A selective inhibitor of the osteoclastic V-H(+)-ATPase prevents bone loss in both thyroparathyroidectomized and ovariectomized rats.

A potent and selective inhibitor of the osteoclastic V-H(+)-ATPase, (2Z,4E)-5-(5,6-dichloro-2-indolyl)-2-methoxy-N-(1,2,2,6, 6-pentamethylpiperidin-4-yl)-2,4-pentadienamide (SB 242784), was evaluated in two animal models of bone resorption. SB 242784 completely prevented retinoid-induced hypercalcemia in thyroparathyroidectomized (TPTX) rats when administered orally at 10 mg/kg. SB 242784 was highly efficacious in the prevention of ovariectomy-induced bone loss in the rat when administered orally for 6 months at 10 mg/kg/d and was partially effective at 5 mg/kg/d. Its activity was demonstrated by measurement of bone mineral density (BMD), biochemical markers of bone resorption, and histomorphometry. SB 242784 was at least as effective in preventing bone loss as an optimal dose of estrogen. There were no adverse effects of compound administration and no effects on kidney function or urinary acidity. Selectivity of the inhibitor was further studied using an in situ cytochemical assay for bafilomycin-sensitive V-H(+)-ATPase using sections of osteoclastoma and numerous other tissues. SB 242784 inhibited the osteoclast enzyme at 1,000-fold lower concentrations than enzymes in any of the other tissues evaluated. SB 242784 demonstrates the utility of selective inhibition of the osteoclast V-H(+)-ATPase as a novel approach to the prevention of bone loss in humans.

Adjuvant aminoglutethimide therapy for postmenopausal patients with primary breast cancer.

Three hundred and twenty-two postmenopausal patients with primary breast cancer and ipsilateral axillary node involvement were randomized to receive aminoglutethimide and hydrocortisone or placebo for 2 years in a double blind randomized trial between April 1980 and March 1985. Two hundred and eighty-six patients were eligible for the study of whom 145 received active drug and 141 received placebo. At the present time significantly fewer patients have relapsed or died without previous relapse in the treatment arm (P = 0.002); 43 of 145 (30%) patients receiving aminoglutethimide have relapsed or died compared with 63 of 141 (40%) of those receiving placebo. Local recurrence is also significantly reduced (P = 0.002) since only 6 patients receiving active treatment developed local recurrence compared to 21 receiving placebo. Side effects were severe enough to necessitate complete withdrawal or reduction of therapy in 27 of 145 (19%) in the treatment arm of the study compared with 21 of 141 (15%) in the placebo arm. A single treatment-related death occurred, due to agranulocytosis. Aminoglutethimide and hydrocortisone therefore delay relapse after surgery for primary breast cancer in postmenopausal women. It is too early to assess any effect on overall survival.

Fluctuation of mineral apposition rate at individual bone-remodeling sites in human iliac cancellous bone: independent correlations with osteoid width and osteoblastic alkaline phosphatase activity.

We investigated the determinants of bone formation at individual remodeling sites (BMUs) in cancellous bone from 8 osteologically normal, sex hormone-replete women with endometriosis. All were tetracycline double-labeled (2, 12, 2, and 4 day regime) before iliac bone biopsy. At each BMU the mineral apposition rate (MAR) was determined conventionally from the distance between label midpoints (MAR 1) and also from the distance between the mineralization front and the trailing edge of the second label (MAR 2). MAR 1 and 2 were compared with within-BMU measurements of osteoid width (O.Wi) and the activities of osteoblastic alkaline phosphatase (AP) and succinic dehydrogenase (SDH, an enzyme in the Krebs cycle), both quantitated by microdensitometry. A total of 143 BMUs were evaluated, of which 88 were satisfactory for all measurements and 132 were satisfactory for all but SDH. There was a weak correlation (r = 0.34) between MAR 1 and 2 at individual sites, with a mean difference of 0.49 micron/day (mean MAR 0.82 micron/day). The mean MAR of individual subjects tended to be either increasing or decreasing (F = 16.1, p < 0.01). In linear regressions, MAR 2 was statistically dependent on O.Wi, AP, and SDH (73% of the variance accounted for). In contrast, MAR 1 was weakly correlated with O.Wi and only 30% of its variance was accounted for by AP, SDH, and O.Wi. The variance in the MAR 2 data was inversely increased (p < 0.01) compared with MAR 1 as the number of days of bone formation represented.(ABSTRACT TRUNCATED AT 250 WORDS)

Certain vitamin D metabolites potentiate the expression of parathyroid hormone bioactivity.

