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We sought to determine if any protective effect of dietary calcium (Ca) or Ca supplements on bone could be at least partially mediated by increased calcitonin (CT) secretion. First we studied 10 healthy premenopausal women (median age, 35.5 years) who were randomized to high or low dietary Ca intake (1752 versus 391 mg elemental Ca per day) for 2 weeks and then crossed over. At the end of each dietary period, blood was drawn on 1 day at 0800, 1200, 1700, and 2000 h to assess diurnal variation of plasma CT levels. CT secretory reserve was assessed on the next day by Ca infusion (2 mg Ca per kg body weight over 5 minutes). Next, we studied 10 healthy premenopausal women who took a low-Ca diet (∼ 400 mg Ca per day) for a 2 week control period. The women were then randomized to high- or low-Ca intake [400 mg dietary Ca ± 1500 mg Ca per day (as supplemental CaCO3)] and then crossed over. At the end of each study period, the diurnal variation in CT was tested on day 1; the CT secretory reserve was assessed on day 3 by an oral Ca load (500 mg as CaCO3)] and on day 5 by Ca infusion. Plasma immunoreactive CT was measured in whole plasma (iCT) and after silica extraction (exCT), predominantly monomeric CT. Neither increased dietary Ca nor Ca supplements affected the diurnal levels of iCT or exCT or augmented plasma CT responses to an oral Ca load. Furthermore, there was no change in secretory reserve for iCT or exCT after Ca infusion. We conclude that short-term Ca administration, in the form of dietary Ca or Ca salts, at the extremes of normal dietary intake and in doses normally used to treat osteoporosis, respectively, does not affect CT secretion in healthy premenopausal women. It seems unlikely that any potential beneficial effects of ingested Ca on the skeleton in women are mediated by CT.

We describe a young woman who acquired a painful, diffuse osteosclerosis of the cervical, thoracic, and lumbar spine, pelvis, and long bones of the legs as an adult. Bone densitometry showed a large increase in apparent bone density. Skeletal radiographs demonstrated progressive endosteal and periosteal thickening of the cortices. A bone scan showed increased uptake of radiolabel. The serum total alkaline phosphatase and 1,25-(OH)2D3 levels were substantially elevated and the immunoreactive PTH was mildly elevated. Bone biopsy showed increased bone turnover, especially on endocortical and intracortical surfaces, but the structural indices were normal. By 4 years after presentation the bone pain had remitted and the serum alkaline phosphatase, 1,25-(OH)2D3, and PTH were normal. No cause for the occurrence of osteosclerosis in this patient could be found.

To study the effect of raloxifene on the response to conjugated estrogen cream or nonhormonal moisturizer in postmenopausal women with preexisting signs of vaginal atrophy.Postmenopausal women with preexisting and untreated vaginal atrophy were enrolled in this parallel, placebo-controlled, randomized study. A total of 187 women were randomized to four treatment groups: daily oral raloxifene (60 mg per day) or a placebo in a double-blind manner plus one application of conjugated estrogen cream (0.5 g) or one applicator full of nonhormonal moisturizer, open label. The conjugated estrogen cream or nonhormonal moisturizer was applied daily for the first 2 weeks, and then twice weekly thereafter for 3 months. Efficacy of treatment regimens on signs and symptoms of vaginal atrophy was evaluated by monitoring objective and subjective parameters.Signs and symptoms of vaginal atrophy improved in all four treatment groups. Raloxifene did not diminish the magnitude of improvement when administered with either vaginal preparation. Conjugated estrogen cream produced a statistically greater improvement in signs (P.05) but not in individual symptoms or overall satisfaction relative to nonhormonal moisturizer.Postmenopausal women with evidence of preexisting vaginal atrophy may use either low-dose conjugated estrogen cream or nonhormonal moisturizer to treat the atrophy concurrently with raloxifene (60 mg per day).

Antiresorptive treatments for postmenopausal osteoporosis have been studied extensively, but due to the volume of published data and lack of head-to-head trials, it is difficult to evaluate and compare their fracture reduction efficacy. The objective of this review is to summarize the results from clinical trials that have fracture as an endpoint and to discuss the factors in study design and populations that can affect the interpretation of the results. Although there are numerous observational studies suggesting that estrogen and hormone replacement therapies may reduce the risk of vertebral and nonvertebral fractures, there is no large, prospective, randomized, placebo-controlled, double-blind clinical trial demonstrating fracture efficacy. The effects of raloxifene, alendronate, risedronate, and salmon calcitonin on increasing bone mineral density (BMD) and decreasing fracture risk have been shown in randomized, placebo-controlled, double-blind clinical trials of postmenopausal women with osteoporosis. Although the increases in lumbar spine BMD vary greatly in these trials, the decrease in relative risk of vertebral fractures is similar among therapies. However, nonvertebral fracture efficacy has not been consistently demonstrated. Combined administration of two antiresorptive therapies results in greater BMD increases, but the effects on fracture risk are unknown. Direct comparisons of clinical trial results should be considered carefully, given the differences in study design and populations. Differences in study design that may influence the efficacy of fracture risk reduction include calcium and vitamin D supplementation, primary fracture endpoints, definition of vertebral deformity or fracture, discontinuation rates, and statistical power. Factors in the study population that may influence fracture efficacy include the age of the population and the proportion of subjects with prevalent fractures. The use of surrogate endpoints such as BMD to predict fracture risk should be approached with caution, as the relationship between BMD changes and fracture risk reduction with antiresorptive therapies is uncertain. Consideration of these results from clinical trials can contribute to clinical judgment in selecting the best treatment option for postmenopausal osteoporosis.

