Trypanosoma lewisi: Pyruvate and transketolase activity in normal and thiamine deficient rats

/nl. J. Biochem. Vol. 16, No. 6, pp. 699-702, 1984 Printed in Great Britain. All rights reserved

0020-711X/84 $3.00 + 0.00 Copyright Q 1984 Pergamon Press I.td

2-RY~~~~S~~~ LEWISI: PYRUVATE AND TRANSKETOLASE ACTIVITY IN NORMAL AND THIAMINE DEFICIENT RATS CLARENCE M. LEE, EDLUE N. MOYO and GEORGIANA F. ABOKO-COLE* Department of Zoology, Howard University, Washington,

DC 20059, U.S.A.

Transketolase and pyruvate changes were studied in rats infected with Trypanosoma tewisi and fed complete, thiamine-deficient and pair-fed control diets. 2. Regardless of the dietary group, marked increases in pyruvate levels were observed in the infected animals. 3. There were no significant differences in erythrocyte transketolase activity of rats given a full complement diet. Significant decreases, however, were observed in the transketolase activity of pair-fed and thiamine deficient rats. The greater decreases occurred in the infected animals. Abstract-l.

Parasite

INTRODUCTION

T. lewisi “E” isolate obtained from England by Dr D. R. Lincicome was used as the experimental organism. This non-pathogenic trypanosome has been maintained in rats by serial passage over the last 20 years (Lincicome and Hill, 1965).

Numerous nutritional studies have shown relationships between the host’s vitamin status, resistance to infection and disturbance of the host’s metabolism (McCoy, 1934; Sadun et al., 1951; Chandler, 1953; Geiman, 1958; Squibb, 1963; Lincicome and Shepperson, 1965; Lincicome and Lee, 1971; Lee and Aboko-Cole, 1975, 1976). The purpose of this paper is to report on the pyruvate and transketolase changes in the blood of rats infected with Trypanosoma Iewisi and fed complete, thiamine-deficient and pair-fed control diets. MATERIALS AND

Experimental infection Pure suspensions of trypanosome cells were prepared as physiological saline suspensions of washed cells obtained from blood drawn by cardiac puncture of anesthestized rats. The trypanosomes were separated from the blood by centrifugation and removed by filter sampler tubes (American Scientific Products, Columbia, Maryland). The cells were then resuspended in an adjusted volume of normal saline so that 1 ml contained lo3 cells, The quantitative estimate of cells was made using the hemacytometer, Toisson’s fluid, red blood cells pipette and a x 200 diluting factor (Lincicome and Watkins, 1963). Twenty-eight days after the initiation of a dietary regimen, one half of the rats in each dietary group were injected intraperitoneally with 1 ml of a pure suspension of trypanosomes (10’ cells per ml) in physiological saline.

METHODS

Experimental hosts Six seventy-two (672) female hundred and SpragueDawley rats, weighing 60 rt 5 g were obtained from the Animals Breeding Laboratories of the National Institute of Health, Bethesda, Maryland. Table 1 shows the distribution, number and initial body weights of rats used in this study, together with dietary groupings that formed the structure of each experiment.

Pyruvute assuys An enzymatic spectrophotometric method adopted from Segal et al. (1956) was used for dete~ination of pyruvic acid. All assay materials and direction for use were supplied commercially (Calbiochem, Los Angeles, California).

Experimental dieis All diets were purchased commercially Biochemicals, Cleveland, Ohio).

(ICN mutational

Housing and feeding qf rats

Red blood cell transketolase activity

Rats were housed individually in sterilized steel meshbottomed hanging cages and fed appropriate diets from metal feedings cups especially designed to minimize spillage. All rats in the control and thiamine deficient groups were allowed to feed ad lib&urn. Rats in the pair-fed group were fed the control (adequate) diet daily in amounts equal to the food consumed by their thiamine deficient paired mates. The daily food intake of each rat was determined by subtracting the amount of food remaining in the cup from the amount given in previous day. The rats were provided water ad libitum. Water bottles and feeding cups were cleaned frequently to minimize algal and bacterial contamination.

Red blood cells were separated from heparinized blood, washed with 0.9% saline, diluted with an equal volume of water and frozen at -40°C overnight. Transketolase activity of the hemolysate was measured by the micromethod of Warnock f 1970, 1975). Statisticai evaluation The data were studied as averages calculated from four experiments. Statistical treatment of the data consisted of calculation of standard deviations and application of Student’s f-test for significance of differences (Snedecor, 1959). Statistical significance was attached to all differences of means having a probability level of 5% or better. RESULTS

*Present address: Center for Preprofessional Education, P.O. Box 1124, Howard University, Washington, DC 20059, U.S.A.

