Combination of nanoindentation and quantitative backscattered electron imaging revealed altered bone material properties associated with femoral neck fragility.

Osteoporotic fragility fractures were hypothesized to be related to changes in bone material properties and not solely to reduction in bone mass. We studied cortical bone from the superior and inferior sectors of whole femoral neck sections from five female osteoporotic hip fracture cases (74-92 years) and five nonfractured controls (75-88 years). The typical calcium content (Ca(Peak)) and the mineral particle thickness parameter (T) were mapped in large areas of the superior and inferior regions using quantitative backscattered electron imaging (qBEI) and scanning small-angle X-ray scattering, respectively. Additionally, indentation modulus (E) and hardness (H) (determined by nanoindentation) were compared at the local level to the mineral content (Ca(Ind)) at the indent positions (obtained from qBEI). Ca(Peak) (-2.2%, P = 0.002), Ca(Ind) (-1.8%, P = 0.048), E (-5.6%, P = 0.040), and H (-6.0%, P = 0.016) were significantly lower for the superior compared to the inferior region. Interestingly, Ca(Peak) as well as Ca(Ind) were also lower (-2.6%, P = 0.006, and -3.7%, P = 0.002, respectively) in fracture cases compared to controls, while E and H did not show any significant reduction. T values were in the normal range, independent of region (P = 0.181) or fracture status (P = 0.551). In conclusion, it appears that the observed femoral neck fragility is associated with a reduced mineral content, which was not accompanied by a reduction in stiffness and hardness of the bone material. This pilot study suggests that a stiffening process in the organic matrix component contributes to bone fragility independently of mineral content.

Sex hormone status may modulate rate of expansion of proximal femur diameter in older women alongside other skeletal regulators.

CONTEXT: Little is known of associations between hip geometry and skeletal regulators. This is important because geometry is a determinant of both hip function and resistance to fracture. OBJECTIVE: We aimed to determine the effects of sex hormone status and other candidate regulators on hip geometry and strength. SUBJECTS AND METHODS: A random sample of 351 women aged 67-79 had two to four hip dual-energy x-ray absorptiometry scans performed over 8 yr of follow-up. Hip structural analysis software was used to measure subperiosteal diameter (PD) and the distance from the center of mass to the lateral cortical margin (d-lat) on three 5-mm-thick cross-sectional regions: narrow neck, intertrochanter, and shaft. Section modulus (Z), bone mineral density (grams per centimeter squared), and an index of bone mineral content (cross-sectional area) were calculated as estimators of bone strength. Serum analytes measured at baseline included SHBG, estradiol, PTH, creatinine, albumin, vitamin D metabolites, and glutamate- and gamma-carboxyglutamate-osteocalcin (OC). A linear mixed model was used to model associations with predictor variables, including testing whether the predictors significantly modified the effect of aging. RESULTS: Aging was associated with increasing PD and d-lat, and higher baseline SHBG significantly modified this effect, in the case of PD, increasing the rates of change at the narrow neck region by 19% for SHBG level 2 sd higher than population mean (P = 0.026). Higher baseline creatinine was independently associated with faster increases in PD and d-lat with aging (P < 0.041). Z declined faster with aging if baseline PTH was higher, and higher albumin had a contrary effect. Z was positively associated with free estradiol and inversely associated with SHBG and glutamate-OC. CONCLUSION: These results show large effects of SHBG on the regulation of proximal femur expansion and bending resistance, probably acting as a surrogate for low bioavailable estrogen. Potentially important effects for fracture resistance in old age were also revealed for PTH, markers related to renal function and the nutritional markers albumin and undercarboxylated OC.

Cytochemical bioassay of parathyroid hormone: characteristics of the assay and analysis of circulating hormonal forms.

