Thyrocalcitonin: ultracentrifugation in gradients of sucrose.

Analysis of thyrocalcitonin, by density-gradient ultracentrifugation at high speed, showed that its molecular weight (5000 to 6000) is considerably less than was heretofore recognized.

Two mutations in the hormone binding domain of the vitamin D receptor cause tissue resistance to 1,25 dihydroxyvitamin D3.

We have identified and characterized two mutations in the hormone binding domain of the vitamin D receptor (VDR) in patients with hereditary vitamin D-resistant rickets. One patient was found to have a premature stop mutation (CAG to TAG) in the hinge region affecting amino acid 149 (Q149X) and the other demonstrated a missense mutation (CGC to CTC) resulting in the substitution of arginine 271 by leucine (R271L) in the steroid binding domain. Eukaryotic expression analyses in CV-1 cells showed the inability of both patients' VDR to induce transcription from the osteocalcin hormone gene response element at 10(-7) M 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Normal transcription levels could, however, be elicited by the missense mutated VDR (R271L) in the presence of 1,000-fold higher 1,25-(OH)2D3 concentrations than needed for the wild-type receptor. This shows that Arg 271 directly affects the affinity of the VDR for its ligand and its conversion to leucine decreases its affinity for 1,25(OH)2D3 by a factor of 1,000. Arg 271 is located immediately 3-prime to a 30 amino acid segment (VDR amino acids 241-270) that is conserved among members of the steroid/thyroid/retinoid hormone receptor superfamily. These results represent the first missense mutation identified in the hormone binding domain of VDR and further define the structure-function relationship of 1,25(OH)2D3 ligand binding to its nuclear receptor.

Mapping the gene causing X-linked recessive idiopathic hypoparathyroidism to Xq26-Xq27 by linkage studies.

Idiopathic hypoparathyroidism has been reported to occur as an X-linked recessive disorder in two multigeneration kindreds. Affected individuals, who are males, suffer from infantile onset of epilepsy and hypocalcemia, which appears to be due to an isolated congenital defect of parathyroid gland development; females are not affected and are normocalcemic. We have performed linkage studies in these two kindreds (5 affected males, 11 obligate carrier females, and 44 unaffected members) and have used cloned human X chromosome sequences identifying restriction fragment length polymorphisms to localize the mutant gene causing this disorder. Our studies established linkage between the X-linked recessive idiopathic hypoparathyroid gene (HPT) and the DXS98 (4D.8) locus, peak LOD score = 3.82 (theta = 0.05), thereby mapping HPT to the distal long arm of the X chromosome (Xq26-Xq27). Multilocus analysis indicated that HPT is proximal to the DXS98 (4D.8) locus but distal to the F9 (Factor IX) locus, thereby revealing bridging markers for the disease. The results of this study will improve genetic counseling of affected families, and further characterization of this gene locus will open the way for elucidating the factors controlling the development and activity of the parathyroid glands.

Regulation by calcium of parathyroid hormone mRNA in cultured parathyroid tissue.

We have examined the effect of changes in the concentration of extracellular calcium on parathyroid hormone mRNA in both short-term (hours) and long-term (days) cultures of bovine parathyroid tissue. Using a 32P-labeled PreProPTH cDNA probe, PTH mRNA was measured by gel blot hybridization of total RNA from tissue slices incubated for 4 h in low (0.5 mM) or high (5 mM) calcium concentrations and also by dot blot hybridization of cytoplasmic RNA extracted from aggregates of partially dispersed cells cultured up to 72 h in low (0.4 mM), normal (1 mM), or high (3 mM) calcium concentrations. PTH mRNA was unchanged over 4 h while high calcium had suppressed PTH secretion. However PTH mRNA did respond during long-term culture. By 24 h in high calcium there was a 50% suppression which was maintained for a further 48 h. PTH mRNA in normal calcium remained unchanged over 72 h while in low calcium it had increased slightly by 48 h. In contrast to the effect seen in cultured bovine parathyroid cells, PTH mRNA in human parathyroid adenoma cells cultured for 48 h in high calcium was decreased by only 10%.

Translational regulation of parathyroid hormone gene expression and RNA: protein interactions.

