Rickets in Nigerian Children: Response to Calcium Supplementation by Tom Thacher* MD, Robert H. Glew** PhD, Christian Isichei*** BM, BCh, Juliana O. Lawson**** BM, BCh, John K. Scariano** BS, Bruce W. Hollis***** PhD, and Dorothy J. VanderJagt** *Department of General Medical Practice, Jos University Teaching Hospital, Jos, Nigeria **Department of Biochemistry and Molecular Biology, University of New Mexico School of Medicine, Albuquerque, New Mexico, USA ***Department of Chemical Pathology, Jos University Teaching Hospital, Jos, Nigeria ****Department of Paediatrics, Jos University Teaching Hospital, Jos, Nigeria *****Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, USA Summary In a previous study of rachitic children in Jos, Nigeria we concluded that inadequate dietary intake of calcium was the primary contributing factor to the development of their rickets. The objective of the present study was to determine the effect of calcium supplementation in 10 children with radiographically and biochemically proven rickets from the same geographical area. Rachitic children were provided with calcium supplements of 1000 mg/day for a period of 3 months. Serum and urine samples were obtained at baseline and at 24 hours, 1 week, 4 weeks, and 12 weeks after initiation of supplementation. Serum calcium, phosphorus, alkaline phosphatase, intact parathyroid hormone, 25-hydroxyvitamin D, and 1,25-dihydroxyvitamin D were measured at each time point. Dietary recalls obtained at two separate times were used to estimate usual daily intakes of calcium and phosphorus. Ten non-rachitic age-matched controls from the same geographical area were recruited for comparison. Nine of 10 rachitic subjects had radiographic evidence of healing after 3 months of calcium therapy. Although serum calcium concentrations returned to control levels, other biochemical data indicated that the rickets of these subjects may have been multifactorial in aetiology, pointing to a possible defect in the synthesis of 25-hydroxyvitamin D.
Introduction Nutritional rickets is a disease of impaired bone mineralization in children and is most often caused by a de®ciency of vitamin D. In many parts of the world vitamin D is obtained from the diet through the consumption of forti®ed foods. In contrast, in many non-industrialized countries, the daily requirement for vitamin D is satis®ed by exposure of the skin to sunlight, which promotes the non-enzymatic conversion of 7dehydrocholesterol to vitamin D in the epidermis.1 Thus, Acknowledgements This work was supported by a Minority International Research Training (MIRT) grant to RHG and DVJ from the Fogarty International Center of the National Institutes of Health. The authors would like to acknowledge Edward R. Garrison, PhD, Department of Biology and Life Sciences, Navajo Community College, Shiprock, NM for assistance with the analyses of dietary intakes. Correspondence: D. J. VanderJagt, Department of Biochemistry and Molecular Biology, University of New Mexico School of Medicine, Room 249, BMSB, Albuquerque, NM 87131, USA. Tel. 505 272 5799; Fax 505 272 6587. E-mail
[email protected]. 202
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vitamin D de®ciency is unexpected in tropical areas of the world, particularly Africa, where there is ample opportunity for children to be exposed to sunlight. In these areas, inadequate calcium intake, excessive excretion of calcium in the urine, intestinal malabsorption, or end-organ resistance to vitamin D may contribute to the development of rickets.2±6 In African populations rickets usually occurs between 1 and 2 years of age, after the child has been weaned and has begun consuming a low-calcium diet. This is in contrast to the aetiology of rickets in North America where rickets usually occurs before 1 year of age in exclusively breastfed infants who receive minimal sunlight exposure.7 Calcium de®ciency rickets has been described in several indigenous African populations2±6 and is characterized by mild hypocalcaemia, secondary hyperparathyroidism, and elevated levels of serum 1,25-dihydroxyvitamin D [1,25(OH)2 -D] in the face of low to normal 25-hydroxyvitamin D [25-(OH)-D] concentrations. In a recent study we conducted in semi-arid northern Nigeria6 where the predominant ethnic groups are Hausa and Fulani, we obtained biochemical evidence that the rickets in that part of the country was caused by calcium de®ciency. Most of the children in northern Nigeria Journal of Tropical Pediatrics
