Ann Nutr Metab 1990;34:104-111
© 1990 S. Karger AG. Basel 0250-6807/90/0342-0104S2.75/0
Comparative Studies on the Effect of Vitamin A, B, and B6 Deficiency on Oxalate Metabolism in Male Rats Sadhna Sharma. Harmeet Sidhu, Ravinder Narula. Swam K. Thind, Ravindra Nath Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh, India
Key Words. Vitamin A • Vitamin B| • Vitamin B6 • Oxalate urolithiasis
Introduction Oxalic acid is a common constituent of mammalian urine. It becomes pathologically significant when present in excess and may amount to increased crystallization of cal cium stones [1,2]. Apart from endogenous oxalate, dietary oxalate is also of paramount importance, since a substantial portion of urinary oxalate is of dietary origin. There fore, the chemical composition of stones is likely to be related to the diet. Even though many pioneer workers com mented that this disease is not caused by one
single factor, nutritional factors particularly, vitamin deficiencies are unquestionably re lated to the genesis of this disease [3, 4], Experimentally, urinary lithiasis can be in duced in laboratory animals by feeding diets deficient in vitamin A, B| or B6. During the last decade, the metabolic role of these vita mins has been extensively studied [5]. Vary ing degrees of clinical and subclinical defi ciency of vitamin A and B6 have been re ported in patients having urinary calculi [4, 6, 7], However, the complete picture of pathological processes which take place in the body as a result of a lack of these nu
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Abstract. The study was conducted to investigate the effect of vitamin A, B, and B6 defi ciency on oxalate metabolism in rats. A significant hyperoxaluria was the common observa tion in all the three vitamin deficiencies (vitamin B6 > vitamin A > vitamin B|). The activities of hepatic glycolate oxidase and glycolate dehydrogenase were markedly enhanced in vitamin-A- and vitamin-B(,-deficient rats. However, lactate dehydrogenase levels re mained unaltered in these deficiencies as compared to their respective pair-fed controls. Vitamin B| deficiency of 4 weeks’ duration could augment the activity of glycolate oxidase only, with no alterations in the glycolate dehydrogenase and lactate dehydrogenase levels. Intestinal oxalate uptake studies revealed increased bio-availability of oxalate from the gut in vitamin-A- and vitamin-B6-deficient rats. Thus, the results suggest the relative contribution of both exogenous as well as endogenous oxalate in the process of calculogenesis under various nutritional stress conditions in rat.
105
Effect of Vitamin Deficiencies on Oxalate Metabolism
Materials and Methods Male Wistar rats (40-50 g body weight) were ac climatized for 5-6 days and were randomly divided into six groups of 8 rats each. The animals were fed their respective dietary regimen for a specified exper imental period: Group I - fed vitamin-A-deficient diet [8] ad libitum for 5 weeks; group II = pair-fed with group I + 150 IU retinyl acetate/rat/day by gas tric intubation; group III = fed vitamin-Br deficient diet [9] ad libitum for 4 weeks; group IV = pair-fed with group III + 100 gg thiamine HCl/rat/day by gas tric intubation; group V= fed vitamin-B6-deficient diet [10] ad libitum for 4 weeks; group VI - pair-fed with group V + 100 pg pyridoxine HCl/rat/day by gas tric intubation. The composition of various experimental diets is given in table 1. Food consumption and weight gain of individual rats were measured every alternate day. At the end of the experimental period, clinical symp toms of vitamin deficiencies became apparent, i.e. scaly skin, loss o f body weight in the vitamin-A-defi cient. acrodynia and alopecia in the vitamin-B6-deficient and anorexia and loss o f weight in the vitaminBr deficient groups. Before sacrificing, the rats were individually placed in metabolic cages with free ac cess to water to collect 24-hour urine samples which were analysed for oxalate [11]. The animals were sacrificed under light ether anaesthesia. The vitamin A status (groups 1 and II) was ascertained biochemically by estimating hepatic vitamin A content [12], The vitamin B, (groups III and IV) and vitamin B6 (groups V and VI) status o f animals was assessed by measuring the haemolvsates for erythrocyte transketolase [13] and erythrocyte ala nine transaminase [14], respectively. Oxalate-Biosynthesizing Enzymes The activities o f various oxalate-biosynthesizing enzymes, i.e. glycolate oxidase (GAO) [ 15], glycolate
Table 1. Composition (g/100 g diet) o f experimen tal diets Ingredienls
Vitamin-Adeficient diet
Casein 20.0 (fat-free) Starch 38.8 Sucrose 30.0 Vitamin mixture 2.2 (deficient in the respective vitamin) Salt mixture 4.0 Ground nut oil 5.0
Vitamin-B|deficient diet 25.0 28.7 23.0 2.2 6.1 15.0
The composition of the vitamin-B6-deficient diet was essentially the same as that o f the vitamin-Br deficient diet except in vitamin mixture; vitamin B6 was deleted and replaced by B|. The casein was obtained commercially from Johnson and Johnson Co.. Bombay, and was made free o f vitamin B| and B^ by repeated washings with ethyl alcohol and water. For the vitamin-A-deficient diet, fat-free casein was obtained by repeated washings initially with water, then acetone, and by drying it at 60 °C. The vitamin mixture was prepared as described in the ICN cata logue (nutritional, biochemical division o f ICN. Life Sciences Group. Cleveland. Ohio, USA. The salt mix ture was prepared as described by Hegested et al.
no].
dehydrogenase (GAD) [16] and lactate dehydroge nase (LDH) [17], were assessed in the postmitochondrial supernatants of 10% w/v tissue homogenates prepared in 0.01 M potassium phosphate buffer, pH 7.1. GAO and LDH were assayed in the liver, while LDH was assayed also in the kidneys. Intestinal Transport o f Oxalate Starting from the ligament of Treitz, an approxi mately 20-cm portion of gut was removed and flushed with ice-cold oxygenated Krebs-Ringer buffer (KRB) containing 140 mM NaCl. 6.0 mM KC1 and 4.0 mM Tris-HCl buffer, pH 7.4. The flushed intestine was everted, and 0.3- to 0.5-cm segments were cut and immersed in oxygenated KRB at 4 °C. The intestinal uptake of oxalate was measured using everted intesti nal rings by the tissue accumulation method using
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trients and which ultimately lead to urinary lithiasis is not clear. The present work has been conducted to assess the effect of vita min A, B| or B6 deficiency in rats on the endogenous biosynthesis of oxalate, its intes tinal absorption and subsequent excretion in the urine.
