Vitamins
Ascorbic Acid Effects on Vitamin D Hormone Metabolism and Binding in Guinea Pigs1 ¡COR H. SERGEEV,
YRIY P. ARKHAPCHEV
AND VLADIMIR B. SPIRICHEV
Institute of Mutation, Academy of Medical Sciences of the USSR, Moscow 109240, USSR
ABSTRACT Ascorbic acid deficiency in guinea pigs fed a vitamin D-re pie te diet caused a moderate reduction of Ca level in serum and bone; 25-hydroxycholecalciferol or 25-hydroxyergocaIciferol (25-OHD) serum concentration tended to decline; renal 25hydroxycholeealciferol-1 -hydroxylase ( 1 -OHase) activity decreased 50%; and 25-hydroxycholecalciferol-24-hydroxylase activity increased 1.6fold. Chromatin 1,25-dihydroxycholecalciferol [1,25 -(OH )2D3]receptor concentration in the intestinal mucosa decreased 20-30%, and the percentage of occupied receptors decreased from 12-15% to 6-8%. Receptor affinity for 1,25-(OH)2D3 did not change (Kd= 0.24-0.26 nmol/L, /Qz = 0.06-0.10 nmol/L), but the cooperativity coefficient decreased from 1.7 to 1.4. Vitamin C deficiency potentiated effects of vitamin D deprivation and impaired a restorative action of vitamin D. It was accompanied by a marked delay in the elevation of 25-OHD concentration in serum as well as decreased 1-OHase activity in kidneys and a lower concentration of occupied 1,25-(OH)2D3 receptors in the intestinal mucosa. The data demonstrate a critical role for ascorbic acid in vitamin D metabolism and binding. J. Nutr. 120:1185-1190, 1990. INDEXING KEY WORDS: •ascorbic acid •vitamin D metabolism •vitamin D receptor •guinea pigs
The vitamin D endocrine system includes three major metabolites, 25-hydroxycholecalciferol (25OHD3),2 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] and 24,25-dihydroxycholecalciferol [24,25-(OH)2D3](1). 25-OHÜ3is a circulating metabolite of cholecalciferol, and 1,25-(OH)2D3is its hormonal form analogous to other steroid hormones (2,3). The intestinal mucosa and bone are the primary target tissues for 1,25-(OH)2D3(1). The hormone enhances Ca absorption in the intestine and résorptionand remodeling of bone. The function and mode of action of the alternative metabolite, 24,25(OH)2D3remain a question (1, 4). Vitamin D endocrine system regulation is achieved via the change of 1,25(OH)2D3and 24,25-(OH)2D3formation rates in kidneys
(1) and binding performance of 1,25-(OH)2U3receptors (3). Ascorbic acid significantly influences steroidogenesis, but the biochemical mechanism of the action re mains unknown (5).Vitamin C deficiency in guinea pigs and humans is accompanied by characteristic changes in bone tissue to some extent similar to those of rickets (6). In view of these facts, we investigated the possible role of ascorbic acid in vitamin D metabolism and receptor binding. To this purpose we have determined serum concentrations of the main circulating vitamin D metabolite, 25-hydroxycholecalciferol or 25-hydroxyergocalciferol (25-OHD), and the activities of renal 1and 24-hydroxylases of 25-OHD3 in guinea pigs given various levels of vitamins C and D. The concentration of unoccupied and endogenously (in vivo) occupied 1,25(OH)2D3receptors in chromatin of the intestinal mucosa were measured; binding kinetics were determined as well. To assess effects of ascorbic acid on vitamin D function, the following indices were measured in guinea pigs: serum Ca concentration and alkaline phosphatase activity, active Ca transport in the small intestine, and Ca and collagen content in bone. The data obtained strongly suggest that ascorbic acid affects the vitamin D endocrine system both at the level of 1,25-(OH)2D3 formation in kidneys and its receptor binding in the target tissue.
'Preliminary results from this study were presented at the 7th Workshop on Vitamin D, Rancho Mirage, CA, 24-29 April 1988. Abbreviations: 1,25-(OH)2D3, 1,25-dihydroxy cholecalciferol; 24,25-(OH)2D3, 24,25-dihydroxycholecalciferol; 25-OHD3, 25-hydro xycholecalciferol; 25-OHD, 25-hydroxycholecalciferol or 25-hydroxyergocalciferol; 1-OHase, 25-OHD3-l-hydroxylase; 24-OHase, 25-OHD3-24-hydroxylase; EDTA, ethylenediaminetetraacetic acid; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; TPCK, L-l-tosilamide-2-phenylethylchloromethyl ketone; AP, alkaline phos phatase; PTH, parathyroid hormone; P¡,inorganic phosphate.