With the development of a sensitive bioassay for the skeletal effects of parathyroid hormone (PTH), it has become possible to investigate the possible interaction between PTH and vitamin D3 metabolites. This assay is based on the stimulation of glucose-6-phosphate dehydrogenase (G6PD) activity in either the hypertrophic chondrocytes of the growth plate or the osteoblasts lining the metaphyseal trabeculae of rat metatarsals. The response to PTH is paralleled by the activity of dibutyryl cAMP. None of the vitamin D3 metabolites tested had any effect on enzyme activity when tested by themselves. However, both 1,25(OH)2D3 and 25(OH)D3 caused a dose-related potentiation of the response to PTH. Neither 1,24,25(OH)3D3 nor 1,25(OH)2D3 26,23-lactone potentiated the response to PTH. Because this potentiation of the response to PTH occurs after only 8 minutes, it is suggested that it represents a nongenomic response to the vitamin D3 metabolites.

Iliac trabecular bone formation predicts radial trabecular bone density changes in type 1 osteoporosis.

In 28 patients with idiopathic or postmenopausal type 1 (spinal crush fracture) osteoporosis, resorption indices and dynamic measurements of trabecular bone formation based on in vivo tetracycline labeling in 7.5 mm transiliac biopsies have been compared with trends in radial cortical and trabecular bone density measured with computed tomography. Positive correlations were observed between trabecular bone density trends in the radius and indices of bone formation in the ilium. These were improved when one of the two resorption indices was included with a formation index in bivariate regressions. Marked interindividual variations in radial bone density trends were also seen in cortical bone. These correlated poorly with trends in trabecular bone. Weak negative relationships between cortical bone trends and indices relating to bone formation and resorption were observed, but a positive association was seen with single-labeled surfaces on iliac trabeculae. If, as has been suggested, there are periodic variations in bone formation, the results suggest that axial and peripheral trabecular bone density trends are synchronized in osteoporosis, perhaps in response to systemic factors, such as circulating hormones.

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)

Monoclonal antibodies with selective reactivity against osteoblasts and osteocytes in human bone.

Monoclonal antibodies (MAb) may provide valuable tools for studying osteoblast differentiation. We therefore raised a panel of MAb reactive with cells of this phenotype using 1,25(OH)2D3-treated human trabecular osteoblast-like cells (HOBS) as the immunogen. Immunohistochemical studies on various tissues, including undecalcified cryostat sections of fetal and adult human bone, identified 11 bone cell-reactive MAb. Of these, 2 demonstrated particularly selective reactivities against osteocytes (OB/M) and osteoblasts (OB/L). These reactivities were also seen in developing bone from rat, rabbit, and marmoset. OB/L and OB/M demonstrated limited reactivity against a small number of human tissues from the extensive panel of substrates tested. Both MAb exhibited reactivity against discrete populations of cells in the large and small intestine. In addition, OB/L reacted with cells in the basal epidermis of skin and OB/M with cells in blood vessel walls. Both antibodies demonstrated reactivity against a variety of cultured osteoblast-like cell lines and other cultured cell types. These MAb may therefore provide a valuable means of studying osteoblast ontogeny.

Vis-à-vis cells and the priming of bone formation.