A cytochemical bioassay for parathyroid hormone (PTH) was used for the characterization of the biological activity of circulating forms of the hormone. PTH-stimulated glucose-6-phosphate dehydrogenase activity in distal convoluted tubule cells was quantitated by integrating microdensitometry and the response to native bovine (b)PTH(1-84) was found to be linear between graded doses of hormone from 5 fg/ml to 5 pg/ml. Synthetic bPTH(1-34) and human (h)PTH(1-34) elicited a parallel and equimolar response; however calcitonin, ACTH, glucagon, epinephrine, vasopressin, and insulin failed to significantly stimulate the enzyme in doses up to 100,000 times greater than the lowest concentration of bPTH used. The assay was capable of distinguishing hormonal activity in normal, hypoparathyroid, and hyperparathyroid human plasma. After gel chromatography, bioactivity in plasma of hyperparathyroid patients with skeletal disease but normal kidney function coeluted mainly with bPTH(1-84), whereas bioactivity in plasma of hyperparathyroid patients with skeletal disease but severe uremia coeluted in approximately equivalent amounts with bPTH(1-84) and hPTH(1-34). Despite the abundance of small molecular-weight bioactivity in the peripheral circulation in uremia, approximately 85% of the bioactivity in the parathyroid venous effluent coeluted with bPTH(1-84). The results therefore demonstrate the sensitivity and specificity of the assay for PTH and its utility in measuring the hormone in human parathyroid disorders. The results furthermore demonstrate the importance of entities cochromatographing with bPTH(1-84) in comprising the circulating bioactive hormone in hyperparathyroidism, and support the concept of a biological role for smaller forms of PTH, at least in chronic renal failure.

Effects of physical activity on evolution of proximal femur structure in a younger elderly population.

INTRODUCTION: For a fixed weight, a wider bone of standardised length is stiffer. But moving the cortices away from the centre of mass risks creating structural (elastic) instability, and hip fractures have been postulated to occur as a consequence of buckling of the thinned supero-lateral femoral neck cortex during a fall. We hypothesised that stereotyped physical activity (e.g., walking) may help conserve bending resistance (section modulus, Z) through redistribution of bone tissue, but it might be at the expense of supero-lateral cortical stability. METHODS: Hip structural analysis (HSA) software applied to DXA scans was used to derive measurements of section modulus and distances of a cross-section's centre of mass from the supero-lateral cortical margin (lateral distance, in cm). DXA scans were obtained on 1361 men and women in the EPIC-Norfolk population-based prospective cohort study. Up to 4 repeat DXA scans were done in 8 years of follow-up. Weight, height and activities of daily living were assessed on each occasion. A detailed physical activity and lifestyle questionnaire was administered at baseline. The lateral distance was measured on three narrow cross-sections with good precision: narrow neck (NN, coefficient of variation 2.6%), intertrochanter (IT) and shaft (S). A linear mixed model was used to assess associations with predictors. RESULTS: Ageing was associated with medial shifting of the centre of mass, so that lateral distance increased. Both greater weight and height were associated with greater lateral distance (P<0.0001). Among physical activity-related variables, walking/cycling for >1 h/day (P=0.025), weekly time spent on moderate impact activity (P=0.003), forced expiratory volume in 1 s (NN and IT, P<0.026) and lifetime physical activity (IT, P<0.0001) were associated with higher lateral distance. However, after adjusting for these variables, activities of daily living scores (NN, P<0.0001) and weekly time spent on low impact hip flexing activities were associated with shorter lateral distance (P=0.001). Greater baseline lateral distance was significantly associated with increased risk of subsequent hip fracture (n=26) in females (P<0.05, all regions) independently of age, height and bone mineral content. CONCLUSION: The age-related shift medially of the centre of mass of the femoral neck and trochanter may have adverse effects on fracture resistance in the event of a fall, so compromising the beneficial effects of walking on fitness, strength and risk of falling. The role of more diverse physical activity patterns in old age that impose loading on the supero-lateral cortex of the femur, involving for example hip flexion and stretching, needs investigation for their ability to correct this medial shifting of the centre of mass.

