Dietary fat may modulate adipose tissue homeostasis through the processes of autophagy and apoptosis.

PURPOSE: Obesity increases the risk of cardiovascular disease, type 2 diabetes mellitus and cancer development. Autophagy and apoptosis are critical processes for development and homeostasis in multicellular organisms and have been linked to a variety of disorders. We aimed to investigate whether the quantity and quality of dietary fat can influence these processes in the adipose tissue of obese people. METHODS: A randomized, controlled trial within the LIPGENE study assigned 39 obese people with metabolic syndrome to 1 of 4 diets: (a) a high-saturated fatty acid diet, (b) a high-monounsaturated fatty acid (HMUFA) diet, and (c, d) two low-fat, high-complex carbohydrate diets supplemented with long-chain n-3 polyunsaturated fatty acids (LFHCC n-3) or placebo (LFHCC), for 12 weeks each. RESULTS: We found an increase in the expression of autophagy-related BECN1 and ATG7 genes after the long-term consumption of the HMUFA diet (p = 0.001 and p = 0.004, respectively) and an increase in the expression of the apoptosis-related CASP3 gene after the long-term consumption of the LFHCC and LFHCC n-3 diets (p = 0.001 and p = 0.029, respectively). CASP3 and CASP7 gene expression changes correlated with HOMA index. CONCLUSION: Our results suggest that the processes of autophagy and apoptosis in adipose tissue may be modified by diet and that the consumption of a diet rich in monounsaturated fat may contribute to adipose tissue homeostasis by increasing autophagy. They also reinforce the notion that apoptosis in adipose tissue is linked to insulin resistance. CLINICAL TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT00429195.

Vitamin E protection of obesity-enhanced vascular calcification in uremic rats.

This study aimed to determine the extent of extraskeletal calcification in uremic Zucker rats, by comparing obese and lean phenotypes, and to evaluate the influence of vitamin E (VitE) on the development of calcifications in both uremic rats and human vascular smooth muscle cells (HVSMCs) cultured in vitro. Zucker rats of lean and obese phenotypes with normal renal function [control (C); C-lean and C-obese groups] and with uremia [5/6 nephrectomy (Nx); Nx-lean and Nx-obese groups] and uremic rats treated with VitE (Nx-lean + VitE and Nx-obese + VitE groups) were studied. Uremic groups were subjected to Nx, fed a 0.9% phosphorus diet, and treated with calcitriol (80 ng/kg ip). The aortic calcium concentration was significantly higher (P < 0.05) in Nx-obese rats (10.0 ± 2.1 mg/g tissue) than in Nx-lean rats (3.6 ± 1.3 mg/g tissue). A decrease in plasma glutathione peroxidase activity was observed in Nx-obese rats compared with Nx-lean rats (217.2 ± 18.2 vs. 382.3 ± 15.5 nmol·min(-1)·ml(-1), P < 0.05). Treatment with VitE restored glutathione peroxidase activity and reduced the aortic calcium concentration to 4.6 ± 1.3 mg/g tissue. The differences in mineral deposition between Nx-lean, Nx-obese, Nx-lean + VitE, and Nx-obese + VitE rats were also evidenced in other soft tissues. In HVSMCs incubated with high phosphate, VitE also prevented oxidative stress and reduced calcium content, bone alkaline phosphatase, and gene expression of core-binding factor-α1. In conclusion, uremic obese rats develop more severe calcifications than uremic lean rats and VitE reduces oxidative stress and vascular calcifications in both rats and cultures of HVSMCs.

[In vitro dynamics of parathyroid hormone secretion regulated by calcium and effects on the cell cycle: parathyroid hyperplasia versus adenoma]. / Dinámica in vitro de la secreción de hormona paratiroidea regulada por calcio y efecto sobre el ciclo celular: adenoma frente a hiperplasia paratiroidea.

AIM: To compare the dynamics of calcium-regulated PTH secretion in vitro from adenomatous versus hyperplastic glands and to investigate the relationship between the parathyroid cell cycle and the calcium-regulated PTH secretion in these glands. MATERIALS AND METHODS: A total of 31 parathyroid glands (8 adenomatous and 23 hyperplastic) from 8 patients with primary hyperparathyroidism and 7 with secondary hyperparathyroidism respectively were studied. For the evaluation of calcium-regulated PTH secretion, small parathyroid pieces of 1 mm were sequentially transferred to wells with varying Ca concentrations: 0.4, 0.6, 0.8, 1, 1.25 and 1.35 or 1.5 mM. PTH concentrations were determined in the medium. For the parathyroid cell cycle studies, parathyroid cells were isolated without the use of enzymes and cell cycle was analyzed using the method described by Vindelov. The nuclei were acquired by flow cytometer and analyzed using the CELLFIT software. RESULTS: In parathyroid tissues from hyperplastic glands, the increase in extracellular calcium produced a decrease in PTH secretion which was apparent with a calcium level as low as 0.8 mM and the maximal inhibition of PTH secretion was obtained with a calcium of 1.25 mM, by the contrary, adenomatous glands required a calcium of 1.2 mM to produce a minimal decrease in PTH secretion. In hyperplastic parathyroid glands but not in parathyroid adenomas there was a significant correlation between the percentage of cells in G0/G1 phase with the set point (r = 0.914; P < 0.005) and the basal serum Ca (r = 0.862; P < 0.02). CONCLUSIONS: The control of the extracellular calcium-PTH release in vitro is less sensitive in parathyroid adenomas than hyperplasic parathyroid glands. In parathyroid hyperplasia the cell proliferation may be regulated by the extracellular calcium concentration (higher calcemia less proliferation).

