Hadad, C.M. and Platz, M.S. 2002. The reaction of triplet flavin with indole. A study
of the cascade of reactive intermediates using density functional theory and time resolved infrared spec-
troscopy. J Am Chem Soc. 124: 7226–34.
Martínez-Frías, M.L. 2008. The biochemical structure and function of methylenetetrahydrofolate reductase
provide the rationale to interpret the epidemiological results on the risk for infants with Down syndrome.
Am J Med Genet A. 146A: 1477–82.
Martini, M.C., Lampe, J.W., Slavin, J.L. and Kurzer, M.S. 1994. Effect of the menstrual cycle on energy and
nutrient intake. Am J Clin Nutr. 60: 895–9.
Mason, C.W., D’Souza, V.M., Bareford, L.M., Phelps, M.A., Ray, A. and Swaan, P.W. 2006. Recognition,
co-internalization, and recycling of an avian riboflavin carrier protein in human placental trophoblasts.
J Pharmacol Exp Ther. 317: 465–72.
Massey, V. 2000. The chemical and biological versatility of riboflavin. Biochem Soc Trans. 28: 283–96.
Mayersohn, M., Feldman, S. and Gibaldi, M. 1969. Bile salt enhancement of riboflavin and flavin mononucleo-
tide absorption in man. J Nutr. 98: 288–96.
Mazzio, E.A. and Soliman, K.F. 2004. Effects of enhancing mitochondrial oxidative phosphorylation with reduc-
ing equivalents and ubiquinone on 1-methyl-4-phenylpyridinium toxicity and complex I-IV damage in
neuroblastoma cells. Biochem Pharmacol. 67: 1167–84. Erratum in Biochem Pharmacol. 67: 1809, 2004.
McCabe, R., Bullenkamp, J., Hansson, L., Lauber, C., Martinez-Leal, R., Rössler, W., Salize, H.J., Svensson,
B., Torres-Gonzalez, F., van den Brink, R., Wiersma, D. and Priebe, S. 2012. The therapeutic relationship
and adherence to antipsychotic medication in schizophrenia. PLoS One. 7: e36080.
McCollum, E.V. and Kennedy, C. 1916. The dietary factors operating in the production of polyneuritis. J Biol
Chem. 24: 491–5.
McCormick, D.B. 1990. Riboflavin. In Present Knowledge in Nutrition, 6th ed., ed. M.L. Brown, 146.
Washington, DC: International Life Sciences Institute.
McMahon, M., Itoh, K., Yamamoto, M., Chanas, S.A., Henderson, C.J., McLellan, L.I., Wolf, C.R., Cavin,
C. and Hayes, J.D. 2001. The Cap‘n’Collar basic leucine zipper transcription factor Nrf2 (NF-E2 p45-
related factor 2) controls both constitutive and inducible expression of intestinal detoxification and glu-
tathione biosynthetic enzymes. Cancer Res. 61: 3299–307.
McNulty, H., McKinley, M.C., Wilson, B., McPartlin, J., Strain, J.J., Weir, D.G. and Scott, J.M. 2002. Impaired
functioning of thermolabile methylenetetrahydrofolate reductase is dependent on riboflavin status: impli-
cations for riboflavin requirements. Am J Clin Nutr. 76: 436–41.
Meints, C.E., Gustafsson, F.S., Scrutton, N.S. and Wolthers, K.R. 2011. Tryptophan 697 modulates hydride
and interflavin electron transfer in human methionine synthase reductase. Biochemistry. 50: 11131–42.
Meisel, P., Amon, I., Hüller, H. and Jährig, K. 1980. Effect of theophylline on the riboflavin-sensitized photo-
degradation of bilirubin in vitro. Biol Neonate. 38: 30–3.
Meister, A. and Anderson, M.A. 1983. Glutathione. Annu Rev Biochem. 52: 711–60.
Menendez, C.E., Hacker, P., Sonnenfeld, M., McConnell, R. and Rivlin, R.S. 1974. Thyroid hormone regula-
tion of glutathione reductase activity in rat erythrocytes and liver. Am J Physiol. 226: 1480–3.
Merlini, G., Bruening, R., Kyle, R.A. and Osserman, E.F. 1990. The second riboflavin-binding myeloma IgG
lambdaDOT. I. Biochemical and functional characterization. Mol Immunol. 27: 385–94.
Merrill, A.H., Jr. and McCormick, D.B. 1980. Affinity chromatographic purification and properties of flavoki-
nase (ATP:riboflavin 5′-phosphotransferase) from rat liver. J Biol Chem. 255: 1335–8.
Merrill, A.H., Jr., Foltz, A.T. and McCormick, D.B. 1981. Formation and mode of action of flavoproteins. Annu
Rev Nutr. 1: 281–317.
Mestdagh, F., De Meulenaer, B., De Clippeleer, J., Devlieghere, F. and Huyghebaert, A. 2005. Protective influ-
ence of several packaging materials on light oxidation of milk. J Dairy Sci. 88: 499–510.
Métayer, S., Seiliez, I., Collin, A., Duchêne, S., Mercier, Y., Geraert, P.A. and Tesseraud, S. 2008. Mechanisms
through which sulfur amino acids control protein metabolism and oxidative status. J Nutr Biochem. 19:
207–15.
Mewies, M., McIntire, W.S. and Scrutton, N.S. 1998. Covalent attachment of flavin adenine dinucleotide
(FAD) and flavin mononucleotide (FMN) to enzymes: the current state of affairs. Protein Sci. 7: 7–20.
Meyer, U.A. 1996. Overview of enzymes of drug metabolism. J Pharmacokinet Biopharm. 24: 449–59.
Miller, S.J. 1989. Nutritional deficiency and the skin. J Am Acad Dermatol. 21: 1–30.
Miller, W.L. 2008. Steroidogenic enzymes. Endocr Dev. 13:1–18.
Miller, W.L. 2011. Role of mitochondria in steroidogenesis. Endocr Dev. 20: 1–19.
Miramar, M.D., Costantini, P., Ravagnan, L., Saraiva, L.M., Haouzi, D., Brothers, G., Penninger, J.M., Peleato,
M.L., Kroemer, G. and Susin, S.A. 2001. NADH oxidase activity of mitochondrial apoptosis-inducing
factor. J Biol Chem. 276: 16391–8.
K15043_C006.indd 257 4/10/2013 4:57:06 PM