0022-3166/90 $3.00 ©1990 American Institute of Nutrition. Received 16 May 1989. Accepted 6 June 1990.
1185 Downloaded from https://academic.oup.com/jn/article-abstract/120/10/1185/4738505 by St Bartholomew's & the Royal London School of Medicine and Denistry user on 20 August 2018
1186
SERGEEV ET AL.
MATERIALS AND METHODS Animals and diets. Male 1.5-mo-old guinea pigs (a colony of the breeding nursery Stolbovaya, Moscow), 130-150 g body weight were used in our experiments. They were fed a vitamin D- and vitamin C-free diet containing 0.6% Ca and 0.6% P (7).3The animals were subdivided into eight groups, each including seven or eight guinea pigs. The animals in the first group were supplied with adequate vitamin D and C (+D+C); the second, only vitamin D (+D-C); the third, only vitamin C (-D+C); and the fourth, neither of these vitamins (-D-C). The animals of the fifth (-D+C) and sixth groups (-D-C) were treated with cholecalciferol for 1d, and the animals of the seventh (-D+C) and eighth (-D-C) groups were treated for 4 d prior to decapitation. The duration of the experiment was 4 wk, and all measurements were made in guinea pigs killed after this period. In all cases tissues were used in assays immediately after the decap itation of animals. Vitamin C was added in the diet of vitamin C-replete guinea pigs (groups 1, 3, 5, and 7) at a level of 0.15%. To avoid a high mortality of vitamin C-deficient guinea pigs (groups 2, 4, 6, and 8), ascorbic acid was added in their diet during the last week of the experiment at a level of 0.005% (8). Cholecalciferol was administered orally in doses of 2 ug per animal every other day (groups 1 and 2) or 2 ug each day of repletion (groups 5, 6, 7 and 8). Metabolism of 25-hydroxyvitamin D3 in kidneys. 25-OHDs metabolism was studied in vitro using renal cortex slices (7, 9). Substrate (25-hydroxycholecalciferol, Upjohn, Kalamazoo, MI) concentration in the re action mixture was saturating—4.3 umol/L plus 0.05 uCi/mL of 25-hydroxy-[26,27-3H]cholecalciferol (spe cific activity 20.6 Ci/mmol, Amersham, Bucks, En gland). Cholecalciferol metabolites, 25-OHD3, 1,25(OH)2D3and 24,25(OH)2D3,were separated by high per formance liquid chromatography (HPLC) as described elsewhere (7, 10). Briefly, preliminary purification of chloroform-methanol extracts and partial separation of cholecalciferol metabolites were performed in the stan dard HPLC column (0.46 x 25 cm) packed with the silylating mixture SIL-PREP,25-40 um (Alltech, Deerfield, IL). The elution mixture was hexane-isopropanol (80:20).The fraction containing 1,25-(OH)2D3and 24,25(OH)2D3was resolved using the column Ultrasphere ODS 5 urn (0.46 x 25 cm) (Beckman, Altex Division, Vienna, Austria) with an elution system composed of methanol-water (90:10). Receptors for l,25-(OH)2D3Ìn the intestine. Unoccu pied and in vivo occupied receptors for 1,25-(OH)2D3in the crude chromatin fraction of the intestinal mucosa were quantified by the method of Hunziker et al. (11, 12)with slight modifications. To determine the concen tration and binding kinetics of unoccupied receptors, aliquots (100 uL, 0.2-0.3 mg protein) of chromatin sus Downloaded from https://academic.oup.com/jn/article-abstract/120/10/1185/4738505 by St Bartholomew's & the Royal London School of Medicine and Denistry user on 20 August 2018