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Table 2. Biochemical assessment o f vitamin A, B, and B6 deficiency in rats Hepatic vitamin A IU/g tissue weight Group I Group II
u C-oxaIate [18]. The extracellular space was mea sured separately by incubating the tissues in the pres ence of ’H-inulin and was subtracted from all uptake values. Protein was estimated in all the tissues by the method of Lowry et ai. [ 19], Student’s t test was used 144.43 ± 11.41 for evaluating the results statistically. 2.76 ±0.10*
Erythrocyte transketolase U/mg protein Group III Group IV
Results 0.32 + 0.02 0 .11 ±0.01*
After the specified experimental period, group II animals exhibited about 95-96% Erythrocyte alanine transaminase depletion of hepatic vitamin A reserves as U/mg protein X 10'6 compared with their respective pair-fed con Group V 46.02 ±2.31 trols, thus indicating the induction of vita Group VI 12.93±1.01* min A deficiency. Similarly, the biochemical assessment of thiamine and pyridoxine defi All values are means ± SEM of 6 -8 animals. One ciency revealed a significant decrease in international unit of vitamin A is described as equiv erythrocyte transketolase (p < 0.001) and alent to 0.344 pg retinyl acetate. One unit o f transke tolase is defined as I pg o f ribose-5-phosphate metab erythrocyte alanine transaminase (p < olized per minute at 37 °C. One unit o f alanine trans 0.001) activity as compared with their re aminase is defined as 1 pmol o f pyruvate formed per spective pair-fed control rats, thus providing minute at 37 °C. * p < 0.001 as compared to pair-fed evidence for the production of these defi controls. ciencies in rats (table 2).
Table 3. Effect o f vitamin A, B| or B6 deficiency on body, liver and kidney weights o f rats Parameter
Pair-fed controls
Vitamin-Adeficient rats
Body weight, g
143.25 ± 5.65
114.25 ±3.00* 96.83 ±2.05
Liver weight, g
3.88 ± 0.19
3.49 ± 0 .1 2
4.12 ±0.18
3.24 ±0.24*
4.78 ±0.23
4.16 ±0.26
Liver weight, 3.49 ±0.12 g/100 g body weight
3 .7 6 ± 0 .1 0
4.42 ±0.21
4.28 ±0.31
4.73 ± 0.20
4.75 ±0.28
Kidney weight, g
1.12 ± 0.02
1.21 ±0.02
0.84 ±0.03
0.75 ±0.02
0.92 ±0.03
0.85 ±0.03
Kidney weight, 1.15 ± 0.03 g/100 g body weight
1.31 ± 0 .0 4
0.88 ±0.05
0.94 ±0.04
0.91 ±0.03
0.99 ±0.02
Vitamin-Bideficient rats
Pair-fed controls
76.76 ±2.89** 100.63 ±3.77
Vitamin-B*deficient rats 85.78±3.46*
All values are means ± SEM of 6 -8 animals. ** p < 0.001, * p < 0.05 as compared to pair-fed controis.
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Pair-fed controls
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Effect of Vitamin Deficiencies on Oxalate Metabolism
Urinary Excretion o f Oxalate A significant decrease in body weights of animals was observed under all vitamindeficient conditions; however, no statisti cally significant changes were observed in the kidney and liver weights (table 3). Rats deficient in vitamin A, B| or B6 pro duced a significant increase of variable de grees in the 24-hour urinary oxalate excre tion (fig. 1). The maximum hyperoxaluria was observed in vitamin-B6-deficient rats (approx. 2-fold) followed by a 1.83-fold in crease in the vitamin-A-deficient group and about 1.34-fold in thiamine-deficient rats as compared with their pair-fed controls. Endogenous Biosynthesis o f Oxalate The specific activity of major oxalate-biosynthesizing enzymes, i.e. GAO and GAD,
Fig. I. Urinary excretion of oxalate in vitamin-Adeficient (■ ). vitamin-B|-deficient ( 0 ) . vitamin-B6deficient (g|) and pair-fed control (Q ) rats. Values are means ± SEM o f 8-10 animals.
o o Ö
A
B,
B6
A
B,
B6
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Fig. 2. Activities of GAO (a). GAD (b) and LDH (c, d) in vitaminA-deficient ( ■ ) , vitamin-Bi-deficient ( 0 ) . vitamin-B6-deficient ( ^ ) and pair-fed control ( □ ) rats. All values are means ± SEM o f 8 -1 0 animals. One unit of GAO is de fined as I nmol o f glvoxylatc pro duced per minute at 37 °C. One unit o f GAD is defined as 1 nmol of oxalate produced per minute at 37 °C. One unit of LDH is defined as change in 0 .1 optical density per minute at 340 nm at 25 °C.
Sharma/Sidhu/Narula/Thind/Nath
108
was significantly increased in the vitamin-Adeficient group as compared to pair-fed con trols (fig. 2). The liver LDH levels remained unaltered in this deficiency, whereas kidney LDH activity was significantly decreased (p < 0.01) in this nutritional disorder. Vitamin B| deficiency of 4 weeks’ dura tion resulted in a significant increase (p < 0.001) in the activity of liver GAO with no change in the activity of GAD. The activities of both liver as well as kidney LDH re mained unaltered under this nutritional dis order (fig. 2). A significant increase in the liver GAO (p < 0.001) and GAD (p < 0.01) was ob served in the vitamin-B6-deficient group compared to pair-fed controls. No differ ences were observed in liver and kidney LDH levels between pyridoxine-deficient rats (group VI) and their pair-fed controls (group V).
