Vitamin D-dependent rickets type II. Defective induction of 25-hydroxyvitamin D3-24-hydroxylase by 1,25-dihydroxyvitamin D3 in cultured skin fibroblasts.

UNLABELLED: 1,25(OH)2D3 induces 25(OH)D3-24-hydroxylase (24-OHase) in cultured skin fibroblasts from normal subjects. We evaluated 24-OHase induction by 1,25(OH)2D3 in skin fibroblasts from 10 normal subjects and from four unrelated patients with hereditary resistance to 1,25(OH)2D or vitamin D-dependent rickets type II (DD II). Fibroblasts were preincubated with varying concentrations of 1,25(OH)2D3 for 15 h and were then incubated with 0.5 microM [3H]25(OH)D3 at 37 degrees C for 30 min; lipid extracts of the cells were analyzed for [3H]24,25(OH)2D3 by high performance liquid chromatography and periodate oxidation. Apparent maximal [3H]24,25(OH)2D3 production in normal cell lines was 9 pmol/10(6) cells per 30 min and occurred after induction with 10(-8) M 1,25(OH)2D3. 24-OHase induction was detectable in normal fibroblasts at approximately 3 X 10(-10) M 1,25(OH)2D3. [3H]24,25(OH)2D3 formation after exposure to 1,25(OH)2D3 was abnormal in fibroblasts from all four patients with DD II. In fibroblasts from two patients with DD II, [3H]24,25(OH)2D3 formation was unmeasurable (below 0.2 pmol/10(6) cells per 30 min) at 1,25(OH)2D3 concentrations up to 10(-6) M. Fibroblasts from the other two patients with DD II required far higher than normal concentrations of 1,25(OH)2D3 for detectable [3H]24,25(OH)2D3 induction. In one, [3H]24,25(OH)2D3 production reached 2.9 pmol/10(6) cells per 30 min at 10(-6) M 1,25(OH)2D3 (30% normal maximum at 10(-6) M 1,25(OH)2D3). In the other, [3H]24,25(OH)2D3 production achieved normal levels, 7.3 pmol/10(6) cells per 30 min after 10(-6) M 1,25(OH)2D3. The two patients whose cells had a detectable 24-OHase induction by 1,25(OH)2D3 showed a calcemic response to high doses of calciferols in vivo. Our current observations correlate with these two patients' responsiveness to calciferols in vivo and suggest that their target organ defects can be partially or completely overcome with extremely high concentrations of 1,25(OH)2D3. The two patients whose cells showed no detectable 24-OHase induction in vitro failed to show a calcemic response to high doses of calciferols in vivo. IN CONCLUSION: (a) the measurement of 24-OHase induction by 1,25(OH)2D3 in cultured skin fibroblasts is a sensitive in vitro test for defective genes in the 1,25(OH)2D effector pathway. (b) This assay provides a useful tool for characterizing the target tissue defects in DD II and predicting response to calciferol therapy.

Evidence of extraocular muscle restriction in autoimmune thyroid disease.

Patients with Graves' disease lacking eye symptoms frequently have abnormal intraocular pressure (IOP) increases on upward gaze (greater than or equal to 3 mm Hg) indicative of apparent subclinical ophthalmopathy. Because of the close relationship between Graves' disease (GD) and Hashimoto's thyroiditis (HT), we examined 30 patients with a history of HT as well as 26 patients with a history of GD, 4 patients with a history of subacute thyroiditis, 1 patient with a history of silent thyroiditis, and 25 normal subjects for the presence of IOP abnormalities at 15 degrees and 25 degrees upgaze. While all of the patients were asymptomatic, had no exophthalmos, and were euthyroid at the time of the exam, Hertel exophthalmometer readings (mean +/- SD) for the patients with GD were significantly higher (P less than 0.005) than those for either the HT patients or normal subjects (17.1 +/- 2.4 vs. 14.5 +/- 2.3 vs. 14.4 +/- 4.2 mm, respectively). At 15 degrees upgaze, IOP abnormalities occurred in 25% and 13% of patients with GD and HT, respectively. At 25 degrees upgaze, these figures rose to 54% for the GD patients and 37% in HT patients. Only 1 of 25 normal subjects had elevated IOP changes on upgaze, as did the 1 patient with silent thyroiditis, but the patients with subacute thyroiditis did not. These data suggest the frequent presence of extraocular muscle restriction in patients with a history of HT as well as in patients with a history of GD. Maximal detection of these IOP abnormalities requires that patients be examined at 25 degrees upgaze. These data support the belief that the autoimmune bases of both GD and HT are closely linked, at least as manifested by eye muscle involvement.

Diabetic ketoacidosis presenting with a normal anion gap.

Historically, diabetic ketoacidosis has been attributed exclusively to the accumulation of ketoacids resulting in wide anion gap metabolic acidosis. An unusual patient with non-insulin-dependent diabetes mellitus who presented with mild hyperglycemia, ketonemia, and acidosis with a normal anion gap is described. No other known causes of hyperchloremic metabolic acidosis were present, and resolution of acidosis followed administration of intravenous insulin. This case illustrates that the finding of a normal anion gap, per se, may not exclude the presence of diabetic ketoacidosis.

Angiotensin-converting enzyme: characteristics in human skin fibroblasts.

