J. Nutr. Sci. Vitaminol., 27, 117-128, 1981

Effect and Fate of Orally Administered

Lactic Acid in Rats

Masami MOROTOMI, Kosei SAKAI, Kazunaga YAZAWA,

Nobuo SUEGARA, Yasuo KAWAI,

and Masahiko MUTAI1

Department of Intestinal Microbiology, YAKULT Institute for Microbiological Research,

1796 Yaho, Kunitachi, Tokyo 186, Japan

(Received July 22, 1980)

Summary We investigated the effect and the fate of an extremely high

amount of orally administered lactic acid in rats. The dosed amount of

lactic acid, 390mg per 200g body weight (30 times higher than that

normally detected in the stomach of rats), was determined from the results

of observation of acute toxicity of lactic acid in rats. Six hours after the

administration of excess lactic acid together with 10ƒÊCi of L-[U-14C]lactic

acid and 10ƒÊCi of D-[U-14C]lactic acid, rats were sacrificed and the pH of

the blood, the amount of lactic acid in each organ, L-lactate dehy

drogenase (LDH) and some other enzyme activities and incorporation of

radioactivities in each fraction of certain organs were measured. The

control rats were given the labeled lactic acid and the same volume of

water in place of cold lactic acid. Significant decrease of blood pH (•¢-

pH=0.14) and increase of blood lactic acid concentration (2-fold) were

observed. However, these differences were no longer observed at 24hr

after the administration. The amount of lactic acid degraded to expired

CO2 was 42.4% in the experimental group, whereas it was 61.3% in the

control group. Radioactivities incorporated into protein and lipid frac

tions in the experimental group were higher than those in the control

group, 3.8 and 4.9 times, respectively. It was suggested that an extremely

high amount of orally administered lactic acid was utilized as an energy

source, and that an excess of lactic acid was incorporated into protein and

lipid in addition to degradation into CO2.

Key Words lactic acid, rats, blood pH, LDH, lactic acidosis

Lactic acid is an important by-product of glycolysis of muscle glycogen. Metabolism of lactic acid under anaerobic conditions has been investigated by various authors (1-4). Recently, studies on lactic acidosis in diseases such as diabetes mellitus and leukemia have been reported (S, 6). In man, lactic acid is

1 諸富正 己,酒 井 孝成,矢 沢一 良,末 柄信 夫,河 合康雄,務 台方彦

117

118 M. MOROTOMI et al .

supplied not only under anaerobiosis during exercise but also from diet , lactic acid drinks, and lactic acid bacteria in the digestive tracts. However, investigation on the

metabolic fate of orally administered lactic acid and its effect on man is rarely

found. The purpose of the present investigation is to determine the effect and the

fate of orally administered lactic acid in rats.

EXPERIMENTAL

Experiment 1: Acute toxicity of lactic acid. Acute toxicity of orally administered lactic acid in rats was preexamined before tracer experiment. Twenty male Fischer 344 rats weighing about 180g were divided into 4 groups of 5 rats each. Three

groups of rats were given 1,300,650 and 130mg of DL-lactic acid respectively per 200g body weight via a stomach tube. The other group designated as a control was

given the same volume (0.5ml) of water instead of lactic acid. The dose of lactic acid employed was settled based on the LD50 in rats (750mg/200g body weight) (7) and the amount of lactic acid detected in the stomach of normal rats (8).

Experiment 2: Tracer experiment. Ten male rats of the Fischer 344 strain

weighing about 180g were divided into 2 groups of 5 rats each and housed in

wire cages in a room at 24•}1•Ž. The animals were supplied with standard rat

pellets (Oriental Yeast Co., Tokyo, Type NMF) and water ad libitum at all times.

Five rats received, during 9:00-10:00 a.m, by a stomach tube, DL-lactic acid of

390mg/200g body weight together with 10ƒÊCi of L-[U-14C]lactic acid and 10ƒÊCi of

D-[U-14C]lactic acid (The Radiochemical Centre, Amersham, spec . act.

