Phosphate supplementation in young men: lack of effect on calcium homeostasis and bone turnover.

OBJECTIVE: To examine the effect of phosphate supplements on calcium homeostasis and bone turnover in young men. DESIGN: Study 1 was a randomised, controlled, cross-over trial of 1000 mg elemental phosphate given for one week, with a standard diet of 800 mg/d each of calcium and phosphorus. Study 2 was an escalating dose study of 0, 1000, 1500 and 2000 mg/d elemental phosphate, each given for one week, with a standard diet of 1000 mg/d each of calcium and phosphate. SETTING: Northern General Hospital, Sheffield. SUBJECTS: Study 1, 10 healthy men ages 19-32 y. Study 2, 12 healthy men ages 19-38 y. INTERVENTIONS: Sodium acid phosphate tablets each containing 500 mg elemental phosphorus (16.1 mmol/d), given with meals. RESULTS: Study 1, administration of 32.2 mmol/d phosphorus resulted in a mean increase of 13.2 mmol/d urinary phosphorus, a mean decrease of 1.1 mmol/d urinary calcium, and a mean increase of 7 ng/mL in serum intact PTH. There were no changes in serum phosphate, osteocalcin or urinary N-telopeptide excretion. Study 2, administration of 64.4 mmol/d phosphorus resulted in a mean increase of 27.2 mmol/d urinary phosphorus, a mean decrease of 2.4 mmol/d urinary calcium, with no change in serum phosphate, PTH or urinary deoxypyridinoline. CONCLUSIONS: Phosphate supplementation of the diet does not affect bone turnover in young men.

Arg21 is the preferred kexin cleavage site in parathyroid-hormone-related protein.

Parathyroid-hormone-related protein (PTHrP) contains several potential sites for proteolytic processing. Although there is considerable evidence for the existence of cleaved products in vivo, little is known about the post-translational processing of PTHrP. We have used purified kexin (Kex2) protease to identify which cleavage sites in recombinant PTHrP(1-141) might be of physiological significance. Cleavage products were identified by N-terminal sequencing. Kex2 preferentially cleaved PTHrP(1-141) carboxy to the triplet arginine site Arg-Arg-Arg21 with a Km of 3.3 +/- 1.7 microM and a kcat of 6 +/- 1.2 s-1. Substitution of alanine for Arg19 resulted in substantially reduced conversion, while no detectable cleavage occurred when alanine was substituted for either Arg20 or Arg21. In contrast, the degree of Kex2 cleavage at Arg21 in PTHrP(1-34) was lower. No detectable cleavage occurred in an unrelated synthetic peptide containing both double and triple arginine sites. Low levels of cleavage also took place carboxy to Lys-Arg97, Lys-Arg105, Arg-Arg106 and Thr-Arg108. Cleavage carboxy to Lys-Arg105, the best of these minor sites, occurred with a Km of 8.4 +/- 2.7 microM and a kcat of 0.8 +/- 0.2 s-1. These studies indicate that the preferred Kex2 cleavage site in PTHrP(1-141) is carboxy to Arg-Arg-Arg21, which effectively destroys its parathyroid-hormone-like biological activity. Cleavage of this site by Kex2-related mammalian convertases in vivo may be an important mechanism for full elaboration of the non-parathyroid-hormone-like paracrine actions of PTHrP in a tissue-specific manner.

Parathyroid hormone-related protein is a factor in normal fish pituitary.

Using antibodies to the amino-terminal region of human parathyroid hormone-related protein (PTHrP) we have demonstrated PTHrP immunoreactivity in pituitaries and plasma of the sea bream (Sparus aurata). Pituitary cells at two distinct locations contained immunodetectable PTHrP; an anterior group in the rostral pars distalis which also contained immunoreactive thyroid stimulating hormone (TSH), and a posterior group lying at the border of the pars intermedia and proximal pars distalis between cells which stained with antibody to human corticotrophin-like intermediate lobe peptide. By Western blot analysis pituitary extracts contained two immunoreactive isoforms of PTHrP, one of 29 kDa and the other of 26 kDa. Media of pituitaries incubated for up to 14 days in Krebs-Ringer bicarbonate also had several isoforms of immunodetectable PTHrP, two of them corresponding to the 29- and 26-kDa molecular forms but there were in addition both larger and smaller molecules. The concentration of PTHrP in sea bream plasma was comparable with levels observed in human subjects with humoral hypercalcaemia of malignancy. There was no reaction between pituitary cells or pituitary extracts and antibody to human parathyroid hormone. Thus sea bream pituitary contains immunoreactive PTHrP, which appears to be released into medium during in vitro incubation and which may be a significant source of plasma immunoreactive PTHrP in vivo.

