ABSTRACTS P-147 to P-184
Posters will be on display throughout the symposium, but will be attended by their presenting authors as follows:
Odd numbers on Friday 11:00 - 12:00, Sunday 11:00 - 12:00
and Monday 10:00 - 11:00
Hormones, Including Estrogen, Vitamin D, PTHrP
BREAST ARTERIAL CALCIFICATION IN PATIENTS ON HEMODIALYSIS
B. Jeren-Strujic*, Z. Borkovic, J. Crnjakovic, J. Gudel, B. Rozman
Dubrava University Hospital, Zagreb, Croatia
Calcification of blood vessel wall is a degenerative process combined with progressive development of atherosclerotic process characteristic for older age group. Patients with chronic renal insufficiency reveal greater incidence of blood vessel calcification in younger age group. Pathological mechanism of calcium and phosphorus, increased Ca/P ratio and secondary hyperparathyroidism have an important role in the development of vascular calcifications. The presence of calcifications in breast arteries (BAC) causes great pain. The aim of our study was to generalize blood vessel calcification in patients on hemodialysis by using mammography. Our study included 54 female patients aged between 28 and 80, mean age X=57±12.4. All patients underwent mammography, biochemical tests and indirect PTH. Mean values were as follows: hemodialysis X=4.2±2.9, Ca/P X=4.8±1.3, PTH X=456.1±487, BAC pos 36.7 (66.7%), BAC neg 18 (33.3%). Control group of healthy patients consisted of 56 females aged between 25 and 80 (X=56.2±8.2). Mammograms revealed no BAC in the control group of patients.
Our results show that calcifications occurred in 66.7% of our patients with 36 mammograms indicating significant BAC. Neither patients' age nor Ca/P ratio are statistically significant for BAC. Statistical significance was observed in the duration of hemodialysis and level of PTH. BAC diagnosed by radiography point to accelerated development of atherosclerosis and remind that atherosclerosis is a potential risk factor for the development of CAD in patients on hemodialysis.
MULTI-LOCUS ASSOCIATION BETWEEN SINGLE NUCLEOTIDE POLYMORPHISMS (SNPS) OF THE CALCIUM-SENSING RECEPTOR (CASR) GENE AND IONIZED CALCIUM IN A POPULATION OF SOUTHERN ITALIANS
A. Scillitani1*, V. Guarnieri1, C. Longo1, S. De Geronimo2, L. A. Moscarella1, C. Battista1, L. D'Agruma1, F. Bertoldo3, S. Minisola2, D. E. C. Cole4
1'Casa Sollievo della Sofferenza' Hospital, San Giovanni Rotondo, Italy
2Instituite of Clinical Science, University 'Umberto I' , Roma, Italy
3Institute of Internal Medicine, University of Verona, Verona, Italy
4Dept. of Laboratory Medicine & Pathobiology, University of Toronto, Toronto ON, Canada
Ionized calcium (iCa) is a quantitative trait subject to genetic influence, maintained in a narrow range through the action of the calcium-sensing receptor (CASR) and the principal calcitropic hormone, PTH. One CASR SNP prevalent in Caucasians (A986S) is significantly associated with iCa in young women, but association with the two neighbouring SNPs, R990G and Q1011E, has not been examined. We therefore studied 166 unrelated adults resident in southern Italy (108 males & 58 females; 45 ± 11 yrs old) recruited either as unrelated controls for a larger study of primary hyperparathyroidism or through a blood donor clinic. Subjects were genotyped by sequencing an amplicon containing all 3 loci; serum ionized calcium, creatinine and PTH were measured by standard techniques. Genotype frequencies for the CASR 986, 990 and 1011 loci were, respectively, 84AA/69AS/13SS, 148RR/16RG/2GG, 158QQ/8QE. There was no evidence of Hardy-Weinberg or linkage disequilibrium. At the A986S locus, subjects with the AA genotype had significantly lower iCa than subjects with one or two S alleles (1.23±.01(SE) vs 1.25±.01 mM, p=.03). However by Q1011E genotype, iCa was lower in QQ genotype subjects than in the QE group (1.24±.01 vs 1.28±.02 mM, p<.01). Finally, at the R990Q locus, iCa was higher in the RR group compared to those with 1 or 2 copies of the 990G allele (1.24±.01 vs 1.22±.01 mM, p<.03). Multiple regression analysis with clinical covariates (age, sex, creatinine, PTH) explaining 15.3% of the total variance shows that 5 composite genotypes together contribute significantly (p<.001) and substantially (74.6% of the explained variance) to the prediction model, with the following corrected iCa means: AARGQQ (1.21±.01), AARRQQ (1.23±.01), ASRRQQ (1.24±.01), SSRRQQ (1.25±.01), AARRQE (1.28±.02). Thus, our data confirm the association between increased iCa and the A986S locus, but suggest that the R990G and Q1011E alleles are also predictive, at least in this sample from southern Italy. Given the significant between-population variations in frequency of variant alleles in this CASR SNP cluster, tri-locus haplotyping may prove to be the most informative in studies of association between variation in CASR and disease.
