The Influence of Ethanol on Splanchnic and Skeletal Muscle Metabolism in Man Lennart Jorfeldt and Anders Juhlin-Dannfelt Splanchnic and leg exchange of ethanol, acetate, glucose, lactate, pyruvate, glycerol, and free fatty acids was studied in five healthy volunteers before and after a 60-min infusion of ethanol. leg and splanchnic blood flows were determined simultaneously using a modiged indicator dilution technique. The blood alcohol concentration obtained was 1.88 mmoles/ liter and splanchnic uptake of ethanol was calculatedto be 1.18 mmoles/min. All subjects showed an acetate uptake over the legs with a mean of 0.25 mmole/min. Splanchnic glucose production was attenuated in four of five subjects after ethanol treatment, and glucose uptake by the legs was significantly reduced. The normal
splanchnic uptake of lactate was changed by ethanol to a release, and the arterial concentration was nearly doubled. Net leg release of lactate decreased significantly. Splanchnic blood flow and oxygen uptake were uninfluenced by ethanol, whereas leg blood flow decreased from a mean of 0.77 to 0.65 liter/min. It is concluded that, following ethanol treatment, (1) a major part of acetate released from the splanchnit region is taken up by the muscles, (2) leg glucose uptake is decreased by a reduction of the same magnitude as the acetate uptake, and (3) leg blood flow is reduced, probably owing to a constriction of muscle vessels.
T
HE LIVER is the main center of ethanol oxidation, and even subtoxic doses of ethanol exert a dramatic influence upon hepatic metabolism. Ethanol combustion hence causes a shift in the hepatic NAD/NADH2 ratio in favor of a reduction considered to be responsible for impaired gluconeogenesis with a potential risk for hypoglycemia. The main part of acetate produced during ethanol oxidation is released from the liver,’ providing a new substrate for the peripheral tissues, and an acetate uptake has also been documented for the human heart2 and the forearm3 (Jorfeldt, unpublished results). Ethanol administration hence influences the blood-borne substrates at the disposal of the muscles, and, in addition to the metabolic changes previously mentioned, small doses of ethanol are also known to decrease the level of the main fuel for the muscles, i.e., the free fatty acids (FFA), probably owing to a reduced lipolysis.4 As a consequence, it was considered attractive to study the pathways through which ethanol-induced changes in liver metabolism influence substrate turnover by skeletal muscle, and a modified flow method was developed for simultaneous measurements of splanchnic and leg blood flows to enable quantitative comparisons of substrate exchange between the liver and the leg. From the Department of Clinical Physiology, Huddinge Hospital, Karolinska Institute, Huddinge. Sweden. Receivedfor publication h4arch 24.1977. Supported by grants from the Swedish Medical Research Council (Project 4139) and from the Karolinska Institute. Reprint requests should be addressed to Dr. Anders Juhlin-Dannfelt. Department of Clinical Physiology, Huddinge Hospital, S-141 86 Huddinge. Sweden. o I978 by Grune & Stratton, Inc. ISSN 00260495.00260495/ 7812701~00I2%02.00~0
Metabolism,Vol. 27, No.
1 (January),1978
97
98
JORFELDT
MATERIALS
AND JUHLIN-DANNFELT
AND METHODS
Subjects Five healthy nonobese male volunteers with a mean weight 81 kg 71-86) in study. The mean of the was yr (range None the subjects made use of beverages, they were to drink alcohol or take the hr the All subjects were also formed of the purpose, risks of study before voluntarily giving formal consent.
Procedure The subjects were studied in the morning after an overnight fast. A Cournand catheter (No. 7) was inserted percutaneously into an antecubital vein and advanced to a right-sided hepatic vein under Ruoroscopic control. Teflon catheters were placed in one femoral and one brachial artery, two femoral veins, and one antecubital vein. Splanchnic and leg blood flows were determined simultaneously on the basis of modifications of the Bradley technique’ and the method described by Jorfeldt and Wahren.6 After a priming dose of I2 mg indocyanine green dye, a constant infusion of dye (1.1 mg/min) was administered in the femoral artery for 20 min, after which four sets of blood samples were taken from the hepatic and femoral veins and the brachial artery with 3-min intervals. Metabolites were sampled from the same vessels 20 and 27 min after the infusion. Expired air was sampled for the determination of pulmonary oxygen uptake and ventilatory exchange ratio. Ethanol was then given intravenously as a 10% solution in 0.45”/, aqueous sodium chloride. The infusion started with a priming dose of 80 mmoles during 10 min followed by a constant infusion at the approximate rate of 1.8 mmoles/min maintained for the remainder of the study. When alcohol had been administered for 40 min the second infusion of indocyanine dye was started, and samples for flow and metabolite determinations were drawn in the same manner as before ethanol treatment.
Analyses Expired air was analyzed by means of the Scholander microtechnique. Oxygen saturation was determined photometrically using an IL 182 CO-Oximeter (Instrumentation Laboratory), which was regularly calibrated by means of an IL 327 tonometer. Hemoglobin concentration was measured using the cyanmet-hemoglobin technique.’ Hematocrit was determined by means of a microcapillary hematocrit centrifuge and corrected for trapped plasma according to Garby and Vuille.8 Electrode techniques were used for the determination of PO, and PcoZ. All blood samples for metabolites were drawn in plastic syringes. Blood for the determination of FFA was transferred into heparinized tubes and centrifuged. Plasma samples of 2.0 ml were taken for extraction of FFA in IO ml of Dole’s mixture, FFA being subsequently determined using the methods of Dole’and Trout et al.” as described elsewhere.” Blood samples for glucose determination were immediately precipitated in perchloric acid (PCA), 0.33 mole/liter, buffered with glycine to pH 2.7 and analyzed during the same day with a glucose oxidase method operating with dicarboxidin as a chromogen (Kabi Glox novum). The remaining metabolites were analyzed by microfluorometry. Blood was precipitated with an equal amount of chilled PCA. 3 moles/liter. After centrifugation the acid extract free from protein was neutralized with 20% KOH. Pyruvate was determined on the day of the experiment; the other determinations took place within I wk from that date and the blood extracts were stored meanwhile at a temperature of at least -20°C. Lactate was determined by a microfluorometric modification of the method of Hohorst et al.” at pH 9.5 with these final cuvette concentrations: glycine and hydrazine 0.36 mole/liter, NAD 0.50 mmole/liter, lactate dehydrogenase (LDH; from rabbit muscle) 200 rkat/liter,* and lactate less than 130 pmoles/liter. The fluorescence intensity was measured before LDH was added and once again 120 min later.
*wmole substrate
converted/set.
