Synthetic Human Parathyroid Hormone Research Articles

Effect of treatment for 6 months with human parathyroid hormone (1-34) peptide in ovariectomized cynomolgus monkeys ( Macaca fascicularis)

A potential negative side effect of intermittent parathyroid hormone (PTH) therapy to treat osteoporosis is the loss of cortical bone concomitant with increased cancellous bone mass. We addressed this issue by studying the effects of PTH on whole-body, axial, and appendicular bone mass in an animal model with haversian cortical bone remodeling. Ovariectomized, young adult female cynomolgus monkeys were assigned to placebo (n = 9) or PTH groups (n = 10). The PTH group received 10 μg/kg synthetic human PTH(1-34) peptide by subcutaneous injection, 3 days/week for 6 months, and the placebo group received vehicle. Multiple endpoints of bone mass, strength, and turnover in the axial and appendicular skeleton were assessed, including dual-energy X-ray absorptiometry (DEXA), quantitative computed tomography (qCT), analysis of serum (calcium, phosphorus, alkaline phosphatase, osteocalcin, and tartrate-resistant acid phosphatase) and urinary (calcium and creatinine) biomarkers, histomorphometry, and biomechanical testing. Compared with placebo-treated animals, PTH-treated monkeys had no change in whole-body bone mass, but a 6.7% increase in spinal areal bone mineral density (aBMD) was observed. Cortical bone mass measured by qCT at appendicular sites was not affected by PTH treatment, but there were significant increases in cancellous bone mass in the proximal tibia, and a similar trend in the distal radius. Small, transient increases in serum and urinary calcium were observed, but there were no treatment-related effects on other biochemical endpoints. Increased bone formation rate (BFR/BV) in the midradius and midfemur was accompanied by a nonsignificant increase in midfemur porosity. Increased vertebral cancellous bone volume (BV/TV) was associated with greater trabecular and interstitial thickness with no effect on wall thickness. Increases in bone strength were observed in both axial (vertebral maximum stress and load at fracture) and appendicular (femoral neck fracture load) skeleton. Together, these results indicate that PTH therapy in the cynomolgus monkey results in a net gain of spinal and appendicular cancellous bone mass with no adverse effect on cortical bone.

Control of hypercalcaemia of parathyroid carcinoma by immunisation

Patients with parathyroid tumours can develop extreme hypercalcaemia and osteitis fibrosa cystica. Clinical features result from the action of parathyroid hormone (PTH) on bone receptors. Because this hormone is produced in microgram quantities, inhibition of its metabolic effects with potent PTH antibodies should be possible. We tested whether an immunisation with synthetic human and bovine PTH peptides could stimulate autoantibodies against PTH. A patient with metastatic parathyroid carcinoma in the lungs and pleura developed severe bone disease and extreme hypercalcaemia that proved resistant to conventional therapy. She was immunised with 200 microg human and bovine PTH peptides and 50 microg human PTH. Booster doses were also given at 4 weeks and 11 weeks. The patient was then seen every week. Antibodies against PTH were produced within 4 weeks of initial immunisation and titres increased with repeated doses of immunogens. Total serum calcium concentrations, which had ranged from 3.5 mmol/L to 4.2 mmol/L over the previous 18 months, fell to between 2.5 mmol/L and 3.0 mmol/L over 6 months of therapy. This fall was accompanied by striking clinical improvement. We believe this is the first use of immunotherapy to control remote, non-metastatic complications of malignant disease. B-cell tolerance to human PTH was broken by immunisation with PTH peptides in adjuvant. This therapeutic approach could be used to control excess hormone production in several types of endocrine tumour and may have applications in other diseases.

Influence of the dosing frequency of parathyroid hormone-(1-38) on its anabolic effect in bone and on the balance of calcium, phosphorus and magnesium.

