Conservacion de Las Enzymas

Physiol Rev
82: 1–18, 2002; 10.1152/physrev.00022.2001.
Conservation of Digestive Enzymes

STEPHEN ROTHMAN, CHARLES LIEBOW, AND LOIS ISENMAN
University of California-San Francisco, San Francisco, California; State University of New York, Buffalo,
New York; and Brandeis University, Waltham, Massachusetts
I. Introduction 1
II. Foundation of the Traditional Viewpoint: the Single Pass 2
III. Membrane Permeability 3
A. Intestinal epithelium 3
B. Transpancreatic transport 4
IV. Circulation Measured In Situ 4
A. Circulation of intact digestive enzymes by tracer analysis 4
B. Magnitude of circulation 5
C. Effect on the plasma pool 7
V. Attempts at Corroboration 8
A. Pancreatic arteriovenous concentration difference 8
B. Intravenous injection of radiolabeled digestive enzyme 9
C. Fate of digestive enzymes in the intestines 11
VI. Rate of Synthesis of Digestive Enzymes Compared With Their Rate of Secretion 12
A. Basal rate of protein secretion 12
B. Enhancing synthesis 13
C. Ribosomes 13
D. Energy requirement 14
E. Summary 14
VII. Membrane Protein Transport 14
VIII. Forms of Circulating Proteins 15
IX. Conservation of Other Digestive Enzymes 15
X. Conclusions 16
Rothman, Stephen, Charles Liebow, and Lois Isenman. Conservation of Digestive Enzymes. Physiol Rev 82:
1–18, 2002; 10.1152/physrev.00022.2001.—The traditional understanding is that an entirely new complement of
digestive enzymes is secreted by the pancreas into the small intestines with each meal. This is thought to be
necessary because, like food itself, these enzymes are degraded during digestion. In this review we discuss
experiments that bring this point of view into question. They suggest that digestive enzymes can be absorbed into
blood, reaccumulated by the pancreas, and reutilized, instead of being reduced to their constituent amino acids in
the intestines. This is called an enteropancreatic circulation of digestive enzymes.
I. INTRODUCTION Not long after the original findings were reported (20,
48), the idea and the evidence were variously affirmed and
In this review we summarize experiments whose im- questioned by additional experimentation (26, 37, 44, 74,
plications were of great interest when they were first 76 –78). Disagreement ensued, and the subject became
reported. They provided unexpected evidence that the controversial (6, 42, 75, 79, 87–90, 94). This was not just
conventional belief that every meal is digested by an because of the surprising nature of the observations, but
entirely new complement of digestive enzymes (Fig. 1) is also because they seemed to require that the proteins
incorrect. The data suggested that instead of being com- cross several cellular membranes in transit, a possibility
pletely degraded in the small bowel with the food they viewed as unlikely if not impossible at the time. In addi-
digest, a large fraction of the digestive enzymes secreted tion, the exact chemical forms of the circulating proteins
by the pancreas are absorbed and recycled in an entero- were not known. Were they modified, and if they so, in
pancreatic circulation (Fig. 1). what way? And, finally, it turned out that the negative
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2 ROTHMAN, LIEBOW, AND ISENMAN
FIG. 1. A: traditional view of the life history of the digestive enzymes secreted by the exocrine pancreas. From this
perspective, these products (open circles) (Ep) are secreted into the duct system of the gland, and through it enter the
small intestines (Ei), where they digest food and are subsequently degraded into their substituent amino acids (AAi) by
auto- and heterocatalytic catalysis (e). The amino acid products are then absorbed across the intestinal epithelium into
blood (AAb). An equivalent amount of amino acid is accumulated by pancreatic tissue (AAp), and the cycle is completed
when these amino acids are incorporated into new proteins (Ep) that are secreted by the pancreas in the ensuing period.
According to this scheme, each meal is digested by a completely new complement of digestive enzymes synthesized
during the interdigestive period. B: an alternative model that is supported by results discussed in this review. From this
perspective, only a small fraction of the digestive enzymes secreted by the pancreas (Ep) into the intestines (Ei) is
degraded during a single cycle of digestion (the degradative cycle is shown by narrow arrows on the right). Most is
absorbed into blood (Eb), accumulated by the pancreas (Ep), and secreted during the next digestive cycle (shown by the
broad arrows on the left). In this scheme, the pancreas does not produce a new complement of digestive enzymes for
each meal but conserves and reutilizes these protein products.
studies were not failed attempts to reproduce the original activity disappears as the chyme passes down the small
observations, but different experiments altogether that bowel into the colon (8, 40, 51, 68). This has been as-
neither proved the circulation hypothesis false nor ex- sumed to be the consequence of enzyme degradation in
plained the affirmative evidence in other ways. transit and it alone.
Given these uncertainties, research on the topic soon This view is grounded in two presumptions. First,
ended, leaving the existence of digestive enzyme conser- because mixtures of activated digestive enzymes, for ex-
vation an unsettled matter, seemingly neither adequately ample, from pancreatic juice or tissue homogenates, left
confirmed nor adequately refuted. Indeed, the evidence standing at room temperature lose their activity over
has never been the subject of retrospective review and time, presumably due to auto- and heterocatalytic proteo-
analysis. This is our intention here some 25 years after the lytic degradation, the same events would occur at least as
initial discoveries. What did the evidence show, what actively in the intestinal lumen. It has been believed,
didn’t it show, and what remains to be learned? though not proven, that the pro- or inactive forms of
proteolytic enzymes such as trypsinogen and proelastase
II. FOUNDATION OF THE TRADITIONAL are completely and essentially instantaneously activated
VIEWPOINT: THE SINGLE PASS when they enter the intestines. This is thought to be due
to the presence of enterokinase, an enzyme secreted by
It may seem surprising, but the traditional under- the intestinal epithelium that splits a particular lysine
standing that the pancreas produces an entirely new com- containing peptide bond near the COOH terminus of
plement of enzymes with each meal is not based on direct trypsinogen. This then produces the active form of this
experimental evidence that this is the case, or even on enzyme, trypsin, which in turn activates the other proteo-
evidence that it is capable of such replacement. Nor is it lytic enzymes in what is referred to as the activation
based on proof that the digestive enzymes are completely cascade. Subsequently, the active proteolytic enzymes
degraded in the small intestines subsequent to their se- digest each other as well as nonproteolytic digestive en-
cretion, thereby requiring their replacement. What is zymes such as amylase and lipase, as the ingested food is
known is that 95–99% or almost all digestive enzyme being degraded.
Physiol Rev • VOL 82 • JANUARY 2002 • www.prv.org