Summary Hyperparathyroidism–jaw tumor syndrome (HPT-JT) is an autosomal dominant disease characterized by the development of multiple parathyroid adenomas and multiple fibro-osseous tumors of the maxilla and mandible. Some families have had affected members with involvement of the kidneys, variously reported as Wilms tumors, nephroblastomas, and hamartomas. The HPT-JT gene ( HRPT2 ) maps to chromosome 1q25–q31. We describe further investigation of two HPT-JT families (K3304 and K3349) identified through the literature. These two expanded families and two previously reported families were investigated jointly for linkage with 21 new, closely linked markers. Multipoint linkage analysis resulted in a maximum LOD score of 7.83 (at recombination fraction 0) for markers D1S2848–D1S191. Recombination events in these families reduced the HRPT2 region to ∼14.7 cM. In addition, two of these four study families (i.e., K3304 and K11687) share a 2.2-cM length of their (expanded) affected haplotype, indicating a possible common origin. Combining the linkage data and shared-haplotype data, we propose a 0.7-cM candidate region for HRPT2.

Bone mass is purportedly reduced by an endogenous or exogenous excess of thyroid hormone or, perhaps, by calcitonin deficiency. Patients who have undergone thyroidectomy could be subject to all of these effects, yet their practical implications in terms of fracture risk are poorly defined. Interpretation is further hampered by the focus on women, where results may be influenced by involutional osteoporosis. Consequently, we assessed the potential for fractures among the 136 Rochester, Minnesota men who underwent thyroidectomy between 1935 and 1979, relative to a group of age-matched control men from the community. With 2194 person-years of follow-up in each group, survival free of any fracture of vertebra, proximal humerus, distal forearm, pelvis, or proximal femur was similar in the two groups (p = 0.23), and the relative risk of any of these fractures for thyroidectomized patients versus their controls was increased only 1.5-fold (95% CI, 0.7-3.2). The difference was entirely accounted for by a statistically significant excess of proximal femur fractures in the men with thyroidectomy. Risk factors for fractures among men with thyroidectomy included greater age at surgery, greater extent of surgery, and the presence of risk factors for secondary osteoporosis. Thus, thyroidectomy, performed mainly for adenoma or goiter, seems to have little overall influence on the risk of age-related fractures in men. However, the association with hip fractures requires further evaluation.

BACKGROUND: Familial hypocalciuric hypercalcemia is a genetically heterogeneous disorder with three variants: types 1, 2, and 3. Type 1 is due to loss-of-function mutations of the calcium-sensing receptor, a guanine nucleotide-binding protein (G-protein)-coupled receptor that signals through the G-protein subunit α11 (Gα11). Type 3 is associated with adaptor-related protein complex 2, sigma 1 subunit (AP2S1) mutations, which result in altered calcium-sensing receptor endocytosis. We hypothesized that type 2 is due to mutations effecting Gα11 loss of function, since Gα11 is involved in calcium-sensing receptor signaling, and its gene (GNA11) and the type 2 locus are colocalized on chromosome 19p13.3. We also postulated that mutations effecting Gα11 gain of function, like the mutations effecting calcium-sensing receptor gain of function that cause autosomal dominant hypocalcemia type 1, may lead to hypocalcemia. METHODS: We performed GNA11 mutational analysis in a kindred with familial hypocalciuric hypercalcemia type 2 and in nine unrelated patients with familial hypocalciuric hypercalcemia who did not have mutations in the gene encoding the calcium-sensing receptor (CASR) or AP2S1. We also performed this analysis in eight unrelated patients with hypocalcemia who did not have CASR mutations. In addition, we studied the effects of GNA11 mutations on Gα11 protein structure and calcium-sensing receptor signaling in human embryonic kidney 293 (HEK293) cells. RESULTS: The kindred with familial hypocalciuric hypercalcemia type 2 had an in-frame deletion of a conserved Gα11 isoleucine (Ile200del), and one of the nine unrelated patients with familial hypocalciuric hypercalcemia had a missense GNA11 mutation (Leu135Gln). Missense GNA11 mutations (Arg181Gln and Phe341Leu) were detected in two unrelated patients with hypocalcemia; they were therefore identified as having autosomal dominant hypocalcemia type 2. All four GNA11 mutations predicted disrupted protein structures, and assessment on the basis of in vitro expression showed that familial hypocalciuric hypercalcemia type 2-associated mutations decreased the sensitivity of cells expressing calcium-sensing receptors to changes in extracellular calcium concentrations, whereas autosomal dominant hypocalcemia type 2-associated mutations increased cell sensitivity. CONCLUSIONS: Gα11 mutants with loss of function cause familial hypocalciuric hypercalcemia type 2, and Gα11 mutants with gain of function cause a clinical disorder designated as autosomal dominant hypocalcemia type 2. (Funded by the United Kingdom Medical Research Council and others.).

Hereditary hyperparathyroidism-jaw tumor syndrome (HPT-JT) is an autosomal dominant disease (OMIM 145001) that has recently been mapped to chromosomal region 1q21-q32 (HRPT2). Here we report two families with HPT-JT syndrome in which adult renal hamartomas or cystic kidney disease were prominent associated features, possibly representing a new phenotypic variant of the HPT-JT syndrome. In the first family, renal lesions were present in five out of six affected individuals, whereas HPT and JT were seen in four and two cases, respectively. In the second family, JT was found in three of the five affected individuals and two affected members also exhibited polycystic kidney disease. The possibility of the latter cosegregating as a separate autosomal dominant gene can not be ruled out. A sex-dependent penetrance of primary HPT, resulting in predominantly male-affected cases was evident in the two families. Twenty microsatellite markers in the HRPT2 region were typed, in addition to markers in the multiple endocrine neoplasia (MEN) types 1 and 2 regions at 11q13 and 10q11. The disease in these two kindreds was linked to five markers in the 1q21-q32 region (logarithm-of-odds scores: 3.2-4.2), whereas linkage to the MEN1 and MEN2 regions was excluded. Meiotic recombinations detected in affected individuals placed the locus telomeric of D1S215, thus narrowing the HRPT2 region from > 60 to approximately 34 centimorgans. Loss of heterozygosity was studied in seven renal hamartomas from two affected individuals in the first family, as well as in a jaw tumor and a parathyroid tumor from the second family. All renal hamartomas showed loss of heterozygosity at the 1q21-q32 region. The losses invariably involved the wild type allele derived from the unaffected parent, suggesting the inactivation of a tumor suppressor gene in this region.