[Dynamics of calcium-regulated PTH secretion in secondary hyperparathyroidism: comparison between "in vivo" vs. "in vitro" responses]. / Dinámica de la secreción de PTH regulada por calcio en el hiperparatiroidismo secundario: comparación de la respuesta in vivo frente a in vitro.

AIM: To compare the dynamics in vivo and in vitro calcium-PTH release of uremic patients with secondary hyperparathyroidism and their hyperplasic parathyroid glands after parathyroidectomy. MATERIALS AND METHODS: Seven patients with secondary HPT and their 23 hyperplasic glands obtained after surgical parathyroidectomy were evaluated. In vivo studies of the PTH secretion curve were obtained by induction of hypocalcemia and hypercalcemia with a continuous endovenous infusion of sodium EDTA and Ca gluconate, respectively. For the in vitro studies, small parathyroid pieces of 1 mm were sequentially transferred to wells with varying Ca concentrations: 0.4, 0.6, 0.8, 1, 1.25 and 1.5 mM. iPTH concentrations were determined in the medium. RESULTS: The in vivo set point did not correlate with the basal, maximal or minimal PTH concentrations, although it correlated significantly with the basal serum Ca concentration (r = 0.62, p < 0.02). Both in vivo and in vitro PTH secretion curves were sigmoidal, although the in vivo set point was higher than the in vitro (1.57 +/- 0.05 vs. 1.27 +/- 0.07 mM, p < 0.001). The degree of maximal PTH inhibition were similar in both circumstances (30.5 +/- 8.1 vs. 33.6 +/- 5.4 %; p = NS) with a significant direct correlation (r = 0.901; p < 0.01). CONCLUSIONS: The in vivo set point of calcium is more closely related to serum calcium concentration than to basal iPTH concentration. Although there are differences between the in vivo and in vitro calcium set point the maximal degree of PTH inhibition was similar in both circumstances.

The calcimimetic R-568 increases vitamin D receptor expression in rat parathyroid glands.

We previously demonstrated that extracellular calcium regulates vitamin D receptor (VDR) expression by parathyroid cells. Since the calcimimetic R-568 potentiates the effects of calcium on the calcium-sensing receptor, it was hypothesized that administration of R-568 may result in increased VDR expression in parathyroid tissue. In vitro studies of the effect of R-568 on VDR mRNA and protein were conducted in cultures of whole rat parathyroid glands and human hyperplastic parathyroid glands. In vivo studies in Wistar rats examined the effect of R-568 and calcitriol alone and in combination. Incubation of rat parathyroid glands in vitro with R-568 (0.001-1 microM) resulted in a dose-dependent decrease in parathyroid hormone (PTH) secretion and an increase in VDR expression (mean +/- SE). Incubation in 1 mM calcium + 0.001 microM R-568 elicited an increase in VDR mRNA (306 +/- 46%) similar to the maximum increase detected with 1.5 mM calcium (330 +/- 42%). In vivo, VDR mRNA was increased after administration of R-568 (168 +/- 9%, P < 0.001 vs. control) or calcitriol (198 +/- 16%, P < 0.001 vs. control). Treatment with R-568 also increased VDR protein in normal rat parathyroid glands and in human parathyroid glands with diffuse, but not nodular, hyperplasia. In conclusion, the present study shows that the calcimimetic R-568 exerts a stimulatory effect on VDR expression in the parathyroid glands of study models and provides additional evidence for the use of calcimimetics in the treatment of secondary hyperparathyroidism.

Both duration and degree of hypercalcemia influence the reduced parathyroid hormone response to hypocalcemia after hypercalcemia.