VITAMIN D STATUS AND ITS RELATIONSHIP WITH BONE MINERAL DENSITY IN HEALTHY ASIAN INDIANS
V. Arya1, R. Bhambri2, M. M. Godbole1, A. Mithal2*
1Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India
2Indraprastha Apollo Hospital, New Delhi, India
Vitamin D plays an important role in calcium homeostasis and skeletal metabolism. The Indian sub-continent is situated between 8.4 degree N and 37.6 degree N latitude and has adequate sunshine throughout the year. Thus, it has been presumed that Indians are vitamin D sufficient. We measured serum 25(OH) D and 1, 25(OH)2 D levels in healthy hospital staff from a hospital in north India. Mean (±SD) age of the subjects was 34.2 ±6.7 years (range 24-53). Serum 25 (OH) D levels were estimated in 92 subjects (67 females and 25 males) and serum 1, 25 (OH)2 D levels in 65 subjects (50 females and 15 males). Serum 25 (OH)D
and 1, 25 (OH)2 D levels were estimated by 125 I radioimmunoassay. Serum intact parathormone (PTH) concentration was measured by IRMA. Bone mineral density was estimated using dual energy X-ray absorptiometer (Hologic QDR 4500A).
There was no significant difference in serum 25(OH) D level between males and females . Only 33.7 percent (31/92) of the subjects had serum 25(OH) D concentrations above 15 nanog/ml and could be classified as vitamin D sufficient. 20.6 percent (19/92) subjects had severe vitamin D deficiency (<5 nanog/ml), 27.2 percent (25/92) subjects had moderate vitamin D deficiency (5-9.9 nanog/ml) while 18.5 percent (17/92) had mild vitamin D deficiency (10-14.9 nanog/ml). Mean (±SD) serum intact PTH, estimated in a limited number of subjects (n=15), was 72.3 ±21.0 pg/ml (range 36-100). There was a significant correlation between daily sun exposure and 25(OH) D levels (r=0.731, p<0.001). Serum 25(OH) D levels correlated with BMD at femoral neck and Ward's triangle (r=0.50, p=0.020 and r=0.46, p=0.037 respectively). There was significant inverse linear correlation between serum 25(OH) D concentration and intact PTH (r= - 0.61, p=0.028).
Our findings show that vitamin D deficiency is common in urban Indian hospital staff. The possible reasons include inadequate sunlight exposure and skin pigmentation in Indians. Low 25(OH) D level is possibly one of the causes of lower bone mineral density amongst Indians.
ALFACALCIDOL* REDUCES THE NUMBER OF FALLERS AND FALLS IN COMMUNITY-DWELLING ELDERLY PROVIDED A MINIMUM TOTAL DAILY INTAKE OF 500MG CALCIUM
*ALPHA-D3 BY TEVA
L. Dukas1*, H. A. Bischoff1, L. S. Lindpaintner1, E. Schacht2, D. Birkner-Binder1, B. Thalmann1, H. B. Stähelin1
1Geriatrische Universitätsklinik, Kantonsspital, Basel, Switzerland
2Metabolic Bone Disease Unit, Universitätsklinik Balgrist, Zurich, Switzerland
Background: Elderly have an up to 50% increased annual risk of falls, associated with increased risk of hip fractures, morbidity, loss of independence, institutionalisation and mortality. Previous studies with vitamin D and calcium or calcitriol (D-hormone) reported a fall frequency reduction among institutionalised or osteopenic elderly women. Data are lacking about this calcitriol effect among community-dwelling elderly, particularly among men. In a 36-week randomised double-blind placebo-controlled intervention trial we studied the effect of Alfacalcidol(1alpha(OH)D3), a calcitriol pro-drug, upon fall risk among community- dwelling elderly.
