Metabolic and Microcirculatory Effects
of Glucagon in Hypovolemic Shock
William Schumer, MD; Burton Miller, MD; Ronald Lee Nichols,
Gerald O. McDonald, MD; Lloyd M. Nyhus, MD, Chicago
Glucagon infused into Macaca rhesus
monkeys in hypovolemic shock significantly
improved splanchnic microcirculatory per-
fusion, increased pH, and decreased phos-
phate, lactate, amino acid, and fatty acid lev-
els as compared to the controls. Further, in
vitro rat liver perfusion studies showed that
glucagon significantly increased lactic acid
extraction, even after reducing oxygen pres-
sure and perfusant flow to a minimum.
Thus, in addition to its cardiotonic and vaso-
dilatory effects, glucagon was shown in this
study to improve perfusion in the splanchnic
microcirculation and to decrease anaerobic
metabolites. These findings support its use
in the treatment of hypovolemic shock.
investigations by Siegel
Recent
'and by Kock and their co-work¬
ers have shown that glucagon has a
salutary effect on deranged hemody-
namics in hemorrhagic shock; specifi¬
cally, it is cardiotonic and vasodila-
tory to the splanchnic vasculature.'·-
Glucagon also induces gluconeogen-
esis and glycogenolysis and inhibits
lypolysis and serum phosphorus pro¬
duction, as shown by Exton and asso¬
ciates.'-7 Since our previous studies
showed that the low flow state de-
Accepted for publication March 20,1973.
From the Department of Surgery, University
of Illinois College of Medicine, Abraham Lincoln
School of Medicine; and the West Side Veterans
Administration Hospital, Chicago.
Read before the 30th annual meeting of the
Central Surgical Association, Toronto, Feb 22,
1973.
Reprint requests to West Side Veterans Ad-
ministration Hospital, 820 S Damen Ave, Chi-
cago 60612 (Dr. Schumer).
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MD;
creases microcirculatory perfusion,
thereby increasing anaerobic metabo-
lism,"-9 we postulated that glucagon
may be beneficial in reversing these
microcirculatory and biochemical al¬
terations.
Methods
Part l.-The first part of this study was
designed to reveal the metabolic and mi¬
crocirculatory effects of glucagon in the in¬
tact animal. Twenty adult, female, Macaca
rhesus monkeys weighing approximately 3
kg were fasted for 18 hours and then
anesthetized with 25 mg/kg pentobarbital
sodium. Number 14 polyethylene catheters
were inserted into the femoral artery for
bleeding and into the femoral vein for in¬
jection or infusion. After a control sample
was drawn, the animal was bled at 100-ml
increments every 15 minutes until 50% of
its volume was withdrawn. One-half hour
later the mean arterial blood pressure, cen¬
tral venous pressure, and heart rate were
measured to ascertain that the animal was
in hypovolemic shock (Table 1). The experi¬
mental group was infused with glucagon
at a constant infusion rate of 18.3^g/
kg/min for six hours, with a limit of 20 ml
of volume. The control group was given 20
ml of saline, the vehicle of the glucagon in-
Table 1.—Hemodynamic Factors in
Macaca Rhesus Monkey in Hypovolemic Shock
Factor
Arterial pressure,
mean
Heart rate
Inferior vena cava pressure
fusions. Before bleeding and at 50% vol¬
ume reduction, blood samples were with¬
drawn to measure anaerobic metabolites
follows: lactate and pyruvate were de¬
as
termined with a Boehringer biochemical
kit; free amino acid nitrogen assays were
performed by Russell's technique;10 free
fatty acids were measured by the method
of Mackenzie and co-workers;" and inorgan¬
ic phosphate levels were determined by the
method of Fiske and SubbaRow'2 (Table 2).
To study the microcirculation, the omen-
tum was mobilzed from a small lateral
flank incision and placed on the microscope
stage by tacking to a cork-bordered slide.
To prevent drying of the omentum, saline-
moistened packs were placed around the
flank incision, and a heat-filtered low-volt¬
age bulb was used as the light source. The
microcirculation was magnified 100 to 360
times. Microphotographs were obtained
before bleeding, at 50% volume reduction,
and after glucagon infusion.