The stimulation of parathyroid hormone (PTH) secretion by hypocalcemia is reduced when hypocalcemia is preceded by hypercalcemia. The present study investigates whether the duration and degree of hypercalcemia influence the reduced PTH response to hypocalcemia after hypercalcemia. In addition, the implication of the arachidonic acid (AA) signaling pathway in this effect is evaluated. The PTH response to hypocalcemia has been studied in a control group and in four groups of rabbits subjected to hypercalcemia for different periods of time (between 30 and 120 min) and at two levels of hypercalcemia (1 x 9 and 2 x 1 mM). AA levels have been measured in parathyroid glands from rabbits subjected to hyper- and hypocalcemia. When compared with controls, rabbits that had been hypercalcemic (2 x 1 mM) for 2 h showed a markedly attenuated PTH response to hypocalcemia (50% of normal PTHmax), rabbits that had been in hypercalcemia (2 x 1 mM) for 75 min had an intermediate PTH response to hypocalcemia (70% of normal PTHmax) and rabbits that had been subjected to either 30 min hypercalcemia of 2 x 1 mM or 120 min hypercalcemia of 1 x 9 mM had a normal PTH response to hypocalcemia. AA levels increased in hypercalcemia and decreased in hypocalcemia; however, no differences were observed at either calcium level in short-time (30 min) versus long-time (120 min) hypercalcemia. In conclusion, the attenuated PTH response to hypocalcemia after hypercalcemia is dependent on both the period of time that the parathyroid glands have been exposed to hypercalcemia and the degree of hypercalcemia. In addition, this reduced PTH response does not seem to be related to changes in the AA signaling pathway.

(99m)Tc-sestamibi scintigraphy and cell cycle in parathyroid glands of secondary hyperparathyroidism.

Double-phase parathyroid MIBI ((99m)Tc-sestamibi) was performed in 27 patients with secondary hyperparathyroidism (SPT). Focal areas of increased uptake were scored for intensity on a three-point scale. All patients underwent subtotal parathyroidectomy (SPTx), and a total of 78 glands were removed at operation. Blood was obtained from the jugular vein before and after SPTx to measure the parathyroid hormone (PTH) levels. The volume and weight of the glands were calculated. The tissue was divided, with one aliquot being used for cell cycle analysis. The nuclei were acquired by flow cytometry and analyzed using CELLEIT software. Cell viability was assessed by flow cytometry and analyzed with LYSIS II software. Positive MIBI uptake was observed in 88.8% of patients. Focal MIBI uptake of one, two, or three glands was observed in 6, 11, and 8 patients, respectively. All patients experienced an 86% decrease in PTH blood level after SPTx compared to that before excision. A correlation was found between the volume of glands and the blood levels of intact PTH (iPTH) (r = 0.5, p < 0.05). A positive correlation was observed between MIBI uptake and the iPTH levels before SPTx (p < 0.01) and between the uptake of MIBI in the parathyroid glands and the cell cycle phases; low-grade uptake correlated with the G(0) phase and higher uptake with G(2)+S phase (r = 7, p < 0.01). No correlation was observed between MIBI uptake and the weight of the glands. MIBI scintigraphy accurately reflects the functional status of the hyperplastic parathyroid glands: Higher uptake grades correlated with the active growth phase. MIBI uptake does not reveal parathyroid enlargement; rather, it identifies the presence of hyperfunctioning autonomous glands. SPTx and total parathyroidectomy with autografting (TPTx+A) are the most widely accepted surgical approaches for patients with SPT. Reoperation for recurrence is necessary in 6% to 15% of cases. MIBI is now considered to be the radionuclide of reference for parathyroid gland scanning, although it is widely accepted that it produces poor results when trying to detect hyperplastic glands.

PTHrP enhances the secretory response of PTH to a hypocalcemic stimulus in rat parathyroid glands.

BACKGROUND: The secretion of parathyroid hormone (PTH) from the parathyroid glands might be regulated by autocrine/paracrine factors, and a feedback regulatory mechanism of PTH on the secretion of PTH has been suggested. Because of the existence of a common receptor between PTH and PTH-related peptide (PTHrP), the aim of the present study was to examine the possible effects of PTHrP 1-40 and 1-86 on PTH secretion in rats. METHODS: In vivo, the effect of PTHrP on Ca++-regulated PTH secretion was examined by the induction of hypocalcemia and hypercalcemia by an infusion of EGTA and Ca++, with and without PTHrP. The eventual effects of PTHrP on the peripheral metabolism of PTH were examined by infusion of human PTH (hPTH) with and without PTHrP. hPTH was measured by an intact hPTH assay not cross reacting with rat PTH or PTHrP. To examine whether near physiological levels of circulating PTH have an autoregulatory effect in vivo on PTH secretion from the parathyroid gland, an acute reduction of the circulating PTH was induced by an acute unilateral parathyroidectomy (UPTX). PTH secretion from the remaining parathyroid gland was followed in response to EGTA-induced hypocalcemia. In vitro investigations on the effect of PTHrP 1-40 on PTH secretion from whole rat parathyroid glands incubated in media containing a calcium concentration of 0.6 or 1.35 mmol/L were performed to confirm whether the effect of PTHrP was directly on the gland. The rat PTH assay was examined for cross reaction with PTHrP. RESULTS: In vivo, the same rate of decrease of plasma Ca++ was induced in the experimental groups. The maximal response of PTH to hypocalcemia (218 +/- 16 pg/mL, N = 6) was significantly enhanced by PTHrP 1-40 (525 +/- 79 pg/mL, N = 6) and by PTHrP 1-86 (465 +/- 29 pg/mL, N = 6, P < 0.001). No effect of PTHrP on PTH secretion was found during normocalcemia or hypercalcemia. UPTX resulted in a 50% reduction of PTH secretion, and no compensatory increase of PTH was observed. PTHrP had no effect on the metabolism of PTH. In vitro, low-Ca++-induced PTH secretion was significantly augmented by 300% (P < 0.01) when the medium contained PTHrP 1-40. PTHrP did not cross react with the PTH assay. CONCLUSIONS: PTHrP significantly enhanced the low-Ca++-stimulated PTH secretion in vivo and in vitro. An autocrine/paracrine role of PTHrP in the parathyroid glands is suggested. An autoregulatory effect of circulating PTH on the PTH secretion from parathyroid glands seems unlikely. The "maximal secretory capacity" of the parathyroid glands induced by hypocalcemia in vivo and in vitro is not the maximum, as PTH secretion can be increased even further, by several-fold.