Bone formation throughout skeletal growth and remodeling always entails deposition of new bone onto a pre-existing mineralized surface. In contrast, the initial deposition of bone in development requires the formation, ex novo, of the first mineralized structure in a nonmineralized tissue. We investigated the cellular events associated with this initial bone formation, with specific reference to the respective role of cartilage and bone cells in bones which form via a cartilage model. The cellular architecture of initial osteogenic sites was investigated by light, confocal, and electron microscopy (EM) in the membranous ossification of fetal calvarial bones (not forming via a cartilage model) and in the membranous ossification of the bony collars of endochondral bones. Bone sialoprotein (BSP), which is expressed during early phases of bone deposition and has been proposed to be involved in the control of both mineral formation and bone cell-matrix interactions, was used as a marker of initial bone formation. We found that at all sites, BSP-producing cells (as identified by intracellular immunoreactivity) are arranged in a characteristic vis-à-vis (face to face) pattern prior to the appearance of the first mineralizing BSP-immunoreactive extracellular matrix. In perichondral osteogenesis, the vis-à-vis pattern comprises osteoblasts differentiating from the perichondrium/periosteum and early hypertrophic chondrocytes located at the lateral aspects of the rudiment. By EM, the first mineral and the first BSP-immunoreactive sites coincide temporally and spatially in the extracellular matrix at the boundary between cartilage and periosteum. We further showed that in an in vitro avian model of chondrocyte differentiation in vitro to osteoblast-like cells, early hypertrophic chondrocytes replated as adherent cells turned on the expression of high levels of BSP in conjunction with the switch to collagen type I synthesis and matrix mineralization. We propose a model for the priming of bone deposition, i.e., the formation of the first bone structure, in which the architectural layout of cells competent to deposit a mineralizing matrix (the vis-à-vis pattern) determines the polarized deposition of bone. For bones forming via a cartilage model, the priming of bone deposition involves and requires cells that differentiate from early hypertrophic chondrocytes.

A number of osteocalcin antisera recognize epitopes on proteins other than osteocalcin in cultured skin fibroblasts: implications for the identification of cells of the osteoblastic lineage in vitro.

Rabbit antisera to bovine osteocalcin were produced independently in two laboratories and their specificities established by western blot analysis. By immunohistochemistry each of the five polyclonal antisera produced an intense cytoplasmic staining in human bone-derived cells. Staining intensity was strongly attenuated by preabsorption of the antisera with osteocalcin. No staining was observed using nonimmune rabbit serum. However, the choice of skin cells as negative controls for osteocalcin synthesis yielded an unexpected positive staining pattern similar to that seen with the bone-derived cells over a range of antiserum dilutions. This was not caused by the uptake of exogenous osteocalcin from the culture medium because a similar pattern of staining was observed when medium was supplemented with osteocalcin-depleted fetal calf serum. Treatment with 1,25-dihydroxyvitamin D3 induced osteocalcin mRNA expression and osteocalcin secretion in cultures of bone-derived cells but not in skin fibroblasts. The results demonstrate that these polyclonal antisera also recognize epitopes shared with other proteins synthesized in culture by skin fibroblasts. Furthermore, three mouse monoclonal antibodies to distinct regions of the osteocalcin molecule show differential staining of human bone-derived cells, skin cells, and osteosarcoma cells (MG63). These observations indicate that the shared epitope residues in the central region of osteocalcin and are consistent with the specific synthesis of osteocalcin by bone cells alone. The observed nonspecificity of many osteocalcin antisera may compromise immunocytochemical studies of the osteoblast phenotype in studies in vitro when based solely on reactivity with inadequately characterized osteocalcin antisera.

Peptide aldehyde inhibitors of cathepsin K inhibit bone resorption both in vitro and in vivo.

We have shown previously that cathepsin K, a recently identified member of the papain superfamily of cysteine proteases, is expressed selectively in osteoclasts and is the predominant cysteine protease in these cells. Based upon its abundant cell type-selective expression, potent endoprotease activity at low pH and cellular localization at the bone interface, cathepsin K has been proposed to play a specialized role in osteoclast-mediated bone resorption. In this study, we evaluated a series of peptide aldehydes and demonstrated that they are potent cathepsin K inhibitors. These compounds inhibited osteoclast-mediated bone resorption in fetal rat long bone (FRLB) organ cultures in vitro in a concentration-dependent manner. Selected compounds were also shown to inhibit bone resorption in a human osteoclast-mediated assay in vitro. Chz-Leu-Leu-Leu-H (in vitro enzyme inhibition Ki,app = 1.4 nM) inhibited parathyroid hormone (PTH)-stimulated resorption in the FRLB assay with an IC-50 of 20 nM and inhibited resorption by isolated human osteoclasts cultured on bovine cortical bone slices with an IC-50 of 100 nM. In the adjuvant-arthritic (AA) rat model, in situ hybridization studies demonstrated high levels of cathepsin K expression in osteoclasts at sites of extensive bone loss in the distal tibia. Cbz-Leu-Leu-Leu-H (30 mg/kg, intraperitoneally) significantly reduced this bone loss, as well as the associated hind paw edema. In the thyroparathyriodectomized rat model, Cbz-Leu-Leu-Leu-H inhibited the increase in blood ionized calcium induced by a 6 h infusion of PTH. These data indicate that inhibitors of cathepsin K are effective at reducing osteoclast-mediated bone resorption and may have therapeutic potential in diseases of excessive bone resorption such as rheumatoid arthritis or osteoporosis.