Misevičienė, L., Anusevičius, Z., Sarlauskas, J., Sevrioukova, I.F. and Cėnas, N. 2011. Redox reactions of the
FAD-containing apoptosis-inducing factor (AIF) with quinoidal xenobiotics: a mechanistic study. Arch
Mohrenweiser, H.W. and Novotny, J.E. 1982. ACP1GUA-1—a low-activity variant of human erythrocyte acid
phosphatase: association with increased glutathione reductase activity. Am J Hum Genet. 34: 425–33.
Mokashi, V., Singh, D.K. and Porter, T.D. 2004. Rat supernatant protein factor-like protein stimulates squalene
monooxygenase and is activated by protein kinase A. Biochem Biophys Res Commun. 316: 688–92.
Moreira, P.I., Custódio, J., Moreno, A., Oliveira, C.R. and Santos, M.S. 2006. Tamoxifen and estradiol interact with
the flavin mononucleotide site of complex I leading to mitochondrial failure. J Biol Chem. 281: 10143–52.
Mowat, C.G., Gazur, B., Campbell, L.P. and Chapman, S.K. 2010. Flavin-containing heme enzymes. Arch
Mulkey, J.P. and Oehme, F.W. 1993. A review of thallium toxicity. Vet Hum Toxicol. 35: 445–53. Erratum in
Vet Hum Toxicol. 35: 511, 1993.
Muñoz, M.A., Pacheco, A., Becker, M.I., Silva, E., Ebensperger, R., Garcia, A.M., De Ioannes, A.E. and
Edwards, A.M. 2011. Different cell death mechanisms are induced by a hydrophobic flavin in human
tumor cells after visible light irradiation. J Photochem Photobiol B. 103: 57–67.
Murataliev, M.B., Feyereisen, R. and Walker, F.A. 2004. Electron transfer by diflavin reductases. Biochim
Biophys Acta. 1698: 1–26.
Murthy, U.S. and Adiga, P.R. 1978. Estrogen-induced synthesis of riboflavin-binding protein in immature
chicks. Kinetics and hormonal specificity. Biochim Biophys Acta. 538: 364–75.
Nabokina, S.M., Subramanian, V.S. and Said, H.M. 2012. Effect of clinical mutations on functionality of the
human riboflavin transporter-2 (hRFT-2). Mol Genet Metab. 105: 652–7.
Nagumo, A., Kamei, T., Sakakibara, J. and Ono, T. 1995. Purification and characterization of recombinant
squalene epoxidase. J Lipid Res. 36: 1489–97.
Naidoo, N. 2009. ER and aging—protein folding and the ER stress response. Ageing Res Rev. 8: 150–9.
Nakamura, M.T. and Nara, T.Y. 2004. Structure, function and dietary regulation of Δ6, Δ5 and Δ9 desaturases.
Ann Rev Nutr. 24: 345–76.
Nakano, E., Mushtaq, S., Heath, P.R., Lee, E.S., Bury, J.P., Riley, S.A., Powers, H.J. and Corfe, B.M. 2011.
Riboflavin depletion impairs cell proliferation in adult human duodenum: identification of potential
effectors. Dig Dis Sci. 56: 1007–19.
Natarajan, S.K. and Becker, D.F. 2012. Role of apoptosis-inducing factor, proline dehydrogenase, and NADPH
oxidase in apoptosis and oxidative stress. Cell Health Cytoskelet. 4: 11–27.
Natraj, U., George, S. and Kadam, P. 1988. Isolation and partial characterization of human riboflavin carrier
protein and the estimation of its levels during human pregnancy. J Reprod Immunol. 13: 1–16.
Nieva, J., Kerwin, L., Wentworth, A.D., Lerner, R.A. and Wentworth, P., Jr. 2006. Immunoglobulins can utilize
riboflavin (Vitamin B2) to activate the antibody-catalyzed water oxidation pathway. Immunol Lett. 103: 33–8.
Norton, W.N., Daskal, I., Savage, H.E., Seibert, R.A. and Lane, M. 1976. Effects of riboflavin deficiency on the
ultrastructure of rat sciatic nerve fibers. Am J Pathol. 85: 651–60.
Norton, W.N., Daskal, I., Savage, H.E., Seibert, R.A. and Lane, M. 1979. Asynchrony of erythroblast matura-
tion induced by riboflavin deficiency. Virchows Arch B Cell Pathol Incl Mol Pathol. 30: 43–51.
Ogura, R., Ueta, H., Hino, Y., Hidaka, T. and Sugiyama, M. 1991. Riboflavin deficiency caused by treatment
with Adriamycin. J Nutr Sci Vitaminol (Tokyo). 37: 473–7.
Ohkawa, H., Ohishi, N. and Yagi, K. 1983a. New metabolites of riboflavin appeared in rat urine. Biochem Int.
6: 239–47.
Ohkawa, H., Ohishi, N. and Yagi, K. 1983b. Occurrence of riboflavinyl glucoside in rat urine. J Nutr Sci
Vitaminol (Tokyo). 29: 515–22.
Ohkawa, H., Ohishi, N. and Yagi, K. 1983c. New metabolites of riboflavin appear in human urine. J Biol Chem.
258: 5623–8.
Ohno, K. and Hirata, M. 1990. Induction of gamma-glutamylcysteine synthetase by prostaglandin A2 in L-1210
cells. Biochem Biophys Res Commun. 168: 551–7.
Oka, M. and McCormick, D.B. 1987. Complete purification and general characterization of FAD synthetase
from rat liver. J Biol Chem. 262: 7418–22.
Olteanu, H. and Banerjee, R. 2001. Human methionine synthase reductase, a soluble P-450 reductase-like
dual flavoprotein, is sufficient for NADPH-dependent methionine synthase activation. J Biol Chem. 276:
35558–63.
O’Morain, C.A. 1990. Does nutritional therapy in inflammatory bowel disease have a primary or an adjunctive
role? Scand J Gastroenterol Suppl. 172: 29–34.
K15043_C006.indd 258 4/10/2013 4:57:06 PM