Part 2.—The second part of the experi¬
ment was designed to show the metabolic
effect of glucagon on an in vitro rat liver
perfusion system, using a technique modi¬
fied after Miller and associates." Sprague-
Dawley rats, each weighing 400 gm, were
fasted overnight. Each was lightly
anesthetized with ether, and the peritoneal
cavity was then opened. The bile duct was
cannulated with a No. 11 polyethylene
Control Experimental
93 ±12 45 ±8
161 ±15 240 ± 21
16 ±6 0
 Table 2.—Metabolic Effect of Glucagon in Hypovolemic Shock*
0% Volume Bled 50% Volume Bled
Control Experimental Control Experimental
Lactate, mg/100 ml 9.80 ± 1.800 4.00 ± 0.700t 104.80 ± 7.600 35.40 ± 3.200t
Amino acids,
mg/100 ml 5.20 ± 0.800 2.80 ± 0.960t 14.80 ± 2.000 6.20±1.200t
Fatty acids,
millimol/liter 180.00 ±32.000 106.00 ± 28.000t 582.00 ± 62.000 210.00 ±40.000t
Inorganic
phosphates,
mg/100 ml 5.10 ±0.900 3.10 ± 0.800t 12.10 ± 1.400 4.80 ± 0.700t
pH 7.38 ± 0.006 7.42 ± 0.008 7.08 ±0.002 7.28 ± 0.008t
Pco2 44.40 ± 3.000 39.00 ± 3.000 28.00 ± 4.000 34.00 ± 5.000t
Microcirculation 4+ 3+
*
Each group contained five monkeys. tP<.01. tP<.05.

Table 3.—Glucagon Effect on Lactate Extraction in In Vitro Prefused Rat Liver*
Control
Perfusion Lactate,
Time, Millimols/lOOgm
min Body Weight
0.349 ±0.150
15 0.240 ± 0.023
30 0.106 ±0.052
60 f-0.264 ± 0.034
120 +0.283 ± 0.034
180 +3.350 ± 0.340
Lactate extraction equals inflow
catheter, and the superior mesenteric vein
was ligated and cannulated with a No. 35
polyethylene catheter entering the portal
vein. During the remaining preparation,
the liver was perfused with Krebs-Ringer
bicarbonate solution through the portal
vein. The liver was then gently dissected
and placed in the perfusion system. Per¬
fusion rates were measured by occluding
the outflow of the perfusion chamber and
measuring the amount of perfusant per
minute in a graduated pipette. The result¬
ing rates varied from 15 to 20 mi/min. Ali¬
quote of the perfusant were used to mea¬
sure lactic acid. Bile production indicated
the viability of the liver; and microscopic
studies, performed at one, two, and three
hours, confirmed its architectural integ¬
rity.
The livers were perfused with 110 ml of
Krebs-Ringer bicarbonate solution contain¬
ing 4% (W/V) albumin. During the infu¬
sion lactate was added to maintain a con¬
centration of 1 millimol/liter. At zero-time,
glucagon was added to the perfusant of
the experimental livers at a dose of 10 SM.
Lactate extraction was measured by sub¬
tracting the amount of the outflow per¬
fusant from that of the inflow at the spe¬
cific times shown in Table 3. The perfusant
was deproteinized with perchloric acid, and
its lactate content was determined. The
decreased, the glucagon-stimulated
liver maintained lactate extraction to
Experimental a more significant extent than did the
Lactate, control. At low perfusion and Po_,, the
Millimols/100 gm control liver produced lactate, but the
pH Body Weight pH
7.480 ± 0.04 0.686 ± 0.034 7.48 ± 0.09 glucagon-treated liver continued to
7.430 ±0.04 0.642 ± 0.024 7.47 ±0.07 extract it. This consequently pro¬
7.300 ± 0.06 0.718 ±0.019 7.32 ±0.04 duced lower lactic acid and hydrogen
7.210 ±0.05 0.542 ±0.018 7.28 ± 0.04 ion concentrations in the experimen¬
6.900 ± 0.07 0.280 ± 0.075 7.28 ±0.05 tal liver perfusant (Table 3). Thus,
6.860 ± 0.05 0.100 ±0.010 7.20 ± 0.04 part 2 showed that, in the presence of
lactate minus outflow lactate. glucagon, increased lactate extrac¬
tion and decreased pH were not
merely the products of increased per¬
oxygen pressure (Po,) was measured in an fusion through the liver, but were in¬
aliquot of the outflow perfusant using an deed a metabolic effect.