Idoxifene: a novel selective estrogen receptor modulator prevents bone loss and lowers cholesterol levels in ovariectomized rats and decreases uterine weight in intact rats.

Idoxifene, a novel selective estrogen receptor modulator, was tested for its effects on bone loss, serum cholesterol, and uterine wet weight and histology in the ovariectomized (Ovx) rat. Idoxifene (0.5 mg/kg x day) completely prevented loss of both lumbar and proximal tibial bone mineral density (BMD). In an intervention study, idoxifene (0.5 and 2.5 mg/kg x day) completely prevented further loss of both lumbar and proximal tibial BMD during a 2-month treatment period commencing 1 month after surgery, when significant loss of BMD had occurred in the Ovx control group. Idoxifene reduced total serum cholesterol, which was maximal at 0.5 mg/kg x day. Idoxifene alone displayed minimal uterotrophic activity in Ovx rats and inhibited the agonist activity of estrogen in intact rats. Histologically, myometrial and endometrial atrophy were observed in both idoxifene and vehicle-treated Ovx rats. In this report, we also provide molecular-based evidence to support the observations in vivo of a novel selective estrogen receptor modulator (SERM) mechanism of action in bone and endometrial cells. Idoxifene is an agonist through the estrogen response element (ERE) and exhibits similar postreceptor effects to estrogen in bone-forming osteoblasts. Idoxifene also stimulates osteoclast apoptosis, and these pleiotropic effects ultimately could contribute to the maintenance of bone homeostasis. However, idoxifene differs from estrogen in a tissue-specific manner. In human endometrial cells, where estrogen is a potent agonist through the ERE, idoxifene has negligible agonist activity. Moreover, idoxifene was able to block estrogen induced gene expression in endometrial cells, which is in agreement with the observation in the intact rat study. In the uterus, idoxifene has a pharmacologically favorable profile, lacking agonist and therefore growth-promoting activity. Together with its cholesterol lowering effect and lack of uterotrophic activity, these data suggest that idoxifene may be effective in the prevention of osteoporosis and other postmenopausal diseases without producing unwanted estrogenic effects on the endometrium.

The metatarsal cytochemical bioassay of parathyroid hormone: validation, specificity, and application to the study of pseudohypoparathyroidism type I.

The most sensitive method for assaying the bioactivity of PTH in unextracted plasma is the renal cytochemical bioassay. However, PTH acts on bone as well as kidney and clinical studies have suggested that the actions of circulating PTH level may be different at the two sites. We developed cytochemical bioassay for PTH based on the stimulation of glucose 6-phosphate dehydrogenase activity in the hypertrophic chondrocytes of the growth plate and the osteoblasts lining the metaphyseal trabeculae of rat metatarsal bones. The index of precision was 0.14 +/- 0.02 (SE) and the interassay variation was 31%. With this assay, plasma bioactive PTH levels in normal subjects and patients with primary hyperparathyroidism ranged from 0.5-18 ng/L and from 27-850 ng/L, respectively. Studies of patients with pseudohypoparathyroidism type I indicated that plasma PTH bioactivity in such patients is greater in the metatarsal bioassay than in the renal bioassay; no such differences were found in normal subjects or patients with primary hyperparathyroidism.

Evaluation of osteoblastic activity by morphometric comparison of alkaline phosphatase cytochemistry vs. tetracycline fluorescence.