Oppenheimer, S.J., Bull, R. and Thurnham, D.I. 1983. Riboflavin deficiency in Madang infants. P N G Med J.
26: 17–20.
Ortega, R.M., Quintas, M.E., Martínez, R.M., Andrés, P., López-Sobaler, A.M. and Requejo, A.M. 1999.
Riboflavin levels in maternal milk: the influence of vitamin B2 status during the third trimester of preg-
nancy. J Am Coll Nutr. 18: 324–29.
Paine, M.J., Garner, A.P., Powell, D., Sibbald, J., Sales, M., Pratt, N., Smith, T., Tew, D.G. and Wolf, C.R. 2000.
Cloning and characterization of a novel human dual flavin reductase. J Biol Chem. 275: 1471–8.
Park, K.J., Lee, C.H., Kim, A., Jeong, K.J., Kim, C.H. and Kim, Y.S. 2012. Death receptors 4 and 5 activate
Nox1 NADPH oxidase through riboflavin kinase to induce reactive oxygen species-mediated apoptotic
cell death. J Biol Chem. 287: 3313–25.
Patel, J.M. and Pawar, S.S. 1974. Riboflavin and drug metabolism in adult male and female rats. Biochem
Pharmacol. 23: 1467–77.
Pedrolli, D.B., Nakanishi, S., Barile, M., Mansurova, M., Carmona, E.C., Lux, A., Gärtner, W. and Mack, M.
2011. The antibiotics roseoflavin and 8-demethyl-8-amino-riboflavin from Streptomyces davawensis are
metabolized by human flavokinase and human FAD synthetase. Biochem Pharmacol. 82: 1853–9.
Pejchal, R., Campbell, E., Guenther, B.D., Lennon, B.W., Matthews, R.G. and Ludwig, M.L. 2006. Structural
perturbations in the Ala→Val polymorphism of methylenetetrahydrofolate reductase: how binding of
folates may protect against inactivation. Biochemistry. 45: 4808–18.
Pelliccione, N., Pinto, J., Huang, Y.P. and Rivlin, R.S. 1983. Accelerated development of riboflavin deficiency
by treatment with chlorpromazine. Biochem Pharmacol. 32: 2949–53.
Pelliccione, N.J., Karmali, R., Rivlin, R.S. and Pinto, J. 1985. Effects of riboflavin deficiency upon prostaglan-
din biosynthesis in rat kidney. Prostaglandins Leukot Med. 17: 349–58.
Pinto J., Huang, Y.P. and Rivlin, R.S. 1987. Mechanisms underlying the differential effects of ethanol upon the
bioavailability of riboflavin and flavin adenine dinucleotide. J Clin Invest. 79: 1343–8.
Pinto, J., Huang, Y.P., McConnell, R.J. and Rivlin, R.S. 1978. Increased urinary riboflavin excretion resulting
from boric acid ingestion. J Lab Clin Med. 92: 126–34.
Pinto, J., Raiczyk, G.B., Huang, Y.P. and Rivlin, R.S. 1986. New approaches to the possible prevention of side
effects of chemotherapy by nutrition. Cancer. 58(Suppl 8): 1911–4.
Pinto, J.T. and Heston, W.D.W. 1999. Prostate specific membrane antigen: a unique folate hydrolase. A review
of recent findings. Prostate J. 1: 15–26.
Pinto, J.T. and Rivlin, R.S. 1979. Regulation of formation of covalently bound flavins in liver and cerebrum by
thyroid hormones. Arch Biochem Biophys. 194: 313–20.
Pinto, J.T. and Rivlin, R.S. 1987. Drugs that promote renal excretion of riboflavin. Drug Nutr Interact. 5:
143–51.
Pinto, J.T., Huang, Y.P. and Rivlin, R.S. 1981. Inhibition of riboflavin metabolism in rat tissues by chlorproma-
zine, imipramine and amitriptyline. J Clin Invest. 67: 1500–6.
Pinto, J.T., Huang, Y.P. and Rivlin, R.S. 1985. Inhibition by chlorpromazine of thyroxine modulation of flavin
metabolism in liver, cerebrum and cerebellum. Biochem Pharmacol. 34: 93–5.
Poirier, Y., Antonenkov, V.D., Glumoff, T. and Hiltunen, J.K. 2006. Peroxisomal beta-oxidation—a metabolic
pathway with multiple functions. Biochim Biophys Acta. 1763: 413–26.
Pollegioni, L., Piubelli, L., Sacchi, S., Pilone, M.S. and Molla, G. 2007. Physiological functions of D-amino
acid oxidases: from yeast to humans. Cell Mol Life Sci. 64: 1373–94.
Poole, L.B., Hall, A. and Nelson, K.J. 2011. Overview of peroxiredoxins in oxidant defense and redox regula-
tion. Curr Protoc Toxicol. 7: 7.9.
Porter, D.H., Cook, R.J. and Wagner, C. 1985. Enzymatic properties of dimethylglycine dehydrogenase and
sarcosine dehydrogenase from rat liver. Arch Biochem Biophys. 243: 396–407.
Porter, F.D. and Herman, G.E. 2011. Malformation syndromes caused by disorders of cholesterol synthesis.
J Lipid Res. 52: 6–34.
Powers, H.J. 2003. Riboflavin (vitamin B2) and health. Am J Clin Nutr. 77: 1352–60.
Powers, H.J., Corfe, B.M. and Nakano, E. 2012. Riboflavin in development and cell fate. Subcell Biochem. 56:
229–45.
Powers, H.J., Weaver, L.T., Austin, S. and Beresford, J.K. 1993. A proposed intestinal mechanism for the effect
of riboflavin deficiency on iron loss in the rat. Br J Nutr. 69: 553–61.
Prasad, P.D., Malhotra, P., Karande, A.A. and Adiga, P.R. 1992. Isolation and characterization of riboflavin
carrier protein from human amniotic fluid. Biochem Int. 27: 385–95.
Prasad, R., Lakshmi, A.V. and Bamji, M.S. 1983. Impaired collagen maturity in vitamins B2 and B6 deficiency-
probable molecular basis of skin lesions. Biochem Med. 30: 333–41.
K15043_C006.indd 259 4/10/2013 4:57:06 PM

Prentice, A.M. and Bates, C.J. 1981. A biochemical evaluation of the erythrocyte glutathione reductase (EC 1.6.4.2)
test for riboflavin status. 2. Dose-response relationships in chronic marginal deficiency. Br J Nutr. 45: 53–65.
Quinton, L.J., Blahna, M.T., Jones, M.R., Allen, E., Ferrari, J.D., Hilliard, K.L., Zhang, X., Sabharwal, V.,
Algül, H., Akira, S., Schmid, R.M., Pelton, S.I., Spira, A. and Mizgerd, J.P. 2012. Hepatocyte-specific
mutation of both NF-κB RelA and STAT3 abrogates the acute phase response in mice. J Clin Invest. Please provide
[Epub ahead of print]. update on the
publication
Raiczyk, G.B. and Pinto, J. 1988. Inhibition of flavin metabolism by adriamycin in skeletal muscle. Biochem status.
Pharmacol. 37: 1741–4.
Ramanujam, V.M., Anderson, K.E., Grady, J.J., Nayeem, F. and Lu, L.J. 2011. Riboflavin as an oral tracer for
monitoring compliance in clinical research. Open Biomark J. 2011: 1–7.
Ranganathan, R., Nicklas, T.A., Yang, S.J. and Berenson, G.S. 2005. The nutritional impact of dairy product con-
sumption on dietary intakes of adults (1995–1996): the Bogalusa Heart Study. J Am Diet Assoc. 105: 1391–400.
Rao, P.N., Crippin, J., Levine, E., Hunt, J., Baliga, S., Balart, L., Anthony, L., Mulekar, M. and Raj, M.H. 2006.
Elevation of serum riboflavin carrier protein in hepatocellular carcinoma. Hepatol Res. 35: 83–7.
Rao, P.N., Levine, E., Myers, M.O., Prakash, V., Watson, J., Stolier, A., Kopicko, J.J., Kissinger, P., Raj, S.G.
and Raj, M.H. 1999. Elevation of serum riboflavin carrier protein in breast cancer. Cancer Epidemiol
Biomarkers Prev. 8: 985–90.
Razeto, A., Mattiroli, F., Carpanelli, E., Aliverti, A., Pandini, V., Coda, A. and Mattevi, A. 2007. The crucial
step in ether phospholipid biosynthesis: structural basis of a noncanonical reaction associated with a
peroxisomal disorder. Structure. 15: 683–92.
Reddy, M.B. and Love, M. 1999. The impact of food processing on the nutritional quality of vitamins and
minerals. Adv Exp Med Biol. 459: 99–106.
Rehder, D.S. and Borges, C.R. 2010. Cysteine sulfenic acid as an intermediate in disulfide bond formation and
nonenzymatic protein folding. Biochemistry. 49: 7748–55.
Rich, P.R. and Maréchal, A. 2010. The mitochondrial respiratory chain. Essays Biochem. 47: 1–23.
Rioux, V., Pédrono, F. and Legrand, P. 2011. Regulation of mammalian desaturases by myristic acid: N-terminal
myristoylation and other modulations. Biochim Biophys Acta. 1811: 1–8.
Rivlin, R.S. 1966. Riboflavin. In Present Knowledge in Nutrition, eds. E.E. Ziegler and L.J. Filer, 167–73.
Washington, DC: ILSI Press.
Rivlin, R.S. 1969. Perinatal development of enzymes synthesizing FMN and FAD. Am J Physiol. 216: 979–82.
Rivlin, R.S. 1970. Medical progress: riboflavin metabolism. N Engl J Med. 283: 463–72.
Rivlin, R.S. 1991. Disorders of vitamin metabolism: deficiencies, metabolic abnormalities and excesses. In
Cecil Textbook of Medicine, 19th ed., eds. J.H. Wyngaarden, L.H. Smith, Jr., J.C. Bennett and F. Plum, F.,
1170–83. Philadelphia: W.B. Saunders.
Rivlin, R.S. 1994. Vitamin deficiency. In Conns’ Current Therapy, ed. R.E. Rakel. 551–9, Philadelphia: W.B.
Saunders.
Rivlin, R.S. and Dutta, P. 1995. Vitamin B2 (riboflavin). Relevance to malaria and antioxidant activity. Nutr
Today. 30: 62–7.
Rivlin, R.S. and Pinto, J.T. 2001. Riboflavin. In Present Knowledge in Nutrition, 8th ed., eds. B. Bowman and
R. Russell, 1313–32. Washington, DC: ILSI Press.
Rivlin, R.S., Menendez, C. and Langdon, R.G. 1968. Biochemical similarities between hypothyroidism and
riboflavin deficiency. Endocrinology 83: 461–9.
Rockett, K.A., Targett, G.A. and Playfair, J.H. 1988. Killing of blood-stage Plasmodium falciparum by lipid
peroxides from tumor necrosis serum. Infect Immun. 56: 3180–3.
Roe, D.A. 1991. Riboflavin deficiency: mucocutaneous signs of acute and chronic deficiency. Semin Dermatol.
10: 293–5.
Roe, D.A., Kalkwarf, H. and Stevens, J. 1988. Effect of fiber supplements on the apparent absorption of phar-
macological doses of riboflavin. J Am Diet Assoc. 88: 211–3.
Roe, D.A., McCormick, D.B. and Lin, R.T. 1972. Effects of riboflavin on boric acid toxicity. J Pharm Sci. 61:
1081–5.
Rohner, F., Zimmermann, M.B., Wegmueller, R., Tschannen, A.B. and Hurrell, R.F. 2007. Mild riboflavin defi-
ciency is highly prevalent in school-age children but does not increase risk for anaemia in Côte d’Ivoire.
Br J Nutr. 97: 970–6.
Ron, D. and Walter, P. 2007. Signal integration in the endoplasmic reticulum unfolded protein response. Nat
Rev Mol Cell Biol. 8: 519–29.
Roy, D.K., Saha, A. and Mukherjee, A.K. 2006. Spectroscopic and thermodynamic study of charge transfer
complex formation between cloxacillin sodium and riboflavin in aqueous ethanol media of varying com-
position. Spectrochim Acta A Mol Biomol Spectrosc. 63: 694–9.
K15043_C006.indd 260 4/10/2013 4:57:06 PM

