Positive effects of anabolic steroids, vitamin D and calcium on muscle mass, bone mineral density and clinical function after a hip fracture. A randomised study of 63 women.

A total of 63 women who had an operation for a fracture of the hip was randomly allocated to one year of treatment either with anabolic steroids, vitamin D and calcium (anabolic group) or with calcium only (control group). The thigh muscle volume was measured by quantitative CT. The bone mineral density of the hip, femur and tibia was assessed by quantitative CT and dual-energy x-ray absorptiometry and of the heel by quantitative ultrasound. Quantitative CT showed that the anabolic group did not lose muscle volume during the first 12 months whereas the control group did (p<0.01). There was less bone loss in the proximal tibia in the anabolic group than in the control group. The speed of gait and the Harris hip score were significantly better in the anabolic group after six and 12 months. Anabolic steroids, even in this moderate dose, given in combination with vitamin D and calcium had a beneficial effect on muscle volume, bone mineral density and clinical function in this group of elderly women.

An assessment of human liver-derived in vitro systems to predict the in vivo metabolism and clearance of almokalant.

The ability of various human derived in vitro systems to predict various aspects of the in vivo metabolism and kinetics of almokalant have been investigated in a multicenter collaborative study. Although almokalant has been withdrawn from further clinical development, its metabolic and pharmacokinetic properties have been well characterized. Studies with precision-cut liver slices, primary hepatocyte cultures, and hepatic microsomal fractions fortified with UDP-glucuronic acid all suggested that almokalant is mainly glucuronidated to the stereoisomers M18a and M18b, which is in good agreement with the results in vivo. Both in vivo and in vitro studies indicate that the formation of M18b dominates over that of M18a, although the difference is more pronounced with the in vitro systems. Molecular modeling, cDNA-expressed enzyme analysis, correlation analysis, and inhibition studies did not clearly indicate which P450 enzymes catalyze the oxidative pathways, which may indicate a problem in identifying responsible enzymes for minor metabolic routes by in vitro methods. All of the in vitro systems underpredicted the metabolic clearance of almokalant, which has previously been reported to be a general problem for drugs that are cleared by P450-dependent metabolism. Although few studies on in vivo prediction of primarily glucuronidated drugs have appeared, in vitro models may consistently underpredict in vivo metabolic clearance. We conclude that in vitro systems, which monitor phase II metabolism, would be beneficial for prediction of the in vivo metabolism, although all of the candidate liver-derived systems studied here, within their intrinsic limitations, provided useful information for predicting metabolic routes and rates.

Optimal processors for images with an arbitrary number of gray levels.

We present a new group of processors, optimal in a maximum-likelihood sense, for target location in images with a discrete number of gray levels. The discrete gray-level distribution can be of any arbitrary form. We compare the performance of the processor derived for five discrete levels with the performance of a processor derived for a continuous Gaussian distribution and show that there are cases when only the processor derived for discrete levels will exhibit satisfactory performance. We give an explanation of this difference based on moment analysis and show how the correlation orders are related to statistical moments of the input scene.

How interactions between drugs and agarose-carrageenan hydrogels influence the simultaneous transport of drugs.

The diffusional transport of a series of small drug molecules (<400 Da) in agarose gel with and without kappa-carrageenan (a negatively charged polysaccharide) is studied. The drug molecules have amphiphile character, the hydrophilic part being a tertiary amine which is the same in all six drugs. The difference in structure resides in the hydrophobic part which give these molecules different properties such as a difference in CMC-values (critical micelle concentration). The transport studies show that the apparent diffusion coefficients (Dapp) of all the drugs in 1% (w/w) agarose gel are almost identical and with a value similar to that in water. These results were anticipated because of the small size of the drugs, the low concentration of agarose, and the lack of interaction between the diffusant and the polymer. In agarose gels also containing 0.02% (w/w) kappa-carrageenan, however, the Dapp-values are significantly decreased for all drugs, except for lidocaine. This lowering of the Dapp is ascribed to the interaction between the drug molecules and kappa-carrageenan. The Dapp-values of the drugs in the gel system containing kappa-carrageenan correlate well with the adsorption isotherms of the same drugs in the drug/kappa-carrageenan/water system obtained previously [1] and the Dapp-values follow the order: chlorpromazine

Influence of the phosphate balance on the activity distribution of 99mTc-hydroxy-methylene diphosphonate. Experimental studies in the mouse.

