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Malate dehydrogenase isoenzymes of liver and plasma in prepatent murine schistosomiasis mansoni
795 TRANSACTIONS OFTHEROYAL
Soc~n’
OP TROPICAL
Malate dehydrogenase
Parasitology
Laboratory,
MEDICINE
AND HYGIENE,
VOL.
74, No.
6, 1980
isoenzymes of liver and plasma in prepatent murine schistosomiasis mansoni
F. M. FERRANTE AND E. H. PIKE Department of Microbiology, New York Medical College, Valhalla, USA
New York 10595,
Host-parasite relationships in murine schistosomiasis have been extensively studied, but only limited research has been directed toward the host’s hepatic metabolism during prepatency. This may be due to the electron microscope (EM) study of STENGER et al. (1967) which demonstrated hepatocyte ultrastructural integrity well into patency. PAGE & KNOWLES (1973) reported generalized hepatocyte ultrastructural changes during prepatency. Aberrant cellular architecture included alteration of the membrane systems of the endoplasmic reticulum and the plasmalemma, and high &d low amplitude swelling of mitochoidria. The initial arbiters of cellular iniurv are disruption of the integrity of mitochondrial membranes and the plasmalemma, with associated changes in the mitochondrial matrices. Such ultrastructural alterations as described by PAGE & KNOWLES, therefore, suggest cellular damage. Malate dehydrogenase (MDH) is a respiratory and gluconeogenic enzyme with isoenzymes compartmentalized between the cytosol and the mitochondrial matrix. Significant increase of mitochondrial MDH isoenzvme CmMDH) in the nlasma is indicative of cell&r damage ‘(GARBUS,- 1971). Ultrastructural changes as described by PAGE 8; KNOWLES would necessitate release of sienificant amounts of mMDH to the blood plasma, wYhich might form the basis of a diagnostic test during prepatent murine schistosomiasis. Methods and Materials 77 of 150 Albany strain-(Nylar) male mice weighing 20 to 22 g were each exposed to 200 cercariae of the Puerto Rican strain of Schistosoma mansoni using the tail immersion technique; 73 served as controls. At weekly intervals from weeks 2 through 6 post-exposuie, 10 control and 10 exposed mice were bled. Blood was collected in heoarinized capillary tubes, centrifuged at 760g at 4”6, plasma separated and stored at - 20°C. Following bleeding, mice were killed and portal perfusion immediately performed. Perfusates from exposed mice were examined for the presence of schistosomes. To determine patency liver squashes from exposed mice were examined at weeks 5 and 6 post-exposure. Each liver was then placed in a tube containing cold ohvsiological saline. The tubes were set in an ice baih andthe livers homogenized with the Willems Polvtron irom range 3 103 to 22 < 1031. ,, using \. three bursts of five seconds each. Homogenates were centrifuged at 780g for 10 min, the supernatants stored at - 2O’C.
Fig. 1, Representative portion of pellet derived from LSs illustrating presence of disrupted membranes and absence of intact mitochondria. Portion of a lipid droplet appears at A. 6000X.
Abbreviations used electron microscope EM liver supernatants LSS malate dehydrogenase MDH LMDH liver malate dehydrogenase activity cytoplasmic malate dehydrogenase cMDH isoenzyme mMDH mitochondrial malate dehydrogenase isoenzyme third malate dehydrogenase isoenzyme xMDH band LMDHI liver malate dehydrogenase isoenzyme distribution PMDHI plasma malate dehydrogenase isoenzyme distribution total liver protein TLP
MDH OF LIVER AND PLASMA IN PREPATENTMURINE SCHISTOSOMIASISMANSONI Samples of liver supernatants (LSs) and plasma were subjected to polyacrylamide disc gel electrophoresis. The gels were stained by tetrazolium reduction (RABINOWITZ & DIETZ, 1967), scanned and the areas quantitated by planimetry. MDH activity in LSs was assayed (SEIGEI. & BING, 1956) and total liver protein (TLP) determined (LOWRY et al., 1951). Data were analysed by analysis of Variance and Student’s t test. In order to determine the status of mitochondrial membranes, standard EM procedures were used to examine the LSs which were centrifuged at 45,000 g for 30 min at 4°C. The pellet was fixed in gluteraldehyde and osmium, alcohol dehydrated, epon embedded, stained and thin sections examined by EM.
p-e4 . .