Rozen, R. 2002. Methylenetetrahydrofolate reductase: a link between folate and riboflavin. Am J Clin Nutr.
76: 301–2.
Sacchi, S., Bernasconi, M., Martineau, M., Mothet, J.P., Ruzzene, M., Pilone, M.S., Pollegioni, L. and Molla,
G. 2008. pLG72 modulates intracellular D-serine levels through its interaction with D-amino acid oxi-
dase: effect on schizophrenia susceptibility. J Biol Chem. 283: 22244–56.
Sacheck, J., Goodman, E., Chui, K., Chomitz, V., Must, A. and Economos, C. 2011. Vitamin D deficiency,
adiposity, and cardiometabolic risk in urban schoolchildren. J Pediatr. 159: 945–50.
Said, H.M., Ortiz, A., Ma, T.Y. and McCloud, E. 1998. Riboflavin uptake by the human-derived liver cells Hep
G2: mechanism and regulation. J Cell Physiol. 176: 588–94.
Said, H.M., Wang, S. and Ma, T.Y. 2005. Mechanism of riboflavin uptake by cultured human retinal pigment
epithelial ARPE-19 cells: possible regulation by an intracellular Ca2+–calmodulin-mediated pathway.
J Physiol. 566(Pt 2): 369–77.
Sandor, P.S., Afra, J., Ambrosini, A. and Schoenen, J. 2000. Prophylactic treatment of migraine with β-blockers
and riboflavin: differential effects on the intensity dependence of auditory evoked cortical potentials.
Headache. 40: 30–5.
Santos de Souza, A.C., Kodach, L., Gadelha, F.R., Bos, C.L., Cavagis, A.D., Aoyama, H., Peppelenbosch,
M.P. and Ferreira, C.V. 2006. A promising action of riboflavin as a mediator of leukaemia cell death.
Apoptosis. 11: 1761–71.
Sauberlich, H.E., Judd, J.H., Jr., Nichoalds, G.E., Broquist, H.P. and Darby, W.J. 1972. Application of the
erythrocyte glutathione reductase assay in evaluating riboflavin nutritional status in a high school student
population. Am J Clin Nutr. 25: 756–62.
Sawada, O., Tshida, T. and Horiike, K. 2001. Frontal gel chromatographic analysis of the interaction of a pro-
tein with self-associating ligands: aberrant saturation in the binding of flavins to bovine serum albumin.
J Biochem. 129: 899–907.
Schafer, F.Q. and Buettner, G.R. 2001. Redox environment of the cell as viewed through the redox state of the
glutathione disulfide/glutathione couple. Free Radic Biol Med. 30: 1191–212.
Schiff, M., Froissart, R., Olsen, R.K., Acquaviva, C. and Vianey-Saban, C. 2006. Electron transfer flavoprotein
deficiency: functional and molecular aspects. Mol Genet Metab. 88: 153–8.
Schmitz, G. and Muller, G. 1991. Structure and function of lamellar bodies, lipid-protein complexes involved
in storage and secretion of cellular lipids. J Lipid Res. 32: 1539–70.
Schoenen, J., Jacquy, J. and Lenaerts, M. 1998. Effectiveness of high-dose riboflavin in migraine prophylaxis.
A randomized controlled trial. Neurology. 50: 466–70.
Schoenen, J., Leneaerts, M. and Bastings, E. 1994. High dose riboflavin as a prophylactic treatment of migraine:
results of an open pilot study. Cephalalgia. 14: 328–9.
Scholte, H.R., Busch, H.F., Bakker, H.D., Bogaard, J.M., Luyt-Houwen, I.E. and Kuyt, L.P. 1995. Riboflavin-
responsive complex I deficiency. Biochim Biophys Acta. 1271: 75–83.
Schönleben-Janas, A., Kirsch, P., Mittl, P.R., Schirmer, R.H. and Krauth-Siegel, R.L. 1996. Inhibition of human
glutathione reductase by 10-arylisoalloxazines: crystalline, kinetic, and electrochemical studies. J Med
Chem. 39: 1549–54.
Selak, M.A., Armour, S.M., MacKenzie, E.D., Boulahbel, H., Watson, D.G., Mansfield, K.D., Pan, Y., Simon,
M.C., Thompson, C.B. and Gottlieb, E. 2005. Succinate links TCA cycle dysfunction to oncogenesis by
inhibiting HIF-alpha prolyl hydroxylase. Cancer Cell. 7: 77–85.
Selhub, J. 1999. Homocysteine metabolism. Annu Rev Nutr. 19: 217–46.
Serganov, A., Huang, L. and Patel, D.J. 2009. Coenzyme recognition and gene regulation by a flavin mono-
nucleotide riboswitch. Nature. 458: 233–7.
Sevrioukova, I.F. 2009. Redox-linked conformational dynamics in apoptosis-inducing factor. J Mol Biol. 390:
924–38.
Sevrioukova, I.F. 2011. Apoptosis-inducing factor: structure, function, and redox regulation. Antioxid Redox
Signal. 14: 2545–79.
Shankar, A.H. 2000. Nutritional modulation of malaria morbidity and mortality. J Infect Dis. 182 (Suppl 1):
S37–53.
Shargel, L. and Mazel, P. 1973. Effect of riboflavin deficiency on phenobarbital and 3-methylcholanthrene
induction of microsomal drug-metabolizing enzymes of the rat. Biochem Pharmacol. 22: 2365–73.
Silva, E. and Edwards, A.M. eds. 2006. Flavins: Photochemistry and Photobiology, 151–182. Cambridge, UK:
RSC Publishing.
Sim, K.G., Hammond, J. and Wilcken, B. 2002. Strategies for the diagnosis of mitochondrial fatty acid
β-oxidation disorders. Clin Chim Acta. 323: 37–58.
Sisson, T.R. 1987. Photodegradation of riboflavin in neonates. Fed Proc. 46: 1883–5.
K15043_C006.indd 261 4/10/2013 4:57:06 PM