Production of parathyroid hormone-related protein by the rat mammary gland in pregnancy and lactation.

Production of parathyroid hormone-related protein by the rat mammary gland in pregnancy and lactation. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E1077-E1085, 1992.--Production of parathyroid hormone-related protein (PTHrP) by the mammary gland of Sprague-Dawley rats has been examined using immunohistochemistry and in situ hybridization to detect PTHrP and PTHrP mRNA, respectively. PTHrP and PTHrP mRNA could be demonstrated in nests of epithelial cells of the developing mammary gland at day 14 of pregnancy and in the epithelial secretory cells lining the alveoli during the latter stages of pregnancy and during lactation. A specific radioimmunoassay was also used to measure the concentration of PTHrP secreted in the milk throughout lactation. The concentration of PTHrP in milk was relatively low initially but increased during the latter stages of lactation, whereas calcium concentrations remained virtually constant throughout lactation. No correlation was found between the concentrations of calcium and PTHrP in rat milk. These results show that PTHrP is present in rat milk and also in mammary tissue before parturition, and therefore it may assist in the development of the mammary gland during pregnancy.

Ontogeny of parathyroid hormone-related protein in the ovine parathyroid gland.

PTH-related protein (PTHrP) has been implicated in calcium regulation during fetal life. In this study the ontogeny of PTHrP was examined in ovine parathyroid glands. Immunohistochemical techniques, Western blot analysis, and a RIA with antisera raised against synthetic fragments of human (h) PTHrP (i.e. 1-34, 1-40, 50-69, and 107-141) were used to detect the presence of immunoreactive PTHrP in parathyroid glands from fetal and neonatal lambs and maternal ewes. Positive immunostaining for PTHrP was observed in fetal (from 116 days of gestation) and lamb (up to 180 days post birth) but not maternal parathyroid glands with the PTHrP(50-69) antiserum. Fetal and lamb parathyroid glands consisted entirely of one cell type in which PTHrP immunoreactivity to PTHrP(50-69) antiserum was found. In contrast, immunoreactivity to PTHrP could not be detected in sections of fetal, lamb, or maternal parathyroid glands with antisera raised against PTHrP(1-34) or PTHrP(107-141). However, PTHrP immunoreactivity in urea/acid extracts of newborn lamb parathyroid glands could be detected by Western blot analysis and RIA with antisera raised against the N-terminal portion of PTHrP. Western blot analysis with the PTHrP(1-34) antisera revealed that urea/acid extracts of newborn lamb parathyroid glands contained a substance with a mol wt of 14.4K, which corresponded in size to that of hPTHrP(1-84). Newborn lamb parathyroid glands contained 0.35 ng PTHrP/micrograms extract, whereas maternal parathyroid glands contained only 0.035 ng PTHrP/micrograms extract when tested in a RIA employing recombinant hPTHrP(1-84) as standard and an antibody raised against hPTHrP(1-40). The detection of immunoreactive PTHrP in the developing ovine parathyroid gland provides further evidence to support the suggestion that PTHrP produced in the parathyroid gland is involved in the normal hormonal regulation of calcium metabolism in the mammalian fetus and neonate.

Parathyroid hormone-related protein in milk and its correlation with bovine milk calcium.

Parathyroid hormone-related protein (PTHrP) has been shown to be present in milk of various mammals. We have assayed PTHrP in milk of various species by radioimmunoassay and estimated the molecular weights by Western blot analysis. PTHrP concentrations in bovine, ovine and human milk were 59.2 +/- 18.5, 74.1 +/- 35.0 and 36.6 +/- 20.7 micrograms/l (mean +/- S.D.) respectively, in pooled samples collected at various stages of lactation. PTHrP in mammalian milk was found to exist in two forms with molecular weights of 17.5 kDa and 21.5 kDa approximating those of PTHrP(1-108) and (1-141) respectively. In comparison, marsupial milk PTHrP appeared as a single low molecular weight form of 14.4 kDa which approximated to that of PTHrP(1-84). We performed a longitudinal study measuring the concentration of PTHrP in milk throughout lactation in cows, and found it to increase with the duration of lactation (r = 0.669, n = 91). We further examined the relationship between the concentration of PTHrP and total calcium in bovine milk, and the differences between these constituents in milk from Friesian and Jersey cows. PTHrP concentrations correlated positively with total milk calcium (r = 0.346, n = 105). The mean milk concentration of PTHrP of the Jerseys was significantly higher than that of the Friesians (52.6 +/- 5.4 micrograms/l compared with 41.0 +/- 4.8 micrograms/l, P less than 0.01), as was the mean milk calcium concentration (30.5 +/- 3.0 mmol/l compared with 26.7 +/- 2.7 mmol/l, P less than 0.01). We therefore postulate that production of PTHrP by the mammary gland may be associated with calcium transport from blood to milk. Also PTHrP may play a role in the development of milk fever in Jerseys which are predisposed to this condition.