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consume diets that are largely devoid of dairy products and are based on staples such as millet and sorghum, both of which are rich in phytates and oxalates,8 substances which chelate calcium and make it unavailable for absorption. In order to test the hypothesis that the rickets in this northern Nigerian population we have been studying is the result of calcium de®ciency, we conducted a 3 month calcium supplementation trial in children with radiographically and biochemically proven rickets. An estimate of their usual intakes of calcium and phosphorus was obtained and serum measurements related to calcium and vitamin D status were monitored during the course of calcium supplementation. Methods Subjects Eighteen children between 1 and 8 years of age who had bone deformities characteristic of rickets (e.g. genu varum, genu valgum, widened wrists) were recruited from within and around Jos, Nigeria. An equal number of age-matched control children without clinical evidence of rickets were also recruited from the General Outpatient Department of the Jos University Teaching Hospital. The control group consisted of children who had returned for follow-up of some acute illness (e.g. malaria, pneumonia) from which they had recovered. Children with rachitic bone deformities underwent radiography of the knees; radiographs were not taken of control children. All radiographs were reviewed by two physicians (TT, JOL) experienced in the diagnosis and management of rickets. Radiographic criteria used to de®ne active rickets were metaphyseal cupping and fraying, and widening of the epiphysis. Radiographic evidence of healing was de®ned as the presence of new calci®cation and reduced widening of the growth plate compared with the baseline radiographs. For each radiograph, both physicians mutually agreed on the interpretation of active rickets and on the presence or absence of improvement. Serum samples and 24 hour urine specimens were obtained from the 10 children with radiographically con®rmed active rickets and their corresponding controls. Following the collection of baseline samples, the rachitic subjects were started on calcium supplements (1000 mg/day elemental calcium divided into two doses; TUMSTM chewable calcium carbonate tablets; SmithKline Beecham, Pittsburgh, PA). Supplementation was continued for 12 weeks. Serum and urine collections were repeated after 24 hours, 7 days, 4 weeks, and 12 weeks of calcium therapy. To facilitate accurate 24-hour urine collections, patients and controls were hospitalized for each sample collection. The existence of renal disease was excluded on the basis of serum creatinine concentration and urine analysis using Combur-10 test strips (Boehringer Mannheim, Germany). Radiographs of the knees were repeated at the end of the 12-week supplementation period. Journal of Tropical Pediatrics
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Each subject was examined and a parent interviewed to obtain information concerning factors which might affect their child's calcium, or vitamin D status, which included the following: duration of breastfeeding, history of preterm delivery, family history of rickets, average daily sunlight exposure, and any occurrence of diarrhoeal disease in the preceding 3 months. The family history was considered positive if any ®rst-degree relatives such as parents or siblings exhibited characteristic leg deformities in childhood. Information about medication use was also obtained, including multivitamin preparations, calcium or vitamin D supplements. Socioeconomic status was determined using the educational level and occupation of the parents, according to a scoring system validated in a Nigerian population.9 Written informed consent was obtained from a parent or guardian of the patient or control subject and approval for the study was obtained from the Ethical Committee of the Jos University Teaching Hospital. Anthropometric measurements The height and weight and occipitofrontal and mid-arm circumferences were obtained for rachitic subjects and controls. Those portions of the body not covered by clothing were noted and the percentage of exposed skin was estimated using the Lund and Browder age-related burn chart. Subjects were also compared to controls with respect to skin pigmentation by colour photographs obtained using standardized background and lighting. Dietary analyses The dietary intake of calcium, phosphorus, and total calories for subjects and controls was estimated by obtaining two 24-hour dietary recalls on separate days. The mineral and caloric intake was calculated using food composition tables designed for African foodstuffs.10;11 The mineral content of foods not available in these tables was determined by atomic adsorption