OBJECTIVE: The purpose was to determine whether changes in the phosphate balance have an influence on the distribution of bone-seeking radiopharmaceuticals. MATERIAL AND METHODS: The biodistribution of 99mTc-HDP in mice, intravenously administered under varying conditions, was assessed by removing different organs and estimating their activity in a scintillation counter. Some experiments were also performed with 99mTc-MDP and 99mTc-DPD. RESULTS: After 1 h and 18 h on phosphate-enriched drinking water, the mice showed a strongly increased uptake in all organs/tissues representing background activity and a decrease in the bone uptake. This pattern changed with time. After 6-8 days of phosphate load, we saw a more favourable distribution with a reduction of the background and whole-body activity. Administration of hPTH 1-34 gave rise to an activity distribution similar to that after 6-8 days on phosphate-enriched water. Changing the phosphate balance had less obvious effects on the distribution of 99mTc-MDP and 99mTc-DPD. CONCLUSION: The activity distribution of bone-seeking radiopharmaceuticals in the mouse is affected by the phosphate balance. The mechanism behind this finding is unknown but it may be partially mediated by PTH. It is possible that changes in the phosphate balance, induced by pharmaceuticals or by dietary changes, may affect the image quality at bone scintigraphy.

Distortion-invariant filter for nonoverlapping noise.

A new heuristic filter based on the optimum filter for disjoint noise developed by Javidi and Wang [J. Opt. Soc. Am. A 11, 2604 (1995)] is presented. In this new filter a number of optimum filters built from single training images are combined linearly by use of the synthetic discriminant function (SDF) approach into a distortion-invariant filter for disjoint noise. Like the traditional SDF approach, this summation technique makes it possible to control the height of the correlation peak easily, for example, if a uniform filter response is needed. The filter is compared with the distortion-invariant version of the optimum filter on images with low contrast and high levels of nonoverlapping clutter. The new filter shows good results, demonstrating that it is, with very simple heuristic methods, possible to improve the performance of distortion-invariant filters for nonoverlapping noise.

A new method for diffusion measurement in polymeric films based on a stacked sheet concept.

PURPOSE: To develop and evaluate a simple, yet well defined, method to measure diffusion in semi-solids, i.e. polymeric materials. METHODS: The method was based on a concept where equivalent discs of polymeric films were cut and stacked on top of each other. The diffusion process was allowed to proceed unidimensionally through the stack of films perpendicular to the film surface. After an appropriate time, the stack was analysed disc by disc with respect to solute content and from the concentration profile so obtained the diffusion coefficient was calculated. RESULTS: An all-in-one device was developed, manufactured in stainless-steel, that cuts circular discs and stores each one successively in a "stack" in the cell compartment. CONCLUSIONS: Data from a silicone based system shows that the method, although simple, is accurate and reproducible.

Estimation of the C-cell numbers in rat thyroid glands using the optical fractionator.