Only since the 1970s have scientific and medical advances brought osteoporosis into sharp focus for physicians and the public, and brought effective treatments. The entire health care and pharmaceutical enterprises are undergoing dramatic changes that will affect how we find, assess, prevent, and treat osteoporosis. These changes and accompanying challenges to therapeutic development in osteoporosis must be understood in order to plan for future policies, payments, and development strategies.

Existing radioimmunoassays for parathyroid hormone (PTH) in rat plasma are based on cross-reactivity of rat PTH (rPTH) with heterologous antisera. We used the synthetic NH2-terminal fragment of rPTH [rPTH-(1-34)] to develop a homologous radioimmunoassay for circulating PTH. An antiserum to rPTH-(1-34) was raised in a goat (G-813), and the same peptide was used as radioligand (125I) and standard. Purification of the label by high-performance liquid chromatography (HPLC) increased specific binding greater than twofold and sensitivity by 50-100%. With a final antiserum dilution of 1:70,000, maximum specific binding of 30-33%, nonspecific binding of 1-5%, and 50-microliters sample additions, the assay detection limit was 1.8-2.5 pmol/l. A midregional fragment of human PTH did not displace 125I-labeled rPTH-(1-34). HPLC of extracts of rat parathyroid glands and hyperparathyroid plasma showed only a single peak of immunoreactivity that eluted 2 min after rPTH-(1-34). Dose dilution curves for rat parathyroid gland extracts, rPTH-(1-34) added to rat plasma, and endogenous rat plasma PTH all paralleled the standard curve. Immunoreactive PTH (irPTH) was detectable in greater than 90% of fasting normal rat plasma and changed appropriately in response to hyper- and hypocalcemia induced by low-calcium and vitamin D-deficient diets, injections of calcium and EDTA, and after thyroparathyroidectomy. The normal range for rat plasma irPTH was less than 2.0-12 pmol/l, in general agreement with bioassay results of others. Thus rPTH-(1-34) is an excellent immunogen for raising antisera to rPTH, and assays incorporating it may be of great value in studying rat parathyroid physiology.

Plasma concentrations of PTH are much lower for a given calcium or phosphorus level in patients with familial benign hypercalcemia (FBH, or familial hypocalciuric hypercalcemia) than in those with primary hyperparathyroidism; these and other data suggest that there might be tissue hypersensitivity to PTH in FBH. To test this hypothesis, we have used cultured dermal fibroblasts from abdominal skin biopsies of six patients with FBH and six age- and sex-matched controls as surrogate PTH-responsive tissues. Cells in 24-well plastic plates were exposed to vehicle, human PTH-(1-34) (10(-10)-10(-7) M), prostaglandin E2 (10(-6) M), or isoproterenol (10(-4) M) for 10 min in the presence of isobutylmethylxanthine, and cellular cAMP was determined by RIA. All cells responded to PTH with dose-dependent increases in cAMP, and all responded strongly to prostaglandin E2 and isoproterenol. There were no consistent or significant differences between control and FBH fibroblasts in maximal responses to the three agonists, and half-maximal stimulation was achieved with about 10(-9) M PTH in both normal and FBH cells. These data are not consistent with increased tissue sensitivity to PTH in FBH.

We have investigated the effects of PTH-induced desensitization on second messenger interactions in the rat osteosarcoma cell line ROS 17/2.8. Adenylate cyclase activation was assessed by accumulation of immunoassayable cAMP, and cytosolic calcium ion ([Ca2+]i) concentrations were measured in adherent perifused cells loaded with the Ca2(+)-sensitive bioluminescent protein aequorin. Preexposure to rat PTH-(1-34) [rPTH-(1-34); 10(-8) M for 48 h, then 10(-7) M for 24 h] dramatically reduced (by 85%) the cAMP response to fresh challenge [2 min; 10(-9)-10(-7) M rPTH-(1-34)], but the peak PTH-induced rise of [Ca2+]i was not diminished significantly (0-20%). Nevertheless, we did observe other changes in the PTH-induced [Ca2+]i response. Exposure of treated cells to (Bu)2cAMP nearly abolished the [Ca2+]i response to PTH (greater than 80% reduction), but had much less effect on the PTH-stimulated [Ca2+]i increment of the naive cells (less than 35% reduction). Treated cells also had a blunted [Ca2+]i response to PTH in the presence of low extracellular calcium (greater than 60% reduction), but in the naive cells, low extracellular Ca2+ did not significantly diminish the peak PTH-induced [Ca2+]i rise, although low extracellular Ca2+ dramatically reduced the area under this [Ca2+]i transient (greater than 50%). Low extracellular Ca2+ had no influence on the peak [Ca2+]i responses of treated cells to bradykinin or prostaglandin F2 alpha. Although the peak PTH-stimulated [Ca2+]i rise of treated cells in normal Ca2+ medium was not significantly attenuated, the time to half-maximum [Ca2+]i concentration was significantly increased (greater than 100%), and the area under the [Ca2+]i transient was diminished. These alterations in the [Ca2+]i response of treated cells were not observed upon challenge with bradykinin or prostaglandin F2 alpha. Thus, 1) the cAMP and [Ca2+]i responses of ROS 17/2.8 cells to rPTH-(1-34) are not obligatorily coupled; 2) the response of naive cells to PTH includes both the release of Ca2+ from intracellular stores and the entry of extracellular Ca2+; and 3) pretreatment of these cells with rPTH-(1-34) augments the dependence on Ca2+ entry during hormone rechallenge. We propose that the preserved PTH-stimulated [Ca2+]i rise in treated cells results partly from loss of cAMP-mediated inhibition of extracellular Ca2+ entry.

