Substituted 2-oxiranecarboxylic acids: a new group of candidate hypoglycaemic drugs.

Drugs to treat diabetes that can be taken orally have long been sought, although the successful management of insulin-dependent diabetes mellitus by simple chemotherapy may be an unachievable goal. The only drugs currently used for the treatment of non-insulin-dependent diabetes have limited effectiveness. In this article Peter Selby and Stanley Sherratt describe the development of a new group of candidate hypoglycaemic drugs, esters of substituted 2-oxiranecarboxylic acids, which merit full clinical evaluation. These drugs are hydrolysed to the free acids which are then converted to their coenzyme A esters in cells. The CoA esters inactivate carnitine palmitoyltransferase I in the outer mitochondrial membrane, thus preventing the excessive oxidation of long-chain fatty acids that occurs in diabetes. This causes a secondary decrease in hepatic gluconeogenesis and an increase in peripheral glucose utilization leading to improved glucose tolerance.

Osteopenia, neurological dysfunction, and the development of Charcot neuroarthropathy.

OBJECTIVE: To determine factors that might be associated with the development of Charcot neuroarthropathy. RESEARCH DESIGN AND METHODS: This cross-sectional prevalence study examined neurological function and bone density in matched groups of neuropathic diabetic patients with and without radiological evidence of Charcot neuroarthropathy. RESULTS: Patients with Charcot neuroarthropathy had a global impairment of neurological function that was significantly greater than that of otherwise matched non-Charcot neuropathic patients. All 17 Charcot patients had evidence of autonomic neuropathy compared with 10 of the control subjects (P = 0.03). The Charcot patients had evidence of reduced bone density in the lower limbs compared with the neuropathic control subjects (P = 0.009), but relatively preserved bone density in the spine (P = 0.4 vs. control subjects). CONCLUSIONS: We conclude that minor trauma in diabetic patients with peripheral neuropathy might result in a fracture in those with a reduced bone density and thus trigger the development of Charcot neuroarthropathy.

Bone loss in celiac disease is related to secondary hyperparathyroidism.

Celiac disease is a major cause of intestinal malabsorption. Previous studies have demonstrated that celiac disease is associated with significant osteoporotic bone loss. These studies have suggested that successful treatment of the malabsorption is associated with amelioration of the bone loss. Such studies have failed to examine bone mass at peripheral skeletal sites which is more likely to be responsive to changes in parathyroid hormone (PTH) in response to calcium malabsorption. We have examined bone density in the lumbar spine, femoral neck, and distal forearm in 35 patients with celiac disease who had been established on gluten-free diet. In addition, the concentrations of PTH and 1,25-dihydroxyvitamin D (1,25(OH)2D) were measured. Bone density was below that expected for the subject's age and gender at all sites. This was most marked in the distal forearm where the bone density was 1.40 SD below expected (p < 0.0001). In the forearm, there was a negative relationship between bone density and PTH concentration (r = -0.49, p = 0.009). In the forearm and lumbar spine, there was a negative relationship between 1,25(OH)2D concentration and bone density. Bone mass was not related to the concentration of 25-hydroxyvitamin D at any of the skeletal sites measured. Bone density is reduced in the peripheral skeleton in celiac disease and this deficit persists despite treatment with apparent normalization at axial skeletal sites. This reduction in bone mass is related to the presence of secondary hyperparathyroidism which should be sought in all patients with treated celiac disease.

Localization of estrogen receptor beta protein expression in adult human bone.

