Phillip W. Lambert

Demonstration of a lack of change in serum 1 alpha,25-dihydroxyvitamin D in response to parathyroid extract in pseudohypoparathyroidism.

Studies were carried out to compare the effects of parathyroid extract (PTE) on serum and urinary calcium (Ca) and phosphorus (P), serum 25-hydroxyvitamin D (25-OHD), serum 24,25-dihydroxyvitamin D (24,25(OH)2D), serum 1 alpha,25-dihydroxyvitamin D (1 alpha,25(OH)2D), and urinary cyclic AMP in two normal subjects, two patients with hypoparathyroidism (HP) and six patients with pseudohypoparathyroidism (PHP), some of whom were on suboptimal treatment with vitamin D. Two of the patients with PHP were studied while on long-term treatment with 1 alpha,25-(OH)2D3. Before PTE, serum 1 alpha, 25(OH)2D was at the lower limit of normal in one patient and was abnormally low in the other five patients. None of these individuals was on treatment with 1 alpha,25(OH)2D3. Serum 25-OHD and 24,25(OH)2D were either increased or at the upper limit of normal in the patients given vitamin D and were normal in the other patients. PTE lowered the serum P and increased the serum 1 alpha,25(OH)2D, serum and urinary Ca, urinary P, and urinary cyclic AMP in the normal subjects and patients with HP. In individual studies, changes in serum 1 alpha,25(OH)2D and serum Ca occurred in parallel before, during, and after PTE. In contrast, PTE had very little effect in the patients with PHP. Whereas there were highly significant positive correlations between serum 1 alpha,25(OH)2D in each of the normal subjects and patients with HP, there were significant correlations in only one of the patients with PHP. An increase in serum Ca in response to PTE was observed in one of the two patients with PHP who were on long-term treatment with 1 alpha,25(OH)2D3. In these individuals, PTE produced only slight increases in serum 1 alpha,25(OH)2D. Serum 25-OHD and 24,25(OH)2D were not changed by PTE in any of the subjects or patients. The results provide evidence that hypocalcemia in HP and PHP arises in part from low circulating 1 alpha,25-(OH)2D, and indicate that the lack of change in serum 1 alpha,25(OH)2D with PTE in patients with PHP is related to impaired renal adenylate cyclase and phosphaturic responses. These and previous results support the idea that diminished renal production of 1 alpha,25(OH)2D, because of a defect in the parathyroid hormone-responsive adenylate cyclase system, may be a contributing factor in the pathogenesis of the abnormal calcium metabolism in PHP.

Vitamin D in plasma: quantitation by a nonequilibrium ligand binding assay.

The concentration of vitamin D was determined in human and bovine plasma samples under various physiological and nonphysiological conditions using a nonequilibrium ligand binding assay. Prior to ligand binding analysis the vitamin D in the plasma organic extracts was purified using chromatographic procedures involving Lipidex-5000 and high performance liquid chromatography. The use of a nonequilibrium assay system greatly increased the sensitivity of our assay allowing for a minimum volume of the initial plasma sample. The vitamin D levels in plasma responded to increased sun exposure as well as to the intoxication with vitamin D3. Analysis of a plasma sample from a vitamin D-deficient patient revealed that lipid interference was not a factor in this ligand binding assay.

Pre- and postoperative studies of plasma calcitonin in primary hyperparathyroidism.

The importance of calcitonin in the homeostatic response to the chronic hypercalcemia of primary hyperparathyroidism is uncertain. To clarify this issue, we have used a new, sensitive radioimmunoassay for human calcitonin to measure basal plasma calcitonin concentrations in 50 patients with primary hyperparathyroidism (32 female, 18 male). We assayed calcium-stimulated calcitonin concentrations preoperatively in 22 of the patients (16 female, 6 male) and postoperatively in 6. Finally, we assayed pentagastrin-stimulated calcitonin concentrations preoperatively in eight of the patients (three female, five male). Plasma calcitonin values after an overnight fast were indistinguishable from those in normal subjects (mean+/-SE, males, 48+/-3 normal and 46+/-5 pg/ml hyperparathyroid, females, 31+/-2 normal and 37+/-3 pg/ml hyperparathyroid.) Among hyperparathyroid patients of both sexes, increases of calcitonin during Ca infusion (15 mg Ca/kg in 4 h) were within normal limits. However, the mean maximal increase of calcitonin was significantly lower in hyperparathyroid than in normal subjects (P < 0.05). In six patients normocalcemic 5-15 mo after parathyroid surgery, fasting plasma calcitonin values were not significantly different, but responses to Ca infusion were greater than preoperatively (Delta calcitonin +/-SE: 13+/-4 preoperatively and 53+/-35 pg/ml postoperatively). The mean maximal increase of calcitonin after pentagastrin (0.5 mug/kg i.v.) was slightly lower than normal in the patients (mean+/-SE, males, 45+/-8 normal and 38+/-10 pg/ml hyperparathyroid, females, 6+/-2 normal and 0 pg/ml hyperparathyroid). Thus, primary hyperparathyroidism is accompanied by normal steady-state concentrations of circulating calcitonin, and normal-to-blunted C-cell responses to pentagastrin or induced hypercalcemia, the response to calcium generally increasing after successful parathyroid surgery. These results clearly show that primary hyperparathyroidism is not characterized by hypercalcitoninemia. The seemingly paradoxical absence of elevated steady-state calcitonin concentrations may be accounted for partly by decreased secretory reserve. However, primary hyperparathyroidism may also be accompanied by an increase in the threshold of sensitivity for calcium stimulation of calcitonin secretion.