pension in TED buffer (10 mmol/L Tris-HCl, 1.5 mmol/L EDTA, 1.0 mmol/L DTT, 0.3 mmol/L PMSF, pH 7.4) were incubated for 4 h at 0-4°Cwith 1,25-dihydroxy[26,27-3H]cholecalciferol (specific activity 180 Ci/mmol, Amersham) in the absence (total binding) or presence (nonspecific binding) of a 250-fold excess of unlabeled 1,25-(OH)2D3(a kind gift from M. Uskokovic, Hoffann-La Roche, Nutley, NJ). [3H]l,2S-(OH}£>3 at in creasing (0.125-2.5 nmol/L) or saturating (2.5 nmol/L) concentrations were used for assay of the chromatin unoccupied 1,25-(OH)2D3receptor levels. To measure occupied 1,25-(OH)2D3receptors, unoccupied binding sites were blocked by preincubation of the chromatin fraction at 0-4°Cwith TPCK (200 umol/L), followed by quantification of occupied receptors by exchange incu bation with [3H]1,25-(OH)2D3 (2.5nmol/L) at 37°C for 30 min (7, 11). The hormone bound to the receptor was separated from the free ligand by the hydroxyapatite assay (11).The washed hydroxyapatite pellets were ex tracted with chloroform-methanol (1:2). Because the 1,25-(OH)2D3binding by its chromatin receptors of the chick intestinal mucosa was character ized by positive cooperativity (12), we used the Hill equation to analyze the kinetics data. The average dis sociation constant of the two binding sites [Ka]as well as the cooperativity coefficient (nH) were calculated from the Hill plot. The dissociation constant of the second binding site (/Cai)was calculated from the asymp tote of the second portion of the Scatchard curve (13), and the maximum concentration (ßmiut) was indicated by the intercept of the Scatchard plot with the x axis. Other methods. Total Ca, inorganic phosphate (P¡), and alkaline phosphatase (EC 3.1.3.1) in serum and Ca and hydroxyproline content in bone (femoral diaphyses) were measured as described elsewhere (10). Active Ca transport in the small intestine was assessed by the ability of duodenal disks to absorb 45Ca(specific activity 10 uCi/mg Ca, V/O Isotop, Moscow) in vitro (14). 25OHD serum concentration was measured by competi tive protein binding assay using the Vitamin D3 Screening Kit (Buhlmann Lab., Basel, Switzerland). Ascorbic and dehydroascorbic acid content in the liver
Composition of the diet (g/kg) was: casein (vitamin-free), 180; cornstarch, 457; glucose, 100; vegetable oil, 70; cellulose, 100; miner als, 50; vitamins, 1.0, choline-HCl, 2.0; CaCO3, 14; K2HPO4, 16; NaH2PO4-2H2O, 10. Vitamins added (g/kg) diet) were: thiamin-HCl, 0.016; riboflavin, 0.016; pyridoxine-HCl, 0.016; calcium pantothenate, 0.04; nicotinic acid, 0.10; folie acid, 0.01; D-biotin, 0.0006; cyanocobalamin, 0.00004; menadione, 0.002; DL-ct-tocopheryl acetate, 0.05; and retinyl palmitate (15,000 lu/kg of the diet). Minerals added (g/kg) diet were: KC1, 28.6, NaCl, 10.4; MgSO4, 8.95; FeSO4 7H2O, 1.61; CuSO4-5H2O, 0.039; NaF, 0.056; CoCl2 6H2O, 0.002; MnSO4 5H2O, 0.028; ZnS04-7H20, 0.22; (NH4)6Mo7O244H2O, 0.002; KI, 0.005. Vi tamin-free casein and vitamin C (L-ascorbic acid) were obtained from NPO Vitamini, Moscow; vitamin D3 (cholecalciferol) was obtained from Serva, FRG.
1187
ASCORBIC ACID AND VITAMIN D HORMONE
and kidneys was determined by the method of Roe et al. (15). Statistics. All results were analyzed by variance analysis. When statistical significance was reached, the groups (3, 5 and 7 vs. 1, and 4, 6 and 8 vs. 2, as well as paired groups 1-2,3-4, 5-6 and 7-8)were compared using the Student-Fisher t test.