We developed and validated a radioimmunoassay for circulating human parathyroid hormone-related peptide (PTHrP), based on a commercial antiserum to the synthetic 1-34 fragment of PTHrP, 125I-Tyr degrees-PTHrP(1-34) as radioligand, and prior extraction of the native peptide from plasma with C-2 cartridges. We determined immunoreactive PTHrP concentrations in plasma samples from 48 healthy persons (mean +/- SD, 3.1 +/- 1.0 pmol/liter; range, less than 2 to 5 pmol/liter), 8 patients with primary hyperparathyroidism, 36 patients with hypercalcemia and a concurrent malignant lesion, and 9 normocalcemic patients with cancer and increased serum levels of carcinoembryonic antigen or prostate-specific antigen. PTHrP was normal in samples from patients with primary hyperparathyroidism (3.2 +/- 1.1 pmol/liter), secondary hyperparathyroidism (2.5 +/- 1.3 pmol/liter), and cancer without hypercalcemia (2.4 +/- 1.0 pmol/liter). In contrast, plasma immunoreactive PTHrP levels were increased (6.0 to 85.0 pmol/liter) in 47% of patients with hypercalcemia and cancer of various types, with or without bone metastatic lesions. Large amounts of PTHrP were also found in conditioned medium from cultured human prostatic carcinoma cells. Thus, PTHrP may be a causative factor for hypercalcemia associated with a malignant lesion in at least half of the cases. Measurement of circulating PTHrP may be of differential diagnostic help in hypercalcemic states.

In an earlier 8-day study, we showed increased immunoreactive PTH (iPTH) levels in young adults fed high phosphorus (P), low calcium (Ca) diets assembled from common grocery foods, a dietary pattern characteristic of teens and young adults. Because animals fed high P, low Ca diets developed secondary hyperparathyroidism and, ultimately, osteopenia, perhaps the typical teen diet may reduce peak bone mass and contribute to osteoporotic fracture later in life. To determine if the elevation in iPTH levels and action persists with chronic intake of this typical diet, we studied the 24-h mineral and hormone responses of 15 young women (18-25 yr of age) to either high P, low Ca or control diets. Each subject served as her own control, first consuming a basal diet (800 mg Ca, 900 mg P) for 28 days; 10 women were then switched to the high P, low Ca test diet (400 mg Ca, 1700 mg P) for 28 days, while the remaining 5 women in the control group continued eating the basal diet. On days 28 and 56, all subjects were studied as inpatients for 24 h, with blood drawn every 4 h and collection of fasting and 24-h urine. Serum iPTH (midregion) and serum intact PTH (2-site immunoradiometric assay) increased significantly [maximal increases of 26% (P less than 0.002) and 36% (P less than 0.004), respectively] after 4 weeks of consuming the test diet, and there was no change in the control group. In contrast to our 8-day study, plasma levels of 1,25-dihydroxyvitamin D did not change in either group. Our findings suggest that this common dietary pattern in young adult women causes persistent alterations in calcium-regulating hormones that could be unfavorable to achieving maximal positive bone balance.

Publisher Summary This chapter discusses familial benign hypocalciuric hypercalcemia (FBHH) and Neonatal Primary Hyperparathyroidism. The FBHH syndrome is inherited in an autosomal dominant fashion and is usually characterized by lifelong asymptomatic hypercalcemia, relative hypocalciuria, and the lack of the classical complications of hypercalcemia, including bone disease and renal stone disease. Some observers have also suggested that FBHH increases the risk of acute pancreatitis. The skeleton in patients with FBHH generally appears to be normal histologically, radiographically, and by mineral densitometry. While FBHH does not appear to threaten skeletal integrity, it does not confer immunity to osteoporosis. The relatively low urinary excretion of calcium in FBHH results from avid renal tubular reabsorption of calcium, and the urinary excretion of magnesium is unexpectedly low given that the serum level is in the upper part of the normal range or, in some cases, mildly elevated. Studies of the FBHH syndrome have almost uniformly suggested a genetic disorder of calcium sensing by the parathyroid gland paired with enhanced renal tubular reabsorption of calcium. PTH secretion persists at normal or slightly elevated values in the face of hypercalcemia, but without obvious disturbances of other calcium-regulating hormone concentrations. These pathophysiologic abnormalities would be most parsimoniously explained by dominant mutations of a single calcium sensing receptor expressed both in parathyroid glands and in kidneys. Neonatal primary hyperparathyroidism can be a mild syndrome representing the neonatal expression of FBHH, or it can present as a much more dramatic disease with high morbidity and even mortality if untreated, which is known as neonatal severe primary hyperparathyroidism (NSHPT).

Publisher Summary This chapter reviews the current state of knowledge about primary hyperparathyroidism (1°HPT) and bone and highlights recent long-term data. Variable degrees of osteopenia are common in patients having 1°HPT and osteoporosis may be evident at the diagnosis of 1°HPT. The skeletal deficits are occasionally severe, but usually of undetermined relationship to the hyperparathyroidism. On average, the decrements of bone mass suggest only about a doubling of fracture risk, an increment not discernible in the small studies done to date. The few prospective studies of fracture risk in 1°HPT were not sufficiently powered to adequately address the issue. Osteopenia may be worst at primarily cortical sites, which would suggest a greater risk of appendicular than of spinal crush fractures. Regardless of site or severity of osteopenia, surgical therapy of 1°HPT causes substantially increased bone mineral density (BMD) at most sites, on the order of 10 to 12%. Increases of such magnitude are rarely seen in therapy of osteoporosis by any other means. Moreover, the increases are larger and may go on for longer periods than could be accounted for by simple filling in of remodeling space. One must reason that decrements of bone mass similar to those seen in 1°HPT increase fracture risk under other circumstances, and assure that restoration of BMD after parathyroid adenomectomy in hyperparathyroid patients should substantially reduce fracture risk. Severe bone disease caused by 1°HPT is rare. As a group, hyperparathyroid patients have mildly to moderately reduced bone mineral density that may be worst for cortical bone, but which has been observed at all sites. Removal of parathyroid adenomas and restoration of normal parathyroid function causes substantial, lasting increases of BMD (averaging 10 to 12%). Gain of bone occurs at all sites, may go on for up to 10 years, and is greatest in patients having the greatest baseline decrements of BMD.