The development of parasitemia with T. lewisi was described earlier 699

in rats inoculated (Lee et al., 1983).

CLARENCEM.

700 Table 1. Average initial diets and inoculated

LEE ef al.

body weight (g f SD) of rats fed a complete, thiamine deficient and pair-fed with Trypanosoma lewisi (numbers of animals employed in parentheses) Experiment

Dietary

Blood pyruvate

2

3

4

61 k 5 (28) 60 + 4 (28)

60 f 4 (28) 60 f 5 (28)

61 f 5 (28) 62 + 6 (28)

60 i 4 (28) 61 i 6 (28)

112 112

61 k 4 (28) 60 f 6 (28)

61 * 6(28) 60 + 5 (28)

60 f 4 (28) 61 f 5 (28)

61 k 5(28) 60 i 6 (28)

112 112

61 f 4 (28) 61 f 5 (28) 168

61 + 5 (28) 60 i 5 (28) 168

61 f 6 (28) 62 k 5 (28) 168

60 i 5 (28) 62 k 6 (28) 168

112 112 672

Erythrocyte

level

Figure 1 shows the average blood pyruvate levels of T. lewisi infected rats fed complete, pair-fed and thiamine deficient diets. In comparison to control rats, infected animals showed higher pyruvate levels which were statistically significant 6 out of 12 assays. Infected rats showed significant increases over the controls for days 35-55. These increases ranged from 9-l 13%. For the same period (days 35-55) trypanosome infected pair-fed control rats demonstrated greater pyruvate levels than non-infected ones, reaching a maximum of 83% by day 45. Thiamine deficient infected rats showed greater pyruvate levels than non-inoculated rats fed the same diet. These were significant from day 30 until day 60. In relation to control values, these differences ranged from 27-124x. 40

Total no. of rats

I

group

Control Noninfected Infected Thiamine Deficient Noninfected Infected Pair-Fed Noninfected Infected Total No. of rats

no.

r-

transketolase

activity

The transketolase activity in erythrocytes of rats given complete, thiamine deficient and pair-fed diets and inoculated with T. lewisi is shown in Fig. 2. Greater transketolase activity was observed in erythrocytes of rats fed the adequate diets than in erythrocytes of thiamine deficient rats. Throughout the 60 days of observation, there were no significant differences in the transketolase activity between infected and uninfected rats on the complete diet. However, the transketolase activity of T. Iewisi infected pair-fed rats was significantly reduced (day 40 to day 60) compared to their non-infected controls. Enzyme activity in rats deprived of thiamine was about one third those of animals with an adequate supply of the vitamin. As in the pair-fed animals, these were significant differences in the transketolase activity between the infected and uninfected animals fed the thiamine deficient diet. These differences were very apparent from day 35 to day 60.

36 0 8 Z

32

z 8

2.8

.A

0

I

I

I

I

I

I

10

20

30

40

50

60

Days Fig. 1. Blood pyruvate levels in rats given complete, thiamine-deficient and pair-fed diets and inoculated with Trypanosoma lewisi. a-averages for rats inoculated with T. lewisi and fed a complete diet; O-averages for uninoculated rats fed a complete diet; O-averages for rats inoculated with T. lewisi and fed a thiamine-deficient diet. x -averages for uninoculates rats fed a thiamine-deficient diet: V-averages for rats inoculated with T. lewisi and fed a pair-fed diet; +-averages for uninoculated rats fed a pair-fed diet.

0

10

20

30

40

50

60

Doys Fig. 2. Erythrocyte transketolase activity in rats given complete, thiamine-deficient and pair-fed diets and inoculated with Trypunosoma lewisi. a-averages for rats inoculated with T. lewisi and fed a complete diet; O-averages for uninoculated rats fed a complete diet; O-averages for rats inoculated with T. lewisi and fed a thiamine-deficient diet; x -averages for uninoculated rats fed a thiamine-deficient diet; V-averages for rats inoculated with T. lewisi and fed a pair-fed diet; +-averages for uninoculated rats fed a pair-fed diet.