The effect of the frequency of administration of synthetic human parathyroid hormone fragment 1-38 [hPTH-(1-38)] on bone formation and on the balance of calcium, phosphorus, and magnesium was investigated in 32 9-week-old female Sprague-Dawley rats, using a randomly complete block design. Rats received subcutaneously during 14 days either the vehicle solution once a day or 50 micrograms hPTH-(1-38)/kg BW once a day at 8:00 a.m., twice a day at 8:00 a.m. and 5:00 p.m. or three times a day at 8:00 a.m., 0:30 p.m., and 5:00 p.m. The balance study was performed during the last 48 h of the hPTH-(1-38) treatment schedule after which femora, tibiae, and lumbar vertebrae were removed for the determination of the dry weight, volume, and contents of Ca, P, Mg, hydroxyproline, and DNA. PTH treatment was associated with a significant increase of the apparent intestinal absorption of Ca, P, and Mg. Mean urinary Ca excretion augmented with the increase of the frequency of dosing. Urinary Ca excretion correlated negatively with the Ca apparent intestinal absorption and with the Ca content of the tibiae in the 2 groups with the highest frequency of dosing. The mean Ca, P, and Mg balances, the mean contents of bone Ca, P, and Mg and the mean bone dry weights were significantly increased with PTH treatment. In contrast to the mean volume of tibiae which was not affected by the PTH administration, the mean volume of the fifth lumbar vertebrae increased with the treatment. With the 2 times and 3 times daily treatments, mean hydroxyproline concentrations in the femora were significantly higher than the control values. An increase of the mean hydroxyproline content of the third lumbar vertebrae was evidenced with the 1 time and 2 times daily treatment, but the mean of the highest frequency of dosing was not different from that of the control group. The DNA content of femoral and of the fourth lumbar vertebrae significantly decreased with the frequency of dosing.

High-level production of recombinant human parathyroid hormone 1-34.

Expression of the synthetic human parathyroid hormone 1-34 [hPTH(1-34)] gene by a gene fusion strategy was demonstrated. hPTH(1-34) was produced at the C terminus of the partner peptides involving amino acids 1 to 97, 1 to 117, or 1 to 139 of a modified Escherichia coli beta-galactosidase by linker peptides containing oligohistidine of different lengths. The fusion proteins in the inclusion bodies were rendered soluble with urea and subjected to site-specific cleavage with the secretory type yeast Kex2 protease. Optimal expression and enzymatic processing were achieved in the fusion protein beta G-117S4HPT, constructed from amino acids 1 to 117 of beta-galactosidase and the linker of HHHHPGGSVKKR. The fusion protein accumulated more than 20% of the E. coli total protein. The hPTH(1-34) was purified up to 99.5% with a good yield of 0.5 g/liter of culture. The purified product was identified as intact hPTH(1-34) by amino acid analysis and N-terminal sequencing.

Synthetic human parathyroid hormone 1-34 vs calcitriol and calcium in the treatment of hypoparathyroidism

To test the hypothesis that treatment with human parathyroid hormone 1-34 (PTH 1-34) can maintain normal serum calcium without hypercalciuria in patients with hypoparathyroidism. Randomized crossover trial lasting 20 weeks. Each 10-week arm consisted of a 2-week inpatient dose-adjustment phase followed by an 8-week outpatient phase. Tertiary care center. A total of 10 patients with hypoparathyroidism were enrolled consecutively over a 15-month period. Half of the patients were prior National Institutes of Health patients, and the other 5 patients were referred from outside physicians. A dose of PTH 1-34 was administered each morning by subcutaneous injection. Calcitriol was given orally twice daily with supplemental calcium carbonate. Serum and urine calcium and phosphorus levels. Once-daily treatment with PTH 1-34 maintained serum calcium in the normal range with decreased urine calcium excretion (P<.05 at 2 weeks and P<.Ol at 10 weeks) compared with calcitriol treatment. Biochemical markers of bone turnover increased significantly (P<.Ol at 10 weeks) during PTH 1-34 treatment. Treatment of hypoparathyroidism with PTH 1-34 reduces urine calcium excretion compared with treatment with calcitriol and calcium.

