Role of Pyridoxine in Oxalate Metabolism RAVINDRA NATH, S W A R A N KAUR THIND, M. S. R. MURTHY, SHAKEEL FAROOQUI, RAJAN GUPTA, A N D HARI K. KOUL Department of Biochemistry Postgraduate Institute of Medical Education and Research Chandigarh, India
INTRODUCTION Oxalic acid, a dicarboxylate widespread in plants, plays no vital function in man and experimental animals. The animal system is endangered by the toxicity of oxalate, as this compound is not metabolized further in the body and is excreted as such in urine. Nonetheless, deposition of calcium oxalate is commonly found in end-state renal diseases owing to retention of oxalate.'22The commonest pathological condition involving oxalate is the formation of calcium oxalate stones in the urinary tract (urolithiasis). Persistent hyperoxaluria is the most common feature in urolithiasis patients. It is well established that nutritional pyridoxine deficiency leads to hyperoxaluria and renal stone formation in experimental animals and In this paper, we discuss the role of pyridoxine in oxalate metabalism and illustrate the molecular mechanism(s) of hyperoxaluria in pyridoxine deficiency using the results of our studies over the past several years.
MATERIALS AND METHODS Pyridoxine deficiency was induced in male Wistar rats (b.w. 35-40 g) by feeding them a vitamin B, deficient diet' for 45 days. The deficiency was biochemically assessed by measuring erythrocyte alanine transaminase (EGPT) and erythrocyte glutamic acid transaminase (EGOT) activities together with percent pyridoxal phosphate stimulation index (percent PALP) of EGPT and EGOT.9 Intestinal oxalate uptake was determined by everted gut rings," as well as by using brush-border membrane vesicles." Oxalate-synthesizing enzymes were assayed as described previously." Assay of EGOT and EGPT was also used as an index of pyridoxine status in human subjects tested for hypero~a1uria.l~
RESULTS AND DISCUSSIONS
Endogenous Oxalate Biosynthesis Major sources of endogenous oxalate include ascorbic acid, glycine, glycolic acid, and glyoxylic acid.I4 The enzymes that are required for oxalate biosynthesis are glycolic acid oxidase (GAO), lactate dehydrogenase (LDH), and glycolic acid dehydro274
NATH et al.: OXALATE METABOLISM
275
genase (GAD). Liver GAO activity is increased in vitamin B, deficiency. This increase was highly significant even when the activity was expressed as units per gram tissue. No change was observed in liver LDH while liver GAD activity also showed a significant increase ( p < 0.05) (TABLE1).
Role of Transaminases and Biosynthetic Enzymes Chronic pyridoxine deficiency leads to the decreased activity of most of the transaminases, including glyoxylate transaminase, whose physiological function was thought to control the glyoxalate pool by converting it to g1y~ine.I~ On this basis, many worker^^^^^^," postulated that glyoxylate, which increases in pyridoxine deficiency, may further be converted to oxalate, thereby resulting in hyperoxaluria, which is a characteristic feature of vitamin B, deficiency. However, many studiesI6,” show that glyoxylate is not an obligatory intermediate in the biosynthesis of oxalate from glycolate or ethylene glycol. Particularly, the conversion of glycolate to oxalate is many-fold higher in vitamin B, deficiency than that of glyoxylate.” Thus the above
Effect of Vitamin B, Deficiency on Activity of Oxalate-Synthesizing Enzymes in the Rat Liver
TABLE 1.
Glycolate Oxidase B, Deficient Units/mgprotein Units/gmtissue
2.64 228
0.24“ f 32‘ t
Lactate Dehydrogenase
Glycolate Dehydrogenase
Pair Fed
B, Deficient
Pair Fed
B, Deficient
1.04 t 0.07 116 t 7
2.63 f 0.23 273 i 23
2.53 f 0.10 284 f 14
4.03 f 5.OOb 4.14 + 0.48
Pair Fed 2.65 2.95
i 2
0.3 0.0
NOTE: Results are expressed as mean f SEM. Number of animals in each group was six to eight. ‘ p < 0.001 as compared to control. b p < 0.05 as compared to control. c p < 0.01 as compared to control.
hypothesis, which was based on an increased glyoxylate pool, could not answer these important questions. Our results, which show a significant increase in liver GAO activity in pyridoxine-deficient rats, are contradictory to the earlier report’* where no changes were noted. G A O is essentially localized in peroxi~omes’~ and can convert glycolate to glyoxylate and further to oxalate in two steps (FIG.1). Another enzyme, D-amino acid oxidase (DAO), which converts glycine to glyoxylate by oxidative deamination, is also present in peroxisome~.’~ It has also been shown that the tissue glycine pool increases in pyridoxine deficiency.” It is likely that the synthesis of glyoxylate by DAO in pyridoxine deficiency remains unchecked due to very low transaminase activity, and this is rapidly oxidized to oxalate by GAO. Glyoxylate may also be oxidized by peroxisomal LDH” to oxalate. GAD in liver may not be very significant in the oxidation of glycolate to oxalate as this enzyme activity tended to decrease in vitamin B, deficiency. Preliminary studies in our laboratory showed that phosphoglycolate can be converted to oxalate faster than glycolate itself. In vitro glycolate is phosphorylated to phosphoglycolate by pyruvate kinase.22
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OXALAT
GLYCINE
I? I
I
I
+ fl
PH OSPHOGLYCOLATE
i
I
KlDNEY
LIVER
INHIBITED IN 86 DEE INCREASED IN 66 DEF.
FIGURE 1. Metabolism of oxalate in pyridoxine-deficient rat liver and kidney. Enzymes: DAO, D amino acid oxidase; TA,transaminase;GAO, glycolic acid oxidase; LDH, lactate dehydrogenase; GAD,glycolic acid dehydrogenase;PK, pyruvate kinase; GR, glyoxylate reductase.