Separation of inhibitory activity from biologically active parathyroid hormone in patients with pseudohypoparathyroidism type I.

Patients with pseudohypoparathyroidism type I have the symptoms of hypoparathyroidism despite elevated levels of immunoreactive parathyroid hormone (PTH). However, the circulating levels of bioactive PTH, as measured in a cytochemical bioassay, are generally within the normal range suggesting that the high levels of immunoreactive PTH are either due to the presence of biologically inactive fragments of parathyroid hormone or to the presence of an 'inhibitor' of PTH bioactivity. Gel-permeation chromatography has been used to fractionate plasma from patients with pseudohypoparathyroidism type I and revealed the presence of high levels of bioactive PTH and of an 'inhibitor'. This inhibitory activity was absent or much lower in plasma from control subjects. These results indicate, therefore, that in pseudohypoparathyroidism type I the expression of the biological activity of PTH at the level of the kidney is affected by the presence of a circulating inhibitor which can be separated from intact PTH by gel-permeation chromatography.

In vivo effect of 1,25-dihydroxycholecalciferol on the proliferation and differentiation of avian chondrocytes.

A combination of immunocytochemistry and in situ biochemistry has been used to determine the in vivo effects of 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] on the proliferation and differentiation of chondrocytes. Chicks were fed a diet supplemented with 1,25-(OH)2D3 (2.5, 5, or 10 micrograms/kg diet) for 3 weeks, and measurements were made in sections of growth plate of chondrocyte proliferation and rate of maturation through the growth plate [using bromodeoxyuridine (BrdUrd) labeling] and also chondrocyte differentiation [assessed by alkaline phosphatase (ALP) activity]. The labeling indices of the control and supplemented chicks were similar (23.1 +/- 1.3 versus 23.2 +/- 1.6%); however, within a 21 h period the BrdUrd-positive cells of the supplemented chicks had moved down the growth plate significantly farther than in the control chicks (71.0 +/- 2.8 versus 52.6 +/- 1.8%). Greater ALP (mean integrated absorbance) activity higher up the growth plate of the supplemented chicks indicated a more differentiated phenotype in cells closer to the epiphyseal junction. Within individual transitional chondrocytes ALP activity in the 10 micrograms/kg supplemented chicks was 26.6 +/- 0.85, which was significantly higher (p < 0.01) than that of the control chicks (19.2 +/- 0.9). These results suggest that 1,25-(OH)2D3 in vivo does not increase the rate of chondrocyte proliferation but accelerates the onset of maturation.

Single cell enzyme activity and proliferation in the growth plate: effects of growth hormone.

Longitudinal growth is a result of proliferation and differentiation of chondrocytes in the growth plate. Growth hormone (GH) stimulates longitudinal growth, and GH receptors have been shown on growth plate chondrocytes, but the effects of GH on chondrocytes of different cell layers are not clear. To study the effect of GH on chondrocyte activity, in situ biochemical techniques were used to measure enzyme activities, which are associated with cell differentiation (alkaline phosphatase [ALP]) and osteoclast activity (tartrate-resistant acid phosphatase [TRAP]), within single cells of the growth plate. Uptake of bromodeoxyuridine (BrdU) was used as a parameter for proliferative activity. In addition, glucose-6-phosphate dehydrogenase (G6PD) was measured since increased proliferation has been associated with increased G6PD activity. The role of GH was studied in a model of isolated GH deficiency (dwarf rat) and complete pituitary deficiency (hypophysectomized rat). Groups of GH-deficient dwarf rats were infused with recombinant human GH in either a continuous or a pulsatile manner, since the pattern of GH secretion is an important regulator of growth in the rat. After 7 days, G6PD activity in proliferative chondrocytes and TRAP activity in osteoclasts was increased, while ALP activity in hypertrophic chondrocytes was decreased. GH not only increased the number of chondrocytes that incorporated BrdU but also the total number of chondrocytes in the proliferative zone; therefore, its ratio, the labeling index (an indicator of proliferative rate), was not increased. The widths of the proliferative and hypertrophic zones were increased by both patterns of GH administration. The width of the resting zone was unaffected by continuous GH but decreased by pulsatile GH. ALP and TRAP activities were, respectively, higher and lower in hypophysectomized rats compared with the GH-deficient animals. Hypophysectomized rats had smaller growth plates than dwarf rats with a disproportionally wide resting zone, which, like BrdU uptake, was not affected by GH. GH treatment resulted in increased TRAP and decreased ALP activity. These results indicate that GH stimulates the commitment of chondrocytes within the resting/germinal layer to a proliferative phenotype (as opposed to stimulating the rate of chondrocyte proliferation) but only in the presence of other pituitary hormones. Furthermore, this study shows that enzyme activities within single chondrocytes and osteoclasts are GH-sensitive. The extent to which these effects are direct or mediated by systemic or local growth factors remains to be clarified.