100mCi/mmol and 21mCi/mmol, respectively). The control rats were given labeled

lactic acid and the same volume of water in place of cold lactic acid in the same

manner. The treated rats were placed in a metabolism cage (Natsume Seisakusho , Type KN-0070, Tokyo), enabling the separate collection of urine , feces and expired 14CO

2. The expired 14CO2 was trapped in a CO2-absorber, monoethanolamine. Scintillation medium contained 1ml of CO2-absorber, 2ml of methyl cellosolve, and 7ml of scintillation cocktail (0.1g of POPOP and 4g of PPO in 1 liter of toluene, referred to as scintillation cocktail A) . The animals were killed by decapitation 6hr after the administration. Blood samples were collected in chilled tubes containing heparin. Total blood volume was calculated as 1/13 of the body weight. The liver, kidney, and brain were removed, weighed, and homogenized in 20ml of 0.15M KCl with a Potter-Elvehjem homogenizer with a Teflon pestle. The gastrointestinal tract was also removed and washed gently with the same solution to separate its contents. Then the tissue was homogenized. The contents were also homogenized together with the washings. Radioactivity was measured using a Packard model 3255 Tri-Garb liquid scintillation spectrometer equipped with automatic external standardization. For the determination of the total radioactivity of organs and tissues (other than that of CO2), 0.2ml of homogenates was transferred into counting vials, and to each vial was added 0.3ml of distilled water and 10ml of scintillation cocktail (mixture of 2 volumes of scintillation cocktail A

J. Nutr. Sci. Vitaminol.

EFFECT AND FATE OF LACTIC ACID 119

and 1 volume of Triton X-100, referred to as scintillation cocktail B). For the determination of the radioactivity of the lipid fraction, 1ml of the homogenates was lyophilized, the lipid then being extracted with a mixture of chloroform and methanol (2:1, v/v). The lipid extracts in the organic solvent were evaporated to dryness using a rotary evaporator. The recovered total lipid was dissolved in the organic solvent and the radioactivity was counted in scintillation cocktail A. For the determination of the radioactivity of the protein fraction, a portion of the homogenate was treated with 20 parts of 10% trichloroacetic acid (TCA), and the radioactivity of the insoluble fraction on the glass filter was counted in scintillation cocktail A. For the determination of the radioactivity of the metabolites of lactic acid, 1ml of each homogenate was lyophilized after deproteinization with barium hydroxide and zinc sulfate. The residues were redissolved in a known volume of 2% NH3 solution, and applied for thin-layer chromatography. TLC plates (Silica gel G) were developed with a mixture of n-hexane-formic acid-water (90:7:3, v/v). The chromatograms of lactate, malate, succinate and fumarate, volatile fatty acids

(VFA), and pyruvate including glucose and amino acids were identified with 0.1% brom thymol blue. Futhermore, pyruvate was extracted from the chromatoplate, concentrated by N2 gas blowing under acidic conditions, and applied for TLC with the same developing system to separate pyruvate from amino acids and glucose. The marked zones were scraped into scintillation vials and 0.5ml of distilled water was added, radioactivity being counted in 10ml of scintillation cocktail B.

Others. The animals were autopsied immediately after death and histological

examination was performed by visual inspection and by microscopy. The tissues

observed were the heart, spleen, liver, kidney, pancreas, brain, testis, thymus,

thyroid, parathyroid gland, suprarenal gland, salivary gland, mesenteric lymph

node, and gastrointestinal tracts.

L- and D-Lactic acid was determined by the method of Gutman and Wahlefeld (9), and Gawehn and Bergmeyer (10), respectively. L-Lactate dehydrogenase (LDH) activity was determined by the method of Gutman and Wahlefeld (9). In the, liver and blood, cholesterol concentration was determined by the method of Few (11). The activity of HMG-CoA (3-hydroxy-3-methylglutaryl

- CoA) reductase in the liver was determined by the method of Kuroda and Endo (12).