Synthetic Human Parathyroid Hormone 1-34 vs Calcitriol and Calcium in the Treatment of Hypoparathyroidism

<h3>Objective.</h3> —To test the hypothesis that treatment with human parathyroid hormone 1-34 (PTH 1-34) can maintain normal serum calcium without hypercalciuria in patients with hypoparathyroidism. <h3>Design.</h3> —Randomized crossover trial lasting 20 weeks. Each 10-week arm consisted of a 2-week inpatient dose-adjustment phase followed by an 8-week outpatient phase. <h3>Setting.</h3> —Tertiary care center. <h3>Patients.</h3> —A total of 10 patients with hypoparathyroidism were enrolled consecutively over a 15-month period. Half of the patients were prior National Institutes of Health patients, and the other 5 patients were referred from outside physicians. <h3>Interventions.</h3> —A dose of PTH 1-34 was administered each morning by subcutaneous injection. Calcitriol was given orally twice daily with supplemental calcium carbonate. <h3>Main Outcome Measures.</h3> —Serum and urine calcium and phosphorus levels. <h3>Results.</h3> —Once-daily treatment with PTH 1-34 maintained serum calcium in the normal range with decreased urine calcium excretion (<i>P</i>&lt;.05 at 2 weeks and<i>P</i>&lt;.01 at 10 weeks) compared with calcitriol treatment. Biochemical markers of bone turnover increased significantly (<i>P</i>&lt;.01 at 10 weeks) during PTH 1-34 treatment. <h3>Conclusions.</h3> —Treatment of hypoparathyroidism with PTH 1-34 reduces urine calcium excretion compared with treatment with calcitriol and calcium.

培養ニワトリ骨髄骨における破骨細胞のアクチンフィラメントに対する副甲状腺ホルモンおよびカルシトニンの影響

Medullary bones dissected from hen femurs at 3 hours (when osteoclasts cease bone resorption ) and 12 hours (when osteoclasts actively resorb bone) after oviposition were cultured in BGJb media containing synthetic human parathyroid hormone (PTH) and synthetic eel calcitonin (CT), respectively, and the effects of PTH and CT on the distributional patterns of actin filaments in the medullary bone osteoclasts were observed. Three hours after oviposition, the osteoclasts displayed strong fluorescence of actin filaments in the form of amorphous bands in the apical region of the cytoplasm, corresponding to clear zones. There was no change in the distributional patterns of actin filaments in osteoclasts after culturing without PTH for 24 hours. However, after culturing with PTH for 24 hours, central parts of the actin bands in the osteoclasts became obscure, and the osteoclasts displayed actin filaments as linear forms perpendicular to the bone surface. On the other hand, 12 hours after oviposition, actin filaments appeared as lines perpendicular to the bone surface in the apical region of the cytoplasm, where ruffled borders had developed. There was no change in the distributional patterns of actin filaments in osteoclasts after culturing without CT for 24 hours. However, after culturing with PTH for 24 hours, the linear actin filaments disappeared and the osteoclasts displayed amorphous bands of actin filaments in the apical region of the cytoplasm. These results suggested that PTH and CT would regulate osteoclastic bone resorption by modifying the organization of actin filaments in osteoclasts

Growth hormone does not enhance the anabolic effect of human parathyroid hormone (1–34) on bone in aging multiparous and virgin rats

In humans, the progressive loss in skeletal bone mass with aging increases the risk of osteoporosis and bone fractures in the elderly. As parathyroid hormone (PTH) and growth hormone (GH) may both be anabolic in bone, we tested if the combination of these hormones would increase bone mass more than either agent alone in aging multiparous and virgin female rats. In four separate studies, female rats (15–18 months old) were treated for 12 or 15 days with synthetic human parathyroid hormone (hPTH 1–34) at 80 μg/kg per day given once daily, alone or combined with ovine GH at 1 mg/kg per day given twice daily. Bone of distal femurs, proximal tibias and lumbar vertebrae, dietary mineral (Ca, P, Mg) excretion and balance and serum chemistry were assessed. PTH alone stimulated bone formation and increased bone mass in three of four experiments and consistently increased serum 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2D 3) and mineral retention. GH alone did not change either bone formation or bone mass, but combined with PTH increased mineral retention and serum 1,25-(OH) 2D 3), more than either hormone alone. Despite this stimulatory effect on Ca retention, GH did not further enhance the anabolic effect of hPTH 1–34 on bone mass of aging multiparous and virgin female rats.