Skalka, H.W. and Prchal, J.T. 1981. Cataracts and riboflavin deficiency. Am J Clin Nutr. 34: 861–3.
Slow, S. and Garrow, T.A. 2006. Liver choline dehydrogenase and kidney betaine-homocysteine methyltrans-
ferase expression are not affected by methionine or choline intake in growing rats. J Nutr. 136: 2279–83.
Smulders, Y.M. and Blom, H.J. 2011. The homocysteine controversy. J Inherit Metab Dis. 34: 93–9.
Soares, M.J., Satyanarayana, K., Bamji, M.S., Jacob, C.M., Ramana, Y.V. and Rao, S.S. 1993. The effect of
exercise on the riboflavin status of adult men. Br J Nutr. 69: 541–51.
Spector, R. 1980. Riboflavin transport in the central nervous system. Characterization and effects of drugs.
J Clin Invest. 66: 821–31.
Spence, J.T. and Tocatlian, J. 1961. The interaction of molybdenum with riboflavin and flavin mononucleotide.
J Am Chem Soc. 83: 816–9.
Stern, K.G. and Holiday, E.R. 1934. Zur konstitution des photo-flavins; versuche in der alloxazine-reihe. Ber
Dtsch Chem Gesellsch. 67: 1104–6.
Stern, L.L., Shane, B., Bagley, P.J., Nadeau, M., Shih, V. and Selhub, J. 2003. Combined marginal folate and
riboflavin status affect homocysteine methylation in cultured immortalized lymphocytes from persons
homozygous for the MTHFR C677T mutation. J Nutr. 133: 2716–20.
Stuart, J., Moat, S.J., Pauline, A.L, Ashfield-Watt, A., Powers, H.J., Newcombe, R.G. and McDowell, I.F.W.
2003. Effect of riboflavin status on the homocysteine-lowering effect of folate in relation to the MTHFR
(C677T) genotype. Clin Chem. 49: 295–302.
Subramanian, S. and Adiga, P.R. 1996a. Hormonal modulation of secretion of immunoreactive riboflavin car-
rier protein by adult rat Leydig cells in vitro. Indian J Biochem Biophys. 33: 274–80.
Subramanian, S. and Adiga, P.R. 1996b. Hormonal modulation of riboflavin carrier protein secretion by imma-
ture rat Sertoli cells in culture. Mol Cell Endocrinol. 120: 41–50.
Subramanian, V.S., Subramanya, S.B., Rapp, L., Marchant, J.S., Ma, T.Y. and Said, H.M. 2011. Differential
expression of human riboflavin transporters -1, -2, and -3 in polarized epithelia: a key role for hRFT-2 in
intestinal riboflavin uptake. Biochim Biophys Acta. 1808: 3016–21.
Sue-Chu, M., Kristensen, S. and Tønnesen, H.H. 2009. Photoinduced color changes in two different qualities
of riboflavin in the solid state and in various tablet formulations photoreactivity of biologically active
compounds. XX. Pharmazie. 64: 428–35.
Sugiyama, M. 1991. Effects of vitamins on chromium(VI)-induced damage. Environ Health Perspect. 92:
63–70.
Susin, S.A., Daugas, E., Ravagnan, L., Samejima, K., Zamzami, N., Loeffler, M., Costantini, P., Ferri, K.F.,
Irinopoulou, T., Prévost, M.C., Brothers, G., Mak, T.W., Penninger, J., Earnshaw, W.C. and Kroemer, G.
2000. Two distinct pathways leading to nuclear apoptosis. J Exp Med. 192: 571–80.
Swanson, M.A., Usselman, R.J., Frerman, F.E., Eaton, G.R. and Eaton, S.S. 2008. The iron-sulfur cluster of
electron transfer flavoprotein-ubiquinone oxidoreductase is the electron acceptor for electron transfer
flavoprotein. Biochemistry. 47: 8894–901.
Switzer, B.R., Stark, A.H., Atwood, J.R., Ritenbaugh, C., Travis, R.G. and Wu, H.M. 1997. Development
of a urinary riboflavin adherence marker for a wheat bran fiber community intervention trial. Cancer
Epidemiol Biomarkers Prev. 6: 439–42.
Tandler, B. and Hoppel, C.L. 1980. Ultrastructural effects of riboflavin deficiency on rat hepatic mitochondria.
Anat Rec. 196: 183–90.
Tandler, B., Erlandson, R.A., Smith, A.L. and Wynder, E.L. 1969. Riboflavin and mouse hepatic cell struc-
ture and function. II. Division of mitochondria during recovery from simple deficiency. J Cell Biol. 41:
477–93.
Taniguchi, M. and Hara, T. 1983. Effects of riboflavin and selenium deficiencies on glutathione and related
enzyme activities with respect to lipid peroxide content of rat livers. J Nutr Vitaminol. 29: 283–92.
Tappel, A.L. 1974. Selenium-glutathione peroxidase and vitamin E. Am J Clin Nutr. 27: 960–5.
Taylor, F.R. 2011. Nutraceuticals and headache: the biological basis. Headache. 51: 484–501.
Tello, D., Balsa, E., Acosta-Iborra, B., Fuertes-Yebra, E., Elorza, A., Ordóñez, Á., Corral-Escariz, M., Soro,
I., López-Bernardo, E., Perales-Clemente, E., Martínez-Ruiz, A., Enríquez, J.A., Aragonés, J., Cadenas,
S. and Landázuri, M.O. 2011. Induction of the mitochondrial NDUFA4L2 protein by HIF-1α decreases
oxygen consumption by inhibiting Complex I activity. Cell Metab. 14: 768–79.
Theorell, H. 1934. Reindarstellung (Kristallisation) des gelben Atmungsfermentes und die reversible Spaltung
desselben. Biochem Z. 272: 155–6.
Theorell, H. 1937. Die freie eiweisskomponente des gelben ferments und ihre kupplung mit Lactoflavinphos
phorsaure. Biochem Z. 290: 293–303.
Thorp, V.J. 1980. Effect of oral contraceptive agents on vitamin and mineral requirements. J Am Diet Assoc.
76: 581–4.
K15043_C006.indd 262 4/10/2013 4:57:06 PM