The activity of cholesterol 7ƒ¿-dehydroxylase in the liver was determined by the

method of Nicolau et al. (13). In the brain, the activities of monoamine oxidase and

glutaminase were determined by the method of Obata et al. (14) and Benzinger et

al. (15), respectively. In the liver and kidney, the activities of glutamate oxaloacetate

transaminase (GOT) and glutamate pyruvate transaminase (GPT) were determined

by the method of Bergmeyer and Bernt (16). The activities of hepatic and renal

glucose-6-phosphatase (G-6-Pase) were determined by the method of Weber and

Cantero (17).

Determination of arterial blood pH. Another 2 groups of animals of 5 rats each were treated with lactic acid in the same way as described in the tracer

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120 M. MOROTOMI et al .

experiment but without labeled lactic acid. Six hours after the administration , the rats were anesthetized with sodium pentobarbital and the arterial blood was taken

from the abdominal aorta. Immediately after the collection of the blood , pH was measured with Hitachi-Horiba pH meter and a combination electrode .

RESULTS

Experiment 1The effect of oral administration of lactic acid on the food intake and body

weight gain of the animals is shown in Fig. I. In the 1,300mg (per 200g body weight) administration group, one rat died, and in the 650mg administration group, two rats died during 24hr after the administration . One day after the lactic acid administration, the concentration of lactic acid in blood was 0.43 and 0.47mg/ml in the control and the 1,300mg administration rats, respectively. After 8 days, the

Fig. 1. Effect of oral administration of lactic acid on the body weight and food intake

of rats. •›, control; •~, group I, administered with 130mg/200g body weight of DL

-lactic acid; •£, group II, 650mg/200g body weight; •  , group III, 1,300mg/200g body weight; •«, administration of lactic acid .

J. Nutr. Sci. Vitaminol.

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122 M. MOROTOMI et al.

same amount of lactic acid was re-administered to the rats. Two rats died in the 1,300mg re-administration group. Dyspnea, snivel, vomiting and abdominal in

fl ation were observed in these animals immediately after the administration . From these results, the amount of lactic acid to administer was settled up 390mg per 200g body weight in the following experiments. This amount of lactic acid was 30

-fold that detected in the stomach of normal rats (8).

Experiment 2Total radioactivity. Degradation of orally administered DL-lactic acid into

expired CO2 is shown in Fig. 2. After 6hr of administration, the percentage of the

given isotope degraded into CO2 was 61.3% in the control animals, whereas 42.4% in the experimental animals. In the control animals, lactic acid was metabolized rapidly into CO2 during the 3hr after administration. Distribution of the total radioactivity and lactate, pyruvate, succinate and fumarate , malate, VFA, amino acid and glucose, protein and lipid fractions in each part of the gastrointestinal tracts, liver, kidney, brain, blood, feces and urine of the experimental and control animals is shown in Table 1. About 91% and 78% of dosed radioactivity were

recovered in the experimental and the control animals , respectively. This difference of recovery seems to be attributed to the difference of recovery of the radioactivity from the stomach contents between the control and the experimental groups , that is, the radioactivity recovered from the stomach contents of the experimental rats was about 37% of dosed amount and 6 times higher than that of the control rats (Table 1). And also, the radioactivity of lactic acid recovered from the stomach contents of the experimental rats was 16 times higher than that of the control rats (Fig. 2). These observations show that efflux from the stomach is slow in the experimental animals. The distribution of the dose% of radioactivity and the amount of each

Fig. 2. Degradation of orally administered lactic acid to expired CO2 and the amount

of 14C-lactic acid remaining in the stomach after 6hr of administration. Mean•}SD,

n=5. -, _??_, control group; ----, _??_, experimental group.