Evidence suggests that the newly described estrogen receptor beta (ER-beta) may be important for estrogen (17beta-estradiol) action on the skeleton, but its cellular localization in adult human bone requires clarification. We addressed this by using indirect immunoperoxidase with a novel affinity purified polyclonal antibody to human ER-beta, raised to hinge domain (D) sequences from the human receptor. Bone was demineralized in 20% EDTA and all biopsy specimens were formalin-fixed and wax-embedded. Vigorous retrieval was essential for ER-beta detection. In sections (5 microm) of benign prostate hyperplasia, used as positive control, clear nuclear immunoreactivity was seen in glandular epithelial cells, with a 1:500 dilution of ER-beta40. For bone sections, optimal antibody dilutions were 1:100-1:250. We found that in normal bone (from graft operations), in fracture callus from both men and women (>25 years old), pagetic bone, osteophytes, and secondary hyperparathyroid bone, all from older patients, ER-beta was expressed clearly in osteoclast nuclei, with little cytoplasmic immunoreactivity. Nuclear immunoreactivity was still prominent in osteoclasts, with antibody diluted 1:500, although it faded in other cells. Osteoblasts, in areas of active bone formation or bone remodeling, also expressed ER-beta, as did some osteocytes. However, hypertrophic chondrocytes were negative, unlike mesenchymal cells, adjacent to the osteogenesis. Megakaryocytes and some capillary blood vessels cells were receptor positive. All ER-beta expression was blocked totally by preincubation of antibody with antigen. We conclude that ER-beta is expressed in cells of osteoblast lineage and in osteoclasts. The latter appear relatively abundant in this receptor and this might provide a means for direct action of estrogen on osteoclasts.

Corticosteroids do not alter the threshold for vertebral fracture.

Corticosteroid use is one of the most important secondary causes of osteoporosis. Generally, it has been believed that in addition to its effect on bone mineral density (BMD), it also causes an alteration in bone quality that means that fractures occur at a lower BMD than might be expected. To establish if this is the case, we have compared the relationship between BMD and vertebral fracture in patients receiving corticosteroids with that in patients who had never received such therapy. Information was gathered on those patients who had been referred to the participating centers and had both BMD measurements and lateral thoracolumbar radiographs. In all, 452 patients (391 female) were identified; of these 82 (63 female) were receiving corticosteroids. There was no significant difference in BMD between the patients on corticosteroids and those with other suspected causes of osteoporosis. Vertebral fractures were present in 53% of patients on steroids compared with 35% of those who had no such treatment (p = 0.0035). The fractures were more likely to be multiple in patients on corticosteroids (p = 0.0042). However, if the relationship between bone density and fracture is investigated by plotting the cumulative prevalence of fracture against the bone density, measured by T score, the median BMD for fractures actually was marginally lower in patients on steroids, -2.74 (95% confidence interval [CI], -2.77 to -2.70) compared with -2.65 (95% CI, -2.66 to -2.65) in those who had not received steroids. Our results fail to support the notion that the fracture threshold is altered in patients on long-term steroids and suggest that the same diagnostic criteria should be used for osteoporosis in patients whether or not they are taking corticosteroid therapy.

Preliminary in situ identification of estrogen target cells in bone.

Although estrogens profoundly influence skeletal growth and maturation, their mechanism of action is still unclear. To identify their target cells in bone, estrogen receptors were located by immunofluorescence using the H222 monoclonal antibody in cryosections (both undecalcified and briefly decalcified) of hyperplastic mandibular condyle (persistent asymmetric mandibular growth) from a 14-year-old girl and radius and ulna from an 18-month-old female pig (epiphyseal fusion) and from a 3-month-old guinea pig (epiphyses open). Bone was removed from the animals at the peak of estrus. The most striking feature in all three species was the high proportion (approximately 50%) of receptor positive osteocytes. Although all sections contained active bone-forming surfaces, we were unable to identify clearly osteoblasts or lining cells that were estrogen receptor positive. In pig bone only, distinctive groups of receptor positive chondrocytes, with a pericellular localization of collagen type 1, were detected above the growth plate but below secondary centers of ossification. This observation suggests that osteocytes are major skeletal estrogen target cells and may be involved in coordinating the response of surface bone cells to the hormone, and further that chondrocytes may be involved in estrogen-induced epiphyseal growth plate fusion.

A negative search for a paramyxoviral etiology of Paget's disease of bone: molecular, immunological, and ultrastructural studies in UK patients.