The aim of this study was to investigate the mechanism by which translation of parathyroid hormone (PTH) mRNA is regulated with regard to the subcellular distribution of PTH mRNA and RNA:protein interactions. Sucrose density ultracentrifugation of RNA from bovine parathyroid cells indicated that there was no evidence for a pool of nonribosomal PTH mRNA, and the extracellular calcium concentration had no effect on polysome size. UV cross-linking studies revealed two proteins in parathyroid cell cytosol which bound specifically to the 5'-untranslated region (UTR) of PTH mRNA with molecular masses of 66 and 68 kD while proteins with apparent molecular masses of 48 and 70 kD bound to the 3'-UTR. In vitro translation assays indicated that parathyroid cell cytosol contains factors that inhibit translation of PTH mRNA. Fractionation of cytosol revealed that this effect was associated with proteins within the molecular mass range 30-90 kD. To determine which sequences in PTH mRNA mediate translational regulation, RNA was synthesized from luciferase gene constructs containing the 5'- and/or 3'-UTR of PTH mRNA, and translated in vitro. Addition of parathyroid cell cytosol reduced the translation of RNA containing the 5'- and 3'-UTR of PTH mRNA by 44 +/- 7% but had no effect on the translation of RNA containing only the luciferase coding region. Translation of RNA containing only the 5'-UTR of PTH mRNA was unchanged; however, cytosol reduced the translation of RNA containing the 3'-UTR by 31 +/- 9%. These data demonstrate a role for RNA:protein interactions in the regulation of PTH synthesis and that translational control is mediated primarily through interactions with the 3'-UTR of PTH mRNA.

Mutations in the vitamin D receptor gene in three kindreds associated with hereditary vitamin D resistant rickets.

Hereditary vitamin D resistant rickets has been associated with a number of mutations within the DNA and ligand binding domains of vitamin D receptors (VDR). The aim of our study was to identify and characterize the causative mutations in three kindreds with this condition. Resistance of 1,25(OH)2D3 was confirmed in cultured skin fibroblasts in which there was no induction of 24-hydroxylase activity; binding of 1,25(OH)2D3 to VDR was undetectable in patients 1 and 2, but normal in patients 3 and 4. The coding region of the VDR gene was sequenced to seek mutations. A mutation in the VDR gene of patient 1 resulted in a STOP codon, patient 2 showed a 56 bp deletion leading to frameshift and premature termination of VDR; a point mutation of A to C lying within the hormone-binding domain was shown for patients 3 and 4, who were siblings. Transactivation studies confirmed that these were functional mutations. Gel shift assays using nuclear extract from patient 3 demonstrated that the mutation that altered a conserved amino acid (glutamine-259) known to be involved in heterodimerization with other nuclear receptors affected protein: protein interactions.

Post-transcriptional regulation of bovine parathyroid hormone synthesis.

Incubation of bovine parathyroid cells for 48 h in 0.4 mmol calcium/l had no significant effect on steady-state preproparathyroid hormone (preproPTH) mRNA levels when compared with cells incubated in 1.0 mmol calcium/l, but low calcium concentrations increased the membrane-bound polysomal content of preproPTH mRNA by 200 +/- 16% (mean +/- S.D.). No preproPTH mRNA was detected on free polysomes. Actinomycin D (5 and 10 micrograms/ml) had no effect on steady-state preproPTH mRNA levels measured in dot-blot assays after 24 h, but reduced levels in cells incubated in 1.0 mmol calcium/l to 54 +/- 16% and 39 +/- 12% of control values respectively after 48 h of incubation. Similarly, in cells incubated in 0.4 mmol calcium/l, actinomycin D (5 and 10 micrograms/ml) reduced steady-state preproPTH mRNA levels to 57 +/- 13% and 45 +/- 5% of control values respectively. Actinomycin D did not prevent the rise in polysomal content of preproPTH mRNA induced in cells by incubation in 0.4 mmol calcium/l, but increased polysomal content in cells incubated in 0.4 and 1.0 mmol calcium/l by 159 +/- 9% and 164 +/- 13% respectively after 48 h. These results demonstrate post-transcriptional regulation of PTH synthesis in cultured bovine parathyroid cells, and suggest that this control involves a protein which may be calcium-sensitive.

Early regulation of c-myc mRNA by 1,25-dihydroxyvitamin D3 in human myelomonocytic U937 cells.

The effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3; 10 nmol/l) on the human monomyelocytic cell line U937 were investigated. Addition of 1,25-(OH)2D3 led to a decrease in cell proliferation which fell at 72 h to 67.8 +/- 4.3% (mean +/- S.E.M.) of control values. The presence of CD14, a surface marker found on mature monocytes/macrophages but not on U937 cells, was detectable as early as 18 h and peaked at 48 h, when 63.6 +/- 4.2% of the cells were positive. However, changes in c-myc mRNA levels were detected earlier, starting within 4 h of exposure to the hormone and being reduced to 38 +/- 8.2% of control values of 24 h. These effects were reversible after removal of the hormone, with the same sequence of events seen following addition of the hormone. There was first an increase in c-myc mRNA levels, starting within 2 h and reaching control values by 24 h. These changes were followed by loss of CD14 which became undetectable after 72 h. Proliferation recovered slowly and incompletely, since it was 81.7 +/- 0.7% of control after 72 h. A constant reciprocal relationship between c-myc mRNA and CD14 levels was found both in the presence and after removal of 1,25-(OH)2D3. Regulation of U937 cell proliferation and maturation by 1,25-(OH)2D3 is thus preceded by early modulation of c-myc mRNA.

Binding of 1,25-dihydroxyvitamin D3 receptors to the 5'-flanking region of the bovine parathyroid hormone gene.

To further define the binding site for receptors for 1,25(OH)2D3 (VDR) in the bovine PTH gene and to study the interactions of transcription factors with VDR, Southwestern and gel shift assays were used. Data from the former indicated binding of VDR to DNA fragments spanning the regions -451 to -348 bp and -668 to -452 bp. Studies using gel shift assays confirmed binding to the -451 to -348 bp fragment and specificity was shown by using excess concentrations of unlabelled -451 to -348 bp fragment to compete for binding, whereas excess unlabelled -347 to +50 bp did not compete. Binding was also observed with the -668 to -452 bp fragment but excess concentrations of unlabelled -668 to -452 or -451 to -348 bp fragments did not compete for binding to radiolabelled fragments. These data indicate the presence of two binding domains within this region; the upstream element having a lower affinity for VDR than the downstream element. In addition, there was no interaction between VDR and consensus sequences for AP1, AP2, AP3 and SP1. The putative vitamin D3 response element (VDRE) contains two similar hexameric steroid response element-like half-sites placed as AGGTCA-related direct repeats. The upstream repeat is at -461 to -456 bp and the downstream element is at -449 to -444 bp. The presence of these half-sites is consistent with our experimental data in which cleavage with SspI at -452 bp resulted in two DNA fragments which bound VDR.

Regulation of preproparathyroid hormone messenger RNA and hormone synthesis in human parathyroid adenomata.

Preproparathyroid hormone (preproPTH) mRNA and PTH secretion were measured in human parathyroid adenomata (n = 8) cultured in 1.0 or 3.0 mmol calcium/l and compared with changes in bovine parathyroid glands (n = 3) as a control. Incubation of bovine glands in 3.0 mmol calcium/l for 24 h resulted in a fall in mRNA levels to 47.3 +/- 21.7% (mean +/- S.D.) compared with cells incubated in 1.0 mmol calcium/l, with a concomitant decrease in secretion to 62.6 +/- 10.8%. These values fell further to 30.1 +/- 15.5% and 42.1 +/- 18.7% respectively after 48-h incubation. One human adenoma responded to high levels of calcium in a similar manner with mRNA levels falling to 44.6 +/- 11.9% and secretion to 30.3 +/- 17.3% within 24 h. However, in the majority of adenomata (seven out of eight), after 24-h incubation in 3.0 mmol calcium/l, mRNA levels fell to 54.1 +/- 14.6% but there was no change in secretion. In two of these adenomata which were cultured for 48 h, there was no suppression of secretion despite mRNA levels having fallen to approximately 60% of control. Incorporation of [35S]methionine into PTH secreted from human adenomatous cells was quantified by densitometry. There was no difference in the amount of radiolabelled PTH secreted from cells incubated in high levels of calcium compared with those in normal levels of calcium. In similar experiments, the effects of high calcium on the synthesis of PTH in bovine cells was assessed by determination of radiolabelled intracellular PTH.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of 1,25-dihydroxyvitamin D3 and cortisol on bovine and human parathyroid cells.