Appropriate public health responses to clusters: the art of being responsibly responsive.

Between 1981 and 1988, the Minnesota Department of Health actively responded to over 400 reports from persons concerned about disease occurrence in their community, school, or workplace. Almost all of these reports involved perceived excesses of cases of cancer. Although there is little potential for identifying unsuspected public health problems or developing new etiologic insights, the Minnesota Department of Health has found that responding to reported clusters is a legitimate and necessary public health activity. To be responsibly responsive to these concerns, the Department has developed four steps to prioritize investigation of reported disease clusters, as well as six criteria for determination of the feasibility of environmental epidemiologic investigations. Approximately 95% of all concerns have been handled within the first two steps of this approach, generally requiring only education, or sometimes examination of readily-available data. Less than 5% of the concerns have required additional data collection and evaluation, and only about 1% have resulted in full-scale epidemiologic studies. Successful conclusions at all levels of this process require that public health officials develop effective communication, maintain objectivity, and provide leadership for controversial and difficult issues.

Immunohistochemical localization of parathyroid hormone-related protein in parathyroid adenoma and hyperplasia.

Parathyroid hormone-related protein (PTHrP) is invoked as the cause of humoral hypercalcaemia of malignancy (HHM); it is contained in the keratinocyte layer of normal skin; and there is evidence that is is produced by fetal parathyroids. Antibodies against synthetic PTHrP peptides have been raised in rabbits and sheep. This immunohistochemical study has found that primary parathyroid adenomata and hyperplastic glands from patients with chronic renal failure stain positively with antisera against PTHrP(1-34) and PTHrP(50-69). Primary hyperplastic glands are negative. No staining with anti-PTHrP(106-141) antiserum could be detected immunohistochemically in any of the parathyroid adenomata or hyperplasia.

Production of parathyroid hormone-related protein by a rat parathyroid cell line.

Parathyroid hormone-related protein (PTHrP), the peptide associated with humoral hypercalcemia of malignancy, has been identified in fetal and adult parathyroid glands. We here report a sub-clone of a rat parathyroid cell line which secretes a single peptide species corresponding in size to PTHrP(1-84). Biological activity of the secretion product was blocked by a specific antiserum against PTHrP, but not by parathyroid hormone (PTH) antiserum. Secretion of PTHrP by these cells was regulated by extracellular calcium in the physiological range. A single messenger RNA species for PTHrP was identified, though PTH mRNA could not be shown in these cells. Hybrid CAT genes containing 700-1000 bp of 5'-flanking DNA from the human PTH or PTHrP genes were transfected into these cells, and the PTHrP gene was expressed at 10-fold higher levels than the PTH gene. These cells thus provide a valuable model system for investigation expression of PTHrP in a non-transformed cell line.

Purification and characterization of recombinant human parathyroid hormone-related protein.

Full-length human parathyroid hormone-related protein (PTHrP-(1-141] as well as a carboxyl-terminal shortened form (PTHrP-(1-108] have been expressed from recombinant DNA-derived clones. These proteins were expressed in Escherichia coli as fusion proteins so that cyanogen bromide cleavage yields the desired product. Both proteins were purified and then characterized by sodium dodecyl sulfate gel electrophoresis, amino-terminal amino acid sequencing, peptide mapping, and mass spectral analysis. Recombinant PTHrP-(1-141), PTHrP-(1-108), synthetic PTHrP-(1-34), and naturally derived PTHrP are all equipotent in the stimulation of cyclic AMP levels in the osteoblast-like cell line UMR 106-01. However, PTHrP-(1-141) and -(1-108) are two to four times more active than PTHrP-(1-34) in the stimulation of plasminogen activator activity from this cell line. PTHrP-(1-141) reacts equipotently with PTHrP-(1-34) in a radioimmunoassay using an antiserum prepared against PTHrP-(1-34). PTHrP-(1-141), -(1-108), and -(1-84) were used as PTHrP-specific mobility standards on sodium dodecyl sulfate gel electrophoresis to determine the approximate length of two forms of naturally derived PTHrP. The data show that PTHrP purified from the lung tumor cell line BEN contains a major form of about 108 amino acids and another form of about 141 amino acids.