Discussion Hyperoxaluria is the common observation in all these vitamin deficiencies, yet their mode of action on oxalate metabolism seems to be quite different. The increased activity of some of the oxalate-biosynthesizing enzymes in vitamin B| deficiency can be attributed to a disturbance in carbohydrate and amino acid metabolism, which makes a significant con tribution towards the oxalate pool in the body. It has been postulated that glycine and presumably other amino acids are being oxi dized for energy by thiamine-deficient rats [20] leading to increased glyoxylate formation which can induce synthesis of GAO to pro duce less harmful oxalate. Also, previous studies from our laboratory [21] demon strated that the activity of a-ketoglutarate, glyoxylate carboligase, a thiamine-pyrophosphate-dependent enzyme, is significantly de creased in thiamine deficiency, thus blocking the conversion of glyoxylate to COj, whereas excess glyoxylate is converted to oxalate by LDH and GAD or excreted as such. In thiamine-deficient animals, GAD does not play any significant role, because glyoxy-
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Fig. 3. Intestinal uptake o f 14C-oxalate in vitaminA-deficient ( ■ ) . vitamin-Br deficient (B ), vitaminB(,-deficient ( ^ ) and pair-fed control ( □ ) rats. Values are means ± SEM of 8-10 observations. The intesti nal rings were incubated in 5.0 ml o f oxygenated buffer containing 0.5 mM of potassium oxalate along with 0.1 pCi/ml o f (U -l4C)oxalate.
Intestinal Absorption o f Oxalate The results (fig. 3) showed a significant increase (p < 0.01) in the uptake of oxalate by intestinal rings in the vitamin-A-deficient group as compared to pair-fed controls. The intestinal uptake of oxalate remained unal tered in the vitamin-B |-deficient group, while vitamin-B6-deficient animals exhib ited a net increase in the oxalate uptake at 0.5 mM oxalate concentration (0.39 ± 0.02 pmol/h/g tissue in B6-deficient animals and 0.26 ± 0.02 pmol/h/g tissue in pair-fed control rats).
late is a potent inhibitor of GAD [14. 15]. The unaltered behaviour of the kidney and liver LDH corroborates well with the find ings of other workers [22, 23]. These obser vations lead to the conclusion that hyperox aluria observed in thiamine deficiency is of endogenous origin with little contribution from dietary sources as indicated by similar intestinal oxalate uptake rates in both thia mine-deficient and pair-fed control groups. Significant hyperoxaluria in pyridoxine deficiency has been reported from several laboratories [24-28]. The increase in the liver GAO levels in vitamin B6 deficiency also suggests that hyperoxaluria in this defi ciency is due to an influence of this vitamin on oxalate metabolism in the liver thus sup porting earlier observations [29]. Pyridoxal phosphate is known to be a cytoplasmic mo dulator of nuclear steroid translocation [3032], while the number and metabolism of peroxisomes is under the control of sex hor mones [33]. Thus, as increased translocation of androgen to nuclei in pyridoxine defi ciency causes an increase in the genesis and metabolism of peroxisomes which contain glycolate oxidase suggesting that pyridoxine regulates the oxalate metabolism via steroid hormones. The unaltered levels of kidney and liver LDH in vitamin B6 deficiency indi cate its less important role in oxalate biosyn thesis. Unlike thiamine deficiency, dietary oxa late is of considerable importance in pro moting hyperoxaluria in vitamin Bfe defi ciency. The alterations in the uptake rates of oxalate in this disorder are attributable to the induction of a new biphasic transport carrier which facilitates its passage across the enterocyte microvillus membrane [34], Though a co-enzyme function of vitamin A, unlike that of many water-soluble vita
109
mins, is not known, it is certain to regulate the activity of a number of enzymes of pro tein and vitamin A metabolism [35]. The accumulation of glycine, tyrosine and trypto phan in vitamin A deficiency, which are im portant endogenous precursors of oxalate, may induce the activity of GAO and GAD. Since vitamin A most probably fulfils multi ple functions at the molecular level, it might be difficult to find a single common mecha nism for the biochemical mode of action of vitamin A deficiency on the hepatic synthe sis of oxalate. The hyperabsorption of oxa late and stimulation of GAO and GAD in vitamin A deficiency may inhibit the activity of kidney LDH, since some iso-enzymes of LDH are known to be sensitive to the higher concentration of its product [36]. The changes in the absorption pattern of oxalate in hypovitaminosis A could be due to the known effect of vitamin A on biologi cal membranes [37, 38]. The low content of the dry matter and physical condition of the small intestine in vitamin-A-deficient ani mals appeared to be a reflection of changes in the thickness of the intestinal wall which may affect the in vitro transfer of various nutrients by the intestine [39]. Thus, the present study emphasizes the role of vitamin A, Bj and B6 deficiencies which may increase oxalate biosynthesis leading to hyperoxaluria. The data suggest that the severity of deficiency required for hyperoxaluria to develop is greater in vita min A and B| deficiencies than in vitamin B6 deficiency. Hyperabsorption of oxalate is an additional factor contributing to urinary ox alate in vitamin A and B6 deficiency, while in thiamine deficiency hyperoxaluria is only of endogenous origin. In the endemic under nourished regions, it is difficult to confine the deficiency to a particualr vitamin; there
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Effect of Vitamin Deficiencies on Oxalate Metabolism
110
fore, overlapping vitamin A. B) and B6 defi ciencies can sufficiently alter the oxalate me tabolism to produce marked hyperoxaluria which is one of the important causative fac tors for calculogenesis.