INFLUENCE
OF
ETHANOL
99
A modification of the method of Biicher13 was used for pyruvate determinations. The final cuvette concentrations were as follows: triethanolamine (pH 7.6) 0.2 mole/liter, NADH 0.07 mmole/liter, LDH 45 pkat/liter, and pyruvate less than 50 ccmoles/liter. The second measurement of fluorescence intensity was performed 10 min after addition of LDH. Glycerol in blood was determined by the method of Wieland,14 the procedure being adapted for microfluorometry. The determinations were consequently effected at pH 9.5 with these final cuvette concentrations: glycine 70 mmoles/liter, hydrazine 0.4 mole/liter, NAD 0.6 mmole/liter, MgS04 7.0 mmoles/liter, glycerokinase 0.8 pkat/liter, glycerol-3-phosphate dehydrogenase 7 pkat/liter, and glycerol less than 14 pmoles/liter. Ethanol in plasma was determined using the method of Biicher and Redetskils adapted for fluorometry with these cuvette concentrations: sodiumpyrophosphate 0.063 mole/liter, semicarbazide 0.015 mole/liter, glycine 0.020 mole/liter, NAD 0.137 mmole/ liter, antidiuretic hormone (ADH) 263 rkat/liter, and ethanol 0.024 mmole/liter. Acetate was measured in plasma using the method of Bergmeyer and Moellering16 adapted for microfluorometry by Wolfe (personal communication). The procedure is outlined here: NADH and LDH were added in order to remove pyruvate from plasma, and the mixture was allowed to react for 5 min. The nucleotides (original and additional) were then removed by absorption of activated charcoal. The charcoal was cleansed with concentrated HCI once and with water five times, and then dried and ground to a powder. The mixture was deproteinized by placing the tube in boiling water for 3 min during continual stirring. The proteins were removed by centrifuging at 13,000 rpm for 30 min at +4”C. The measurements were performed at pH 7.85 with these final cuvette concentrations: triethanolamine 0.2 mole/liter, MgClz 0.003 mole/liter, L-malate 0.01 mole/liter NAD 1.0 mmole/liter, coenzyme A (CoA) 0.046 mmole/liter, ATP 2.7 mmoles/liter, phosphotransacetylase 19 ckat/liter, citrate synthase 10 pkatlliter, LDH 10 Fkat/liter, malate dehydrogenase 31 pkatlliter, acetate kinase 59 pkatjiter, and acetate less than 0.2 mmole/ liter. The readings were taken prior to and 120 min after adding the acetate kinase. The pyridine nucleotides, the ATP, and the enzymes were all obtained from Boehringer Mannheim Corp. The concentrations of the metabolites were estimated from standard curves. The contents of ethanol and acetate in whole blood samples were calculated with the assumption that the two substances are equally distributed in plasma and blood water phase. All determinations were carried out in duplicate with an error of the methods expressed as the coefficient of variation of single determinations as follows: glucose l.O’A, lactate 1.6%. pyruvate 4.0%. glycerol 2.8%. FFA 7.6x, ethanol 4.2%. and acetate 4.4%. Recovery was checked in every batch of samples and was found to be within the range of 93x-103%. Standard statistical methods were used” in analyses of the data, resorting to the paired t test when applicable.
Table I.
Oxygen
Uptake,
Respiratory
Exchange Ratio, Leg Blood Flow, Estimated
Splanchnic Blood Flow (ESBF), and Arterial
Concentrations
Control Oxygen uptake Respiratory Leg blood
(ml/min)
exchange flow,
ratio
in two
legs (liters/min)
ESBF (lite.rs/min) Arterial
(Mean
f SEM) After Ethanol
299 f
295 +
10
0.03
0.06
0.80
f
10
f
0.06’
0.77
f
0.08
0.65
f
0.07t
1.22
f
0.13
1.12
f
0.09
concentrations
Ethanol
(mmoles/liter)
-
1.89
& 0.14
Acetate
(mmoles/liter)
-
0.85
f
0.08
Glucose
(mmoles/liter)
Lactate
(mmoler/liter)
4.72
& 0.10
4.62
f
0.12
0.49
f
0.07
0.89
f
0.07$ 0.002*
Pyruvate
(mmoles/liter)
0.052
f
0.004
0.042
f
Glycerol
(mmoles/liter)
0.040
f
0.005
0.031
f
0.004*
0.41
f
0.06
0.37
f
0.04
FFA (mmoles/liter)
*Significantly
different
from
the control
value,
tSignificantl~
different
from
the control
value,
p < 0.05. b < 0.01.
$Signifkantly
different
from
the control
value,
p < 0.001
JORFELDT
100
AND JUHUN-DANNFELT
Table 2. Splanchnic Uptake (Mean f SEM, mmoler/min) Control
After Ethanol
Ethanol
-
1.18 f 0.10
Acetate
-
GlUCOS.3
lactate Pyruvote
-0.93
1.00 f 0.15 f 0.25
-0.50
f 0.07
0.11 f 0.03
-0.08
f 0.03t
-0.004
zk 0.007
0.021 f 0.001t
Glycerol
0.035 f 0.006
0.025 f 0.005*
Oxygen
3.15 f 0.37
2.97 f 0.31
*Significantly different from the control value, p < 0.05. tsignificantly different from the control value, p < 0.01.
RESULTS
The results are summarized in Tables l-3. The dye concentration in the recirculating blood was 27% of the concentration in femoral venous blood from the infused leg (1.93 f 0.24 versus 7.18 + 0.57 mg/liter) (Fig. 1). The dye concentrations were identical in arterial blood and femoral venous blood drawn from the leg not infused. Estimated splanchnic blood flow (ESBF) was unchanged, as was the arterialhepatic vein oxygen difference, which resulted in an unaltered splanchnic oxygen uptake. In all subjects there was a small decrease in leg blood flow, from a mean of 0.77 to 0.65 liter/min (p < 0.01). The arterial-femoral venous difference increased (p < 0.05), and, as a consequence, the calculated leg oxygen uptake did not change. Total-body oxygen uptake was uninfluenced by ethanol treatment. The respiratory exchange ratio showed a slight attenuation (p < 0.05). Before alcohol infusion no significant amounts of ethanol could be detected in blood. After a 1-hr intravenous infusion blood ethanol levels averaged 1.89 •t 0.14 mmoles/ liter (0.10 g/liter). Splanchnic uptake was calculated as 1.18 + 0.10 mmoles/ min. No significant arterial-femoral venous difference across the leg was found. Arterial concentration of acetate rose from insignificant values to a mean of 0.85 f 0.08 mmole/liter. Splanchnic release was 1.00 •t 0.15 mmole/min, and it was estimated to be about 80% of ethanol uptake. In all subjects there was a positive arteriovenous difference for acetate across the leg, and the uptake amounted to 0.25 f 0.07 mmole/min. Arterial glucose concentration as well as splanchnic production showed a small but insignificant decrease. Leg uptake, however, decreased significantly (p < 0.05). Ethanol infusion changed the normal splanchnic uptake of lactate to a release Table 3. leg Uptake (Mean Control Acetate Glucose lactate
-
Pyruvate
-0.001
Glycerol
-0.025
Oxygen
After Ethanol 0.25 f 0.07
0.12 f 0.04 -0.11
f SEM, mmoles/min)
f 0.02 f 0.003 f 0.011
1.99 f 0.21
*Significantly different from the control value, p < 0.05.
0.06 f 0.03* -0.05
f 0.03*
0.001 f 0.002 -0.016 f 0.007 1.96 f 0.32
101
INFLUENCE OF ETHANOL
cont., mg/l fv
infused leg
v fv
.
contralateral
leg
b a D hv
.