Incubation of bovine parathyroid cells with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) decreased both preproparathyroid mRNA levels and parathyroid hormone (PTH) secretion. There was a fall to 56.6 +/- 13.7% (mean +/- S.E.M.) and 65.1 +/- 9.3% in mRNA levels and PTH secretion respectively at 1 nmol 1,25-(OH)2D3/l, and 41.1 +/- 13.6% and 42.0 +/- 12.1% at 10 nmol 1,25-(OH)2D3/l after 24 h. After 48 h in 0.1 nmol 1,25-(OH)2D3/l, mRNA levels had fallen to 35.3 +/- 12.6% and PTH secretion to 32.1 +/- 5.0%. In human adenomatous cells, however, incubation with 1,25-(OH)2D3 (10 nmol/l) had no effect on either mRNA levels or PTH secretion even after 48 h. This lack of sensitivity of adenomatous cells to 1,25-(OH)2D3 was not due to an absence of receptors (3847 +/- 39 receptors/ng cytosolic protein in adenomatous cells compared with 4068 +/- 371 in bovine cells) or receptors being of low affinity. Cortisol (1 mumol/l) caused a reduction in the number of receptors for 1,25-(OH)2D3 in bovine parathyroid cells of approximately 20% within 24 h of incubation, but no change in affinity. This decrease was accompanied by abolition of the response to 1,25-(OH)2D3 and was reversible, in that withdrawal of cortisol for the final 24 h of incubation was sufficient for the response to return, the number of receptors having returned to control values. These results suggest that only a small percentage of receptors for 1,25-(OH)2D3 in bovine parathyroid cells may be functional at any one time.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of homologous immunological assays for human parathyroid hormone.

Antisera to a trichloroacetic-acid precipitate of human parathyroid hormone (PTH) were produced in goats. Two of these antisera (G36 and G31) were of high affinity, and the bovine and porcine hormones were less reactive. Synthetic peptides containing the amino-terminal region of human PTH reacted with both antisera; the 1--34 peptide (PTH-(1--34)), with the sequence proposed by Niall, Sauer, Jacobs, Keutmann, Segre, O'Riordan, Aurbach & Potts in 1974, was more reactive than that having the sequence proposed by Brewer, Fairwell, Ronan, Sizemore & Arnaud in 1972. The antisera were further characterized with a number of other native and synthetic fragments of human PTH and reacted poorly with fragments from the carboxy-terminal region of the molecule. Since the amino-terminal fragments did not account for all the immunoreactivity, it is assumed that the antisera had some recognition sites for the central part of the molecule. Highly purified human PTH-(1==84) was labelled with 125I and radioimmunoassays were developed using this tracer and antiserum G36. To avoid the problems associated with labelling human PTH with 125I, a labelled antibody assay was developed with G36 and an immunoadsorbent consisting of human PTH-(1--34) (sequence of Niall et al.) coupled to cellulose. A sensitive homologous amino-terminal specific assay was developed in this way.

Lack of effect on ovariectomy on the metabolism of vitamin D and intestinal calcium-binding protein in female rats.

The metabolism of 25-hydroxycholecalciferol (25-(OH)D3), plasma concentration of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) and the amount of calcium-binding protein (CaBP) in duodenal mucosa were determined in ovariectomized rats and were compared with data observed in normal age-matched cyclic rats. Sephadex LH-20 and high-pressure liquid chromatography were used for the study of the metabolism of 25-(OH)D3. The concentration of 1,25-(OH)2D3 in plasma and prolactin in serum were measured by radioimmunoassay. Calcium-binding protein in duodenal mucosa was determined immunologically using electroimmunodiffusion. The results showed that the lack of ovarian hormones and low prolactin levels observed in ovariectomized rats did not promote a significant change in the metabolism of 25-(OH)D3, in the levels of 1,25-(OH)2D3 in the circulation or in the amount of CaBP in duodenal mucosa. It is possible that the regulation of 25-(OH)D3 by sex hormones is restricted to the state of calcium stress such as during egg-laying in birds or pregnancy and lactation in mammals.

Region-specific immunoassays for parathyroid hormone.