J. Nutr. Sci. Vitaminol.

EFFECT AND FATE OF LACTIC ACID 123

Fig. 3. Metabolic fate of orally administered lactic acid in the control and the experimental group. The dose % of radioactivity and the amount of each fraction expressed as degraded lactic acid in the whole body are shown, calculated from the results shown in Table 1.

fraction expressed as degraded lactic acid in the whole body are shown in Fig. 3,

calculated from the results shown in Table 1. There was a difference in the manner

of the disposal of lactic acid between the experimental and the control animals,

although some problems may remain in comparing the fate of lactic acid between

the control and the experimental groups at 6hr after the administration, because the

amount of expired CO2 reached its plateau at 3hr after the administration in the

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124 M. MOROTOMI et al .

Fig. 4. The amount of lactic acid in the digestive tracts of the control and the

experimental group. _??_, L-lactic acid; _??_ , D-lactic acid.

Fig. 5. The amount of lactic acid in the blood, liver , brain, kidneys, and urine of the control and the experimental group, _??_, L-lactic acid; _??_

, D-lactic acid.

control group.

By the histological examination, bleeding and necrosis of the stomach and the

liver were observed in the rats orally administered an excess of lactic acid . There

were no obvious histological changes in the other organs. The blood pH was

7.50•}0.02 in the control animals and 7.36•}0.03 (significantly different from the

J. Nutr. Sci. Vitaminol.

EFFECT AND FATE OF LACTIC ACID 125

Fig. 6. Specific activity of L-lactate dehydrogenase of the liver, blood, brain, and

digestive tracts of the control and the experimental group.

control, p<0.01) in the experimental animals, respectively. The blood pH seemed to be somewhat higher than actual arterial pH because of brief exposure to air during the collection of the blood. Figures 4 and 5 show the amount of lactic acid in the organs and tissues in the control and the experimental groups. The concentration of lactic acid in the blood of the experimental group was 2.2-fold higher than that of the control group. The amount of lactic acid in the brain and the kidney in the experimental group was 3.1-fold higher than that of the control group. The amount of hepatic lactic acid was similar in both groups. The amount of lactic acid in the stomach contents of the experimental group was 50-fold higher than that of the control group.

Figure 6 shows the activity of LDH in the organs and tissues in both groups.

There was no significant difference in the activity of LDH between the control and

the experimental groups.

Table 2 shows the activities of glucose-6-phosphatase (G-6-Pase), GPT, and GOT of the liver and the kidney in both groups. The activities of hepatic and renal G-6-Pase in the control group were 3-fold higher than those of the experimental

group. GOT and GPT activities in the experimental group were 2- to 3-fold higher than those of the control group. The average activity of L-glutaminase in the control

group was 2-fold higher than that of the experimental group, but there was no significant difference between the two groups. The activity of monoamine oxidase was at similar levels in both groups.

Concentrations of plasma cholesterol in the control and the experimental

animals were 102.4•}8.7 and 123.9•}25.0mg/dl, respectively. There was signifi

cantly difference (p<0.001) in the concentrations of liver cholesterol in the control

(2.47•}0.24mg/g) and the experimental (5.04•}0.28mg/g) groups. The incorpo

ration of radioactivities into cholesterol fraction was examined. Small amounts of

radioactivites, about 0.1% of dosed amount, were detected in the cholesterol

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126 M . MOROTOMI et al.

Table 2. The activities of G-6-Pase, GPT and GOT in the liver and kidney of the

control and the experimental rats.

1) ƒÊg Pi/(min• mg protein). 2) ƒÊmol NAD/(min• mg protein). G-6-Pase, glucose-6

- phosphatase; GPT, glutamate pyruvate transaminase; GOT, glutamate oxaloacetate

transaminase. Data show mean•}standard deviation, a Significantly different from the

control, p<0.05. b Significantly different from the control, p<0.001.

fraction of the liver of both the control and the experimental rats. The activity of

hepatic HMG-CoA reductase, the rate-limiting enzyme of cholesterol biosynthesis,

in the experimental groups was about 2-fold higher than that of the control group

but there was no significance. The activity of hepatic cholesterol 7ƒ¿-hydroxylase, the

rate limiting enzyme of bile acid biosynthesis (cholesterol catabolism), was similar

in both groups.