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.

Preliminary characterization of porcine bone marrow stromal cells: skeletogenic potential, colony-forming activity, and response to dexamethasone, transforming growth factor beta, and basic fibroblast growth factor.

Neonatal pig bone marrow stromal cells (PBMSC) were tested in vivo and in vitro to establish their use as a large-animal model for the study of skeletogenesis. When implanted in diffusion chambers in athymic mice for 6-8 weeks, both freshly isolated pig bone marrow and passage 2 PBMSC formed partially mineralized cartilage, bone-like material, and fibrous tissue. The cartilage showed metachromatic, perilacunar staining with toluidine blue and safronin O, alcian blue staining for chondroitin and keratan sulfate, and intense immunostaining for type II collagen. Osteocalcin was immunolocalized to the mineralized regions, consistent with the formation of bone. Alkaline phosphatase was primarily observed in cell layers at boundaries between tissue types. Unstimulated monolayer cultures of PBMSC produced type I but not type II collagen, responded to dexamethasone (10(-8) M) with a 1.7-fold increase in alkaline phosphatase activity, and were stimulated to divide by basic fibroblast growth factor (1.5-fold; EC50 1 ng/ml). Transforming growth factor beta (TGF-beta) blocked both dexamethasone-induced alkaline phosphatase expression (EC50, 1 ng/ml of TGF-beta) and the mitogenic effects of bFGF (EC50 0.06 ng/ml of TGF-beta). When incubated for 10-14 days in medium containing dexamethasone, beta-glycerophosphate and ascorbate PBMSC formed mineralized nodules. Calcification occurred in the middle of the aggregates and was associated with intensely alkaline phosphatase positive cells and a dense type I collagen-rich matrix. PBMSC also displayed colony-forming unit-fibroblastic activity, with approximately 1 in 80 of the plated cells formed colonies > 128 cells over 14-21 days. PBMSC therefore mimic the known activities of stromal cells from other species, including the human, suggesting that they are a valid model for skeletal research.

Bone remodeling at the endocortical surface of the human femoral neck: a mechanism for regional cortical thinning in cases of hip fracture.