Paget's disease of bone is a common bone disease characterized by increased and disorganized bone remodeling at focal sites throughout the skeleton. The etiology of the disease is unresolved. A persistent viral infection has long been suggested to cause the disease. Antigen and/or nucleic acid sequences of paramyxoviruses (in particular measles virus [MV], canine distemper virus [CDV], and respiratory syncytial virus [RSV]) have been reported in pagetic bone by a number of groups; however, others have been unable to confirm this and so far no virus has been isolated from patients. Here, we reexamined the question of viral involvement in Paget's disease in a study involving 53 patients with established disease recruited from seven centers throughout the United Kingdom. Thirty-seven patients showed clear signs of active disease by bone scan and/or histological assessment of the bone biopsy specimens and 12 of these had not received any therapy before samples were taken. Presence of paramyxovirus nucleic acid sequences was sought in bone biopsy specimens, bone marrow, or peripheral blood mononuclear cells using reverse-transcription polymerase chain reaction (RT-PCR) with a total of 18 primer sets (7 of which were nested), including 10 primer sets (including 3 nested sets) specifically for MV or CDV. For each patient at least one sample was tested with all primer sets by RT-PCR and no evidence for the presence of paramyxovirus RNA was found in any patient. In 6 patients, bone biopsy specimens with clear histological evidence of active disease tested negative for presence of measles and CDV using immunocytochemistry (ICC) and in situ hybridization (ISH). Intranuclear inclusion bodies, similar to those described by others previously, were seen in pagetic osteoclasts. The pagetic inclusions were straight, smooth tubular structures packed tightly in parallel bundles and differed from nuclear inclusions, known to represent MV nucleocapsids, in a patient with subacute sclerosing panencephalitis (SSPE) in which undulating, diffuse structures were found, arranged loosely in a nonparallel fashion. In the absence of amplification of viral sequences from tissues that contain frequent nuclear inclusions and given that identical inclusions are found in other bone diseases with a proven genetic, rather than environmental, etiology, it is doubtful whether the inclusions in pagetic osteoclasts indeed represent viral nucleocapsids. Our findings in this large group of patients recruited from throughout the United Kingdom do not support a role for paramyxovirus in the etiology of Paget's disease.

Increased catabolism of 25-hydroxyvitamin D in patients with partial gastrectomy and elevated 1,25-dihydroxyvitamin D levels. Implications for metabolic bone disease.

Serum vitamin D metabolites and PTH were measured in seven subjects with a history of previous partial gastrectomy (PGX) and metabolic bone disease. The elimination t1/2 of [3H]25-hydroxyvitamin D3 ([3H]25OHD3) in serum was assessed after an iv pulse dose of 5 microCi [26,27-3H]25OHD3. Median serum 25OHD3 was 37.5 (27.5-101.3) nmol/L, [normal range (NR) 10.8-58.5 nmol/L], mean serum 1,25-dihydroxyvitamin D [1, 25-(OH)2D3] was raised at 175 +/- 72 pmol/L, (NR 48-120 pmol/L) and mean PTH was also high, 67 +/- 27 ng/L, (NR 10-60 ng/L). Serum t1/2 [3H]25OHD3 ranged from 10.9-21.2 days. A strong negative correlation existed between t1/2 [3H]25OHD3 and serum 1,25-(OH)2D3 [Spearman's rank correlation coefficient (r = -0.82, P = 0.002)] and PTH [Spearman's rank correlation coefficient (r = -0.81, P = 0.001)]. Four subjects who had high initial PTH concentrations (60-115 ng/L) and elevated 1,25-(OH)2D levels (162-300 pmol/L) were reassessed after calcium supplementation to suppress secondary hyperparathyroidism (2 degrees HPT). In this subgroup, after-treatment PTH fell from 82 +/- 24 to 52 +/- 24 ng/L (mean +/- SD), not significant; 1,25-(OH)2D fell from 210 +/- 61 to 116 +/- 28 pmol/L, P = 0.015; and t1/2 [3H]25OHD3 increased from 13.2 +/- 1.9 to 18.9 +/- 3.1 days, P = 0.012. Patients with PGX and evidence of 2 degrees HPT with elevated 1,25-(OH)2D have a reduced t1/2 [3H]25OHD3, and this may explain the increased susceptibility of the subjects to osteomalacia. Calcium supplementation suppresses 2 degrees HPT, increases t1/2 [3H]25OHD3 and may protect against PGX osteoporosis and osteomalacia.

Calcium requirement--a reappraisal of the methods used in its determination and their application to patients with osteoporosis.