CONSERVATION OF DIGESTIVE ENZYMES 3
The second presumption is that the epithelium of the digestive enzymes. As a consequence, if digestive en-
small bowel is completely impervious to proteins. If this zymes could be absorbed from the intestines into the
were true, then the only way that the digestive enzymes bloodstream, they could be taken up by the cells of the
could escape the intestinal enclosure would be by their pancreas and be secreted into the intestines where they
movement aborally into the large bowel. Because almost would be reutilized.
all activity is lost during this passage, some sort of inac- If such a process existed for substantial quantities of
tivation, presumably due to proteolytic degradation, digestive enzymes, they might be recirculated in a manner
would have to occur. Throughout the 20th century there similar to bile salts. Bile salts are released into the gut
was confidence, bordering on certainty, that biomem- from the liver and gallbladder where they emulsify fat.
branes were impermeable to protein molecules. Not only After fat absorption takes place, the unneeded bile salts
was there no direct experimental evidence for this con- are absorbed across the ileum, reaccumulated by the
clusion, but there was considerable evidence to the con- liver, conserved, and secreted again with the next cycle of
trary for the epithelium of the small intestines. It ap- digestion. This is called the enterohepatic circulation. The
peared to be permeable to a variety of protein molecules, liver only manufactures a small fraction of the bile salt
including various digestive enzymes (1– 4, 9, 13, 22, 32–34, pool de novo each day, and the possibility that digestive
41, 52, 53, 66, 99 –101). These results, however, were enzymes, which are far more energetically costly to man-
judged to be artifactual, pathological, quantitatively triv- ufacture, indeed are among the most costly biomolecules
ial, or exceptions to a general rule of impermeability. to synthesize, might be involved in a similar conservation
Whatever the evidence, the intestines of adult animals process was intriguing.
were widely believed to be impermeable to proteins, in- The capacity to conserve digestive enzymes was first
cluding digestive enzymes. tested in the mid 1970s. Two types of experiment were
This conclusion was reinforced by a third presump- carried out. In the first, the permeability of the relevant
tion. It was believed that if digestive enzymes, particularly membranes (pancreatic and intestinal) to particular di-
active proteolytic enzymes, entered the bloodstream, gestive enzymes was assessed in vitro. Did processes
massive systemic pathology would result, including the exist that would allow for a circulation? Second, given
uncontrolled “digestion” of the body’s organs. This would their existence, could circulation be demonstrated in in-
of course be life threatening, and because digestion nor- tact animals?
mally occurs in the absence of such a cataclysm, it was
safe to assume that digestive enzymes were not absorbed
III. MEMBRANE PERMEABILITY
into blood.
However, two unavoidable facts argued against this
conclusion. First, digestive enzymes, including the vari- A. Intestinal Epithelium
ous active proteolytic forms, were normal, even apprecia-
ble, constituents of blood (5, 7, 10, 11, 15, 17, 18, 36, 38, 39, The ileum was found to be permeable to the two
43, 73, 93, 96, 97, 98, 104). Although their presence might digestive enzymes that were tested, chymotrypsinogen
primarily reflect release directly from the pancreas into and amylase, using everted sacs of intestine (48) as well
the bloodstream, their intestinal absorption could not be as pieces of intestine as membranes separating two halves
excluded. Indeed, it was suggested by the presence of of an incubation (Ussing) chamber (21) to measure trans-
active proteolytic digestive enzyme. Second, blood port. After labeled chymotrypsinogen was added to the
plasma contained a variety of enzyme inhibitors, particu- medium bathing everted sacs of rabbit ileum (the mucosal
larly protease inhibitors, whose presence could, and in- compartment), it appeared in the internal medium of the
deed seemed designed to, prevent or mitigate pathologi- sac (the serosal compartment) at a rate some 10 times
cal occurrences that might occur pursuant to the that seen for a control protein, albumin (48). Similarly,
absorption of active enzyme. In fact, the second most amylase (55 kDa) appeared in the serosal medium of an
abundant plasma protein after albumin was a trypsin Ussing chamber soon after being added to its mucosal
inhibitor, ␣1-antitrypsin. compartment (21). In this case, the enzyme was trans-
It was the realization that digestive enzymes might be ported at a rate some two to three orders of magnitude
absorbed into blood that gave rise to the idea of an greater than seen for the control molecule, the much
enteropancreatic circulation. It had been discovered that smaller polysaccharide inulin (⬃9 kDa). In this study,
some of these proteins could be transported from the enzyme activity, rather than radioactivity, was followed to
interstitial space of pancreatic tissue across the basolat- ensure that the molecule had not been degraded en route.
eral surface of its secretion cells (29, 30, 45, 47), and it Amylase was transported intact across the epithelium in
was appreciated that material accumulated by these cells both directions, but was preferentially absorbed by an
might subsequently be secreted into the gland’s duct sys- active transport process (Fig. 2) (21).
tem and enter the intestinal lumen along with endogenous It was possible to estimate the absorptive capacity of

When radiolabeled forms of these enzymes were

added to the medium bathing the gland, providing access
to its interstitial fluid compartment, they appeared in
ductal fluid over time. Like the ileal epithelium, the pan-
creatic epithelial layer was permeable to these proteins.
Also, like the intestines, transport seemed to be selective.
For example, labeled chymotrypsinogen was secreted at
20 times the rate observed for the control protein albumin
(48). Again, the secretion of the exogenous material was
also measured as enzyme activity (31) to demonstrate that
the enzymes were transported across the gland intact and
functional.
The rate of transpancreatic transport could be sub-
stantial, greatly surpassing basal rates of endogenous se-
cretion. For example, maximal transpancreatic amylase
secretion was some 15 times the rate of unstimulated
endogenous secretion, or ⬃20% of a maximal endogenous
response to pharmacological stimulants. For chymotryp-
sinogen, transpancreatic secretion was 7.5 times the un-
stimulated endogenous rate, or ⬃30% of a maximal re-
sponse. This was in the absence of stimulants such as the
FIG. 2. Amylase transport in the mucosal to serosal direction
hormone cholecystokinin (CCK) or acetylcholine that
(M3 S) across ileal epithelium in vitro. The appearance of amylase in were found to greatly enhance transpancreatic transport
the serosal medium is shown as a function of time after its addition to as well as endogenous secretion (28, 48).
the mucosal bathing fluid, either in the presence or absence of oxygen
(95% O2-5% CO2 or 95% N2-5% CO2). The results show active amylase Transport across the pancreas was discovered to be
absorption. [Modified from Goetze and Rothman (21).] the result of protein movement through the gland’s secre-
tory epithelial or acinar cells, not extracellularly via para-
cellular shunts (31, 55–57, 92). For example, when chy-
the intestines from these measurements. With the use of motrypsinogen was added to the bathing medium several
conservative assumptions (that transport only occurs hours before applying a maximal cholinergic stimulus, the
across ileal epithelium and that the maximal rate possible response elicited by the stimulant subsequently was more
was being observed under the particular conditions of the than three times the maximal response seen without pre-
study), 50 – 65% of the amylase secreted by the rabbit incubation (31) (Fig. 3). This salutary effect was the result
pancreas in response to maximal stimulation would be of the prior uptake and storage of the exogenous enzyme
absorbed over a comparable period of time (21). Thus, at by the secretion cells. If endogenous amylase pools were
least as a first-order approximation, the potential existed labeled with radioactive amino acids, the addition of un-
to transport sufficient quantities of amylase across the labeled amylase to the medium bathing the gland not only
intestinal epithelium to support a significant conservation reduced the specific radioactivity of the secreted amylase
of this enzyme. by some 90%, meaning that ⬃90% was of exogenous ori-
gin, but produced a large (72%) absolute reduction in the
secretion of endogenous (labeled) protein (31). This was
B. Transpancreatic Transport the result of the mixing of exogenous and endogenous
enzyme in the cell and their subsequent competition for
Likewise, the potential for transport across the pan- exit from it.
creatic epithelium, i.e., from the interstitial spaces of the
gland to its duct system, was assessed. An organ culture
IV. CIRCULATION MEASURED IN SITU
preparation of rabbit pancreas was used for this purpose.
The rabbit pancreas is extremely thin and can be studied
whole in vitro without vascular perfusion (80, 86). The A. Circulation of Intact Digestive Enzymes
whole organ is removed from the animal and placed in an by Tracer Analysis
incubation chamber where it is bathed by a physiological
salt solution. The gland’s exocrine secretion can be col- A variety of studies had established that intestinal
lected pure and undiluted by cannulating the pancreatic and pancreatic epithelia were permeable to digestive en-
duct. As in the intestinal studies, amylase and chymotryp- zymes and that this permeability was of sufficient magni-
sinogen were the test molecules. tude to accommodate a conservation process. Although

site of its original secretion to follow its normal route. In

the third situation, the secreted fluid was collected and
injected into circulatory system via a large vein, rather
than being allowed to enter the intestinal lumen. All of
this was done in the continued presence of a maximal
cholinergic stimulus to drive protein secretion.
In the absence of enzyme circulation, there was no
reason to expect that the diversion of the secreted pro-
teins from the animal would influence the subsequent rate
of protein secretion by the gland. That would be deter-
mined by the cholinergic agonist acting locally on the
secretion cells, and there was no basis for thinking that
the removal of already secreted protein from the body
would affect its presence. For the same reason, reintro-
ducing the secreted enzymes into the intestines, or inject-
ing them into the bloodstream, was not expected to affect
FIG. 3. The transpancreatic transport of chymotrypsinogen (ChTg)
under basal conditions (basal), in the presence of 1 mg/ml chymotryp-
sinogen A (⫹ChTg), after the administration of acetyl-␤-methylcholine
(1 mg/100 ml bath fluid) (⫹Mch), and after the administration of Mch
subsequent to a 2-h preincubation in 1 mg/ml chymotrypsinogen A
(ChTg ⫹ Mch). Values are given (as ␮mol substrate split/min by secre-
tion) for the peak 10-min collection period, except for “basal.” [Modified
from Isenman and Rothman (31).]
the fact that the pertinent membranes were permeable