Methods: 378 Swiss community-dwelling women (191) and men (187), 70-years old and older, were randomised to receive either 1ug capsule of Alfacalcidol or placebo daily. Serum calcitropic hormones were measured by radioimmunoassay at baseline and 12-weekly. Falls and dietary calcium intake were assessed by questionnaires. We used multivariate-controlled logistic regression models to assess the risk to be a faller, according to treatment groups and by medians of total calcium intake. Results presented are from ITT analyses.
Results: Baseline calcidiol and calcitriol serum levels were within normal ranges.
Compared to placebo, the 36-weeks treatment with Alfacalcidol was associated with a significant reduction in number of fallers (OR 0.45, 95%CI 0.21-0.97, p=0.04) in participants with a total calcium intake of more than 512mg/day. This association was not observed in participants with a total calcium intake of less than 512mg/day (OR 1.00, 95%CI 0.47-2.11, p=N.S.)(Figure). Similar results were found for number of falls.
Independent of calcium intake, Alfacalcidol treatment was also associated with a significant 37.9% reduction of iPTH serum levels (p<.0001). We observed no cases of clinically relevant hypercalcemia.
Conclusion: Among community-dwelling elderly with a minimum total daily calcium intake of 500mg, Alfacalcidol treatment significantly and safely reduces the number of fallers and falls.
DIETARY PHOSPHORUS RESTRICTION REGULATION OF RENAL 25-HYDROXYVITAMIN D3 1-ALPHA HYDROXYLASE IN RATS: EFFECT OF AGE
M. S. Wong, W. P. Lai*, P. Y. Cheung, S. C. L. Lo
The Open Laboratory of Chirotechnology, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hum, Kowloon, Hong Kong, P.R. China
Age-related decrease in renal 25-hydroxyvitamin D3 1-alpha hydroxylase (1- OHase) activity results in a decrease in renal 1,25(OH)2D3 production. However, the underlying mechanism for it is still unclear. Dietary phosphorus restriction (LPD) is a potent stimulus for renal 1-OHase production. Previous study in our laboratory showed that adult rats (3 months of age) failed to increase renal 1,25(OH)2D3 production in response to LPD. In the present study, we aim to determine if there is an age-related change in expression of renal 1-OHase protein in response to LPD and determine if the stability of renal 1-OHase protein alter with age or LPD feeding in rats. Young and adult male Sprague Dawley rats were fed either normal phosphate (P) diet (0.65% P, 0.60% Ca) or low phosphate diet (0.1% P, 0.60% Ca) for 0-7 days (D0- D7). Serum P level decreased while serum1,25(OH)2D3 levels increased in both young and adult rats in response to low P diet feeding. Upon feeding with 5 days of LPD, the expression of 1-OHase protein increased significantly by 2.5 fold in young rats (p<0.001); whereas, no significant increase in 1-OHase was observed in adult rats. Young and adult rats fed either normal or low P diet for 5 days were then injected with cycloheximide (0.3mg/100g of body weight), a protein synthesis inhibitor, to determine the effect of age and diet on in vivo stability of 1-OHase protein. In young
rats, LPD stabilized the expression of 1-OHase protein and prolonged its half-life. The basal expression of 1-OHase protein in adult rats was higher and appears to be more stable than that in young rats under normal P condition. Moreover, LPD did not alter stability of protein in adult rats. The results indicated that the regulation of renal 1- OHase protein by LPD was different between young and adult rats. LPD increased renal 1-OHase protein expression in young rats, at least in part by increasing the stability of renal 1-OHase. The stability of renal 1-OHase protein in adult rats was higher than in young rats and did not further increase in response of LPD feeding.