oxygen electrode. The perfusant and Po,
were gradually decreased by slowing the Comment
variable pump and decreasing oxygen to
the oxygenator, simulating hypoperfusion Glucagon, a pancreatic polypeptide
and anoxia to the liver. produced by the alpha cells of the is¬
lets of Langerhans, has been shown
to stimulate gluconeogenesis in the
Results
isolated perfused liver." In normal an¬
The results of part 1 of the study imals it is able to convert lactic, ami-
shown in Table 2. Over the six-
are no, and fatty acids to glucose, but it
hour investigation period, glucagon- does not affect the conversion of fruc¬
treated monkeys showed a significant
tose to glucose. This indicates that
decrease in serum concentrations of glucagon acts in the glycolytic path¬
lactate, amino acids, fatty acids, way at the pyruvate to phosphoenol-
phosphates, and an increase in pH pyruvate reaction. It has been sug¬
and arterial carbon dioxide pressure gested that this enhanced gluconeo¬
as compared to the controls. Micro- genesis is mediated by the activation
photographs of the omenta revealed of cyclic adenosine monophosphate
a significantly increased micro¬ (AMP). Exton and Park observed that
circulatory flow in glucagon-treated exogenous cyclic AMP stimulates
monkeys as compared to the controls gluconeogenesis in the perfused liver,
(Fig 1).
suggesting that this nucleotide me¬
Part 2 of the study indicated that, diates the action of glucagon in liver
under equal perfusion rates during gluconeogenesis. They found that cy¬
the first 15 minutes, the presence of clic AMP produced changes in the cel¬
glucagon in the perfusant resulted in lular levels of gluconeogenic inter¬
increased lactate extraction (Fig 2). mediates similar to those produced
As the perfusant and Po, were by glucagon. Furthermore, glucagon

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 Fig 1.—Left, Control. Note stream lines and capillary circulation. Glucagon effect. Note return of stream lines in arterioles with in¬
Center, 50% volume bled. Note absence of stream lines in arteri- complete restoration of flow (original magnification 300).
oles, dilatation of venioles, and stagnant flow in capillaries. Right,

rapidly produced increases in intra-

cellular cyclic AMP." Kock and associ¬
ates studied the vasodilatory effect of
glucagon in normal dogs and found Control, N=6
that blood flow was increased in the " Experiment, N=10
splanchnic vasculature." In our study
we have shown this effect in hypo¬
volemic monkeys. We have previously
reported that in the shocked animal
the gastrointestinal tract is a most
prolific producer of lactic acid.17'
Therefore, as evidenced by our pres¬
ent study, glucagon has a significant
effect in increasing splanchnic blood
flow, thereby decreasing anaerobic
metabolism.
The above studies support the use
of glucagon in low flow states based
on its beneficial hemodynamic ef¬
fects. There is, however, an important Fig 2.—Glucagon effect on lactate extraction in in vitro perfused rat liver.
metabolic rationale as well: glucagon
appears to stimulate the conversion
of lactic acid, amino acids, and fatty concept was further supported by the the liver and induced lactate extrac¬
acids to glucose in low flow states. microcirculatory studies: glucagon in¬ tion during the three hours of the ex¬
Part 1, the in vivo experiment, indi¬ creased microcirculatory perfusion in periment. By stimulating gluconeo¬
cated that glucagon decreased serum the shocked monkey. In part 2, the in genesis from lactate, amino, and free
lactic, amino, and fatty acid levels vitro experiment, we found that de¬ fatty acids," glucagon produced a re¬
and thus decreased the metabolic aci- creasing perfusant flow and Po, re¬ duction of the acid gluconeogenic sub¬
dosis of anaerobiosis. It is possible duced lactate extraction. Indeed, the strates. Studies by Schröder and asso¬
that the hemodynamic effects of liver .continued to produce lactic acid ciates indicated that the liver is
glucagon—cardiotonic and splanchnic as the Po, and perfusion were re¬ active in causing the hyperlactemia
vasodilation-decreased anaerobic duced to a minimum. Glucagon, how¬ of low flow states either by failing to
metabolism in nonvital tissue. This ever, prevented lactate production by clear lactate from the blood or by ac-

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 tually producing lactate.1" The liver's
contribution to lactic acidosis is re¬
lated to its rate of oxygen consump¬
tion: as this rate decreases, so does
the liver's ability to metabolize lac¬
tate. Numerous investigators have
shown that liver glycogen is com¬
pletely and rapidly utilized, and al¬
though hyperglycemia occurs in the
early stages of shock, hypoglycemia
characterizes the final stages.1719 The
organism must therefore depend on
the gluconeogenic organs-liver, kid-
1. Siegel JH, et al: The effect of gluca-
gon infusion on cardiovascular function in
the critically ill. Surg Gynecol Obstet
131:505-515, 1970.