suggested that circulation would occur given the oppor-
tunity, it was important to demonstrate in situ that it did.
To test for circulation, labeled chymotrypsinogen was
instilled into the upper duodenum of anesthetized rabbits,
and secretory fluid was simultaneously collected from the
cannulated pancreatic duct to monitor for its appearance
(Fig. 4). Shortly after intestinal instillation, a small
amount of labeled protein (48) (Fig. 4) was recovered in
ductal fluid. Electrophoretic profiles established that the
label was still associated with chymotrypsinogen and had
not been reincorporated into the various endogenous pro-
teins secreted by the gland.
B. Magnitude of Circulation
Although this confirmed the existence of an entero-

pancreatic circulation, a different approach was needed
to assess its magnitude and hence the possibility of con-
servation. To make such a determination, the secretion of
digestive enzymes by the pancreas of anesthetized rabbits
was followed in three different experimental situations
(Fig. 5) (20). In the first situation, secretion was collected
directly from the duct system of the gland via a catheter FIG. 4. The circulation of labeled chymotrypsinogen. Top: drawing
and permanently removed from the animal. Equal shows the path followed by labeled bovine chymotrypsinogen (solid
circles) after its instillation into the duodenum (1), through its absorp-
amounts of albumin were instilled into the intestines in- tion (2), pancreatic uptake (3), and subsequent secretion (4). Bottom:
stead of the secreted enzymes. In the second experimen- graph shows the concentration of the labeled chymotrypsinogen as it
tal situation, after removal of a small aliquot for the appeared in pancreatic secretion after its instillation into the small
intestines. Electrophoresis demonstrated that the label remained asso-
measurement of its protein content, the secreted fluid was ciated with chymotrypsinogen. [Modified from Liebow and Rothman
immediately returned to the intestinal lumen close to the (48).]

and allowing it to be recycled. Furthermore, at steady

state, it would be immaterial whether the material had
been added back to the intestinal lumen or injected di-
rectly into blood. The effect would be roughly the same
either way, since only the location of reintroduction
would have differed.
As it turned out, all three conditions, diversion, in-
testinal instillation, and intravenous injection, altered se-
cretion by the gland and in the particular ways predicted
by the circulation hypothesis. When the secreted material
was diverted from the gut, protein secretion by the gland
declined in a roughly exponential fashion (Fig. 5). Both
reinstillation into the intestines and intravenous injection
of the secreted material ameliorated this decline and to
approximately the same degree (Fig. 5).
Perhaps the most convincing observation was that
protein secretion by the pancreas could be restored when
pancreatic fluid was injected into blood. This made no
sense in terms of a single pass mechanism. Why would
digestive enzyme in blood increase the rate of protein
secretion? The fact that the effect of intestinal instillation
and intravenous injection were comparable seemed to
rule out the possibility that digestive enzymes in the in-
testines had stimulated secretion by means of some sort
of intestinal nervous or hormonal action.
The efficiency of circulation could be estimated by
comparing the rate of secretion with diversion to that
after readministration of the secreted material (either
intestinal instillation or vascular injection). When the
amount readministered was plotted against the increase
in pancreatic secretion, input was related to output by a
linear function with a slope of 0.92 (Fig. 6). That is, the
FIG. 5. The bulk circulation of digestive enzyme. Top: drawing
shows three experimental conditions: 1) pancreatic secretion (open efficiency of circulation was 92%. In accordance with the
circles) diverted from the animal via a catheter, 2) pancreatic secretion theory, the data fit the same function whether the material
returned to the duodenum after a sample is removed for measurement of was added to the intestines or injected into the blood-
its protein contents, and 3) pancreatic secretion injected intraveneously.
Intestinal instillation and intravenous injection were carried out every 30 stream.
min. A cholinergic agonist (methylcholine chloride) and the peptide
hormone secretin were injected at half-hourly intervals for the duration
of the experiment, starting at time 0. Bottom: graph shows the amount
of protein in pancreatic secretion after the 3 different treatments. The
instillation or intravenous injection of the secreted protein (2 and 3)
enhanced subsequent protein secretion two- to threefold compared with
the diverted control (1). [Modified from Goetze and Rothman (20).]
secretion. From the single pass perspective, whatever one

might do to the material after it was secreted, removing it
from the animal, letting it enter the duodenum, injecting it
into blood, or anything else that we might think of, would
not be expected to affect the ensuing rate of secretion.
On the other hand, if enzyme conservation occurred,
under the right circumstances protein secretion would be
altered by each of these manipulations. It would be re-
duced if sufficient circulating enzyme was removed from FIG. 6. The relationship between the intestinal instillation or intra-
the animal by its diversion. Whereas the reinstillation or venous injection of digestive enzyme and the amount recirculated. Data
from both instilled and injected studies form a single function with a
intravenous injection of the same material would amelio- slope of 0.92; that is, 92% of the instilled or injected material is recircu-
rate that reduction by reintroducing the diverted enzyme lated. [Modified from Goetze and Rothman (20).]

Circulation seemed to be a process of substantial organs and tissues of the body was assessed and com-
magnitude for the particular conditions of study. We un- pared with that observed before stimulation (61). Amy-
derline this phrase to stress the importance of appreciat- lase distribution was appraised by measuring the en-
ing the circumstances in which circulation was examined. zyme’s activity, rather than by means of its physical
The results demonstrated, and only demonstrated, that separation or by radioactive labeling, to ensure that the
when secretion is driven by maximal cholinergic stimula- distribution of active enzyme was being surveyed.
tion for some 5 h in animals that were previously fasted The cholinergic stimulant was known to produce a
overnight, circulation was substantial. A period of maxi- significant reduction in the amylase content of the rat
mal, prolonged stimulation was chosen to ensure deple- pancreas and to increase amylase secretion into the in-
tion of tissue enzyme contents, and the animals were testines by at least an order of magnitude over unstimu-
fasted overnight to ensure the presence of substantial lated rates (81). Under these conditions, the decrease in
enzyme stores in the pancreas initially and to provide a the enzyme contents of the gland is roughly equal to the
more or less food-free intestinal tract. amount it secretes. If recirculation occurred efficiently,
One would not expect to see a similar effect in any then reducing the tissue content of digestive enzyme
and all circumstances, notably in the absence of substan- should add approximately the same amount of protein to
tial and prolonged stimulation of secretion to mobilize the circulating pools and should be measurable there.
large amounts of product for circulation, or in the ab- After an overnight fast (again, to maximize tissue
sence of an appropriate fast to maximize pancreatic en- content and minimize food in the small bowel), over 99%
zyme contents and provide an empty small bowel. In of the amylase activity in the rat was found in three
other conditions, for example during unstimulated secre- compartments: the pancreas with 92.1%, the intestines
tion or after short periods of stimulation, the amount (mucosa plus contents) with 5.9%, followed by the plasma
circulating might be small and difficult to measure. And if (plus interstitial fluid) with 1.3% (61). How did things
the animals were not fasted, the presence of food in the change after a 3-h course of stimulation? According to the
intestines might delay circulation in an uncharacterized, single pass hypothesis, the amylase lost from the pan-
meal-dependent manner as food substrate competed with creas should be found exclusively in the intestines, pri-
the absorptive process for the enzymes. marily in the small intestines, minus whatever degrada-
tion had taken place.
C. Effect on the Plasma Pool Remarkably, after 3 h of continuous highly aug-
mented secretion into the small intestines, no significant
Although these experiments provided convincing ev- elevation in amylase activity was seen at this location
idence for bulk circulation, they did not actually follow (Fig. 7). That copious amounts of the enzyme had entered
the movement of the secreted products through the rele- the small bowel could be confirmed by large increases in
vant compartments, most importantly into and through amylase activity seen in the cecum (two orders of mag-
blood. This was subsequently shown in awake rats. As in nitude above controls levels) and colon (61). Why wasn’t
the rabbit studies just discussed, rats were given a max- there a comparable, or even a greater increase, in the
imal cholinergic stimulus for an extended period of time small intestines? This could be explained in three ways.
(3 h), after which the distribution of amylase in various The secreted amylase might have simply passed into the
FIG. 7. Change in the percentage of the

whole body distribution of amylase activity
attributable to the pancreas, small intestine
(intestines), and blood plus interstitial fluid
(blood) after 3 h of cholinergic stimulation
(depletion, stipled bars) and a subsequent
2-h period of recovery (solid bars). Note that
at 3 h (depletion) amylase lost from the pan-
creas is in great part found in blood, not the
small intestines. Similarly, the recovery of
pancreatic amylase content is accompanied
by an equivalent loss of amylase from blood.
[Modified from Miyasaka and Rothman
(61).]