Liver Peroxisomes Glyoxylate that is produced within the peroxisomes may have a different metabolism than glyoxylate produced in cytosol.16 This difference may be explained by the assumption that the peroxisomal membrane does not allow the transport of glyoxylate but does allow the transport of glycolate.” Glyoxylate, if administered from outside, may be decarboxylated by glyoxylate carboligase in the condensation reaction with a-ketoglutarate at a faster rate even though some of it may be converted to oxalate by cytoplasmic LDH.’ This exogenous glyoxylate is unable to enter the peroxisomes. However, glyoxylate produced within the peroxisomes may not be able to leave this organelle and is subsequently oxidized to oxalate. This particular pathway may increase several-fold in vitamin B, deficiency owing to increased GAO activity, as a result of a probable increase in the number of liver peroxisomes. Kidney Enzymes The kidney enzymes, however, present a picture which is altogether different from that of the liver. Kidneys do not possess the activity of G A O in their peroxisomes, but they do have the activity of DAO.I9 Glyoxylate reductase is also present in the kidney and can reduce glyoxylate to glycolate using N A D H + H + .In pyridoxine deficiency the kidney glyoxylate pool increases because of the reduced transaminase activity. This increased glyoxylate may in turn be reduced to glycolate by glyoxylate reductase or by peroxisomal LDH. Once glycolate is produced, it can be rapidly oxidized directly to oxalate without forming enzyme-free glyoxylate as intermediate by GAD. GAD activity in the kidney is greatly enhanced in pyridoxine deficiency (TABLE2). Subcellular localization of this enzyme is not known. Nevertheless, GAD can act on glycolate, whether the latter is produced in peroxisomes or in cytosol. The role played by cytoplasmic kidney LDH is not clear. Probably, it has no functional significance as far
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as pyridoxine deficiency-induced hyperoxaluria is concerned, as no significant increase in LDH activity was observed in this deficiency (TABLE2). If this enzyme were to be active in oxidizing glyoxylate, the exogenously administered glyoxylate could have been significantly converted to oxalate in vitamin B, deficiency.”
Role of Sex Hormones Even though no clear evidence is available on the effect of pyridoxine deficiency on the levels of testosterone, it is likely that this vitamin must have some role in regulating testosterone level which in turn regulates G A O activity. Another possibility for the increase in GAO activity could be a decreased turnover of this enzyme in vitamin B, deficiency, but this is not yet confirmed. Preliminary studies in our laboratory showed that while the K,,, for glycolate of GAO remains unaltered, there is an increase in V,,, in vitamin B, deficiency, suggesting thereby an increase in the net enzyme content.*’ However, GAD activity does not seem to be under the control of testosterone, and its activity followed a pattern opposite to that of GAO. The factors regulating this enzyme activity need further elucidation. In conclusion, it may be speculated that the increased synthesis of oxalate in vitamin B, deficiency may have two origins, one from liver and another from kidney, which involve separate and probably independent metabolic pathways (FIG.1). The individual contributions of these two organs towards the excretion of urinary oxalate in vitamin B, deficiency need to be further studied.
Intestinal Absorption of Oxalate Oxalate absorption from the gut is merely a passive diffusion phenomenon in man and experimental animals. The effects of pyridoxine deficiency on uptake of oxalate by everted intestinal sacs (TABLE3) revealed that the uptake rate of oxalate is elevated significantly ( p < 0.001) in vitamin B,-deficient rats as compared to animals in the control group. Data on the rate of oxalate uptake from the intestine in control and vitamin B,-deficient rats in a concentration range of 0.1-6.0 m M oxalate are given in FIGURE2. Mucosal oxalate concentration increased linearly in the control group and at no point was saturation of oxalate uptake detected, suggesting a diffusion process for oxalate uptake by intestine. Carrier-mediated saturation kinetics was observed in vitamin B,-deficient rats between 0.1 and 0.8 m M oxalate concentrations. On further
Effect of Vitamin B, Deficiency on Activity of Oxalate-Synthesizing Enzymes in the Rat Kidney
TABLE 2.
Lactate Dehydrogenase B, Deficient Pair Fed Units/mg protein Units/gm tissue
3.7 + 0.08 312 * 8
NOTE: Results expressed as mean ‘ p < 0.05.
3.32 f 0.10 330 + 18
Glycolate Dehydrogenase B, Deficient Pair Fed 1.102 + 0.04“ 0.202 c 0.05 0.112 + 0.02 1.093 f 0.33“
+ SEM. Number of animals in each group was six to eight.
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TABLE 3. Effect of Vitamin B, Deficiency on Oxalate Uptake from Rat Intestinea
Grouo Control Pair Fed Vitamin B, Deficient
Oxalate Uptake (wmollhr gm tissue)
.
0.26 t 0.02 0.27 + 0.06 0.41 -+ 0.02b
=Values are mean SE of six to eight observations. b p < 0.001 as compared to the control or pair fed group.
increasing the mucosal oxalate concentrate (up to 6 m M ) , the absorption increased linearly suggesting a passive diffusion process a t higher oxalate concentrations. However, at all concentrations of oxalate tested, oxalate uptake in vitamin B,-deficient rats was significantly greater than that observed in control rats, as is evident from the slope of the data (0.60 in control to 0.84 in vitamin B,-deficient rats). The oxalate transport carrier has a K , of 1.25 mmoles with a V,,, of 1.84 mol . hr-lg wet wt-’. We also investigated oxalate uptake using isolated brush-border membrane vesicles (BBMV) in control and vitamin B,-deficient rats,” because it was suggested that BBM are likely to be more specific and sensitive models for elucidating a mechanism of oxalate absorption as has been shown for other nutrient^.^^.^^ The oxalate absorption was studied by measuring oxalate uptake a t different concentrations of substrate (0.11.0 m M ) using BBM. The data presented in FIGURE3 shows involvement of a transport ligand in the oxalate uptake process in vitamin B,-deficient rats a t concentrations of 0.1-0.6 mM. However, oxalate uptake increased linearly with the increased concentrations of oxalate in the medium a t all concentrations above 0.6 m M (0.61mM). In comparsion to this biphasic transport of oxalate in pyridoxine deficiency, the pair-fed rats exhibited a simple passive diffusion process, which is evident from the linear relationship between the oxalate uptake and oxalate concentration in the medium. Data presented in FIGURE 3 (insert) reveals an apparent affinity constant ( K , ) and maximal influx rate (VmaX) for oxalate as 0.86 m M a n d 18.24 nm/mg protein
2ol FIGURE 2. Oxalate uptake from control (0) and vitamin B,deficient ( 0 )rat intestinal gut rings. Each point is the mean t SE of eight to ten observations. Vertical bars represent SE. Insert: Oxalate uptake at higher concentrations. WCn \-t
3
1
05
10
OXALATE ( m M )
NATH et al.: OXALATE METABOLISM
279
DEF'ICIENT
0
.2
.4
fi
-8
1-0
OXALATE CONCENTRATON (mM)
FIGURE 3. Oxalate uptake into BBM vesicles of control ( 0 )and vitamin B,-deficient (A)rats. Insert shows Lineweaver-Burk plot of the uptake by vitamin B,-deficient BBM.