References 1 Howard JE, Thomas WC: Control of crystalliza tion in urine. Am J Med 1968;45:693-699. 2 Williams HE. Smith LH. Jr: A new genetic variant of primary' hyperoxaluria. N Engl J Med 1968; 278:233-238. 3 Nath R. Thind SK. Murthy MSR. Talwar HS. Farooqui S: Molecular aspects o f idiopathic uro lithiasis. Mol Aspects Med 1984;7:1-176. 4 Anasuya A: Nutritional factors in urolithiasis: Re cent studies; in Singh PP, Pendse AP (eds): Mul tidimensional Approach to Urolithiasis. Udaipur, Agrawal Printers. 1987. pp 99-109. 5 Hagler L. Herman RH: Oxalate metabolism I-V. Am J Clin Nutr 1973:26:758-765, 882-889, 1006-1010, 1073-1079, 1242-1250. 6 Broadus AE, Horst RL. Littledike ET. MahafTey JE, Rasmussen H: Primary hyperparathyroidism with intermittent hypercalcemia: Serial observa tions and simple diagnosis by means o f oral cal cium tolerance test. Clin Endocrinol 1980:15: 225-245. 7 Murthy MSR. Talwar HS, Thind SK. Nath R. Rajendran L, Bapna BC: Effect o f pyridoxine sup plementation on recurrent stone formers. Tnt J Clin Pharmacol Ther Toxicol 1982:20:434-437. 8 Lakshmanan MR. Jungalwala FB. Cama HR: Me tabolism and biological potency o f 5,6-monoepoxy vitamin A aldehyde in the rat. Biochem J 1965:95:27-34. 9 Thomas MR. Kirksey A: Influence o f pyridoxine supplementation on vitamin B6 levels in milk of rats deficient in vitamin. J Nutr 1976; 106:509— 514. 10 Hegested DM. Mills RC, Elvehjam CA. Hart EB: Choline in the nutrition of chicks. J Biol Chem 1941;138:459-466. 11 Hodgkinson A, Williams A: An improved colori metric procedure for urinary oxalate. Clin Chim Acta 1972:36:127-132.
12 Dugan RE, Frigerio NA, Siebert JM: Colorimetric determination of vitamin A and its derivatives with trifluoroacetic acid. Anal Chem 1964:36: 114-117. 13 Boni L, Kieckens L, Hendrikx A: An evaluation of a modified erythrocyte transketolase assay for as sessing thiamine nutritional adequacy. J Nutr Sci Vitaminol 1980:26:507-514. 14 Kishi H, Folkares K: Improved and effective as say of the glutamic oxaloacetic transaminase by the coenzyme apoenzyme system (CAS) principle. J. Nutr Sci Vitaminol 1976;22:225-234. 15 Fry DW, Richardson KE: Isolation and character ization o f glycolic acid dehydrogenase from hu man liver. Biochim Biophys Acta 1979:567:482— 491. 16 Fry DW. Richardson KE: Isolation and character ization o f glycolic acid oxidase from human liver. Biochim Biophys Acta 1979:568:135-144. 17 Liao LL, Richardson KE: Inhibition of oxalate biosynthesis in isolated perfused liver by DL-phenyl lactate and n-heptonate. Arch Biochem Bio phys 1973;154:68-75. 18 Alvardo F, Mahmood A: Cotransport of organic solutes and sodium ions in the small intestine. A general model: Amino acid transport. Biochemis try- 1974:13:2882-2890. 19 Lowry OH. Rosebrough NJ. Farr AL. Randall RS: Protein measurement with the Folin phenol re agent. J Biol Chem 1951;193:265-275. 20 Liang CC: Alternative metabolic pathway of rats suffering from thiamine deficiency. J Nutr Sci Vitaminol 1976:22(suppl):47-50. 21 Sidhu H. Gupta R. Thind SK, Nath R: Oxalate metabolism in thiamine-deficient rats. Ann Nutr Metab 1987;31:354-361. 22 Gubler CJ: Enzyme studies in thiamine deficien cy. Int J Vitam Nutr Res 1968:38:287-303. 23 Park DW, Gubler CJ: Studies on the physiological functions o f thiamine deficiency. 5. Effects o f thi amine deprivation and thiamine antagonists on blood pyruvate and lactate and activity of lactate dehydrogenase and its isoenzymes in blood and tissues. Biochim Biophys Acta 1969; 177:537— 543. 24 Faber SR, Feitter WW, Bleiler RE. Ohlson MA. Hodges RE: The effects o f an induced pyridoxine and pantothenic acid deficiency on excretion of oxalic acid and xanthurenic acid in the urine. Am J Clin Nutr 1963:12:406-412.
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Effect o f Vitamin Deficiencies on Oxalate Metabolism
35 Bondi A. Dror Y, Nir I: Effect o f vitamin A defi ciency on some enzymes involved in protein and vitamin A metabolism. Nutr Metab 1982; 15:246251. 36 Thind SK, Nath R: Lactate dehydrogenase isoen zymes in the rat kidney and bladder in experimen tal urolithiasis. Int Res Commun Ser Med Sci 1977;5:429. 37 DeLuca LM. Bhat PV, Sasak W, Adamo S: Bio synthesis o f phosphoryl and glycosyl phosphoryl derivatives of vitamin A in biological membranes. Fed Proc 1979:38:2535-2539. 38 Stillwell W, Ricketts M: Effects of trans-retinol on the permeability of egg lecithin liposomes. Bio chem Biophys Res Commun 1980;97:148-153. 39 Varnell TR: Effect of avitaminosis A and dietaryregime on intestinal transfer and hepatic concen tration o f free amino acids. J Vitam Nutr Res 1972;42:271-277.
