Establishment of the second international standards for porcine and human calcitonins: report of the international collaborative study.

The biological potency of calcitonins in clinical use in long-term treatment of Paget's disease of bone and, increasingly, in osteoporosis is usually expressed international units defined by the relevant World Health Organization international reference preparation. The international reference preparations for porcine and human calcitonins were ampouled in 1970 and stocks are now exhausted. Replacement standards were ampouled in 1989 and have been evaluated and calibrated by an international collaborative study comprising 16 laboratories in 12 countries. Evaluations included high-performance liquid chromatography and in vitro bioassay; calibration of each new ampouled preparation in terms of its international reference preparation was by in vivo rat hypocalcaemia bioassay. On the basis of the results of the study and with the agreement of the participants, replacement standards were established by the Expert Committee on Biological Standardization of the World Health Organization in 1991: the international standard for porcine calcitonin (ampoule code 89/540), with an assigned potency of 0.8 international units per ampoule, and the international standard for human calcitonin, with an assigned potency of 17.5 international units per ampoule. Both international standards appeared to be sufficiently stable to serve as the international standards for in vivo biological assays. Comparison of the two species of calcitonin in the same hypocalcaemia assay showed that they were approximately equipotent when the doses were given intravenously but that the human peptide was four- to sixfold more potent than porcine calcitonin when doses were given subcutaneously, emphasizing the need to compare "like with like".

Human parathyroid peptide treatment of vertebral osteoporosis.

Previous studies have shown that treatment with daily injections of human parathyroid peptide (hPTH) 1-34 increase axial cancellous bone mass partially at the expense of peripheral cortical bone. In the present work the same hPTH 1-34 regime given for 12 months has been combined with oestrogen or nandrolone therapy to control peripheral bone resorption. Spinal and iliac cancellous (but not cortical) bone increased by 40%-50% above initial values while no perceptible changes occurred in radial cortical or cancellous bone. The evidence of radiokinetic and histomorphometric studies performed before and in the last months of treatment suggested that bone remodeling had proceeded through a transient anabolic phase with increased activation, but that activation had become normal after 11-12 months in the cancellous bone of the ilium whereas it continued to be raised elsewhere in the skeleton. It is concluded that in combination with oestrogens, hPTH peptides given daily injections hold great promise for the treatment of patients with osteoporosis who have already lost substantial amounts of spinal cancellous bone.

Bone remodeling in hip fracture.

Hip fracture incidence has shown strong upward secular trends in many societies with wide differences in age adjusted incidence between nations. Falls and reduced physical activity have emerged as the strongest risk factors in epidemiological studies, while clinical investigations have pointed to secondary hyperparathyroidism as an important candidate cause of the loss of femoral cortical bone in old age. Until recently there have been few studies performed directly on the region of interest in the proximal femur. Non-invasive methodology using 85Sr has now been developed by our group for measuring bone formation and (with concurrent serial DXA densitometry) resorption in the femoral neck. Bone turnover averaged about 8% annually in controls. A group of younger cases of femoral fracture showed similar indices of total and regional bone formation to a control group; but their resorption was higher. To further investigate this, a femoral neck bone biopsy technique has been developed which can be applied to fracture cases treated by arthroplasty. Preliminary studies have established that the anatomical asymmetry of the neck in cross-section is considerable and imposes restraints on the interpretation of smaller or incomplete femur biopsies. Prospects are quite good that, in the absence of tetracycline pre-labeling, mineralization can be studied by assessment of alkaline phosphatase-positive surfaces in cryostat sections. Moreover, such sections will permit study of other anatomically localized metabolic activities as well as antigen expression and osteocyte viability. Candidate mechanisms for the regional decline in bone quality as well as bone mass in subjects suffering hip fracture can now be investigated more effectively.

International collaborative study by in vitro bioassays of the first international standard for porcine inhibin.