large bowel from the small intestines, as the elevated reciprocal fashion (61) (Fig. 7). That is, the rate at which
levels there might suggest. Alternatively, it might have amylase disappeared from blood was correlated with its
been degraded or otherwise inactivated in the small in- reappearance in the pancreas. It was not distinct from it,
testines. Finally, it may have been absorbed. as we might expect if we were dealing with two unrelated
As it happened, it was clear that the missing amylase events, for example, if the disappearance of amylase from
had not simply passed through. Although enzyme levels in blood was the consequence of its breakdown in the liver
the cecum and colon were greatly elevated, they still only or its excretion via the kidney, while its appearance in the
represented a tiny fraction of the activity found at the pancreas was due to the synthesis of new amylase.
same time in the small bowel even without any elevation
(⬍1%) (61). Nor did it seem likely that degradation ac-
V. ATTEMPTS AT CORROBORATION
counted for the missing amylase activity. Although the
rate of amylase degradation in the small intestines is
Taken together, the observations just outlined pro-
unknown, it must be far lower than its rate of secretion.
vided a compelling case for the conservation and circula-
How else could the enzyme be of use in digestion? What-
tion of digestive enzymes. It was not long before the
ever its rate, if we assume that degradation is increased in
phenomenon was corroborated (24, 37). However, almost
proportion to the increase in amylase secreted, we would
as quickly there were reports that questioned its existence
nonetheless expect to see a substantial, indeed a propor-
(44, 74, 78). As noted at the outset, these negative obser-
tional increase in amylase activity. And, of course, how-
vations were not failed attempts to reproduce the findings
ever much degradation may have occurred, how do we
that we have just discussed, such as the effect of the
explain the fact that increases at least proportionately as
diversion, reinstillation, and intravenous injection of se-
great as those seen in the large bowel and cecum were not
creted protein on pancreatic function, but entirely differ-
observed in the small intestines?
ent experiments that focused in particular on one predic-
This left the third possibility. Amylase was absorbed.
tion of the circulation hypothesis. For circulation to
This could be assessed simply by looking for the missing
occur, enzyme in blood must be taken up by and trans-
amylase in blood or in tissues that might be accessed
ported across pancreatic tissue into the gland’s duct sys-
through the bloodstream. When plasma levels were mea-
tem. Two subsidiary hypotheses, or what were thought to
sured, much of the missing amylase activity was found.
be subsidiary hypotheses, of this understanding were
Plasma and interstitial fluid amylase now represented
tested.
⬃13% of the total amylase contents of the body, an order
of magnitude increase above the ⬃1% seen before stimu-
lation. It was now the second largest amylase pool after A. Pancreatic Arteriovenous Concentration
the pancreas, and more than double the intestines (61). Difference
Most importantly, the increase accounted for most of the
amylase that had been secreted by the pancreas (Fig. 7). The first hypothesis was that if uptake occurs, diges-
Because it was known that amylase is also secreted tive enzymes in plasma should be extracted during the
directly from the pancreas into blood (28, 60), the ele- passage of blood through the pancreatic capillary bed. As
vated blood levels might have been the consequence of a consequence, they should be present at lower concen-
this process, not the absorption of enzyme. But this made trations in venous blood leaving the gland than in arterial
little sense, since in this case we would have to conclude blood entering it. When such measurements were made,
that almost all of the amylase secreted by the gland after venous blood had a higher, not a lower, amylase concen-
cholinergic stimulation had been directed into the blood- tration (44). This meant that the enzyme was being re-
stream, not the gut, and this was observably false (28, 81). leased from the pancreas into blood, not withdrawn from
The occurrence of circulation was further validated it, as predicted. From this observation it was concluded
by what happened when the stimulant was withdrawn. that digestive enzymes are not taken up by the pancreas
Two hours after its withdrawal, the percentage of amylase from blood and as such cannot cross the gland. Of course,
in the pancreas had recovered to values close to those if they cannot cross the gland, then their circulation
found in fasted rats (61) and once again contained ⬃90% through the pancreas is not possible.
of the total in the body. This rapid replenishment was However, what was observed only applied to the
consistent with microscopic studies in which recovery in particular conditions that were examined. One could not
the volume density (the volume of the cell occupied) of generalize, as was done, from it to all states and circum-
zymogen granules (the structures in which the digestive stances. Given that digestive enzymes equilibrate across
enzymes are stored) occurred over roughly the same pe- the basolateral membrane of the acinar cell (29, 30, 45), it
riod of time for the same conditions in the same species could not be concluded that there would always be a net
(81). Whatever the rate of recovery, as pancreatic levels of movement of amylase from tissue to blood whatever the
amylase increased, its level in plasma fell in a roughly physiological state. This turned out to be particularly

important since the researchers had infelicitously chosen known. Only then can we calculate how much unlabeled
to look for net amylase extraction from blood under the material is moved for a given amount of radioactivity.
most unfavorable conditions possible. They examined Such knowledge was lacking in these studies and conse-
fasted dogs under general anesthesia. In this circum- quently it could not be assumed, as it was, that the radio-
stance, the pancreas does not secrete significant quanti- labeled material traced the movement of endogenous sub-
ties of digestive enzymes into the intestines. The amount stances. The fact that only small, seemingly negligible
of enzyme secreted and made available for circulation quantities of labeled protein appeared in pancreatic se-
would be expected to be one to two orders of magnitude cretion said nothing about the movement of unlabeled
less than during active secretion. Hence, given the exis- protein.
tence of an ongoing release of digestive enzymes from the On entering the systemic circulation, either as a re-
pancreas into blood (28), it was not surprising that a net sult of intravenous injection or absorption, marker mole-
uptake of enzyme by the pancreas was not seen. The cules like the labeled digestive enzymes that were used,
judgement that there would never be a net uptake of first equilibrate with their endogenous forms in plasma,
amylase by pancreatic tissue because of what was ob- and, where they exit the capillary beds, in the various
served in this particular circumstance was like conclud- interstitial fluid compartments of the body. That is to say
ing that because most passengers on the New York sub- the labeled digestive enzymes would not be “attracted” to
way are heading from the Boroughs to Manhattan at 8 A.M. the pancreas as if it were some sort of magnet but would
that this would also be true at 5 P.M. be dispersed broadly throughout the body. Only after this
broad distribution takes place can there be any sizable
clearance by the pancreas.
B. Intravenous Injection of Radiolabeled As a consequence, one would expect that only a
Digestive Enzyme small percentage of the labeled material would end up in
the interstitial spaces of the pancreas initially and be
The second prediction was that if radiolabeled diges- available for uptake and transport across the glandular
tive enzymes were injected into the systemic circulation epithelium. We can estimate how much from the mass of
they would be accumulated by the pancreas and subse- the pancreas in relation to the total soft tissue mass of the
quently found in its exocrine secretion. Two groups (74, body, assuming that the size of the interstitial space is a
76 –78) tested this hypothesis, and both reported that little relatively constant proportion of the fractional mass of all
if any of the injected material was secreted by the gland soft tissues and that most capillary beds are equally per-
for the times and circumstances they examined. Like the meable to these substances. Calculated in this way, the
workers who measured arteriovenous concentration dif- interstitial space of the pancreas would contain ⬃0.15% of
ference, they concluded that transport of digestive en- the injected aliquot after its initial equilibration through-
zymes across the pancreas does not occur and hence that out the extracellular spaces of the animal. The actual
their circulation through this gland is not possible. number might be somewhat smaller or larger depending
The supposition seemed to be that if the circulation on the suitability of the assumptions used in making the
hypothesis was correct, then most, or at least a large calculation, but even if it were an order of magnitude
percentage, of the injected material would appear forth- larger, only a small percentage of the injected material
with in exocrine secretion as the gland rapidly cleared the would be found in the interstitial spaces of the pancreas
plasma of the labeled protein, much like the kidney clears initially. Given a value of 0.15%, finding 3– 4% of the in-
it of glucose. One group thought that the recovery of 3– 4% jected label in pancreatic secretion is impressive.
of the injected material in pancreatic secretion was too In any event, as said, only after its broad dispersion
small to be considered meaningful. throughout the body can the labeled protein be effectively
The use of radiolabeled enzymes in these studies was redistributed to the pancreas and other tissues that might
predicated on the understanding that the radioactive ma- accumulate it. If, for the sake of estimation, we assume
terial “traced” the movement of the bulk of unlabeled that this redistribution occurs by the same diffusive and
digestive enzyme; that is, if little labeled enzyme crossed flow processes that led to the initial distribution, then it
the pancreatic epithelium, then the same could be said of would occur more slowly to the degree that the exoge-
unlabeled digestive enzymes of endogenous origin. This is nous (labeled) material is diluted by its endogenous (un-
called tracer kinetics, and for it to apply two things must labeled) forms in blood and the various interstitial spaces.
be known. First, the labeled material must have equili- The greater the dilution, the longer redistribution would
brated with the unlabeled form of the substance in all take.
relevant compartments, in this case in the various extra- For the case at hand, we would expect a considerable
cellular pools of the body. Second, the ratio of the labeled dilution because the concentration of digestive enzymes
to the unlabeled material, referred to as specific radioac- in the relevant endogenous compartments is substantial,
tivity (SRA), in each of these compartments must be in the tens to hundreds of nanograms per milliliter range