MOLECULAR ACTION OF GINSENOSIDE RG1 IN HUMAN BREAST CANCER (MCF-7) CELLS
W. F. Chen1*, Y. K. Chan1, D. A. Guo2, M. S. Wong1
1The Open Laboratory of Chirotechnology, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR, Peoples Republic of China
2School of Pharmaceutical Sciences, Peking University, Beijing 100083, Peoples Republic of China
Phytoestrogens are currently receiving considerable attention as a potential alternative therapy for reducing the incidence of cancer and protecting against osteoporosis. Elucidating the detailed mechanism can help us to understand the potentially benefit and risk of ginseng consumption. Ginsenoside Rg1 is a steriodal saponin of high abundance in ginseng. Our previous studies have demonstrated that Rg1 is a potent phytoestrogen. It can act like an estrogen analog in stimulating human breast cancer cell growth as well as in the activation of estrogen response element- luciferase activity in HeLa cell. However, our results also indicated that there is no direct interaction between Rg1 and estrogen receptor (ER) alpha, suggesting that Rg1 may activate ER via a ligand-independent pathway. Since there is accumulating evidence that there is a cross talk between ER pathway and IGFIR pathway in ER- positive breast cancer cells, we hypothesize that Rg1 might mediate its action via the cross-talk with IGF-I receptor mediated pathway similar to the action of estrogen.
In this study, we examined the effect of different concentrations of Rg1 on the protein expression of IGF-IR, IRS-1, Shc and ER alpha using western blotting and the effect of Rg1 on IRS-1 and ER alpha mRNA levels using semiquantitative RT-PCR in human breast cancer MCF-7 cells. Rg1 treatment increased IGF-IR and IRS-1 protein level in a time and dose dependent manner (P<0.05) in MCF-7 cells. In contrast, no apparent change was found on Shc protein level (P>0.05). Co-treatment of MCF-7 cells with the pure ER antagonist ICI 182,780 completely abrogated the stimulatory effects of Rg1 on IGF-IR and IRS-1 protein level, indicating that these effects were specific and directly mediated through ER. Treatment of MCF-7 cells with Rg1 or17beta-estradiol for 48h increased IRS-1 mRNA level and these effects were also abrogated by ICI 182,780. 17Beta-estradiol, but not Rg1, decreased the expression of ER alpha protein and mRNA. In conclusion, these data provide the first evidence that the IGF-IR pathway is involved in the proliferative effect of ginsenoside Rg1 in MCF- 7 cells and caution is warranted for the consumption of ginseng products in women who suffered from ER-positive breast cancer.
DOWNREGULATION OF HUMAN 25-DIHYDROXYVITAMIN D3 1ALPHA-HYDROXYLASE PROMOTER ACTIVITY BY NFKAPPAB VIA MULTIPLE NFKAPPAB RESPONSE ELEMENTS
R. Ebert1*, M. Jovanovic1, D. Schneider1, J. Adamski2, F. Jakob1
1Clinical Research Unit, Orthopaedic Department, University of Wuerzburg, Wuerzburg, Germany
2Institute of Experimental Genetics, GSF-National Research Center for Environment and Health, Neuherberg, Germany
The secosteroid hormone 1,25-dihydroxyvitamin D3 is the major regulator of systemic calcium metabolism and plays an important role in cell differentiation. 1,25- dihydroxyvitamin D3 has also been implicated as an immunomodulator, which acts through its own receptor (VDR). In autoimmune diseases like rheumatoid arthritis for example, levels of 1,25-dihydroxyvitamin D3 are reduced and levels of tumor necrosis factor alpha (TNFalpha), which is a potent activator of the transcription factor nuclear factor kappa B (NFkappaB), are enhanced. Here we report on the characterization of putative NFkappaB elements within the promoter of human 1alpha-hydroxylase, the enzyme which converts 25-hydroxyvitamin D3 to the active hormone 1,25- dihydroxyvitamin D3.
A 1413 bp fragment of the promoter of human 25-hydroxyvitamin D3 1a- hydroxylase was amplified from a BAC clone, sequenced and cloned into the luciferase reporter vector pGL2-basic and pGL3-basic. 5' deletion constructs were created and cloned into pGL3-basic. The full-length promoter fragment was screened for putative NFkappaB sites. The farmost 3' located NFkappaB site was mutated by site directed mutagenesis. Reportergene constructs were transfected into HEK-293 cells and luciferase activity was measured. NFkappaB responsiveness was analyzed by cotransfection of p50 and p65 expression plasmids.