Publisher Summary This chapter discusses familial benign hypocalciuric hypercalcemia (FBHH) and Neonatal Primary Hyperparathyroidism. The FBHH syndrome is inherited in an autosomal dominant fashion and is usually characterized by lifelong asymptomatic hypercalcemia, relative hypocalciuria, and the lack of the classical complications of hypercalcemia, including bone disease and renal stone disease. Some observers have also suggested that FBHH increases the risk of acute pancreatitis. The skeleton in patients with FBHH generally appears to be normal histologically, radiographically, and by mineral densitometry. While FBHH does not appear to threaten skeletal integrity, it does not confer immunity to osteoporosis. The relatively low urinary excretion of calcium in FBHH results from avid renal tubular reabsorption of calcium, and the urinary excretion of magnesium is unexpectedly low given that the serum level is in the upper part of the normal range or, in some cases, mildly elevated. Studies of the FBHH syndrome have almost uniformly suggested a genetic disorder of calcium sensing by the parathyroid gland paired with enhanced renal tubular reabsorption of calcium. PTH secretion persists at normal or slightly elevated values in the face of hypercalcemia, but without obvious disturbances of other calcium-regulating hormone concentrations. These pathophysiologic abnormalities would be most parsimoniously explained by dominant mutations of a single calcium sensing receptor expressed both in parathyroid glands and in kidneys. Neonatal primary hyperparathyroidism can be a mild syndrome representing the neonatal expression of FBHH, or it can present as a much more dramatic disease with high morbidity and even mortality if untreated, which is known as neonatal severe primary hyperparathyroidism (NSHPT).

Background In postmenopausal women, raloxifene hydrochloride has favorable effects on bone and lipid metabolism and does not stimulate reproductive tissues. The studies reported herein evaluated the long-term (3-year) effects of raloxifene treatment on bone mineral density (BMD), serum lipid levels, and drug tolerability in healthy postmenopausal women. Methods A total of 1145 healthy European and North American postmenopausal women aged 45 through 60 years were enrolled in 2 parallel, double-blind, randomized, placebo-controlled trials of identical design and randomly assigned to receive raloxifene hydrochloride, 30, 60, or 150 mg, or placebo daily; all groups received 400 to 600 mg of elemental calcium. Assessments included measurements for BMD by dual-energy x-ray absorptiometry, markers of bone turnover, and serum lipid levels. Results Lumbar spine BMD changed from baseline to 36 months as follows: placebo (mean percentage change + SE), −1.32% +0.22%; raloxifene, 30 mg, 0.71% +0.23%; raloxifene, 60 mg, 1.28% +0.23%; and raloxifene, 150 mg, 1.20% +0.24%. Comparable BMD changes were observed in the hip and total body. Biochemical markers of bone turnover were suppressed by raloxifene to normal premenopausal ranges through 3 years. Serum low-density lipoprotein cholesterol was reduced 7% to 12% below baseline through 3 years. Study withdrawals due to any reason (37%) and withdrawals due to adverse events (14%) were not different among groups. The only significant adverse effect of therapy was hot flashes (25% in the 60-mg raloxifene group vs 18% in the placebo group); hot flashes were typically reported as mild and were not associated with study withdrawal (1.7% for 60-mg raloxifene vs 2.4% for placebo). Conclusions Raloxifene preserves BMD at important skeletal sites, lowers serum low-density lipoprotein cholesterol levels, and has a tolerability profile comparable to placebo. These results indicate a favorable benefit-risk profile of raloxifene for long-term use in healthy postmenopausal women.

Estrogen, one of several sex steroid hormones, mediates its actions through the estrogen receptor. The estrogen receptor (ER) has two subtypes, ER alpha and ER beta, each of which predominates in specific tissues and organs. Cofactor proteins interact with the ER to maximize ligand-dependent transactivation of target-gene promoters. The estrogen response element is the final step in estrogen-mediated gene regulation, and current research is focused on alternate response elements. The resulting biologic action can vary according to the specific type of ER, cofactor milieu, response element, and ligand. Selective estrogen receptor modulators (SERMs) exhibit tissue-specific estrogen agonist or antagonist activity. The SERM raloxifene, which binds to ER and targets a distinct DNA element, may distinguish agonist vs antagonist activity by ER subtype and has unique activity among other SERMs because of its molecular conformation. Phytoestrogens, a potential alternative to hormone replacement therapy and for cancer prevention, do not consistently mimic estrogen's activity. Different types of phytoestrogens have different potencies, and taking high-dose supplements after menopause may not emulate the apparent benefits of lifelong consumption of phytoestrogen-rich diets. In conclusion, the complexity of estrogen action--through different ER subtypes, with various cofactors, on alternate response element--is further enhanced by ligands with selective estrogen activity. Additional research is needed to elucidate these pathways and the resulting biological effects.

Publisher Summary This chapter elucidates that the laboratory analyses is useful in the diagnosis of bone and calcium metabolic disorders. It elaborates upon serum that plays an important role in the diagnosis of bone and calcium metabolic disorders. Serum Calcium is measured to detect hypercalcemia or hypocalcemia and is not directly related to bone turnover rates. Serum Phosphorous is measured to detect effects of poor phosphorus intake, or phosphate retention in renal failure. Serum Parathyroid Hormone (PTH) is measured to detect hyper or hyposecretion of PTH for pathophysiologic diagnosis of hypercalcemia and hypocalcemia. Serum vitamin D metabolites are useful in the measurement of 25-hydroxyvitamin D, predominant circulating metabolite, and the best reflection of either nutritional vitamin D deficiency or vitamin D excess. Serum Parathyroid Hormone Related Protein (PTHrP) is measured to diagnose humoral hypercalcemia of malignancy resulting from hypersecretion of PTHrP by neoplasm, only after determining serum PTH suppressed. The excretion of calcium and phosphorus in urine varies with diet and is measured to evaluate suspected renal calcium leak in osteoporosis, and intestinal malabsorption. The strategy for pathophysiologic diagnosis of hypercalcemia and hypocalcemia are discussed here. The guidelines to interpret the serum chemistry panel in hyper calcemia and hypocalcemia in the context to calcium, phosphorous, creatinine, albumin, globulins, chloride, and cholesterol are summarized.