25,26-Dihydroxycholecalciferol: A precursor in the renal synthesis of 25-hydroxycholecalciferol-26,23-lactone

Abstract Both rachitic 1,25-dihydroxycholecalciferol-treated and normal chicks produced detectable quantities of 25-hydroxycholecalciferol-26,23-lactone, although renal homogenates from rachitic chicks failed to produce this compound. The addition of either 25-hydroxycholecalciferol or 25,26-dihydroxycholecalciferol to the renal homogenates led to the production of the lactone, although more lactone resulted when 25,26-dihydroxycholecalciferol was used. When tritiated 25-hydroxycholecalciferol was added to rachitic 1,25-dihydroxycholecalciferol-treated or normal chick renal homogenates, an unidentified tritiated vitamin D metabolite distinct from 25-hydroxycholecalciferol-26,23-lactone was produced.

An Improved Method for the Measurement of 1,25-(OH)2D3 in Human Plasma

Here we report a highly sensitive and convenient ligand binding assay for the determination of 1,25(OH)2D3 in small volumes of human plasma. This method involves: (1) extraction of vitamin D3 and its metabolites using methanol-methylene chloride with separation of phases by centrifugation; (2) gel chromatography and high pressure liquid chromatography for the quantitative isolation of 1,25-(OH)2D3; and (3) a sensitive ligand binding assay for 1,25-(OH)2D3 employing cytosol receptor from the intestinal mucosa of rachitic chicks. Using modified rachitogenic chick diets allows early (less than 4 wks) harvesting of active receptor for 1,25-(OH)2D3 in high yield. The method includes a rapid and effective procedure for stable and long-term storage of the active cytosol receptor. A convenient dextran-charcoal means is used for the separation of receptor bound from free 1,25-(OH)2D3 resulting in the achievement of a lower (less than 5%) background (i.e., nonspecific binding) than reported for other 1,25-(OH)2D3 assays. Analysis of this receptor shows it to be a saturable, single class of binding sites with a dissociation constant (Kd) of approximately 3.7 x 10-11. The final recovery of 1,25-(OH)2D3 following extraction and chromatography is 80 +/- 3% and triplicate determinations can be made on a 3 ml plasma sample. The ligand binding assay routinely detects less than or equal to 5pg of 1,25-(OH)2D3 per assay tube and the inter- and intraassay variation, based on repeated determinations of 1,25-(OH)2D3 in pooled normal human plasma, is less than 5%. Preliminary studies indicate that our methodology will permit measurement of plasma 1,25-(OH)2D3 levels in all normal subjects and in pathophysiologic states where 1,25-(OH)2D3 levels may be below or above normal values. 1,25-(OH)2D3 values (pg/ml +/- SEM) in human plasma obtained from both normals and patients with various untreated calcium homeostatic disorders were: normals = 33.5 +/- 1.8; end-stage chronic renal failure = 5.1 +/- 1.2; primary hypoparathyroidism = 18.3 +/- 2.8; primary hyperparathyroidism = 61.4 +/- 7.1; and hyperthyroidism with associated hypercalcemia = 42.1 +/- 8.4.