2. Kock NG, Tibblin S, Schenk WG Jr:
Modification by glucagon of the splanchnic
vascular responses to activation of the
sympathicoadrenal system. J Surg Res
11:12-17, 1971.
3. Exton JH, Park CR: Control of gluco-
neogenesis in the perfused liver of normal
and adrenalectomized rats. J Biol Chem
240:PC955-PC957, 1965.
4. Exton JH, et al: Gluconeogenesis in
the perfused liver: The effects of fasting,
alloxan diabetes, glucagon, epinephrine,
adenosine 3\m='\,\5\m='\-monophosphate and insulin.
Am J Med 40:709-715, 1966.
5. Exton JH, Park CR: Control of gluco-
neogenesis in liver: I. General features of
gluconeogenesis in the perfused livers of
rats. J Biol Chem 242:2622-2636, 1967.
6. Exton JH, Park CR: Control of gluco-
neogenesis in liver: II. Effects of glucagon,
catecholamines, and adenosine 3\m='\,5\m='\-mono-
phosphate on gluconeogenesis in the per-
fused rat liver. J Biol Chem 243:4189-4196,
Benjamin F. Rush, MD, Newark, NJ: Dr.
Schumer's laboratory still shows that sign
of ultimate affluence, that is, shock experi¬
ments done in the primate. I will try not to
let my envy affect the tenor of my re¬
marks.
Past experiments evaluating drugs that
cause vasodilatation alone in shock therapy
have at best shown equivocal results, the
potential improvement in perfusion pro¬
vided by vasodilatation being offset by the
accompanying drop in perfusion pressure.
On the other hand, a drug like glucagon,
which not only causes vasodilatation but at
the same time increases cardiac output, al¬
lows perfusion pressure to be maintained
despite the reduced resistance and affords
a great therapeutic promise.
The second set of experiments in this pa¬
per suggests that there is an improvement
in metabolism as a direct metabolic effect
However, there is a slight quibble, since
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ney, and heart—to produce glucose
from lactic, amino, and fatty acids.
Glucagon appears to stimulate gluco¬
neogenesis, and as shown by our liver
perfusion studies, it can do so in the
anoxic and poorly perfused liver.
Most notable is the effect of gluca¬
gon on serum inorganic phosphates.
Increases in inorganic phosphates
have been considered to indicate de¬
creases in energy phosphorylates,
such as adenosine triphosphate
(ATP). Glucagon decreases serum
References
1968.
7. Exton JH, Corbin JG, Harper SC:
Control of gluconeogenesis in liver: V. Ef-
fects of fasting, diabetes, and glucagon on
lactate and endogenous metabolism in the
perfused rat liver. J Biol Chem 247:4996\x=req-\
5003, 1972.
8. Schumer W, Durrani KM: Study of
the effects of norepinephrine on micro-
circulation of the dog omentum in oligemic
shock. Ann Surg 158:982-989, 1963.
9. Schumer W, Lee DK, Jones B: The
physiologic effect of vasodilators on the
omentum of the dog in low flow states.
Angiology 16:140-147, 1965.
10. Russell J: Note on the colorimetric
determination of amino nitrogen. J Biol
Chem 156:469-477, 1944.
11. Mackenzie RD, et al: Rapid color-
imetric micromethod for free fatty acids. J
Lipid Res 8:589-597, 1967.
12. Fiske CH, SubbaRow Y: Phospho-
creatine. J Biol Chem 81:629-679, 1929.
13. Miller LL, et al: The dominant role
of the liver in plasma protein synthesis: A
direct study of the isolated perfused rat
liver with the aid of lysine-\g=e\-C14. J Exp
Discussion
one has to ask whatwas the distribution of
the perfusate, were cells being perfused in
the treated liver, which were not being
perfused in the untreated liver, despite the
fact that the flow rates were the same?