spectrophotometry (unpublished data). Biochemical analyses All serum and urine samples were frozen and stored at 708C until they were transported on dry ice to the University of New Mexico, Albuquerque, NM. The followinganalyses wereperformed onserum:total calcium, phosphorus, magnesium, intact parathyroid hormone (PTH), 25-(OH)-D, 1,25-(OH)2 -D, alkaline phosphatase, creatinine, albumin, and prealbumin. The concentrations of calcium, creatinine, and deoxypyridinoline were measured in urine samples. Serum calcium, phosphorus, alkaline phosphatase, and creatinine measurements were performed as described previously.6 Serum magnesium concentrations were measured colorimetrically using bound `calmagite' (Sigma Diagnostics, St Louis, MO). Prealbumin concentrations were determined using radial immunodiffusion (The Binding Site Limited, Birmingham, UK). Intact PTH, 25-(OH)-D, and 1,25-(OH)2 -D in serum were measured by radioimmunoassay methods (INCSTAR, 203
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Stillwater, MN). The urinary deoxypyridinoline levels were determined using an enzyme immunoassay method (Pyrilinks-DÒ ) purchased from Metra Biosystems (Mountain View, CA). Statistical analyses Statistical analyses were performed using NCSS, version 6.0 (Kaysville, UT). The Mann±Whitney U-test was used to compare the ordered categories of socioeconomic status. The paired t-test was used to compare mean values of continuous variables between rachitic subjects and controls for those variables that were normally distributed. The Wilcoxon signed-rank test for difference in medians was used for those variables that were not normally distributed. A p value of less than 0.05 was considered signi®cant. Unless otherwise noted, values are reported as means 6 1 standard deviation. Results Clinical characteristics There were no signi®cant differences between the anthropometric characteristics of the rachitic subjects and controls (Table 1). The nutritional status of the rachitic and control children appeared to be similar as judged by their serum albumin and prealbumin concentrations; both groups of subjects were at the lower limit of the reference range for serum prealbumin concentration (109±273 mg/l).12 The rachitic and control children were weaned at approximately the same age (Table 2). Although the rachitic children started walking at a slightly later age than the controls, the difference was not signi®cant. Whereas the families of six of 10 experimental children had a family history of rickets, only one of 10 families of the control subjects had a positive history. Dietary intake The mean caloric intakes for the rachitic and control groups prior to supplementation were 118 6 39 kcal/kg and 115 6 47 kcal/kg, respectively and the difference in the mean values was not statistically signi®cant. The median calcium intake for rachitic children was 191 (128±461) mg/day and was not signi®cantly different from that of the control group, 169 (128±551) mg/day ( p 0:4). Both the rachitic children and controls had calcium intakes well below the recommended daily allowance of 800 mg/day for children.13 The median daily intakes of phosphorus for the rachitic children and control subjects were not signi®cantly different [783 (324±1024) mg/day vs. 780 (199±1547) mg/day, respectively; p 0:95]. The mean ratios of calcium to phosphorous in the diets of the rachitic and control subjects were identical (0:35 6 0:11 vs. 0:35 6 0.32, p 0:97). Biochemical analyses Prior to supplementation, there were statistically signi®cant differences between the serum calcium levels of 204
the rachitic and control groups (8:7 6 0:7 mg/dl vs. 9:8 6 0:5 mg/dl, p 0:003) (Table 3). The mean serum calcium concentration for the rachitic subjects was at the lower end of the reference range for children (8.7 mg/ dl12 ) and seven of the 10 rachitic children had a serum calcium level below this value (Fig. 1). Twenty-four hours after the ®rst calcium supplements were administered, the serum calcium levels increased in seven out of 10 rachitic children. Following 12 weeks of calcium therapy, there was no statistically signi®cant difference in the serum calcium levels for the rachitic and control subjects (9:5 6 0:6 mg/dl vs. 9:8 6 0:5, p 0:25, Table 3). In contrast, the mean serum phosphorus concentration of the rachitic children remained signi®cantly lower than that of the control group throughout the course of the study. The average serum alkaline phosphatase activity of the rachitic subjects at baseline was 2.8-fold greater than that of the control subjects but decreased by 30 per cent after 12 weeks of calcium supplementation (Table 3).