Reversal of Secondary Hyperparathyroidism by Cimetidine in Chronically Uremic Dogs

Chronic cimetidine therapy has been shown to suppress circulating concentrations of immunoreactive parathyroid hormone (iPTH) in hemodialysis patients. To evaluate the long-term metabolic effects of cimetidine treatment, we studied seven chronically uremic dogs for 20 wk. The dogs were studied under metabolic conditions before, during, and after cimetidine therapy. iPTH fell progressively in the five treated dogs from 536+/-70 muleq/ml (mean+/-SE) (nl < 100 muleq/ml) before treatment to 291+/-25 muleq/ml at 12 wk (P < 0.001) and 157+/-32 muleq/ml at 20 wk (P < 0.001). The control dogs showed no consistent change in iPTH. The fall in iPTH was not associated with a change in serum ionized calcium. However, serum phosphorus decreased from 5.7+/-0.9 mg/dl to 3.4+/-0.2 mg/dl by the 20th wk (P < 0.05). By contrast, the serum concentration of 1,25-dihydroxycholecalciferol increased in all treated dogs from 33.4+/-4.3 pg/ml to 51.8+/-2.4 pg/ml during treatment (P < 0.01). Calcium balance was negative in all seven dogs before cimetidine (-347+/-84 mg/72 h) and remained so in the control dogs; it became positive in the five treated dogs after 12 wk (1,141+/-409 mg/72 h) (P < 0.05). Phosphorus balance, 24-h fractional phosphate excretion, and creatinine clearance remained unchanged. Pooled samples of serum obtained during the control and 20th wk of therapy were fractionated by gel filtration and the eluates assayed for immunoreactivity. The decrease in iPTH was associated with a decrease in all the immunoreactive species, indicating suppression of parathyroid gland secretion. These observations indicate that cimetidine suppressed circulating concentration of biologically active parathyroid hormone. A probable net decrease in the loss of phosphorus from bone to blood ensued, resulting in a fall in serum phosphorus. This may have stimulated synthesis of 1,25-dihydroxycholecalciferol and led to a positive calcium balance, thereby maintaining the serum ionized calcium concentration. The maintenance of phosphate balance, despite suppression of iPTH by cimetidine, indicates that factors other than hyperparathyroidism relate to phosphate homeostasis in chronically uremic dogs.

Plasma immunoreactive calcitonin in lung cancer.

We have measured plasma calcitonin in 135 untreated eucalemic men with lung cancer and a control/smoker population. Calcitonin levels were determined by radioimmunoassay and validated by immunoextraction. Plasma immunoreactive calcitonin moieties were purified by immunoadsorbent chromatography, treated with mercaptoethanol and urea, and characterized by gel filtration. Artifacts in human calcitonin radioimmunoassays of cancer-patient plasmas were detected by parallel plasma incubations in a salmon calcitonin radioimmunoassay system which does not detect human calcitonin and by immunoprecipitation of tracer at the end of radioimmunoassay incubations. Heating fresh plasmas to 65 degrees C for 1.5 hours reduced radioimmunoassay artifacts without loss of calcitonin moieties. Such characterization of hypercalcitoninemia in each of the histopathological types of lung cancer has raised some important questions about the interpretation of plasma calcitonin radioimmunoassay measurements in lung cancer. Based on inhibition of tracer-antibody binding, plasma calcitonin seemed to be elevated in 18% (14/80) of basal plasma samples obtained from patients with epidermoid or with anaplastic lung cancer. Unequivocal hypercalcitoninemia (heat stable, causing no inhibition of antibody-tracer binding in the salmon calcitonin radioimmunoassays, and immunoextractable with human calcitonin antibodies) was not found in any of the apparently hypercalcitoninemic plasmas from persons with epidermoid or anaplastic lung cancer. By contrast, unequivocal hypercalcitoninemia was found in 27% (15/55) of plasmas from patients with small cell carcinoma or adenocarcinoma. Most of the immunoreactive calcitonin recovered from small cell and adenocarcinoma lung cancer plasmas with unequivocally elevated calcitonin is much larger than calcitonin monomer.

Assay for Multiple Vitamin D Metabolites

During the past decade there have been major advances in our knowledge of vitamin D metabolism and its role in both physiologic and pathophysiologic situations of calcium (Ca) homeostasis. Critical to these advances has been the development of ligand-binding assays for a wide spectrum of vitamin D3 metabolites. Separate ligand-binding assays have been reported for vitamin D,1,2 25-OHD,3–11 24,25-(OH)2D,12–15 25,26-(OH)2D,16–18 and 1,25-(OH)2D.19–35 More recently, methodology has evolved for the measurement of two or more vitamin D metabolites in a single, relatively small plasma or serum sample.23,28,34–42