p M
C-
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i ÃŽ
RESULTS Guinea pigs fed a vitamin D-depleted diet demon strated typical biochemical indices of that vitamin deficiency: 25-OHD serum concentration fell to the minimal detectable level and hypocalcemia, hypophosphatemia, increased serum alkaline phosphatase activ ity, and a significant decrease of active Ca transport in the small intestine (Tables 1 and 2, group 3 as compared to group 1) were observed. The changes in bone tissue were typical for osteoid accumulation:mineralization inhibition without collagen content alterations. These impairments were accompanied by signifi cantly enhanced 25-OHD3-1 -hydroxylase (1-OHase) and a tendency for reduced 25-OHD3-24-hydroxylase (24OHase) activities in kidneys (Table 2, group 3 vs. group 1). The concentration of occupied 1,25-(OH)2D3 recep tors in the chromatin of the intestinal mucosa fell to the minimal detectable level whereas the concentration of that hormone's unoccupied receptors tended to decline (Table 3, group 3 vs. group 1). Affinity and cooperativity coefficients of 1,25-(OH)2D3 receptors remained un changed (Table 3). Guinea pigs fed a vitamin C-depleted diet, demon strated a profound deficiency of this vitamin. The ascor bic acid concentration in the liver of vitamin C-depleted (group 2) and vitamin C-replete (group 1) guinea pigs was 13.8 ±0.7 vs. 75.5 ±23.7 ug/g and in kidneys was 12.3 ±0.7 vs. 48.8 ±15.0 ug/g; the dehydroascorbic acid content in groups 2 and 1 was 7.0 ±0.5 vs. 37.0 ±15.0 Ug/g in the liver and < 0.5 vs. 35.0 ±16.0 ug/g in kidneys. Impairments of Ca metabolism in vitamin C-deficient guinea pigs were similar to, but less marked than, those in vitamin D deficiency. Signs of impairment included a moderate hypocalcemia, a tendency for re duced Ca absorption in the small intestine and signifi cantly reduced bone mineralization (Table 1, group 2 vs. group 1). The hydroxyproline content in bone tissue was significantly but not dramatically lower, possibly be cause of the delay of collagen degradation. Vitamin C deficiency resulted in pronounced impair ments of vitamin D metabolism (Table 2, group 2 vs. group 1): 25-OHD serum concentration decreased by 33% (not significant because of the high variation); 1-OHase activity in kidneys was 49% of the control value, and 24-OHase activity increased 1.6-fold. The concentration of unoccupied 1,25-(OH)2D3 receptors in the intestinal mucosa (Table 3, group 2) dropped 20Downloaded from https://academic.oup.com/jn/article-abstract/120/10/1185/4738505 by St Bartholomew's & the Royal London School of Medicine and Denistry user on 20 August 2018
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Ascorbic Acid Effects on Vitamin D Hormone Metabolism and Binding in Guinea Pigs1 ¡COR H. SERGEEV,
YRIY P. ARKHAPCHEV
AND VLADIMIR B. SPIRICHEV
Institute of Mutation, Academy of Medical Sciences of the USSR, Moscow 109240, USSR
ABSTRACT Ascorbic acid deficiency in guinea pigs fed a vitamin D-re pie te diet caused a moderate reduction of Ca level in serum and bone; 25-hydroxycholecalciferol or 25-hydroxyergocaIciferol (25-OHD) serum concentration tended to decline; renal 25hydroxycholeealciferol-1 -hydroxylase ( 1 -OHase) activity decreased 50%; and 25-hydroxycholecalciferol-24-hydroxylase activity increased 1.6fold. Chromatin 1,25-dihydroxycholecalciferol [1,25 -(OH )2D3]receptor concentration in the intestinal mucosa decreased 20-30%, and the percentage of occupied receptors decreased from 12-15% to 6-8%. Receptor affinity for 1,25-(OH)2D3 did not change (Kd= 0.24-0.26 nmol/L, /Qz = 0.06-0.10 nmol/L), but the cooperativity coefficient decreased from 1.7 to 1.4. Vitamin C deficiency potentiated effects of vitamin D deprivation and impaired a restorative action of vitamin D. It was accompanied by a marked delay in the elevation of 25-OHD concentration in serum as well as decreased 1-OHase activity in kidneys and a lower concentration of occupied 1,25-(OH)2D3 receptors in the intestinal mucosa. The data demonstrate a critical role for ascorbic acid in vitamin D metabolism and binding. J. Nutr. 120:1185-1190, 1990. INDEXING KEY WORDS: •ascorbic acid •vitamin D metabolism •vitamin D receptor •guinea pigs