Regulation of urinary phosphate excretion in camels

Urinary inorganic phosphorus (P) excretion was measured in 16 adult female non-pregnant, non-lactating daily watered camels. They were randomly divided into four groups of four animals. Groups 1, 2, and received either an i.v. infusion of phosphate or synthetic human parathyroid hormone (PTH) or PTH-related peptide (PTHrP), respectively. The fourth group was used as a control. Intravenous P loading induced a significant increase in phosphatemia and in P renal clearance. Both PTH and PTHrP increased calcemia and decreased phosphatemia. They had no significant effect on urinary calcium excretion, but they increased P renal clearance and phosphaturia. Thus, the regulation of urinary P excretion in normally watered camels looks similar to that already described in other ruminants.

Cardiac electrophysiologic effects of parathyroid hormone in the guinea pig.

The cardiac electrophysiologic effects of parathyroid hormone (PTH) were studied in three different experimental models. Purified synthetic human parathyroid hormone (hPTH 1-84), at a dose of 30 ng/g body weight (BW), prolonged the QT interval of surface ECGs in intact guinea pigs. To further clarify the mechanisms of the QT prolongation caused by hPTH, the action potentials of guinea pig papillary muscle were recorded by conventional microelectrode techniques. hPTH (10(-7)M) prolonged the action potential duration without changing the action potential amplitude, resting potential, maximal upstroke velocity of the action potential, or maximal repolarization rate. hPTH affected only the plateau phase, in a concentration-dependent fashion. The half-maximum concentration was 6.8 x 10(-8)M for action potential duration at 30% repolarization (APD30), 3.2 x 10(-8)M for APD50, and 2.1 x 10(-8)M for APD90. In isolated single guinea pig ventricular cells, the patch clamp method was used to record the slow inward calcium current (ICa) and delayed rectifier potassium current (IK) responsible for the action potential plateau phase. hPTH increased the peak amplitude of ICa at + 10mV by an average of 42%, and increased IK at + 10mV by 85%. These results indicate that, in ventricular muscle, hPTH prolongs the action potential duration and QT interval by the balance of the prolongation effect on APD by enhancing the slow inward calcium current and the shortening effect on APD by enhancing the delayed rectifier potassium current.

Comparison of the effects of intermittent and continuous administration of human parathyroid hormone(1–34) on rat bone

We compared the effects of synthetic human parathyroid hormone (1–34) (hPTII[1–34]) on rat bones when administered by intermittent injection and by continuous infusion for 4 weeks. Continuous infusion of hPTH(I-34) caused a dose-dependent decrease in the dry weight of the femur and a hyperparathyroidism-like condition in the high-dose group (214 ng/kg/h). In this group, although bone morphology was quite abnormal, the metaphyseal trabecular bone volume increased, whereas the epiphyseal trabecular bone volume tended to decrease, enhancing both bone resorption and formation. The diaphyseal cortical bone of the tibia became markedly porous, and this appeared to be the main cause of the decrease in total bone weight. Conversely, the dry weight of the femur increased in a dose-dependent manner in the intermittent-injection groups. Neither porosity of the cortical bone nor abnormal morphology of the trabecular bone was observed. The volume of the metaphyseal trabecular bone increased while retaining its normal morphology. The epiphyseal trabecular bone formation obviously increased without increase in the number of osteoclasts. In other words, it was postulated that the total weight of bone increased because intermittent injection Df hPTH(1–34) increased the trabecular bone volume without loss of the cortical bone volume. These results show that either mode of treatment with hPTH(1–34) continuously accelerated bone formation, whereas the continuous infusion was essential for persistently and markedly enhanced acceleration of bone resorption. It appears that the increase in bone volume with intermittent injection of hPTH(1–34) resulted from the prominent manifestation of its bone-formative action rather than from its bone-resorptioe action. Thus, we think that intermittent administration of hPTH(1–34) is useful for bone formation in the treatment of osteoporosis.

