Acta physiol. scand. 1974. 91. 76-82 Institute of Physiology, Vnivenity of Aarhus, Denmark

The Effect of Glycerol on the Postexercise Lactate Clearance By

CHRISTIAS OLSEN and EBBE STH.%XGE PETERSEN

Received 26 Sovember 1973

Abstract

OI.sES: CHR. and E. s. PETEnsEs. Tht. cffcct of g1)Wrol 011 the postexercise lactate clt.arapicc. . h a physiol. scand. 1974. 91. 76-82.

The effert of increased glycerol concentrations induced by peroral intake of glycerol on the concentrations of lactate and pyruvate ill blood was investigated in 7 young adults at rest and after exercise. 10 niin of work on a bicycle ergometer induced a sniall but statistically signifi- cant rise in the glycerol concentration. The concentration of lactate at rest was significantly higher after than before glycerol intake. The lactatelpyruvate ratio was significantly higher after than before glycerol intake both at rest and after 15 min of recovery and onwards. The metabolic clearance of lactate after exercise was reduced by the glycerol intake to about 70 '/o of the control value. The effect of glycerol on the metabolic clearance of lactate was the same whether estimated at rest or after exercise. These observations are in accordance with the concept. that glycerol metabolism influences the lactatelpyruvate ratio in the liver and thereby reduces the clearance of lactate.

Ethanol has been found to inhibit gluconeogenesis from glycerol by the perfused liver (Krebs 1968.1: also in man ethanol has been found to inhibit glycerol metabolism (,Lundquisr c*t 01. 1965). In both these conditions the hepatic content of L-3-glycerophosphate was increased, probably through an increase in the extra- mitochondria1 N.\DH,/N.AD' ratio ! Krebs 1968, Robinson and Xewsholme 1969).

Similarly cthanol has been found 10 inhibit the conversion of lactate to glucose in the perfured rat liver I Krebs ct 01. 1969 b) , and in man following infusion of "C-labelled lactate i Kreisberg. Owen and Siegal 1971 arid .severe exercise (Krebs cf 01. 1969 a ) . Also in these cases the underlying inechanism coiild be a reduction of NAD' to NADH driving the reaction

Lactic acid + X.\D'#Pyruvic acid I N.\DH + H' towards the left. Glycerol is metabolized predominantly by the liver ( Larsen 1963, Borchgrevink

mid Have1 1963). while the small intestine [,Saunders and Dawson 1962. Holt 1964), the kidney and probably other tissues as well play a minor role (Robinson and Scwsholnie 1969 I . The initial steps in the gluconeogenesis from glycerol are:

76

GLYCEROL AND LACTATE ELIMINATION 77 Glycerol + ATP+L-3-glycerophosphate + ADP L-3-glycerophosphate + NAD'edihydroxycaetone phosphate + NADH. I t might consequently be expected that reduced pyridinenucleotide generated by

the metabolism of glycerol (cf. Williamson et al. 1969)-as by ethanol-might result in impairment of lactate uptake. This situation may be relevant at physiologically raised glycerol concentrations in the postexercise period. Lipolysis with increasing concentrations of free fatty acids-and glycerol-is an important energy yielding factor not only in work of long duration (Rodahl, Miller and Issekutz 1964), but also in relatively short lasting work (Keul, Doll and Keppler 1968).

In the present work we have consequently studied the clearance of lactate in the postexercise period with or without artificially raised blood glycerol concentrations. A preliminary report has been presented (Olsen and Strange Petersen 1973 a) .

A study of the changes in the lactate concentration and in the ratio of lactate to pyruvate (L/P) in arterial and venous blood draining a resting limb after i.v. injec- tion of sodium- (L + ) -lactate and after exercise has been reported previously (Olsen and Strange Petersen 1973 b) .

Methods The subjects were 9 healthy students, 8 men and 1 woman aged 22-28 years. The subjects came to the laboratory in the morning. They had been instructed not to take any alcohol in 24 h and not to exercise on the morning of the experiment. The experiments started at least 2 h after a light breakfast and 1 h rest on a couch.