per 20 min, respectively, for pyridoxine-deficient rat intestinal BBM, confirming our earlier observations using gut rings. The difference in values of K,,, and V,,, in the two experiments", " is obligatory because of the presence of a number of nonspecific processes using gut rings. We also studied the effect of the protein synthesis inhibitors cycloheximide and actinomycin-D on intestinal uptake of oxalate in control and vitamin B,-deficient rats (FIG.4). While actinomycin D and cycloheximide treatment completely reversed the carrier-mediated uptake process in B,-deficient rats, the uptake process was not at all affected in control rats, suggesting that the transport carrier may be a protein. To characterize the transport ligand in terms of binding
- O'*0.6 1I
0
/
02
04
06
08
1-0
OXALATE ( m M )
FIGURE 4. Effect of actinomycin D on the intestinal uptake of oxalate. Oxalate uptake from control (0),vitamin B,-deficient (0)and vitamin B, actinomycin D treated (0)rats. Values are Used with permission). mean SEM of eight observations. (From Farooqui et
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affinity for the substrate, we studied binding of oxalate to intestinal BBM of both vitamin B,-deficient as well as control animals at zero osmotic space. Oxalate bound specifically to BBM prepared from pyridoxine-deficient rats but not to BBM of control rats2, FIGURE5 shows a Scatchard plot characterizing the relationship between concentration of oxalate and its binding to intestinal BBM of pyridoxine-deficient rats. The results suggest induction of two binding systems for oxalate comprised of a low-affinity site and a high-affinity site. The high-affinity site had a Kd of 24.29 nmolar but had a limited capacity to bind 30 pmoles oxalate/mg protein, while the low-affinity site had a Kd of 487.5 nmolar but possessed the capacity to bind more than 156 pmoles oxalate/mg protein. Several kinetic properties of the high-affinity site were studied. Competition of other dicarboxylates, namely, malate, pyruvate, oxaloacetate, glyoxylate, and parabonate with oxalate revealed that the affinity order was oxalate > parabonate > glyoxylate > oxaloacetate, demonstrating a high specificity of oxalate
0
20
LO
60
80
100
120
140
153
OXALATE BOUND p rnoles/rng PROTEIN
FIGIJRE 5. Scatchard Plot of oxalate binding to intestinal BBM of vitamin B,-deficient rats.
binding to BBM in pyridoxine deficiency. Functional isolation of transport carriers has been imperative in their biochemical characterization. To achieve this objective, we recently isolated the carrier protein for oxalate from pyridoxine-deficient BBM using deoxycholate as detergent." FIGURE6 shows the chromatographic profile of B,deficient BBM after incubation with oxalic acid containing trace amounts of ['4C]oxalate. The effluent fractions were monitored for protein (by measuring O.D. a t 280 nm as well as for ['4C]oxalate. A single oxalate binding peak was obtained. The molecular weight was determined by calibrating the gel coIumn with known standards. The oxalate-bound protein (OBP) was eluted at about the 90-kDa fraction (data not shown). The OBP peak was pooled, and oxalate was removed by dialysis for 48 hours at 4OC.Thus, we obtained a protein (OBP), which on isolation from BBM, bound oxalate. The appropriate question at this stage is whether OBP is capable of binding oxalate
NATH et al.: OXALATE METABOLISM
281 C a l M N CWRACTERISTICS;
DIMENSIONS.(2.0 x 67.5 Cm)
0.7
30
” 10.6 25 1‘I
0.5
TOTALBED MClMEzU93Bml FLWRATE. 28 mllhr VCXDVOUJME - L O ml
E 0.4 B
w 0.3
u
a 0.2
8
m
5 4
0.1
a
25
50 75 100 125 EFFLUENT VOLUME (ml) --+
I
FIGURE 6. Chromatographic profile of proteins, isolated from vitamin B,-deficient rat intestinal brush-border membrane on Sephadex G-75 column, eluted with 0.2 Mphosphate buffer, pH 7.4.
when it is present in the lipid bilayer. To address this question, we incorporated BBM-isolated OBP into phosphatidyl choline : cholesterol (9 : 1 molar ratio) liposomes. The data presented in FIGURE7 reveals that proteoliposomes had 80-90% increased binding to oxalate as compared to respective control liposomes, which lacked OBP. Thus we demonstrated the capability of OBP to bind oxalate even when incorporated into liposomes, which mimics the natural membrane. The studies on the
EXPERIMENT
EXPERIMENT
I
TI
FIGURE 7. Oxalate binding to liposornes in the presence and absence of oxalate-binding protein (OBP).2q
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functional reconstitution of OBP into liposomes has encouraged us to undertake the detailed kinetic studies of oxalate uptake into liposomes after incorporation in OBP. Further experiments are in progress. On the basis of the above results, we postulate that a high-affinity, oxalate-binding protein is induced in intestinal BBM in pyridoxine deficiency. This protein may be involved in the carrier-mediated process of oxalate uptake in vitamin B, deficiency that leads to enhanced intestinal absorption of dietary oxalate.
Generalized Hypothesis of Hyperoxaluria in Vitamin B, Dejciency Oxalic acid is an end-product of metabolic pathways, is not further degraded in animals and man, and is excreted as such.' In our studies we observed hyperoxaluria following pyridoxine deficiency. As shown in the results, intestinal absorption of dietary oxalate as well as enzymes of the oxalate biosynthetic pathway are elevated. Since pyridoxine is a cofactor in transaminase reactions, it was therefore proposed that following pyridoxine deficiency, a decrease in the glycine-glyoxylate transaminase reaction would increase the glyoxylate pool (FIG.8). The increase in the glycolatemetabolizing enzymes GAO and GAD following pyridoxine deficiency can explain the increased production of oxalate from glycolate and glycolate precursors, that is, ethylene glycol, xylitol, and ethanolamine, thus increasing the body oxalate pool. The situation becomes critical as the result of the increased uptake of oxalate owing to the induction of a transport carrier for oxalate in the intestinal brush-border membrane in B, deficiency. Oxalate is then transported to blood for its excretion through the kidneys, resulting in hyperoxaluria.