UNLABELLED: Endocortical remodeling and wall thickness (W.Th.) were measured in femoral neck bone from 12 female fracture cases (81.3 +/- 1.5 years) and 12 sex-matched controls (81.9 +/- 1.9 years). Regionally, osteoid and eroded surface were increased, whereas W.Th. was reduced. These processes likely contribute to cortical bone loss seen in hip fracture. INTRODUCTION: Because periosteal expression of alkaline phosphatase was similar between cases and controls, we hypothesized that the mechanism causing the marked femoral neck cortical thinning associated with hip fracture may be net endocortical bone loss. METHODS: Twelve female cases of femoral neck fracture (mean age = 81.3 +/- 1.5 years) and 12 age- and sex-matched postmortem controls (mean age = 81.9 +/- 1.9 years) were included in the study. Samples of their femoral neck bone were embedded in methyl methacrylate, sectioned at 10 microm, and stained with Solochrome cyanine R and Goldner's trichrome for the detection of osteoid (%OS/BS) and resorption surfaces (%ES/BS) respectively. In addition, wall thickness (W.Th.) and lamellar thickness (Lm.Th.) data were also collected from identifiable endocortical bone packets as a measure of formative potential. RESULTS AND CONCLUSIONS: %OS/BS was significantly elevated in the anterior (control = 3.4 +/- 0.7: fracture = 11.0 +/- 2.3; p = 0.0001), inferior (3.4 +/- 1.0: 9.9 +/- 3.0; p = 0.0009), and posterior quadrants (3.2 +/- 0.8: 9.1 +/- 2.3; p = 0.0021). Only for anterior region was increased %ES/BS demonstrated in the fracture group (2.8 +/- 0.6: 5.3 +/- 0.7; p = 0.055). W.Th. (mm) was reduced only in the inferior region of the fracture cases (control = 33.7 +/- 1.2: fracture = 30.6 +/- 0.9; p = 0.013), whereas Lm.Th. was also reduced inferiorly (control = 2.7 +/- 0.08: fracture = 2.5 +/- 0.08; p = 0.042). These data suggest that an endocortical remodeling imbalance involving reduced bone formation within inferior region coupled with elevated anterior resorption may make an important contribution to the cortical thinning observed in cases of femoral neck fracture.

Structure of the femoral neck in hip fracture: cortical bone loss in the inferoanterior to superoposterior axis.

Although bone mass is a contributory risk factor for hip fracture, its distribution about the femoral neck is also important. Femoral neck biopsies were obtained from 13 females with intracapsular hip fracture (fracture: mean age 74.3 +/- 2.3 years [SEM]) and 19 cadaveric samples (control: 9 males and 10 females 79.4 +/- 1.7 years) and the areas of cortical and cancellous bone were quantitated in octants. In the control group, although males had larger bones than females, the proportions of cortical and cancellous bone were not different (p > 0.05) between the genders. The total amount of bone, as a proportion of bone + marrow, was significantly reduced in the fractures compared with the female controls (%Tt.Ar: fracture 27.83 +/- 1.18, female control 33.62 +/- 1.47; p = 0.0054). Reductions in cortical bone area occurred in all regions but particularly in the inferior, inferoanterior, and anterior octants (p < 0.05). There were no differences between cases and controls in the regional amount of cancellous bone (all regions, p > 0.178). Marked reductions in mean cortical bone width between the fracture and female control group occurred in the anterior, inferoanterior (31%), and superoposterior (25%) regions. Representing cortical widths as simple Fourier functions of the angle about the center of area (R2adj = 0.79) showed in the cases that there was preservation of the cortical bone in the inferior region, with the proportional loss of cortical bone being greatest in the inferoanterior and superoposterior regions. It is concluded that loss of cortical, rather than cancellous, bone predominates in cases of femoral neck fracture. This loss occurs primarily along the inferoanterior to superoposterior axis. As this axis bears the greatest strain during a fall, it is hypothesized that specific thinning of the cortex in these regions leads to an exaggerated propensity to fracture in those so affected, above that resulting from an equivalent general decrease in bone mass.

Cortical remodeling following suppression of endogenous estrogen with analogs of gonadotrophin releasing hormone.