Effect of a low calcium dialysate on parathyroid hormone secretion in diabetic patients on maintenance hemodialysis.

Diabetic patients on maintenance dialysis often are characterized by a relative parathyroid hormone (PTH) deficiency and a form of renal osteodystrophy with low bone turnover known as adynamic bone. The goal of the present study was to determine whether a reduction in the dialysate calcium concentration would increase the predialysis (basal) PTH and maximal PTH level. Thirty-three diabetic maintenance hemodialysis patients with basal PTH values less than 300 pg/ml were randomized to be dialyzed with either a regular (3.0 mEq/liter or 3.5 mEq/liter, group I) or low (2.25 mEq/liter or 2.5 mEq/liter, group II) calcium dialysate for 1 year. At baseline and after 6 months and 12 months of study, low (1 mEq/liter) and high (4 mEq/liter) calcium dialysis studies were performed to determine parathyroid function. At baseline, basal (I, 126+/-20 vs. II, 108+/-19 pg/ml) and maximal (I, 269 pg/ml+/-40 pg/ml vs. II, 342 pg/ml+/-65 pg/ml) PTH levels were not different. By 6 months, basal (I, 98+/-18 vs. II, 200+/-34 pg/ml, p = 0.02) and maximal (I, 276 pg/ml+/-37 pg/ml vs. II, 529 pg/ml+/-115 pg/ml; p = 0.05) PTH levels were greater in group II. Repeated measures analysis of variance (ANOVA) of the 20 patients who completed the entire 12-month study showed that only in group II patients were basal PTH (p = 0.01), maximal PTH (p = 0.01), and the basal/maximal PTH ratio (p = 0.03) different; by post hoc test, each was greater (p < 0.05) at 6 months and 12 months than at baseline. When study values at 0, 6, and 12 months in all patients were combined, an inverse correlation was present between basal calcium and both the basal/maximal PTH ratio (r = -0.59; p < 0.001) and the basal PTH (r = -0.60; p < 0.001). In conclusion, in diabetic hemodialysis patients with a relative PTH deficiency (1) the use of a low calcium dialysate increases basal and maximal PTH levels, (2) the increased secretory capacity (maximal PTH) during treatment with a low calcium dialysate suggests the possibility of enhanced parathyroid gland growth, and (3) the inverse correlation between basal calcium and both the basal/maximal PTH ratio and the basal PTH suggests that the steady-state PTH level is largely determined by the prevailing serum calcium concentration.

Effect of phosphate on parathyroid hormone secretion in vivo.

Alterations in phosphate homeostasis play an important role in the development of secondary hyperparathyroidism in renal failure. Until recently, it was accepted that phosphate retention only increased parathyroid hormone (PTH) secretion through indirect mechanisms affecting calcium regulation and calcitriol synthesis. However, recent in vitro studies have suggested that phosphate may directly affect PTH secretion. Our goal was to determine whether in vivo an intravenous phosphate infusion stimulated PTH secretion in the absence of changes in serum calcium. Three different doses of phosphate were infused intravenously during 120 minutes to increase the serum phosphate concentration in dogs. Sulfate was also infused intravenously as a separate experimental control. A simultaneous calcium clamp was performed to maintain a normal ionized calcium concentration throughout all studies. At the lowest dose of infused phosphate (1.2 mmol/kg), serum phosphate values increased to approximately 3 mM, but PTH values did not increase. At higher doses of infused phosphate (1.6 mmol/kg and 2.4 mmol/kg), the increase in serum phosphate to values of approximately 4 mM and 5 mM, respectively, was associated with increases in PTH, even though the ionized calcium concentration did not change. Increases in PTH were not observed until 30-60 minutes into the study. These increases were not sustained, since by 120 minutes PTH values were not different from baseline or controls despite the maintenance of marked hyperphosphatemia. During the sulfate infusion, serum sulfate values increased by approximately 3-fold, but no change in PTH values were observed. In conclusion, an acute elevation in serum phosphate stimulated PTH secretion in the intact animal, but the magnitude of hyperphosphatemia exceeded the physiologic range. Future studies are needed to determine whether PTH stimulation is more sensitive to phosphate loading in states of chronic phosphate retention. Moreover, the mechanisms responsible for the delay in PTH stimulation and the failure to sustain the increased PTH secretion need further evaluation.