4 types of experiments were carried out: 1. 7 subjects worked for 10 min on an electrically braked bicycle ergometer (Elema Schonan-

der) at a load previously determined to give a steady state heart rate of 170-180 beats per minute. When the work period had ended the subjects were placed on a couch in supine position.

2. This type of experiment was similar to the first with the one exception that the 7 sub- jects were given glycerol orally before work started, and at intervals during the recovery period.

3. 3 subjects were given glycerol orally as above, but did not exercise, and spent the whole experimental period of 2 1 / 2 h resting on the couch.

4. 7 subjects spent the whole experimental period resting on the couch without glycerol load.

In the experiments of type 2 and 3 the subjects drank 25-50 g glyceroI in 25 m1 water 1 /2 h before work started. 5 g glycerol in 10 ml water was subsequently given with 20 min intervals to maintain a concentration of about 8 mM.

The glycerol used was redestilled (Merck 4094)-and free from ethanol. Venous blood was collected through a polythene catheter inserted through an antecubital

vein to the avillary vein. Arterialized capillary blood was taken from the hyperemized ear lobe (in the following these samples will be referred to as arterial (cf. Siggaard-Andersen 1966)). Simultaneous samples were taken as indicated in results.

Arterial blood dripped freely into tubes with iced perchloric acid; similarly venous blood was precipitated immediately after sampling.

Lactate and pyruvate concentrations in both venous and arterial blood samples were measured by an enzymatic, fluorimetric micromethod (Olsen 1971 ).

Glycerol concentrations (in venous plasma only) were determined according to the methcd of Wieland (1957) as modified by Larsen (1963). Statistics: P-values have been calculated from t-tests based on paired comparisons except for the U P ratios on which Wilcoxon's signed-rank test was employed (Sokal and Rohlf 1969).

CHRISTIAN OLSEN A N D EBBE STRANGE PETERSEN

GLYCEROL ATE

78 LA<

tnhi

1

I

Fig. 1. Results of one experi- ment in which glycerol was given but no work was per- formed. The points to the left of the break in the time axis represent samples taken before gl)-cerol was given. Time 0 is 30 min after glycerol in- take. Lactate (open circles) and L / P ratio (filled circles) \<ere measured in arterial blood. GI>-cerol (dots) was measured in venous plasma.

T m L t . I. T h e t-ffcct of qlycerol o n the mean arterial concentrations (:S.E.) of Lactate, Pyruvate, and 12;P ratio a t rest ( n = 10).

Control Glycerol

Lactatr 11111 0.63 0 07 o 91 + o m P -.. 0.01

s s

P i 0.005

P\ l l l \ 111 [ I l l l l 0 . 0 7 7 - 0 011 0.084 ;L 0 009

1 , P ratio 8.5 11.0

'r \ H I . F 11. The influencr o f the experimental condition on the mean arterial concentrations ( - : ~ S . E . , of Lactate. Pyru\-ate, and LIP ratio at rest. Sample I 1 was taken 30 min after sannplr I. i n = i'i

I I1

Lactatr ( m \ I 0.S5 I 0.05 0.52 iO.05

Pyruvate ! i n l I i o .079~,0 .013 0.072 50.01 3

I. P ratio 7.9 7.8

Results

'Three restins subjects ivere given glycerol orally. The concentrations of glycerol and lactate, and the lactate/pyruvate ratios in one of these esperiments are shown in figure 1. The glycerol concentration at time 0 (that is 30 min after the intake of ;I0 g glycerol I NXS 0.46 m M cornpaired to 0.20 mM before glycerol was taken. After this the concentration started to rise more rapidly to reach a plateau of about 10 mR1 in about 7.5 min. This pattern of changes in glycerol concentration follow-

GLYCEROL A N D LACTATE ELIMINATION 79 ing oral intake was also seen in the two other subjects who did not work, and in the 7 subjects who were given glycerol and subsequently worked for 10 min.