We estimated the total number of calcitonin-immunoreactive C-cells in rat thyroid gland using the optical fractionator, the unbiased stereological method for estimation of numbers. It was necessary first to use a fixative composed of formalin, acetic acid, and ethanol to distinctly visualize the C-cells. The 40-microm-thick sections had to adhere to chromalum-gelatin-coated Superfrost Plus glass slides, and the immunostaining technique had to stain the C-cells evenly throughout the whole sections. Because the C-cells were irregularly distributed in the thyroid tissues, their counting required screening of about 500 fields per lobe, but the number of C-cells counted need not be high, about 90 per lobe. We estimated that rats have 185,000 +/- 42,000 C-cells (mean +/- SD; n - 7). The C-cell population did not differ significantly between the two lobes of a given rat, but it varied markedly among rats. The biological differences among the animals contributed 83% to the observed variability, whereas the methodological uncertainty contributed 17%. The serum levels of calcitonin and calcium were not closely correlated to the C-cell numbers. Our results indicate that variability in C-cell experiments can be reduced most effectively by increasing the number of animals used. However, the similar C-cell frequency found in the two thyroid lobes of each rat allows the use of one uniformly sampled lobe for quantification and the other lobe for further analysis.

Different effects on the expression of CYP7 and CYP27 in rabbit liver by cholic acid and cholestyramine.

It was recently reported that both cholesterol 7 alpha-hydroxylase CYP7 and sterol 27-hydroxylase CYP27 are subject to negative feedback control by bile acids in isolated rat hepatocytes and rat liver (Twisk et al., (1995) Biochem. J. 305, 505-511; Vlahcevic et al. XIII International Bile Acid Meeting, September 30-October 2, 1994, Abstract 17). In the present work a series of experiments was performed to study whether there is a coordinate regulation of CYP7 and CYP27 also in rabbit liver. Treatments of rabbits with cholic acid or cholestyramine resulted in marked suppression and induction, respectively, of CYP7 mRNA. In contrast, there were no significant effects on the CYP27 mRNA expression, amount of CYP27 protein or mitochondrial 27-hydroxylase activity. Thus, there is an apparent difference between rat and rabbit with respect to regulation of CYP27 by bile acids. It is concluded that, in rabbit, there is little or no coordinate regulation of CYP7 and CYP27 at a transcriptional level, and that CYP27 is not subject to a negative feedback control by bile acids neither at a transcriptional nor at a posttranscriptional level.

Serum calcitonin forms and concentrations in young and elderly healthy females.

To further investigate the role of calcitonin (CT) in normal physiology we studied circulating forms and the secretion after "calcium clamp" in young and elderly healthy females. Heterogeneity of CT in serum was disclosed after immunoextraction, fast protein liquid chromatography, and radioimmunoassay in young (27 +/- 3 years; mean +/- SD, n = 6) and elderly females (69 +/- 6 years, n = 11). Three distinct molecular forms appeared with approximate mol wt of 30, 10, and 3-4 kDa. All young women studied had considerable amounts of circulating monomer-like CT whereas several elderly had undetectable or low levels. The influence of age on basal and calcium stimulated, immunoextracted CT in serum was also studied in young (26 +/- 4 years; mean +/- SD, n = 13) and elderly (63 +/- 6 years; n = 12) healthy females. The calcium stimulation was carried out by means of the standardized calcium clamp method, where calcium was kept on a presettled level at 1.45 mmol/liter (+/- 2%) for 60 minutes. CT was immunoextracted from serum in all series of experiments with a polyclonal antiserum directed against the mid- and carboxyterminal region of the CT molecule, and the amount of extracted CT was determined by radioimmunoassay using another polyclonal antiserum against the carboxyterminal portion. After calcium infusion, the increase in CT was significantly higher in young women than in elderly (P < 0.05). At basal conditions, the CT levels were not significantly different but slightly higher in young than in elderly females. In conclusion, several elderly women lack monomer-like calcitonin in serum in contrast to young women.(ABSTRACT TRUNCATED AT 250 WORDS)

Liver mitochondrial cytochrome P450 CYP27 and recombinant-expressed human CYP27 catalyze 1 alpha-hydroxylation of 25-hydroxyvitamin D3.