The effects of estrogen suppression on osteonal remodeling in young women was investigated using transiliac biopsies (eight paired biopsies + four single pre; three single post biopsies) taken before and after treatment for endometriosis (6 months) with analogs of gonadotrophin releasing hormone (GnRH). Estrogen withdrawal increased the proportion of Haversian canals with an eroded surface (106%, p = 0.047), a double label (238%, p = 0.004), osteoid (71%, p = 0.002), and alkaline phosphatase (ALP) 116%, p = 0.043) but not those showing tartrate-resistant acid phosphatase (TRAP) activity (p = 0.25) or a single label (p = 0.30). Estrogen withdrawal increased TRAP activity in individual osteoclasts in canals with diameters greater than 50 microns (p = 0.0089) and also the number of osteons with diameters over 250 microns (p = 0.049). ALP activity in individual osteoblasts was increased but not significantly following treatment (p = 0.051). Wall thickness was significantly correlated with osteon diameter (p < 0.001). In a separate group of patients (four pairs + one post biopsy) on concurrent treatment with tibolone, there was no significant increase in the osteon density, cortical porosity, median canal diameter, or the markers of bone formation and resorption. Enzyme activities and numbers of active canals were also not increased with the concurrent treatment, but there was still an increase in the osteon diameter. As previously shown for cancellous bone, estrogen withdrawal increased cortical bone turnover. We have now shown that resorption depth within Haversian systems was also increased with treatment. The enhanced TRAP activity in individual osteoclasts supports the concept that osteoclasts are more active following estrogen withdrawal in agreement with theoretical arguments advanced previously. Understanding the cellular and biochemical mechanisms responsible for increased depth of osteoclast resorption when estrogen is withdrawn may allow the development of new strategies for preventing postmenopausal bone loss.

Intracapsular hip fracture and the region-specific loss of cortical bone: analysis by peripheral quantitative computed tomography.

Generalized bone loss within the femoral neck accounts for only 15% of the increase in intracapsular hip fracture risk between the ages of 60 and 80 years. Conventional histology has shown that there is no difference in cancellous bone area between cases of intracapsular fracture and age and sex-matched controls. Rather, a loss of cortical bone thickness and increased porosity is the key feature with the greatest change occurring in those regions maximally loaded during a fall (the inferoanterior [IA] to superoposterior [SP] axis). We have now reexamined this finding using peripheral quantitative computed tomography (pQCT) to analyze cortical and cancellous bone areas, density, and mass in a different set of ex vivo biopsy specimens from cases of intracapsular hip fracture (female, n = 16, aged 69-92 years) and postmortem specimens (female, n = 15, aged 58-95 years; male, n = 11, aged 56-86 years). Within-neck location was standardized by using locations at which the ratio of maximum to minimum external diameters was 1.4 and at more proximal locations. Cortical widths were analyzed using 72 radial profiles from the center of area of each of the gray level images using a full-width/half-maximum algorithm. In both male and female controls, cancellous bone mass increased toward the femoral head and the rate of change was gender independent. Cancellous bone mass was similar in cases and controls at all locations. Overall, cortical bone mass was significantly lower in the fracture cases (by 25%; p < 0.001) because of significant reductions in both estimated cortical area and density. These differences persisted at locations that are more proximal. The mean cortical width in the cases was significantly lower in the IA (22.2%;p = 0.002) and inferior regions (19%;p < 0.001). The SP region was the thinnest in both cases and controls. These data confirm that a key feature in the etiology of intracapsular hip fracture is the site-specific loss of cortical bone, which is concentrated in those regions maximally loaded during a fall on the greater trochanter. An important implication of this work is that the pathogenesis of bone loss leading to hip fracture must be by a mechanism that varies in its effect according to location within the femoral neck Key candidate mechanisms would include those involving locally reduced mechanical loading. This study also suggests that the development of noninvasive methodologies for analyzing the thickness and estimated densities of critical cortical regions of the femoral neck could improve detection of those at risk of hip fracture.

Bioactivity of parathyroid hormone and parathyroid hormone-like peptide: agonist and antagonist activities of amino-terminal fragments as assessed by the cytochemical bioassay and in situ biochemistry.