Results At autopsy livers of infected mice were noted to be grossly enlarged when compared to controls. Perfusates of infected mice contained either developing or adult worms as the infection progressed. Eggs and granulomata were found in liver squashes beginning at week 5. EM studies of LSs revealed membrane fragments with no intact mitochondria visible. Liver MDH activity (LMDH) decreased as mouse age increased. There was no disparity in activity between infected and control mice (Table I). Infected mice TLP values were different at weeks 3 and 5 when compared to controls (Table I). Significant differences in isoenzyme distribution obtained from the LSs of infected and control mice occurred at weeks 2, 5 and 6 without correlative changes in the plasma (Table I). There was a substantial increase of mMDH and a decrease of the cytoplasmic isoenzyme (cMDH) obtained from the LSs of infected mice at weeks 2 and 5 without a concomitant increase or decrease of the plasma MDH isoenzyme distribution (PMDHJ). A strong third band (xMDH) was obtained at all time intervals from the LSs of all mice. This band was demonstrated in the plasma solely at week 3 and then in only 50 “/b of both infected and control mice. Significant intra-week differences between infected and control mice were established for TLP and liver MDH isoenzyme distribution (LMDHI). Analysis of variance revealed no significant interweek differences between the two groups for either parameter over all time intervals. Discussion Release of MDH into extramitochondrial spaces paralled the occurrence of mitochondrial swelling (RENDON & PACKER, 1976). An indicator of severe cellular injury was shown to be the release of mMDH from tissue to blood (GARBUS, 1971). In the present study, there was no significant difference in mMDH plasma levels from schistosome-infected and control mice. This suggested non-discernible liver pathology as judged by this parameter under the conditions of the experiment. GONDE-de1 PINO et al. (1966) were unable to demonstrate any percentage difference in LMDHI of infected and control mice at 7 weeks postinfection. Significant differences in LMDHI were demonstrated at weeks 2, 5 and 6 in infected mice
797
F. M. FERRANTE AND E. H. PIKE when compared to controls in the present study. Unlike the findings of CONnE-de1 PIN0 at 7 weeks post-infection, a third isoenzyme band could be consistently separated and quantitated in the LSs at weeks 2 through 6. The appearance in the plasma of the anomalous third band, xMDH, was also reported by CONDEde1 EINO. Disc electrophoresis of all plasma analyses in that study produced streaking and tailing, preventing quantitation of the third band. In the present work, xMDH appeared in the plasma solely at week 3 in 50”,1 of the animals, but in readily discernible bands. Stable variations of the folding of the polypeptide chains of enzymes may give rise to alterations in the three-dimensional structure, generating isomeric forms. The existence of such isomers or “conformational isoenzymes” for MDH has long been postulated. Upon electrophoresis, the isomers will resolve into distinct sub-bands. The number of sub-bands obtained is a function of the sensitivity of the technique, species examined, ontogeny of that species and the particular tissue examined (WILKINSON, 1970). It is conceivable that disc electrophoresis as used in this study allowed only partial separation on MDH sub-bands, readily explaining the appearance of a third band, at least in liver. The present work concurs with the findings of CONDE-de1 PIN0 et al. (1966) that the methods employed are not suitable for PMDHI analysis because of the anomalous results obtained. Isoelectric focusing (Ross, 1976) or elution and chromatographic analysis may reveal the exact nature of the third MDH band. Schistosomes metabolize large amounts of glucose as their prime energy source. Enlodgement of developing schistosomes within the mesenteric vasculature ensures a ready supply. As the liver is an important site for gluconeogenesis and regulation of blood sugar levels, developing schistosomes may induce changes in hepatic carbohydrate metabolism. While this work suggests no change in LMDH or isoenzyme distribution, further elucidation of the biochemical effects of maturing schistosomes upon the enzymatic processes of gluconeogenesis may lead to the development of diagnostic tests. These would be valuable during prepatency, as presagers of impending oviposition, Acknowledgements The authors express their appreciation to Dr. L. K. Eveland, Downstate Medical Center SUNY, for the infected mice, to A. M. Snow for preparation
of the electron micrographs, to Drs. J. Hagedoorn and L. Herman for their reading and to the Alumni Association of NYMC for fellowship support for F. M. Ferrante. References Conde-de1 Pino, E., Perez-Vilar, M., CintronRivera, A. A. & Seneriz, R. (1966). Studies in Schistosoma mansoni I. Malic and lactic dehydrogenase of adult worms and cercariae. Experimental Parasitology, 18, 320-326. Garbus, J. (1971). Serum malate dehydrogenase isoenzymes as indicators of severe cellular injury. Clinica
et Chimica
Acta,
35, 502-504.