BRUSH
ETHYLENE GLYCOL
BORDER
BASOLATERAL
.X. Y. I- .ITni .-.
BLOOD
GLY O X YLATE 5
.
-
!
I t
INTESTINAL MUCOSAL
d INCREASED CONTRIBUTION
T0
BODY POOL
OXALATE
0
---
OXALATE
a
9
LIVER 6 KIDNEY
ENDOGENWS BIOSYNTHESIS
I /k
INHlBlTlON
FIGURE 8. Proposed mechanism of hyperoxaluria in vitamin B, deficiency.
NATH et al.: OXALATE METABOLISM
283
Therapeutic Role of Vitamin B6 on Hyperoxafziria To test the above hypothesis, we studied the effect of pyridoxine supplementation in hyperoxaluric patient^.'^ Supplementation of pyridoxine in human subjects with hyperoxaluria (at a dose of 10 mg pyridoxine HC1/70 kg body weight/day) significantly ( p < 0.001) decreased oxalate excretion after 90 days of pyridoxine supplementation. Administration of pyridoxine also improved the pyridoxine status of patients, as evidenced by the percent pyridoxal phosphate stimulation index of their erythrocyte transaminase activities. A positive correlation was observed between pyridoxine status as assessed by activities of erythrocyte G P T and GOT and extent of hyperoxaluria (TABLE 4).
Effect of Pyridoxine Supplementation on Urinary Oxalate and Erythrocyte Transaminase Activity in Hyperoxaluric Patients
TABLE 4.
Supplementation Period (Days) 0 15 30 90 180
Urinary Oxalate (mg/24 hr) 65.8 r 8.5" 55.0 * 11.3 57.7 f 9.9 38.2 * 7.2' 22.2 * 5.3b
Percentage Activation by Added Pyridoxal-5-Phosphate EGOT
EGPT
32 24 25 23 6
47 28 28 16 11
"Mean SEM. b p < 0.001 as compared to 0 day.
CONCLUSIONS We conclude that hyperoxaluria as observed in pyridoxine deficiency arises from increased endogenous oxalate biosynthesis, as a result of reduced transaminase activities and increased activities of oxalate biosynthetic enzymes (GAO and GAD) and increased absorption of dietary oxalate, due to induction of oxalate-transporting protein in intestinal brush-border membrane. Further supplementation of pyridoxine has been found to be beneficial in reducing hyperoxaluria in idiopathic stone formers as well as in experimentally induced hyperoxaluric rats, thus supporting the proposed hypothesis.
REFERENCES 1. HODGKINSON, A. 1977. Oxalic acid in biology and medicine. Academic Press. London. 2. BOER,P., L. L. VAN,R. J. HENE& E. J. DORHOUT MEES. 1984. Am. J. Kidney Dis. 4 118. 3. GERSHOFF, S. N. & F. F. FARAGALLA. 1959. J. Biol. Chem. 234 2391. 4. GERSHOFF, S. N. 1964. Vitam. Horm. 2 2 581. 5. HANNET,B., D. W. THOMAS, A. H. CHALMERS, A. M. ROFE,J. B. EDWARDS & R. G. EDWARDS. 1977. J. Nutr. 107: 458. J. D. & S. N. GERSHOFF.1979. J. Nutr. 109 171. 6. RIBAYA.
284 7. 8. 9. 10. 1I . 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
ANNALS NEW YORK ACADEMY OF SCIENCES LILIEN,0. M., W. S. H. HAMMOND, D. J. KRAURS, A. ELBANDAWI & J. E. SCHOONMAKER. 1981. Invest. Urol. 18: 451. PRASAD, R., V. LYALL& R. NATH.1982. Ann. Nutr. Metab. 2 6 324. KISHI,H. & R. FOLKARES. 1976. J. Nutr. Sci. Vitaminol. 22225. FAROOQUI, S., R. NATH,S. K. THIND& A. MAHMOOD. 1984. Biochem. Med. 3 2 34. KOUL,H. K., R. GUPTA,S. K. THIND& R. NATH.1989. In Urolithiasis Research. R. Nath & S. K. Thind, Eds.: 221. Ashish Publishers. New Delhi, India. MURTHY,M. S. R., H. S. TALWAR, S. K. THIND& R. NATH.1982. Ann. Nutr. Metab. 2 6 201. MURTHY, M. S. R., S. FAROOQUI, H. S. TALWAR, S. K. THIND,R. NATH,L. RAJENDRAN & B. C. BAPNA.1982. Int. J. Clin. Pharmacol. Ther. Tox. 20(9): 434. HANGLER, L. & R. H. HEMAN.1973. Am. J. Clin. Nutr. 2 6 882. WILL,E. J. & 0.L. M. BIJOVET.1979. Metabolism28: 542. ROFE,A. M. & J. B. EDWARDS. 1978. Biochem. Med.20 323. 1965. J. Biol. Chem. 2 4 0 1889. RUNYAN, T. J. & S. N. GERSHOFF. RICHARDSON, K. E. 1967. Toxicol. Appl. Pharmacol. 1 0 40. MASTERS,C. J. & R. S. HOLMES.1977. Physiol. Rev. 57: 816. M. A. 1964. Vitam. Horm. 2 4 561. WILLIAMS, & R. W. E. WATTS.J. Biochem. 82: 221. GIBBS,D. A,, S. HAUSCHILDT KAYNE,F. J. 1974. Biochem. Biophys. Res. Commun. 5 9 8. NATH,R., S. K. THIND,M. S. R. MURTHY, H. S. TALWAR & S. FAROOQUI. 1984. Mol. Asp. Med. 7(1/2): 80. V. & A. N. RADHAKRISHNAN. 1980. Biochem. Pharmacol. 2 9 713. GANAPATHY, G., M. KESSLER,M. HOSANG,J. WEBBER& U. SCHMIDT. 1984. Biochim. SEMENZER, Biophys. Acta 779: 343. KOUL,H. K., S. K. THIND& R. NATH.1988. Proceedings 1st International Conference on Biomembranes in Health and Disease. In press. KOUL,H. K., R. GUPTA,S. K. THIND& R. NATH.1988. Abstr. A 52 Urol. Res. I 6 187. FAROOQUI, S., A. Mahmood, R. Nath & S. K. Thind. 1981. Ind. J. Exp. Biol. 19 551. KOUL,H. K., S. K. THIND& R. NATH.1988. Proceedings of the First National Symposium & Workshop on Liposome Research. New Delhi, India. Abstr. 3.