Alkaline phosphatase (ALP) activity was used as a novel histomorphometric index of osteoblastic surfaces involved in mineralization. The enzyme cytochemical reaction was done on sections of low temperature processed, glycol methacrylate (GMA) embedded bone biopsies from 39 patients with various types of renal osteodystrophy (age 48 +/- 12 yrs; 19 males, 20 females) who had received tetracycline labelling. Sets of three serial sections were obtained from each tissue block: the 1st section (2 microns thick) was stained with Methylene blue Azure 11 for morphology; the 2nd section (2 microns thick) was used for ALP cytochemistry; the 3rd section was left unstained for UV microscopy. ALP positive osteogenic cells on bone surfaces displayed either of two distinct morphologies: a) typical plump, 'active' osteoblasts, and b) flat, elongated cells otherwise indistinguishable from 'bonelining cells'. These ALP+ flat cells were in contact with sites of active osteoid and mineral deposition and also codistributed with tetracycline labels outside of, and in continuity with, osteoid seams. Flat lining cells which were ALP negative were never associated with labels. Therefore, ALP activity also provided an objective criterion for differentiating two different 'phenotypes' among flat bone lining cells (ALP+ and ALP-), associated or not associated with matrix mineralization, respectively. The following histomorphometric variables were measured: Ob.S/BS, OS/BS, MS/BS and ALP.S/BS. Ob.S/BS, OS/BS and MS/BS were different in different types of ROD. However, OS/BS always exceeded MS/BS which, in turn, always exceeded Ob.S/BS. ALP.S/BS exceeded OS/BS in controls, mixed ROD and hyperparathyroidism, whereas the reverse occurred in osteomalacia and aplastic bone, due to the abundance of ALP lining cells over nonmineralizing surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)

1-substituted 4-aryl-5-pyridinylimidazoles: a new class of cytokine suppressive drugs with low 5-lipoxygenase and cyclooxygenase inhibitory potency.

A series of 1-alkyl- or -aryl-4-aryl-5-pyridinylimidazoles (A) were prepared and tested for their ability to bind to a recently discovered protein kinase termed CSBP and to inhibit lipopolysaccharide (LPS)-stimulated TNF production in mice. The kinase, CSBP, appears to be involved in a signaling cascade initiated by a number of inflammatory stimuli and leading to the biosynthesis of the inflammatory cytokines IL-1 and TNF. Two related imidazole classes (B and C) had previously been reported to bind to CSBP and to inhibit LPS-stimulated human monocyte IL-1 and TNF production. The members of the earlier series exhibited varying degrees of potency as inhibitors of the enzymes of arachidonic acid metabolism, PGHS-1 and 5-LO. Several of the more potent CSBP ligands and TNF biosynthesis inhibitors among the present series of N-1-alkylated imidazoles (A) were tested as inhibitors of PGHS-1 and 5-LO and were found to be weak to inactive as inhibitors of these enzymes. One of the compounds, 9 (SB 210313) which lacked measureable activity as an inhibitor of the enzymes of arachidonate metabolism, and had good potency in the binding and in vivo TNF inhibition assays, was tested for antiarthritic activity in the AA rat model of arthritis. Compound 9 significantly reduced edema and increased bone mineral density in this model.

Giemsa as a fluorescent stain for mineralized bone.

We present evidence for a previously unrecognized differential staining effect of Giemsa solution in fluorescence microscopy. The effect consists of selective fluorescent staining of mineralized bone (and elastic fibers) in tissue sections and, like the classical Romanowsky effect, is based on the differential binding of Eosin Y to tissue structures in the presence of Azur II and Methylene Blue. This effect opens the way to new applications of the Giemsa solution in fluorescence microscopy and in confocal fluorescence microscopy.

Microcytophotometric analysis of human osteoclast metabolism: lack of activity in certain oxidative pathways indicates inability to sustain biosynthesis during resorption.

It has been proposed that highly biosynthetic cells oxidize fatty acids to generate ATP while maintaining high levels of glucose metabolism through the glycolytic and pentose shunt systems to supply biosynthetic intermediates. We investigated the metabolic strategies and substrate for ATP production in the osteoclast. We used in situ quantitative microcytophotometric techniques to determine the maximal activity of the pentose shunt (glucose-6-phosphate dehydrogenase; G6PD), the glycolytic pathway (glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase; G3PD and LDH), fatty acid oxidation (beta-hydroxyacyl dehydrogenase; HOAD), and the Krebs cycle (succinate dehydrogenase; SDH) in human osteoclasts in situ, and related these enzyme activities to the degree of involvement of the cells in resorption. Unlike other highly biosynthetic cells, such as chondrocytes and macrophage polykaryons, osteoclasts associated with bone resorption were deficient in G3PD, LDH, and G6PD activity. However, osteoclasts did demonstrate a capacity for fatty acid oxidation which increased in cells apposed to the bone surface. The lack of significant glycolytic and pentose shunt activity in the osteoclast provides good evidence that resorbing osteoclasts, unlike phagocytosing macrophage polykaryons, have the metabolic characteristics of cells with greatly reduced capabilities of de novo mRNA synthesis but which do maintain high rates of ATP production. The possibility that the loss of glycolytic activity is a prelude to cell death is discussed.