Sequence analyses revealed 4 base exchanges and 3 base deletions compared to the published sequence which were identically found in 5 control persons. In silico promoter analyses revealed 18 putative NFkappaB sites, 11 of which were found to bind NFkappaB in EMSA experiments. Cotransfection of NFkappaB p50 and p65 subunits resulted in dramatic reduction of the wildtype promoter activity as well as a series of 5' deletion constructs. Destruction by mutation of the farmost 3' located NFkappaB response element resulted in marked upregulation of promoter activity.
We show here a negative regulation of the 1alpha-hydroxylase promoter in HEK- 293 cells. This would be in line with clinical findings in inflammatory diseases, where low 1,25(OH)2 vitamin D3 levels are coincident with high levels of proinflammatory cytokines involving the NFkappaB signaling pathway. Downregulation of 1alpha- hydroxylase could as well contribute to the pathogenesis of inflammation associated osteoporosis and osteopenia.
STIMULATION OF BONE MATRIX SYNTHESIS BY PHYTOESTROGENS IN NORMAL AND IMMORTALIZED HUMAN PERIOSTEAL CELLS
H. Tullberg-Reinert1*, R. Bruggisser2, U. Dürmüller1, W. Schaffner2, G. Jundt1
1Institute of Pathology, Kantonsspital Basel/University Clinics, CH 4003 Basel, Switzerland
2Institute of Pharmaceutical Biology, University of Basel, CH 4056 Basel, Switzerland
The decrease of endogenous estrogen production is claimed to be a major cause of rapidly progressing postmenopausal osteoporosis. This process is multifactorial and it involves increased activity of bone resorbing osteoclasts and decreased bone formation by osteoblasts.
In the present study normal human periosteal cells and their immortalized derivatives were chosen to test the effects of 17beta estradiol (E2) and the two phytoestrogens genistein (gen) and kaempferol (kae) on selected parameters of in vitro bone formation.
As evidenced by immunocytochemistry(IHC) , both the normal and the immortalized cells expressed typical markers of the osteoblast lineage: cbfa1, alkaline phosphatase (ALP), collagen type I and III, osteocalcin, osteonectin and osteopontin and the vitamin D receptor. Furthermore, they constituatively expressed osteoprotegerin (OPG), an inhibitory soluble ligand of osteoclast activation as well as estrogen receptor alpha and estrogen receptor beta, thus being a valuble functional cellular model for testing estrogenic compounds. Functional tests included counting of cell numbers and the measurement of alkaline phosphatase (p-nitrophenyl phosphate assay), collagen synthesis by a Sirius Red based microassay (Tullberg and Jundt, 1999) and mineralization by an Alizarin Red-based micromethod (Stanford et al. 1995).
Upon treatment with either E2 (0.1nM) or kae (10microM) the cells upregulated collagen type I and OPG (IHC). Furthermore gen stimulated ALP whereas kae elevated the synthesis of collagen, indicating that both phytoestrogens may have different mode of actions.
Our periosteal cell model thus appears to be a suitable tool to screen for bone forming activity of phytoestrogens and plant extracts.
CALCIUM AND VITAMIN-D THERAPY (SUPPLEMENTATION) DRAMATICALLY IMPROVE THE BONE MINERAL DENSITY IN PATIENTS WITH NUTRITIONAL OSTEOMALACIA.
H. R. Joshi1*, P. Varthakavi2, P. Merchant2, R. A. Sengupta2, J. Dholye2, B. Jhankaria2, V. Ayyar2
1Karnataka Institute of Medical Sciences, Hubli, Karnataka, India
2Topiwala National Medical College And B Y L Nair Municipal Hospital, Mumbai Central, Mumbai, India
Severe Nutritional Osteomalacia is still commonly encountered in India.Subclinical vitamin-D deficiency is widespread in the urban population.There is a paucity of data regarding bone mineral density(BMD)and bone biochemistry in Nutritional Osteomalacia and its response to therapy.
We studied 16 patients(all women)with nutritional osteomalacia and 10 age,sex and BMI matched controls.The patients were followed up for a period of 6 months on treatment.
Patients were divided into two groups.One group received vitamin-D as cholecalciferol and another group as calcitriol.Both groups received calcium supplement as 1 gm calcium carbonate per day.