The predominant variety of familial benign hypocalciuric hypercalcemia (FBHH) is FBHH(3q), which is associated with presumed inactivating mutations of the cell surface calcium receptor (CaR) gene on chromosome 3q13.3-q21. We sought mutations of the CaR gene in FBHH by direct sequencing of PCR-amplified genomic DNA from 14 affected families: 8 mapped to 3q13, 1 mapped to chromosome 19p, and 5 unmapped. We sequenced the entire coding region of the gene (exons 2-7) in one or two affected members of each family and found six point mutations that altered one amino acid, cosegregated with hypercalcemia, and were absent in more than 100 unaffected persons. Four mutations were unique (S53P, D215G, S657Y, and P748R), and two had been reported previously (P55L and R185Q). Of four mutant CaR proteins expressed in Xenopus oocytes, three were deficient in extracellular Ca2+-induced signaling. No CaR mutations were found in eight families, including the one mapped to chromosome 19p. Three benign polymorphisms occurred in the COOH-terminal region of the CaR protein in 10%, 15%, and 30% of more than 100 unaffected persons. Thus, FBHH-causing CaR mutations were clustered in the NH2-terminal extracellular and membrane-spanning regions of the receptor protein. We suggest that these are important functional domains, probably for calcium binding and signal transduction, respectively. Finally, mutations in regulatory or intronic regions of the CaR gene may also underlie many cases of FBHH.

Inactivating mutations of the parathyroid cell calcium receptor (CaR) gene cause one form of familial benign/hypocalciuric hypercalcemia, and in homozygous form, cause neonatal severe primary hyperparathyroidism with parathyroid hyperplasia. Thus, we postulated that partial or total loss of CaR function might contribute to calcium insensitivity or even stimulate cell proliferation in sporadic parathyroid adenomas (PAds). To examine this possibility, we sought loss of heterozygosity (LOH) for markers flanking the CaR locus (3cen-3q21) in 35 PAds. We used 16 highly-polymorphic PCR-based markers in paired normal and tumor DNA, extracted from slices of archived surgical specimens. Nineteen to 24 of the DNA pairs were informative with at least one marker. In two informative pairs, we found LOH for markers D3S1303, D3S1267, or D3S1269, which are tightly-linked with and flank the CaR locus. In one tumor, deletion mapping confined the lost area between D3S1271 and D3S1238 (41.7 centimorgans, cM). In the other tumor, LOH spanned most of chromosome 3, ranging at least from D3S1307 to D3S1311 (271.4 cM). LOH was confirmed by repetition of the experiments and quantified by phosphorimaging. Thus, we found LOH encompassing the CaR locus in approximately 10% of sporadic PAds. These data are consistent with the hypothesis that loss of CaR function may occur in PAds, with functional consequences for calcium sensitivity and cell proliferation.

To the Editor. —Dr Sorum's 1 Commentary is spot on. When I call my attorney, I can hear the meter clicking in the background, and the bill arrives within days after the telephone call. The time is overdue for physicians to receive compensation for the extraordinary amount of "free services" that we render over the wire. The time consumed is real time, the judgments require expertise, the advice given has consequences, and the courts have established our liability for the consequences of those calls. Even as an academic physician with a limited practice, I find myself spending an extraordinary amount of time in the evening and on weekends making telephone calls to patients, either because the patients are unreachable during the workday or because I simply have no other time to make the calls. The burden certainly would be lighter were I to receive modest compensation. I hope Sorum's proposal

The human PTH/PTH-related peptide (PTH/PTHrP) receptor could be involved in hereditary disorders of PTH or PTHrP action. Knowledge of the gene's chromosomal location would allow studies linking it to specific disease traits. Therefore, we mapped the human PTH/PTHrP receptor gene by polymerase chain reaction of human/rodent somatic cell hybrid panels using oligonucleotide primers designed to amplify a portion of the gene from genomic DNA. The PTH/PTHrP gene was unambiguously assigned to the short arm of human chromosome 3, in the region designated 3p21.1-p24.2. Analysis of a second chromosome 3-specific mapping panel suggests that the gene is located near the 3p21.2-p21.3 boundary. The availability of highly polymorphic markers located in this region will permit exploration of the PTH/PTHrP receptor locus in genetic linkage searches for the causes of bone, calcium, and other potential disorders.

A 16-year-old girl sought medical attention at the Mayo Clinic because of a 4.5-kg weight loss, hypercalcemia, and a pelvic mass. Preoperatively, the level of the beta-subunit of human chorionic gonadotropin was 147 IU/liter. After a brief period for observation and hydration, abdominal exploration revealed a stage III dysgerminoma; total abdominal hysterectomy and bilateral salpingo-oophorectomy were performed. Within the dysgerminoma, syncytial giant cells expressed human chorionic gonadotropin-positive immunostaining in the cytoplasm. Postoperatively, the value of the beta-subunit of human chorionic gonadotropin decreased rapidly. The patient received whole-abdomen irradiation 4 weeks postoperatively, after which the level of calcium returned to normal. The patient has been free of disease for more than 7 years.

