Validation of urinary calcium isotope excretion from bone for screening anabolic therapies for osteoporosis.

SUMMARY: Urinary excretion of calcium tracers in labeled individuals decreases in response to antiresorptive therapy, providing a tool to rapidly screen potential therapies. Using teriparatide, we demonstrate in this study that anabolic therapy also decreases tracer excretion, confirming that this method can also be used to screen potential anabolic therapies. INTRODUCTION: Changes in urinary excretion of calcium tracers from a labeled skeleton may be a rapid and sensitive method to screen potential therapies for osteoporosis. This method has been used to screen antiresorptive therapies, but the effect of anabolic therapies on tracer excretion is unknown. METHODS: Eight-month-old female Sprague Dawley rats (n = 11) were given 50 µCi (45)Ca iv. After a 1-month equilibration period, baseline urinary (45)Ca excretion and total bone mineral content (BMC) were measured. Rats were then treated with 30 µg/kg teriparatide sc per day, a bone anabolic agent, for 80 days. Urine was collected throughout the study and analyzed for (45)Ca and total Ca, and BMC was measured at the beginning and end of the study. RESULTS: Teriparatide decreased urinary (45)Ca excretion by 52.1 % and increased BMC by 21.7 %. The change in bone calcium retention as determined by the ratio of (45)Ca to total Ca excretion in urine from day 6 through 15 of teriparatide treatment was significantly correlated (p = 0.036) with the change in BMC after 80 days of teriparatide treatment. CONCLUSION: Urinary excretion of calcium tracers from labeled bone is an effective method to rapidly screen potential anabolic therapies for osteoporosis.

A randomized trial of vitamin D3 supplementation in children: dose-response effects on vitamin D metabolites and calcium absorption.

CONTEXT: Changes in serum vitamin D metabolites and calcium absorption with varying doses of oral vitamin D3 in healthy children are unknown. OBJECTIVE: Our objective was to examine the dose-response effects of supplemental vitamin D3 on serum vitamin D metabolites and calcium absorption in children living at two U.S. latitudes. DESIGN: Black and white children (n = 323) participated in a multisite (U.S. latitudes 34° N and 40° N), triple-masked trial. Children were randomized to receive oral vitamin D3 (0, 400, 1000, 2000, and 4000 IU/d) and were sampled over 12 weeks in winter. Serum 25-hydroxyvitamin D (25(OH)D) and 1,25-dihydroxyvitamin D (1,25(OH)2D) were measured using RIA and intact PTH (iPTH) by immunoradiometric assay. Fractional calcium absorption was determined from an oral stable isotope 44Ca (5 mg) in a 150-mg calcium meal. Nonlinear and linear regression models were fit for vitamin D metabolites, iPTH, and calcium absorption. RESULTS: The mean baseline 25(OH)D value for the entire sample was 70.0 nmol/L. Increases in 25(OH)D depended on dose with 12-week changes ranging from -10 nmol/L for placebo to 76 nmol/L for 4000 IU. Larger 25(OH)D gains were observed for whites vs blacks at the highest dose (P < .01). Gains for 1,25(OH)2D were not significant (P = .07), and decreases in iPTH were not dose-dependent. There was no dose effect of vitamin D on fractional calcium absorption when adjusted for pill compliance, race, sex, or baseline 25(OH)D. CONCLUSION: Large increases in serum 25(OH)D with vitamin D3 supplementation did not increase calcium absorption in healthy children living at 2 different latitudes. Supplementation with 400 IU/d was sufficient to maintain wintertime 25(OH)D concentrations in healthy black, but not white, children.

Soft tissue calcification in the Ossabaw miniature pig: experimental and kinetic modeling studies.