INTRODUCTION Oxalic acid, a dicarboxylate widespread in plants, plays no vital function in man and experimental animals. The animal system is endangered by the toxicity of oxalate, as this compound is not metabolized further in the body and is excreted as such in urine. Nonetheless, deposition of calcium oxalate is commonly found in end-state renal diseases owing to retention of oxalate.'22The commonest pathological condition involving oxalate is the formation of calcium oxalate stones in the urinary tract (urolithiasis). Persistent hyperoxaluria is the most common feature in urolithiasis patients. It is well established that nutritional pyridoxine deficiency leads to hyperoxaluria and renal stone formation in experimental animals and In this paper, we discuss the role of pyridoxine in oxalate metabalism and illustrate the molecular mechanism(s) of hyperoxaluria in pyridoxine deficiency using the results of our studies over the past several years.
MATERIALS AND METHODS Pyridoxine deficiency was induced in male Wistar rats (b.w. 35-40 g) by feeding them a vitamin B, deficient diet' for 45 days. The deficiency was biochemically assessed by measuring erythrocyte alanine transaminase (EGPT) and erythrocyte glutamic acid transaminase (EGOT) activities together with percent pyridoxal phosphate stimulation index (percent PALP) of EGPT and EGOT.9 Intestinal oxalate uptake was determined by everted gut rings," as well as by using brush-border membrane vesicles." Oxalate-synthesizing enzymes were assayed as described previously." Assay of EGOT and EGPT was also used as an index of pyridoxine status in human subjects tested for hypero~a1uria.l~
RESULTS AND DISCUSSIONS
Endogenous Oxalate Biosynthesis Major sources of endogenous oxalate include ascorbic acid, glycine, glycolic acid, and glyoxylic acid.I4 The enzymes that are required for oxalate biosynthesis are glycolic acid oxidase (GAO), lactate dehydrogenase (LDH), and glycolic acid dehydro274
NATH et al.: OXALATE METABOLISM
275
genase (GAD). Liver GAO activity is increased in vitamin B, deficiency. This increase was highly significant even when the activity was expressed as units per gram tissue. No change was observed in liver LDH while liver GAD activity also showed a significant increase ( p < 0.05) (TABLE1).
Role of Transaminases and Biosynthetic Enzymes Chronic pyridoxine deficiency leads to the decreased activity of most of the transaminases, including glyoxylate transaminase, whose physiological function was thought to control the glyoxalate pool by converting it to g1y~ine.I~ On this basis, many worker^^^^^^," postulated that glyoxylate, which increases in pyridoxine deficiency, may further be converted to oxalate, thereby resulting in hyperoxaluria, which is a characteristic feature of vitamin B, deficiency. However, many studiesI6,” show that glyoxylate is not an obligatory intermediate in the biosynthesis of oxalate from glycolate or ethylene glycol. Particularly, the conversion of glycolate to oxalate is many-fold higher in vitamin B, deficiency than that of glyoxylate.” Thus the above
Effect of Vitamin B, Deficiency on Activity of Oxalate-Synthesizing Enzymes in the Rat Liver
TABLE 1.
Glycolate Oxidase B, Deficient Units/mgprotein Units/gmtissue
2.64 228
0.24“ f 32‘ t
Lactate Dehydrogenase
Glycolate Dehydrogenase
Pair Fed
B, Deficient
Pair Fed
B, Deficient
1.04 t 0.07 116 t 7
2.63 f 0.23 273 i 23
2.53 f 0.10 284 f 14
4.03 f 5.OOb 4.14 + 0.48
Pair Fed 2.65 2.95
i 2
0.3 0.0
NOTE: Results are expressed as mean f SEM. Number of animals in each group was six to eight. ‘ p < 0.001 as compared to control. b p < 0.05 as compared to control. c p < 0.01 as compared to control.
hypothesis, which was based on an increased glyoxylate pool, could not answer these important questions. Our results, which show a significant increase in liver GAO activity in pyridoxine-deficient rats, are contradictory to the earlier report’* where no changes were noted. G A O is essentially localized in peroxi~omes’~ and can convert glycolate to glyoxylate and further to oxalate in two steps (FIG.1). Another enzyme, D-amino acid oxidase (DAO), which converts glycine to glyoxylate by oxidative deamination, is also present in peroxisome~.’~ It has also been shown that the tissue glycine pool increases in pyridoxine deficiency.” It is likely that the synthesis of glyoxylate by DAO in pyridoxine deficiency remains unchecked due to very low transaminase activity, and this is rapidly oxidized to oxalate by GAO. Glyoxylate may also be oxidized by peroxisomal LDH” to oxalate. GAD in liver may not be very significant in the oxidation of glycolate to oxalate as this enzyme activity tended to decrease in vitamin B, deficiency. Preliminary studies in our laboratory showed that phosphoglycolate can be converted to oxalate faster than glycolate itself. In vitro glycolate is phosphorylated to phosphoglycolate by pyruvate kinase.22
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OXALAT
GLYCINE
I? I
I
I
+ fl
PH OSPHOGLYCOLATE
i
I
KlDNEY
LIVER
INHIBITED IN 86 DEE INCREASED IN 66 DEF.
FIGURE 1. Metabolism of oxalate in pyridoxine-deficient rat liver and kidney. Enzymes: DAO, D amino acid oxidase; TA,transaminase;GAO, glycolic acid oxidase; LDH, lactate dehydrogenase; GAD,glycolic acid dehydrogenase;PK, pyruvate kinase; GR, glyoxylate reductase.