Immunoassays specific for limited regions of bovine parathyroid hormone were developed in four ways. With the heterogeneous antisera produced by immunizing with intact bovine parathyroid hormone (BPTH 1-84), the specificity of radioimmunoassays could be enhanced by presaturating either with an amino-terminal (BPTH 1-34) or carboxy-terminal (BPTH 53-84) fragment. Then, the antibodies which had not been neutralized reacted exclusively with the opposite end of the molecule, even using [125I]BPTH 1-84 as tracer. With some antisera, the appropriate fragment and intact hormone reacted identically. However, with other antisera, the fragment reacted less well than the intact hormone, possibly because these antisera contain antibodies reacting with the middle of the molcule. Using the labelled fragment ([125I]BPTH 1-34) as tracer, with heterogeneous antisera, radioimmunoassays specific for the amino-terminal region were obtained. With one antiserum, BPTH 1.34 reached identically with the intact hormone, but with another antiserum, the fragment was more reactive than the intact molecule. A region-specific radioimmunoassay was also developed using antibodies produced by immunization with a fragment of the hormone. An antiserum raised against BPTH 1-34 had high affinity for the amino-terminal fragement, but reacted less well with the intact hormone. Immunoradiometric assays, specific for the amino- or carboxy-terminal regions, developed by using immunoadsorbents consisting of a fragment (either BPTH 1-34 or BPTH 53-84) coupled to cellulose. These were used to fractionate 125I-labelled antibodies. With some of these selected antibodies, the appropriate fragment was of lower reactivity than the intact hormone. This may have been due to the presence of an incomplete antigenic site on the fragment, or to conformational differences between the fragment and the corresponding region of the intact hormone. With other selected antibodies the fragment and the intact molecule reacted identically. Careful selection of antisera and of technique is necessary to obtain an assay in which a fragment and the intact hormone behave identically.

Purification of human parathyroid hormone: recent studies and further observations.

During the isolation of human parathyroid hormone there is an extensive loss of immuno-assayble hormone over the successive extraction steps, due in part to the presence of fragments that are soluble in 4% trichloroacetic acid. These fragments are derived from both the amino- and carboxyl-terminal regions of the hormone. The hormonal fractions precipitated with trichloroacetic acid were further purified by gel filtration and ion-exchange chromatography. At the final ion-exchange purification step, some preparations of the hormone eluted in multiple fractions. When the various components were characterized separately by immunoassay, amino acid composition, enzymic cleavage and partial sequence analysis, they were found to be closely comparable, although the most acidic fraction contained a blocked terminal amino group. Extraction of a number of batches of tissue permitted revision of the amino acid composition of human parathyroid hormone. Biosynthetic studies with labelled amino acids confirmed the absence of tyrosine and the presence of phenylalanine and threonine and localized these residues to definite regions of the molecule.

Immunological properties of synthetic human parathyroid hormone 53--84 fragment.

The immunological properties of a synthetic peptide comprising the carboxyl-terminal 53--84 region of human parathyroid hormone (PTH) have been studied. The immunoreactivity of the synthetic human PTH-(53--84) peptide paralleled that of a 53--84 fragment of the native human hormone prepared by enzymic digestion, in both a standard radioimmunoassay, which was not region-specific, and also a radioimmunoassay specific for the carboxyl-terminal region of PTH. However, in both types of radioimmunoassay the synthetic human PTH-(53--84) peptide was four to five times more reactive than the native human PTH-(53--84) fragment.

Binding of the receptor for 1,25-dihydroxyvitamin D3 to the 5'-flanking region of the bovine parathyroid hormone gene.

Receptors for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) were prepared from bovine parathyroid glands and incubated with fragments of DNA of the 5'-flanking region of the bovine parathyroid hormone (PTH) gene covering 1700 base pairs (bp) upstream of the initiation site. In filter binding assays, incubation of the DNA fragment spanning -700 to +50 bp with 200 micrograms cytosolic protein gave 288 +/- 63% (mean +/- S.D.) of binding in the absence of protein. In contrast, there was no significant reaction with the -1350 to -700 bp fragment, nor was there binding of the receptor to a fragment of DNA covering the coding region of the PTH gene. Substitution of bovine serum albumin for the receptor preparation did not induce binding to the -700 to +50 bp fragment. The receptor-binding site was further defined to -700 to -100 bp as deletion of the -100 to +50 bp did not reduce receptor binding. Reaction of receptors further purified by sucrose density ultracentrifugation with a monoclonal antibody in immunoblots revealed a single species with a molecular mass of approximately 50,000 Da, which was absent in preparations of cos-1 cells. Autoradiography following incubation of receptors immobilized on nitrocellulose filters with the -700 to +50 bp fragment indicated a single reactive band coincident with the band in the immunoblot. The DNA fragment did not bind to filters containing preparations of cos-1 cells. Extraction of the receptors in the presence or absence of 1,25-(OH)2D3 (4 nmol/l) or the presence of KCl (150 mmol/l) in the incubation medium had no significant effect on DNA binding to the protein in this assay. (ABSTRACT TRUNCATED AT 250 WORDS)

Carcinoid tumour of stomach and primary hyperparathyroidism: a new association.