INTRANASAL HYPOCALCEMIC EFFECT OF MONO-PEGYLATED SALMON CLACITONINS
K. C. Lee*, Y. S. Yoo, D. H. Na
College of Pharmacy, SungKyunKwan University, Suwon City 440-746, Korea
The extremely low bioavailability of nasally administered salmon calcitonin (sCT) has been considered mainly due to degradation by proteolytic enzymes in the nasal mucosa. We reported recently that the covalent attachment of polyethylene glycol (PEG) to sCT has been shown to significantly increase the stability of sCT in tissue homogenates, probably due to protection from proteolytic enzymes by PEG molecules.
To evaluate the hypocalcemic effect of polyethylene ethylene glycol-conjugated salmon calcitonins (PEG-sCT) in rats, mono-PEGylated sCTs (mono-PEG-sCTs) and unmodified sCT were administered via the intranasal route and serum calcium levels were measured by colorimetric assay using o-cresolphthalein.
Mono-PEG-sCTs were prepared with different sizes of succinimidyl succinate monomethoxy PEG molecules (PEG2K, PEG5K, PEG12K) and characterized by high-performance liquid chromatography and matrix assisted laser desorption ionization-time of freight (MALDI-TOF) mass spectrometry.
Nasal instillation of mono-PEG2K-sCT at a dose of 2 IU/kg resulted in sustained reduction in serum calcium levels over 8 hr, with a maximum reduction of 13% after 6 hr of application. Whereas, unmodified sCT showed a transient decrease in serum calcium levels, with the maximum reduction (5 %) observed after 30 min of administration. The overall reduction in serum calcium levels expressed as the net change in AUC relative to control in 8 hr were 11.9, 4.6, and 2.6% for mono-PEG2K- , mono-PEG5K-, and mono-PEG12K-sCT, respectively, compared to 3.2% for unmodified sCT.
The relative bioavailability of nasal administered 2 IU/kg of mono-PEG2K-sCT was approximately 4-folds higher than after intranasal administration of unmodified sCT, and the absolute bioavailability was approximately 91% of intravenously injected sCT in 8 hr.
And intranasal absorption of mono-PEG-sCTs was inversely related to the molecular weights of the PEG attached. Out of the PEGylated sCTs examined, mono- PEG2K-sCT showed the most pronounced hypocalcemic effect.
In conclusion, the intranasal delivery of mono-PEG-sCTs in achieving sustained calcium lowering effects could be an alternative clinical effective therapy for variety of bone diseases.
POSTMENOPAUSAL OSTEOPOROSIS AND SERUM LEVELS OF 25(OH)D3 IN GREECE
A. E. Georgiadis*, S. Zervoudis, E. Koukli, D. Babiolakis, C. Vogiatzi
Osteoporosis Center, Gynecological Hosp, LITO, Athens, Greece
During the last 10yrs some articles in the literature claim that people living in South Europe have lower serum levels of 25(OH)D3 than people of North Europe (1) and some experts believe that this could be the cause of a chronic progressive osteopenia and subsequently osteoporosis in women during the postmenopausal periode.
In order to study this issue, we have measured the serum 25(OH)D3 (Assay of (Biomedica.co.at/vitamind) at fall (Sept-Nov 2002) of 153 postmenopausal women MeanAge=62±6 yrs) with similar demographic characteristics and PTH and all others exams for bone metabolism eg Calcium,Phosphoros,Alkal Phosphatase etc) between normal limits.
We classified them in 2 groups, according to their bone mass (Lumbar Spine+Femoral Neck, measured with DEXA (Hologic Delphi). Group A of 45 women had normal bone mass. Group B of 108 women had osteopenia (T<-2,0) and/or osteoporosis (T<-2,5) in one or both regions and were under treatment with Calcitonin or Alendronate or Risendronate and were taking regularly Calcium 600mg/d and 400 UI/d of Vit D3 for more than 6 ms.
Our results showed that the Group A had a mean serum 25(OH)D3 at 28,8±6,8 ng/ml and at Group B the mean serum level of 25(OH)D3 was 16,9±5,03 ng/ml. This difference was significant (p<0,05).
Our results emphasize 3 points: a. Osteopenic-osteoporotic postmenopausal women have hypovitaminosis D(<20ng/ml)(2)b.In these patients the regular treatment with Calcium 600mg/d and 400 UI/d Vit.D3 can not resolve this problem and c. at fall the serum levels of 25(OH)D3 of healthy postmenopausal greek women are normal even without vitamin D supplements.