The mean glycerol concentrations before and 30 rnin after the intake of glycerol in the 10 subjects were 0.21 mM and 2.95 mM respectively.

Fig. 1 further shows that although the glycerol concentration at time 0 had only risen moderately, both the lactate concentration and the L/P ratio had approximate- ly doubled; these two parameters remained at this new level without further changes in spite of the further increase in glycerol concentration.

Mean values of lactate, pyruvate, and the L/P ratio in arterial blood before and 30 rnin after oral glycerol intake in the 10 expts. are presented in Table I. Whilst the change in pyruvate concentration was not significant, both the lactate concen- trationand the L/P ratio were significantly higher in the glycerol than in the control experiments.

In resting conditions which were similar except that no glycerol was given, the lactate and pyruvate concentrations and the L/P ratio in two blood samples taken at 30 min interval were almost identical (Table 11).

The glycerol intake did not influence significantly on the pyruvate concentrations at any time during the recovery period after exercise. The L/P ratio (like at rest) was highest in the glycerol experiments, the difference, however, was not significant during the first 15 rnin of recovery.

Table I11 shows that the maximal lactate concentrations reached in the two types of experiments were very similar The heart rate changes, too, were almost the same on the two occasions, the average resting heart rate being 74 and 67, respectively, and the steady state value during work 179 and 177, respectively.

The mean rate of lactate removal can be evaluated by inspection of Table 111; the decline in concentration of lactate during the first 30 rnin after work was rough- ly idenrical in the two types of experimental situation; during the later phase of recovery, from 50 rnin onwards, however, the concentrations were consistently and significantly higher in the glycerol than in the control experiments.

On plotting the logarithms of the lactate concentrations against time, the points representing the last 5 samples describe a fairly well defined linear fall. Assuming this exponential decline in lactate concentration to represent the metabolic removal of lactate by conversion to glucose and by oxidation, the rates of these metabolic processes can be roughly estimated by the half-times. The half-time in the control experiments was about 40, and in the glycerol experiments about 60 min.

Similar half-times were obtained whether arterial or venous concentrations were used indicating that this latter part of the curve represents metabolic changes only.

The average changes in glycerol concentration induced by work in the controI experiments are shown in Fig. 2. The concentration started to increase during the work period; towards the end of work the concentrations were significantly higher ( P < 0.01) than at rest. Maximal concentrations were seen 5-10 rnin after the end of work, and the values were not yet normalt a t the end of the experiments about 1 1/2 h after work.

80 CHRISTIAN OLSEN AND EBBE STRANGE PETERSEN

'TABLE 111. The effect of glycerol on the mean arterial Concentrations (5S.E.) of lactate (rnnt) in the recovery period after exercise.

l l inutes after esercise 0 3 10 15 20

Control n = T 7 29=0.39 i.34:0.89 4.58=0.60 3.54: 0.64 2.56+0.39

Glyccrol n ~ 7 b 73,0 65 b 01+0.86 4.43+0.67 3 .76 i0 .73 2.5610.36

P A S s 5 NS XS NS

Discussion

We ha1.e found the rate of removal of lactate produced during a 10 minute period of fairly heavy kvork significantly diminished when blood glycerol concentrations had been raised by oral intake of glycerol. This observation parallels that of Krebs c , t al. ( 1969 a I of the effect of alcohol on lactate clearance.

Resting arterial lactate concentrations ivere significantly increased following glycerol intake : this increase appeared to be caused by glycerol as the experimenta1 condition by itself did not induce any such changes ! c f . Table I and 11).

Lactate rrmoval is generally considered to involve at least two different mecha- nisms. a diffusion into body water. and metabolic removal. mainly by reconversion to glucose. but also by direct oxidation.

Lactate removal during the first 3 0 4 0 min of recovery in our experiments was not affected by glycerol. The major cause of the fall in lactate concentration in this period heirig diffusion. and as glycerol is not likely to affect either the distribution volume or the rate of diffusion. this is not unexpected.