Certainly the significance in changes in
lactate, pH, and other metabolic factors in
the presence of glucagon are most impres¬
sive.
We have had a recent opportunity to
treat a patient in hypovolemic shock with
glucagon, with fairly impressive results.
This was a patient who had a period of
bleeding following a chest procedure, who
had reached that very unhappy state
where the pressure on the right side of the
heart was rising rapidly, where the heart
had not responded to the use of isoprotere-
nol hydrochloride (Isuprel). Glucagon was
then given, with a prompt drop in pressure
on the right side of the heart and ultimate
recovery of the patient. This leads to a
inorganic phosphates, suggesting
that it increases ATP production and
hexose-phosphorylating reactions.
Thus, we may conclude that gluca¬
gon is a pharmacologie agent, which
is cardiotonic, vasodilatory in the
splanchnic microvasculature, and ca¬
pable of metabolically decreasing
anaerobic metabolites—the major
contributor to low flow metabolic
acidosis.
Med 94:431-453, 1951.
14. Kock NG, Tibblin S, Schenk WG Jr:
Hemodynamic responses to glucagon: An
experimental study of central, visceral and
peripheral effects. Ann Surg 171:373-379,
1970.
15. Schumer W: Lactate studies of the
dog in oligemic shock. J Surg Res 8:491\x=req-\
494, 1968.
16. Schr\l=o"\derR, et al: The role of the
liver in the development of lactic acidosis
in low flow states. Postgrad Med J 45:566\x=req-\
570, 1969.
17. Gutman A, Shafrir E: Metabolic in-
fluences on enzymes of glycogen synthesis
and breakdown in adipose tissue. Am J
Physiol 207:1215-1220, 1964.
18. Metzger B, Helmreich E, Glaser L:
The mechanism of activation of skeletal
muscle phosphorylase A by glycogen. Proc
Nat Acad Sci 57:994-1001, 1967.
19. Stoner HB: Studies on the mecha-
nism of shock: The quantitative aspects of
glycogen metabolism after limb ischaemia
in the rat. Br J Exp Pathol 39:635-651,
1958.
fairly pragmatic comment: we received
shortly thereafter a note from the hospital
administrator indicating that we had used
$900 worth of glucagon on this patient, and
what about that?
Robert E. Condon, MD, Milwaukee:
This study by Dr. Schumer and his col¬
leagues has accomplished two things of
major importance. First, the experiments
confirm in shocked primates the he¬
modynamic effect of glucagon in decreas¬
ing splanchnic resistance and increasing
splanchnic blood flow. Second, the experi¬
ments clearly demonstrate that glucagon
decreases the production of lactate in
shock by recruiting new aerobic metabolic
pathways. This second observation is most
exciting and represents, I believe, the be¬
ginning of important new investigative ef¬
forts by his group in the field of metabo¬
lism and shock.
I have one question and a further com-
 ment of caution. The question: What vol¬
ume of saline was administered to the
monkeys during these experiments? Fifty
percent of the blood volume of a rhesus
monkey is of the order of 150 or 200 ml.
Therefore, administration of just a few
hundred milliliters of saline could effect re¬
suscitation of the shocked animals. At first
glance, it would seem that this matter is
well-controlled since presumably equiva¬
lent volumes were administered to control
monkeys and to those treated with gluca¬
gon. But things are not so simple as they
may seem. The administration of glucagon
in the doses used in these experiments re¬
sults in a marked shift of circulating vol¬
ume to the splanchnic region at the ex¬
pense of circulation to the kidneys, brain,
and other peripheral vascular beds. There¬
fore, the administration of even modest
volumes of fluid together with glucagon
might produce much greater splanchnic
flow than would occur in controls given the
same volume of saline but no glucagon.
The microcirculatory effects observed in
these experiments might then be due both
to volume resuscitation as well as to gluca¬
gon, and if so, would imply that fluid ad¬
ministration concomitantly with glucagon
would be needed. I'd appreciate comment
regarding this aspect of the presentation.