A lyophilized preparation of inhibin from porcine ovarian follicular fluid, ampoule code 86/690, was made internationally available as a research standard for in vitro bioassays in 1987. A study involving ten participants in eight countries assessed the stability and suitability of this research standard to serve as an international standard. Each of the participants used in vitro assays, the majority of which depended upon the inhibition of release of follicle-stimulating hormone from dispersed rat anterior pituitary cells. The research standard 86/690 was compared with coded ampoules of 86/690 stored under conditions of accelerated thermal degradation and with inhibins from different species. Intra- and interlaboratory variation for estimates of potency of a coded duplicate ampoule of the research standard provided the basis for comparisons of non-identical inhibins, but the fourfold variability of potency estimates for identical ampoules was such that no conclusions about the differences seen for non-identical inhibins could be made. Predictions of stability from consensus estimates of potency of ampoules that have undergone accelerated thermal degradation indicated that the research standard had satisfactory stability. On the basis of this study, the research standard 86/690 was deemed sufficiently stable and suitable to serve as a standard for in vitro bioassays and was established by the World Health Organization Expert Committee on Biological Standardization as the First International Standard for Porcine Inhibin. The possible presence, in biological extracts (standard or sample), of other bioactive proteins, such as activin and follistatin, complicates the quantitative interpretation of bioassay data. A standard of highly purified human inhibin is now required as a standard for immunoassays used for clinical research purposes; sufficient quantities of recombinant human inhibins have recently been donated for ampouling and evaluation by bio- and immunoassay in the subsequent phase of the standardization of inhibins.

Relationship between the location of osteoblastic alkaline phosphatase activity and bone formation in human iliac crest bone.

It is not feasible to use in vivo tetracycline double labeling to study bone formation in biopsies taken during the emergency fixation of fractures. We therefore compared the trabecular localization and extent of osteoblastic alkaline phosphatase (AP) perimeters with tetracycline and osteoid perimeters in iliac crest biopsies from 7 women with postmenopausal osteoporosis and 13 women without metabolic bone disease. Fresh biopsies were chilled to -70 degrees C, and triplicate serial unfixed undecalcified cryostat sections were cut and reacted for AP, stained for osteoid, or mounted unstained. At individual remodeling sites, the mineralizing perimeter (M.Pm) was measured as the extent of a double or single label accompanied by greater than or equal to 1 lamella of osteoid and greater than or equal to 1 lamella of mineralized matrix between the mineralization front and the adjacent label. Osteoid perimeters (O.Pm) and AP perimeters (AP.Pm) were also measured. In each biopsy there was good agreement between the location of AP and bone formation (kappa statistic, range 0.71-1.0). The overall sensitivity and specificity of AP as an indicator of the location of bone formation were 0.963 and 0.902, respectively. At the level of the basic multicellular unit, in those samples in which greater than 3 active BMUs were found, there was (1) significant positive correlation between the M.Pm and both AP.Pm and AP-positive O.Pm (except 1 patient) and (2) no significant difference between the M.Pm and AP-positive O.Pm (17 of 18 patients and 18 of 18 patients at the tissue level).(ABSTRACT TRUNCATED AT 250 WORDS)

Bone remodelling does not decline after menopause in vertebral fracture osteoporosis.

There is considerable current interest in whether activators of bone remodelling, such as IL-1 and other cytokines, are involved in the pathogenesis of osteoporosis. We have therefore studied indices relating to remodelling activation in 50 patients with postmenopausal vertebral osteoporosis and 12 with hip fracture osteoporosis in comparison with 25 age- and sex-matched controls. Because of uncertainty regarding the accuracy of current biochemical markers of bone formation with respect to the estimation of whole body rates of bone formation, a 85Sr-based radioisotopic method was used. This method was previously validated by comparison with data obtained after double in vivo labelling of transiliac biopsies taken nearly simultaneously. Bone resorption was estimated from urinary hydroxyproline data. Controls selected for their continued good health showed a progressive and statistically highly significant decline in indices of bone formation with time after menopause. No such decline was seen in the vertebral fracture patients (P less than 0.005). There were no hip fracture patients within 10 years of menopause so this statistical test could not be applied appropriately to them. The hydroxyproline data were consistent with the suggestion arising from the bone formation data that remodelling declines progressively after menopause in the controls but not in the vertebral fracture patients. The data also suggested that these two fracture groups were in more negative calcium balance than the controls, this being particularly marked in the hip fracture cases. Plasma osteocalcin data correlated moderately well with the kinetic measurements of bone formation. It is concluded that vertebral fracture osteoporosis is associated with prolongation of menopausal levels of bone remodelling which is inappropriate by comparison with healthy controls.