Effects of infusion of parathyroid hormone and primary hyperparathyroidism on formation and breakdown of type I collagen

The influence of chronic and acute exposure to parathyroid hormone (PTH) on formation and breakdown of type I collagen, using two recently developed radioimmunoassays for serum PICP (the carboxyterminal propeptide of type I procollagen) and serum ICTP (the carboxyterminal telopeptide of type I collagen), have been evaluated. Fasting morning values were obtained from 18 women with primary hyperparathyroidism (HPT) and an equal number of age-matched, healthy controls. A 24-hour infusion of synthetic human parathyroid hormone (PTH 1-38) was performed in 14 healthy females. The patients with HPT had higher values for serum ICTP than the controls (6.0 +/- 3.0 and 4.1 +/- 2.1 micrograms/liter; P < 0.05), whereas the serum PICP concentrations were not different (170 +/- 72 and 151 +/- 65 micrograms/liter; n.s.). During infusion of PTH in healthy subjects, there was an increase of the serum ICTP concentrations (from 3.6 +/- 1.3 to 4.4 +/- 1.8 micrograms/liter; P < 0.001) whereas those of serum PICP decreased (from 185 +/- 78 to 118 +/- 42 micrograms/liter; P < or = 0.0001). The increase of serum ICTP during infusion of PTH was positively related to the increase of serum calcium and other indices of bone resorption, i.e., fasting urinary excretions of hydroxyproline and calcium. The decrease of serum PICP was also related to the changes of serum ICTP and hydroxyproline in urine, serum calcium, and alkaline phosphatase but not to osteocalcin, an established marker of osteoblastic activity. The findings support the fact that serum ICTP is a valuable method for evaluating bone resorption and is also easy to perform.(ABSTRACT TRUNCATED AT 250 WORDS)

Natriuresis and cyclic GMP stimulation by parathyroid hormone 1-34 in man

Synthetic human parathyroid hormone (PTH 1-34) was infused into seven healthy adults and their renal responsiveness examined. Blood and urine were collected at time zero and at 30 and 60 min after the infusion of PTH 1-34 (15µg in 50 ml of glucose solution infused in 10 min). A highly significant and parallel rise in both cyclic GMP and sodium excretion was found. No variation in blood atrial natriuretic factor concentration could be detected thereby suggesting that cyclic GMP is directly stimulated by PTH 1-34 and may play a role in the natriuretic effect of PTH.

Modulation of the Anabolic Effect of Synthetic Human Parathyroid Hormone Fragment-(1-34) in the Bone of Growing Rats by Variations in the Dosage Regimen

1. The influence of the time of the day of the administration of synthetic human parathyroid hormone fragment-(1-34) [hPTH-(1-34)] on its anabolic effect in bone was investigated in 23 60-day-old female Wistar rats. Rats were randomly assigned to the groups vehicle control morning, hPTH-(1-34)-treated morning, vehicle control afternoon and hPTH-(1-34)-treated afternoon, and once daily received a subcutaneous injection of 8 micrograms of hPTH-(1-34)/100 g body weight for 11 days. The increase in net intestinal calcium absorption and the increase in calcium balance were not influenced by the time of day of hPTH-(1-34) treatment. Four days after cessation of treatment, the net intestinal calcium absorption and calcium balance in hPTH-(1-34)-treated rats were not different from those of the control rats. 2. Modulation of the anabolic effect by variation of the hPTH-(1-34) dosage regimen was investigated in 43 60-day-old female Wistar rats. Rats were randomly assigned to the groups vehicle control, environmental control, 8 micrograms of hPTH-(1-34)/100 g body weight every 3 days for 24 days, 8 micrograms of hPTH-(1-34)/100 g body weight every 2 days for 16 days, 8 micrograms of hPTH-(1-34)/100 g body weight every day for 8 days, 4 micrograms of hPTH-(1-34)/100 g body weight twice a day for 8 days and 2.7 micrograms of hPTH-(1-34)/100 g body weight three times a day for 8 days. In all cases, the total dose of hPTH-(1-34) received was 64 micrograms/100 g body weight.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of recombinant human (1-84) or synthetic human (1-34) parathyroid hormone on the skeleton of adult osteopenic ovariectomized rats