4.0 -
2.0 -
1 I 0-k
K
z---s----:
1
I
24
20
d
I
I
I
I
20
2% minutes
Control
24
I
0
20 minutes
Ethanol
Fig. 1. lndocyanine dye concentration in the hepatic contralateral leg, and an artery in one subject.
vein,
femoral
veins of the infused
and
in all subjects, and the arterial concentration almost doubled (0.49 f 0.07 versus 0.89 f 0.07 mmole/liter, p < 0.001) (Fig. 2). The femoral venous concentration increased from 0.64 f 0.05 to 0.96 f 0.06 mmole/liter (p < 0.01). The net leg release of lactate decreased significantly (p < 0.05). Before ethanol treatment there was no significant exchange of pyruvate over the splanchnic bed, but after ethanol administration all subjects had a significant uptake (p < 0.01). Leg exchange was uninfluenced, but arterial concentration fell from 0.052 f 0.004 to 0.042 f 0.002 mmole/liter (p < 0.05). The femoral venous concentration showed a decrease of the same magnitude (0.054 f 0.002 versus 0.041 f 0.003 mmole/liter,p < 0.01). The arterial concentration of FFA after the 1-hr ethanol treatment was slightly but not significantly lowered. The net exchanges across the leg and the splanchnic bed were unchanged. Alcohol caused a fall in arterial glycerol concentration from 0.040 to 0.031 mmole/liter (p < 0.05). No significant exchange across the leg was found either before or during ethanol administration. The uptake over the splanchnic region fell from 0.035 to 0.025 mmole/min after ethanol (p < 0.05). DISCUSSION
Most alcohol-induced metabolic changes in the body can be attributed to hepatic metabolism being disturbed by oxidation of ethanol. When investigating the influence of alcohol on skeletal muscle metabolism it therefore seemed to be of great interest to study the splanchnic metabolism at the same time so as to enable quantitative comparisons between these two regions. Both splanchnic and leg blood flows have to be determined for such studies, and separate tech-
JORFELDT
102
AND JUHLIN-DANNFELT
GLUCOSE Art
Splanchmc
cone
mmol/l 5.0
mmol/min
‘I
mmol / min 0.04,
l-
1
mmol/ 0.02,
‘- ‘7’
-004
E
1sg uptall mmol/min 0.3
*1 t
‘&’
C
prod..
min
‘- I&* /’
J
-002-
C
E
C
E
Fig . 2 . Arterial concentration and splanchnic and leg exchange of glucow, lactate, and pyruvate in the control situation (C) and after ethanol administration (E).
niques are available for each. The present study shows that the procedure can be adjusted to allow simultaneous measurements of blood flow from both areas. For this purpose a lower infusion rate and a longer infusion period were used than is normally the case in leg blood flow determinations. This modification presumably may result in relatively higher dye concentrations in recirculating blood and thus exert an unfavorable influence on the concentration differences between femoral venous and recirculating blood, but the results from this study (Fig. 1) showed that there was still a favorable relationship at rest. However, the procedure would not be easily applicable when leg blood flow is increased, as during exercise. As the arterial dye concentration was shown to agree with the concentration in the contralateral vein, there was apparently no need for an additional arterial catheter. Calculations of substrate exchange require a steady-state situation. The changes in arterial concentration were small for all metabolites for which regional exchange calculations were made, except acetate and ethanol. In the case of alcohol, the infusion started with a primary dose to achieve the blood concentration required, after which the dose was intended to maintain a constant concentration. An almost constant arterial concentration was maintained for more than 50 min before the measurements. This period is about 10 times the estimated mean transit time if a splanchnic water flow of 1 liter and a distribu-
INFLUENCE
OF ETHANOL
103
tion volume of 5 liters is assumed and a steady state is consequently obtained. Concerning acetate, other studies have shown that intravenous ethanol administration involves a rapid increase in blood concentration,’ and the splanchnic acetate exchange therefore may be considered to be in a steady state. The mean transit time for acetate and ethanol across the leg is, however, prolonged, since the blood flow at rest is low when compared to leg water content. Nevertheless, no significant arterial-venous difference for ethanol was observed after a 50-min ethanol infusion. A minor part of the arterial-venous difference for acetate might however be due to a nonsteady state. Alcohol administration has been considered to exert a vasodilatory effect on blood vessels in the skin.ls This contrasts with its reported effect of retarding muscle blood flow, probably by direct local action.‘9*20The decrease in total leg blood flow found in this study could hence have been caused by vasoconstriction in skeletal muscle. The flow method could not be used to determine whether the decrease was counterbalanced to some extent by an increased cutaneous blood flow. Conflicting results have been reported for the effect of ethyl alcohol on ESBF. Mendeloff *l demonstrated a consistent increase in splanchnic blood flow during infusion of ethanol and concluded that the effect could be ascribed to a lowered peripheral resistance of the splanchnic bed. Stein et a1.22found an increased flow but noted that it was proportional to the effect on the systemic circulation. On the other hand, Castenfors et a1.23and Lundquist et al.’ found no effect at all or even a slight decrease in ESBF after ethanol treatment. The latter results are in agreement with those of this study. One reason for the discrepancies could be that the dose of ethanol varied; the route of administration may also have influenced the flow. On administration of ethanol, blood concentration of acetate quickly increases and reaches a steady-state level, as previously discussed. The concentration is independent of the ethanol concentration within wide limits,24 and the production of acetate is counterbalanced by an uptake in peripheral tissues. Forsander et a1.25showed that when rats were given ‘Glabeled ethanol radioactive carbon dioxide was formed when blood perfused the hind quarters of the animals. Lindeneg et al.2 exhibited acetate utilization by the human myocardium. Lundquist et al.3 and Jorfeldt (unpublished results) found an uptake of acetate in the forearm. The uptake of acetate registered in this study was 0.25 mmole/min in the legs, corresponding to 25% of the splanchnic release. When assuming that 70% of the leg volume consists of skeletal muscle and that the total muscle mass of the human body comprises 40% of body weight,26 it seems that about 50% of the acetate released from the liver is taken up by the muscles. The fractional uptake of acetate in the leg was 43x, which is in accordance with values from the forearm (Jorfeldt, unpublished results). If a complete oxidation of released acetate to carbon dioxide and water were at hand, the combustion could account for 25% of the leg and 15% of the total-body oxygen uptake. It has been shown in a recent study, however, that only 50% of acetate is oxidized to carbon dioxide and water in the hind quarters of the rat.*’ Several cases of severe hypoglycemia have been reported after ethanol ingestion, especially in chronic alcoholics and children.28 A prerequisite is, however,
104
JORFELDT AND JUHLIN-DANNFELT