The short term effect of peripherally administered brain-gut peptides on gastric acid secretion in rats.

Certain brain gut-peptides are known to either stimulate or inhibit gastric acid secretion in several species after direct injection into the central nervous system. However there is inconsistency of published results on the gastric acid secretory response to some of these peptides after peripheral administration in different experimental systems. Seven peptides, namely neurotensin (NT), substance P, cholecystokinin (CCK), thyrotropin releasing hormone (TRH), human calcitonin (hCT), rat calcitonin-gene-related peptide (rCGRP) and bombesin, all known to modulate gastric acid secretion after central administration, were initially screened for activity after peripheral (subcutaneous) injection of 10 micrograms/kg body weight in a single rat model. Peptides showing an effect were retested at lower doses. Despite the inherent variability of the gastric acid secretory response in the non-anaesthetized pylorus ligated rat, a standardized experimental design confirmed that reproducible and statistically valid results could be obtained. The technical feasibility of using a one hour collection period as might be appropriate for short acting peptides was demonstrated by the significant dose dependent inhibitory activity of salmon calcitonin. In this model, NT and substance P had no significant effect on either volume or concentration of acid secreted, CCK showed a slight stimulation of acid output, and TRH, hCT, rCGRP and bombesin all inhibited acid output; CGRP and bombesin were active at 10 and 100-fold lower doses. The potent and inhibitory activity of bombesin in this system is in disagreement with other publications reporting no effect or variable stimulatory effect in rats. Time and dose dependent responses in our rat system indicate that this apparent discrepancy may be explained by the short duration of action of bombesin.

International Standards for salmon calcitonin, eel calcitonin, and the Asu1-7 analogue of eel calcitonin: calibration by international collaborative study.

Regulatory specifications in most countries require that the potency of salmon calcitonin (sCT) clinical products be expressed in International Units (IU) defined by the World Health Organization (WHO) International Standard. The first ampouled standard was prepared in 1972 and has been distributed world-wide since then. A batch of ampoules to serve as the replacement standard is now required. Other piscine calcitonins, eel calcitonin (eCT) and an amino-suberic acid analogue of eCT (Asu1-7 eCT) are now clinical products in some countries and international standards are required for these peptides which are similar to, but not identical with, sCT. This paper describes the preparation of three new ampouled standards and their biological calibration by international collaborative study comprising 17 participants from 10 countries. Following the recommendations in the final report of the collaborative study, the 2nd International Standard (IS) for sCT, the 1st IS for eCT and the 1st IS for Asu1-7 eCT were recently established by WHO, each with an assigned potency in IU, and are now available for issue.

Rapid periarticular bone loss in rheumatoid arthritis. Possible promotion by normal circulating concentrations of parathyroid hormone or calcitriol (1,25-dihydroxyvitamin D3).

For approximately 2 years, bone loss was measured in women with early stages of rheumatoid arthritis (RA) and in control subjects, using serial computed tomography and dual photon absorptiometry. Rapid trabecular bone loss from the distal radius was observed in the RA patients but not the controls. The bone loss correlated with initial plasma levels of parathyroid hormone and 1,25-dihydroxyvitamin D3 (calcitriol) concentrations. It has been suggested that these humoral factors may interact with cytokines or other mediators produced in the adjacent wrist joint. Losses of the cortical bone of the radial midshaft and the lumbar spine were modest and were comparable in the 2 groups. Indices relating to both bone formation and bone resorption predicted bone loss at these 2 sites, but changes in the parathyroid hormone and calcitriol concentrations did not.

Specific inhibition of basal gastric acid secretion by salmon calcitonin in rats does not necessarily involve a central pathway.