Effect of calcitriol and age on recovery from hypocalcemia in hemodialysis patients.

Calcitriol is used to treat hyperparathyroidism in hemodialysis patients. Calcitriol treatment, either through a reduction in parathyroid hormone (PTH) levels or direct effect on bone, decreases the osteoblast and osteoclast surface and bone formation rate. Our study of 13 hemodialysis patients was designed to evaluate whether calcitriol treatment changed the rate of spontaneous recovery from hypocalcemia induced by a low-calcium dialysis. Calcitriol treatment decreased basal PTH levels from 614 +/- 84 to 327 +/- 102 pg/mL (P < 0.001) and maximal PTH levels from 1,282 +/- 157 to 789 +/- 161 pg/mL (P < 0.001), but the rate of serum ionized calcium recovery from hypocalcemia did not change. When the 13 patients were separated based on the median age of 64 years, the predialysis serum ionized calcium level was less in the younger (group I, 44 +/- 6 years; n = 6) than older (group II, 68 +/- 1 years; n = 7) patients (1.05 +/- 0.03 v 1.22 +/- 0.03 mmol/L, respectively; P < 0.01) despite similar basal (group I, 595 +/- 122 pg/mL v group II, 629 +/- 96 pg/mL) and maximal (group I, 1,114 +/- 299 pg/mL v group II, 1,425 +/- 141 pg/mL) PTH levels. Before calcitriol treatment, the rate of serum ionized calcium recovery from induced hypocalcemia was greater (P < 0.05) for similar PTH levels in the older than younger patients. After calcitriol treatment, despite a similar reduction in PTH levels, the rate of calcium recovery increased (P < 0.05) in the younger patients but did not change in the older patients. We also observed that toward the end of the low-calcium hemodialysis, PTH values decreased even though serum ionized calcium level continued to decline when the rate of calcium reduction slowed. In addition, hysteresis, defined as a lower PTH value during the recovery from hypocalcemia than during the induction of hypocalcemia for the same serum calcium concentration, was present during the spontaneous recovery from hypocalcemia. In conclusion, in the hemodialysis patient: (1) age appeared to affect the bone response to PTH and calcitriol treatment, (2) the PTH response to hypocalcemia was affected by a deceleration in the rate of calcium decrease, and (3) hysteresis of the PTH response to hypocalcemia occurred during the spontaneous recovery from hypocalcemia.

Effect of rate of calcium reduction and a hypocalcemic clamp on parathyroid hormone secretion: a study in dogs.

BACKGROUND: The parathyroid hormone (PTH) calcium curve is used to evaluate parathyroid function in clinical studies. However, unanswered questions remain about whether PTH secretion is affected by the rate of calcium reduction and how the maximal PTH response to hypocalcemia is best determined. We performed studies in normal dogs to determine whether (a) the rate of calcium reduction affected the PTH response to hypocalcemia and (b) the reduction in PTH values during a hypocalcemic clamp from the peak PTH value observed during the nadir of hypocalcemia was due to a depletion of stored PTH. METHODS: Fast (30 min) and slow (120 min) ethylenediamine-tetraacetic acid (EDTA) infusions were used to induce similar reductions in ionized calcium. In the fast EDTA infusion group, serum calcium was maintained at the hypocalcemic 30-minute value for an additional 90 minutes (hypocalcemic clamp). To determine whether the reduction in PTH values during the hypocalcemic clamp represented depletion of PTH stores, three subgroups were studied. Serum calcium was rapidly reduced from established hypocalcemic levels in the fast-infusion group at 30 and 60 minutes (after 30 min of a hypocalcemic clamp) and in the slow-infusion group at 120 minutes. RESULTS: At the end of the fast and slow EDTA infusions, serum ionized calcium values were not different (0.84 +/- 0.02 vs. 0.82 +/- 0.03 mM), but PTH values were greater in the fast-infusion group (246 +/- 19 vs. 194 +/- 13 pg/ml, P < 0.05). During the hypocalcemic clamp, PTH rapidly decreased (P < 0.05) to value of approximately 60% of the peak PTH value obtained at 30 minutes. A rapid reduction in serum calcium from established hypocalcemic levels at 30 minutes did not stimulate PTH further, but also PTH values did not decrease as they did when a hypocalcemic clamp was started at 30 minutes. At 60 minutes, the reduction in serum calcium increased (P < 0.05) PTH to peak values similar to those before the hypocalcemic clamp. The reduction in serum calcium at 120 minutes in the slow EDTA infusion group increased PTH values from 224 +/- 11 to 302 +/- 30 pg/ml (P < 0.05). CONCLUSIONS: These results suggest that (a) the reduction in PTH values during the hypocalcemic clamp may not represent a depletion of PTH stores. (b) The use of PTH values from the hypocalcemic clamp as the maximal PTH may underestimate the maximal secretory capacity of the parathyroid glands and also would change the analysis of the PTH-calcium curve, and (c) the PTH response to similar reductions in serum calcium may be less for slow than fast reductions in serum calcium.