From -1.0 min onivards the decline in lactate concentration was significantly slower when glycerol concentrations were high indicating an impaired metabolism of lactate. The difference in rate of metabolism in the two experiments has been quantified by treating the latter parts of the lactate clearance curves as monoex- ponential and calculating the half-times. Thus roughly estimated glycerol reduced the metabolic rate of con\-ersion of lactate to 40/60 or 67 Cyk of the control value.

Assuming the production of lactate in the tissues a t rest to be unaffected by glycerol. and lactate concentrations to reflect a steady state between production and metabolic turn-o\-er of lactate. the ratio of the lactate concentration a t rest with or without raised glycerol concentration should similarly reflect the extent to which lactate metabolism is inhibited by glycerol. This ratio was i f f . Table I ) 0.63/0.91 or 0.69. a closely value similar to that estimated from the half-times.

The suggested mechanism underlying this finding is that an increased glycerol metabolism largely taking place in the liver causes a rise in the cytoplasniatic NADH/NAD' ratio. This change again is reflected by an increased L/P ratio in the cytoplasm, and in the blood leaving the liver (Schimassek 1963).

An increased lactate concentration and L/P ratio in hepatic tissue and in the blood of stall-ed rats following injection of glycerol has been reported by William-

GLYCEROL AND LACTATE ELIMINATION 81

25 30 35 50 65 80 95

2.21+0.24 1.71h0.15 1.41 1 0 . 1 8 1.15k0.06 0.8310.09 0.63k0.02 0.62+0.04 2.0810.27 1.87&0.21 1.6610.15 1.33k0.08 1.24h0.14 0.99k0.05 0.91 +0.05 NS NS NS < 0.005 < 0.025 < 0.005 < 0.005

son et al. (1969). Also in the perfused rat liver an initial increase of lactate output and of L/P ratio following glycerol has been observed (Olsen, unpublished). In the present work both arterial lactate and L/P ratio were increased during the period of raised glycerol concentration at rest and during recovery from exercise in agree- aent with this concept.

Lundquist et al. (1965) did not find the L/P ratio in hepatic venous blood significantly changed during glycerol infusion, and claimed that the hepatic lactate consumption was unaffected by glycerol. In a later report (Tygstrup, Winkler and Lundquist 1971), however, an increase in the hepatic venous L/P ratio from 24 to 33 was observed in 3 expts.

The level of glycerol concentration reached, following oral glycerol intake in our experiments, was very high. The results presented in Fig. 1, however, seem to in- dicate that already at a glycerol concentration of slightly above twice the resting concentration-;and perhaps at an even lower value-the maximum change in both lactate concentration and L/P ratio is manifest.

A question to be raised now is whether the normally occurring changes in glycerol concentration during work are sufficiently large to affect lactate removaI. In our short work periods clear and significant increments in glycerol concentration were observed (Fig. 2 ) . The mean change from rest to maximum was 0.07 mM, or approximately 32 $'& (range 7-132 %).

Other workers employing longer exercise periods have reported changes of up to 600 "/o (Havel, Pernow and Jones 1967, Johnson et al. 1969, Keul, Doll and Haralambie 1970). Both Johnson et al. (1969) and Keul et al. (1970) found greater glycerol changes in trained than in untrained subjects.

GLYCEROL I 1

mMI I I

J i i Fig. 2. Changes in glycerol con- centration during and after work

Bars indicate 2 S.E. 0 10 30 50 70 90 Minubs in the control experiments. n = 7. < 1WoRK:

6-743003. Acta physiol. scand. Vol. 91: I

82 CHRISTIAN OLSEN AND EBBE STRANGE PETERSEN

It appears therefore reasonable to believe that glycerol concentrations during work exceeding a certain intensity and duration may reach levels where lactate metabolism may be inhibited.

This study was supported by the Danish Medical Research Council. We are indebted to Miss Lis i Hansen and Mn. Kirsten Klosterqiard for valuable technical assistance. -

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