Liver Peroxisomes Glyoxylate that is produced within the peroxisomes may have a different metabolism than glyoxylate produced in cytosol.16 This difference may be explained by the assumption that the peroxisomal membrane does not allow the transport of glyoxylate but does allow the transport of glycolate.” Glyoxylate, if administered from outside, may be decarboxylated by glyoxylate carboligase in the condensation reaction with a-ketoglutarate at a faster rate even though some of it may be converted to oxalate by cytoplasmic LDH.’ This exogenous glyoxylate is unable to enter the peroxisomes. However, glyoxylate produced within the peroxisomes may not be able to leave this organelle and is subsequently oxidized to oxalate. This particular pathway may increase several-fold in vitamin B, deficiency owing to increased GAO activity, as a result of a probable increase in the number of liver peroxisomes. Kidney Enzymes The kidney enzymes, however, present a picture which is altogether different from that of the liver. Kidneys do not possess the activity of G A O in their peroxisomes, but they do have the activity of DAO.I9 Glyoxylate reductase is also present in the kidney and can reduce glyoxylate to glycolate using N A D H + H + .In pyridoxine deficiency the kidney glyoxylate pool increases because of the reduced transaminase activity. This increased glyoxylate may in turn be reduced to glycolate by glyoxylate reductase or by peroxisomal LDH. Once glycolate is produced, it can be rapidly oxidized directly to oxalate without forming enzyme-free glyoxylate as intermediate by GAD. GAD activity in the kidney is greatly enhanced in pyridoxine deficiency (TABLE2). Subcellular localization of this enzyme is not known. Nevertheless, GAD can act on glycolate, whether the latter is produced in peroxisomes or in cytosol. The role played by cytoplasmic kidney LDH is not clear. Probably, it has no functional significance as far
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as pyridoxine deficiency-induced hyperoxaluria is concerned, as no significant increase in LDH activity was observed in this deficiency (TABLE2). If this enzyme were to be active in oxidizing glyoxylate, the exogenously administered glyoxylate could have been significantly converted to oxalate in vitamin B, deficiency.”
Role of Sex Hormones Even though no clear evidence is available on the effect of pyridoxine deficiency on the levels of testosterone, it is likely that this vitamin must have some role in regulating testosterone level which in turn regulates G A O activity. Another possibility for the increase in GAO activity could be a decreased turnover of this enzyme in vitamin B, deficiency, but this is not yet confirmed. Preliminary studies in our laboratory showed that while the K,,, for glycolate of GAO remains unaltered, there is an increase in V,,, in vitamin B, deficiency, suggesting thereby an increase in the net enzyme content.*’ However, GAD activity does not seem to be under the control of testosterone, and its activity followed a pattern opposite to that of GAO. The factors regulating this enzyme activity need further elucidation. In conclusion, it may be speculated that the increased synthesis of oxalate in vitamin B, deficiency may have two origins, one from liver and another from kidney, which involve separate and probably independent metabolic pathways (FIG.1). The individual contributions of these two organs towards the excretion of urinary oxalate in vitamin B, deficiency need to be further studied.
Intestinal Absorption of Oxalate Oxalate absorption from the gut is merely a passive diffusion phenomenon in man and experimental animals. The effects of pyridoxine deficiency on uptake of oxalate by everted intestinal sacs (TABLE3) revealed that the uptake rate of oxalate is elevated significantly ( p < 0.001) in vitamin B,-deficient rats as compared to animals in the control group. Data on the rate of oxalate uptake from the intestine in control and vitamin B,-deficient rats in a concentration range of 0.1-6.0 m M oxalate are given in FIGURE2. Mucosal oxalate concentration increased linearly in the control group and at no point was saturation of oxalate uptake detected, suggesting a diffusion process for oxalate uptake by intestine. Carrier-mediated saturation kinetics was observed in vitamin B,-deficient rats between 0.1 and 0.8 m M oxalate concentrations. On further
Effect of Vitamin B, Deficiency on Activity of Oxalate-Synthesizing Enzymes in the Rat Kidney
TABLE 2.
Lactate Dehydrogenase B, Deficient Pair Fed Units/mg protein Units/gm tissue
3.7 + 0.08 312 * 8
NOTE: Results expressed as mean ‘ p < 0.05.
3.32 f 0.10 330 + 18
Glycolate Dehydrogenase B, Deficient Pair Fed 1.102 + 0.04“ 0.202 c 0.05 0.112 + 0.02 1.093 f 0.33“
+ SEM. Number of animals in each group was six to eight.
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TABLE 3. Effect of Vitamin B, Deficiency on Oxalate Uptake from Rat Intestinea
Grouo Control Pair Fed Vitamin B, Deficient
Oxalate Uptake (wmollhr gm tissue)
.
0.26 t 0.02 0.27 + 0.06 0.41 -+ 0.02b
=Values are mean SE of six to eight observations. b p < 0.001 as compared to the control or pair fed group.
increasing the mucosal oxalate concentrate (up to 6 m M ) , the absorption increased linearly suggesting a passive diffusion process a t higher oxalate concentrations. However, at all concentrations of oxalate tested, oxalate uptake in vitamin B,-deficient rats was significantly greater than that observed in control rats, as is evident from the slope of the data (0.60 in control to 0.84 in vitamin B,-deficient rats). The oxalate transport carrier has a K , of 1.25 mmoles with a V,,, of 1.84 mol . hr-lg wet wt-’. We also investigated oxalate uptake using isolated brush-border membrane vesicles (BBMV) in control and vitamin B,-deficient rats,” because it was suggested that BBM are likely to be more specific and sensitive models for elucidating a mechanism of oxalate absorption as has been shown for other nutrient^.^^.^^ The oxalate absorption was studied by measuring oxalate uptake a t different concentrations of substrate (0.11.0 m M ) using BBM. The data presented in FIGURE3 shows involvement of a transport ligand in the oxalate uptake process in vitamin B,-deficient rats a t concentrations of 0.1-0.6 mM. However, oxalate uptake increased linearly with the increased concentrations of oxalate in the medium a t all concentrations above 0.6 m M (0.61mM). In comparsion to this biphasic transport of oxalate in pyridoxine deficiency, the pair-fed rats exhibited a simple passive diffusion process, which is evident from the linear relationship between the oxalate uptake and oxalate concentration in the medium. Data presented in FIGURE 3 (insert) reveals an apparent affinity constant ( K , ) and maximal influx rate (VmaX) for oxalate as 0.86 m M a n d 18.24 nm/mg protein
2ol FIGURE 2. Oxalate uptake from control (0) and vitamin B,deficient ( 0 )rat intestinal gut rings. Each point is the mean t SE of eight to ten observations. Vertical bars represent SE. Insert: Oxalate uptake at higher concentrations. WCn \-t
3
1
05
10
OXALATE ( m M )
NATH et al.: OXALATE METABOLISM
279
DEF'ICIENT
0
.2
.4
fi
-8
1-0
OXALATE CONCENTRATON (mM)
FIGURE 3. Oxalate uptake into BBM vesicles of control ( 0 )and vitamin B,-deficient (A)rats. Insert shows Lineweaver-Burk plot of the uptake by vitamin B,-deficient BBM.