Angiotensin-converting enzyme, although most prominent in vascular endothelium, has been identified in numerous tissues. Recent studies have indicated that several hormones, including glucocorticoids and thyroid hormone, may affect the activity of this enzyme. In the present study, angiotensin-converting enzyme was examined in homogenates of cultured human skin fibroblasts. Angiotensin-converting enzyme activity was measured by a radiometric assay using [Glycine-1-14C] Hippuryl-L-histidyl-L-leucine (1.1 mmol/L) as substrate, and was expressed as nmol hippuric acid formed per minute/mg protein. Angiotensin-converting enzyme was identified in all five cell strains tested, and the activity observed was 0.97 +/- 0.18 nmol/min/mg protein (mean +/- SE). The optimum pH was between 6.9 and 7.6, and optimum temperature was 37 degrees C, with loss of activity of 55 degrees C and higher. Buffer strength was optimized at Tris 0.025 mol/L, and 1.0 mol/L NaCl. Activity increased linearly with protein concentration and with time, and the Km = 1.14 mmol/L. The most potent inhibitor of fibroblast ACE was captopril (SQ 14,225) with an IC50 = 10(-10) mol/L; other inhibitors included SQ 20,881, EDTA, and phenanthroline. Competitive substrates included angiotensin-I, substance P, and bradykinin. Four hormones, T3 (10(-9)-10(-7) mol/L), 1,25 (OH)2D3 (10(-8)-10(-7) mol/L), dexamethasone (10(-7)-10(-6) mol/L), and a synthetic androgen, R1881 (10(-8)-10(-7) mol/L) were incubated with cells for 72 hours. In all incubations, there was no significant effect on cellular ACE activity induced by any agent. Angiotensin-converting enzyme activity in serum free media was less than 1% of cell activity and was unaltered by hormone treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Whole cell and nuclear androgen uptake in skin fibroblasts from infertile men.

In order to reexamine the hypothesis that a high percentage of infertile men with oligo/azoospermia have androgen resistance due to androgen receptor abnormalities, both whole cell and nuclear uptake of [3H]R1881 (a synthetic, nonmetabolizable androgen) were measured in intact, dispersed fibroblasts cultured from pubic skin biopsy specimens of 15 men selected because of infertility associated with varying degrees of oligozoospermia. Eight men had sperm densities less than or equal to 2 X 10(6)/ml; 7 were greater than 2 X 10(6)/ml. Serum levels of FSH and LH were elevated in the severely oligo/azoospermic group, but normal in the other infertile men; concentrations of testosterone, estradiol, and prolactin were normal in both groups. The controls were six normal, age-matched, fertile males. There was no difference in binding capacity or dissociation constant for androgen uptake either into whole cells (3940 +/- 940 [mean +/- SE] sites/cell vs. 4700 +/- 1120 sites/cell, P = NS) or into nuclei (1360 +/- 340 sites/cell vs. 1460 +/- 340 sites/cell, P = NS) of the fibroblasts from the patients vs. the controls, respectively. Furthermore, there was no correlation between patient sperm densities and fibroblast whole cell or nuclear uptake binding capacities. Finally, there was no difference in any androgen binding parameter when only the fibroblasts from the men with severe oligozoospermia or azoospermia were compared with the controls. The authors conclude that the infertility of men with severe testicular germ cell depletion cannot be accounted for by a quantitative androgen receptor abnormality in their pubic skin fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Simulation of a prolactin-secreting adenoma by an intrasellar craniopharyngioma.

We report here an unusual case of a 21-year-old woman who presented with amenorrhea, galactorrhea, and hyperprolactinemia (66 to 81 ng/ml) secondary to an unsuspected intrasellar craniopharyngioma. The results of preoperative endocrine testing were compatible with the presence of a prolactin-secreting adenoma. Moreover, high resolution computed tomographic scanning revealed an uncalcified hypodense sellar mass that did not enhance after contrast administration, a feature shared by many prolactinomas. This report illustrates that amenorrhea, galactorrhea, mild to moderate hyperprolactinemia, and a sellar mass should not automatically be attributed to the presence of a prolactin-secreting adenoma.

Prevalence of increased intraocular pressure in Graves' disease--evidence of frequent subclinical ophthalmopathy.

Graves' exophthalmos is frequently associated with elevated intraocular pressure on upgaze. Eighty patients with Graves' disease were evaluated prospectively by applanation tonometry to assess the prevalence of ophthalmopathy in this disorder. Whereas 21 (26 per cent) of the 80 patients had exophthalmos, 61 (76 per cent) had abnormal intraocular pressure (delta greater than or equal to 3 mm Hg). All patients with exophthalmos had elevated intraocular pressure on upgaze; 40 (68 per cent) of 59 patients without proptosis had abnormal pressure readings. The mean interval between the onset of Graves' disease and this study was 6.3 +/- 1.0 years (S.E.M.) for those patients who had exaggerated positional changes in intraocular pressure, as compared with 3.0 +/- 1.0 years for those with normal intraocular pressure (P less than 0.005). All but 1 of 15 patients in whom the diagnosis of Graves' disease had been documented 10 or more years earlier had increased intraocular pressure on upgaze. We conclude that Graves' ophthalmopathy is more common than is recognized clinically and that eye involvement is an inevitable complication of the disease.