Received: September 22, 1989 Accepted: November 10. 1989 Prof. Ravindra Nath Department of Biochemistry Postgraduate Institute of Medical Education and Research Chandigarh 160012 (India)
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25 Gershoff SN: Production of urinary calculi in vitamin B6 deficient male, female and castrated male rats. J Nutr 1970; 100:117-122. 26 Hauschildt S. Rudolph R, Feldheim W: Oxalatstoffwechsel und Thiamin-Pyridoxin-Versorgung bei der Ratte. Int J Vitam Nutr Res 1972:42:457467. 27 Murthy MSR. Farooqui S. Talwar HS. Thind SK, Nath R: Effects o f pyridoxine on sodium glycolate induced hyperoxaluria in rats. Biochem Med 1982;26:77-84. 28 Sidhu H, Gupta R. Farooqui S, Thind SK, Nath R: Absorption o f glyoxylate and oxalate in thia mine and pyridoxine deficient rats. Biochem Int 1986:12:71-79. 29 Varalakshmi P. Richardson KE: The effects of vitamin B6 deficiency and hepatectomy on the synthesis o f oxalate from glycolate in the rats. Biochim Biophys Acta 1983;757:1-7. 30 Anonymous: Does pyridoxal phosphate have a non-coenzymic role in steroid hormone action? Nutr Rev 1980:38:93-95. 31 Anonymous: The function o f vitamin B
© 1990 S. Karger AG. Basel 0250-6807/90/0342-0104S2.75/0
Comparative Studies on the Effect of Vitamin A, B, and B6 Deficiency on Oxalate Metabolism in Male Rats Sadhna Sharma. Harmeet Sidhu, Ravinder Narula. Swam K. Thind, Ravindra Nath Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh, India
Key Words. Vitamin A • Vitamin B| • Vitamin B6 • Oxalate urolithiasis
Introduction Oxalic acid is a common constituent of mammalian urine. It becomes pathologically significant when present in excess and may amount to increased crystallization of cal cium stones [1,2]. Apart from endogenous oxalate, dietary oxalate is also of paramount importance, since a substantial portion of urinary oxalate is of dietary origin. There fore, the chemical composition of stones is likely to be related to the diet. Even though many pioneer workers com mented that this disease is not caused by one
single factor, nutritional factors particularly, vitamin deficiencies are unquestionably re lated to the genesis of this disease [3, 4], Experimentally, urinary lithiasis can be in duced in laboratory animals by feeding diets deficient in vitamin A, B| or B6. During the last decade, the metabolic role of these vita mins has been extensively studied [5]. Vary ing degrees of clinical and subclinical defi ciency of vitamin A and B6 have been re ported in patients having urinary calculi [4, 6, 7], However, the complete picture of pathological processes which take place in the body as a result of a lack of these nu
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Abstract. The study was conducted to investigate the effect of vitamin A, B, and B6 defi ciency on oxalate metabolism in rats. A significant hyperoxaluria was the common observa tion in all the three vitamin deficiencies (vitamin B6 > vitamin A > vitamin B|). The activities of hepatic glycolate oxidase and glycolate dehydrogenase were markedly enhanced in vitamin-A- and vitamin-B(,-deficient rats. However, lactate dehydrogenase levels re mained unaltered in these deficiencies as compared to their respective pair-fed controls. Vitamin B| deficiency of 4 weeks’ duration could augment the activity of glycolate oxidase only, with no alterations in the glycolate dehydrogenase and lactate dehydrogenase levels. Intestinal oxalate uptake studies revealed increased bio-availability of oxalate from the gut in vitamin-A- and vitamin-B6-deficient rats. Thus, the results suggest the relative contribution of both exogenous as well as endogenous oxalate in the process of calculogenesis under various nutritional stress conditions in rat.
105
Effect of Vitamin Deficiencies on Oxalate Metabolism
Materials and Methods Male Wistar rats (40-50 g body weight) were ac climatized for 5-6 days and were randomly divided into six groups of 8 rats each. The animals were fed their respective dietary regimen for a specified exper imental period: Group I - fed vitamin-A-deficient diet [8] ad libitum for 5 weeks; group II = pair-fed with group I + 150 IU retinyl acetate/rat/day by gas tric intubation; group III = fed vitamin-Br deficient diet [9] ad libitum for 4 weeks; group IV = pair-fed with group III + 100 gg thiamine HCl/rat/day by gas tric intubation; group V= fed vitamin-B6-deficient diet [10] ad libitum for 4 weeks; group VI - pair-fed with group V + 100 pg pyridoxine HCl/rat/day by gas tric intubation. The composition of various experimental diets is given in table 1. Food consumption and weight gain of individual rats were measured every alternate day. At the end of the experimental period, clinical symp toms of vitamin deficiencies became apparent, i.e. scaly skin, loss o f body weight in the vitamin-A-defi cient. acrodynia and alopecia in the vitamin-B6-deficient and anorexia and loss o f weight in the vitaminBr deficient groups. Before sacrificing, the rats were individually placed in metabolic cages with free ac cess to water to collect 24-hour urine samples which were analysed for oxalate [11]. The animals were sacrificed under light ether anaesthesia. The vitamin A status (groups 1 and II) was ascertained biochemically by estimating hepatic vitamin A content [12], The vitamin B, (groups III and IV) and vitamin B6 (groups V and VI) status o f animals was assessed by measuring the haemolvsates for erythrocyte transketolase [13] and erythrocyte ala nine transaminase [14], respectively. Oxalate-Biosynthesizing Enzymes The activities o f various oxalate-biosynthesizing enzymes, i.e. glycolate oxidase (GAO) [ 15], glycolate
Table 1. Composition (g/100 g diet) o f experimen tal diets Ingredienls
Vitamin-Adeficient diet
Casein 20.0 (fat-free) Starch 38.8 Sucrose 30.0 Vitamin mixture 2.2 (deficient in the respective vitamin) Salt mixture 4.0 Ground nut oil 5.0
Vitamin-B|deficient diet 25.0 28.7 23.0 2.2 6.1 15.0
The composition of the vitamin-B6-deficient diet was essentially the same as that o f the vitamin-Br deficient diet except in vitamin mixture; vitamin B6 was deleted and replaced by B|. The casein was obtained commercially from Johnson and Johnson Co.. Bombay, and was made free o f vitamin B| and B^ by repeated washings with ethyl alcohol and water. For the vitamin-A-deficient diet, fat-free casein was obtained by repeated washings initially with water, then acetone, and by drying it at 60 °C. The vitamin mixture was prepared as described in the ICN cata logue (nutritional, biochemical division o f ICN. Life Sciences Group. Cleveland. Ohio, USA. The salt mix ture was prepared as described by Hegested et al.