a fasting period of several days with depletion of the liver glycogen stores.2g In this study (Fig. 2) splanchnic glucose production decreased after an overnight fast in four of five subjects, but the fall was not statistically significant. However, glucose uptake by the legs did decrease (p < 0.05), and this attenuation of the glucose uptake is in agreement with results obtained in dogs by Lockner et al *,3owho found a 25% decrease in peripheral glucose utilization. The decrease in splanchnic glucose production was counterbalanced by a lowered uptake in the legs, resulting in an unchanged arterial concentration. The inhibition of peripheral glucose utilization could hence not be ascribed to a decreased blood concentration, but is probably caused by ethanol or one of its metabolic products. It may be explained by the circumstance that activation of acetate reduced the concentration of free coenzyme A in a degree that limited its incorporation into pyruvate oxidizing systems. 3’ The acetate uptake could thus theoretically act as a substitute for the decreased glucose uptake, but this could not be established with the methods applied in this study. It may be concluded, however, that the total oxidative metabolism of the leg is not converted by ethanol, as the leg oxygen uptake was identical in the control and ethanol situations. The abovementioned results differ from those of Lundquist et al.,’ who found an increased arterial glucose concentration and an increased glucose uptake by the forearm. This might be explained by the fact that they administered alcohol orally as beer and not as an infusion of ethanol. It has been shown by Jorfeldt (unpublished results) that the results for the leg are valid even for the forearm on administration of ethanol as a constant intravenous infusion. It is now well established that ethanol inhibits hepatic gluconeogenesis owing to a decrease of the NAD/NADH* ratio in the liver.*‘v3*The changed redox potential of the liver causes the normal uptake of lactate to shift to a release, in this study doubling the arterial concentrations (Fig. 2). The net release of lactate from the legs decreased significantly, probably owing to the increased arterial lactate concentration, as described by Jorfeldt.33 As previously described34 the splanchnic uptake of pyruvate rose significantly after ethanol treatment (Fig. 2) and could thus be used for reduction to lactate in order to reestablish the redox potential in the liver. Ethanol has been reported to inhibit glycerol uptake by the liver.35 In this study splanchnic uptake highly correlated with the splanchnic inflow (the product of arterial concentration and ESBF), and the regression line (y = 0.83 0.01; r = 0.96,~ < 0.01) was identical whether alcohol was infused or not. The arterial concentration of glycerol was lower after ethanol administration, probably owing to a diminished lipolysis, and the splanchnic uptake was consequently lower (p < 0.05). It may be concluded, however, that the major effect of alcohol on hepatic gluconeogenesis is caused by the abolished lactate uptake, which can be calculated to account for about 50% of the gluconeogenic capacity. REFERENCES 1. Lundquist F, Tygstrup N, Winkler K, et al: Ethanol metabolism and production of free
acetate in the human liver. J Clin Invest 41: 955-961, 1962
INFLUENCE
OF ETHANOL
2. Lindeneg P, Mellemgaard K, Fabricius J, et al: Myocardial utilization of acetate, lactate and free fatty acids after ingestion of ethanol. Clin Sci 27:427-435, 1964 3. Lundquist F, Sestoft L, Damgaard SE, et al: Utilization of acetate in the human forearm during exercise after ethanol ingestion. J Clin Invest 52:3231-3235, 1973 4. Lieber CS, Leevy CM, Stein SW, et al: Effect of ethanol on plasma free fatty acids in man. J Lab Clin Med 59:826-832,1962 5. Bradley E: Measurements of hepatic blood flow: Methods in medical research, in Potter VR (ed): Methods in Medical Research. Chicago, Year Book, 1948, p 199 6. Jorfeldt L, Wahren J: Leg blood flow during exercise in man, Clin Sci 41:459-473, 1971 7. Drabkin DL, Austin JH: Spectrophotometric studies II. Preparations from washed blood cells: Nitric oxide hemoglobin and sulfhemoglobin. J Biol Chem 112:51-65, 1935 8. Garby L, Vuille JC: The amount of trapped plasma in a high speed microcapillary hematocrit centrifuge. Stand J Clin Lab Invest 13642-645, 1961 9. Dole VP: A relation between non-esterified fatty acids in plasma and the metabolism of ‘glucose. J Clin Invest 35:150-154, 1956 IO. Trout DL, Ester EH, Friedberg SJ: Titration of free fatty acids of plasma: A study of current methods and new modification. J Lipid Res 1:199-202, 1960 11. Have1 J, Naimark A, Borchgrevink F: Turnover rate and oxidation of free fatty acids of blood plasma in man during exercise: Studies during continuous infusion of palmitate-I-C 14. J Clin Invest 42:1054-1063, 1963 12. Hohorst HJ, Kreutz FH, Btlcher TH: Ueber Metabolitgehalte und Metabolitkoncentrationen in der Leber der Ratte. Biochem Z 332: 18-46, 1959 13. Bticher T, in Bergmeyer HU (ed): Methods of Enzymatic Analysis (ed I). Academic, New York, 1965, p 253 14. Wieland 0: Eine enzymatische Methode zur Bestimmung von Glycerin. Biochem Z 329: 313-319,1957 15. BUcher T, Redetzski H: Eine spezifische photometrische Bestimmung von Athylalkohol auf fermentativen Wege. Klin Wochenschr 29: 615-616, 1951 16. Bergmeyer HU, Moellering H: Enzymatische Bestimmung von Acetat. Biochem Z 344:167-189, 1966 17. Snedecor GW, Corchran WG: Statistical Methods (ed 6). Ames, Iowa, Iowa State University Press, 1967
105
18. Cook EN, Brown GE: The vasodilating effects of ethyl alcohol on the peripheral arteries. Clinic 31:449-452, 1932 19. Fewings JD, Hanna JJD, Walsh JA, et al: The effects of ethyl alcohol on the blood vessels of the hand and forearm in man. Br J Pharmacol Chemother 27:93-106, 1966 20. Graf K, Strom G: Effect of ethanol ingestion on arm blood flow in healthy young men at rest and during leg work. Acta Pharmacol Toxicol 17:115-120, 1960 21. Mendeloff AJ: Effect of intravenous infusions of ethanol upon estimated hepatic blood flow in man. J Clin Invest 33:1298-1302, 1954 22. Stein SW, Lieber CS, Leevy CM, et al: The effect of ethanol upon systemic and hepatic blood flow in man. Am J Clin Nutr 13:68-74, 1963 23. Castenfors H, Hultman E, Josephson B: Effect of intravenous infusions of ethyl alcohol on estimated hepatic blood flow in man. J Clin Invest 39:776-781, 1960 24. Lundquist F: The concentration of acetate in blood during alcohol metabolism in man. Acta Physiol Stand 175:97, 1960 25. Forsander 0, Rgiha N, Suomalainen H: Oxydation des Athylalkohols in isolierter Leber und isoliertem Hinterkijrper der Ratte. Hoppe SeylersZ Physiol Chem 318:1-5, 1960 26. Andres R, Cader G, Zierler KL: The quantitatively minor role of carbohydrate in oxidative metabolism by skeletal muscle in intact man in the basal state. Measurements of oxygen and glucose uptake and carbon dioxide and lactate production in the forearm. J Clin Invest 35:671-682, 1956 27. Karlsson N, Fellenius E, Kiessling KH: The metabolism of acetate in the perfused hindquarter of the rat. Acta Physiol Stand 93:391400, 1975 28. Freinkel N, Singer DL, Arky RA, et al: Alcohol hypoglycemia. I. Carbohydrate metabolism of patients with clinical alcohol hypoglycemia and the experimental reproduction of the syndrome with pure ethanol. J Clin Invest 42:1112-1133, 1963 29. Freinkel N. Cohen A, Arky RA, et al: Alcohol hypoglycemia. II A. Postulated mechanism of action based on experiments with rat liver slices. J Clin Endocrinol25:76-94, 1965 30. Lockner A, Wulff J, Madison L: Ethanolinduced hypoglycemia. I. The acute effects of ethanol on hepatic glucose output and peripheral glucose utilization in fasted dogs. Metabolism l&l-18,1967 31. Lundquist F: Production and utilization
JORFELDT
106 of free acetate in man. Nature 1962 32. Krebs
193578-579,
HA: The effects of ethanol on the metabolic activities of the liver. Adv Enzyme Regul6:467-489, 1968 33. Jorfeldt L: Metabolism of L(+)-lactate in human skeletal muscle during exercise. Acta Physiol Stand [Suppl] 338:45-55, 1970 34. Wolfe BM, Have1 JR, Marliss EB, et al:
AND JUHLIN-DANNFELT
Effects of a 3-day fast and of ethanol on splanchnic metabolism of FFA, amino acids and carbohydrates in healthy young men. J Clin Invest 57:329-340. 1976 35. Lundquist F, Tygstrup N, Winkler K, et al: Glycerol metabolism in the human liver: Inhibition by ethanol. Science 150:616-617, 1965
splanchnic uptake of lactate was changed by ethanol to a release, and the arterial concentration was nearly doubled. Net leg release of lactate decreased significantly. Splanchnic blood flow and oxygen uptake were uninfluenced by ethanol, whereas leg blood flow decreased from a mean of 0.77 to 0.65 liter/min. It is concluded that, following ethanol treatment, (1) a major part of acetate released from the splanchnit region is taken up by the muscles, (2) leg glucose uptake is decreased by a reduction of the same magnitude as the acetate uptake, and (3) leg blood flow is reduced, probably owing to a constriction of muscle vessels.