Fig 1. Serum calcium concentrations for the rachitic subjects during the course of calcium supplementation. The upper panel shows the serum calcium levels for two subjects who had received calcium supplements or a multivitamin prior to entry into the study. The serum calcium levels of the remaining subjects (n 8) are shown in the lower panel. Serum calcium concentrations are given for the control subjects for comparison. Journal of Tropical Pediatrics
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Noteworthy, however, was the failure of the serum alkaline phosphatase activity of the rachitic subjects to decrease to the level of the control subjects by the end of the supplementation period, at which time there was unequivocal radiographic evidence of healing in nine of the 10 rachitic children. At baseline, using paired analysis, no signi®cant difference was found in the serum concentrations of PTH between the rachitic subjects and age-matched controls ( p 0:72, Table 3). However, after 1 week of calcium therapy, the serum PTH concentrations in the rachitic children fell to levels which were signi®cantly lower than those of the controls ( p 0:03, Fig. 2). Interestingly, the serum PTH levels increased from a median value of 2.1 pg/ml at 1 month to a median level of 55 pg/ml after 12 weeks of calcium supplementation ( p 0:03). Furthermore, the median PTH concentrations of the rachitic subjects following 3 months of calcium therapy was approximately twice that of the controls ( p 0:003). The rachitic children had serum 25-(OH)-D concentrations that were approximately half those of the control group (9:6 6 4:5 ng/ml vs. 20:9 6 2:9 ng/ml, p < 0:001). At baseline, eight of the 10 rachitic children had serum 25-(OH)-D concentrations below the lower limit of the reference range for children (15 ng/ml) and the levels did not change during the course of calcium supplementation (Fig. 3). Furthermore, both the rachitic and control subjects had serum 1,25-(OH)2 concentrations that were more than twice the upper limit of the reference range for children of that age (20±60 pg/ml) (Table 3). As was observed with 25-(OH)-D, there was no signi®cant changes in the serum 1,25-(OH)2 levels in the rachitic
Fig 2. Serum concentrations of PTH for rachitic subjects during the course of calcium supplementation. Serum levels of PTH for control subjects are given for comparison. Journal of Tropical Pediatrics
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Fig. 3. Serum concentrations of 25-(OH)-D for rachitic subjects during the course of calcium supplementation. Serum levels of 25-(OH)-D in control subjects are given for comparison. children in repsonse to calcium supplementation (Fig. 4). Serum concentrations of magnesium for the rachitic subjects did not change during the course of calcium supplemenation (1.89 meq /l and 2.01 meq/l for baseline and 12 weeks, respectively) and were within the reference range for children of that age (1.4±1.9 meq/l). The
Fig: 4. Serum concentrations of 1,25-(OH)2 -D for rachitic subjects during the course of calcium supplementation. Serum levels of 1,25-(OH)2 -D for control subjects are given for comparison. 205
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urinary excretion of deoxypyridinoline, a marker of bone resorption,14 decreased markedly during supplementation from 84:4 6 27:7 nmol/mmol creatinine at baseline to 48:2 6 16:1 nmol/mmol creatinine after 12 weeks of therapy, a level which was not signi®cantly different from that of the controls (52:2 6 18:9 nmol/mmol). Discussion Understanding the pathogenesis of rickets in any population has clear and obvious therapeutic implications. Based on the results of studies of rachitic children in Nigeria conducted by us6 and other investigators,4;5 it appears that rickets in Nigeria may have more than one cause, depending on geographic and ethnic factors. Okunofua and co-workers4 concluded that rickets in southern Nigeria is due primarily to calcium de®ciency. In contrast, in a later study in the same city in Nigeria, Oginni and co-workers5 concluded that while the rickets was caused mainly by calcium de®ciency, vitamin D de®ciency was a contributing factor in one of their subjects and end-organ resistance to vitamin D could not be ruled out for two patients with very high levels of 1,25-(OH)2 -D (> 1400 pmol/l). In the present study of 10 rachitic children in northern Nigeria, nine out of 10 rachitic subjects showed radiographic evidence of healing following a supplementation regimen of 1000 mg calcium per day for 12 weeks, during which time the serum calcium levels of all 10 children were restored to control