Parathyroid hormone-related protein: isolation, molecular cloning, and mechanism of action.

Many factors, such as interleukin 1, TGF alpha, tumor necrosis factor alpha and beta, and PGs, have been implicated in etiological roles in HHM (Martin and Mundy, 1987). Much interest in the past has also centered upon the likelihood of ectopic secretion of PTH in this condition. We have purified a protein (PTHrP) implicated in HHM from a human lung cancer cell line (BEN). Full-length cDNA clones have been isolated and were found to encode a prepropeptide of 36 amino acids and a mature protein of 141 amino acids. Eight of the first 13 amino acids were identical with human PTH, although antisera directed to the NH2 terminus of PTHrP do not recognize PTH; this homology is not maintained in the remainder of the molecule. PTHrP therefore represents a previously unrecognized hormone, possibly related to the PTH gene by a gene duplication mechanism. In support of this notion, the PTHrP gene has been localized to the short arm of chromosome 12; it is believed that chromosome 11, containing the PTH gene, and chromosome 12 are evolutionarily related. In addition, the human PTHrP gene has been isolated, characterized, and shown to have a similar intron--exon organization as the PTH gene. It is possible that the original ancestral gene is indeed the PTHrP gene; resolution of this question awaits studies in lower species. Peptides synthesized to the predicted protein sequence have enabled detailed structure-function studies that have identified NH 2-terminal sequences to be responsible for the biological effects of the molecule. Antibodies raised against the various synthetic peptides have led to the immunohistochemical localization of PTHrP in many human squamous cell carcinomas as well as in a subpopulation of keratinocytes of normal skin. The availability of these antibodies has opened the way for the development of a radioimmunoassay to detect PTHrP in the sera of cancer patients at risk of developing hypercalcemia. The recent characterization of PTHrP-like activity in the ovine fetus suggests some physiological function for PTHrP. It is possible that PTHrP, as the fetal counterpart of PTH, has the role of maintaining the maternal-fetal calcium gradient. The isolation and characterization of PTHrP have added to our understanding of the mechanisms of hypercalcemia and may contribute to the understanding of other metabolic bone diseases, such as osteoporosis and Paget's disease. Finally, and perhaps most importantly, PTHrP may play a hitherto unrecognized role in normal cell physiology.

Prevalence of hypertension and associated risk factors among diabetic individuals. The Three-City Study.

A cross-sectional study was conducted from 1979 through 1982 in three rural Minnesota cities to describe the natural history of diabetes mellitus. Detailed abstracts of the medical records of physician-defined diabetic individuals were used to construct medical profiles. As part of the effort, the date of physician-defined hypertension was identified and used to calculate hypertension prevalence. The crude hypertension prevalence was 56.8% for women and 33.99% for men, with an overall crude prevalence of 47.2%. No hypertensive patients were identified among diabetic patients less than 15 yr old, and most hypertensive diabetic patients were greater than 70 yr old. Hypertension prevalence in diabetic individuals demonstrated a highly significant trend with age (P less than .01). Compared with the general population, diabetic individuals had a significantly higher prevalence of hypertension (P less than .01), largely explained by the higher prevalence in women. Variables known to be associated with hypertension risk in the general population were also significantly associated with hypertension among diabetic individuals. Older age, being female, and increased body mass index were strongly associated with hypertension in this study population. Diabetic individuals with hypertension were six times more likely to have renal disease than those without hypertension. The level of blood glucose control, diabetes treatment (i.e., insulin or oral hypoglycemics versus diet), and diabetes duration were not associated with hypertension.

Parathyroid hormone-related protein of malignancy: active synthetic fragments.