The purpose of this study was to evaluate the dose-effect relationship of recombinant human PTH [hPTH-(1-84)] and synthetic human PTH [sPTH-(1-34)] for the skeleton of estrogen-deplete osteopenic rats. Ex vivo densitometry of regionalized whole femurs and histomorphometry of proximal tibial cancellous bone were the end points. Retired breeder female rats, aged 6-7 months, were used. Ten were killed at baseline, and the rest were ovariectomized (OVX). On day 42, a pre-PTH treatment OVX group was killed. The rest were then treated by daily sc injection with hPTH (0, 1.55, 15.5, or 155 micrograms/kg BW.day) or sPTH (0.55, 5.5, or 55 micrograms/kg BW.day) and killed on day 70. The level of cancellous bone mineral was lower in pretreatment OVX rats than at baseline. It was higher in rats treated with 15.5-155 micrograms/kg.day hPTH and 5.5-55 micrograms/kg.day sPTH than in pretreatment and vehicle-treated OVX rats. Cancellous bone volume was also lower both 42 and 70 days after OVX. Although hPTH did not affect cancellous bone volume, treatment with 5.5-55 micrograms/kg.day sPTH caused higher bone volume than in either pretreatment or vehicle-treated OVX rats. Trabecular number declined after OVX and did not change with PTH treatment. In contrast, trabecular thickness declined after OVX, but was higher after 15.5-155 micrograms/kg.day hPTH and 5.5-55 micrograms/kg.day sPTH treatment. In OVX rats, the amount of mineralizing surface was greater by day 42 and fell toward control levels by day 70. It was greater in rats treated with 15.5-155 micrograms/kg.day hPTH or 5.5-55 micrograms/kg.day sPTH than in vehicle-treated OVX rats. On a molar basis, sPTH was modestly more potent than hPTH. Osteoclast surface was not affected by PTH treatment. Treating estrogen-deplete osteopenic adult rats for 28 days with 15.5-155 micrograms/kg.day hPTH or 5.5-55 micrograms/kg.day sPTH increases trabecular thickness, but not trabecular number, to cause a rise in bone mass. The extent of mineralizing surface rises without a change in resorption surface. The marked rise in mineralizing surfaces suggests that extended in vivo treatment with PTH activates osteogenic precursor cells near once quiescent surfaces to become osteoblasts.

Effect of parathyroid-hormone-related protein on sodium-dependent phosphate transport in renal brush border membrane vesicles in rats. Comparison with parathyroid hormone.

Parathyroid-hormone-related protein (PTHrP) has significant homology with parathyroid hormone (PTH) in the amino-terminal region. We compared the effects of synthetic human PTHrP [hPTHrP(1-34)] on Na(+)-dependent phosphate transport with those of synthetic human PTH [hPTH(1-34)]. Intravenous administration of hPTHrP(1-34) in parathyroidectomized rats caused hypercalcemia, hypophosphatemia and phosphaturia to the same degree as hPTH(1-34). In kinetic studies using renal brush border membrane vesicles, the apparent Km and Vmax of phosphate transport were identical for the two peptides [hPTHrP(1-34): Km 27.6 +/- 0.8 microM, Vmax 3.78 +/- 0.04 nmol/mg protein, hPTH(1-34): Km 29.6 +/- 0.9 microM, Vmax 3.04 +/- 0.90 nmol/mg protein]. Both hPTHrP(1-34) and hPTH(1-34) at a supramaximal dose were equipotent in eliciting a 12-fold increase in cyclic adenosine 3',5'-monophosphate (cAMP) production in the kidney. These results indicate that, in addition to the similar effect of hPTHrP(1-34) and hPTH(1-34) on serum calcium levels and urinary phosphorus and nephrogenous cAMP excretion in vivo, these two peptides have similar effects of Na(+)-dependent phosphate transport in brush border membrane vesicles in vitro.