UNLABELLED: Calcium (Ca) deposition into vascular tissue was measured in Ossabaw miniature pigs with and without metabolic syndrome (MetS) using Ca tracer kinetics and coronary atherosclerosis measured with intravascular ultrasound. Pigs with MetS had higher Ca uptake into coronary arteries than lean pigs. INTRODUCTION: Ca deposition into arteries is a common disease in humans. The Ossabaw pig develops MetS when fed an atherogenic diet. The aim of this study was to measure Ca deposition into arteries of lean vs. MetS pigs. METHODS: Male pigs were fed for 5 months with chow diet (healthy, lean; n = 7) or atherogenic diet (n = 8) consisting of chow supplemented with 2 % cholesterol, 43 % kcal from fat, and 20 % kcal from fructose. Pigs were verified to have MetS by obesity, insulin resistance, impaired glucose tolerance, dyslipidemia, and hypertension. Two pigs received 50 nCi of (41)Ca i.v. and blood was drawn frequently for 24 h, and 2, 3, 6, 8, 10, 15, 20, and at sacrifice at 28 days after injection. Peripheral arteries were biopsied four times per pig over the 28th day and coronary artery sampled at sacrifice. Tissues were analyzed for (41)Ca:Ca. A compartmental model was used to estimate rates of Ca deposition into the arteries. RESULTS: The MetS swine had higher (41)Ca and atherosclerosis in coronary arteries than lean pigs. CONCLUSIONS: This pig model is a suitable model for studying vascular calcification in humans.

Antiresorptive effects of phytoestrogen supplements compared with estradiol or risedronate in postmenopausal women using (41)Ca methodology.

INTRODUCTION: Reduction of ovarian estrogen secretion at menopause increases net bone resorption and leads to bone loss. Isoflavones have been reported to protect bone from estrogen deficiency, but their modest effects on bone resorption have been difficult to measure with traditional analytical methods. METHODS: In this randomized-order, crossover, blinded trial in 11 healthy postmenopausal women, we compared four commercial sources of isoflavones from soy cotyledon, soy germ, kudzu, and red clover and a positive control of oral 1 mg estradiol combined with 2.5 mg medroxyprogesterone or 5 mg/d oral risedronate (Actonel) for their antiresorptive effects on bone using novel (41)Ca methodology. RESULTS: Risedronate and estrogen plus progesterone decreased net bone resorption measured by urinary (41)Ca by 22 and 24%, respectively (P < 0.0001). Despite serum isoflavone profiles indicating bioavailability of the phytoestrogens, only soy isoflavones from the cotyledon and germ significantly decreased net bone resorption by 9% (P = 0.0002) and 5% (P = 0.03), respectively. Calcium absorption and biochemical markers of bone turnover were not influenced by interventions. CONCLUSIONS: Dietary supplements containing genistein-like isoflavones demonstrated a significant but modest ability to suppress net bone resorption in postmenopausal women at the doses supplied in this study over a 50-d intervention period.

Soy isoflavones do not affect bone resorption in postmenopausal women: a dose-response study using a novel approach with 41Ca.

INTRODUCTION: The purpose of this 3-way crossover study was to identify the effective dose of soy protein isolate enriched with isoflavones for suppressing bone resorption in postmenopausal women using a novel, rapid assessment of antibone resorbing treatments. METHODS: Thirteen postmenopausal women (>or=6 yr since menopause) were predosed with 41Ca iv. After a 200-d baseline period, subjects were given 43 g soy protein/d that contained 0, 97.5, or 135.5 mg total isoflavones in randomized order. The soy protein isolate powder was incorporated into baked products and beverages. Each 50-d intervention phase was preceded by a 50-d pretreatment phase for comparison. Serum isoflavone levels and biochemical markers were measured at the end of each phase. Twenty-four-hour urine samples were collected approximately every 10 d during each phase for 41Ca/Ca analysis by accelerator mass spectrometry. RESULTS: Serum isoflavone levels reflected the amount of isoflavones consumed in a dose-dependent manner. None of the isoflavone levels had a significant effect on biochemical markers of bone turnover, urinary cross-linked N teleopeptides of type I collagen and serum osteocalcin, or bone turnover as assessed by urinary 41Ca/Ca ratios. CONCLUSIONS: Soy protein with isoflavone doses of up to 135.5 mg/d did not suppress bone resorption in postmenopausal women. This is the first efficacy trial using the novel technique of urinary 41Ca excretion from prelabeled bone.

