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Original Studies |
Departments of Medicine (E.M.A, D.A.M., N.H.B.) Pediatrics (B.W.H.), and Pharmacology (N.H.B.), Medical University of South Carolina; Ralph H. Johnson Veteran Affairs Medical Center (N.H.B.), Charleston, South Carolina 29401; Mayo Clinic (R.K.), Rochester, Minnesota 55905
Address all correspondence and requests for reprints to: Norman H. Bell, M.D., Ralph H. Johnson Veterans Affairs Medical Center, 109 Bee Street, Charleston, South Carolina 29401-5799.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study was carried out to determine vitamin D status and whether the vitamin D-endocrine system is altered in Asian Indians living in the southern United States.
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Materials and Methods |
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Twenty-seven normal Caucasian and eighteen normal Asian Indian men and women ranging in age from 20–40 yr were studied. Subjects were admitted to the General Clinical Research Center of the Medical University of South Carolina for 2.5 days and were placed on a constant fluid intake and a diet that was estimated to contain 400 mg calcium per day, 900 mg phosphorus per day, 110 mEq sodium, 60 mEq potassium, and 200 IU vitamin D. Five of the Asian Indian men and women were admitted for a second study in which 25(OH)D3, 40–80 µg per day, was given orally for 7 days, including a 2.5 day admission on the same regimen. Daily fasting blood and 2 consecutive 24-h urine samples were obtained for measurement of serum total and ionized calcium, phosphate, creatinine, vitamin D, 25(OH)D, 1,25-dihydroxyvitamin D [1,25(OH)2D], intact PTH, osteocalcin, urinary calcium, phosphorus, and creatinine. Serum and urinary calcium (15), phosphorus (16), and creatinine (17) were determined by automated colorimetric analyses. Creatinine clearance was calculated by standard methods, and serum vitamin D (18), 25(OH)D (19), 1,25(OH)2D (20), osteocalcin (21), and intact PTH (22) were determined by radioimmunoassay. The studies were conducted over a period of 1 calendar year in the 2 groups.
Skin fibroblast studies
In 5 normal Asian Indian and 10 normal Caucasian men and women, activity of 25(OH)D-24-hydroxylase in cultured skin fibroblasts was measured. Both studies were approved by the Medical University of South Carolina Office for Protection from Research Risks. All subjects gave their written informed consent.
Materials
25(OH)D3, 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] and 1,25(OH)2D3 were generously provided by Hoffman-LaRoche (Nutley, NJ). [26(27)-methyl-3H]25-OHD3 (3330 Gbq) and [26(27)-methyl-3H]-24, 25(OH)2D3 (3330 Gbq) were purchased from Amersham (Arlington Heights, IL), and silica Bond-Elut cartridges were purchased from Analytichem International (Harbor City, CA). Cell culture materials were purchased from GIBCO (Grand Island, NY), and HPLC grade solvents were from Baxter Diagnostics (McGaw Park, IL). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) materials were purchased from Bio-Rad (Melville, NY), and Western analysis reagents were from Amersham. All chemicals were of the purest grade available commercially.
Cell culture
Monolayer fibroblast cultures were established from 3 mm punch skin biopsies of the inner forearm and grown in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% FCS, 1 mM sodium pyruvate, 23 mM HEPES, 25 mM glucose, 4 mM glutamine, 0.1 mM nonessential amino acids, penicillin/streptomycin (100 U/100 µg per mL) and FungizoneR (0.25 µg/mL). At confluency, cells were detached from flasks by trypsinization (0.25% trypsin/0.5 mM EDTA in normal saline) and seeded into 6-well plates at a density of 104 cells/mL in growth medium. Cells were used between the 3rd and 10th passages.
After 6–8 days in culture, the activity of 24-hydroxylase in confluent monolayers of skin fibroblasts after treatment with 1,25(OH)2D3 for 6 h was measured. Briefly, culture medium was replaced with fresh growth medium containing 1% FCS and graded concentrations of 1,25(OH)2D3 in ethanol (0.02–0.04%, final concentration), and the plates were incubated at 37 C in 5% CO2 for 6 h. For determination of kinetic constants, cells were treated with 10 nM 1,25(OH)2D3 for 6 h before incubating with graded concentrations of 25(OH)D3 (0.1–4 µM). At the end of the incubation period, the medium was removed, and monolayers were washed twice with serum-free DMEM growth medium. Cells were then incubated with 25(OH)D3 (in ethanol, 0.1% final concentration for dose-response and 0.01–0.2% for kinetic analysis) for 30 min at 37 C in 5% CO2, and the reaction was stopped by adding 1 mL acetonitrile. 3H-24,25-(OH)2D3 was added to each well to estimate recovery, and the mixtures were subjected to solid-phase extraction as previously described (23).