The vitamin D endocrine system includes three major metabolites, 25-hydroxycholecalciferol (25OHD3),2 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] and 24,25-dihydroxycholecalciferol [24,25-(OH)2D3](1). 25-OHÜ3is a circulating metabolite of cholecalciferol, and 1,25-(OH)2D3is its hormonal form analogous to other steroid hormones (2,3). The intestinal mucosa and bone are the primary target tissues for 1,25-(OH)2D3(1). The hormone enhances Ca absorption in the intestine and résorptionand remodeling of bone. The function and mode of action of the alternative metabolite, 24,25(OH)2D3remain a question (1, 4). Vitamin D endocrine system regulation is achieved via the change of 1,25(OH)2D3and 24,25-(OH)2D3formation rates in kidneys
(1) and binding performance of 1,25-(OH)2U3receptors (3). Ascorbic acid significantly influences steroidogenesis, but the biochemical mechanism of the action re mains unknown (5).Vitamin C deficiency in guinea pigs and humans is accompanied by characteristic changes in bone tissue to some extent similar to those of rickets (6). In view of these facts, we investigated the possible role of ascorbic acid in vitamin D metabolism and receptor binding. To this purpose we have determined serum concentrations of the main circulating vitamin D metabolite, 25-hydroxycholecalciferol or 25-hydroxyergocalciferol (25-OHD), and the activities of renal 1and 24-hydroxylases of 25-OHD3 in guinea pigs given various levels of vitamins C and D. The concentration of unoccupied and endogenously (in vivo) occupied 1,25(OH)2D3receptors in chromatin of the intestinal mucosa were measured; binding kinetics were determined as well. To assess effects of ascorbic acid on vitamin D function, the following indices were measured in guinea pigs: serum Ca concentration and alkaline phosphatase activity, active Ca transport in the small intestine, and Ca and collagen content in bone. The data obtained strongly suggest that ascorbic acid affects the vitamin D endocrine system both at the level of 1,25-(OH)2D3 formation in kidneys and its receptor binding in the target tissue.
'Preliminary results from this study were presented at the 7th Workshop on Vitamin D, Rancho Mirage, CA, 24-29 April 1988. Abbreviations: 1,25-(OH)2D3, 1,25-dihydroxy cholecalciferol; 24,25-(OH)2D3, 24,25-dihydroxycholecalciferol; 25-OHD3, 25-hydro xycholecalciferol; 25-OHD, 25-hydroxycholecalciferol or 25-hydroxyergocalciferol; 1-OHase, 25-OHD3-l-hydroxylase; 24-OHase, 25-OHD3-24-hydroxylase; EDTA, ethylenediaminetetraacetic acid; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; TPCK, L-l-tosilamide-2-phenylethylchloromethyl ketone; AP, alkaline phos phatase; PTH, parathyroid hormone; P¡,inorganic phosphate.
0022-3166/90 $3.00 ©1990 American Institute of Nutrition. Received 16 May 1989. Accepted 6 June 1990.
1185 Downloaded from https://academic.oup.com/jn/article-abstract/120/10/1185/4738505 by St Bartholomew's & the Royal London School of Medicine and Denistry user on 20 August 2018
1186
SERGEEV ET AL.
MATERIALS AND METHODS Animals and diets. Male 1.5-mo-old guinea pigs (a colony of the breeding nursery Stolbovaya, Moscow), 130-150 g body weight were used in our experiments. They were fed a vitamin D- and vitamin C-free diet containing 0.6% Ca and 0.6% P (7).3The animals were subdivided into eight groups, each including seven or eight guinea pigs. The animals in the first group were supplied with adequate vitamin D and C (+D+C); the second, only vitamin D (+D-C); the third, only vitamin C (-D+C); and the fourth, neither of these vitamins (-D-C). The animals of the fifth (-D+C) and sixth groups (-D-C) were treated with cholecalciferol for 1d, and the animals of the seventh (-D+C) and eighth (-D-C) groups were treated for 4 d prior to decapitation. The duration of the experiment was 4 wk, and all measurements were made in guinea pigs killed after this period. In all cases tissues were used in assays immediately after the decap itation of animals. Vitamin C was added in the diet of vitamin C-replete guinea pigs (groups 1, 3, 5, and 7) at a level of 0.15%. To avoid a high mortality of vitamin C-deficient guinea pigs (groups 2, 4, 6, and 8), ascorbic acid was added in their diet during the last week of the experiment at a level of 0.005% (8). Cholecalciferol was administered orally in doses of 2 ug per animal every other day (groups 1 and 2) or 2 ug each day of repletion (groups 5, 6, 7 and 8). Metabolism of 25-hydroxyvitamin D3 in kidneys. 