The accepted practice of calculating the dietary calcium requirement from the relationship between calcium balance and intake was recently criticized on mathematical grounds. To try to determine the appropriate calcium intake in patients with osteoporosis, I examined the relationships between calcium intake, output, and balance in a group of such patients. Calcium-balance studies were performed on 25 patients with untreated osteoporosis: intake (x +/- SEM) was 30.34 +/- 3.02 mmol/d, output was 30.59 +/- 2.69 mmol/d, and calcium balance was -0.25 +/- 0.64 mmol/d. There was a significant correlation between intake and both output and balance (r = 0.98, P < 0.001 and r = 0.61, P < 0.01, respectively). Intake and output became equal at 31.7 (95% CI 27.9, 35.5) mmol/d. This value is the same as that obtained when the conventional method of calculation is used. Furthermore, if random values for calcium output are assigned to the observed values of intake it can be demonstrated that the same values will always be obtained by both methods The only difference is that the calculation based on balance and intake results in a narrower confidence interval.

Immunofluorescent localization of estrogen receptor-alpha in growth plates of rabbits, but not in rats, at sexual maturity.

Estrogens are considered essential to the mechanism for closure of epiphyses in both males and females. The mechanism for this, however, is still unclear. It is likely that estrogen acts directly on growth plate chondrocytes, but the localization of the cells expressing the estrogen receptor (ER) has yet to be ascertained. Moreover, in rodents, growth plates remain open well into adult life. Whether the distribution of estrogen target cells in rodent epiphyses differs from that in other species, is also unclear. We therefore compared localization of estrogen target cells (denoted by ER-alpha protein expression) in species in which growth plates fuse, with that in rodents. Thus, we have investigated ER-alpha protein expression in femoral growth plates from male and female rabbits, just at sexual maturity (6 months), when growth plate fusion was just commencing, and in rats of equivalent developmental stage (9 weeks). ER-alpha was detected in undecalcified cryosections by immunofluorescence with 1D5 monoclonal antibody, raised to human ER-alpha; uterine sections were positive controls. ER-alpha-positive cells were localized to the proliferative/early hypertrophic zone of male and female rabbits. By contrast, cells in the similar region of the mature rat growth plate were ER-alpha-negative in both genders, although receptor could be readily detected in uteri of mature female rats. In growth plates of immature male and female rats (6 weeks), however, ER-alpha was clearly expressed by cells of the proliferative/early hypertrophic zone, but was barely detectable in uteri from immature females. Our findings support the view that estrogen may act directly on the growth plate and, in species in which there is epiphyseal fusion, may thus have a role in this process. If ER-alpha expression is lost at sexual maturity, as in rodents, growth plates may remain open into adulthood. Our results also show the changes in ER-alpha expression in growth plates of maturing rats may be opposite to that in the uterus and raise the possibility that receptor expression may be controlled differently in reproductive and skeletal tissues.

Osteoporosis in hypogonadal men: role of decreased plasma 1,25-dihydroxyvitamin D, calcium malabsorption, and low bone formation.

To investigate the pathogenesis of osteoporosis in male hypogonadism we have investigated a heterogeneous group of 13 men with hypogonadism: 7 men (median age 60, range 31-79) with two or more vertebral crush fractures and 6 men (median age 61.5, range 28-76) without vertebral fractures. The group with crush fractures had trabecular and cortical osteoporosis as assessed by Singh grade, iliac crest trabecular bone volume, and metacarpal cortical area/total area. This was accompanied by an altered trabecular architecture with a reduction in number of trabeculae but no change in trabecular width, which contrasts with age-related bone loss in men where there is no reduction in trabecular number but thinning of trabeculae. The fracture group had significantly lower plasma 1,25-dihydroxyvitamin D [1,25(OH)2D] concentrations than the nonfracture group, and this was associated with malabsorption of calcium. Irrespective of the presence or absence of osteoporosis, treatment with testosterone led to a significant increase in total and free plasma 1,25(OH)2D and an improvement in calcium absorption measured with radiocalcium and by balance techniques. In addition, urine biochemistry, metabolic balance studies, and bone biopsy suggest that skeletal retention of calcium and bone formation are increased by testosterone treatment. We conclude that male hypogonadism causes both cortical and trabecular osteoporosis and altered trabecular architecture. A major risk factor for the development of osteoporosis is reduction in plasma 1,25(OH)2D, leading to malabsorption of calcium and reduced bone formation.