no].
dehydrogenase (GAD) [16] and lactate dehydroge nase (LDH) [17], were assessed in the postmitochondrial supernatants of 10% w/v tissue homogenates prepared in 0.01 M potassium phosphate buffer, pH 7.1. GAO and LDH were assayed in the liver, while LDH was assayed also in the kidneys. Intestinal Transport o f Oxalate Starting from the ligament of Treitz, an approxi mately 20-cm portion of gut was removed and flushed with ice-cold oxygenated Krebs-Ringer buffer (KRB) containing 140 mM NaCl. 6.0 mM KC1 and 4.0 mM Tris-HCl buffer, pH 7.4. The flushed intestine was everted, and 0.3- to 0.5-cm segments were cut and immersed in oxygenated KRB at 4 °C. The intestinal uptake of oxalate was measured using everted intesti nal rings by the tissue accumulation method using
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trients and which ultimately lead to urinary lithiasis is not clear. The present work has been conducted to assess the effect of vita min A, B| or B6 deficiency in rats on the endogenous biosynthesis of oxalate, its intes tinal absorption and subsequent excretion in the urine.
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Table 2. Biochemical assessment o f vitamin A, B, and B6 deficiency in rats Hepatic vitamin A IU/g tissue weight Group I Group II
u C-oxaIate [18]. The extracellular space was mea sured separately by incubating the tissues in the pres ence of ’H-inulin and was subtracted from all uptake values. Protein was estimated in all the tissues by the method of Lowry et ai. [ 19], Student’s t test was used 144.43 ± 11.41 for evaluating the results statistically. 2.76 ±0.10*
Erythrocyte transketolase U/mg protein Group III Group IV
Results 0.32 + 0.02 0 .11 ±0.01*
After the specified experimental period, group II animals exhibited about 95-96% Erythrocyte alanine transaminase depletion of hepatic vitamin A reserves as U/mg protein X 10'6 compared with their respective pair-fed con Group V 46.02 ±2.31 trols, thus indicating the induction of vita Group VI 12.93±1.01* min A deficiency. Similarly, the biochemical assessment of thiamine and pyridoxine defi All values are means ± SEM of 6 -8 animals. One ciency revealed a significant decrease in international unit of vitamin A is described as equiv erythrocyte transketolase (p < 0.001) and alent to 0.344 pg retinyl acetate. One unit o f transke tolase is defined as I pg o f ribose-5-phosphate metab erythrocyte alanine transaminase (p < olized per minute at 37 °C. One unit o f alanine trans 0.001) activity as compared with their re aminase is defined as 1 pmol o f pyruvate formed per spective pair-fed control rats, thus providing minute at 37 °C. * p < 0.001 as compared to pair-fed evidence for the production of these defi controls. ciencies in rats (table 2).
Table 3. Effect o f vitamin A, B| or B6 deficiency on body, liver and kidney weights o f rats Parameter
Pair-fed controls
Vitamin-Adeficient rats
Body weight, g
143.25 ± 5.65
114.25 ±3.00* 96.83 ±2.05
Liver weight, g
3.88 ± 0.19
3.49 ± 0 .1 2
4.12 ±0.18
3.24 ±0.24*
4.78 ±0.23
4.16 ±0.26
Liver weight, 3.49 ±0.12 g/100 g body weight
3 .7 6 ± 0 .1 0
4.42 ±0.21
4.28 ±0.31
4.73 ± 0.20
4.75 ±0.28
Kidney weight, g
1.12 ± 0.02
1.21 ±0.02
0.84 ±0.03
0.75 ±0.02
0.92 ±0.03
0.85 ±0.03
Kidney weight, 1.15 ± 0.03 g/100 g body weight
1.31 ± 0 .0 4
0.88 ±0.05
0.94 ±0.04
0.91 ±0.03
0.99 ±0.02
Vitamin-Bideficient rats
Pair-fed controls
76.76 ±2.89** 100.63 ±3.77
Vitamin-B*deficient rats 85.78±3.46*
All values are means ± SEM of 6 -8 animals. ** p < 0.001, * p < 0.05 as compared to pair-fed controis.
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Pair-fed controls
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Effect of Vitamin Deficiencies on Oxalate Metabolism
Urinary Excretion o f Oxalate A significant decrease in body weights of animals was observed under all vitamindeficient conditions; however, no statisti cally significant changes were observed in the kidney and liver weights (table 3). Rats deficient in vitamin A, B| or B6 pro duced a significant increase of variable de grees in the 24-hour urinary oxalate excre tion (fig. 1). The maximum hyperoxaluria was observed in vitamin-B6-deficient rats (approx. 2-fold) followed by a 1.83-fold in crease in the vitamin-A-deficient group and about 1.34-fold in thiamine-deficient rats as compared with their pair-fed controls. Endogenous Biosynthesis o f Oxalate The specific activity of major oxalate-biosynthesizing enzymes, i.e. GAO and GAD,
Fig. I. Urinary excretion of oxalate in vitamin-Adeficient (■ ). vitamin-B|-deficient ( 0 ) . vitamin-B6deficient (g|) and pair-fed control (Q ) rats. Values are means ± SEM o f 8-10 animals.
o o Ö
A
B,
B6
A
B,
B6
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Fig. 2. Activities of GAO (a). GAD (b) and LDH (c, d) in vitaminA-deficient ( ■ ) , vitamin-Bi-deficient ( 0 ) . vitamin-B6-deficient ( ^ ) and pair-fed control ( □ ) rats. All values are means ± SEM o f 8 -1 0 animals. One unit of GAO is de fined as I nmol o f glvoxylatc pro duced per minute at 37 °C. One unit o f GAD is defined as 1 nmol of oxalate produced per minute at 37 °C. One unit of LDH is defined as change in 0 .1 optical density per minute at 340 nm at 25 °C.