Humoral hypercalcemia of malignancy (HHM) is at least partly caused by tumor secretion of PTH-related peptide (PTHrP), but there is growing evidence for cosecretion with PTHrP of other bone-resorbing peptides, such as the cytokine interleukin-1 alpha (IL-1 alpha). Administration of PTHrP in vivo and in vitro generally mimics the actions of PTH itself, with increases in both resorption and formation of bone. However, bone in HHM is characterized by uncoupling of bone turnover, with increased resorption and decreased formation. We performed experiments to determine whether IL-1 alpha might alter the effects of PTHrP and produce uncoupling. Thus, we administered to 100-g male rats by sc osmotic minipumps synthetic PTHrP-(1-34) alone (2 micrograms/100 g/day), recombinant IL-1 alpha alone (1.5 micrograms/100 g/day), both peptides together at the previous doses, or vehicle only. We infused 5 groups of 12 rats each (PTHrP, IL-1 alpha, PTHrP plus IL-1 alpha, ad libitum fed control, and controls pair-fed to the PTHrP plus IL-1 alpha group) for 14 days. At the end of the study, blood and urine were taken for chemical measurements, and tibias and femurs were harvested for histomorphometry and extraction of RNA from periosteal cells. As expected, PTHrP induced hypercalcemia, relative hypophosphatemia, phosphaturia, and reduced bone mass. Osteoblast number was increased, but osteoclast number was not. Indices of bone formation were unchanged or reduced. The dose of IL-1 alpha chosen had no statistically significant effect, except for reduced longitudinal bone growth, but when combined with PTHrP, IL-1 alpha reduced hypercalcemia, hypophosphatemia, and phosphaturia. In contrast to the blood and urine effects, IL-1 alpha did not interact significantly with PTHrP's effect on bone measurements. Northern analysis of periosteal cell mRNA showed that PTHrP reduced expression of osteocalcin, but not glyceraldehyde-3-phosphate dehydrogenase; IL-1 alpha had no additional effect. These data suggest that 1) continuously administered PTHrP alone may induce uncoupled bone turnover with decreased cortical bone formation; 2) IL-1 alpha appears to inhibit strongly the renal effects of PTHrP and weakly (if at all) its actions on bone and, thus, to decrease its hypercalcemic, phosphaturic, and hypophosphatemic actions; and 3) cosecretion of IL-1 alpha, and possibly other peptide cytokines, with PTHrP may modify the clinical expression of HHM.

Over the last 25 years, the perceived clinical spectrum of primary hyperparathyroidism (HPT) has changed dramatically from a disorder characterized by severe bone and renal disease to one typically manifested by few or mild symptoms and little evidence of organ damage. Reasons for this change in spectrum include changing demographics (primary HPT is primarily a disease of the middle-aged and elderly), diffusion of medical knowledge leading to a higher index of suspicion, and improved clinical laboratory technology (especially inexpensive and accurate determination of serum calcium and parathyroid hormone). In the first 343 cases of primary HPT seen at the Massachusetts General Hospital, 57% had renal stones, 23% had hyperparathyroid bone disease, and less than 1% had no symptoms. By contrast, studies dating from the availability of automated serum calcium measurement found renal stones and hyperparathyroid bone disease in less than 5% of cases, and about half of cases had few or no symptoms. Most patients with primary HPT today have mild, nonspecific symptoms, such as weakness, fatigue, and mental depression, and such signs as arterial hypertension and osteopenia, and detection of their hypercalcemia is generally serendipitous. The mildness and slow progression seen in many cases of primary HPT has resulted in much controversy about appropriate management.

The cause of hypercalcemia in familial benign hypercalcemia (FBH; also called familial hypocalciuric hypercalcemia) is unclear, although it is PTH dependent. It is also uncertain how plasma PTH levels are related to the severity of biochemical abnormalities in FBH. Because the PTH-related peptide (PTHrP) has many PTH-like actions, it might have a role in the hypercalcemia of FBH. Thus, we studied 29 patients with FBH from 11 families, 29 age- and sex-matched controls, and 42 patients with primary hyperparathyroidism (1 degree HPT), measuring PTH with a highly sensitive two-site immunochemiluminometric assay and the hypercalcemic tumor factor PTH-related peptide (PTHrP) with an extraction/concentration RIA. Plasma PTH values were elevated in 86% of 1 degree HPT patients (36 of 42), but in only 20% of FBH patients, (6 of 29). Plasma PTHrP was elevated in 1 FBH patient, and the group mean value was normal. Plasma PTH was positively correlated with calcium (Ca) in 1 degree HPT (r = 0.66; P less than 0.0001) and in FBH (r = 0.53; P less than 0.004), but the slopes of the regressions were markedly different: 1 degree HPT, 6.72; FBH, 1.61 (P less than 0.0001). There was a negative correlation between PTH and phosphorus (P) in 1 degree HPT (r = -0.39; P less than 0.01) and in FBH (r = -0.41; P less than 0.03), but, again, the slopes differed greatly: 1 degree HPT, -6.57; FBH, -1.95 (P less than 0.0001). There were no correlations between PTHrP and Ca or between PTH and PTHrP. The sums and products of PTH and PTHrP were not better correlated with Ca than PTH alone. Thus, PTH values are lower at given Ca and P levels in patients with FBH than in those with 1 degree HPT, suggesting that PTH is more effective in raising Ca and lowering P in FBH than in 1 degree HPT. The enigma of FBH remains: what molecular defect can simultaneously cause parathyroid cell insensitivity to Ca, enhanced renal tubular reabsorption of Ca, increased renal rejection of P, and enhanced or retained sensitivity to PTH?

The roles of parathyroid hormone (PTH) and calcitonin (CT) in the pathogenesis of familial benign hypercalcemia (FBH, or hypocalciuric hypercalcemia) are uncertain. Thus we performed studies in 26 patients with FBH, 12 patients with primary hyperparathyroidism (HPT), and 20 normal volunteers, to answer these questions: are plasma levels of intact or biologically active PTH frequently elevated in FBH? Is plasma intact PTH nonsuppressible during calcium infusion? Is there blunting of the C cell CT response to calcium infusion as occurs in primary HPT? We used three methods for measurement of PTH: a mid region-specific radioimmunoassay (iPTH, antiserum GP-1M), an extraction-concentration bioassay (bioPTH, stimulation of cAMP generation in osteoblastlike cells), and a two-site immunoradiometric assay (IRMA) for intact PTH. PTH levels were significantly elevated in primary HPT by all three methods, but mean PTH was normal in FBH and 85–92% of values overlapped the normal range. During 5 minute calcium infusions (2 mg Ca2+ per kg) iPTH values fell little, but bioPTH and intact PTH fell sharply in all three groups. Mean calcium-induced decreases of intact and bioPTH were indistinguishable from normal in FBH, but PTH levels generally remained elevated at 5 minutes in primary HPT. In FBH basal and postinfusion CT levels were normal. The data show that, in the majority of patients with FBH, PTH concentrations and bioactivity in blood are within the normal range and are suppressed rapidly to very low levels with further increases of calcium. The data suggest that the abnormality of parathyroid function in FBH differs from that in primary HPT. There was no deficiency of CT or C cell responsiveness in FBH, another difference from primary HPT. Nonetheless, in 8–15% of cases, measurement of PTH could not discriminate FBH from primary HPT.