Tillotson, J.A. and Karcz, M.S., Jr. 1977. Urinary riboflavin metabolites in the rat. J Nutr. 107: 1269–76.
Tisdall, F.F., McCreary, J.F. and Pearce, H. 1943. The effect of riboflavin on corneal vascularization and symp-
toms of eye fatigue in R.C.A.F. personnel. Can Med Assoc J. 49: 5–13.
Tokutomi, S., Matsuoka, D. and Zikihara, K. 2008. Molecular structure and regulation of phototropin kinase by
blue light. Biochim Biophys Acta. 1784: 133–42.
Tontisirin, K., Tejavej, A., Siripoonya, P., Satayasai, V., Gjernsritrakul, C. and Valaiphatchara, U. 1989. Effect
of phototherapy on nutrients utilization in newborn infants with jaundice. J Med Assoc Thai. 72 Suppl
1: 177–82.
Toogood, H.S., Leys, D. and Scrutton, N.S. 2007. Dynamics driving function: new insights from electron trans-
ferring flavoproteins and partner complexes. FEBS J. 274: 5481–504.
Torchetti, E.M., Bonomi, F., Galluccio, M., Gianazza, E., Giancaspero, T.A., Iametti, S., Indiveri, C. and Barile,
M. 2011. Human FAD synthase (isoform 2): a component of the machinery that delivers FAD to apo-
flavoproteins. FEBS J. 278: 4434–49.
Torchetti, E.M., Brizio, C., Colella, M., Galluccio, M., Giancaspero, T.A., Indiveri, C., Roberti, M. and Barile,
M. 2010. Mitochondrial localization of human FAD synthetase isoform 1. Mitochondrion. 10: 263–73.
Townsend, D.M. 2007. S-Glutathionylation: indicator of cell stress and regulator of the unfolded protein
response. Mol Interv. 7: 313–24.
Trachewsky, D. 1978. Aldosterone stimulation of riboflavin incorporation into rat renal flavin coenzymes and
the effect of inhibition by riboflavin analogues on sodium reabsorption. J Clin Invest. 62: 1325–33.
Trachewsky, D., Oakes, M.L. and Morris, D.J. 1985. Induction of flavokinase (EC 2.7.1.26) by aldosterone in
the rat kidney. Endocrinology. 116: 879–88.
Traunmüller, F., Ramharter, M. and Lagler, H. 2003. Normal riboflavin status in malaria patients in Gabon. Am
J Trop Med Hyg. 68: 182–5.
Tsibris, J.C.M., McCormick, D.B. and Wright, L.D. 1965. Studies on donor-acceptor complexes relating to
the intramolecular association of the riboflavin and adenosine moieties of flavin-adenine dinucleotide.
Tsuji, T., Fukuwatari, T., Sasaki, S. and Shibata, K. 2010. Urinary excretion of vitamin B1, B2, B6, niacin, pan-
tothenic acid, folate, and vitamin C correlates with dietary intakes of free-living elderly, female Japanese.
Nutr Res. 30: 171–8.
Tzagoloff, A., Jang, J., Glerum, D.M. and Wu, M. 1996. FLX1 codes for a carrier protein involved in maintain-
ing a proper balance of flavin nucleotides in yeast mitochondria. J Biol Chem. 271: 7392–7.
Van de Perre, P. 2003. Transfer of antibody via mother’s milk. Vaccine. 21: 3374–6.
van den Boom, A., Serra-Majem, L., Ribas, L., Ngo, J., Pérez-Rodrigo, C., Aranceta, J. and Fletcher, R. 2006.
The contribution of ready-to-eat cereals to daily nutrient intake and breakfast quality in a Mediterranean
setting. J Am Coll Nutr. 25: 135–43.
van Herwaarden, A.E. and Schinkel, A.H. 2006. The function of breast cancer resistance protein in epithelial
barriers, stem cells and milk secretion of drugs and xenotoxins. Trends Pharmacol Sci. 27: 10–6.
van Herwaarden, A.E., Wagenaar, E., Merino, G., Jonker, J.W., Rosing, H., Beijnen, J.H. and Schinkel, A.H.
2007. Multidrug transporter ABCG2/breast cancer resistance protein secretes riboflavin (vitamin B2)
into milk. Mol Cell Biol. 27: 1247–53.
Van Veldhoven, P.P. 2010. Biochemistry and genetics of inherited disorders of peroxisomal fatty acid metabo-
lism. J Lipid Res. 51: 2863–95.
Veitch, K., Draye, J.P., Vamecq, J., Causey, A.G., Bartlett, K., Sherratt, H.S. and Van Hoof, F. 1989. Altered
acyl-CoA metabolism in riboflavin deficiency. Biochim Biophys Acta. 1006: 335–43.
Vergani, L., Barile, M., Angelini, C., Burlina, A.B., Nijtmans, L., Freda, M.P., Brizio, C., Zerbetto, E. and
Dabbeni-Sala, F. 1999. Riboflavin therapy. Biochemical heterogeneity in two adult lipid storage myopa-
thies. Brain 122(Pt 12): 2401–11.
Visweswariah, S.S. and Adiga, P.R. 1987. Isolation of riboflavin carrier proteins from pregnant human and
umbilical cord serum: similarities with chicken egg riboflavin carrier protein. Biosci Rep. 7: 563–71.
Vlaming, M.L., Lagas, J.S. and Schinkel, A.H. 2009. Physiological and pharmacological roles of ABCG2
(BCRP): recent findings in Abcg2 knockout mice. Adv Drug Deliv Rev. 61: 14–25.
Wacker, J., Frühauf, J., Schulz, M., Chiwora, F.M., Volz, J. and Becker, K. 2000. Riboflavin deficiency and
preeclampsia. Obstet Gynecol. 96: 38–44.
Waguespack, S. G., Rich, T., Grubbs, E., Ying, A. K., Perrier, N.D., Ayala-Ramirez, M. and Jimenez, C. 2010.
A current review of the etiology, diagnosis, and treatment of pediatric pheochromocytoma and paragan-
glioma. J Clin Endocrinol Metab. 95: 2023–37.
Wanders, R.J., Romeyn, G.J., Schutgens, R.B. and Tager, J.M. 1989. L-pipecolate oxidase: a distinct peroxi-
somal enzyme in man. Biochem Biophys Res Commun. 164: 550–5.
K15043_C006.indd 263 4/10/2013 4:57:06 PM