It is well established that salmon calcitonin (sCT), given either by central (intracerebroventricular) or peripheral (parenteral) injection is a potent inhibitor of gastric acid secretion. It is generally believed that this effect of sCT is mediated by the central nervous system based on reports that the effective peripheral dose is up to 1000 times greater than the effective dose administered centrally. We have reexamined this hypothesis by carrying out a number of independent experiments in two laboratories on the effect of sCT, given either by intracerebroventricular or by subcutaneous injection, on basal gastric acid secretion in unanaesthetized rats. Statistical evaluation of the data showed the reproducibility of the effective inhibitory dose ranges of sCT despite the inherent variability of the pylorus ligated rat system (Shay test). The effective dose range for sCT given centrally was between three- and ten-fold lower than that for peripherally administered sCT. There is no published evidence that a significant amount of peripherally administered sCT passes through the blood-brain barrier to relevant areas of the brain and this is confirmed in our study by autoradiography of serial sections of brain following intravenous administration of radioiodinated sCT. It therefore appears that the inhibitory effect of sCT, given centrally or peripherally, may not necessarily be mediated by a common pathway in the central nervous system. Our results also show that the inhibitory effect of peripherally administered sCT is lost when rats are treated with cysteamine to deplete somatostatin, thus implicating somatostatin as a peripheral mediator.

Human growth hormone treatment of hypophysectomized rats increases the proportion of type-1 fibres in skeletal muscle.

The studies describe alterations after hypophysectomy in the proportion of the type-1 and type-2 fibres in rat skeletal muscles, and the effects of replacement treatment with pituitary human (h) GH. Cytochemical analysis of myosin ATPase, succinate dehydrogenase and lactate dehydrogenase activities in sections of rat hind limb muscles were used as markers of fibre type and revealed that hypophysectomy reduced the proportion of type-1 fibres by 50% in soleus and in extensor digitorum longus muscles. This reduction in the proportion of type-1 fibres was accompanied by the appearance of transitional fibres (type 2C/1B). Following seven daily injections of hGH (60 mIU/day) to hypophysectomized rats, the proportion of type-1 fibres in both soleus and in extensor digitorum longus was increased with a concomitant reduction in the number of transitional fibres. After 11 days of treatment, all these transitional fibres had reverted back to type-1 fibres. Only hGH was observed to elicit this effect; injections of other pituitary hormones had no effect on the proportions of these transitional fibres. These alterations in fibre type occurred more rapidly than the changes reported after prolonged electrical stimulation of muscle or following extended exercise. These findings suggest that hypophysectomy and GH injection can result in a rapid alteration in the fibre composition of skeletal muscle, which may have important implications in terms of the resistance to fatigue and speed of contraction of the muscle.

Anomalies in the assay of parathyroid hormone in normocalcaemic patients with renal stone disease.

A group of 22 normocalcaemic patients with renal stone disease were retrospectively studied using seven different immunoassays for parathyroid hormone (PTH). N-terminal immunoradiometric assays and an intact hormone immunochemiluminometric assay sometimes gave results which fell above the normal range. These results were at variance with values obtained in three region-specific radioimmunoassays and an immunoradiometric assay for the intact hormone. Since these atypical samples contained normal levels of biologically active PTH the elevated immunoassay results were not thought to relate to the normal circulating hormone but rather to interference in the immunometric assays. The interfering substance was thought to be a large molecule which could not be removed by charcoal and did not dilute out in parallel to the PTH standard curve.

Periodic courses of human 1-34 parathyroid peptide alternating with calcitriol paradoxically reduce bone remodelling in spinal osteoporosis.

In an attempt to achieve an anabolic response in both axial and peripheral bone, we treated twelve patients with osteoporosis using human 1-34 parathyroid peptide given discontinuously. The peptide was given as seven daily subcutaneous injections followed by 21 days' treatment with 0.25 mg calcitriol orally. This regime was repeated cyclically for at least sixteen cycles, of which the first four were at a lower dose of hPTH 1-34 than used subsequently. The results of treatment were monitored by kinetic, densitometric, histomorphometric and biochemical studies performed before and during treatment. Two patients developed hPTH 1-34 binding in their plasma during treatment: this was presumed to be due to the development of antibodies. The remainder, instead of increasing their indices of bone turnover as judged by iliac bone histomorphometry, were found to have consistent reductions in trabecular resorption surfaces. The other indices of bone formation and resorption measured showed no change or comparable reductions. The small increases seen in total body calcium were consistent with 'in-filling' of deleted basic multicellular units (BMUs). Because there is no evidence that calcitriol alone causes comparable reductions in activation of bone remodelling in osteoporosis, interruption of treatment with hPTH 1-34 after 7 days may have led to a failure of the activation mechanism to proceed to the resorption stage, with a consequent overall reduction in remodelling activity. This type of treatment regime, with its calcitonin-like effect, might be effective in reducing net bone loss due to imbalance between bone formation and resorption at the BMU level, particularly in patients with increased numbers of BMUs ('high turnover' osteoporosis).(ABSTRACT TRUNCATED AT 250 WORDS)

Biological activities of synthetic human parathyroid hormone (PTH) 1-84 relative to natural bovine 1-84 PTH in two different in vivo bioassay systems.