There is doubt about concentrations of circulating calcitonin gene-related peptide (CGRP) and the value of plasma CGRP measurements in the detection and follow-up of medullary thyroid carcinoma (MTC). Thus, we developed an immunochemiluminometric sandwich assay for CGRP using antibodies purified from a polyclonal antiserum against human CGRP. The assay was sensitive (limit of detection, 0.4 pmol/L; multiply by 3.7892 to derive nanograms per L) and highly specific [no cross-reaction with human calcitonin (CT)]. Normal plasma CGRP values ranged from less than 0.4 to 4.5 pmol/L (median, 0.8; n = 31), with 61% having detectable levels. Values in samples from patients with MTC were elevated: unoperated patients (n = 10), 4.7-137 pmol/L (median, 7.1); and operated patients with gross persistent or recurrent tumor (n = 14), 4.7-171 pmol/L (median, 23.2). In contrast, CGRP values were normal in 78% of nine postoperative patients with elevated CT, but no detectable tumor (range, less than 0.4 to 6.3 pmol/L; median, 1.6). CGRP levels increased after pentagastrin injection in MTC patients, but less than did CT values. Cultured MTC cells in vitro secreted large amounts of CGRP, and rat nerve root ganglia, human osteoblasts, and microvessel endothelial cells secreted lesser amounts. We conclude that CGRP circulates in normal plasma, but at very low levels. Plasma CGRP concentrations are frequently high in patients with MTC, but primarily in those with gross tumor or metastases. Plasma CT assay is the preferable test for MTC, but CGRP assay deserves prospective study for a possible role in predicting gross metastasis.

Specific insulin-like growth factor-binding proteins (IGFBPs) that may enhance or inhibit insulin-like growth factor (IGF) action are produced in various tissues. In the present study we demonstrated that IGFBPs are synthesized and secreted by rat osteoblast-like cells (UMR 106-01). PTH and PTH-related peptide (PTHrP) were potent stimuli for IGFBP production by UMR cells, whereas GH, IGF-I, insulin, epidermal growth factor, and T3 had little or no effect. A maximal 8- to 30-fold increase in IGFBP production was attained at 10(-7)-10(-6) M PTH and PTHrP, with a half-maximal effect at approximately 10(-9) M. By Western blot analysis, PTH and PTHrP markedly and selectively increased the production of 29,000 mol wt (Mr) and, to a lesser extent, 24,000 Mr IGFBPs. Agents that elevate intracellular cAMP by different mechanisms [(Bu)2cAMP, forskolin, and isobutylmethylxanthine] mimicked the effect of PTH and PTHrP on IGFBP synthesis. In comparison, PTH did not stimulate IGFBP production in fibroblasts and ROS 17/2.8 cells, which secrete IGFBPs of 42,000, 38,000, 34,000, 28,000, and 24,000 Mr, but not of 29,000 Mr. The PTH-responsive IGFBPs from UMR cells were nonglycosylated proteins with preferential affinity for IGF-I over IGF-II. These IGFBPs were not immunoprecipitated with antisera against rat IGFBP-2 or human IGFBP-1. Thus, PTH and PTHrP increase the production in UMR 106-01 cells of discrete IGFBP forms with Mr of 29,000 and 24,000 through a cAMP-mediated mechanism, independent of IGF-I synthesis. Taken with the known effects of PTH on IGF production in bone cells, the data suggest that PTH and PTHrP may modulate local IGF action in bone through the regulation of specific IGFBP availability.

Calcitonin assays are essential tools for research into calcitonin (CT) and C-cell physiology and pathophysiology. Several existing radio immunoassays for CT, based on polyclonal antisera, are generally reliable for detection and follow-up of patients having medullary thyroid carcinoma. Occasionally, however, these assays suffer from problems of specificity and sensitivity. Two-site immunometric techniques for CT offer potential for great improvement in sensitivity, specificity, and turnaround time over those based on classical competitive-binding radioimmunoassays.

PTH-related peptide (PTHrP) may be a major cause of the humoral hypercalcemia of malignancy. The circulating form of PTHrP is unknown, but mRNA analysis of tumor tissue suggests that multiple forms of PTHrP may exist. Therefore, we examined the ability of the full 141-amino acid protein as well as 2 amino-terminal fragments, PTHrP-(1-34) and PTHrP-(1-74), to increase cytosolic calcium ion concentrations ([Ca2+]i; assessed by aequorin luminescence) and stimulate cAMP accumulation in osteoblast-like rat osteosarcoma cells (ROS 17/2.8). PTH and all PTH-related peptides examined increased [Ca2+]i and cAMP in a concentration-dependent manner. The [Ca2+]i response to PTHrP-(1-34) closely resembled that to rat PTH-(1-34); both peptides produced biphasic responses. However, the responses to the longer PTHrP fragments generally were not biphasic. There were no significant differences among the three PTHrP forms in increasing [Ca2+]i or stimulating cAMP accumulation, although PTHrP-(1-74) was consistently weaker than the other two PTHrP peptides. PTHrP-(1-34) was more potent than rPTH-(1-34), which, in turn, was more potent than human PTH-(1-34) in increasing [Ca2+]i. However, PTHrP-(1-34) was not consistently more potent than either human PTH-(1-34) or rat PTH-(1-34) in stimulating cAMP accumulation. The inhibitory PTH analog bovine PTH-(3-34) attenuated both cAMP and [Ca2+]i responses to PTHrP-(1-34), but bovine PTH-(7-34) only reduced the [Ca2+]i response. Our data are generally consistent with PTHrP's acting through the PTH receptor, but differences in the effects of inhibitory PTH analogs on PTH and PTHrP action suggest as yet unexplained complexities, such as the existence of a PTH/PTHrP receptor family.