Wang, R.J. and Nixon, B.T. 1978. Identification of hydrogen peroxide as a photoproduct toxic to human cells
in tissue-culture medium irradiated with “daylight” fluorescent light. In Vitro. 14: 715–22.
Wanner, R.L. 1960. Effects of commercial processing of milk and milk products on their nutrient content. In
The Nutritional Evaluation of Food Processing, eds. R.S. Harris and H.V. Loesecke, 173. New York:
John Wiley.
Warburg, O. and Christian, W. 1932. Uber ein neues oxydationsferment und sein absorptionsspetrum. Biochem
Z. 254: 438–58.
Warburg, O. and Christian, W. 1938. Isolierung der prosthetischen gruppe der d-aminosiureoxydase. Biochem
Z. 298: 150–68.
Warburg, O., Geissler, A.W. and Lorenz, S. 1968. Wirkung von riboflavin und luminoflavin auf wachsende
krebszellen. Z Klin Chem Klin Biochem. 6: 467–8.
Waterham, H.R., Koster, J., Romeijn, G.J., Hennekam, R.C., Vreken, P., Andersson, H.C., FitzPatrick, D.R.,
Kelley, R.I. and Wanders, R.J. 2001. Mutations in the 3beta-hydroxysterol Delta24-reductase gene cause
desmosterolosis, an autosomal recessive disorder of cholesterol biosynthesis. Am J Hum Genet. 69:
685–94.
Watson, C.D. and Ford, H.C. 1988. High-affinity binding of riboflavin and FAD by immunoglobulins from
normal human serum. Biochem Int. 16: 1067–74.
Weimar, W.R. and Neims, A.H. 1975. Physical and chemical properties of flavins: binding of flavins to protein
and conformational effects; biosynthesis of riboflavin. In Riboflavin, ed. R.S. Rivlin, 1–47. New York:
Plenum Press.
Werner, R., Manthey, K.C., Griffin, J.B. and Zempleni, J. 2005. HepG2 cells develop signs of riboflavin defi-
ciency within 4 days of culture in riboflavin-deficient medium. J Nutr Biochem. 16: 617–24.
West, D.W. and Owen, E.C. 1969. The urinary excretion of metabolites of riboflavine by man. Br J Nutr. 23:
889–98.
White, H.B., 3rd. 1996. Sudden death of chicken embryos with hereditary riboflavin deficiency. J Nutr. 126(4
Suppl):1303S–7S.
Williams, E.A., Rumsey, R.D.E. and Powers, H.J. 1996. Cytokinetic and structural responses of the rat small
intestine to riboflavin depletion. Br J Nutr. 75: 315–24.
Williams, R.E and Bruce, N.C. 2002. ‘New uses for an Old Enzyme’ – the Old Yellow Enzyme family of flavo-
enzymes. Microbiology. 148: 1607–14.
Winters, L.R., Yoon, J.S., Kalkwarf, H.J., Davies, J.C., Berkowitz, M.G., Haas, J. and Roe, D.A. 1992 Riboflavin
requirements and exercise adaptation in older women. Am J Clin Nutr. 56: 526–32.
Wittig-Silva, C., Whiting, M., Lamoureux, E., Lindsay, R.G., Sullivan, L.J. and Snibson, G.R. 2008. A random-
ized controlled trial of corneal collagen cross-linking in progressive keratoconus: preliminary results.
J Refract Surg. 24: S720–5.
Wollensak, G. 2006. Crosslinking treatment of progressive keratoconus: new hope. Curr Opin Ophthalmol.
17: 356–60.
Wollensak, G., Spoerl, E. and Seiler, T. 2003. Riboflavin/ultraviolet-a-induced collagen crosslinking for the
treatment of keratoconus. Am J Ophthalmol. 135: 620–7.
Xia, C., Panda, S.P., Marohnic, C.C., Martásek, P., Masters, BS. and Kim, J.J. 2011. Structural basis for human
NADPH-cytochrome P450 oxidoreductase deficiency. Proc Natl Acad Sci USA. 108: 13486–91.
Yagi, K., Nakagawa, Y., Suzuki, O. and Ohishi N. 1976. Incorporation of riboflavin into covalently-bound fla-
vins in rat liver. J Biochem. 79: 841–3.
Yamada, K., Chen, Z., Rozen, R. and Matthews, R.G. 2001. Effects of common polymorphisms on the proper-
ties of recombinant human methyltetrahydrofolate reductase. Proc Natl Acad Sci USA. 98: 14853–8.
Yamada, K., Gravel, R.A., Toraya, T. and Matthews, R.G. 2006. Proc Natl Acad Sci USA. 103: 9476–81. Please provide
article title.
Yamaguchi, S., Orii, T., Suzuki, Y., Maeda, K., Oshima, M. and Hashimoto, T. 1991. Newly identified forms
of electron transfer flavoprotein deficiency in two patients with glutaric aciduria type II. Pediatr Res. 29:
60–3.
Yamamoto, S., Inoue, K., Ohta, K.Y., Fukatsu, R., Maeda, J.Y., Yoshida, Y. and Yuasa, H. 2009. Identification
and functional characterization of rat riboflavin transporter 2. J Biochem. 145: 437–43.
Yanagawa, N., Shih, R.N., Jo, O.D. and Said, H.M. 2000. Riboflavin transport by isolated perfused rabbit renal
proximal tubules. Am J Physiol Cell Physiol. 279: C1782–6.
Yang, C.S. 1974. Alterations of the aryl hydrocarbon hydroxylase system during riboflavin depletion and reple-
tion. Arch Biochem Biophys. 160: 623–30.
Please provide
Yang, L., Hu, X. and Xu, L. 2012. Impact of methylenetetrahydrofolate reductase (MTHFR) polymorphisms
update on the on methotrexate-induced toxicities in acute lymphoblastic leukemia: a meta-analysis. Tumour Biol. Apr
publication
status. 20. [Epub ahead of print].
K15043_C006.indd 264 4/10/2013 4:57:06 PM

Yang, Q., Bailey, L., Clarke, R., Flanders, W.D., Liu, T., Yesupriya, A., Khoury, M.J. and Friedman, J.M. 2012.
Prospective study of methylenetetrahydrofolate reductase (MTHFR) variant C677T and risk of all-cause
and cardiovascular disease mortality among 6000 US adults Am J Clin Nutr. 95: 1245–53.
Yao, Y., Yonezawa, A., Yoshimatsu, H., Masuda, S., Katsura, T. and Inui, K. 2010. Identification and compara-
tive functional characterization of a new human riboflavin transporter hRFT3 expressed in the brain.
J Nutr. 140: 1220–6.
Yates, C.A., Evans, G.S. and Powers, H.J. 2001. Riboflavin deficiency: early effects on post-weaning develop-
ment of the duodenum in rats. Br J Nutr. 86: 593–9.
Yates, C.A., Evans, G.S., Pearson, T. and Powers, H.J. 2003. Absence of luminal riboflavin disturbs early post-
natal development of the gastrointestinal tract. Dig Dis Sci. 48: 1159–64.
Yazdanpanah, B., Wiegmann, K., Tchikov, V., Krut, O., Pongratz, C., Schramm, M., Kleinridders, A.,
Wunderlich, T., Kashkar, H., Utermöhlen, O., Brüning, J.C., Schütze, S. and Krönke, M. 2009. Riboflavin
kinase couples TNF receptor 1 to NADPH oxidase. Nature. 460: 1159–63.
Yonezawa, A., Masuda, S., Katsura, T. and Inui, K. 2008. Identification and functional characterization of a
novel human and rat riboflavin transporter, RFT1. Am J Physiol Cell Physiol. 295: C632–41.
Yoshida, H. 2009. ER stress response, peroxisome proliferation, mitochondrial unfolded protein response and
Golgi stress response. IUBMB Life. 61: 871–9.
Yu, M.W. and Fritchie, C.J., Jr. 1975. Interaction of flavins with electron-rich metals. J Biol Chem. 250: 946–51.
Zaniolo, K., St-Laurent, J.F., Gagnon, S.N., Lavoie, J.C. and Desnoyers, S. 2010. Photoactivated multivitamin
preparation induces poly(ADP-ribosyl)ation, a DNA damage response in mammalian cells. Free Radic
Biol Med. 48: 1002–12.
Zempleni, J., Galloway, J.R. and McCormick, D.B. 1996. Pharmacokinetics of orally and intravenously admin-
istered riboflavin in healthy humans. Am J Clin Nutr. 63: 54–66.
Zencirci, B. 2010. Comparison of the effects of dietary factors in the management and prophylaxis of migraine.
J Pain Res. 3: 125–30.
Zhang, J., Zhang, W., Zou, D., Chen, G., Wan, T., Zhang, M. and Cao, X. 2002. Cloning and functional char-
acterization of ACAD-9, a novel member of human acyl-CoA dehydrogenase family. Biochem Biophys
Res Commun. 297: 1033–42.
Zhu, H., Qiu, H., Yoon, H.-W., Huang, S. and Bunn, H.F. 1999. Identification of a cytochrome b-type NAD(P)
H oxidoreductase ubiquitously expressed in human cells. PNAS. 96: 14742–7.
Ziegler, P., Briefel, R., Ponza, M., Novak, T. and Hendricks, K. 2006. Nutrient intakes and food patterns of tod-
dlers’ lunches and snacks: influence of location. J Am Diet Assoc. 106(Suppl 1): S124–34.
Zolotushko, J., Flusser, H., Markus, B., Shelef, I., Langer, Y., Heverin, M., Björkhem, I., Sivan, S. and Birk,
O.S. 2011. The desmosterolosis phenotype: spasticity, microcephaly and micrognathia with agenesis of
corpus callosum and loss of white matter. Eur J Hum Genet. 19: 942–6.
Zou, L., Li, L. and Porter, T.D. 2011. 7-Dehydrocholesterol reductase activity is independent of cytochrome
P450 reductase. J Steroid Biochem Mol Biol. 127: 435–8.
K15043_C006.indd 265 4/10/2013 4:57:06 PM

K15043_C006.indd
View publication stats 266 4/10/2013 4:57:06 PM

Chapterreference 12

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Chapterreference 12

Uploaded by

Copyright:

Available Formats

See discussions, stats, and author profiles for this publication at: https://www.researchgate.