25-OHDs metabolism was studied in vitro using renal cortex slices (7, 9). Substrate (25-hydroxycholecalciferol, Upjohn, Kalamazoo, MI) concentration in the re action mixture was saturating—4.3 umol/L plus 0.05 uCi/mL of 25-hydroxy-[26,27-3H]cholecalciferol (spe cific activity 20.6 Ci/mmol, Amersham, Bucks, En gland). Cholecalciferol metabolites, 25-OHD3, 1,25(OH)2D3and 24,25(OH)2D3,were separated by high per formance liquid chromatography (HPLC) as described elsewhere (7, 10). Briefly, preliminary purification of chloroform-methanol extracts and partial separation of cholecalciferol metabolites were performed in the stan dard HPLC column (0.46 x 25 cm) packed with the silylating mixture SIL-PREP,25-40 um (Alltech, Deerfield, IL). The elution mixture was hexane-isopropanol (80:20).The fraction containing 1,25-(OH)2D3and 24,25(OH)2D3was resolved using the column Ultrasphere ODS 5 urn (0.46 x 25 cm) (Beckman, Altex Division, Vienna, Austria) with an elution system composed of methanol-water (90:10). Receptors for l,25-(OH)2D3Ìn the intestine. Unoccu pied and in vivo occupied receptors for 1,25-(OH)2D3in the crude chromatin fraction of the intestinal mucosa were quantified by the method of Hunziker et al. (11, 12)with slight modifications. To determine the concen tration and binding kinetics of unoccupied receptors, aliquots (100 uL, 0.2-0.3 mg protein) of chromatin sus Downloaded from https://academic.oup.com/jn/article-abstract/120/10/1185/4738505 by St Bartholomew's & the Royal London School of Medicine and Denistry user on 20 August 2018
pension in TED buffer (10 mmol/L Tris-HCl, 1.5 mmol/L EDTA, 1.0 mmol/L DTT, 0.3 mmol/L PMSF, pH 7.4) were incubated for 4 h at 0-4°Cwith 1,25-dihydroxy[26,27-3H]cholecalciferol (specific activity 180 Ci/mmol, Amersham) in the absence (total binding) or presence (nonspecific binding) of a 250-fold excess of unlabeled 1,25-(OH)2D3(a kind gift from M. Uskokovic, Hoffann-La Roche, Nutley, NJ). [3H]l,2S-(OH}£>3 at in creasing (0.125-2.5 nmol/L) or saturating (2.5 nmol/L) concentrations were used for assay of the chromatin unoccupied 1,25-(OH)2D3receptor levels. To measure occupied 1,25-(OH)2D3receptors, unoccupied binding sites were blocked by preincubation of the chromatin fraction at 0-4°Cwith TPCK (200 umol/L), followed by quantification of occupied receptors by exchange incu bation with [3H]1,25-(OH)2D3 (2.5nmol/L) at 37°C for 30 min (7, 11). The hormone bound to the receptor was separated from the free ligand by the hydroxyapatite assay (11).The washed hydroxyapatite pellets were ex tracted with chloroform-methanol (1:2). Because the 1,25-(OH)2D3binding by its chromatin receptors of the chick intestinal mucosa was character ized by positive cooperativity (12), we used the Hill equation to analyze the kinetics data. The average dis sociation constant of the two binding sites [Ka]as well as the cooperativity coefficient (nH) were calculated from the Hill plot. The dissociation constant of the second binding site (/Cai)was calculated from the asymp tote of the second portion of the Scatchard curve (13), and the maximum concentration (ßmiut) was indicated by the intercept of the Scatchard plot with the x axis. Other methods. Total Ca, inorganic phosphate (P¡), and alkaline phosphatase (EC 3.1.3.1) in serum and Ca and hydroxyproline content in bone (femoral diaphyses) were measured as described elsewhere (10). Active Ca transport in the small intestine was assessed by the ability of duodenal disks to absorb 45Ca(specific activity 10 uCi/mg Ca, V/O Isotop, Moscow) in vitro (14). 25OHD serum concentration was measured by competi tive protein binding assay using the Vitamin D3 Screening Kit (Buhlmann Lab., Basel, Switzerland). Ascorbic and dehydroascorbic acid content in the liver
Composition of the diet (g/kg) was: casein (vitamin-free), 180; cornstarch, 457; glucose, 100; vegetable oil, 70; cellulose, 100; miner als, 50; vitamins, 1.0, choline-HCl, 2.0; CaCO3, 14; K2HPO4, 16; NaH2PO4-2H2O, 10. Vitamins added (g/kg) diet) were: thiamin-HCl, 0.016; riboflavin, 0.016; pyridoxine-HCl, 0.016; calcium pantothenate, 0.04; nicotinic acid, 0.10; folie acid, 0.01; D-biotin, 0.0006; cyanocobalamin, 0.00004; menadione, 0.002; DL-ct-tocopheryl acetate, 0.05; and retinyl palmitate (15,000 lu/kg of the diet). Minerals added (g/kg) diet were: KC1, 28.6, NaCl, 10.4; MgSO4, 8.95; FeSO4 7H2O, 1.61; CuSO4-5H2O, 0.039; NaF, 0.056; CoCl2 6H2O, 0.002; MnSO4 5H2O, 0.028; ZnS04-7H20, 0.22; (NH4)6Mo7O244H2O, 0.002; KI, 0.005. Vi tamin-free casein and vitamin C (L-ascorbic acid) were obtained from NPO Vitamini, Moscow; vitamin D3 (cholecalciferol) was obtained from Serva, FRG.