DISCUSSION

Ritz and Heidland (S) reported that lactic acidosis was defined as a state of metabolic acidosis (i.e. arterial pH<7.3) due to an increase in the blood concentration of lactic acid (i.e.>2.0mEq/liter). However, Oliva (6) reported that the concentration of lactic acid of arterial blood increased to 8.0-22mEq/liter (13- to 36-fold higher than normal) during exercise. Oliva (6) also reported that the concentration of lactic acid of arterial blood was increased 3.2- to 56.6-fold in cardiovascular failure, 4.9- to 6.5-fold in acute hypoxemia, more than 3.1-fold in severe anemia, 20- to 40-fold in leukemia, and 17- to 50-fold in diabetes mellitus. In our experiment, the pH of arterial blood in the experimental group after 6hr of administration was 7.36, and the concentration of lactic acid in arterial blood in the experimental group (11.0mEq/liter) was increased to 2-fold of that in the control

group (5.1mEq/liter). Therefore, according to Ritz and Heidland's criteria, a tendency of lactic acidosis was observed. However, this increase of lactic acid in the blood was temporary and less striking than the increase of lactic acid under such diseases reported by Oliva (6), although we administered an extremely high dose of lactic acid in an acid form (the pH of the administered lactic acid solution was about 1.0). The dose level in this study was approximately more than 100 times higher than

J. Nutr. Sci. Vitaminol.

EFFECT AND FATE OF LACTIC ACID 127

the expected intake in humans from lactic acid drinks. Drury and Wick (18) reported that in the constant injection method, 65% of the dosed lactic acid was degraded to expired CO2 in rabbits. In our experiments, 61.3 and 42.4% of the dosed lactic acid in the control and the experimental group were degraded to expired CO2 for 6 hr. Brooks et al. (2) showed that for 90 min, 75% of the dosed 1-14C-lactate was degraded to expired CO2 in rats . This difference from our result

(61.3%) may depend on different labeling at the carbon position of lactic acid and the route of administration. The amount of lactic acid degraded to expired CO2 for

6hr was calculated as 8.0mg in the control group and 165.4mg in the experimental

group, and then 1.1 and 21.6mmol of ATP were formed in the control and the experimental groups, respectively. In the control group, the radioactivities were

mainly detected in the lipid, amino acid and glucose, and protein fractions apart

from in the expired CO2. In the experimental group, the radioactivities were mainly

detected in the lactate, lipid, protein, and amino acid and glucose fractions apart

from in the expired CO2.

There were no striking differences of the LDH activities between the two

groups, although the isozymes of the LDH were not examined. There remain some problems in comparing enzyme activities of the liver in the experimental group with those in the control, because bleeding and necrosis of the liver were observed in the experimental group. The hepatic HMG-CoA reductase activity of the experimental

group was somewhat higher than that of the control group, and it was also observed that plasma and hepatic cholesterol levels of the experimental group were higher

than those of the control group. The radioactivities of protein fractions of the liver,

blood, brain, kidney, and gastrointestinal walls in the experimental group were

higher than those in the control group. The GOT and GPT activities of the liver and

the kidneys in the experimental group were higher than those of the control group,

suggesting that the excess amount of dosed lactic acid was incorporated into amino

acids via TCA cycle members and pyruvate by transamination, and then into

protein from amino acids. The G-6-Pase activities of the liver and the kidneys in the experimental group were lower than those in the control group, suggesting that the

gluconeogenesis system was not stimulated by the orally administered lactic acid in this experiment. The L-glutaminase and monoamine oxidase activities of the brain

in the experimental group did not differ from those in the control group. This may

suggest that orally dosed lactic acid in this experiment had no effect on detoxifying

activities of the brain of the rats.