Riboﬂavin (Vitamin B2)

Chapter · July 2013

Aging and hypertension View project

Impact of Organoselenium Compounds on Prostate Cancer View project

The user has requested enhancement of the downloaded file.

K15043_C006.indd 191 4/10/2013 4:56:58 PM

6.7.1.2 Complex II and the Citric Acid Cycle......................................................... 223

K15043_C006.indd 192 4/10/2013 4:56:58 PM

Flavin adenine dinucleotide (FAD)

K15043_C006.indd 193 4/10/2013 4:56:59 PM

of riboflavin, which is intracellularly phosphorylated first to FMN and then pyrophosphorylated

6.3.3 Coenzymic Derivatives of Riboflavin

6.3.4 Covalently Bound Flavins

K15043_C006.indd 194 4/10/2013 4:56:59 PM

K15043_C006.indd 195 4/10/2013 4:56:59 PM

6.3.5 Reactivity of Isoalloxazine Ring

K15043_C006.indd 196 4/10/2013 4:57:00 PM

6.4 Dietary and Supplemental Sources of Riboflavin

6.4.2 Effects of Food Preparation and Processing

K15043_C006.indd 197 4/10/2013 4:57:00 PM

K15043_C006.indd 198 4/10/2013 4:57:00 PM

6.4.3 Forms of Dietary Riboflavin and Luminal Metabolism

K15043_C006.indd 199 4/10/2013 4:57:00 PM

6.5.2 Riboflavin Transport, Plasma Binding, and Carrier Proteins

K15043_C006.indd 200 4/10/2013 4:57:00 PM

6.5.2.2 Other Specialized Riboflavin Transport Proteins

K15043_C006.indd 201 4/10/2013 4:57:00 PM

6.5.2.3 Transmembrane Riboflavin Efflux Protein

K15043_C006.indd 202 4/10/2013 4:57:00 PM

6.5.2.4 Riboflavin-Binding Proteins in Plasma

K15043_C006.indd 203 4/10/2013 4:57:00 PM

6.5.2.5 Specific Riboflavin Carrier Proteins

K15043_C006.indd 204 4/10/2013 4:57:00 PM

K15043_C006.indd 205 4/10/2013 4:57:00 PM

6.5.3 Riboflavin Storage and Turnover of Flavin Coenzymes

Thyroid hormones (T4, T3)

K15043_C006.indd 206 4/10/2013 4:57:00 PM

K15043_C006.indd 207 4/10/2013 4:57:00 PM

After phosphorylation, a fraction of FMN becomes bound to FMN-binding domains within a

6.5.3.2 Cytosolic FAD Synthetase

6.5.3.3 Mitochondrial FAD Synthetase

K15043_C006.indd 208 4/10/2013 4:57:00 PM

K15043_C006.indd 209 4/10/2013 4:57:00 PM

K15043_C006.indd 210 4/10/2013 4:57:01 PM

K15043_C006.indd 211 4/10/2013 4:57:01 PM

K15043_C006.indd 212 4/10/2013 4:57:01 PM

6.5.4.2 Drugs and Riboflavin Excretion

6.5.4.3 Urinary Metabolites of Riboflavin

K15043_C006.indd 213 4/10/2013 4:57:01 PM

major derivatives include 7-α-hydroxymethylriboflavin (10%–15%), 8α-sulfonylriboflavin (5%–10%),

K15043_C006.indd 214 4/10/2013 4:57:01 PM

K15043_C006.indd 215 4/10/2013 4:57:01 PM

6.6.1 Metabolic Adaptations to Riboflavin Deficiency

Riboflavin sufficiency FMN

K15043_C006.indd 216 4/10/2013 4:57:02 PM

6.6.2 Organelle Adaptations to Riboflavin Deficiency

K15043_C006.indd 217 4/10/2013 4:57:02 PM

6.6.3 Clinical Signs of Riboflavin Deficiency

K15043_C006.indd 218 4/10/2013 4:57:02 PM

K15043_C006.indd 219 4/10/2013 4:57:02 PM

K15043_C006.indd 220 4/10/2013 4:57:02 PM

6.7 Major Metabolic Processes Utilizing Flavin Coenzymes

6.3.3 Coenzymic Derivatives of Riboflavin

6.3.4 Covalently Bound Flavins

6.3.5 Reactivity of Isoalloxazine Ring

6.4 Dietary and Supplemental Sources of Riboflavin

6.4.2 Effects of Food Preparation and Processing

6.4.3 Forms of Dietary Riboflavin and Luminal Metabolism

6.5.2 Riboflavin Transport, Plasma Binding, and Carrier Proteins

6.5.2.2 Other Specialized Riboflavin Transport Proteins

6.5.2.3 Transmembrane Riboflavin Efflux Protein

6.5.2.4 Riboflavin-Binding Proteins in Plasma

6.5.2.5 Specific Riboflavin Carrier Proteins

6.5.3 Riboflavin Storage and Turnover of Flavin Coenzymes

6.5.3.2 Cytosolic FAD Synthetase

6.5.3.3 Mitochondrial FAD Synthetase

6.5.4.2 Drugs and Riboflavin Excretion

6.5.4.3 Urinary Metabolites of Riboflavin

6.6.1 Metabolic Adaptations to Riboflavin Deficiency

6.6.2 Organelle Adaptations to Riboflavin Deficiency

6.6.3 Clinical Signs of Riboflavin Deficiency

6.7 Major Metabolic Processes Utilizing Flavin Coenzymes

6.7.1 Flavoenzyme Complexes: Electron Transport Chain and Citric Acid Cycle

6.7.1.2 Complex II and the Citric Acid Cycle

6.7.1.3 Other Flavoenzymes Involved in Electron Shuttles

6.7.2 Flavoenzymes Involved in Lipid Metabolism

6.7.2.2 Cytochrome b5 Reductase and Fatty Acid Desaturation

6.7.2.3 Flavoenzymes, Cholesterogenesis, and Steroidogenesis

6.7.2.3.4 Flavoproteins and Cytochrome P450 Heme Proteins in

6.7.2.4 Flavoenzymes and Formation of Ether Lipids and Sphingolipids

6.9 The Unfolded Protein Response

6.12 Riboflavin and Cell Signaling Factors

6.13 Extended Use and Medical Applications for Riboflavin

6.13.1.1 Mechanisms of Riboflavin Photosensitization

6.13.1.3 Photosensitization for Neonatal Jaundice

6.13.1.4 Photosensitization and Blood-Borne Pathogens

6.13.1.5 Photosensitization and Treatment of Corneal Disorders (Keratoconus)

6.13.2 Treatment of Migraine and Other Uses with Riboflavin

6.14 Requirements and Assessment