per 20 min, respectively, for pyridoxine-deficient rat intestinal BBM, confirming our earlier observations using gut rings. The difference in values of K,,, and V,,, in the two experiments", " is obligatory because of the presence of a number of nonspecific processes using gut rings. We also studied the effect of the protein synthesis inhibitors cycloheximide and actinomycin-D on intestinal uptake of oxalate in control and vitamin B,-deficient rats (FIG.4). While actinomycin D and cycloheximide treatment completely reversed the carrier-mediated uptake process in B,-deficient rats, the uptake process was not at all affected in control rats, suggesting that the transport carrier may be a protein. To characterize the transport ligand in terms of binding
- O'*0.6 1I
0
/
02
04
06
08
1-0
OXALATE ( m M )
FIGURE 4. Effect of actinomycin D on the intestinal uptake of oxalate. Oxalate uptake from control (0),vitamin B,-deficient (0)and vitamin B, actinomycin D treated (0)rats. Values are Used with permission). mean SEM of eight observations. (From Farooqui et
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affinity for the substrate, we studied binding of oxalate to intestinal BBM of both vitamin B,-deficient as well as control animals at zero osmotic space. Oxalate bound specifically to BBM prepared from pyridoxine-deficient rats but not to BBM of control rats2, FIGURE5 shows a Scatchard plot characterizing the relationship between concentration of oxalate and its binding to intestinal BBM of pyridoxine-deficient rats. The results suggest induction of two binding systems for oxalate comprised of a low-affinity site and a high-affinity site. The high-affinity site had a Kd of 24.29 nmolar but had a limited capacity to bind 30 pmoles oxalate/mg protein, while the low-affinity site had a Kd of 487.5 nmolar but possessed the capacity to bind more than 156 pmoles oxalate/mg protein. Several kinetic properties of the high-affinity site were studied. Competition of other dicarboxylates, namely, malate, pyruvate, oxaloacetate, glyoxylate, and parabonate with oxalate revealed that the affinity order was oxalate > parabonate > glyoxylate > oxaloacetate, demonstrating a high specificity of oxalate
0
20
LO
60
80
100
120
140
153
OXALATE BOUND p rnoles/rng PROTEIN
FIGIJRE 5. Scatchard Plot of oxalate binding to intestinal BBM of vitamin B,-deficient rats.
binding to BBM in pyridoxine deficiency. Functional isolation of transport carriers has been imperative in their biochemical characterization. To achieve this objective, we recently isolated the carrier protein for oxalate from pyridoxine-deficient BBM using deoxycholate as detergent." FIGURE6 shows the chromatographic profile of B,deficient BBM after incubation with oxalic acid containing trace amounts of ['4C]oxalate. The effluent fractions were monitored for protein (by measuring O.D. a t 280 nm as well as for ['4C]oxalate. A single oxalate binding peak was obtained. The molecular weight was determined by calibrating the gel coIumn with known standards. The oxalate-bound protein (OBP) was eluted at about the 90-kDa fraction (data not shown). The OBP peak was pooled, and oxalate was removed by dialysis for 48 hours at 4OC.Thus, we obtained a protein (OBP), which on isolation from BBM, bound oxalate. The appropriate question at this stage is whether OBP is capable of binding oxalate
NATH et al.: OXALATE METABOLISM
281 C a l M N CWRACTERISTICS;
DIMENSIONS.(2.0 x 67.5 Cm)
0.7
30
” 10.6 25 1‘I
0.5
TOTALBED MClMEzU93Bml FLWRATE. 28 mllhr VCXDVOUJME - L O ml
E 0.4 B
w 0.3
u
a 0.2
8
m
5 4
0.1
a
25
50 75 100 125 EFFLUENT VOLUME (ml) --+
I
FIGURE 6. Chromatographic profile of proteins, isolated from vitamin B,-deficient rat intestinal brush-border membrane on Sephadex G-75 column, eluted with 0.2 Mphosphate buffer, pH 7.4.
when it is present in the lipid bilayer. To address this question, we incorporated BBM-isolated OBP into phosphatidyl choline : cholesterol (9 : 1 molar ratio) liposomes. The data presented in FIGURE7 reveals that proteoliposomes had 80-90% increased binding to oxalate as compared to respective control liposomes, which lacked OBP. Thus we demonstrated the capability of OBP to bind oxalate even when incorporated into liposomes, which mimics the natural membrane. The studies on the
EXPERIMENT
EXPERIMENT
I
TI
FIGURE 7. Oxalate binding to liposornes in the presence and absence of oxalate-binding protein (OBP).2q
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functional reconstitution of OBP into liposomes has encouraged us to undertake the detailed kinetic studies of oxalate uptake into liposomes after incorporation in OBP. Further experiments are in progress. On the basis of the above results, we postulate that a high-affinity, oxalate-binding protein is induced in intestinal BBM in pyridoxine deficiency. This protein may be involved in the carrier-mediated process of oxalate uptake in vitamin B, deficiency that leads to enhanced intestinal absorption of dietary oxalate.
Generalized Hypothesis of Hyperoxaluria in Vitamin B, Dejciency Oxalic acid is an end-product of metabolic pathways, is not further degraded in animals and man, and is excreted as such.' In our studies we observed hyperoxaluria following pyridoxine deficiency. As shown in the results, intestinal absorption of dietary oxalate as well as enzymes of the oxalate biosynthetic pathway are elevated. Since pyridoxine is a cofactor in transaminase reactions, it was therefore proposed that following pyridoxine deficiency, a decrease in the glycine-glyoxylate transaminase reaction would increase the glyoxylate pool (FIG.8). The increase in the glycolatemetabolizing enzymes GAO and GAD following pyridoxine deficiency can explain the increased production of oxalate from glycolate and glycolate precursors, that is, ethylene glycol, xylitol, and ethanolamine, thus increasing the body oxalate pool. The situation becomes critical as the result of the increased uptake of oxalate owing to the induction of a transport carrier for oxalate in the intestinal brush-border membrane in B, deficiency. Oxalate is then transported to blood for its excretion through the kidneys, resulting in hyperoxaluria.