Parathyroid hormone-like peptide (PLP) is elaborated from certain tumors and is thought to play a role in the etiology of humoral hypercalcemia of malignancy. The amino-terminal portion of this peptide has a sequence homology with parathyroid hormone PTH. We have compared the agonist potency of the synthetic human amino-terminal 1-34 peptide [human (h)PLP-(1-34)] with that of intact PTH and its amino terminal fragment [hPTH-(1-34)] in the renal and metatarsal cytochemical bioassays (CBA). Furthermore, the antagonist activity of the truncated amino terminal molecule [hPLP-(3-34)] has been compared to that of [Norleu8.18,Tyr34]bovine PTH-(3-34)NH2, and we have also tested their ability to stimulate enzyme activities thought to be associated with bone formation and resorption. In the renal CBA, both PLP-(1-34) and hPTH-(1-34) were equipotent with intact hPTH. In the metatarsal CBA, although the two amino-terminal peptides were equipotent, they elicited an earlier response than the intact PTH molecule. In both assay systems the truncated PLP analog [hPLP-(3-34)] was a more potent antagonist of both PTH and PLP activity than was [Norleu8.18,Tyr34]bovine PTH-(1-34)NH2. In acute studies, hPLP-(1-34) and hPTH-(1-34) stimulated alkaline phosphatase and glucose 6-phosphate dehydrogenase activity in osteoblasts to a similar extent, and both peptides stimulated tartrate-resistant acid phosphatase and succinate dehydrogenase activity in osteoclasts. Longer exposure to the peptides resulted in stimulation of enzyme activity in osteoclasts but not osteoblasts, although there was no difference in potency between the two molecules.

Acute stimulation of thyroidal NAD+ kinase, NADPH reoxidation, and peroxidase activities by physiological concentrations of thyroid stimulating hormone acting in vitro: a quantitative cytochemical study.

Quantitative cytochemical techniques have been employed in a study of some of the acute effects of low doses (0.01----1 mU/liter) of TSH on the metabolism of guinea pig thyroid segments maintained in nonproliferative organ culture. The enzymes involved in the synthesis of NADP+ (NAD+ kinase), its reduction by the pentose-shunt (glucose 6-phosphate dehydrogenase), and its reoxidation both by the microsomal electron chain (diaphorase activity) and by participation in other cellular processes, have been examined. The effect of TSH on peroxidase activity has also been studied. After 10 min stimulation with TSH (1 mU/liter) there was a 60% increase in NAD+ kinase activity which preceded changes in the microsomal reoxidation of NADPH (up 33% by 30 min). There were no changes in the activity of glucose 6-phosphate dehydrogenase. There was a sustained rise in peroxidase activity which reached 129% over control after 30 min. This is the first in vitro demonstration of an acute stimulation of peroxidase and kinase activities by physiological concentrations of TSH. NADPH reoxidation after stimulation with TSH was such that the ratio of NADPH reoxidized via the microsomal respiratory pathway (diaphorase, hydrogen pathway 1) relative to that available for cytosolic utilization (hydrogen pathway 2) increased compared to the unstimulated controls. We suggest that increased NADP+ production (via NAD+ kinase activity) and the preferential shuttling of the NADPH for reoxidation via the microsomal respiratory pathway, coupled with greatly stimulated peroxidase activity, may be important regulators of the control of thyroglobulin iodination and hence thyroid hormone production.

Parathyroid hormone responses to calcium stress during eggshell calcification.

PTH bioactivity during the egg-laying cycle of the chicken has been measured by the cytochemical bioassay for PTH. During the period of eggshell calcification, PTH bioactivity is elevated. After completion of the shell the plasma PTH falls to a low level but is slightly raised again 2 h after ovulation. These results indicate that changes in bioactive PTH play an important role in the calcium metabolism of the chicken during this physiological calcium stress.

Inhibition of parathyroid hormone bioactivity by human parathyroid hormone (PTH)-(3-84) and PTH-(8-84) synthesized in Escherichia coli.