A cytochrome P450 catalyzing 1 alpha-hydroxylation of 25-hydroxyvitamin D3 was purified from pig liver mitochondria. It also catalyzed 27-hydroxylation of 25-hydroxyvitamin D3 and 25-hydroxylation of vitamin D3. The ratio between the 1 alpha-, 27-, and 25-hydroxylase activities remained essentially constant during the purification. Substrates for sterol 27-hydroxylase CYP27 inhibited and a monoclonal antibody raised against CYP27 immunoprecipitated the 1 alpha-, 27-, and 25-hydroxylase activities. Apparently homogeneous preparations of CYP27 from pig and rabbit liver mitochondria catalyzed 1 alpha-hydroxylation. Human liver mitochondrial CYP27 was expressed from its cDNA in Escherichia coli. The nucleotide sequence encoding the N terminus of CYP27 was modified in the first eight codons to achieve expression in E. coli. The purified recombinant-expressed CYP27 reconstituted with the electron-transferring system of adrenal mitochondria catalyzed 1 alpha-hydroxylation of 25-hydroxyvitamin D3. Expression of unmodified CYP27 cDNA in simian COS cells confirmed the 1 alpha-hydroxylase activity toward 25-hydroxyvitamin D3.

Sensitive time-resolved fluoroimmunoassay of salmon calcitonin.

A two-site assay was developed by use of the "dissociation and enhancement lanthanide fluoroimmunoassay" (DELFIA) technique for determination of salmon calcitonin (SCT) in serum after administration to osteoporotic patients. Polyclonal antibodies were produced in rabbits immunized with SCT coupled to ovalbumin. After affinity purification, the antibodies were used both as immobilized capture antibodies and as Eu-chelate-labeled signal antibodies. A sensitive assay with a detection limit of 1.1 pmol/L was achieved, and no cross-reaction with human calcitonin was observed. The intra- and interassay CVs were < 12% (n = 10) and < 15% (n = 4), respectively. Analytical recovery of SCT added to serum was 91% +/- 3% (mean +/- SD, n = 4). SCT was measurable in all the samples from eight osteoporotic patients after subcutaneous SCT administration. We conclude that this new sensitive and specific two-site DELFIA can reliably measure SCT in serum.

Blood sampling procedures influence serum calcitonin concentrations in rats.

The aim of this study was to determine whether serum calcitonin (S-CT) in rats is influenced by the method of taking blood samples. Sampling during halothane anaesthesia, after repeated administration of anaesthesia after a 14-day interval, and sampling without the use of anaesthetics (i.e. after the rats were made unconscious by stunning), resulted in different S-CT values (P < or = 0.001), whereas Ca2+ levels were not affected. In thyroidectomized rats, the S-CT values after stunning were not significantly different whereas those in sham-operated rats were different (P < or = 0.01). The possibility that anaesthesia may suppress stunning-induced changes in S-CT was explored in three other groups of rats subjected to halothane anaesthesia, stunning and stunning under halothane anaesthesia respectively. Although the S-CT level was highest after stunning and lowest in halothane-anaesthetized rats (P < or = 0.001), anaesthesia did not suppress the effect of stunning on S-CT. In conclusion the effect of sampling procedures must be considered in studies on the levels of S-CT in rats.

Calcium homeostasis in normal pregnancy and puerperium. A longitudinal study.

Calcium homeostasis was longitudinally followed in serum and urine throughout normal pregnancy and the puerperium in 23 healthy women. From the 14th week of gestation, samples were obtained every fourth week until the 38th week. Post partum samples were obtained on the fifth day and after eight weeks. In the serum the total calcium decreased continuously during pregnancy. The ionized calcium and phosphate levels remained unchanged and within the reference interval for non-pregnant women. The alkaline phosphatase level progressively increased and high levels were found at term. The magnesium and hematocrit values remained below, whereas the calcitonin level remained just above the reference interval throughout pregnancy. The parathyroid hormone was low initially and increased towards term but within the reference interval. The urine excretion of calcium was constantly high, close to the upper reference limit, and renal function was slightly improved. At the last sampling eight weeks after delivery, all values were within normal limits for non-pregnant women. Calcium homeostasis is considerably changed during pregnancy and non-pregnant reference limits are not often valid.