Lowry, 0. H., Rosenbrough, N. J., Farr, L. & Randall, R. J. (1951). Protein measurement with Folin phenol reagent. Journal of Biological Chemistry, 193, 265-275. Page, C. R. & Knowles, W. J. (1973). Experimental mansoni : hepatic schistosomiasis prepatent cytopathology. American Society of Parasitologists, 48th Annual Meeting, Toronto, Abstract. Rabinowitz, Y. & Dietz, A. A. (1967). Malic and lactic dehydrogenase isoenzymes of normal and leukemic leukocytes separated on glass bead columns. Blood, 29, 182-195. Rendon, A. & Packer, L. (1976). Correlation of mitochondrial swelling and localization of malate dehydrogenase activity. In: Mitochondria. Packer, L. & Gbmez-Puyou, A. (Editors), New York: Academic Press, pp. 151-154. Ross, G. C. (1976). Isoenzymes in Schistosoma spp. : LDH, MDH and acid phosphatases separated by isoelectric focusing in polyacrylamide gel. Comparative Biochemistry and Physiology, 55B, 343346.
Siegel, A. & Bing, R. J. (1956). Plasma enzyme activity in myocardial infarction in dog and man. Proceedings of the Society for Experimental and Medicine, 91, 604-607.
Biology
Stenger, R. J., Warren, K. S. & Johnson, E. A. (1967). An electron microscope study of the liver parenchyma and schistosome pigment in murine hepatosplenic schistosomiasis mansoni. American Journal of Tropical Medicine and Hygiene, 16, 473-482.
Wilkinson, J. H. (1970). Isoenzymes. (2nd edition). Philadelphia : J. B. Lippincott Company.
Accepted
for
publication
19th October,
1979.
Soc~n’
OP TROPICAL
Malate dehydrogenase
Parasitology
Laboratory,
MEDICINE
AND HYGIENE,
VOL.
74, No.
6, 1980
isoenzymes of liver and plasma in prepatent murine schistosomiasis mansoni
F. M. FERRANTE AND E. H. PIKE Department of Microbiology, New York Medical College, Valhalla, USA
New York 10595,
Host-parasite relationships in murine schistosomiasis have been extensively studied, but only limited research has been directed toward the host’s hepatic metabolism during prepatency. This may be due to the electron microscope (EM) study of STENGER et al. (1967) which demonstrated hepatocyte ultrastructural integrity well into patency. PAGE & KNOWLES (1973) reported generalized hepatocyte ultrastructural changes during prepatency. Aberrant cellular architecture included alteration of the membrane systems of the endoplasmic reticulum and the plasmalemma, and high &d low amplitude swelling of mitochoidria. The initial arbiters of cellular iniurv are disruption of the integrity of mitochondrial membranes and the plasmalemma, with associated changes in the mitochondrial matrices. Such ultrastructural alterations as described by PAGE & KNOWLES, therefore, suggest cellular damage. Malate dehydrogenase (MDH) is a respiratory and gluconeogenic enzyme with isoenzymes compartmentalized between the cytosol and the mitochondrial matrix. Significant increase of mitochondrial MDH isoenzvme CmMDH) in the nlasma is indicative of cell&r damage ‘(GARBUS,- 1971). Ultrastructural changes as described by PAGE 8; KNOWLES would necessitate release of sienificant amounts of mMDH to the blood plasma, wYhich might form the basis of a diagnostic test during prepatent murine schistosomiasis. Methods and Materials 77 of 150 Albany strain-(Nylar) male mice weighing 20 to 22 g were each exposed to 200 cercariae of the Puerto Rican strain of Schistosoma mansoni using the tail immersion technique; 73 served as controls. At weekly intervals from weeks 2 through 6 post-exposuie, 10 control and 10 exposed mice were bled. Blood was collected in heoarinized capillary tubes, centrifuged at 760g at 4”6, plasma separated and stored at - 20°C. Following bleeding, mice were killed and portal perfusion immediately performed. Perfusates from exposed mice were examined for the presence of schistosomes. To determine patency liver squashes from exposed mice were examined at weeks 5 and 6 post-exposure. Each liver was then placed in a tube containing cold ohvsiological saline. The tubes were set in an ice baih andthe livers homogenized with the Willems Polvtron irom range 3 103 to 22 < 1031. ,, using \. three bursts of five seconds each. Homogenates were centrifuged at 780g for 10 min, the supernatants stored at - 2O’C.