Human PTH (hPTH)-(3-84) and hPTH-(8-84) were synthesized in Escherichia (E.) coli when the cells were transformed with a multicopy plasmid in which the transcription of human preproPTH cDNA is directed by the E. coli lac promoter. PTH fragments were extracted from cells and purified by reverse phase HPLC. PTH bioactivity and PTH antagonist activity were estimated in a renal cytochemical bioassay. hPTH-(3-84) and hPTH-(8-84) exhibited less than 1% and less than 0.1%, respectively, of the biological activity of synthetic hPTH-(1-84). hPTH-(8-84) had 1% of the PTH inhibitory activity of synthetic [Nle8,18,Tyr34]bovine PTH-(3-34)amide, whereas hPTH-(3-84) was 100 times more active as a PTH inhibitor than the synthetic bovine PTH-(3-34) analog. The latter has so far been recognized as the most potent PTH antagonist in vitro. A 5-fold molar excess of hPTH-(3-84) over hPTH-(1-84) completely blocked the biological action of intact hPTH-(1-84) in the renal cytochemical bioassay. These findings suggest that the carboxyl-terminal portion of the intact hPTH-(1-84) molecule contributes importantly to inhibitor potency.

Biological properties of synthetic human parathyroid hormone: effect of deamidation at position 76 on agonist and antagonist activity.

Recent studies have suggested a role for the carboxyl-terminus of PTH in the binding of the molecule to renal and skeletal receptors, but the functional significance of this binding remains uncertain. We have investigated the possible role of this region by examining the effect of substituting the asparagine residue at position 76 of the native human molecule [Asn76]hPTH-(1-84) with an aspartate residue, [Asp76] hPTH-(1-84) on activity in both renal and skeletal cytochemical (CBA) and adenylate cyclase (AC) bioassays. In the renal CBA, [Asp76]hPTH-(1-84) was considerably less potent than [Asn76]hPTH-(1-84) and produced dose-dependent inhibition of the bioactivity of intact bovine (b) PTH-(1-84), bPTH-(1-34), and [Asn76]hPTH-(1-84). [Asp76]hPTH-(39-84) inhibited the response to intact PTH to a lesser extent, whereas [Asp76]hPTH-(53-84) had no antagonistic activity. In the metatarsal CBA, [Asp76]hPTH-(1-84) inhibited the response to intact PTH, but was less potent than in the renal CBA. In both renal (OK) and skeletal (UMR) cell AC assays [Asp76]hPTH-(1-84) and [Asn76]hPTH-(1-84) were equipotent agonists. Therefore, the CBAs are much more sensitive to modification of the carboxyl end of the molecule than AC assays. The antagonist properties of [Asp76]hPTH-(1-84) appeared to be mediated by phosphodiesterase activation as theophylline abolished the antagonism of this analog. These studies indicate that generation of PTH analogs, modified at the carboxyl-terminal region as well as at the amino-terminus, may be useful for developing potent PTH antagonists.

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.

Inhibition of cytochemical bioactivity of parathyroid hormone by plasma in pseudohypoparathyroidism type I.

Despite the high circulating levels of immunoreactive PTH in patients with pseudohypoparathyroidism type I (PSPI) the levels of bioactive PTH (bioPTH) have been found to be close to the normal range. To elucidate this dissociation, we have studied the recovery of the biological activity of bovine PTH added to the plasma of patients with either PSPI, or with hypoparathyroidism (PTX), primary hyperparathyroidism (HPT) or of normal subjects. In PSPI (n = 10) the recovery of biological activity was 5.6% +/- 3.6 (mean +/- SEM) whereas in PTX (n = 7), in HPT (n = 4) and in normal subjects (n = 8) it was 79% +/- 5, 75% +/- 9 and 68% +/- 4, respectively. In another PSPI patient, who had undergone total parathyroidectomy, bioPTH was undetectable but the recovery from the plasma of added PTH was 84%. Thus we have found inhibition of PTH bioactivity by plasma of PSPI patients which was absent after parathyroidectomy.