Bone mineral density during long-term prophylaxis with heparin in pregnancy.

OBJECTIVE: We studied the effect of long-term heparin treatment on bone mass during pregnancy. STUDY DESIGN: Thromboprophylaxis with heparin was given to 39 women during pregnancy for a mean of 28 weeks and for an average of 6 weeks post partum. Bone mineral density measured with single-photon absorptiometry of the distal and ultradistal parts of the forearm was determined at the time of the start of heparin treatment (mean, twelfth week of gestation), immediately post partum, and on average 7 weeks post partum. The mean dosage of heparin was 17,300 IU/day. A control group of 34 normal pregnant women was studied for comparison. RESULTS: In women treated with heparin, there was almost a 5% reduction in trabecular bone during pregnancy (p < 0.01) and an insignificant recovery post partum. There were no significant changes in bone mass during pregnancy or in the puerperium in the control group. CONCLUSION: Long-term treatment with heparin during pregnancy is associated with bone loss, but indications of reversible changes are observed.

Denser trabecular bone in women with endometrial carcinoma.

Bone mineral density (BMD) was measured in the distal radius of patients with endometrial carcinoma (EC). The patients were classified into two subgroups depending on whether earlier hormonal replacement therapy (HRT) was given. Two groups of women were recruited as controls: patients with post-menopausal bleeding for non-malignant reasons (hospital controls) and healthy women, free of gyn-ecologic symptoms (non-hospital controls). The BMD was significantly higher in the cancer patients and also in the hospital controls than in the non-hospital controls. When several possible confounding factors were checked for in a multivariate analysis, BMD still differed between the groups. This could lend support to the hypothesis that patients with EC may have an altered endogenous endocrine status which eventually affects their bone mass. The results also stress the importance of using strictly defined, healthy women as controls.

Selective inhibition of mitochondrial 27-hydroxylation of bile acid intermediates and 25-hydroxylation of vitamin D3 by cyclosporin A.

It was demonstrated recently that cyclosporin A blocks bile acid synthesis in cultured rat and human hepatocytes by specific inhibition of chenodeoxycholic acid formation. The site of inhibition was found to be the 27-hydroxylation of cholesterol catalysed by the liver mitochondrial 27-hydroxylase [Princen, Meijer, Wolthers, Vonk and Kuipers (1991) Biochem J. 275, 501-505]. In this paper the mechanism by which cyclosporin A blocks mitochondrial 27-hydroxylation was further investigated. It is shown that cyclosporin A inhibited 27-hydroxylation of bile acid intermediates, depending on their polarity. In isolated rat liver mitochondria, 27-hydroxylation of cholesterol was dose-dependently blocked by the drug, giving half-maximal inhibition at 4 microM, whereas 27-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol was not affected. A similar observation was made using electrophoretically homogeneous cytochrome P-450(27) isolated from rabbit liver mitochondria, excluding the possibility that cyclosporin A interfered with transport of substrates into the mitochondrion. Kinetic studies showed that inhibition of the 27-hydroxylation of cholesterol by cyclosporin A was of a non-competitive type. The drug also inhibited the 25-hydroxylase activity towards vitamin D3, catalysed by the same enzyme preparation, to the same extent as 27-hydroxylation of cholesterol. These results suggest that cyclosporin A may interfere with binding of cholesterol, but not of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, to the active site of the enzyme. These data provide an explanation for the selective inhibition of chenodeoxycholic acid synthesis.

Plasma calcitonin, IGF-I levels and vertebral bone mineral density in hyperprolactinemic women during bromocriptine treatment.