1187
ASCORBIC ACID AND VITAMIN D HORMONE
and kidneys was determined by the method of Roe et al. (15). Statistics. All results were analyzed by variance analysis. When statistical significance was reached, the groups (3, 5 and 7 vs. 1, and 4, 6 and 8 vs. 2, as well as paired groups 1-2,3-4, 5-6 and 7-8)were compared using the Student-Fisher t test.
p M
C-
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i ÃŽ
RESULTS Guinea pigs fed a vitamin D-depleted diet demon strated typical biochemical indices of that vitamin deficiency: 25-OHD serum concentration fell to the minimal detectable level and hypocalcemia, hypophosphatemia, increased serum alkaline phosphatase activ ity, and a significant decrease of active Ca transport in the small intestine (Tables 1 and 2, group 3 as compared to group 1) were observed. The changes in bone tissue were typical for osteoid accumulation:mineralization inhibition without collagen content alterations. These impairments were accompanied by signifi cantly enhanced 25-OHD3-1 -hydroxylase (1-OHase) and a tendency for reduced 25-OHD3-24-hydroxylase (24OHase) activities in kidneys (Table 2, group 3 vs. group 1). The concentration of occupied 1,25-(OH)2D3 recep tors in the chromatin of the intestinal mucosa fell to the minimal detectable level whereas the concentration of that hormone's unoccupied receptors tended to decline (Table 3, group 3 vs. group 1). Affinity and cooperativity coefficients of 1,25-(OH)2D3 receptors remained un changed (Table 3). Guinea pigs fed a vitamin C-depleted diet, demon strated a profound deficiency of this vitamin. The ascor bic acid concentration in the liver of vitamin C-depleted (group 2) and vitamin C-replete (group 1) guinea pigs was 13.8 ±0.7 vs. 75.5 ±23.7 ug/g and in kidneys was 12.3 ±0.7 vs. 48.8 ±15.0 ug/g; the dehydroascorbic acid content in groups 2 and 1 was 7.0 ±0.5 vs. 37.0 ±15.0 Ug/g in the liver and < 0.5 vs. 35.0 ±16.0 ug/g in kidneys. Impairments of Ca metabolism in vitamin C-deficient guinea pigs were similar to, but less marked than, those in vitamin D deficiency. Signs of impairment included a moderate hypocalcemia, a tendency for re duced Ca absorption in the small intestine and signifi cantly reduced bone mineralization (Table 1, group 2 vs. group 1). The hydroxyproline content in bone tissue was significantly but not dramatically lower, possibly be cause of the delay of collagen degradation. Vitamin C deficiency resulted in pronounced impair ments of vitamin D metabolism (Table 2, group 2 vs. group 1): 25-OHD serum concentration decreased by 33% (not significant because of the high variation); 1-OHase activity in kidneys was 49% of the control value, and 24-OHase activity increased 1.6-fold. The concentration of unoccupied 1,25-(OH)2D3 receptors in the intestinal mucosa (Table 3, group 2) dropped 20Downloaded from https://academic.oup.com/jn/article-abstract/120/10/1185/4738505 by St Bartholomew's & the Royal London School of Medicine and Denistry user on 20 August 2018
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