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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
Vol. 27, No. 2, 1981
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.
Tab
le
1.
Rad
ioac
tivity
of
each
fra
ctio
n in
ce
rtai
n tis
sues
in
the
cont
rol
and
the
expe
rim
enta
l ra
ts.
Mea
n va
lues
n=
5.
a Pe
rcen
tage
to
th
e do
sed
amou
nt. b
Pe
rcen
tage
to
th
e to
tal
coun
t.
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
Vol. 27, No. 2, 1981
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
Vol. 27, No. 2, 1981
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.
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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
Vol. 27, No. 2, 1981
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
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J. Nutr. Sci. Vitaminol.
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