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was significantly increased in the vitamin-Adeficient group as compared to pair-fed con trols (fig. 2). The liver LDH levels remained unaltered in this deficiency, whereas kidney LDH activity was significantly decreased (p < 0.01) in this nutritional disorder. Vitamin B| deficiency of 4 weeks’ dura tion resulted in a significant increase (p < 0.001) in the activity of liver GAO with no change in the activity of GAD. The activities of both liver as well as kidney LDH re mained unaltered under this nutritional dis order (fig. 2). A significant increase in the liver GAO (p < 0.001) and GAD (p < 0.01) was ob served in the vitamin-B6-deficient group compared to pair-fed controls. No differ ences were observed in liver and kidney LDH levels between pyridoxine-deficient rats (group VI) and their pair-fed controls (group V).
Discussion Hyperoxaluria is the common observation in all these vitamin deficiencies, yet their mode of action on oxalate metabolism seems to be quite different. The increased activity of some of the oxalate-biosynthesizing enzymes in vitamin B| deficiency can be attributed to a disturbance in carbohydrate and amino acid metabolism, which makes a significant con tribution towards the oxalate pool in the body. It has been postulated that glycine and presumably other amino acids are being oxi dized for energy by thiamine-deficient rats [20] leading to increased glyoxylate formation which can induce synthesis of GAO to pro duce less harmful oxalate. Also, previous studies from our laboratory [21] demon strated that the activity of a-ketoglutarate, glyoxylate carboligase, a thiamine-pyrophosphate-dependent enzyme, is significantly de creased in thiamine deficiency, thus blocking the conversion of glyoxylate to COj, whereas excess glyoxylate is converted to oxalate by LDH and GAD or excreted as such. In thiamine-deficient animals, GAD does not play any significant role, because glyoxy-
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Fig. 3. Intestinal uptake o f 14C-oxalate in vitaminA-deficient ( ■ ) . vitamin-Br deficient (B ), vitaminB(,-deficient ( ^ ) and pair-fed control ( □ ) rats. Values are means ± SEM of 8-10 observations. The intesti nal rings were incubated in 5.0 ml o f oxygenated buffer containing 0.5 mM of potassium oxalate along with 0.1 pCi/ml o f (U -l4C)oxalate.
Intestinal Absorption o f Oxalate The results (fig. 3) showed a significant increase (p < 0.01) in the uptake of oxalate by intestinal rings in the vitamin-A-deficient group as compared to pair-fed controls. The intestinal uptake of oxalate remained unal tered in the vitamin-B |-deficient group, while vitamin-B6-deficient animals exhib ited a net increase in the oxalate uptake at 0.5 mM oxalate concentration (0.39 ± 0.02 pmol/h/g tissue in B6-deficient animals and 0.26 ± 0.02 pmol/h/g tissue in pair-fed control rats).
late is a potent inhibitor of GAD [14. 15]. The unaltered behaviour of the kidney and liver LDH corroborates well with the find ings of other workers [22, 23]. These obser vations lead to the conclusion that hyperox aluria observed in thiamine deficiency is of endogenous origin with little contribution from dietary sources as indicated by similar intestinal oxalate uptake rates in both thia mine-deficient and pair-fed control groups. Significant hyperoxaluria in pyridoxine deficiency has been reported from several laboratories [24-28]. The increase in the liver GAO levels in vitamin B6 deficiency also suggests that hyperoxaluria in this defi ciency is due to an influence of this vitamin on oxalate metabolism in the liver thus sup porting earlier observations [29]. Pyridoxal phosphate is known to be a cytoplasmic mo dulator of nuclear steroid translocation [3032], while the number and metabolism of peroxisomes is under the control of sex hor mones [33]. Thus, as increased translocation of androgen to nuclei in pyridoxine defi ciency causes an increase in the genesis and metabolism of peroxisomes which contain glycolate oxidase suggesting that pyridoxine regulates the oxalate metabolism via steroid hormones. The unaltered levels of kidney and liver LDH in vitamin B6 deficiency indi cate its less important role in oxalate biosyn thesis. Unlike thiamine deficiency, dietary oxa late is of considerable importance in pro moting hyperoxaluria in vitamin Bfe defi ciency. The alterations in the uptake rates of oxalate in this disorder are attributable to the induction of a new biphasic transport carrier which facilitates its passage across the enterocyte microvillus membrane [34], Though a co-enzyme function of vitamin A, unlike that of many water-soluble vita
109
mins, is not known, it is certain to regulate the activity of a number of enzymes of pro tein and vitamin A metabolism [35]. The accumulation of glycine, tyrosine and trypto phan in vitamin A deficiency, which are im portant endogenous precursors of oxalate, may induce the activity of GAO and GAD. Since vitamin A most probably fulfils multi ple functions at the molecular level, it might be difficult to find a single common mecha nism for the biochemical mode of action of vitamin A deficiency on the hepatic synthe sis of oxalate. The hyperabsorption of oxa late and stimulation of GAO and GAD in vitamin A deficiency may inhibit the activity of kidney LDH, since some iso-enzymes of LDH are known to be sensitive to the higher concentration of its product [36]. The changes in the absorption pattern of oxalate in hypovitaminosis A could be due to the known effect of vitamin A on biologi cal membranes [37, 38]. The low content of the dry matter and physical condition of the small intestine in vitamin-A-deficient ani mals appeared to be a reflection of changes in the thickness of the intestinal wall which may affect the in vitro transfer of various nutrients by the intestine [39]. Thus, the present study emphasizes the role of vitamin A, Bj and B6 deficiencies which may increase oxalate biosynthesis leading to hyperoxaluria. The data suggest that the severity of deficiency required for hyperoxaluria to develop is greater in vita min A and B| deficiencies than in vitamin B6 deficiency. Hyperabsorption of oxalate is an additional factor contributing to urinary ox alate in vitamin A and B6 deficiency, while in thiamine deficiency hyperoxaluria is only of endogenous origin. In the endemic under nourished regions, it is difficult to confine the deficiency to a particualr vitamin; there
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Effect of Vitamin Deficiencies on Oxalate Metabolism
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fore, overlapping vitamin A. B) and B6 defi ciencies can sufficiently alter the oxalate me tabolism to produce marked hyperoxaluria which is one of the important causative fac tors for calculogenesis.