BRUSH
ETHYLENE GLYCOL
BORDER
BASOLATERAL
.X. Y. I- .ITni .-.
BLOOD
GLY O X YLATE 5
.
-
!
I t
INTESTINAL MUCOSAL
d INCREASED CONTRIBUTION
T0
BODY POOL
OXALATE
0
---
OXALATE
a
9
LIVER 6 KIDNEY
ENDOGENWS BIOSYNTHESIS
I /k
INHlBlTlON
FIGURE 8. Proposed mechanism of hyperoxaluria in vitamin B, deficiency.
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283
Therapeutic Role of Vitamin B6 on Hyperoxafziria To test the above hypothesis, we studied the effect of pyridoxine supplementation in hyperoxaluric patient^.'^ Supplementation of pyridoxine in human subjects with hyperoxaluria (at a dose of 10 mg pyridoxine HC1/70 kg body weight/day) significantly ( p < 0.001) decreased oxalate excretion after 90 days of pyridoxine supplementation. Administration of pyridoxine also improved the pyridoxine status of patients, as evidenced by the percent pyridoxal phosphate stimulation index of their erythrocyte transaminase activities. A positive correlation was observed between pyridoxine status as assessed by activities of erythrocyte G P T and GOT and extent of hyperoxaluria (TABLE 4).
Effect of Pyridoxine Supplementation on Urinary Oxalate and Erythrocyte Transaminase Activity in Hyperoxaluric Patients
TABLE 4.
Supplementation Period (Days) 0 15 30 90 180
Urinary Oxalate (mg/24 hr) 65.8 r 8.5" 55.0 * 11.3 57.7 f 9.9 38.2 * 7.2' 22.2 * 5.3b
Percentage Activation by Added Pyridoxal-5-Phosphate EGOT
EGPT
32 24 25 23 6
47 28 28 16 11
"Mean SEM. b p < 0.001 as compared to 0 day.
CONCLUSIONS We conclude that hyperoxaluria as observed in pyridoxine deficiency arises from increased endogenous oxalate biosynthesis, as a result of reduced transaminase activities and increased activities of oxalate biosynthetic enzymes (GAO and GAD) and increased absorption of dietary oxalate, due to induction of oxalate-transporting protein in intestinal brush-border membrane. Further supplementation of pyridoxine has been found to be beneficial in reducing hyperoxaluria in idiopathic stone formers as well as in experimentally induced hyperoxaluric rats, thus supporting the proposed hypothesis.
REFERENCES 1. HODGKINSON, A. 1977. Oxalic acid in biology and medicine. Academic Press. London. 2. BOER,P., L. L. VAN,R. J. HENE& E. J. DORHOUT MEES. 1984. Am. J. Kidney Dis. 4 118. 3. GERSHOFF, S. N. & F. F. FARAGALLA. 1959. J. Biol. Chem. 234 2391. 4. GERSHOFF, S. N. 1964. Vitam. Horm. 2 2 581. 5. HANNET,B., D. W. THOMAS, A. H. CHALMERS, A. M. ROFE,J. B. EDWARDS & R. G. EDWARDS. 1977. J. Nutr. 107: 458. J. D. & S. N. GERSHOFF.1979. J. Nutr. 109 171. 6. RIBAYA.
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ANNALS NEW YORK ACADEMY OF SCIENCES LILIEN,0. M., W. S. H. HAMMOND, D. J. KRAURS, A. ELBANDAWI & J. E. SCHOONMAKER. 1981. Invest. Urol. 18: 451. PRASAD, R., V. LYALL& R. NATH.1982. Ann. Nutr. Metab. 2 6 324. KISHI,H. & R. FOLKARES. 1976. J. Nutr. Sci. Vitaminol. 22225. FAROOQUI, S., R. NATH,S. K. THIND& A. MAHMOOD. 1984. Biochem. Med. 3 2 34. KOUL,H. K., R. GUPTA,S. K. THIND& R. NATH.1989. In Urolithiasis Research. R. Nath & S. K. Thind, Eds.: 221. Ashish Publishers. New Delhi, India. MURTHY,M. S. R., H. S. TALWAR, S. K. THIND& R. NATH.1982. Ann. Nutr. Metab. 2 6 201. MURTHY, M. S. R., S. FAROOQUI, H. S. TALWAR, S. K. THIND,R. NATH,L. RAJENDRAN & B. C. BAPNA.1982. Int. J. Clin. Pharmacol. Ther. Tox. 20(9): 434. HANGLER, L. & R. H. HEMAN.1973. Am. J. Clin. Nutr. 2 6 882. WILL,E. J. & 0.L. M. BIJOVET.1979. Metabolism28: 542. ROFE,A. M. & J. B. EDWARDS. 1978. Biochem. Med.20 323. 1965. J. Biol. Chem. 2 4 0 1889. RUNYAN, T. J. & S. N. GERSHOFF. RICHARDSON, K. E. 1967. Toxicol. Appl. Pharmacol. 1 0 40. MASTERS,C. J. & R. S. HOLMES.1977. Physiol. Rev. 57: 816. M. A. 1964. Vitam. Horm. 2 4 561. WILLIAMS, & R. W. E. WATTS.J. Biochem. 82: 221. GIBBS,D. A,, S. HAUSCHILDT KAYNE,F. J. 1974. Biochem. Biophys. Res. Commun. 5 9 8. NATH,R., S. K. THIND,M. S. R. MURTHY, H. S. TALWAR & S. FAROOQUI. 1984. Mol. Asp. Med. 7(1/2): 80. V. & A. N. RADHAKRISHNAN. 1980. Biochem. Pharmacol. 2 9 713. GANAPATHY, G., M. KESSLER,M. HOSANG,J. WEBBER& U. SCHMIDT. 1984. Biochim. SEMENZER, Biophys. Acta 779: 343. KOUL,H. K., S. K. THIND& R. NATH.1988. Proceedings 1st International Conference on Biomembranes in Health and Disease. In press. KOUL,H. K., R. GUPTA,S. K. THIND& R. NATH.1988. Abstr. A 52 Urol. Res. I 6 187. FAROOQUI, S., A. Mahmood, R. Nath & S. K. Thind. 1981. Ind. J. Exp. Biol. 19 551. KOUL,H. K., S. K. THIND& R. NATH.1988. Proceedings of the First National Symposium & Workshop on Liposome Research. New Delhi, India. Abstr. 3.