Effect of calcium or 25OH vitamin D3 dietary supplementation on bone loss at the hip in men and women over the age of 60.

Dietary supplements that prevent bone loss at the hip and that can be applied safely in the elderly are likely to reduce hip fractures. A daily dietary supplement of 750 mg calcium or 15 microg 25OH vitamin D3 on bone loss at the hip and other sites, bone turnover and calcium-regulating hormones were studied over 4 yr in elderly volunteers using a randomized, double-blind, placebo-controlled trial. Bone mineral density (BMD) was measured by dual x-ray absorptiometry and bone structure by radiographs. Calcium biochemistry and bone turnover markers were measured in blood and urine. The 316 women entering the trial had a mean age of 73.7 yr and the 122 men of 75.9 yr. Baseline median calcium intake was 546 mg/day, and median serum 25OH vitamin D3 was 59 nmol/L. On placebo, loss of BMD at total hip was 2% and femoral medulla expansion was 3% over 4 yr. Calcium reduced bone loss, secondary hyperparathyroidism, and bone turnover. 25OH vitamin D3 was intermediate between placebo and calcium. Fracture rates and drop-out rates were similar among groups, and there were no serious adverse events with either supplement. A calcium supplement of 750 mg/day prevents loss of BMD, reduces femoral medullary expansion, secondary hyperparathyroidism, and high bone turnover. A supplement of 15 microg/day 25OH vitamin D3 is less effective, and because its effects are seen only at low calcium intakes, suggests that its beneficial effect is to reverse calcium insufficiency.

Fluoride treatment increased serum IGF-1, bone turnover, and bone mass, but not bone strength, in rabbits.

We hypothesized that fluoride partly acts by changing the levels of circulating calcium-regulating hormones and skeletal growth factors. The effects of oral fluoride on 24 female, Dutch-Belted, young adult rabbits were studied. The rabbits were divided into two study groups, one control and the other receiving about 16 mg fluoride/rabbit/day in their drinking water. After 6 months of fluoride dosing, all rabbits were euthanized and bone and blood samples were taken for analyses. Fluoride treatment increased serum and bone fluoride levels by over an order of magnitude (P < 0.001), but did not affect body weight or the following serum biochemical variables: urea, creatinine, phosphorus, total protein, albumin, bilirubin, SGOT, or total alkaline phosphatase. No skeletal fluorosis or osteomalacia was observed histologically, nor did fluoride affect serum PTH or Vitamin D metabolites (P > 0.4). BAP was increased 37% (P < 0.05) by fluoride; serum TRAP was increased 42% (P < 0.05); serum IGF-1 was increased 40% (P < 0.05). Fluoride increased the vertebral BV/TV by 35% (P < 0.05) and tibial ash weight by 10% (P < 0.05). However, the increases in bone mass and bone formation were not reflected in improved bone strength. Fluoride decreased bone strength by about 19% in the L5 vertebra (P < 0.01) and 25% in the femoral neck (P < 0. 05). X-ray diffraction showed altered mineral crystal thickness in fluoride-treated bones (P < 0.001), and there was a negative association between crystal width and fracture stress of the femur (P < 0.02). In conclusion, fluoride's effects on bone mass and bone turnover were not mediated by PTH. IGF-1 was increased by fluoride and was associated with increased bone turnover, but was not correlated with bone formation markers. High-dose fluoride treatment did not improve, but decreased, bone strength in rabbits, even in the absence of impaired mineralization.

Reduced rates of skeletal remodeling are associated with increased bone mineral density during the development of peak skeletal mass.