(28). The amount of labeled protein moved per mole of with a variety of other tissues. For example, the liver
total digestive enzyme would be reduced in proportion to contained about an order of magnitude more per unit
this dilution. For example, given the same forces driving tissue mass. However, with the passage of time, the
movement, if the ratio of labeled to unlabeled protein amount in the pancreas increased greatly, indeed magni-
(SRA) was reduced by two orders of magnitude during an tudinally, while that in other tissues either decreased or
initial period of equilibration of say 10 min, it would take only increased modestly (Table 1). By 7 h after the injec-
1,000 min, or ⬃16 h, for redistribution to occur. Dilution tion, the pancreas had the highest concentration of any
by its unlabeled form would in this sense “trap” the la- tissue examined, and within an hour its contents had
beled material in the animal’s extracellular spaces. Con- increased some 24-fold and were higher than that found in
sequently, it is not justified to conclude, as was done, that the liver. The second largest increase was for intestinal
because little tracer surfaced in pancreatic secretion in tissue (Table 1), which presumably for the most part
the short run, that more would not be found there in due comes from the pancreas. If we factor this in, at 7 h the
course. increase was 43-fold.
Moreover, the appearance of labeled protein in pan- The authors thought that the presence of labeled
creatic tissue does not guarantee its immediate secretion. protein in the pancreas was due to the uptake of labeled
Exogenous digestive enzymes taken up by the acinar cell amino acids previously released from the injected protein
mix with its endogenous stores (31) just as they do in the elsewhere in the body and its subsequent incorporation
extracellular spaces of the body and are diluted by them. into the endogenous protein pool of the gland. Their
Except that the dilution in this case is far greater because evidence for this conclusion was that when they collected
the pancreas contains these proteins at very high concen- secretion from the pancreatic duct of rats after the intra-
trations (mM). In any event, once again the labeled pro- venous injection of a mixture of radiolabeled guinea pig
tein would be “trapped,” unable to leave the acinar cell in digestive enzymes, only radiolabeled rat protein was
any quantity due to its severe competitive disadvantage in found in secretion as estimated by two-dimensional gel
relation to the far more abundant unlabeled enzyme re- electrophoresis.
serves. In fact, even the complete turnover of the cellular Although the amount of labeled protein in secretion
enzyme pool would not guarantee its expulsion, because was small and the distinction between rat and guinea pig
it would remain, at all times, at a severe competitive spots in the electrophoretogram uncertain in some re-
disadvantage for exit from the cell. Its release could only spects, at least some of the labeled protein appeared to
be ensured if the gland was fully depleted of its protein comigrate with unlabeled rat proteins, suggesting that
contents during active, stimulant-induced exocrine secre- they might also be rat proteins. Two measurements were
tion. needed to validate this interpretation. The first was a
Although it seemed unremarkable to these workers, control in which small amounts of labeled guinea pig
when they made observations over an extended period, a protein (similar to the amount of labeled protein found in
substantial redistribution of the labeled material to the secretion) would be admixed with larger amounts of un-
pancreas was actually seen, just as predicted by the cir- labeled rat digestive enzyme (the same amount placed on
culation hypothesis (Table 1) (76). Shortly after injection the gel in the experiment) and subjected to electro-
of the radiolabeled enzyme (15 min), the pancreas con- phoretic separation. In this case, where small amounts of
tained a low concentration of labeled protein compared labeled guinea pig proteins were known to be present,
were distinct guinea pig spots seen and were rat spots
devoid of label? The second measurement is to show that
TABLE 1. Tissue levels of radioactivity after injection of all cellular label is associated with rat protein and that
[35S]methionine-labeled pancreatic exocrine proteins guinea pig protein is not in the acinar cell. Neither mea-
into the blood circulation surement was provided then or subsequently, and as a
result the authors’ claim was not experimentally substan-
TCA-Insoluble Radioactivity, Change From
dpm/100 mg tissue ⫻ 10⫺2 15 Min, % tiated.
There was another issue. As in the studies of arterio-
Organ or Tissue 15 min 60 min 420 min 60 min 420 min
venous concentration difference, the researchers had
Kidney 311 170 60 ⫺56 ⫺81 only studied the unstimulated glands of anesthetized an-
Lung 18 24 17 ⫹33 ⫺6 imals. As said, under these conditions, the pancreas only
Spleen 30 47 37 ⫹57 ⫹23
Liver 29 48 41 ⫹66 ⫹41
secretes small amounts of protein. Thus once again tissue
Skeletal muscle 2 5 9 ⫹250 ⫹450 uptake as well as transpancreatic transport was being
Intestine 3 29 46 ⫹967 ⫹1,533 sought in a setting in which we would expect both to be
Pancreas 3 72 83 ⫹2,400 ⫹2,767
minimal.
Times (in min) refer to time after injection. [Modified from Rohr Finally and significantly, accepting their results as
and Scheele (76).] presented, they neither explained away nor otherwise