From these results, it is assumed that oral intake of lactic acid from foods

habitually taken by humans may have no toxic effect in healthy adults but may be

degraded rapidly to expired CO2. More detailed experimental research on the effect

of lactic acid in infants and the aged is necessary.

REFERENCES

1) Rowell, L. B., Kraning, K. K., Evans, T. O., Kennedy, J. W., Blackmon, J. R., and Kusumi, F. (1966): Splanchnic removal of lactate and pyruvate during prolonged

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128 M. MOROTOMI et al.

exercise in man. J. Appl. Physiol., 21, 1773-1783.2) Brooks, G. A., Brauner, K. E., and Cassens, R. G. (1973): Glycogen synthesis and

metabolism of lactic acid after exercise. Am. J. Physiol., 224, 1162-1166.3) Issekutz, B., Shaw, W. A. S., and Issekutz, T. B. (1975): Effect of lactate on FFA and

glycerol turnover in resting and exercising dogs. J. Appl. Physiol., 39, 349-353.4) Hubbard, J. L. (1973): The effect of exercise on lactate metabolism. J. Physiol, 231,

1-18.5) Ritz, E., and Heidland, A. (1977): Lactic acidosis. Clin. Nephrol., 7, 231-240.6) Oliva, P. B. (1970): Lactic acidosis. Am. J. Med., 48, 209-225.7) in The Merck Index, 9th. ed., Merck and Co., Inc., Rahway, N. J., p. 700.8) Yokokura, T., Yajima, T., and Hashimoto, S. (1977): Effect of organic acid on

gastrointestinal motility of rat in vitro. Life Sci., 21, 59-62.9) Gutmann, I., and Wahlefeld, A. W. (1974): L-(+)-Lactate. Determination with lactate

dehydrogenase and NAD, in Methods of Enzymatic Analysis, Vol. 3, ed, by Bergmeyer, H. U., and Gawehn, K., Academic Press, Inc., New York and London, pp. 1464-1468.

10) Gawehn, K., and Bergmeyer, H. U. (1974): D-(-)-Lactate, in Methods of Enzymatic

Analysis, Vol. 3, ed. by Bergmeyer, H. U., and Gawehn, K., Academic. Press, Inc., New

York and London, pp. 1492-1495.

11) Few, J. D. (1965): Some new steroids color reactions. Analyst, 90, 134-146.

12) Kuroda, M., and Endo, A. (1977): Inhibition of in vitro cholesterol synthesis by fatty

acids. Biochim. Biophys. Acta, 486, 70-81.

13) Nicolau, G., Shefer, G., Salen, G., and Mosbach, E. H. (1974): Determination of

hepatic cholesterol 7ƒ¿-hydroxylase activity in man. J. Lipid Res., 15, 146-151.

14) Obata, F., Ushiwata, A., and Nakamura, Y. (1971): Spectrophotometric assay of monoamine oxidase using 2,4,6-trinitrobenzen-l-sulfonic acid. J. Biochem., 69, 349-354.

15) Benzinger, T., Kitzinger, C., Hews, R., and Burton, K. (1959): Free energy changes of the glutaminase reaction and the hydrolysis of the terminal pyrophosphate bonds of adenosine triphosphate. Biochem. J., 71, 400-407.

16) Bergmeyer, H. H., and Bernt, E. (1974): Glutamate-oxaloacetate transaminase, in Methods of Enzymatic Analysis, Vol. 2, ed. by Bergmeyer, H. U., and Gawehn, K., Academic Press, Inc., New York and London, pp. 727-733.

17) Weber, G., and Cantero, A. (1955): Glucose-6-phosphatase activity in normal,

precancerous, and neoplastic tissues. Cancer Res., 15, 105-108.18) Drury, D. R., and Wick, A. N. (1956): Metabolism of lactic acid in the intact rabbit.

Am. J. Physiol., 184, 304-308.

J. Nutr. Sci. Vitaminol.