Peptides corresponding to the amino-terminal region of the parathyroid hormone-related protein (PTHrP) of humoral hypercalcemia of malignancy were synthesized. A 34-amino acid peptide, PTHrP(1-34), was two to four times more potent than bovine or human PTH(1-34) in bioassays promoting the formation of adenosine 3',5'-monophosphate (cAMP) and plasminogen activator activity in osteogenic sarcoma cells and adenylate cyclase activity in chick kidney membranes. Like parathyroid hormone itself, in which the activity resides in the first 34 residues, PTHrP peptides of less than 30 residues from the amino terminus showed substantially reduced activity. PTHrP(1-34) had only 6% of the potency of bovine PTH(1-34) in promoting bone resorption in vitro. PTHrP(1-34) strongly promoted the excretion of cAMP and phosphorus and reduced the excretion of calcium in the isolated, perfused rat kidney consistent with the symptoms seen in malignant hypercalcemia.

A parathyroid hormone-related protein implicated in malignant hypercalcemia: cloning and expression.

Humoral hypercalcemia of malignancy is a common complication of lung and certain other cancers. The hypercalcemia results from the actions of tumor factors on bone and kidney. We report here the isolation of full-length complementary DNA clones of a putative hypercalcemia factor, and the expression from the cloned DNA of the active protein in mammalian cells. The clones encode a prepro peptide of 36 amino acids and a mature protein of 141 amino acids that has significant homology with parathyroid hormone in the amino-terminal region. This previously unrecognized hormone may be important in normal as well as abnormal calcium metabolism.

Parathyroid hormone-related protein purified from a human lung cancer cell line.

A protein with biological activities similar to parathyroid hormone (PTH) has been purified from serum-free culture medium obtained from a human lung cancer cell line (BEN). A major protein band of 18 kDa was obtained on NaDodSO4/polyacrylamide gels, with faint bands at 35 kDa and 67 kDa. Biological activity was associated only with the 18-kDa band. Amino acid sequence analysis of the material purified by HPLC revealed that 8 of the 16 residues were identical with those of human PTH. Antibody raised to a corresponding synthetic peptide recognized the PTH-related material but showed less than 1% cross-reactivity with human PTH amino-terminal peptides. BEN cells contained PTH DNA, but not PTH messenger RNA, indicating involvement of another gene. The purified PTH-related protein had a specific biological activity approximately equal to 6 times greater than that of bovine PTH(1-34). The PTH-related protein may have a role in the syndrome of humoral hypercalcemia of malignancy.

Feasibility study of a statewide pathology-based cancer surveillance system in Minnesota. I. Information characteristics.

The Minnesota Department of Health has completed a 2-year feasibility study comparing the completeness and accuracy of information from pathology-based cancer ascertainment with that of the traditional surveillance method based on hospital discharge records. Overall, for incident cancers, the primary site designation of the pathology-based system was correct for 94.5% of the cancers, and the histologic designation was correct for 97.0% of the cancers. For prevalent cancers the accuracy of both site and histology designation was inadequate at 81.0 and 76.8% respectively. Pathology-based ascertainment was more complete than discharge-based surveillance (98.4% vs. 96.6%), which reflected the growing number of cancers diagnosed in hospital outpatient departments and medical clinics. The major limitation of the pathology-based system was the inability to determine from written pathology reports whether the cancer was newly diagnosed. However, when asked, pathologists correctly determined the incidence status for approximately 75% of the cancers. In light of the results of the feasibility study, Minnesota is implementing a pathology-based system as a cost-effective, scientifically valid method to meet the state's current and future needs for cancer surveillance.

Incidence, prevalence, and mortality of diabetes mellitus in Wadena, Marshall, and Grand Rapids, Minnesota: the Three-City Study.

The Minnesota Department of Health (MDH) in concert with the Centers for Disease Control (CDC) conducted population-based studies of diabetes mellitus in three Minnesota communities. The use of hospital and clinic records alone for case ascertainment purposes would have missed 16% of study-eligible diabetic individuals. Thus, studies that use only hospital or clinic records may present a biased view of the natural history of diabetes. Physician-defined diabetes without additional diagnostic review yielded a prevalence of 1.6% (age standardized to the 1970 US Caucasian population). Diabetes incidence was 117 per 100,000 (age standardized to the same population). These results compare well with those of a Mayo Clinic population-based study in Rochester, Minnesota, and indicate the utility of physician-defined diabetes as an epidemiologic case definition. Despite differences in population size and structure as well as different medical care systems, diabetes incidence, prevalence, and mortality in these communities was remarkably similar.