accounted for what at the time was the already substan- aspirates from different portions of the gut taken at dif-
tial published evidence, some of which we have dis- ferent times. As expected, with the passage of time the
cussed, that digestive enzymes are taken up by pancreatic presence of the three enzymes in the upper small bowel,
tissue from its interstitial space and transported across or duodenum, first increased and then decreased, return-
the gland in rabbits. In a contemporaneous dialogue in ing at variable rates over a 3- to 6-h period to values
print about some of these issues (6, 42, 75, 79, 87–90, 94) similar to those seen before the meal. But almost every-
we argued that the “falsifying” studies were equivocal and thing else that was observed was not this straightforward.
incomplete and subject to interpretations more benign to As the chyme moved aborally, through the jejunum to
the circulation hypothesis than had been allowed. We still the ileum, only 1% of the lipase activity was still present,
believe this to be true. but over 20% of the trypsin and some 75% of the amylase
made the passage without being either degraded or ab-
sorbed. The authors explained these large differences as
C. Fate of Digestive Enzymes in the Intestines being due to variations in the resistance of the different
enzymes to degradation and additionally for amylase, to
As mentioned at the outset, the traditional under- the protective effect of its binding to the starch substrate,
standing that the digestive enzymes are degraded in the although they presented no evidence to support either
small bowel along with their substrates (the ingested food suggestion. The observed differences might just as well
and the other digestable contents of the small intestines) have reflected differences in the rate and location of the
was not the result of the direct measurement of degrada- absorption of the three enzymes. In any event, and signif-
tion and its correlation to the loss of enzymatic activity. If icantly, it turned out that for all of the enzymes, what
degradation is the fate of all digestive enzyme secreted passed into the lower small bowel was apparently intact
into the bowel subsequent to the ingestion of a meal, then or almost intact enzyme, not substantially degraded prod-
such measurements would show that over time the dis- ucts.
appearance of enzymatic activity in various aboral loca- If one looks at the concentration of enzyme (amount
tions was accompanied by the concomitant and quantita- per unit volume of aspirate), although lipase concentra-
tively equivalent appearance of small peptides and amino tion decreased by some 97% between the duodenum and
acids liberated by that degradation. ileum as measured by activity, its immunoreactivity was
Making such measurements admittedly presents sig- retained. That is, the enzyme appeared to be essentially
nificant problems, but an attempt was made by research- intact or at least sufficiently intact to react with its anti-
ers to assess the situation in a simpler way. They com- body. Trypsin, on the other hand, had become less immu-
pared observed levels of enzyme activity in the intestines noreactive but retained its enzymatic activity, suggesting
to the amount of the enzyme bound in a radioimmunoas- nondegradative structural change. Finally, amylase con-
say (40). As enzyme is degraded, the peptide fragments centration increased.
produced by degradation would either be unable or be- The authors believed that they could exclude absorp-
come increasingly unable to bind to the antibody. The tion from consideration because the disappearance of
parallel loss of enzymatic activity and immunoreactivity immunoreactivity and enzymatic activity did not occur in
would be consistent with the loss of activity by enzyme parallel for either lipase or trypsin. As said, they under-
degradation. Perhaps this can be appreciated with a stood that for every absorbed molecule both its enzymatic
counter example. If enzymatic activity disappeared, but activity and immunoreactivity should disappear from the
not immunoreactivity, we would have to conclude that gut in concert, and this was not seen. However, this
some event other than degradation had produced the explanation holds only if one does not consider alter-
reduction in enzyme activity. ations to the molecules that might occur before their
The researchers who carried out this work under- absorption. For example, given that nondegradative
stood that the parallel disappearance of enzymatic activ- chemical modification takes place in the intestines, al-
ity and immunoreactivity could be the consequence of tered, but still intact, molecules might be absorbed as well
enzyme absorption as well as degradation. In this case, as as or even in preference to the unaltered molecule.
enzyme is absorbed both activity and immunoreactivity In the final analysis, one could not conclude from this
would also disappear. Consequently, the authors con- study that the disappearance of enzyme activity was the
cluded from their measurements that secreted digestive result of degradative processes alone, as was done. Not
enzyme was degraded, not absorbed, but careful exami- only didn’t the data provide reason to choose between
nation of their data reveals something else. degradative and absorptive explanations for the disap-
Experiments were carried out on human subjects pearance of enzyme activity, remarkably there was no
after the ingestion of a meal comprised solely of starch. proof of enzyme degradation at all. In fact, the mainte-
Enzymatic activity and immunoreactivity of lipase and nance of enzyme activity (trypsin and amylase) or immu-
trypsin, and the activity of amylase, were measured in noreactivity (lipase) in the ileum seemed to argue against

it. What was observed was exactly what would be ex- A. Basal Rate of Protein Secretion
pected if absorption had taken place: the disappearance
of enzyme from the bowel in the continued presence of One way to estimate the rate of synthesis is from the
intact digestive enzyme within it. rate of secretion seen in the relative absence of exoge-
nous nervous and hormonal stimuli, that is, “unstimu-
lated” or “basal” secretion. Under these circumstances
VI. RATE OF SYNTHESIS OF DIGESTIVE tissue contents remain unchanged despite ongoing low
ENZYMES COMPARED WITH THEIR levels of secretion. As such, what is being secreted is
RATE OF SECRETION being quantitatively replaced. If replacement is due to
new synthesis, then the rate of secretion is equal to the
Another way to approach the question of the conser- rate of synthesis.
vation of digestive enzymes is to assess the capability of If a significant fraction of new digestive enzyme does
the exocrine pancreas to manufacture sufficient quanti- not successfully negotiate the posttranslational phase of
ties of these proteins to replace those it secretes (88). If production, that is, is degraded without ever being se-
the gland is not capable of meeting this need, then a creted, this method underestimates the true synthetic
conservation of digestive enzymes is not merely sug- rate. However, this is not the rate in which we are really
gested, but required. Over time, shortfall would build on interested. Rather, what we want to know is how much
shortfall, deficiency on deficiency, until the gland was new protein is available to be secreted to replace previ-
ously released protein.
depleted of its enzyme contents and unable to respond to
In the fasted anesthetized rat, secretion in the ab-
stimuli.
sence of exogenous stimuli ranges from ⬃0.25 to 1.0 mg
The most common method of measuring protein
protein䡠g tissue⫺1䡠h⫺1 (81). Tissue levels of enzyme are
synthesis, following the rate of incorporation of radio-
constant at rates of secretion in this range, and hence they
active amino acids into protein molecules, although provide a good estimate of the “effective” rate of protein
useful for comparing synthetic rate under different con- synthesis, the amount of new protein available for secre-
ditions is not satisfactory for quantitative measure- tion under these conditions. On the basis of these values,
ments of the sort needed. There are, however, other between 6 and 24 mg protein are manufactured each day
ways to assess the sufficiency of synthesis. Next we per gram of tissue, or some 4 –35% of the amount secreted
consider three approaches, each dependent on differ- (Table 2). With the assumption of the highest estimate for
ent variables. synthetic rate and lowest for daily secretion, synthesis is
First we must settle on a value for protein secretion still only about one-third of that needed.
to compare to estimates of synthetic capacity. As we have
already discussed in another context, the question is not
whether synthesis is capable of replacing what is secreted TABLE2. Estimates of the rate of protein synthesis
under some circumstances, but under all circumstances, by the exocrine pancreas
particularly when rates of secretion are high and the gland
Estimated Range
is partially or fully depleted of its enzyme contents, as is
frequently the case. The important question is whether Based On: Protein 䡠 g tissue⫺1 䡠 day⫺1 Tissue pool/day, %
under these conditions the recovery of tissue digestive
Basal secretion 6–24 9–34
enzyme content is due solely to new synthesis? Product of ribosome
Using data from fasted anesthetized rats (25, 61, 81), number and time for
synthesis of peptide
and extrapolating to daily rates of secretion, we estimate chain 8–17 11–24
the amount of protein secreted by the pancreas to be Oxygen consumption 5–10 7–14
between 70 and 140 mg protein䡠g tissue⫺1䡠day⫺1. We have Stimulant enhanced 7–29 10–41
assumed that secretion occurs at low or unstimulated Rate of secretion 70–140 100–200
rates for 75% of the day and at highly elevated rates for
25%. Under these conditions, tissue digestive enzyme Percent of tissue pool per day is based on 70 mg protein/g tissue
(that found in rat pancreas after an overnight fast). Basal secretion is
stores would turn over, in one way or another, once to based on the equivalence of basal secretion to the effective rate of
twice daily based on the observed digestive enzyme con- synthesis. Product of ribosome number and time for synthesis of peptide
chain is based on 106 attached ribosomes per cell and a synthetic rate of
tents of 70 mg protein/g tissue in rats after an overnight 3 min/chain. Oxygen consumption compares oxygen consumption to the
fast (25). This range is conservative, that is, biased toward cost of peptide bond synthesis. Stimulant enhanced refers to elevations
the possibility of synthetic sufficiency. Actual 24-h rates over basal rate (given in “basal secretion”) of 50% for 5 h (based on the
experimental literature). Rate of secretion is given for comparison. It is
of secretion in awake animals fed ad libitum would likely based on 75% at the basal rate (0.25–1.0 mg 䡠 g⫺1 䡠 h⫺1) and 25% at 10 –20
be larger, not smaller. mg 䡠 g⫺1 䡠 h⫺1 (estimated from data in the rat, Refs. 25, 61, 81).