levels. Furthermore, calcium supplementation markedly decreased the urinary excretion of deoxypyridinoline in all 10 of the rachitic subjects (Table 3). The parents of the rachitic children reported a decrease in complaints of pain and muscle weakness, which also corroborates the effectiveness of the calcium supplementation. However, even though calcium supplements alone corrected their rickets, the biochemical data we obtained for these children indicate that the aetiology of their bone disease may be multifactorial. Although the serum calcium levels of the rachitic children in our study promptly returned to control levels within 1 week of calcium therapy, their 1,25-(OH)2 -D levels did not change throughout the course of the study. Considering that the half-life of 1,25-(OH)2 -D is 3±6 hours,15 12 weeks of calcium supplementation should have provided more than suf®cient time for the serum 1,25-(OH)2 -D concentrations to normalize. Noteworthy is the observation that both the rachitic and control subjects had elevated 1,25-(OH)2 -D levels in the face of PTH concentrations that were in the normal range. Pettifor and co-workers2 reported that the supranormal 1,25-(OH)2 -D levels present in the rachitic subjects they studied returned to normal within 2 months of calcium treatment. Similarly, Oginni and co-workers16 reported a substantial decline in the mean serum 1,25-(OH)2 -D concentrations from 1345 to 967 pmol/l in four rachitic children in southern Nigeria after 10 days of therapy with 900 mg calcium per day. In addition, they reported a 206
decline in the mean serum 25-(OH)-D concentration from 88 to 29.6 pmol/l in the same subjects. Clearly, the regulation of 1,25-(OH)2 -D is different in the children in our present study. While the serum levels of PTH in the rachitic children were not signi®cantly different from those of the controls at baseline (Table 3), they were inappropriately low in light of their serum calcium levels. Because the diets in this region contain substances that chelate not only calcium but other divalent cations as well, it is possible that the rachitic subjects in our study were also de®cient in magnesium. Magnesium has been reported to be required for PTH secretion.17;18 The rise in PTH levels observed in the rachitic children between 1 and 3 months of calcium supplementation (Fig. 4) was probably not the result of a change in their magnesium status because the calcium preparation we administered to them did not contain magnesium. PTH synthesis and release is also controlled by the extracellular calcium concentrations and by negativefeedback inhibition by 1,25-(OH)2 .9 The unexpected and marked rise in serum PTH concentrations in the rachitic children following normalization of serum calcium levels in the presence of persistently elevated 1,25(OH)2 concentrations is suggestive of some alteration in the calcium/vitamin D/parathyroid system in these children. In addition, the low levels of 25-(OH)-D in the rachitic children in our study indicate that they were vitamin D de®cient. This de®ciency could have resulted from either decreased synthesis or increased metabolic clearance of 25-(OH)-D. Low levels of 25-(OH)-D are seen in a variety of clinical conditions. Individuals with persistently elevated concentrations of 1,25-(OH)2 -D inactivate and excrete 25-(OH)-D at increased rates,20 resulting in an acquired vitamin D de®ciency. The fact that the serum 1,25-(OH)2 -D levels of the controls in our study were elevated and comparable to those of the rachitic children, while their 25-(OH)-D serum concentrations were within the normal range, leads us to conclude that the rachitic children in the present study do not have an acquired vitamin D de®ciency due to elevated concentrations of 1,25-(OH)2 -D. Alternatively, the de®cient levels of 25-(OH)-D in the rachitic subjects in our study could have been caused by decreased activity of the enzyme which converts vitamin D to 25-(OH)-D. Such a defect in two young brothers of Nigerian ancestry was recently described by Casella and co-workers.21 The two brothers presented with mild hypocalcaemia, hypophosphataemia, serum 25-(OH)-D levels indicative of vitamin D de®ciency, and elevated serum 1,25-(OH)2 -D concentrations. Although the rickets in the two patients studied by Casella and co-workers was cured by treatment with vitamin D, pharmacological doses of the vitamin were required to maintain normal serum 25-(OH)-D concentrations. While we were able to correct the rickets in the children we studied with calcium supplements alone, the data we obtained on these children are most consistent with the hypotheses Journal of Tropical Pediatrics
