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1 June 1997 D-Lactate Is Present in Much Larger Amount than L-Lactate in Cephalopods and Gastropods
Shinji Ohmori, Yuko Ohsaki, Shinsuke Akagi, Chie Kondoh, Michi Kawase, Toshie Nagai
Author Affiliations +
Abstract

It has long been known that only L-lactic acid is found in animals and that D-lactic acid is produced in microbial organisms. During the course of study of D-lactate formation from methylglyoxal (the methylglyoxal bypass) in animals, we found that D-form is mainly present in Octopus vulgaris and very little L-lactate was found in octopus muscle. There is an inverse relationship between octopus and normal animals for concentrations of D-and L-lactate. The activities of D-lactate dehydrogenase (D-LDH) was predominantly found in octopus muscle, while L-LDH activity was scarcely detected. Methylglyoxal was the best substrate for D-lactate formation in octopus foot homogenate and pyruvate was the second best substrate. It was also found that D-lactate is present in much higher amounts than the L-form in some animals or plants.

INTRODUCTION

D-Lactate was thought to be an intermediate of glucose metabolism in organisms from 1913 to about 1933 (Dakin and Dudley, 1913; Neuberg, 1913; Neuberg and Kobel, 1928, 1930; Vogt, 1929). Ever since Embden, Meyerhof and others established the glycolytic pathway (Embden et al., 1933; Meyerhof and Lohman, 1934), D-lactate has been forgotten. The presence of D-lactate in fermented products by lactobacilli is well known, but whether D-lactate is normally present in animals and plants has not been known and the physiological meaning of its absence or its presence in them has not been described in any textbook. In microbes, D-lactate is mainly formed via methylglyoxal (MG) from triosephosphates as shown in Fig. 1. In order to study this metabolic pathway in higher plants and animals we first developed quantification methods for D-lactate (Ohmori and Iwamoto, 1988; Ohmori et al., 1991) and MG (Ohmori et al., 1987a,b). Thereafter, we investigated the plasma level of D-lactate in human after exercise (Kondoh et al., 1992b; Ohmori and Iwamoto, 1988). D-Lactate in blood plasma increased 3 to 3.6 times even after a short period of exercise such as 5 min-running; however, MG content in red blood cells decreased to 13% of the initial levels. Next we measured concentration of D-lactate and its related metabolites in liver, blood and muscle of diabetic and starved rat (Kondoh et al., 1992a; Ohmori and Iwamoto, 1988; Ohmori et al., 1987a,b). These rats had significantly higher levels of D-lactate in plasma, liver and skeletal muscle compared with the control group (Kondoh et al., 1992a). Successively, we studied the age-related changes in the levels of D-lactate and its related compound in rat tissues. The hepatic levels of D-or L-lactate, MG, pyruvate and inorganic phosphate markedly decreased during aging process (Kawase et al., 1995). Recently, rats were fed a diet containing 0.064% 3′-methyl-4-dimethylaminoazobenzene (a hepato-carcinogen) for 21 weeks. After the start of the diet hepatic contents of MG and D-lactate increased to 7 and 3 times that of the control, respectively (Kawase et al., 1996). Up to present, we have studied the biochemistry of D-lactate in mammals, however, we wanted to study further the biochemistry of D-lactate in lower animals and plants. At first we studied the distribution of D-lactate in various organisms, which can be easily available. These results are reported in this paper.

Fig. 1

Proposed pathway for the formation of D-lactate in octopus.

i0289-0003-14-3-429-f01.gif

MATERIALS AND METHODS

Chemicals

Hydrazine sulfate, o-phenylenediamine and rabbit muscle aldolase were purchased from Wako Pure Chemicals (Osaka, Japan). Fructose-6-phosphate and γ-glycerophosphate dehydrogenase (γ-GDH) from rabbit muscle, pig heart LDH, phosphoenolpyruvate, and NAD+ were obtained from Oriental Yeast (Tokyo, Japan). Lithium L-and D-lactate, triosephosphate isomerase from rabbit muscle, fructose-1,6-diphosphate and S-lactoyl-glutathione (S-LacGS) were from Sigma Chemicals (St. Louis, Mo., USA). D,L-6,8-Thioctamide was purchased from Tokyo Kasei Kogyo (Tokyo, Japan). MG was prepared just before use by hydrolysis of the dimethylacetal (Aldrich, Milwaukee, Isc., USA) (Kellum et al., 1978). D-Lactate dehydrogenase (D-LDH) from Staphylococcus sp. and diaphorase from Clostridium kluiveri were kindly supplied by Amano Pharmaceutical (Nagoya, Japan). Reduced glutathione (GSH) was kindly supplied by Senju Pharmaceutical (Itami, Japan).

Animals and plants

Octopus, squid, turbo and all vegetables were purchased from appropriate local stores. Other marine animals were kindly provided by the Ushimado Marine Laboratory of Okayama University and African giant snail was kindly supplied by Prof. H. Takeuchi, the medical school of Gifu University. All animals were alive before the experiment.