Measurement of parathyroid hormone in horses.

Measurement of parathyroid hormone (PTH) in horses was performed on plasma samples using 2 immunoradiometric assays: a human intact PTH assay and a rat amino-terminal PTH assay. The assays were validated by assessment of their precision, sensitivity and specificity, and also by evaluating PTH changes in the horse in response to variation in blood ionised calcium. Intra- and inter-assay variance, precision and sensitivity were similar for both human and rat assays; however, the rat assay was slightly more precise and sensitive than the human assay. Both assays detected an increase in PTH levels in the horse when blood ionised calcium was decreased and a decline in PTH concentration with hypercalcaemia. Measurement of PTH concentration in samples from healthy horses with the human assay yielded a mean (+/-s.e.) value of 31.3+/-4.1 pg/ml. When using the rat assay, PTH values were 44.1+/-5.3 pg/ml. Plasma samples held for up to 3 months at -20 degrees C did not show a significant change in PTH concentration. In conclusion, the human intact PTH and the rat amino-terminal assays detected equine PTH and can be used for measurement of this hormone in horses. Quantification of equine PTH using these assays will allow more precise diagnosis of a variety of disorders affecting mineral metabolism in horses.

High phosphate level directly stimulates parathyroid hormone secretion and synthesis by human parathyroid tissue in vitro.

Phosphate retention plays an important role in the pathogenesis of secondary hyperparathyroidism in patients with renal failure. In in vitro studies, high extracellular phosphate levels directly stimulate PTH secretion in rat and bovine parathyroid tissue. The present study evaluates the effect of high phosphate levels on the secretion of PTH and the production of prepro PTH mRNA in human hyperplastic parathyroid glands. The study includes parathyroid glands obtained from patients with primary adenomas and from hemodialysis and kidney-transplant patients with diffuse and nodular secondary hyperplasia. The experiments were performed in vitro using small pieces of parathyroid tissue. The ability of high calcium levels to decrease PTH secretion was less in adenomas than in secondary hyperplasia; among the secondary hyperplasia, nodular was less responsive to an increase in calcium than diffuse hyperplasia. In diffuse hyperplasia, PTH secretion was increased in response to 3 and 4 mM phosphate compared with 2 mM phosphate, despite a high calcium concentration in the medium; prepro PTH mRNA levels increased after incubation in 4 mM phosphate. Similar results were obtained with nodular hyperplasia, except that the elevation of PTH secretion in response to 3 mM phosphate did not attain statistical significance. In adenomas, high calcium concentrations (1.5 mM) did not result in inhibition of PTH secretion, independent of the phosphate concentration, and the prepro PTH mRNA was not significantly increased by high phosphate levels. In conclusion, first, the PTH secretory response to an increase in calcium concentration is less in nodular than diffuse hyperplasia; second, high phosphate levels directly affect PTH secretion and gene expression in patients with advanced secondary hyperparathyroidism.

Mineral metabolism in healthy geriatric dogs.

To study mineral metabolism in geriatric dogs, parathyroid hormone, calcitriol, ionised calcium, phosphorus, blood urea nitrogen and creatinine were evaluated in 35 geriatric dogs (> 10 years) and in 20 young adult dogs (2-5 years). Parathyroid hormone levels were within the normal range in both groups, but values (mean +/- SEM) were greater in the old dogs (34.8 +/- 3.6 vs 21.2 +/- 2.3 pg ml(-1), P=0.005). Calcitriol and ionised calcium were similar in the two groups, and the values for both parameters were within the normal reference range. Plasma phosphorus levels were in the normal range in both groups but tended to be greater in the older dogs (P=0.09). While blood urea nitrogen was similar in the two groups, creatinine levels (mean +/- SEM) were higher in the young dogs (82.2 +/- 3.5 vs 101.7 +/- 4.4 micromol litre(-1)). Even when the dogs were matched for weight, plasma creatinine concentration was still greater in the younger dogs. In conclusion, an increase in parathyroid hormone without changes in calcium, phosphorus and calcitriol has been identified in geriatric dogs.

The effect of a high phosphorus diet on the parathyroid cell cycle.