Combined tamoxifen and anabolic steroid as primary treatment for breast carcinoma in the elderly.

Fifty-one consecutive patients aged over 70 years with localized breast carcinoma received primary treatment with tamoxifen and nandrolone decanoate. Results were compared to a control group of 51 age, tumour volume and nodal status-matched patients selected from a cohort who had primary treatment with tamoxifen alone. One year after commencement of treatment 31 (60%) patients treated with tamoxifen and nandrolone showed regression of their tumour, compared to 24 (47%) in the control group. Twenty-two (44%) patients had side effects attributable to nandrolone. Only four (8%) patients continued nandrolone for more than 12 months, and at 3 years after commencement of treatment 21 (40%) patients from each group had tumour regression. We argue that nandrolone should not be used as primary treatment in breast carcinoma, but may have a role to play in patients who fail to respond to tamoxifen.

Assessment of breast cancer size: a comparison of methods.

Two hundred women presenting with primary breast carcinoma were studied to find the most accurate single or combination of methods to assess breast tumour size. Correlations of the maximum clinical, mammographic and ultrasound tumour diameter were made with maximum histological diameter. Tumour size could be assessed clinically in all 200 patients, and overestimated the size of small tumours and underestimated large tumours (P less than 0.001). Mammographic measurement, which was possible in 145 (72.5%), underestimated the size of large tumours (P less than 0.01). Only 100 women underwent ultrasound examination (size assessed in 86%) and this modality tended to underestimate the size of all tumours (P less than 0.05). All methods of measurement showed similar correlations with histological size. Stepwise linear regression showed that the most accurate and practical estimation could be made using the formula: Histological size = 0.5 x mammographic size + 0.5 x clinical size. We conclude that clinical measurement of breast cancer size is as accurate as that from mammography or ultrasound. Accuracy can be improved by the use of a simple formula of both clinical and mammographic measurements.

Pancreaticogastrostomy after pancreaticoduodenectomy.

Although the mortality following pancreaticoduodenectomy has fallen and is now below 5%, overall 14% of patients develop a leak at the pancreatic anastomosis. This complication carries a 24% mortality rate when pancreaticojejunostomy is the method of reconstruction. In order to reduce the incidence of this complication, pancreaticogastrostomy can be performed following pancreaticoduodenectomy. A total of 41 patients underwent this operation between 1968 and 1989. The indications for operations were carcinoma of the head of the pancreas (n = 19), carcinoma of the ampulla (n = 12), carcinoma of the lower end of the common bile duct (n = 6), chronic pancreatitis (n = 3) and one patient with a nonfunctioning islet cell tumour. One patient developed a pancreatic fistula which closed spontaneously in 5 days. This patient is alive and well 36 months after operation. Pancreaticogastrostomy with pancreatic duct to gastric mucosa anastomosis is recommended as a safe and straight-forward method of reconstruction following pancreaticoduodenectomy.

Interactions between calciotropic hormones.

The metatarsal cytochemical bioassay (CBA) for parathyroid hormone (PTH) was adapted to study interactions between PTH and certain vitamin D metabolites. Thus, while they had no effect in the system alone, both 1,25(OH)2D3 and 25(OH)D3 caused a dose-dependent potentiation of PTH-stimulated glucose 6-phosphate dehydrogenase activity in the hypertrophic chondrocytes of the rat metatarsal. 1,25(OH)2D3 was about 1000 times more potent than 25(OH)D3. Specificity is indicated by the lack of a similar effect when either oestradiol or 1,24,25(OH)3D3 or a lactone derivative of 1,25(OH)2D3 was used. Furthermore, the rapidity of the effect of 1,25(OH)2D3 and 25(OH)D3, within 8 minutes, favours a membranophilic mechanism rather than the conventional nuclear mechanism of steroid hormone action.