Two related studies were conducted to assess the associations between markers of skeletal modeling and remodeling in healthy children. Members of monozygotic twin pairs, aged 6-14, enrolled in a clinical trial of calcium supplementation, were studied at the end of the period of supplementation and for 3 years thereafter. Supplemented children had significantly higher rates of gain in bone mineral density (BMD) (+3% on average) during the period of supplementation accompanied by significantly lower concentrations of serum osteocalcin (OC, -15%). During postsupplement follow-up, both differences in BMD and OC disappeared. Black females, age matched to the baseline ages of the white children, had significantly lower serum concentrations of both OC and tartrate-resistant acid phosphatase (TRAP) at all ages and higher BMDs. When stratified on serum TRAP concentrations, regardless of race, children with lower concentrations had significantly higher BMDs, and no racial differences were apparent. In regression models accounting for 70-80% of the variability in BMD in children, body size and TRAP, but not race, remained significantly associated with BMD. The skeletal advantages seen with calcium supplementation and black race appear to be associated with reduced rates of skeletal turnover. Given that markers of turnover during growth reflect both skeletal modeling and remodeling, and there is no apparent advantage to reduced skeletal modeling, it seems probable that reduced remodeling is the factor that accounts for the increases in bone mass.

Calcium absorption from apple and orange juice fortified with calcium citrate malate (CCM).

OBJECTIVE: Determine calcium (Ca) absorption from Ca fortified orange and apple juice. METHODS: Absorbability was assessed by measuring 45Ca absorption in healthy women (mean age 57 years, n = 57/group) and whole body 47Ca retention in adult female beagle dogs (n = 6/group) and young adult male rats (n = 6/group). Women received 6.24 mmol (250 mg) Ca as calcium citrate malate fortified orange juice (CCM-OJ) or apple juice (CCM-AJ). Dogs received 3.12 mmol (125 mg) Ca as CCM-OJ or CCM-AJ. Rats were administered 0.15 mmol (6 mg) Ca as either milk, CCM-OJ, or CCM-AJ. Additional 47Ca whole body retention experiments in rats measured the effects of differences in the carbohydrate and organic acid contents of the juices on Ca absorption. RESULTS: Mean +/- SEM percent Ca fractional absorption was greater (p < 0.003) in women who consumed CCM-AJ (42 +/- 2%) than those who consumed CCM-OJ (36 +/- 1%). Ca retention in dogs was 15 +/- 1% for CCM-OJ and 29 +/- 2% for CCM-AJ (p < 0.001). Ca retention was significantly different (p < 0.05) in rats administered milk (42 +/- 2%), CCM-OJ (52 +/- 2%), or CCM-AJ (61 +/- 2%). By manipulating the carbohydrate and organic acid concentrations of test solutions to mimic the composition of Ca fortified juices, we found that the greater fructose and lower organic acid content of apple juice accounted for its greater Ca absorbability. CONCLUSIONS: CCM fortified versions of orange and apple juice have high Ca absorbability and are potentially important vehicles for increasing dietary Ca intake. The greater Ca absorption from CCM-AJ compared with CCM-OJ is accounted for by differences in the carbohydrate and organic acid content of the juices. These data suggest that by modifying common beverage ingredients, products with even greater Ca absorbability could be formulated.

Photoprotective action of emollients in ultraviolet therapy of psoriasis.

An in vitro technique was used to measure the monochromatic protection factors of all emollients available on prescription. The action spectra for ultraviolet erythema and erythema in psoralen-sensitized skin were used to calculate, for each emollient, erythema protection factors relevant to UVB phototherapy and psoralen photochemotherapy, respectively. Of the 40 products tested, 22 (55%) had a UVB erythema protection factor > 1.2 at an application density of 2 microliters/cm2, and 31 (78%) at an application density of 4 microliters/cm2. Fewer products, 25% at 2 microliters/cm2 and 50% at 4 microliters/cm2, had a psoralen erythema protection factor > 1.2. A protection factor of 1.2 is equivalent to a reduction in ultraviolet dose of 17%, and is thus likely to be of clinical importance. These results allow a choice of emollient products which may improve response by increasing transmission of radiation through psoriasis scale without a concomitant decrease in transmission due to a sunscreening action.

Elimination of glutathione-induced protection from hyperbaric hyperoxia by acivicin.