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proposed by Casella and colleagues;21 that is, they were de®cient in 25-hydroxylase activity which converts vitamin D to 25-(OH)-D. However, limiting quantities of sera prevented us from determining serum levels of vitamin D in the present study. In summary, it is likely that the bone disease of the rachitic children we studied in Northern Nigeria was multifactorial in aetiology. In addition to a diet that provides inadequate calcium, these children may also have a defect in the pathway of vitamin D metabolism, speci®cally the 25-hydroxylation of vitamin D. Furthermore, because six of the 10 rachitic children in our study had a family history of rickets, we raise the possibility that their putative 25-hydroxylase de®ciency may be hereditary rather than acquired. Nevertheless, this assertion should be tempered by the fact that family members share many environmental factors. Since the sequence of the isolated human cDNA for the 25hydroxylase is known,22 the possibility of mutations in the 25-hydroxylase can be investigated. Such studies are in progress. References 1. Webb AR, Holick MF. The role of sunlight in the cutaneous production of vitamin D. Annu Rev Nutr 1988; 8: 375±99. 2. Pettifor JM, Ross FP, Travers R, Glorieux FH, DeLuca HF. Dietary calcium de®ciency: a syndrome associated with bone deformities and elevated 1,25-dihydroxyvitamin D concentrations. Metab Bone Dis Rel Res 1981; 2: 301±5. 3. Bhimma R, Pettifor JM, Coovadia HM, Moodley M, Adhikari M. Rickets in black children beyond infancy in Natal. S Afr Med J 1995; 85: 668±72. 4. Okonofua F, Gill DS, Alabi ZO, Thomas M, Bell JL, Dandona P. Rickets in Nigerian children: a consequence of calcium malnutrition. Metabolism 1991; 40: 209±13. 5. Oginni LM, Worsfold M, Oyelami A, Sharp CA, Powell DE, David MWJ. Etiology of rickets in Nigerian children. J Pediatr 1996; 128: 692±4. 6. Walter EA, Scariano JK, Easington CR, et al. Rickets and protein malnutrition in northern Nigeria. J Trop Pediatr 1997; 43: 98±102. 7. Bhowmick SK, Johnson KR, Rettig KR. Rickets caused by vitamin D de®ciency in breast-fed infants in the southern United States. Am J Dis Child 1991; 145: 127±30.
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8. Odumodu CU. Antinutrient content of some locally available legumes and cereals in Nigeria. Trop Geogr Med 1992; 44: 260±3. 9. Olunsaya O, Okpepe E, Ezimokhai M. The importance of social class in voluntary fertility control in a developing country. W Afr J Med 1985; 4: 205±11. 10. Agricultural Extension and Research Liaison Services. Food Composition Tables for Use in Africa. AERLS, Ahmadu Bello University, Zaria; 1985. 11. Latham MC. Human Nutrition in Tropical Africa. FAO Food and Nutrition Series. Food and Agricultural Organization of the United Nations, Rome; 1979: No 11: 264±76. 12. Soldin SJ, Hicks JM, eds. Pediatric Reference Ranges. AACC Press, Washington, DC; 1995. 13. Food and Nutrition Board. Recommended Dietary Allowances. National Academy Press, Washington, DC; 1989. 14. Daniloff GY, Hesley RP, Ju J, Evans BJ, Seyedin SM. An assay for deoxypyridinoline: a highly speci®c marker of bone-resorption. J Bone Miner Res 1993; 8 (S1): S357. 15. Holick MF. Vitamin D: photobiology, metabolism and clinical applications. In: DeGroot LJ, ed., Endocrinology. Saunders, Philadelphia; 1995; 990±1014. 16. Oginni LM, Worsfold M, Sharp CA, Oyelami OA, Powell DE, Davie MJW. Plasma osteocalcin in healthy Nigerian children and in children with calcium-de®ciency rickets. Calcif Tissue Int 1996; 59: 424±7. 17. Webster PO. Magnesium. Am J Clin Nutr 1987; 45: 1305± 12. 18. Fatemi S, Ryzen E, Flores J, Endres DB, Rude RK. Effect of experimental human magnesium depletion on parathyroid hormone secretion and 1,25-dihydroxyvitamin D metabolism. J Clin Endocrinol Metab 1991; 73: 1067±72. 19. Naveh-Many T, Friedlander MM, Mayer H, Silver J. Calcium regulates parathyroid hormone messenger ribonucleic acid (mRNA), but not calcitonin mRNA in vivo in the rat. Dominant role of 1,25-dihydroxyvitaminn D. Endocrinol 1989; 125: 275±80. 20. Clements MR, Davies M, Fraser DR, Lumb GA, Mawer EB, Adams PH. Metabolic inactivation of vitamin D is enhanced in primary hyperparathyroidism. Clin Sci 1987; 73: 659±64. 21. Casella SJ, Reiner BJ, Chen TC, Holick MF, Harrison HE. A possible defect in 25-hydroxylation as a cause of rickets. J Pediatr 1994; 124: 929±32. 22. Guo Y-D, Strugnell S, Back DW, Jones G. Transfected human liver cytochrome P-450 hydroxylates vitamin D analogs at different side chain positions. Proc Natl Acad Sci USA 1993; 90: 8668±72.
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