Hyperprolactinemia is associated with decreased bone mineral density, which may be caused by the hypogonadism and hypoestrogenicity noticed in patients with hyperprolactinemia. Since calcitonin inhibits the bone resorption, and insulin-like growth factor I (IGF-I) has important anabolic effects on the skeleton, lack of one or both peptides may contribute to the development of osteopenia. We therefore measured the plasma calcitonin and IGF-I levels in nine women with hyperprolactinemia caused by a prolactin-producing pituitary tumor. The calcium-stimulated C-cell reactivity was studied by measuring calcitonin in plasma during a calcium clamp before and after normalization of serum prolactin during treatment with bromocriptine. Basal CT levels were measurable but lower than in healthy controls. Basal IGF-I levels and calcium-stimulated plasma calcitonin were normal in the hyperprolactinemic state and similar to the calcitonin and IGF-I levels during bromocriptine treatment. The serum prolactin levels decreased (p < 0.001) and the serum estradiol levels increased (p < 0.001). The bone mineral density of the lumbar spine increased significantly during treatment. Thus, basal plasma CT levels are slightly reduced in hyperprolactinemic women. However, the reversible osteopenia in hyperprolactinemic women is less likely to be caused by inhibited IGF-I secretion or by deficient CT levels since the CT response to calcium is normal. In addition, bromocriptine treatment with normalization of prolactin levels is beneficial for the bone mineral content in this condition.

No effects of piroxicam on osteopenia and recovery after Colles' fracture. A randomized, double-blind, placebo-controlled, prospective trial.

In a randomized double-blind study involving 42 postmenopausal women with a displaced Colles' fracture, we investigated whether piroxicam, a nonsteroid anti-inflammatory drug, can reduce posttraumatic osteopenia and improve the rate of recovery. In an earlier study [3] we found a bone-sparing effect caused by piroxicam after external fixation of the rabbit hindleg. The patients were treated with a below-elbow paster slab for 4 weeks after the reduction. The bone mineral content of the forearm bones was measured with a single-photon absorptiometer 8 weeks after the fracture. There was a mean 7% bone mineral decrease in the radius and 5% in the ulna among the patients treated with piroxicam versus 10% in the radius and 7% in the ulna in the placebo group. However, this difference was not significant. Piroxicam did not decrease the rate of fracture healing. The patients who received piroxicam had significantly less pain during plaster treatment, but there was no difference in the rate of functional recovery between the groups.

Calcium homeostasis in pregnancy during long-term heparin treatment.

OBJECTIVE: To investigate the effect of subcutaneous heparin treatment on calcium homeostasis in pregnancy. DESIGN: A longitudinal case-control observational study. SETTING: Department of Obstetrics and Gynaecology, Karolinska Hospital, Stockholm, Sweden. SUBJECTS: 36 pregnant women with previously verified thromboembolic complications and 23 healthy pregnant control women similar in age, parity, weight, and smoking habit. INTERVENTIONS: Thromboprophylaxis during pregnancy and 6 weeks post partum was given with subcutaneous heparin twice daily to the 36 women with a history of thromboembolic complications, 16 received an average dose of 24,500 IU/day and 20 a mean dose of 17,300 IU/day. Venous blood and urine samples were obtained every 4 weeks. MAIN OUTCOME MEASURES: Serum concentrations of total calcium, ionized calcium, calcitonin and urinary calcium. RESULTS: Women on high-dose heparin treatment showed significantly higher concentrations of total and ionized calcium and of calcitonin in serum and significantly lower concentrations of calcium in urine than did 23 normal pregnant controls. The differences were most pronounced in the third trimester. The results obtained in the low-dose heparin group were between those in the high-dose and the control groups. At 8 weeks postpartum there were no significant differences between the heparin-treated women and the controls. No significant differences were found during pregnancy in haematocrit, liver or renal function, serum levels of albumin, phosphate, magnesium, alkaline phosphatase, parathyroid hormone or urinary cyclic AMP. CONCLUSIONS: Heparin treatment during pregnancy results in changes in calcium homeostasis and a dose-dependent response is suggested.