B. Enhancing Synthesis long, or 15 h, only 5 mg of new protein would be pro-

duced. For recovery to be complete in this case, synthesis
Couldn’t this shortfall be made up by increases in the would have to remain elevated for more than 4 days.
rate of synthesis after periods of active secretion? The Obviously, this is far too long for animals like rats and
evidence does not support this possibility. There are nu- humans that feed daily or more frequently.
merous reports, in an abundant literature (for example, The replenishment of tissue contents normally oc-
Refs. 12, 23, 26, 35, 54, 62– 65, 70, 103), of elevations in curs within a few hours of the cessation of active secre-
synthetic rate after the administration of stimulants of tion. For the circumstances described above in rats, it
secretion. However, the effect is modest, generally in the takes just 2 h to reach levels that are roughly equivalent to
range of 25–50%. Sometimes no enhancement is seen or those seen in fasted animals (25, 61). Even if it took 15 h,
even a decrease, whereas on the other hand, the largest or a full 24 h, synthesis could not account for restocking
increase ever reported is about a doubling (54). at the increases in rate that have been observed.
Whatever value we might settle on, it would not be
nearly enough to meet the need. We should understand
that without boosting synthetic rate, even a gland that is C. Ribosomes
only slightly depleted of its enzyme contents during peri-
ods of active secretion would be unable to recover what Another means of estimating the rate of synthesis is
it lost whatever the rate of synthesis might be. When to calculate the product of the time it takes to manufac-
secretion returns to low, “unstimulated” rates after a pe- ture a single peptide chain and the number of ribosomes
riod of active secretion, protein manufacture once again actively engaged in the task, since proteins are manufac-
equals the rate of secretion, and consequently no accu- tured in a one to one correspondence to ribosomes. It
mulation of enzyme can occur. takes several minutes to produce single peptide chains in
As it happens, during periods of active secretion the eukaryotic cells (49, 50, 67, 69, 71, 106). More particularly,
enzyme content of pancreatic tissue declines substan- it takes ⬃3 min to manufacture a molecule of pancreatic
tially, sometimes dramatically. For example, after 3 h of ribonuclease or amylase (67, 71). The number of ribo-
highly augmented secretion in the awake rat, tissue con- somes in the secretion (acinar) cells of the pancreas
tent decreases by ⬃50% (25, 61). For synthesis to account engaged in digestive enzyme synthesis can be estimated
for the recovery under such circumstances, it must be by counting those attached to the endoplasmic reticulum
greatly elevated. The rate of recovery would be propor- in electron micrographs. This number is thought to be the
tional to the increase in synthetic rate. For example, if same, or almost the same, as the number of digestive
secretion was elevated by an order of magnitude in re- enzyme chains being manufactured based on the under-
sponse to a meal, as commonly occurs, synthesis would standing that 1) digestive enzymes are produced exclu-
have to be increased by an order of magnitude for recov- sively on ribosomes attached to the membranes of the
ery to occur in the same time frame as loss from the endoplasmic reticulum (ER); 2) all of these ribosomes are
gland. If synthesis only increased by 50%, a relatively high actively engaged in protein manufacture; and finally 3) the
value, it would take 20 times as long to replace what was great majority are busy manufacturing digestive enzymes,
lost as to lose it. More particularly, if secretion was mag- not other proteins such as membrane proteins, proteins
nitudinally elevated for 3 h and returned to the unstimu- destined for the ER itself or for organelles such as lyso-
lated state immediately thereafter, it would take 60 h, or somes. These discernments are widely believed to be true,
2.5 days, to replace that lost. but whether they are or are not, by presuming them true,
Even this is unrealistically optimistic, because it as- we bias our estimate toward higher synthetic rates.
sumes that synthetic rate remains elevated for 60 h until The ER is a striking feature of the pancreatic acinar
recovery is complete, and this is not seen. Synthetic en- cell and occupies some 20% of the cell’s volume (16). It
hancement occurs primarily in the presence of stimulants, appears to be comprised of numerous elongated, ribo-
that is, during periods of tissue depletion, not recovery, some-studded membrane sacs that are stacked upon each
and in any event only lasts for a few hours. For the other. By making some simple assumptions about the
example given above, the rat pancreas loses ⬃35 mg of its arrangement of ribosomes on the membranes and how
digestive enzyme contents (50%) after 3 h of active secre- the ER sacs fill the volume of the cell they occupy, the
tion (25, 61). If the rate of synthesis were elevated by 50%, number of ribosomes present can be calculated. Specifi-
and secretion returned to its unstimulated rate with re- cally, and to further bias our estimate toward higher
moval of the stimulant, synthesis would only have pro- synthetic rates, we assume that the membranes are ar-
vided about a milligram of new protein, or just a few ranged in orderly rows and that the ribosomes attached to
percent of the 35 mg needed for the recovery of tissue them are packed densely in a rectilinear array, separated
contents. Even if the elevation in synthesis lasted well from each other by the diameter of a single ribosome. We
beyond withdrawal of the stimulant, say five times as chose a 60-nm spacing between sacs based on what is

seen in electron microscopic images. Given this geome- the gland’s mass as a percentage of the total mass of the
try, there are about a million attached ribosomes per cell. animal (59), indicating that at least in the rat, the pancreas
If the stacking of ER membrane is not this regular or the is about as metabolically active as other tissues on aver-
placement of ribosomes on their surface not this dense, or age.
both, as appears to be the case, then this is an overesti- To replace the 70 mg/g tissue digestive enzyme pool
mate, and hence overestimates synthetic rate, again bias- of the fasted rat once a day would require 238 J or almost
ing the result in favor of synthetic sufficiency. 10 times the available energy. Thus putting the cell’s other
With the use of 3 min as the time required to synthe- needs aside, ⬃10% of the digestive enzyme pool can be
size an average digestive enzyme chain, 20 protein mole- replaced daily, or ⬃5–10 mg protein can be synthesized
cules would be manufactured per hour per attached ribo- per gram of tissue each day, or ⬃4 –15% of our estimate of
some assuming that there is no significant delay between the daily rate of secretion (Table 2). With the assumption
the manufacture of one chain and the next on a given of the highest estimate for synthesis and the lowest for
ribosome (any delay would of course reduce the rate of secretion, we would still have a shortfall of some seven-
synthesis). Given a million attached ribosomes per cell, fold.
⬃1.2 ⫻ 1016 protein molecules䡠h⫺1䡠g tissue⫺1 would be
manufactured. Applying Avogadro’s number and convert-
E. Summary
ing to mass (assuming an average molecular mass of 30
kDa for the digestive enzymes), 1 g of tissue is capable of
Although calculations such as these always present
producing ⬃0.67 mg of digestive enzymes per hour. Given
uncertainties, and must to some degree be considered
the various assumptions made in making this calculation,
speculative, it is hard to envision errors that would be of
half this value, or 0.34 mg䡠g⫺1䡠h⫺1, is probably a more
sufficient magnitude to invalidate the conclusion that syn-
realistic, but still a generous, estimate. This amounts to
thesis alone is not able to replace secreted protein under
8 –16 mg protein synthesized䡠day⫺1䡠g tissue⫺1, or about
circumstances in which active secretion occurs. The cal-
6 –24% of the amount we estimate is secreted per day
culations are based on published and well-documented
(Table 2). Once again, there is a substantial shortfall.
experimental measurements, and moreover, to mitigate
Even if we use our highest estimate for synthesis and
error, wherever possible we have tried to choose values
lowest for secretion, the shortfall is fourfold.
that would maximize synthetic rate and minimize secre-
tory capacity to bias our numbers toward synthetic suffi-
D. Energy Requirement ciency. The fact that three independent means of estimat-
ing the rate of synthesis, each depending on different
variables, yield quite similar results (5–10, 8 –16, and 6 –24
A final way to judge synthetic rate is to ask whether
mg䡠g⫺1䡠day⫺1), suggests that the rate of synthesis in these
the pancreas can support the replacement of the digestive
glands is within this range. If so, this is far less than any
enzyme pool from an energetic perspective? That is, does
reasonable estimate for the rate of secretion from actively
it generate enough energy to replace what it loses by new
secreting glands. The conclusion that synthesis is unable
synthesis? As said, protein synthesis is among the most
to replace what is secreted is reinforced by the fact that to
costly biological reactions. The energy required to form
restock depleted glands by means of synthesis alone re-
the peptide bond has been estimated to be ⬃3.5 kJ/g
quires an enhancement, in either magnitude or duration,
protein (58, 59, 72, 95, 102). The pancreas’ available en-
of synthetic rate that is well beyond anything that has
ergy has been measured experimentally as oxygen con-
been observed.
sumption (14, 19). Metabolism is primarily oxidative in
Whatever uncertainty there may be, these estimates
this gland, and values for oxygen consumption reflect its
cannot be taken, either singly or together, as support for
energy utilization relatively well. Thus, knowing the cost
the view that the gland is capable of meeting its need for
of synthesis and the available energy, we can estimate
digestive enzymes solely by new synthesis. To the con-
how much protein can be manufactured by the pancreas
trary, they provide support for the conservation of diges-
per unit time, assuming, incorrectly of course, that all of
tive enzymes and buttress the experimental evidence for
the energy is used for this purpose.
such a process presented above.
Oxygen consumption in the pancreas is ⬃0.01– 0.02
ml䡠g tissue⫺1䡠min⫺1 at standard temperature and pressure
in mammals (14, 19) or 14 –28 ml oxygen䡠g tissue⫺1䡠24 VII. MEMBRANE PROTEIN TRANSPORT
h⫺1. With the assumption that this in the main reflects
glucose oxidation, 1.2 J would be produced for every In section I we mentioned that some of the initial
milliliter of oxygen consumed, and as a consequence, the hesitancy to accept the existence of a conservation of
pancreas would generate somewhere between 17 and 34 digestive enzymes was due to the widely held belief that
J䡠day⫺1䡠g tissue⫺1. This value is roughly in proportion to biological membranes were impermeable to protein mol-