T
HE LIVER is the main center of ethanol oxidation, and even subtoxic doses of ethanol exert a dramatic influence upon hepatic metabolism. Ethanol combustion hence causes a shift in the hepatic NAD/NADH2 ratio in favor of a reduction considered to be responsible for impaired gluconeogenesis with a potential risk for hypoglycemia. The main part of acetate produced during ethanol oxidation is released from the liver,’ providing a new substrate for the peripheral tissues, and an acetate uptake has also been documented for the human heart2 and the forearm3 (Jorfeldt, unpublished results). Ethanol administration hence influences the blood-borne substrates at the disposal of the muscles, and, in addition to the metabolic changes previously mentioned, small doses of ethanol are also known to decrease the level of the main fuel for the muscles, i.e., the free fatty acids (FFA), probably owing to a reduced lipolysis.4 As a consequence, it was considered attractive to study the pathways through which ethanol-induced changes in liver metabolism influence substrate turnover by skeletal muscle, and a modified flow method was developed for simultaneous measurements of splanchnic and leg blood flows to enable quantitative comparisons of substrate exchange between the liver and the leg. From the Department of Clinical Physiology, Huddinge Hospital, Karolinska Institute, Huddinge. Sweden. Receivedfor publication h4arch 24.1977. Supported by grants from the Swedish Medical Research Council (Project 4139) and from the Karolinska Institute. Reprint requests should be addressed to Dr. Anders Juhlin-Dannfelt. Department of Clinical Physiology, Huddinge Hospital, S-141 86 Huddinge. Sweden. o I978 by Grune & Stratton, Inc. ISSN 00260495.00260495/ 7812701~00I2%02.00~0
Metabolism,Vol. 27, No.
1 (January),1978
97
98
JORFELDT
MATERIALS
AND JUHLIN-DANNFELT
AND METHODS
Subjects Five healthy nonobese male volunteers with a mean weight 81 kg 71-86) in study. The mean of the was yr (range None the subjects made use of beverages, they were to drink alcohol or take the hr the All subjects were also formed of the purpose, risks of study before voluntarily giving formal consent.
Procedure The subjects were studied in the morning after an overnight fast. A Cournand catheter (No. 7) was inserted percutaneously into an antecubital vein and advanced to a right-sided hepatic vein under Ruoroscopic control. Teflon catheters were placed in one femoral and one brachial artery, two femoral veins, and one antecubital vein. Splanchnic and leg blood flows were determined simultaneously on the basis of modifications of the Bradley technique’ and the method described by Jorfeldt and Wahren.6 After a priming dose of I2 mg indocyanine green dye, a constant infusion of dye (1.1 mg/min) was administered in the femoral artery for 20 min, after which four sets of blood samples were taken from the hepatic and femoral veins and the brachial artery with 3-min intervals. Metabolites were sampled from the same vessels 20 and 27 min after the infusion. Expired air was sampled for the determination of pulmonary oxygen uptake and ventilatory exchange ratio. Ethanol was then given intravenously as a 10% solution in 0.45”/, aqueous sodium chloride. The infusion started with a priming dose of 80 mmoles during 10 min followed by a constant infusion at the approximate rate of 1.8 mmoles/min maintained for the remainder of the study. When alcohol had been administered for 40 min the second infusion of indocyanine dye was started, and samples for flow and metabolite determinations were drawn in the same manner as before ethanol treatment.
Analyses Expired air was analyzed by means of the Scholander microtechnique. Oxygen saturation was determined photometrically using an IL 182 CO-Oximeter (Instrumentation Laboratory), which was regularly calibrated by means of an IL 327 tonometer. Hemoglobin concentration was measured using the cyanmet-hemoglobin technique.’ Hematocrit was determined by means of a microcapillary hematocrit centrifuge and corrected for trapped plasma according to Garby and Vuille.8 Electrode techniques were used for the determination of PO, and PcoZ. All blood samples for metabolites were drawn in plastic syringes. Blood for the determination of FFA was transferred into heparinized tubes and centrifuged. Plasma samples of 2.0 ml were taken for extraction of FFA in IO ml of Dole’s mixture, FFA being subsequently determined using the methods of Dole’and Trout et al.” as described elsewhere.” Blood samples for glucose determination were immediately precipitated in perchloric acid (PCA), 0.33 mole/liter, buffered with glycine to pH 2.7 and analyzed during the same day with a glucose oxidase method operating with dicarboxidin as a chromogen (Kabi Glox novum). The remaining metabolites were analyzed by microfluorometry. Blood was precipitated with an equal amount of chilled PCA. 3 moles/liter. After centrifugation the acid extract free from protein was neutralized with 20% KOH. Pyruvate was determined on the day of the experiment; the other determinations took place within I wk from that date and the blood extracts were stored meanwhile at a temperature of at least -20°C. Lactate was determined by a microfluorometric modification of the method of Hohorst et al.” at pH 9.5 with these final cuvette concentrations: glycine and hydrazine 0.36 mole/liter, NAD 0.50 mmole/liter, lactate dehydrogenase (LDH; from rabbit muscle) 200 rkat/liter,* and lactate less than 130 pmoles/liter. The fluorescence intensity was measured before LDH was added and once again 120 min later.
*wmole substrate
converted/set.