Homogenization and extraction

Most biological samples were homogenized at 10,000 rpm for 5 min with a Waring blender in 5 volumes of the ice-cold 10 mM potassium phosphate (pH 7.0). Livers were homogenized at 4°C for 30 s in a glass Teflon homogenizer. Fresh baker's yeast was boiled in 100°C for 15 min and centrifuged at 700 × g for 15 min.

Determination of metabolite concentrations

D-Lactate and pyruvate were determined by our methods (Ohmori and Iwamoto, 1988). 3-Methyl-2-quinoxalinol generated from pyruvate which was also derived D-lactate was measured with a Gasukurokogyo liquid chromatograph, model 572 (Tokyo, Japan) with a Gasukurokogyo UV-detector, model 502s. L-Lactate concentration was measured by the UV method (F-kit, Boehringer, Mannheim, FRG), when a large enough quantity of sample had to be assayed. MG was measured as 6,7-dichloro-2-methylquinoxaline by our GLC-method with electron capture detection (Ohmori et al., 1987a). A Shimadzu gas chromatograph, model GC-4CMPEE (Kyoto, Japan) equipped with a 63Ni electron capture detector was used. Inorganic phosphate was determined by the colorimetric method of Lanzetta et al. (1979). Measurements of glycerol were performed using an UV detection kit (Boehringer). GSH and oxidized glutathione (GSSG) were measured by a modification of the method of Griffith (Matsumoto et al., 1996). Glucose was determined at 505 nm using a kit (glucose CII-Testwako, Wako Pure Chemicals, Osaka, Japan). Protein concentrations were measured by the biuret method (Beisenherz et al., 1953).

Enzyme assays

Activities of glycerokinase (GK), 6-phosphofructokinase (PFK), pyruvate kinase (PK), aldolase, glyoxalase I, and glyoxalase II were determined by methods previously described in the literatures (Bergmeyer, 1983; Gracy et al., 1970; Kemp, 1975; Backer, 1955; Valentine and Tanaka, 1966), respectively. The L-and D-LDH activity were measured by the UV method of Vassault (1983).

Configuration of lactate in octopus foot

Octopus foot was boiled for 10 min and the boiled foot was cut into small portions by scissors. The cut tissues were homogenized by a mixer with the boiled water. The homogenate was further boiled for 5 min. The filtrate from the boiled homogenate was passed through a Dowex 50 w ×8 (H+-form) column. The effluent and the washing by 50% methanol from the column were combined and concentrated under reduced pressure. The concentrate was extracted by n-hexane and the water layer was automatically extracted with ether for 40 hr. After the ether layer was evaporated under reduced pressure, the residue was analyzed by a chiral column, Sumichiral OA-5000 (150 × 4.6 mm) (Sumika Chemical Analysis Service, Ltd. Osaka, Japan). The column was eluted by 2 mM copper sulfate containing 5% acetonitrile at flow rate 1.0 ml/min and built in a Shimadzu liquid chromatography, Model LC-6A (Kyoto, Japan). All runs were performed at room temperature and the eluate was monitored at 254nm.

D-Lactate formation from pyruvate in octopus foot homogenate

Octopus feet (8.0 g) were homogenized with Waring blender in 5 volumes 50 mM potassium phosphate (pH 7.0). The homogenate was centrifuged at 6,000 × g for 15 min. The supernatant was dialyzed at 4°C for 2 hr against 5 l of 50 mM potassium phosphate (pH 7.0). The composition of the reaction mixture was as follows: 150 to 600 μl of 1 mM sodium pyruvate, 100 μl of 1 mM NADH, 100 μl of 10 mM nicotinamide, 100 μl of the dialyzed supernatant, and 50 mM potassium phosphate (pH 7.0) for a final volume of 1.0 ml. After the mixture was incubated at 25°C for 30 min, 3 ml of methanol was added and the mixture was centrifuged at 3,000 × g for 15 min. The precipitate was extracted with 4 ml of 70% methanol (methanol: water, 7:3, v/v) and the suspension was centrifuged as above. One ml of the combined extract supernatants was evaporated using a Savant Speed Vac Concentrator (Model SUC-100H, New York). The residue was assayed for D-lactate by our method (Ohmori and Iwamoto, 1988; Ohmori et al., 1991).