High extracellular phosphorus directly increases parathyroid hormone (PTH) secretion and gene transcription. The present study evaluates the effect of high phosphorus diet on the parathyroid cell cycle in rats with normal renal function. Rats were divided into two groups, receiving either a high phosphorus diet (HPD, P=1.2%) or a normal phosphorus diet (NPD, P=0.6%). The dietary calcium content was 0.6% in both diets. Rats were pair fed and sacrificed on days 0, 1, 5, 10 and 15 after initiation of the diet. The parathyroid glands were removed and parathyroid cells dispersed for evaluation of cell cycle and apoptosis by flow cytometry. Serum calcium, phosphorus, PTH and calcitriol were measured. As compared with NPD, the ingestion of a HPD resulted in an increased number of cells in the S phase of the cell cycle from day 1 to 10 (1.2+/-0.09% vs 0.6+/-0.04% for day 1, 1.2+/-0.11% vs 0.6+/-0.06% for day 5 and 1.0+/-0.09% vs 0.5+/-0.04% for day 10, P<0.01). By day 15, the percentage of cells in the S phase in NPD and HPD were not different. In the rats fed the HPD, serum PTH increased significantly from day 5 through 15 (P<0.01). Parathyroid cell apoptosis was minimal and unaffected by the diet. At day 15, the parathyroid gland size in HPD was increased by 27% as compared with NPD (P<0.05). This increase should be attributed to cell proliferation since parathyroid cell size remained unchanged. Serum calcitriol and calcium were not significantly different in the two groups. In HPD, an increase in serum phosphorus was observed only on day 1. The results show that an HPD results in the stimulation of the parathyroid cell cycle independently of changes in calcium and calcitriol.

The PTH-calcium relationship during a range of infused PTH doses in the parathyroidectomized rat.

To establish the PTH dosage that maintains normal mineral homeostasis in the PTX rat, a series of doses of rat 1-34 PTH were infused via a subcutaneously implanted miniosmotic pump. The doses were 0, 0.011, 0.022, 0.044, and 0.11 microg/100 g/hour. After 48 hours, serum calcium ranged from 5.56 +/- 0.02 to 16.29 +/- 0.25 mg/dl, ANOVA P < 0.001, and serum phosphorus from 12.49 +/- 0.03 to 5.33 +/- 0.34 mg/dl, ANOVA P < 0.001. By post hoc test, the serum calcium level was different (P < 0.05) at every PTH dose; the serum phosphorus level was different (P < 0.05) at every PTH dose except between the two highest doses. The PTH dosage that produced a normal serum calcium (10.09 +/- 0.10 mg/dl) and phosphorus (6.90 +/- 0.18 mg/dl) was 0.022 microg/100 g/hour. The relationship between increasing doses of PTH and both serum calcium and phosphorus was curvilinear and the calcium-phosphorus product was remarkably constant from a serum calcium of 7-13 mg/dl. The increase in serum calcium and the decrease in serum phosphorus were more rapid at lower than at higher PTH doses so that for both, an asymptote was reached. At the highest serum calcium values, the calcium-phosphorus product increased and in individual rats, an increase in serum phosphorus was associated with a decrease in serum calcium. In summary, this study shows that (1) for rat 1-34 PTH, the normal replacement dose in the PTX rat with normal renal function on a normal diet is 0.022 microg/100 g/hour; (2) the relationship between PTH and both serum calcium and phosphorus is curvilinear, and an asymptote is reached for both; and (3) the calcium-phosphorus product is remarkably constant as the serum calcium increases from 7 to 13 mg/dl and only increased during marked hypercalcemia when serum phosphorus did not decrease further or even tended to increase.

Evidence for adaptation of the entire PTH-calcium curve to sustained changes in the serum calcium in haemodialysis patients.

BACKGROUND: Based on in vitro studies, the set point of calcium has often been considered to represent an intrinsic property of parathyroid gland function. However, in the dialysis patient, the serum calcium does not consistently reflect the magnitude of hyperparathyroidism; in addition, little information is available on whether the PTH-calcium curve is modified by sustained changes in the serum calcium. The present study in haemodialysis patients was designed to evaluate whether the set point of calcium and the dynamics of PTH secretion were modified by sustained changes in the serum calcium. METHODS: To accomplish the goal of the study and obtain a wide range of changes in the serum calcium, haemodialysis patients were dialysed with either a 1.75 mM (group I) or a 1.25 mM (group II) calcium dialysate for 2 weeks, and were then changed to a 1.25 mM (group I) or a 1.75 mM (group II) calcium dialysate for an additional 2 weeks. At the end of the first and second 2-week periods, low and high calcium studies were performed to obtain PTH-calcium curves. RESULTS: In group I, the serum ionized calcium decreased with the lower calcium dialysate (P < 0.02) and the set point of calcium was reduced (P < 0.02); in group II, the serum calcium did not change and the set point of calcium was not modified. When both groups were evaluated together, the delta serum calcium correlated directly with the delta set point of calcium (r = 0.87, P < 0.001) and inversely with the delta PTH (r = -0.73, P < 0.005); at the same time, an inverse correlation was observed between the delta PTH and the delta set point of calcium (r = -0.67, P < 0.01). Moreover, the delta serum calcium correlated with both the delta ratio of basal/maximal PTH (r = -0.71, P < 0.005) and the change in predialysis serum calcium necessary to maximally stimulate PTH (r = 0.84, P < 0.001); these latter two are indicators of the position of PTH along the PTH-calcium curve. Finally, in group I the entire PTH-calcium curve shifted to the left on the 1.25 mM calcium dialysate as compared with the 1.75 mM calcium dialysate. CONCLUSION: The findings of the present study indicate that: (1) the set point of calcium followed sustained changes in the serum calcium independently of PTH secretion, and (2) the parathyroid gland was able both to adjust the position of PTH secretion on the PTH-calcium curve and to adapt PTH secretion to the existing serum calcium concentration.