Purification of 24,25(OH)2D3 produced by skin fibroblasts in monolayer culture was carried out by passage through Bond Elut C18 cartridges. Cartridges were prewashed successively with 2 mL each of isopropanol, methanol and water. Samples (2 ml) were then applied to the cartridges by dumping and were washed successively under vacuum (200 mm Hg), with 2 mL water, 5 mL methanol: water (60:40, v/v), 5 mL hexane:methylene chloride (99:1, v/v), and eluted with 6 mL hexane: isopropanol (95.5:4.5, v/v). The fractions were dried under N2 and redissolved in 160 µL hexane/isopropanol (88:12, v/v) and analyzed by HPLC (ZORBAXR SIL, 4.6 mm ID x 25 cm, column packed with 5 m. silica, Dupont) following ultraviolet detection. Columns were eluted isocratically at a rate of 2 mL/min with hexane/isopropanol (88:12, v/v) as the mobile phase (23).
Mitochondrial 24-hydroxylase cytochrome P-450 protein was purified by a method similar to one described previously (24). Confluent monolayer cultures in T75 flasks were treated with 10 nM 1,25(OH)2D3 (in ethanol, 0.2% final concentration) in DMEM, as constituted above but containing 1% FCS, for 6 h. Control flasks were treated with ethanol only. At the end of the incubation period, cells were removed by treatment with trypsin/EDTA (0.25% per 0.5 mM) and washed twice in serum-free DMEM. The cell suspension was then centrifuged at 1,200 x g for 10 min and the medium discarded. Cells were resuspended in Tris-EDTA buffer (50 mM Tris. HCl, 2 mM EDTA, pH 8.0) and homogenized (Tekmar Model TR-10, 70%; Tekmar Co, Cincinnati, OH) for 30 sec. Triton X-100 (2% final) and 2-mercaptoethanol (10 mM final) were added, and the suspensions were subjected to sonic oscillations for 3 min with a microtip (Fisher Sonic Demembrator Model 300, setting 40). The suspension was passed through a 22 gauge needle and centrifuged at 800 x g for 10 min. The supernatant was removed and centrifuged at 12,000 x g for 15 min. A dull white pellet containing mitochondria was recovered and dissolved in Tris-EDTA buffer (50 mM Tris.HCl, 2 mM EDTA, 10 mM 2-mercaptoethanol, and 0.1% sodium dodecyl sulfate, pH 8.0). The protein solution was concentrated by using Centricon-10 (Amicon Inc., Beverly, MA), and concentrations of samples were determined with Coomassie protein assay reagent and bovine serum albumin as standard (25).
Electrophoresis of purified P-450 protein was carried out in mini gels of 10% acrylamide with the Laemmli buffer system (26). Gels were calibrated with a standard molecular weight mixture (Rainbow mix, Amersham RPN 756) containing myosin (Mr; 220,000), phosphorylase b (Mr; 97,400), bovine serum albumin (Mr; 67,000), ovalbumin (Mr; 46,000), carbonic anhydrase (Mr; 30,000), soybean trypsin inhibitor (Mr; 21,500), and lysosyme (Mr; 14,300). After electrophoretic separation, the proteins were transferred from the unstained gels to Hybond-ECL nitrocellulose membrane and blotted according to the described protocol (Amersham). The immobilized P-450 protein was conjugated with the polyclonal antibody raised against mitochondrial cytochrome P-450 25(OH)D3-24-hydroxylase (24), followed by incubation with horseradish peroxidase-labeled antirabbit (HAR) immunoglobulin and was detected by enhanced chemiluminescence with ECLR reagents.
Diet history
Diet history of calcium intake was obtained with a questionnaire by a trained nutritionist.
Statistical analysis
Results were analyzed by Student’s nonpaired t test, correlation, and stepwise multiple regression analysis. Results are given as mean ± SE.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Serum total calcium, vitamin D, 25(OH)D and osteocalcin, urinary
calcium, and phosphorus and creatinine clearance were significantly
lower, and serum immunoreactive intact PTH and 1,25(OH)2D
were significantly higher in the Asian Indian than in the Caucasian men
and women (Table 1
). There was no
correlation between body weight and creatinine clearance. There was no
difference in serum ionized calcium, phosphorus, urinary sodium, and
potassium between the two groups. Multivariate regression analysis
showed that age and sex but not race were significant determinants
of serum osteocalcin. Race was a significant determinant of serum
25(OH)D, and race and sex were significant determinants of urinary
calcium (Table 2). Administration of
25(OH)D3 produced significant increases in serum 25(OH)D
and urinary calcium in the Asian Indians, but did not change either
serum immunoreactive intact PTH or serum 1,25(OH)2D (Table 3).