Three cases of carcinoid tumour of the stomach associated with primary hyperparathyroidism had the clinical and pathological features of a pluriglandular syndrome. Two of the patients showed multiple small polypoid carcinoids in the non-antral stomach, in conjunction with a parathyroid adenoma in one and parathyroid hyperplasia in the other case. One of these patients was also suffering from pernicious anaemia. A third patient had a large metastasising carcinoid arising in the gastric body and a parathyroid adenoma. Immunohistochemical stains for PGP 9.5 were positive in the carcinoids of all three cases. In all cases the carcinoids showed immunoreactivity for gastrin. A positive family history of endocrine hyperplasia and neoplasia was established in one case. It is suggested that patients with gastrointestinal carcinoids and their families should be evaluated for hyperparathyroidism, and patients with hyperparathyroidism presenting with upper gastrointestinal symptoms should undergo endoscopy to rule out gastric carcinoid tumours.

Scintillation proximity assay for calcitriol in serum without high pressure liquid chromatography.

A rapid isolation step for 1,25-dihydroxyvitamin D3 without high pressure liquid chromatography (HPLC) and a sensitive radioimmunoassay (RIA) have been developed. The time required for extraction and isolation with a combination of Extrelut-1-minicolumns and Sep-Pak silica cartridges from as little as 0.5 ml serum is only 2 h. The assay can be counted after 8 h of incubation. It is performed in the vial that collects the eluate, thus eliminating transfer losses and errors. No separation of bound and free hormone is necessary before beta-counting in the scintillation proximity assay. The detection limit of the assay is 2.7 ng/l. The intra-assay coefficients of variation are 7.3% and 5.2% for samples with calcitriol concentrations of 31 and 148 ng/l, respectively. The inter-assay coefficients of variation are 11.3%, 13.3% and 16.1% for low (16 ng/l), medium (30 ng/l) and high (148 ng/l) control pool samples, respectively. Normal values for calcitriol range from 32 to 80 ng/l. Elderly subjects, patients with reduced kidney function and pregnant women were also evaluated for their calcitriol levels. This assay correlates well with a RIA employing HPLC prepurification and charcoal separation of bound/free calcitriol (r = 0.94).

The effect of hemodialysis, vitamin D metabolites and renal transplantation on the skeletal demineralization associated with renal osteodystrophy: a computerized histomorphometric analysis.

Twenty-three patients with end-stage renal failure treated by hemodialysis or transplantation were followed for up to 10 years. Sequential full thickness iliac crest bone biopsies were obtained to assess the effects on bone disease of hemodialysis, treatment with 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] and 24,25-dihydroxycholecalciferol [24,25-(OH)2D3] and renal transplantation. The biopsies were analyzed by a computerized histomorphometric technique which allowed accurate measurements of calcified bone and osteoid areas. Serum aluminum and parathyroid hormone concentrations were also monitored. Hemodialysis was associated with a loss of calcified bone and an increase in osteoid areas. The progressive bone loss was arrested but not reversed following treatment with either 1,25-(OH)2D3 or 24,25-(OH)2D3. Osteoid area was unchanged or reduced following treatment with 1,25-(OH)2D3 in all but three patients who had serum aluminum concentrations in excess of 5 mumol/l. 24,25-(OH)2D3 was not effective in reducing osteoid area, and combined treatment with 1,25 and 24,25-(OH)2D3 had no effect beyond that expected with 1,25-(OH)2D3 alone. Bone biopsies showed loss of calcified bone and an increase in osteoid areas one year and more after successful renal transplantation in five patients. Nineteen of the 23 patients developed serum aluminum concentrations greater than 3 mumol/l, probably because of the use of oral aluminum hydroxide as a phosphate binding agent. In these patients serum parathyroid hormone concentrations greater than 600 pg/ml appeared to prevent the development of osteopenia.