Glutathione (GSH) administered intraperitoneally significantly prolongs the time to initial seizure and survival time of rats exposed to hyperbaric hyperoxia (HBO). Acivicin is an antitumor antibiotic that is an inhibitor of gamma-glutamyl transpeptidase (GGT), an enzyme necessary for the breakdown and transport across cell membranes of GSH. To determine whether acivicin treatment alters GSH-induced protection from HBO, rats were dosed with 25 mg/kg of acivicin or vehicle 1 h before O2 exposure at an inspired O2 fraction of 1.0 at 4 ATA. Immediately before exposure, rats received GSH (1 mmol/kg) or vehicle. Time to seizure and time to death were recorded during exposure by direct observation. In separate groups of rats on the same dosing schedule, plasma GSH, renal GGT, and brain GGT were measured 15 min after the GSH injection without HBO exposure and 100 min after the beginning of HBO exposure. Renal GGT was decreased to 2.5% of control and brain GGT to 37% of control in the acivicin-dosed rats. Plasma GSH increased 3-fold in rats given acivicin alone, 52-fold in rats given GSH alone, and 84-fold in rats receiving both acivicin and GSH. Rats dosed with GSH alone had significantly prolonged times to seizure and death compared with all other groups. Rats dosed with GSH after receiving acivicin were not protected from HBO despite the large increase in plasma GSH that occurred in these animals. GSH treatment did not increase tissue GSH in lung, liver, or brain at 160 or 200 min of exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium supplementation and increases in bone mineral density in children.

BACKGROUND: Increased dietary intake of calcium during childhood, usually as calcium in milk, is associated with increased bone mass in adulthood; the increase in mass is important in modifying the later risk of fracture. Whether the increase is due to the calcium content of milk, however, is not certain. METHODS: We conducted a three-year, double-blind, placebo-controlled trial of the effect of calcium supplementation (1000 mg of calcium citrate malate per day) on bone mineral density in 70 pairs of identical twins (mean [+/- SD] age, 10 +/- 2 years; range, 6 to 14). In each pair, one twin served as a control for the other; 45 pairs completed the study. Bone mineral density was measured by photon absorptiometry at two sites in the radius (at base line, six months, and one, two, and three years) and at three sites in the hip and in the spine (at base line and three years). RESULTS: The mean daily calcium intake of the twins given placebo was 908 mg, and that of the twins given calcium supplements was 1612 mg (894 mg from the diet and 718 mg from the supplement). Among the 22 twin pairs who were prepubertal throughout the study, the twins given supplements had significantly greater increases in bone mineral density at both radial sites (mean difference in the increase in bone mineral density: midshaft radius, 5.1 percent [95 percent confidence interval, 1.5 to 8.7 percent]; distal radius, 3.8 percent [95 percent confidence interval, 1.4 to 6.2 percent]) and in the lumbar spine (increase, 2.8 percent [95 percent confidence interval, 1.1 to 4.5 percent]) after three years; the differences in the increases at two of three femoral sites approached significance (Ward's triangle in the femoral neck, 2.9 percent; greater trochanter, 3.5 percent). Among the 23 pairs who went through puberty or were postpubertal, the twins given supplements received no benefit. CONCLUSIONS: In prepubertal children whose average dietary intake of calcium approximated the recommended dietary allowance, calcium supplementation increased the rate of increase in bone mineral density. If the gain persists, peak bone density should be increased and the risk of fracture reduced.

ADP-ribosyltransferase activity of pertussis toxin and immunomodulation by Bordetella pertussis.