Pediatric use of synthetic human parathyroid hormone hPTH 1–34 infusion test in hypoparathyroidism

Pediatric use of synthetic human parathyroid hormone hPTH 1–34 infusion test in hypoparathyroidism

Effect of parathyroid hormone on release of interleukin 1 and interleukin 6 from cultured mouse osteoblastic cells

This study was undertaken to examine the possibility that parathyroid hormone promotes the production of interleukin 1 and 6 in MC3T3-E1 osteoblastic cells derived from mouse calvaria. The cells were incubated in serum-free medium with or without synthetic human parathyroid hormone(1–34). The concentrations of interleukin 1 and 6 in the culture medium were determined by using specific bioassay. The cells cultured without parathyroid hormone for 24 hr released both of interleukins, and parathyroid hormone stimulated the release in a dose-dependent manner. When the cells were cultured with 10 −6 M parathyroid hormone, the release of both interleukins from the cells remained higher than control up to 144 hr. These results suggest that interleukin 1 and 6 release stimulated by parathyroid hormone may be involved in the bone resorbing activity of the hormone.

Fast protein sequence determination with matrix‐assisted laser desorption and ionization mass spectrometry

Measurement and identification of digested peptides by matrix-assisted laser desorption and ionization mass spectrometry (LDI-MS) is demonstrated. Synthetic human parathyroid hormone, pTH (1-34), with a molecular mass of 4117.8 Da was digested with carboxypeptidases Y and B and the sequence of 14 amino acids from the C-terminus of the peptide was determined by analyzing the molecular mass of the truncated peptides. Furthermore, a tryptic digestion of pTH (1-34) was carried out and a molecular mass map of pTH (1-34) was obtained. With the results of the proteolytic digestion a rapid confirmation of the amino-acid sequence of the protein was possible. It is shown that the results of the tryptic digestion can be used for the unambiguous identification of the amino acid residues Lys and Arg, which cannot be distinguished with a mass spectrometer because of their equal nominal masses. Several advantages of amino acid sequence determination by the combination of digestion and LDI-MS are obvious: high sensitivity in the low pmol range, fast digestion time due to high enzyme/substrate ratios, quantification is unnecessary because the amino acids are identified by their molecular mass differences, the low chemical expenditure for the digestions and the accuracy of the sequence determination. Measurements with LDI-MS are fast: sample preparation and the measurement take only a few min. The mass determination and amino acid sequence is completely unimpaired by amino acid contaminations or impurities in the sample. The sensitivity of the method is in the low pmol to fmol range and thus comparable to other analytical methods.

Skeletal responsiveness to parathyroid hormone in healthy females: relationship to menopause and oestrogen replacement

In order to directly evaluate the role of parathyroid hormone (PTH) and its interaction with oestrogens for postmenopausal bone loss, studies were performed where synthetic human (1-38) PTH was infused s.c. over 24 h in 15 healthy females. Measurements were made of serum electrolytes, PTH and biochemical indices of bone turnover: serum osteocalcin and alkaline phosphatase and fasting urinary hydroxyproline and calcium. During the infusion of PTH there were significant increases of serum calcium (15%), fasting urinary calcium (55%) and hydroxyproline (80%) but a reduction of the serum osteocalcin concentrations (15%). There were significant relations between the calcaemic response and the increases of urinary calcium and hydroxyproline and the two latter were also closely related. There was, however, no correlation between the responses to PTH for the formative vs the resorptive indices. Postmenopausal women displayed greater increases of serum calcium and fasting urinary hydroxyproline indicating greater skeletal sensitivity to exogenous PTH. Following treatment with oestrogens the indices of skeletal responsiveness were reversed towards the premenopausal values. The findings demonstrate that during short-term infusion of PTH there is a dissociation between bone resorption and formation and, furthermore, that the menopause is associated with an enhanced skeletal sensitivity for PTH.