Detection of canine distemper virus in 100% of Paget's disease samples by in situ-reverse transcriptase-polymerase chain reaction.

Previous evidence implicating paramyxoviruses in the aetiopathology of Paget's disease of bone has been controversial. While several groups have demonstrated the presence of paramyxoviruses using electron microscopy, immunohistochemistry, and molecular biological techniques, others have found no evidence of viruses using reverse transcriptase-polymerase chain reaction (RT-PCR). We have previously provided evidence that canine distemper virus (CDV) is present in approximately 65% of samples of pagetic bone, using in situ hybridization and RT-PCR; however, these results have been criticized. To further investigate the possible Role of CDV, we have now developed the technique of in situ-RT-PCR (IS-RT-PCR) to examine for the presence of CDV-nucleocapsid (CDV-N) ribonucleic acid (RNA) in pagetic bone. Control samples consisted of uninvolved sites from patients with the disease, normal bone, and several active remodeling states. IS-RT-PCR was optimized to detect CDV-N using distemper-infected vero cells. The specificity of the technique was confirmed using vero cells infected with CDV, which showed amplified signal following IS-RT-PCR, and cells infected with measles virus (MV), in which no positive signal for CDV was detected by IS-RT-PCR. Following conventional in situ hybridization, CDV-N was detectable in 10 of 15 pagetic bone samples. However, after five, and particularly 10, cycles of IS-RT-PCR, CDV-N was found in all 15 samples. There was no evidence of CDV in four samples from uninvolved sites from pagetic patients, or in any of the other control samples. In this study, using the novel technique of IS-RT-PCR, CDV was found to be present in 100% of pagetic samples examined. There was no evidence of the virus in any of the control samples, including samples of bone from uninvolved sites from patients with Paget's disease. These results provide additional proof that CDV is present within pagetic bone and further support the hypothesis that paramyxoviruses are involved in the etiopathology of Paget's disease.

Bone histomorphometry in adult patients with cystic fibrosis.

STUDY OBJECTIVE: Low bone mineral density is a common complication of cystic fibrosis (CF), and recent studies have implicated vitamin D insufficiency as a significant etiologic factor. The aim of this study was to establish whether there was bone biopsy evidence of vitamin D deficiency osteomalacia in patients with CF and to document the general histomorphometric characteristics of CF bone. PATIENTS AND METHODS: A retrospective descriptive and histomorphometric study of postmortem L2/L3 vertebral bone biopsy specimens was undertaken on tissue from 11 posttransplant CF patients and 4 nontransplanted CF patients. Control data were derived from postmortem bone specimens from 15 young adults. RESULTS: Bone from all CF patients was characterized by severe osteopenia in both trabecular and cortical bone. At the cellular level, there was decreased osteoblastic and increased osteoclastic activity. The reduction in osteoblastic activity was due to both a decrease in osteoblast number and a decrease in the biosynthetic potential of osteoblasts. The osteoclastic changes were due to an increase in the number of osteoclasts. The increase in osteoclasis and the uncoupling of osteoblastic and osteoclastic activity resulted in an increase in resorptive surfaces. Although there were few significant differences between the transplanted and nontransplanted CF groups, both cortical and trabecular bone mass tended to be lower after transplantation. None of the CF undecalcified biopsy specimens showed osteoid parameters characteristic of vitamin D deficiency osteomalacia. CONCLUSIONS: CF patients have an unusual and complex pattern of cellular changes within bone that are not typical of vitamin D deficiency osteomalacia.

Changes in plasma sodium concentration in women at the menopause.

A significant increase in the mean plasma sodium concentration and in the fasting urinary Na/Cr ratio was observed in healthy women at the menopause. Both of these changes were reversed by administering oestrogens. None of the above effects appeared to be due to alterations in sodium intake or in renal glomerular function. The most likely cause of the increase in plasma sodium concentration at the menopause appeared to be a decrease in plasma volume since there were also significant increases in the mean plasma albumin and total protein concentrations.

Osteoporosis in adults with cystic fibrosis.

Osteoporosis is prevalent in adults with CF Longitudinal data have not been collected and so the natural history is unknown. The aetiology is not known. There are no published randomized controlled trials evaluating treatments for osteoporosis in CF patients.