Pertussis toxin is produced by the causative agent of whooping cough, Bordetella pertussis, and is an adenosine diphosphate (ADP)-ribosyltransferase capable of covalently modifying and thereby inactivating many eukaryotic G proteins involved in cellular metabolism. The toxin is a principal determinant of virulence in whooping cough and is a primary candidate for an acellular pertussis vaccine, yet it is unclear whether the ADP-ribosyltransferase activity is required for both pathogenic and immunoprotective activities. A B. pertussis strain that produced an assembled pertussis holotoxin with only 1 percent of the ADP-ribosyltransferase activity of the native toxin was constructed and was found to be deficient in pathogenic activities associated with B. pertussis including induction of leukocytosis, potentiation of anaphylaxis, and stimulation of histamine sensitivity. Moreover, this mutant strain failed to function as an adjuvant and was less effective in protecting mice from intracerebral challenge infection. These data suggest that the ADP-ribosyltransferase activity is necessary for both pathogenicity and optimum immunoprotection. These findings bear directly on the design of a nontoxic pertussis vaccine.

Osteomalacia in Paget's disease treated with short term, high dose sodium etidronate.

Eleven patients with Paget's disease treated with sodium etidronate 20 mg/kg/day for two and four weeks showed significant reductions in plasma alkaline phosphatase activity and urinary hydroxyproline excretion, both of which are biochemical markers of bone turnover. After four weeks of treatment, however, histological examination of iliac crest biopsy samples showed that despite a rapid reduction in bone resorption there was an appreciable mineralisation defect; even after only two weeks' treatment the abnormalities in bone formation persisted for up to 10 weeks. The adverse effects of sodium etidronate on mineralisation cannot be dissociated from its beneficial effect on resorption even when it is given for short periods.

A prospective trial of the effect of vitamin D supplementation on metacarpal bone loss in elderly women.

The effect on cortical bone loss of treating elderly women with 15,000 IU vitamin D2 weekly was evaluated by sequential radiographic morphometry of the metacarpals. One hundred nine randomly selected women aged 65-74 yr were studied for 2 yr. The women were randomly allocated to control or treated groups taking placebo or vitamin D2 capsules. Hand radiographs and blood samples were obtained at the beginning and end of the trial. Plasma 25-hydroxyvitamin D was measured by radio-competitive protein binding assay. Comparing the treated and control groups, vitamin D treatment significantly raised the plasma 25-hydroxyvitamin D levels (p less than 0.001) and reduced the rate of cortical bone loss (p less than 0.01). The placebo had no measurable effect on the plasma levels.

Threshold sensitivity and frequency specificity in auditory brainstem response audiometry.

Frequency-specific electric response audiometry can be performed on difficult to test young children if the child is sedated and proper choices are made of acoustic stimuli and recording parameters, although certain compromises are necessary. A very satisfactory sedative is secobarbital, administered intramuscularly in doses related to the weight of the child. As stimuli we recommend '2-1-2' tone bursts at 500, 1 000, 2 000, and 4 000 Hz: i.e., with a rise and fall of two periods and a plateau of one period of the modulated tone. A very robust and sensitive response that is not significantly modified by the sedation and is effective for all four frequencies is the P6-SN10 of the early brainstem sequence. To record this complex favorably requires a bandpass input filter of the Butterworth type with pass-band (at -3 dB) from 50 to 1 700 Hz and rejection rates of 24 dB/octave. With this combination, polarity of stimulus is unimportant and sweep time, rate of stimulation and number of responses averaged may be selected for convenience and simplicity. A routine that requires about an hour of testing time is described and the necessary correction factors are given for estimating a child's behavioral pure-tone thresholds. We believe that our threshold estimates are generally correct within 10 dB, and are sufficiently frequency-specific for proper selection of a hearing aid.

Inhibition of calcium oxalate crystallisation by pentosan polysulphate in control subjects and stone formers.

In vitro studies showed that sodium pentosan polysulphate (SPP) is an active inhibitor of calcium oxalate (CaOx) crystal growth and agglomeration and that it acts by increasing the negative zeta potential on the surface of CaOx crystals. Oral administration of SPP to control subjects, recurrent stone formers and patients with primary hyperoxaluria resulted in an overall increase of 8% (P less than 0.01) in the polyanionic inhibition of CaOx crystallisation in urine as measured by the zeta potential. SPP could provide a novel approach to the medical prevention of recurrent CaOx stone disease.