INFLUENCE
OF
ETHANOL
99
A modification of the method of Biicher13 was used for pyruvate determinations. The final cuvette concentrations were as follows: triethanolamine (pH 7.6) 0.2 mole/liter, NADH 0.07 mmole/liter, LDH 45 pkat/liter, and pyruvate less than 50 ccmoles/liter. The second measurement of fluorescence intensity was performed 10 min after addition of LDH. Glycerol in blood was determined by the method of Wieland,14 the procedure being adapted for microfluorometry. The determinations were consequently effected at pH 9.5 with these final cuvette concentrations: glycine 70 mmoles/liter, hydrazine 0.4 mole/liter, NAD 0.6 mmole/liter, MgS04 7.0 mmoles/liter, glycerokinase 0.8 pkat/liter, glycerol-3-phosphate dehydrogenase 7 pkat/liter, and glycerol less than 14 pmoles/liter. Ethanol in plasma was determined using the method of Biicher and Redetskils adapted for fluorometry with these cuvette concentrations: sodiumpyrophosphate 0.063 mole/liter, semicarbazide 0.015 mole/liter, glycine 0.020 mole/liter, NAD 0.137 mmole/ liter, antidiuretic hormone (ADH) 263 rkat/liter, and ethanol 0.024 mmole/liter. Acetate was measured in plasma using the method of Bergmeyer and Moellering16 adapted for microfluorometry by Wolfe (personal communication). The procedure is outlined here: NADH and LDH were added in order to remove pyruvate from plasma, and the mixture was allowed to react for 5 min. The nucleotides (original and additional) were then removed by absorption of activated charcoal. The charcoal was cleansed with concentrated HCI once and with water five times, and then dried and ground to a powder. The mixture was deproteinized by placing the tube in boiling water for 3 min during continual stirring. The proteins were removed by centrifuging at 13,000 rpm for 30 min at +4”C. The measurements were performed at pH 7.85 with these final cuvette concentrations: triethanolamine 0.2 mole/liter, MgClz 0.003 mole/liter, L-malate 0.01 mole/liter NAD 1.0 mmole/liter, coenzyme A (CoA) 0.046 mmole/liter, ATP 2.7 mmoles/liter, phosphotransacetylase 19 ckat/liter, citrate synthase 10 pkatlliter, LDH 10 Fkat/liter, malate dehydrogenase 31 pkatlliter, acetate kinase 59 pkatjiter, and acetate less than 0.2 mmole/ liter. The readings were taken prior to and 120 min after adding the acetate kinase. The pyridine nucleotides, the ATP, and the enzymes were all obtained from Boehringer Mannheim Corp. The concentrations of the metabolites were estimated from standard curves. The contents of ethanol and acetate in whole blood samples were calculated with the assumption that the two substances are equally distributed in plasma and blood water phase. All determinations were carried out in duplicate with an error of the methods expressed as the coefficient of variation of single determinations as follows: glucose l.O’A, lactate 1.6%. pyruvate 4.0%. glycerol 2.8%. FFA 7.6x, ethanol 4.2%. and acetate 4.4%. Recovery was checked in every batch of samples and was found to be within the range of 93x-103%. Standard statistical methods were used” in analyses of the data, resorting to the paired t test when applicable.
Table I.
Oxygen
Uptake,
Respiratory
Exchange Ratio, Leg Blood Flow, Estimated
Splanchnic Blood Flow (ESBF), and Arterial
Concentrations
Control Oxygen uptake Respiratory Leg blood
(ml/min)
exchange flow,
ratio
in two
legs (liters/min)
ESBF (lite.rs/min) Arterial
(Mean
f SEM) After Ethanol
299 f
295 +
10
0.03
0.06
0.80
f
10
f
0.06’
0.77
f
0.08
0.65
f
0.07t
1.22
f
0.13
1.12
f
0.09
concentrations
Ethanol
(mmoles/liter)
-
1.89
& 0.14
Acetate
(mmoles/liter)
-
0.85
f
0.08
Glucose
(mmoles/liter)
Lactate
(mmoler/liter)
4.72
& 0.10
4.62
f
0.12
0.49
f
0.07
0.89
f
0.07$ 0.002*
Pyruvate
(mmoles/liter)
0.052
f
0.004
0.042
f
Glycerol
(mmoles/liter)
0.040
f
0.005
0.031
f
0.004*
0.41
f
0.06
0.37
f
0.04
FFA (mmoles/liter)
*Significantly
different
from
the control
value,
tSignificantl~
different
from
the control
value,
p < 0.05. b < 0.01.
$Signifkantly
different
from
the control
value,
p < 0.001
JORFELDT
100
AND JUHUN-DANNFELT
Table 2. Splanchnic Uptake (Mean f SEM, mmoler/min) Control
After Ethanol
Ethanol
-
1.18 f 0.10
Acetate
-
GlUCOS.3
lactate Pyruvote
-0.93
1.00 f 0.15 f 0.25
-0.50
f 0.07
0.11 f 0.03
-0.08
f 0.03t
-0.004
zk 0.007
0.021 f 0.001t
Glycerol
0.035 f 0.006
0.025 f 0.005*
Oxygen
3.15 f 0.37
2.97 f 0.31
*Significantly different from the control value, p < 0.05. tsignificantly different from the control value, p < 0.01.
RESULTS
The results are summarized in Tables l-3. The dye concentration in the recirculating blood was 27% of the concentration in femoral venous blood from the infused leg (1.93 f 0.24 versus 7.18 + 0.57 mg/liter) (Fig. 1). The dye concentrations were identical in arterial blood and femoral venous blood drawn from the leg not infused. Estimated splanchnic blood flow (ESBF) was unchanged, as was the arterialhepatic vein oxygen difference, which resulted in an unaltered splanchnic oxygen uptake. In all subjects there was a small decrease in leg blood flow, from a mean of 0.77 to 0.65 liter/min (p < 0.01). The arterial-femoral venous difference increased (p < 0.05), and, as a consequence, the calculated leg oxygen uptake did not change. Total-body oxygen uptake was uninfluenced by ethanol treatment. The respiratory exchange ratio showed a slight attenuation (p < 0.05). Before alcohol infusion no significant amounts of ethanol could be detected in blood. After a 1-hr intravenous infusion blood ethanol levels averaged 1.89 •t 0.14 mmoles/ liter (0.10 g/liter). Splanchnic uptake was calculated as 1.18 + 0.10 mmoles/ min. No significant arterial-femoral venous difference across the leg was found. Arterial concentration of acetate rose from insignificant values to a mean of 0.85 f 0.08 mmole/liter. Splanchnic release was 1.00 •t 0.15 mmole/min, and it was estimated to be about 80% of ethanol uptake. In all subjects there was a positive arteriovenous difference for acetate across the leg, and the uptake amounted to 0.25 f 0.07 mmole/min. Arterial glucose concentration as well as splanchnic production showed a small but insignificant decrease. Leg uptake, however, decreased significantly (p < 0.05). Ethanol infusion changed the normal splanchnic uptake of lactate to a release Table 3. leg Uptake (Mean Control Acetate Glucose lactate
-
Pyruvate
-0.001
Glycerol
-0.025
Oxygen
After Ethanol 0.25 f 0.07
0.12 f 0.04 -0.11
f SEM, mmoles/min)
f 0.02 f 0.003 f 0.011
1.99 f 0.21
*Significantly different from the control value, p < 0.05.
0.06 f 0.03* -0.05
f 0.03*
0.001 f 0.002 -0.016 f 0.007 1.96 f 0.32
101
INFLUENCE OF ETHANOL
cont., mg/l fv
infused leg
v fv
.
contralateral
leg
b a D hv
.