A reduced PTH response to hypocalcemia after a short period of hypercalcemia: a study in dogs.

The relationship between PTH and calcium is best represented as a sigmoidal curve. In the normal animal and human, basal PTH is positioned at approximately 25% of maximal PTH and responds rapidly to small changes in calcium in either direction. Since PTH secretion is designed to respond to either hypo- or hypercalcemia, the study was performed to evaluate whether the parathyroid gland would respond differently to hypocalcemia when the reduction in serum calcium was initiated from sustained hypercalcemia with maximal PTH suppression. Nine dogs were studied and the experimental protocol consisted of two separate parts in which the same dogs were used and the order of study was randomly assigned. For the hypercalcemic part, calcium chloride was infused intravenously to increase serum calcium to between 1.60 and 1.70 mM at 30 minutes and then continued for another 90 minutes to clamp the serum calcium at this level. Sodium EDTA was then infused to lower the serum calcium at a constant rate to less than 0.85 mM. For the normocalcemic part, 5% dextrose in water was infused for two hours to control for fluid volume and time, and then EDTA was infused to lower the serum calcium at a constant rate to less than 0.85 mM. The results show that for the same serum calcium concentration at every 0.05 mM decrement in serum calcium below normal, PTH was less in the hypercalcemic than the normocalcemic dogs (P < 0.02). During the induction of hypocalcemia in the normocalcemic dogs, a characteristic sigmoidal curve was observed in which a small decrease in the serum calcium induced a brisk increase in PTH and a maximal PTH level was rapidly attained; however, during the induction of hypocalcemia in the hypercalcemic dogs, the increase in PTH was progressive, but linear and it was not certain that a maximal PTH level was attained. In conclusion, a sustained period of hypercalcemia resulted in a decreased PTH response to hypocalcemia and reduced the efficiency of the sigmoidal PTH-calcium relationship. Whether the mechanism for this difference in PTH secretion is due to secretory products, modification of the calcium receptor, or changes in intercellular communication among parathyroid cells deserves further study.

Hysteresis of the PTH-calcium curve during hypocalcemia in the dog: effect of the rate and linearity of calcium decrease and sequential episodes of hypocalcemia.

Several studies have shown the presence of hysteresis of the parathyroid hormone (PTH)-calcium relationship in both normal humans and hemodialysis patients; for hypocalcemia, hysteresis is defined as a lower PTH level for the same serum calcium during the recovery from than the induction of hypocalcemia. However, some have questioned whether hysteresis is only a function of the rate and/or direction of change in calcium, and others have proposed that hysteresis is due to depletion of PTH stores. To address these issues, two groups of dogs were studied. To induce hypocalcemia, sodium EDTA (50 mg/kg) was infused either over 60 (termed slow) or 30 (termed fast) minutes; immediately after the cessation of the ethylenediamine tetracetate (EDTA) infusion, calcium chloride (0.75 mEq/kg) was infused over 60 or 30 minutes, respectively, to correct the hypocalcemia. The EDTA infusion produced a linear decrease in serum calcium by progressively increasing the infusion rate at regular intervals. A second cycle of hypocalcemia and recovery using the same protocol was started immediately after the completion of the first cycle. To determine whether a nonlinear decrease in the serum calcium affected the PTH response to hypocalcemia, a third group of dogs, termed superfast, was studied; in this group, EDTA was infused for 30 minutes at a constant rate of 50 mg/kg. The hysteretic loops of PTH produced by the two sequential slow and fast cycles and the superfast cycle during the induction of and recovery from hypocalcemia were similar. Moreover, the maximal PTH level for the two sequential slow and fast cycles and the superfast cycle was not different even though the rates of calcium decrease varied and the calcium decrease was nonlinear in the superfast cycle. In conclusion, (1) since hysteresis was reproducible and the maximal PTH was not different during two sequential cycles of hypocalcemia, hysteresis is not due to PTH depletion; (2) the PTH-calcium curve is not affected by the rate at which hypocalcemia is induced; and (3) the maximal PTH level is not influenced by either the rate or linearity of the reduction in serum calcium.