Fig. 1, Representative portion of pellet derived from LSs illustrating presence of disrupted membranes and absence of intact mitochondria. Portion of a lipid droplet appears at A. 6000X.
Abbreviations used electron microscope EM liver supernatants LSS malate dehydrogenase MDH LMDH liver malate dehydrogenase activity cytoplasmic malate dehydrogenase cMDH isoenzyme mMDH mitochondrial malate dehydrogenase isoenzyme third malate dehydrogenase isoenzyme xMDH band LMDHI liver malate dehydrogenase isoenzyme distribution PMDHI plasma malate dehydrogenase isoenzyme distribution total liver protein TLP
MDH OF LIVER AND PLASMA IN PREPATENTMURINE SCHISTOSOMIASISMANSONI Samples of liver supernatants (LSs) and plasma were subjected to polyacrylamide disc gel electrophoresis. The gels were stained by tetrazolium reduction (RABINOWITZ & DIETZ, 1967), scanned and the areas quantitated by planimetry. MDH activity in LSs was assayed (SEIGEI. & BING, 1956) and total liver protein (TLP) determined (LOWRY et al., 1951). Data were analysed by analysis of Variance and Student’s t test. In order to determine the status of mitochondrial membranes, standard EM procedures were used to examine the LSs which were centrifuged at 45,000 g for 30 min at 4°C. The pellet was fixed in gluteraldehyde and osmium, alcohol dehydrated, epon embedded, stained and thin sections examined by EM.
p-e4 . .
Results At autopsy livers of infected mice were noted to be grossly enlarged when compared to controls. Perfusates of infected mice contained either developing or adult worms as the infection progressed. Eggs and granulomata were found in liver squashes beginning at week 5. EM studies of LSs revealed membrane fragments with no intact mitochondria visible. Liver MDH activity (LMDH) decreased as mouse age increased. There was no disparity in activity between infected and control mice (Table I). Infected mice TLP values were different at weeks 3 and 5 when compared to controls (Table I). Significant differences in isoenzyme distribution obtained from the LSs of infected and control mice occurred at weeks 2, 5 and 6 without correlative changes in the plasma (Table I). There was a substantial increase of mMDH and a decrease of the cytoplasmic isoenzyme (cMDH) obtained from the LSs of infected mice at weeks 2 and 5 without a concomitant increase or decrease of the plasma MDH isoenzyme distribution (PMDHJ). A strong third band (xMDH) was obtained at all time intervals from the LSs of all mice. This band was demonstrated in the plasma solely at week 3 and then in only 50 “/b of both infected and control mice. Significant intra-week differences between infected and control mice were established for TLP and liver MDH isoenzyme distribution (LMDHI). Analysis of variance revealed no significant interweek differences between the two groups for either parameter over all time intervals. Discussion Release of MDH into extramitochondrial spaces paralled the occurrence of mitochondrial swelling (RENDON & PACKER, 1976). An indicator of severe cellular injury was shown to be the release of mMDH from tissue to blood (GARBUS, 1971). In the present study, there was no significant difference in mMDH plasma levels from schistosome-infected and control mice. This suggested non-discernible liver pathology as judged by this parameter under the conditions of the experiment. GONDE-de1 PINO et al. (1966) were unable to demonstrate any percentage difference in LMDHI of infected and control mice at 7 weeks postinfection. Significant differences in LMDHI were demonstrated at weeks 2, 5 and 6 in infected mice
797