4.0 -
2.0 -
1 I 0-k
K
z---s----:
1
I
24
20
d
I
I
I
I
20
2% minutes
Control
24
I
0
20 minutes
Ethanol
Fig. 1. lndocyanine dye concentration in the hepatic contralateral leg, and an artery in one subject.
vein,
femoral
veins of the infused
and
in all subjects, and the arterial concentration almost doubled (0.49 f 0.07 versus 0.89 f 0.07 mmole/liter, p < 0.001) (Fig. 2). The femoral venous concentration increased from 0.64 f 0.05 to 0.96 f 0.06 mmole/liter (p < 0.01). The net leg release of lactate decreased significantly (p < 0.05). Before ethanol treatment there was no significant exchange of pyruvate over the splanchnic bed, but after ethanol administration all subjects had a significant uptake (p < 0.01). Leg exchange was uninfluenced, but arterial concentration fell from 0.052 f 0.004 to 0.042 f 0.002 mmole/liter (p < 0.05). The femoral venous concentration showed a decrease of the same magnitude (0.054 f 0.002 versus 0.041 f 0.003 mmole/liter,p < 0.01). The arterial concentration of FFA after the 1-hr ethanol treatment was slightly but not significantly lowered. The net exchanges across the leg and the splanchnic bed were unchanged. Alcohol caused a fall in arterial glycerol concentration from 0.040 to 0.031 mmole/liter (p < 0.05). No significant exchange across the leg was found either before or during ethanol administration. The uptake over the splanchnic region fell from 0.035 to 0.025 mmole/min after ethanol (p < 0.05). DISCUSSION
Most alcohol-induced metabolic changes in the body can be attributed to hepatic metabolism being disturbed by oxidation of ethanol. When investigating the influence of alcohol on skeletal muscle metabolism it therefore seemed to be of great interest to study the splanchnic metabolism at the same time so as to enable quantitative comparisons between these two regions. Both splanchnic and leg blood flows have to be determined for such studies, and separate tech-
JORFELDT
102
AND JUHLIN-DANNFELT
GLUCOSE Art
Splanchmc
cone
mmol/l 5.0
mmol/min
‘I
mmol / min 0.04,
l-
1
mmol/ 0.02,
‘- ‘7’
-004
E
1sg uptall mmol/min 0.3
*1 t
‘&’
C
prod..
min
‘- I&* /’
J
-002-
C
E
C
E
Fig . 2 . Arterial concentration and splanchnic and leg exchange of glucow, lactate, and pyruvate in the control situation (C) and after ethanol administration (E).
niques are available for each. The present study shows that the procedure can be adjusted to allow simultaneous measurements of blood flow from both areas. For this purpose a lower infusion rate and a longer infusion period were used than is normally the case in leg blood flow determinations. This modification presumably may result in relatively higher dye concentrations in recirculating blood and thus exert an unfavorable influence on the concentration differences between femoral venous and recirculating blood, but the results from this study (Fig. 1) showed that there was still a favorable relationship at rest. However, the procedure would not be easily applicable when leg blood flow is increased, as during exercise. As the arterial dye concentration was shown to agree with the concentration in the contralateral vein, there was apparently no need for an additional arterial catheter. Calculations of substrate exchange require a steady-state situation. The changes in arterial concentration were small for all metabolites for which regional exchange calculations were made, except acetate and ethanol. In the case of alcohol, the infusion started with a primary dose to achieve the blood concentration required, after which the dose was intended to maintain a constant concentration. An almost constant arterial concentration was maintained for more than 50 min before the measurements. This period is about 10 times the estimated mean transit time if a splanchnic water flow of 1 liter and a distribu-
INFLUENCE
OF ETHANOL
103
tion volume of 5 liters is assumed and a steady state is consequently obtained. Concerning acetate, other studies have shown that intravenous ethanol administration involves a rapid increase in blood concentration,’ and the splanchnic acetate exchange therefore may be considered to be in a steady state. The mean transit time for acetate and ethanol across the leg is, however, prolonged, since the blood flow at rest is low when compared to leg water content. Nevertheless, no significant arterial-venous difference for ethanol was observed after a 50-min ethanol infusion. A minor part of the arterial-venous difference for acetate might however be due to a nonsteady state. Alcohol administration has been considered to exert a vasodilatory effect on blood vessels in the skin.ls This contrasts with its reported effect of retarding muscle blood flow, probably by direct local action.‘9*20The decrease in total leg blood flow found in this study could hence have been caused by vasoconstriction in skeletal muscle. The flow method could not be used to determine whether the decrease was counterbalanced to some extent by an increased cutaneous blood flow. Conflicting results have been reported for the effect of ethyl alcohol on ESBF. Mendeloff *l demonstrated a consistent increase in splanchnic blood flow during infusion of ethanol and concluded that the effect could be ascribed to a lowered peripheral resistance of the splanchnic bed. Stein et a1.22found an increased flow but noted that it was proportional to the effect on the systemic circulation. On the other hand, Castenfors et a1.23and Lundquist et al.’ found no effect at all or even a slight decrease in ESBF after ethanol treatment. The latter results are in agreement with those of this study. One reason for the discrepancies could be that the dose of ethanol varied; the route of administration may also have influenced the flow. On administration of ethanol, blood concentration of acetate quickly increases and reaches a steady-state level, as previously discussed. The concentration is independent of the ethanol concentration within wide limits,24 and the production of acetate is counterbalanced by an uptake in peripheral tissues. Forsander et a1.25showed that when rats were given ‘Glabeled ethanol radioactive carbon dioxide was formed when blood perfused the hind quarters of the animals. Lindeneg et al.2 exhibited acetate utilization by the human myocardium. Lundquist et al.3 and Jorfeldt (unpublished results) found an uptake of acetate in the forearm. The uptake of acetate registered in this study was 0.25 mmole/min in the legs, corresponding to 25% of the splanchnic release. When assuming that 70% of the leg volume consists of skeletal muscle and that the total muscle mass of the human body comprises 40% of body weight,26 it seems that about 50% of the acetate released from the liver is taken up by the muscles. The fractional uptake of acetate in the leg was 43x, which is in accordance with values from the forearm (Jorfeldt, unpublished results). If a complete oxidation of released acetate to carbon dioxide and water were at hand, the combustion could account for 25% of the leg and 15% of the total-body oxygen uptake. It has been shown in a recent study, however, that only 50% of acetate is oxidized to carbon dioxide and water in the hind quarters of the rat.*’ Several cases of severe hypoglycemia have been reported after ethanol ingestion, especially in chronic alcoholics and children.28 A prerequisite is, however,
104
JORFELDT AND JUHLIN-DANNFELT