Pyruvate and transketolase in T. lewisi infected DISCUSSION

Numerous authors have demonstrated that the lack of thiamine interfered with the host carbohydrate metabolism (Lu, 1939; Sober et al., 1940; Wright and Scott, 1954; Brin, 1962; Warnock, 1970; Wood et al., 1980). According to these investigators, during deficiency, pyruvate accumulated in the blood; a-ketoglutarate failed to be decarboxylated and transketolase activity was progressively depressed. Blood pyruvate levels in the rats used in this study were similar to those reported by other workers (Robinson, 1966; Thompson and Wooton, 1970). Uninfected rats fed an adequate diet showed average pyruvate levels of 1.08 f 0.08 mg/lOO ml, but when given a thiamine deficient diet, pyruvate levels were markedly increased by an average of 49%. Compared to the adequately fed animals, the average level of blood pyruvate in pair-fed uninfected rats increased only by 3’;/,. Infection with T. lewisi produced further elevation in blood pyruvate. In adequately fed rats, trypanosomes produced more than a 19% increase in blood pyruvate. T. lewisi infected pair-fed control exhibited a 16% increase and thiamine deficient rats had about 46% increase. The marked increase in blood pyruvate of infected animals may be accounted for by the presence of additional pyruvate produced by T. lewisi. Searle and Reiner (1941) reported that T. lewisi was capable of producing pyruvate. In addition, Coleman and Von Brand (1957) showed that pyruvate may be a metabolic end product of several trypanosomes and thus can cause an increase in the blood pyruvate levels of rats during infection. In addition to the changes in levels of pyruvate, the nutritional deficiency seems to enhance a marked decrease in the erythrocyte tranketolase activity. These results correspond with those of previous investigators (Brin et al., 1958, 1960; Warnock, 1970). Animals fed the adequate diet failed to show any significant differences in the activity of the enzyme. Both pair-fed and thiamine deficient animals infected with T. lewisi showed a reduction in enzyme activity based upon the sedoheptulose formation. In the thiamine deficient rats, both infected and noninfected animals showed a reduction in the activity of transketolase starting about day 5 after initiation of a dietary regimen; after day 30, the infected thiamine deficient animals showed a greater reduction in transketolase activity. Results obtained in the erythrocyte transketolase studies gave evidence that blood of infected rats contained less thiamine than that of non-infected rats. In both inanition controls and thiamine deficient animals, the reduction in transketolase activity appeared to be directly related to the parasitemias. Rama Rao and Sirsi (1956) studies thiamine levels of chickens infected with Plasmodium gallinaceum and found a decrease in thiamine levels in the blood. On the other hand, infections with Plasmodium berghei showed an increase in thiamine content of infected spleen and erythrocytes (Singer, 1961). Singer (1961) also reported an increase in the thiamine content in the liver and blood rats infected with TYypanosoma rhodesiense, and suggested that

rats

701

blood thiamine appeared to be related to parasitemia. These changes were observed for both normally fed and thiamine deficient animals, though those of the latter group were of a lower level. Even though the present study presents additional evidence supporting the general hypothesis (Lincicome, 1963, 1971) that parasitism is fundamentally a chemical relationship between a dependent cell/organism and its organic environment, it is obvious that the nutritional relationship of protozoans to the well-being of the host should be investigated to a greater extent. Critical analyses of parasitic action in specific deficient states may be complicated by interrelationships of other vitamins and/or hormones. Results of this study must therefore be interpreted cautiously especially in the light of information linking thiamine with riboflavin and pyridoxine (Ferrebee and Weissman, 1943; Rabinowitz and Snell, 1951; Chiao and Peterson, 1956; Sakuragi and Kummerow, 1957; Lewin and Brown, 1963). REFERENCES

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Searle D. S. and Reiner L. (1941) The role of carbon dioxide in the glucose metabolism of Trypanosoma lewisi. J. biol. Chem. 41, 563-571. Segal S., Blair A. E. and Wyngaarden J. B. (1956) An enzymatic spectrophotometric method for the determination of pyruvic acid in blood. J. lab. clin. Med. 48, 137-143. Singer I. (1961) Tissue thiamine changes in rats with experimental Trypanosomiasis or Malaria. Exp. Parasit. 11, 391401. Snedecor G. W. (1959) Statistical Methods. Iowa State College Press, Ames. Sober H. A., Lipton M. A. and Elvenjem C. A. (1940) Relation of thiamine to citric acid metabolism. J. biol. Chem. 134, 605-616. Squibb R. L. (1963) Avian disease virus and nutrition relationship. IV. Effect of B complex vitamins on growth and mortality of white leghorn chicks infected with Newcastle disease virus. Poultry Sci. 42, 941-944. Thompson R. H. and Wooton I. D. P. (1970) Biochemical Disorders in Human Disease. Academic Press, New York. Warnock L. G. (1970) A new approach to erythrocyte transketolase measurement. J. Nutr. 100, 1957-1962. Wamock L. G. (1975) Transketolase activity of blood hemolysate: a useful index for diagnosing thiamine deficiency. Clin. Chem. 21, 432436. Wood B.. Giisbers A.. Goode A.. Davis S.. Mulholland J. and Breen- K. (1980) A study of partial thiamine restriction in human volunteers. Am. J. clin. Nutr. 33, 848-86 1. Wright R. C. and Scott E. M. (1954) Pyruvate and alphaketoglutarate metabolism in thiamine deficiency. J. biol. Chem. 206, 725-733.