F. M. FERRANTE AND E. H. PIKE when compared to controls in the present study. Unlike the findings of CONnE-de1 PIN0 at 7 weeks post-infection, a third isoenzyme band could be consistently separated and quantitated in the LSs at weeks 2 through 6. The appearance in the plasma of the anomalous third band, xMDH, was also reported by CONDEde1 EINO. Disc electrophoresis of all plasma analyses in that study produced streaking and tailing, preventing quantitation of the third band. In the present work, xMDH appeared in the plasma solely at week 3 in 50”,1 of the animals, but in readily discernible bands. Stable variations of the folding of the polypeptide chains of enzymes may give rise to alterations in the three-dimensional structure, generating isomeric forms. The existence of such isomers or “conformational isoenzymes” for MDH has long been postulated. Upon electrophoresis, the isomers will resolve into distinct sub-bands. The number of sub-bands obtained is a function of the sensitivity of the technique, species examined, ontogeny of that species and the particular tissue examined (WILKINSON, 1970). It is conceivable that disc electrophoresis as used in this study allowed only partial separation on MDH sub-bands, readily explaining the appearance of a third band, at least in liver. The present work concurs with the findings of CONDE-de1 PIN0 et al. (1966) that the methods employed are not suitable for PMDHI analysis because of the anomalous results obtained. Isoelectric focusing (Ross, 1976) or elution and chromatographic analysis may reveal the exact nature of the third MDH band. Schistosomes metabolize large amounts of glucose as their prime energy source. Enlodgement of developing schistosomes within the mesenteric vasculature ensures a ready supply. As the liver is an important site for gluconeogenesis and regulation of blood sugar levels, developing schistosomes may induce changes in hepatic carbohydrate metabolism. While this work suggests no change in LMDH or isoenzyme distribution, further elucidation of the biochemical effects of maturing schistosomes upon the enzymatic processes of gluconeogenesis may lead to the development of diagnostic tests. These would be valuable during prepatency, as presagers of impending oviposition, Acknowledgements The authors express their appreciation to Dr. L. K. Eveland, Downstate Medical Center SUNY, for the infected mice, to A. M. Snow for preparation
of the electron micrographs, to Drs. J. Hagedoorn and L. Herman for their reading and to the Alumni Association of NYMC for fellowship support for F. M. Ferrante. References Conde-de1 Pino, E., Perez-Vilar, M., CintronRivera, A. A. & Seneriz, R. (1966). Studies in Schistosoma mansoni I. Malic and lactic dehydrogenase of adult worms and cercariae. Experimental Parasitology, 18, 320-326. Garbus, J. (1971). Serum malate dehydrogenase isoenzymes as indicators of severe cellular injury. Clinica
et Chimica
Acta,
35, 502-504.
Lowry, 0. H., Rosenbrough, N. J., Farr, L. & Randall, R. J. (1951). Protein measurement with Folin phenol reagent. Journal of Biological Chemistry, 193, 265-275. Page, C. R. & Knowles, W. J. (1973). Experimental mansoni : hepatic schistosomiasis prepatent cytopathology. American Society of Parasitologists, 48th Annual Meeting, Toronto, Abstract. Rabinowitz, Y. & Dietz, A. A. (1967). Malic and lactic dehydrogenase isoenzymes of normal and leukemic leukocytes separated on glass bead columns. Blood, 29, 182-195. Rendon, A. & Packer, L. (1976). Correlation of mitochondrial swelling and localization of malate dehydrogenase activity. In: Mitochondria. Packer, L. & Gbmez-Puyou, A. (Editors), New York: Academic Press, pp. 151-154. Ross, G. C. (1976). Isoenzymes in Schistosoma spp. : LDH, MDH and acid phosphatases separated by isoelectric focusing in polyacrylamide gel. Comparative Biochemistry and Physiology, 55B, 343346.
Siegel, A. & Bing, R. J. (1956). Plasma enzyme activity in myocardial infarction in dog and man. Proceedings of the Society for Experimental and Medicine, 91, 604-607.
Biology
Stenger, R. J., Warren, K. S. & Johnson, E. A. (1967). An electron microscope study of the liver parenchyma and schistosome pigment in murine hepatosplenic schistosomiasis mansoni. American Journal of Tropical Medicine and Hygiene, 16, 473-482.
Wilkinson, J. H. (1970). Isoenzymes. (2nd edition). Philadelphia : J. B. Lippincott Company.
Accepted
for
publication
19th October,
1979.