a fasting period of several days with depletion of the liver glycogen stores.2g In this study (Fig. 2) splanchnic glucose production decreased after an overnight fast in four of five subjects, but the fall was not statistically significant. However, glucose uptake by the legs did decrease (p < 0.05), and this attenuation of the glucose uptake is in agreement with results obtained in dogs by Lockner et al *,3owho found a 25% decrease in peripheral glucose utilization. The decrease in splanchnic glucose production was counterbalanced by a lowered uptake in the legs, resulting in an unchanged arterial concentration. The inhibition of peripheral glucose utilization could hence not be ascribed to a decreased blood concentration, but is probably caused by ethanol or one of its metabolic products. It may be explained by the circumstance that activation of acetate reduced the concentration of free coenzyme A in a degree that limited its incorporation into pyruvate oxidizing systems. 3’ The acetate uptake could thus theoretically act as a substitute for the decreased glucose uptake, but this could not be established with the methods applied in this study. It may be concluded, however, that the total oxidative metabolism of the leg is not converted by ethanol, as the leg oxygen uptake was identical in the control and ethanol situations. The abovementioned results differ from those of Lundquist et al.,’ who found an increased arterial glucose concentration and an increased glucose uptake by the forearm. This might be explained by the fact that they administered alcohol orally as beer and not as an infusion of ethanol. It has been shown by Jorfeldt (unpublished results) that the results for the leg are valid even for the forearm on administration of ethanol as a constant intravenous infusion. It is now well established that ethanol inhibits hepatic gluconeogenesis owing to a decrease of the NAD/NADH* ratio in the liver.*‘v3*The changed redox potential of the liver causes the normal uptake of lactate to shift to a release, in this study doubling the arterial concentrations (Fig. 2). The net release of lactate from the legs decreased significantly, probably owing to the increased arterial lactate concentration, as described by Jorfeldt.33 As previously described34 the splanchnic uptake of pyruvate rose significantly after ethanol treatment (Fig. 2) and could thus be used for reduction to lactate in order to reestablish the redox potential in the liver. Ethanol has been reported to inhibit glycerol uptake by the liver.35 In this study splanchnic uptake highly correlated with the splanchnic inflow (the product of arterial concentration and ESBF), and the regression line (y = 0.83 0.01; r = 0.96,~ < 0.01) was identical whether alcohol was infused or not. The arterial concentration of glycerol was lower after ethanol administration, probably owing to a diminished lipolysis, and the splanchnic uptake was consequently lower (p < 0.05). It may be concluded, however, that the major effect of alcohol on hepatic gluconeogenesis is caused by the abolished lactate uptake, which can be calculated to account for about 50% of the gluconeogenic capacity. REFERENCES 1. Lundquist F, Tygstrup N, Winkler K, et al: Ethanol metabolism and production of free
acetate in the human liver. J Clin Invest 41: 955-961, 1962
INFLUENCE
OF ETHANOL
2. Lindeneg P, Mellemgaard K, Fabricius J, et al: Myocardial utilization of acetate, lactate and free fatty acids after ingestion of ethanol. Clin Sci 27:427-435, 1964 3. Lundquist F, Sestoft L, Damgaard SE, et al: Utilization of acetate in the human forearm during exercise after ethanol ingestion. J Clin Invest 52:3231-3235, 1973 4. Lieber CS, Leevy CM, Stein SW, et al: Effect of ethanol on plasma free fatty acids in man. J Lab Clin Med 59:826-832,1962 5. Bradley E: Measurements of hepatic blood flow: Methods in medical research, in Potter VR (ed): Methods in Medical Research. Chicago, Year Book, 1948, p 199 6. Jorfeldt L, Wahren J: Leg blood flow during exercise in man, Clin Sci 41:459-473, 1971 7. Drabkin DL, Austin JH: Spectrophotometric studies II. Preparations from washed blood cells: Nitric oxide hemoglobin and sulfhemoglobin. J Biol Chem 112:51-65, 1935 8. Garby L, Vuille JC: The amount of trapped plasma in a high speed microcapillary hematocrit centrifuge. Stand J Clin Lab Invest 13642-645, 1961 9. Dole VP: A relation between non-esterified fatty acids in plasma and the metabolism of ‘glucose. J Clin Invest 35:150-154, 1956 IO. Trout DL, Ester EH, Friedberg SJ: Titration of free fatty acids of plasma: A study of current methods and new modification. J Lipid Res 1:199-202, 1960 11. Have1 J, Naimark A, Borchgrevink F: Turnover rate and oxidation of free fatty acids of blood plasma in man during exercise: Studies during continuous infusion of palmitate-I-C 14. J Clin Invest 42:1054-1063, 1963 12. Hohorst HJ, Kreutz FH, Btlcher TH: Ueber Metabolitgehalte und Metabolitkoncentrationen in der Leber der Ratte. Biochem Z 332: 18-46, 1959 13. Bticher T, in Bergmeyer HU (ed): Methods of Enzymatic Analysis (ed I). Academic, New York, 1965, p 253 14. Wieland 0: Eine enzymatische Methode zur Bestimmung von Glycerin. Biochem Z 329: 313-319,1957 15. BUcher T, Redetzski H: Eine spezifische photometrische Bestimmung von Athylalkohol auf fermentativen Wege. Klin Wochenschr 29: 615-616, 1951 16. Bergmeyer HU, Moellering H: Enzymatische Bestimmung von Acetat. Biochem Z 344:167-189, 1966 17. Snedecor GW, Corchran WG: Statistical Methods (ed 6). Ames, Iowa, Iowa State University Press, 1967
105
18. Cook EN, Brown GE: The vasodilating effects of ethyl alcohol on the peripheral arteries. Clinic 31:449-452, 1932 19. Fewings JD, Hanna JJD, Walsh JA, et al: The effects of ethyl alcohol on the blood vessels of the hand and forearm in man. Br J Pharmacol Chemother 27:93-106, 1966 20. Graf K, Strom G: Effect of ethanol ingestion on arm blood flow in healthy young men at rest and during leg work. Acta Pharmacol Toxicol 17:115-120, 1960 21. Mendeloff AJ: Effect of intravenous infusions of ethanol upon estimated hepatic blood flow in man. J Clin Invest 33:1298-1302, 1954 22. Stein SW, Lieber CS, Leevy CM, et al: The effect of ethanol upon systemic and hepatic blood flow in man. Am J Clin Nutr 13:68-74, 1963 23. Castenfors H, Hultman E, Josephson B: Effect of intravenous infusions of ethyl alcohol on estimated hepatic blood flow in man. J Clin Invest 39:776-781, 1960 24. Lundquist F: The concentration of acetate in blood during alcohol metabolism in man. Acta Physiol Stand 175:97, 1960 25. Forsander 0, Rgiha N, Suomalainen H: Oxydation des Athylalkohols in isolierter Leber und isoliertem Hinterkijrper der Ratte. Hoppe SeylersZ Physiol Chem 318:1-5, 1960 26. Andres R, Cader G, Zierler KL: The quantitatively minor role of carbohydrate in oxidative metabolism by skeletal muscle in intact man in the basal state. Measurements of oxygen and glucose uptake and carbon dioxide and lactate production in the forearm. J Clin Invest 35:671-682, 1956 27. Karlsson N, Fellenius E, Kiessling KH: The metabolism of acetate in the perfused hindquarter of the rat. Acta Physiol Stand 93:391400, 1975 28. Freinkel N, Singer DL, Arky RA, et al: Alcohol hypoglycemia. I. Carbohydrate metabolism of patients with clinical alcohol hypoglycemia and the experimental reproduction of the syndrome with pure ethanol. J Clin Invest 42:1112-1133, 1963 29. Freinkel N. Cohen A, Arky RA, et al: Alcohol hypoglycemia. II A. Postulated mechanism of action based on experiments with rat liver slices. J Clin Endocrinol25:76-94, 1965 30. Lockner A, Wulff J, Madison L: Ethanolinduced hypoglycemia. I. The acute effects of ethanol on hepatic glucose output and peripheral glucose utilization in fasted dogs. Metabolism l&l-18,1967 31. Lundquist F: Production and utilization
JORFELDT
106 of free acetate in man. Nature 1962 32. Krebs
193578-579,
HA: The effects of ethanol on the metabolic activities of the liver. Adv Enzyme Regul6:467-489, 1968 33. Jorfeldt L: Metabolism of L(+)-lactate in human skeletal muscle during exercise. Acta Physiol Stand [Suppl] 338:45-55, 1970 34. Wolfe BM, Have1 JR, Marliss EB, et al:
AND JUHLIN-DANNFELT
Effects of a 3-day fast and of ethanol on splanchnic metabolism of FFA, amino acids and carbohydrates in healthy young men. J Clin Invest 57:329-340. 1976 35. Lundquist F, Tygstrup N, Winkler K, et al: Glycerol metabolism in the human liver: Inhibition by ethanol. Science 150:616-617, 1965