Synthetic 1-84 human PTH (hPTH) peptides (with either asparagine) or aspartic acid at position 76) were compared with natural bovine PTH (bPTH) in three in vivo bioassays. Surprisingly, in the chick hypercalcemia bioassay, the human 1-84 peptides were approximately 3 times more potent on a molar basis than bPTH. In contrast, in an in vivo mouse kidney cAMP accumulation bioassay, these human peptides were 3-6 times less potent than bPTH. This low potency of synthetic hPTH relative to bPTH in the renal cAMP assay is in accordance with published relative potency estimates for natural extracted hPTH in in vitro rat renal membrane adenylate cyclase assays. The human and bovine 1-84 peptides were weakly active in an in vivo mouse calvaria cAMP accumulation system, producing a shallow dose-response curve which was not suitable for any quantitative estimates of potency. In contrast, both human and bovine 1-34 fragments were highly active in stimulating accumulation of cAMP in calvaria thus emphasizing the qualitative differences between 1-84 PTH and the 1-34 fragment of both species of PTH. Despite the homology between human and bovine 1-84 PTH, they have markedly different quantitative biological effects on hypercalcemia in chicks and in vivo renal cAMP accumulation in mice. Any estimate of the biological potency of human 1-84 PTH, relative to bovine 1-84 PTH, will need to be defined in terms of the nature and species of the biological test system.

Bone turnover in early rheumatoid arthritis. 1. Biochemical and kinetic indexes.

Biochemical, hormonal, and kinetic indexes of bone turnover were measured in 17 ambulant female patients with rheumatoid arthritis (RA) of recent onset (mean disease duration 14.2 months) and 19 controls. Mean serum osteocalcin concentration and 85Sr accretion rates were reduced and mean urinary hydroxyproline-creatinine ratios were increased in RA, but these differences were not significant compared with control values. Mean total body potassium (TBK), an index of skeletal muscle mass, was significantly reduced in RA, and the ratio of observed to predicted TBK correlated with indexes of bone formation. No abnormality of skeletal metabolism could be shown in early RA, but reduced rates of bone formation associated with diminished muscle mass may influence the development of osteopenia later in the disease.

Decreased responsiveness to chronic salmon calcitonin treatment in rat kidney and calvaria studied using quantitative enzyme cytochemistry.

Growing rats were treated with daily im doses of salmon calcitonin (sCT) (2, 15 and 100 IU/kg) for various times (1, 4 and 24 weeks). The effects on intracellular enzyme activities in bone and kidney were monitored using quantitative cytochemical methods previously developed for the identification of specific target tissue responses to calcitonins. The basal alkaline phosphatase activities in both kidney and bone were decreased by long-term treatment at all time periods and doses tested. No change was noted in basal Ca ATPase activities in kidney after treatment. The capacity of target tissues in chronically treated and control rats to respond to an acute iv dose of sCT was also compared. Acute provocation tests in treated and control rats showed that the renal alkaline phosphatase response was decreased in the rats receiving long-term treatment. Moreover, the direction of response was reversed in chronically treated rats when bone alkaline phosphatase and renal Ca-dependent ATPase activity was measured after acute provocation with sCT, i.e. bone alkaline phosphatase was stimulated instead of being inhibited and renal Ca ATPase was inhibited instead of being stimulated. The application of quantitative cytochemical techniques has demonstrated intracellular changes in enzyme activities in both kidney and bone. The impaired sCT responsiveness can be detected at shorter times of treatment (1 week) and lower doses (2 IU/kg) than has previously been possible by measurement of indices of mineral metabolism in plasma or urine.