Finally, I must express reservations
about the clinical use of glucagon at this
time in the treatment of shock. Our own
studies of experimental hemorrhagic pan¬
creatitis (which is a form of hypovolemic
shock) have failed to demonstrate any ben¬
eficial effect of glucagon on the mortality
of this disorder. Further, the studies of
Fredlund et al {Arch Surg 105:615, 1972)
have demonstrated that glucagon adminis¬
tration in hepatic-artery-ligated animals
results in increased hepatic necrosis as
compared with controls, a finding com¬
pletely contrary to the result expected in
view of the known hemodynamic effects of
glucagon in the splanchnic circulation. I
believe that our understanding of the
pharmacology of glucagon is not yet suffi¬
ciently complete to warrant its routine use
at this time in patients in shock.
John H. Siegel, MD, Buffalo: I am very
impressed with Dr. Schumer's paper, be-
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cause what he is trying to do is to look at
the pharmacologie treatment of the cellu¬
lar mechanisms of shock. This study is
very important, but it raises a lot of ques¬
tions that need to be answered by more
thorough work.
With regard to the use of glucagon, the
human studies we have done would at least
confirm the general outlines of the very
careful and systematic study that Dr.
Schumer has done.
In the evaluation of oxygen consumption
before and after glucagon infusion in con¬
trol patients and in patients with a wide
range of shock conditions including sepsis,
cirrhotic liver disease, and patients with
hypovolemic shock, you can see on the
average that the largest number of pa¬
tients, especially those in shock states, had
significant increases in total oxygen con¬
sumption during the shock process, within
about an hour of the institution of gluca¬
gon infusion.
This increase in oxygen consumption is
heavily dependent on the fact that the
drug has a strong cardiac inonotropic ef¬
fect and increases cardiac output. This
marked increase in total perfusion un¬
doubtedly affects the splanchnic blood flow
on man as it does in experimental animals.
Finally, it is important to demonstrate
that one should think about drugs with re¬
gard to how they act and with special con¬
sideration as to their synergistic effect
The interesting thing about glucagon is
that it directly affects and increases the
production of high-energy phosphate com¬
pounds such as 3.5 cyclic AMP, as do the
catecholamines. However, it has been well-
demonstrated that these two types of
agents act through different receptor sites,
and it is possible to actually block one type
of receptor without blocking the other.
We had a patient in shock with severe
sepsis due to intra-abdominal abscesses. In
evaluating the cardiac index, you can see
the patient initially becomes hyper-
dynamic and gradually falls into a state of
abnormal oxygen consumption (B state
sepsis). The patient's clinical course then
changed into a hypodynamic shock C state
sepsis episode from which she was resusci¬
tated, and then maintained on continuous
infusion of both isuprel and glucagon with
a return of a high cardiac output. Even
though treatment with isuprel was contin¬
ued, when the glucagon alone was discon¬
tinued there was a marked fall in cardiac
output. This fall was due to a marked de¬
pression in myocardial contractility which,
when recognized, was quickly alleviated by
reinstitution of the glucagon, with return
of cardiac output to previous levels.
The point here is that one can use these
agents synergistically so that one can use
small, relatively nontoxic, doses of isuprel
and of glucagon to get a combination ef¬
fect which is of great importance in im¬
proving the circulation. Indeed, Dr. Schu¬
mer's work suggests the glucagon may
have some specific effects not shared by the
catecholamines.
With regard to its use in humans, I can
only say that I have personally used gluca¬
gon in more than 300 patients, either alone
or in combination with isoproterenol. With
the exception of occasional episodes of hy-
perglycemia and concomitant hypokalemia,
which must be evaluated and treated, I
have not detected any specific toxic effect
of this drug.
I have seen no evidence that glucagon
produces arrhythmias or attributes to re¬
nal dysfunction. In fact, most of our pa¬
tients have had increased renal output
once treatment with glucagon was started.
The detailed myocardial studies we have
done have shown that'it increases cardiac
function and that it can be safely adminis¬
tered for prolonged periods of time.
Dr. Schumer: Concerning the question
of whether we perfused the liver com¬
pletely, the microscopic and ultramicroscop¬
io studies showed no changes in the liver,
and bile production was maintained. The
experience of other investigators, such as
Exton, Park, and Williamson, revealed
perfusion studies can be used as valid mod¬
els for the study of metabolic changes. I
would like to emphasize the point that the
controls and the experiments were done
simultaneously. Dr. Condon, glucagon was
combined with 20 ml of saline, so that no
animal received more than 20 ml of saline.