ecules. Because conservation required the transport of Active enzymes might be temporarily inactivated or
digestive enzymes across both intestinal and pancreatic otherwise transformed in the intestines before their cir-
epithelia, minimally across four separate membranes, culation. In this regard, the pancreas secretes substantial
conservation seemed unattainable. Vesicle mechanisms amounts of enzyme inhibitors, most notably pancreatic
could have been invoked; however, the properties of trypsin inhibitor, along with its digestive enzymes. We
transport that were observed, for example, its equilibrat- have no idea what, if anything, these inhibitors do in the
ing nature, suggested direct membrane permeation. intestines. Certainly, trypsin inhibitor might inactivate
Since the first reports of a membrane permeability to trypsin. But if so, to what end? Of course, there are many
protein molecules in the pancreas about 30 years ago (46), others potential means of chemically altering the diges-
a large body of experimental evidence has been published tive enzymes, including, but not limited to, covalent mod-
establishing the direct transport of many different protein ifications. For example, they might be phosphorylated,
molecules across as many biological membranes. Beyond sulfonated, chlorinated, or leader or activation peptides
providing evidence that such processes exist, these stud- reattached in some way. The experiments by Layer et al.
ies have made it clear that membrane protein transport is (40) discussed above provide proof that some sort of
ubiquitous and central to cellular life. Without these pro- nondegradative modification occurs in the intestinal lu-
cesses to sort proteins to their various assigned locations, men to whatever purpose.
to the mitochondrion, the nucleus, the peroxisome, the Modification may also occur pursuant to transport
chloroplast, as well as to allow proteins to exit the cell, across the intestinal epithelium, or after the proteins en-
cellular life as we know it would not exist. Movement ter the bloodstream. As discussed, protease inhibitors are
occurs variously by the passage of proteins through large present at high concentrations in plasma, and we would
membrane channels, through the lipid barrier, or by expect active proteolytic enzymes to bind to them,
means of interactions with special membrane “carrier” thereby reducing the risk of a generalized proteolytic
proteins. We will not review this evidence here; however, degradation. Perhaps these inhibitors also serve as “car-
we direct readers to older reviews that deal with mem- rier” molecules presenting the circulating enzymes to the
brane transport of proteins in the pancreas (82– 84, 91), a pancreas. Finally, when the enzymes return to the pan-
relatively recent general review of the subject by the creas, they may be chemically modified before their re-
authors (27), as well as the first series of monographs ever secretion. Although it is thought that each digestive en-
published on this topic (85). zyme has a particular established structure before its
secretion, and that whatever variations exist reflect the
VIII. FORMS OF CIRCULATING PROTEINS presence of gene-based isoenzymes, work with mass spec-
trometry suggests a multiplicity of forms of amylase and
Hesitancy to accept the existence of an enteropan- other digestive enzymes with only slightly different masses
creatic circulation was also due to the complete lack of (105). Moreover, and significantly, the forms in secretion are
knowledge of the molecular forms of the circulating spe- not identical to those in the zymogen granule.
cies. For example, were they proenzymes, active en-
zymes, inactivated enzymes, or all of these? And if mole-
cules were modified, how and where did this happen? IX. CONSERVATION OF OTHER
This also posed a chicken and egg problem. Before it DIGESTIVE ENZYMES
made any sense to try to determine the forms of the
circulating species, one had to be convinced that circula- It is natural to ask whether other digestive enzymes,
tion actually occurred. And if the fact that nothing was like salivary amylase or gastric pepsin, might be con-
known about the forms of the circulating species engen- served? After all, the same issues apply. Although there
dered skepticism about circulation itself, there seemed no has been very little research to explore these possibilities,
point in attempting to determine the nature of events that Miyasaka and Rothman, in studies discussed above (61),
did not occur. found that when blood levels of pancreatic amylase were
Consequently, research along these lines was not greatly elevated subsequent to stimulation of pancreatic
carried out, and all we have is speculation as fodder for enzyme secretion in rats, the amylase contents of both the
future work. We can start such speculation by asking parotid and submandibular glands were also greatly ele-
whether each and every proteolytic enzyme is activated vated, with the parotid gland elevated by two orders of
immediately after its secretion into the intestinal tract in magnitude. This remarkable uptake of pancreatic amylase
any and all physiological circumstances? As noted, there from blood suggests the possibility that subsequently it
is no evidence that this is the case, only widely held belief. might be secreted into the mouth, though measurements
Knowing the circumstances of activation might be a first of secretion were not made. In a related study, Fredette
step in helping us understand the relative roles of pro- and and Rothman (unpublished data) found that the adminis-
activated enzymes. tration of isoproterenol to rabbits to stimulate the secre-

tion of amylase into the gland’s duct system also leads to 6. BEYNON RJ AND KAY J. Enteropancreatic circulation of digestive
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traveling down the gastrointestinal tract by means of an Chem 361: 633– 640, 1980.
esophagostomy, plasma amylase levels fell by ⬃50%. 8. BORGSTROM B, DAHLQVIST A, LUNDH G, AND SJOVALL J. Studies of
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1521–1536, 1957.
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not merely occur but is a necessary agency. rat. Endocrinology 65: 118 –123, 1959.
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respiratory activity and histological changes in isolated pancreatic
recommend it. Cells and organisms do not have unlimited tissue. Am J Physiol 157: 278 –282, 1949.
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has crucial selective advantage and can be an important
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determinant in the evolution of species. The traditional postnatal rat pancreas. Am Zool 17: 932, 1977.
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complement of digestive enzymes is manufactured for chromatographic studies of isoamylases in human serum, milk and
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G238 –G246, 1980.
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Physiol Rev

82: 1–18, 2002; 10.1152/physrev.00022.2001.

Conservation of Digestive Enzymes

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When radiolabeled forms of these enzymes were

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site of its original secretion to follow its normal route. In

the fact that the pertinent membranes were permeable

Although this confirmed the existence of an entero-

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and allowing it to be recycled. Furthermore, at steady

secretion. From the single pass perspective, whatever one

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FIG. 7. Change in the percentage of the

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B. Enhancing Synthesis long, or 15 h, only 5 mg of new protein would be pro-

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Physiol Rev • VOL 82 • JANUARY 2002 • www.prv.org

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Physiol Rev • VOL 82 • JANUARY 2002 • www.prv.org

Downloaded from www.physiology.org/journal/physrev by ${individualUser.givenNames} ${individualUser.surname} (190.085.172.107) on October 8, 2018.

Physiol Rev • VOL 82 • JANUARY 2002 • www.prv.org

Downloaded from www.physiology.org/journal/physrev by ${individualUser.givenNames} ${individualUser.surname} (190.085.172.107) on October 8, 2018.

Physiol Rev • VOL 82 • JANUARY 2002 • www.prv.org

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