Bibliography:(1).Lips et al.,J.Endocr.Metab.86:1212-1221,2001.(2).Holick M.F.et al.,Clin.Lab.Med.20(3):569,2000.
CORRECTION OF SECONDARY HYPERPARATHYROIDISM DUE TO VITAMIN D DEFICIENCY IN ADULT OSTEOPENIC PATIENTS
S. O. Mazurenko*, O. H. Mazurenko, E. A. Bajanov
Center of Osteoporosis and Metabolic Bone Diseases, Sokolov's Hospital, Saint Petersburg, Russia
Secondary hyperparathyroidism is quite common in eldery people, especially in winter time. Lack of sun light exposure and vitamin D deficiency, decline in renal function with aging are the common causes of this phenomenon. However, we noticed that in the North-West of Russia (Saint Petersburg) the secondary hyperparathyroidism is also seen in relatively young adult people with normal renal function.
The level of intact parathormone (iPTH)was analysed in 290 osteopenic patients, after evaluation by dual energy x-ray absorptiometry. 26 patients (8,9%) 4 males and 22 females aged from 40 to 64 years (mean 55,4 ±6,8)had level of iPTH higher than 7,7 picomol/l (mean 10,7 ±4,1), ranging from 8,1 to 20 picomol/l. Serum ionized calcium, osteocalcin, 25-hydroxyvitamin D were measured. The studied patients had normal renal function, evaluated by creatinine clearance, low levels of 25- hydroxyvitamin D (< 30 nanomol/l)and low or low-normal levels of ionized calcium. Multiple regression analysis was performed. Serum iPTH correlated negatively with levels of serum 25-hydroxyvitamin D (p<0.01), and positively with the levels of osteocalcin (p<0,001). Alfacalcidol therapy at a dosage of 0.5 micrig/day plus carbonate calcium (1200 millig/day) was initiated.
As a result of the therapy a significant inhibition of iPTH was observed in 6 weeks, and osteocalcin levels in 12 weeks. We conclude that secondary hyperparathyroidism due to vitamin D deficiency is a common cause ot the progression of osteoporosis, and therapy with active forms of vitamin D quickly restore the balance of bone metabolism.
OSTEOPROTEGERIN (OPG) AND RECEPTOR ACTIVATOR OF NUCLEAR FACTOR-KB LIGAND (RANKL) MRNA LEVELS IN PRIMARY HYPERPARATHYROIDISM: EFFECT OF PARATHYROIDECTOMY AND ASSOCIATION WITH BONE METABOLISM
L. S. Stilgren*, E. Rettmer, L. Hegedus, H. Beck-Nielsen, B. Abrahamsen
Dept. of Endocrinology, Odense University Hospital, Denmark
The effect of parathyroid hormone (PTH) on the production of OPG and RANKL in bone in vivo is unknown. Most in vitro studies indicate that PTH decreases OPG and increases RANKL. In primary hyperparathyroidism (PHPT), hypersecretion of PTH leads to enhanced bone resorption and -formation with increased risk of fracture. Patients with PHPT are cured by surgery, resulting in normalisation of PTH levels and bone metabolism, but the effects on OPG production are unknown.
Objective: To asses the expression of OPG and RANKL mRNA in bone before and after successful parathyroidectomy (PTX) in patients with PHPT.
Population and methods: Twenty-four patients referred to our clinic for evaluation and treatment of PHPT. A Bordier bone biopsy was done before surgery (n=24) and 12 months after PTX (n=21). Biopsies were frozen in liquid nitrogen and RNA extracted using Trizol. A competitive RT-PCR assay for OPG and RANKL mRNA using artificial cDNA standards was developed and used for quantification. In addition, the results were normalised for GAPDH mRNA content.
Results: The OPG/RANKL gene expression ratio in iliac bone was significantly lower before surgery compared with after (p=0.04). The ratios of OPG/GAPDH (p=0.27) and RANKL/GAPDH (p=0.85) alone were not significantly altered. There was no correlation with serum PTH. Markers of bone turnover decreased significantly after surgery. There was a significant correlation between RANKL/GAPDH mRNA before surgery and serum osteocalcin (rho=0.54; p=0.006) but not serum P1NP (rho=0.36; p=0.09) or P1CP (rho =0.36; p=0.12). Moreover a significant correlation between urinary U-NTX and RANKL/GAPDH mRNA (rho=0.42; p=0.04) were found. The correlations between bone turnover markers and OPG/GAPDH and OPG/RANKL were not significant.