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12 Dugan RE, Frigerio NA, Siebert JM: Colorimetric determination of vitamin A and its derivatives with trifluoroacetic acid. Anal Chem 1964:36: 114-117. 13 Boni L, Kieckens L, Hendrikx A: An evaluation of a modified erythrocyte transketolase assay for as sessing thiamine nutritional adequacy. J Nutr Sci Vitaminol 1980:26:507-514. 14 Kishi H, Folkares K: Improved and effective as say of the glutamic oxaloacetic transaminase by the coenzyme apoenzyme system (CAS) principle. J. Nutr Sci Vitaminol 1976;22:225-234. 15 Fry DW, Richardson KE: Isolation and character ization o f glycolic acid dehydrogenase from hu man liver. Biochim Biophys Acta 1979:567:482— 491. 16 Fry DW. Richardson KE: Isolation and character ization o f glycolic acid oxidase from human liver. Biochim Biophys Acta 1979:568:135-144. 17 Liao LL, Richardson KE: Inhibition of oxalate biosynthesis in isolated perfused liver by DL-phenyl lactate and n-heptonate. Arch Biochem Bio phys 1973;154:68-75. 18 Alvardo F, Mahmood A: Cotransport of organic solutes and sodium ions in the small intestine. A general model: Amino acid transport. Biochemis try- 1974:13:2882-2890. 19 Lowry OH. Rosebrough NJ. Farr AL. Randall RS: Protein measurement with the Folin phenol re agent. J Biol Chem 1951;193:265-275. 20 Liang CC: Alternative metabolic pathway of rats suffering from thiamine deficiency. J Nutr Sci Vitaminol 1976:22(suppl):47-50. 21 Sidhu H. Gupta R. Thind SK, Nath R: Oxalate metabolism in thiamine-deficient rats. Ann Nutr Metab 1987;31:354-361. 22 Gubler CJ: Enzyme studies in thiamine deficien cy. Int J Vitam Nutr Res 1968:38:287-303. 23 Park DW, Gubler CJ: Studies on the physiological functions o f thiamine deficiency. 5. Effects o f thi amine deprivation and thiamine antagonists on blood pyruvate and lactate and activity of lactate dehydrogenase and its isoenzymes in blood and tissues. Biochim Biophys Acta 1969; 177:537— 543. 24 Faber SR, Feitter WW, Bleiler RE. Ohlson MA. Hodges RE: The effects o f an induced pyridoxine and pantothenic acid deficiency on excretion of oxalic acid and xanthurenic acid in the urine. Am J Clin Nutr 1963:12:406-412.
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35 Bondi A. Dror Y, Nir I: Effect o f vitamin A defi ciency on some enzymes involved in protein and vitamin A metabolism. Nutr Metab 1982; 15:246251. 36 Thind SK, Nath R: Lactate dehydrogenase isoen zymes in the rat kidney and bladder in experimen tal urolithiasis. Int Res Commun Ser Med Sci 1977;5:429. 37 DeLuca LM. Bhat PV, Sasak W, Adamo S: Bio synthesis o f phosphoryl and glycosyl phosphoryl derivatives of vitamin A in biological membranes. Fed Proc 1979:38:2535-2539. 38 Stillwell W, Ricketts M: Effects of trans-retinol on the permeability of egg lecithin liposomes. Bio chem Biophys Res Commun 1980;97:148-153. 39 Varnell TR: Effect of avitaminosis A and dietaryregime on intestinal transfer and hepatic concen tration o f free amino acids. J Vitam Nutr Res 1972;42:271-277.
Received: September 22, 1989 Accepted: November 10. 1989 Prof. Ravindra Nath Department of Biochemistry Postgraduate Institute of Medical Education and Research Chandigarh 160012 (India)
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25 Gershoff SN: Production of urinary calculi in vitamin B6 deficient male, female and castrated male rats. J Nutr 1970; 100:117-122. 26 Hauschildt S. Rudolph R, Feldheim W: Oxalatstoffwechsel und Thiamin-Pyridoxin-Versorgung bei der Ratte. Int J Vitam Nutr Res 1972:42:457467. 27 Murthy MSR. Farooqui S. Talwar HS. Thind SK, Nath R: Effects o f pyridoxine on sodium glycolate induced hyperoxaluria in rats. Biochem Med 1982;26:77-84. 28 Sidhu H, Gupta R. Farooqui S, Thind SK, Nath R: Absorption o f glyoxylate and oxalate in thia mine and pyridoxine deficient rats. Biochem Int 1986:12:71-79. 29 Varalakshmi P. Richardson KE: The effects of vitamin B6 deficiency and hepatectomy on the synthesis o f oxalate from glycolate in the rats. Biochim Biophys Acta 1983;757:1-7. 30 Anonymous: Does pyridoxal phosphate have a non-coenzymic role in steroid hormone action? Nutr Rev 1980:38:93-95. 31 Anonymous: The function o f vitamin B