RESULTS AND DISCUSSION

The distribution of D-lactate and its related metabolites in some organisms

As can be seen in Table 1, the most striking finding is that lactic acid in octopus was almost all in the D-form. Other cephalopods and gastropods tested had relatively high amounts of D-lactate. However, since turbo is an exception to the general observation tested here, we are widely studying the D-lactate content in these kinds of animal. Surprisingly, some plants also have exceedingly high levels of the D-form. In Fig. 1, two routes for the D-lactate formation are shown, one is the route via MG from triosephosphates, the other is the Embden Meyerhof pathway via phosphoenolpyruvate. As can be seen in the figure, the former is less effective to produce energy and the latter is energy-productive. D-Lactate and its related metabolites shown in Fig. 1 were determined in octopus foot muscle and rat tissues and the determined values are listed in Table 2. As can be seen from this table, D-lactate concentration in octopus was much higher than those of rat tissues and also than the concentration of L-lactate in rat tissues. On the other hand, L-lactate level in octopus was very low. The configuration of the intrinsic lactate in octopus foot was confirmed by the chiral column. L-and D-Lactate were eluted at retention times of 12 and 15 min, respectively. The latter was found to be produced in much larger quantities. Glutathione level was lower in octopus muscle than in rat tissues. The present report describes for the first time the contents of D-lactate and the MG bypass-related compounds in octopus.

Table 1

D-Lactate and L-lactate in various organisms

i0289-0003-14-3-429-t01.gif

Table 2

Concentrations of D-lactate and its related metabolites in octopus

i0289-0003-14-3-429-t02.gif

Some enzyme activities related to the D-lactate formation

In Table 3, enzyme activities related to the D-lactate formation are listed. PK and PFK are regulating enzymes in the glycolytic pathway in most organisms. The GK activity was also assayed, because it was found to play an important role for the D-lactate formation in rat liver (Kondoh et al., 1994). Aldolase is the enzyme of the division point of the MG bypass and the glycolysis. These enzyme activities in octopus resembled those in rat skeletal muscle more than those in liver except for the D-LDH and L-LDH activity which was scarcely detected in octopus. From Tables 2 and 3 or Fig. 1, higher level of D-lactate in octopus muscle (in Tables 1 and 2) can be understandable, that is, D-lactate appears to be produced from both the MG bypass and the Embden Meyerhof pathway in octopus, while D-lactate must be formed only from the MG bypass in rat, because it possesses no D-LDH.

Table 3

Enzyme activities related to the D-lactate formation

i0289-0003-14-3-429-t03.gif

The D-lactate formation in the homogenate of octopus

As a next problem, we want to know which route is major for the D-lactate formation in octopus. As shown in Fig. 2, production of D-lactate from pyruvate was investigated using homogenate of octopus foot. D-Lactate was formed from 300 μM pyruvate with a 27% yield, which corresponded to an 81% yield on a NADH-added basis. In Fig. 3, the formation of D-lactate from pyruvate, MG and S-LacGS by the homogenate of octopus is shown. Yields were 43.9, 61.2 and 75.9% from pyruvate, MG and S-LacGS, respectively. From the results described here, it is thought that D-lactate is formed from both MG and pyruvate in octopus and the rate of the formation is more favorable from MG than from pyruvate at substrate concentrations of 150 μM.

Fig. 2

D-Lactate formation from pyruvate in octopus foot homogenate. Octopus feet were homogenized and the homogenate was centrifuged. The supernatant was dialyzed. Various amounts of pyruvate were incubated with NADH in the supernatant and D-lactate formed was determined. Details of the method are written in Materials and Methods.

i0289-0003-14-3-429-f02.gif

Fig. 3

D-Lactate formation from pyruvate, methylglyoxal and S-lactoylglutathione in octopus foot homogenate. The incubation condition was described in Materials and Methods with the exception that substrates used were 150 μl of 1 mM sodium pyruvate, methylglyoxal, or S-lactoylglutathione. GSH was added in concentration of 0.5 mM to the incubation mixture for methylglyoxal.

i0289-0003-14-3-429-f03.gif

The presence of high levels of MG in octopus may be due to low levels of GSH in the organism (Table 2).

In conclusion, many people have the common thought that L-lactate is the physiological lactate, however, as reported here, D-lactate is predominantly present in some organisms.

Recently, we isolated two forms of D-LDH from octopus foot muscle. They had the same molecular mass of 38 kDa on SDS-PAGE and were monomer. Sequencing of genes of two types of D-LDH revealed that the homology between this D-LDH and known L- and D-LDH was very low. These results will be soon published.

Acknowledgments

We are grateful to Dr. J. Shiozawa of Max-Planck-Institute of Biochemistry (Munich) for improvement in English style.

We thank Prof. M. Yamamoto and Dr. Ohtsu (the Ushimado Marine Laboratory of Okayama University) for collection of marine animals.

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Shinji Ohmori, Yuko Ohsaki, Shinsuke Akagi, Chie Kondoh, Michi Kawase, and Toshie Nagai "D-Lactate Is Present in Much Larger Amount than L-Lactate in Cephalopods and Gastropods," Zoological Science 14(3), 429-434, (1 June 1997). https://doi.org/10.2108/zsj.14.429
Received: 18 February 1997; Accepted: 1 March 1997; Published: 1 June 1997
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