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. Emax of
25(OH)D-24-hydroxylase activity was significantly higher in Asian
Indians than Caucasians (5.46 ± 0.31 vs. 2.82 ±
0.41 nM 24,
25(OH)2D3/105 cells/30 min,
P = 0.0010) and EC50 was not different in
the two groups (0.45 ± 0.03 vs. 0.60 ± 0.05
nM, P = 0.0665; Fig. 1a). Whereas
Vmax was significantly higher in Asian Indians than
Caucasians (8.54 ± 0.40 VS. 3.71 ± 0.47
nM 24, 25(OH)2D3/105
cells/30 min, P < 0.001), KM was not
different in the two groups (0.60 ± 0.07 vs. 0.65
± 0.12 µM, Fig. 1b). Representative kinetic constants
(KM/Vmax), measured in individual subjects over
the various passages are 1) 0.35 ± 0.03/6.18 ± 0.52; 2)
0.33 ± 0.06/6.98 ± 0.82; 3) 0.73 ± 0.07/8.51 ±
0.42; 4) 0.53 ± 0.02/9.92 ± 0.59; 5) 0.75 ±
0.22/9.28 ± 0.92 for Asian Indians; and 1) 0.38 ±
0.08/3.91 ± 0.24; 2) 0.48 ± 0.08/3.25 ± 0.63; 3)
0.30 ± 0.04/3.71 ± 0.44; 4) 0.61 ± 0.11/3.49 ±
0.63; 5) 0.28 ± 0.02/5.05 ± 0.31 for Caucasians. Thus,
activity of 25(OH)D-24-hydroxylase in skin fibroblasts is significantly
higher in cultured skin fibroblasts from Asian Indians compared with
Caucasians.
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). Both
1,25(OH)2D3-treated and untreated cells
expressed this protein. Under the conditions of the experiment, there
was no apparent quantitative difference in the amount of protein in
response to the treatment.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We postulate that diminished intake, diminished dermal production of vitamin D, and possibly increased activity of 25(OH)D-24-hydroxylase may be responsible for low serum 25(OH)D in Asian Indians. The present study shows that the Vmax for 25(OH)D-24-hydroxylase is significantly higher in Asian Indians than in Caucasians. Whether there is an associated increase in the metabolic clearance of 25(OH)D, and whether this could contribute to the low serum 25(OH)D in Asian Indians is not known. Clearly, the low serum vitamin D could result in a diminished production rate of 25(OH)D because of diminished availability of substrate. The data from Western blot analysis of mitochondrial P-450 proteins suggest that enhanced activation of 25(OH)D-24-hydroxylase, by 1,25(OH)2D3 in fibroblasts from Asian Indians compared with Caucasians apparently was not due to an increase in the quantity of enzyme protein.
Our previous studies demonstrated that African-Americans have a low serum 25(OH)D with similar alteration of the vitamin D-endocrine system (27). Rickets sometimes occurs in newborn African-American infants (28, 29). However, in contrast to Asian Indians and Pakistanis, the bone disease does not occur in African-American children, and osteomalacia does not occur in African-American adults as a consequence of vitamin D deficiency.
African-Americans have a greater bone mass than Caucasians beginning early in life (30, 31), and this reduces the incidence of osteoporosis and fractures (32, 33). As indicated by bone histomorphometry after double-tetracycline labeling, African-American men and women have a lower rate of skeletal remodeling than Caucasians (34). Whether the greater bone mass and diminished rate of skeletal remodeling prevent the development of osteomalacia in African-Americans despite alteration of vitamin D metabolism is not known. Previous studies indicate that, when body mass index and area of projection of x-ray beam are considered, bone mass of Asian Indians and Caucasians is not different (35). The incidence of hip fractures of Asian Indians and Caucasians in Britain also is not different (36).
Body weight and creatinine clearance were significantly lower in the Asian Indians. However, there was no correlation between them. There was no difference in urinary sodium and potassium in the two groups.
Our findings confirm that the vitamin D-endocrine system is altered in Asian Indians, and that the decrease in serum 25(OH)D and urinary calcium with secondary hyperparathyroidism and increase in serum 1,25(OH)2D result from deficiency of 25(OH)D. Our previous studies in obese Caucasians and nonobese African Americans demonstrated that reduction in serum 25(OH)D and urinary calcium can be corrected by 25(OH)D3 (27, 37, 38, 39). In these two groups 25(OH)D3 can also lower serum 1,25(OH)2D and urinary cyclic AMP, an index of PTH secretion (38, 39). In Asian Indians on the other hand, although the administration of 25(OH)D3 did increase serum 25(OH)D and urinary calcium, it failed to change circulating intact PTH and 1,25(OH)2D. It is possible that mild secondary hyperparathyroidism in Asian Indians may not be reversible due to life-long stimulation produced by vitamin D depletion or deficiency.
In summary, our studies show that compared with Caucasians circulating vitamin D, 25(OH)D, urinary calcium and serum osteocalcin are low in Asian Indians and are associated with secondary hyperparathyroidism. Furthermore, administration of 25(OH)D3 increases serum 25(OH)D and urinary calcium but does not lower serum intact PTH or 1,25(OH)2D and 25(OH)D-24-hydroxylase activity in cultured skin fibroblasts is markedly increased in Asian Indians living in the southern United States. Thus, Asian Indians who reside in the United States may be at risk for developing vitamin D deficiency, rickets, and osteomalacia.
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Footnotes |
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Received August 4, 1997.
Revised September 25, 1997.
Accepted October 2, 1997.
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) subjects. Kinetic constants, KM and Vmax,
determined from the Eadie-Hofstee plots (inset b) are,
0.60 ± 0.07 µM and 8.54 ± 0.40 nM
24,25(OH)2D3/105 cells/30 min,
respectively, for Asian Indians and 0.65 ± 0.12 µM
and 3.71 ± 0.47 nM
24,25(OH)2D3/105 cells/30 min for
Caucasians.