Conclusion: The OPG/RANKL mRNA ratio in bone increased significantly after PTX in patients with PHPT, though a correlation with serum PTH levels was not observed. RANKL, but not OPG, mRNA in bone correlated significantly with biochemical markers of bone turnover. This supports the hypothesis that locally produced RANKL affects bone turnover in the hyperparathyroid state, but the influence of OPG is less clear. Endogenous PTH levels appear to exert only a small influence on RANKL and OPG production in human bone in vivo.
DAILY ADMINISTRATION OF 1 MCG ALFACALCIDOL HAS A LOW INCIDENCE OF HYPERCALCEMIA AND HYPERCALCIURIA IN GREEK OSTEOPENIC AND OSTEOPOROTIC FEMALE PATIENTS
Gr. Skarantavos1*, F. Kaplanoglou2, A. Loutriotis3, E. Tsiantoula2, G. Lyritis1
1Research Laboratory on Diseases of the Meusculoskeletal System, University of Athens, KAT Hospital, Greece
2Rheumatology Clinic, KAT Hospital, Greece
36th Ortopaedic Clinic, Voula Hospital, Voula, Greece
Aim: A number of studies, mainly of Japanese origin, support that the administration of high alfacalcidol dosages results in osteoproductive effects in bones. In these studies, the incidence of hypercalcemia and hypercalciuria appears to be negligent. In western literature there are few similar studies, with controversial results. In the present study, we aimed to investigate the effect of daily 1 mcg alfacalcidol administration to postmenopausal osteoporotic and osteopenic Greek women, on calciaemia and calciuria.
Materials and Methods: 709 women participated in the study. All the patients were postmenopausal, aged from 40 to 79 years old (av. 61,93, sd 8,04), with a T score lower than (-1,5 and received no other treatment for osteoporosis. Exclusion criteria were thyroid disease, GI surgery, corticosteroid p.o. administration, antiepileptics and diuretics, chronic diseases, mobility disturbances and all other diseases affecting bone turnover. All patients were subject to laboratory examinations for Ca, P, alkaline phosphatase, T3, T4, TSH, and 24-hour urine for Ca, P and creatinine. Patients presenting pathological levels were excluded from the study. A question on basic educational skills was also included. All patients received 1 mcg alfacalcidol daily in the morning for a period of 6 months. All examinations were repeated at 3 and 6 months. Adverse effects and discontinuation of treatment were recorded.
Results: Out of the 709 women initially included, 574 concluded the study. One woman discontinued treatment following GI complaints. Out of the 574 women 2.4% presented hypercalciuria (Ca 24-hours > 240 mg) at 3 months and 1.9% at 6 months, while hypercalcemia (serum Ca>10,2 mg/ml) was observed at 3 and 6 months at percentages of 2% and 1.7%. An increase of total alkaline phosphatase by 1,94% was also observed, which is not statistically significant, but is an indication of osteoproductive activity.
Conclusions: The administration of 1 mcg alfacalcidol (Alpha D3, Gerolymatos, TEVA) in Greek postmenopausal osteoporotic and osteopenic women is not associated with danger of hypercalcemia or hypercalcuria, and probably has osteoproductive activity, an effect which must be further confirmed by future studies.
ELEVATED LEVELS OF PTHRP IN PLASMA OF WOMEN WITH UTERINE LEIOMYOMAS
V. Paspaliaris1*, G. M. Anderson1, B. Liedtke1, C. Wood1,2
1Kurlama Pty Ltd, Melbourne, Australia
2Swinburne University Hospital, Melbourne, Australia
Uterine leiomyomas (fibroids or myomas), benign tumours of the human uterus, are the single most common indication for hysterectomy. They are clinically apparent in up to 25% of women and cause significant morbidity,including prolonged or heavy menstrual bleeding, pelvic pressure or pain, and, in rare cases,reproductive dysfunction. It has been demonstrated that parathyroid hormone-related protein (PTHrP) acts as a local cell growth modifier and a suppressor of myometrial contraction in an autocrine/paracrine fashion on the uterus and may have a similar role on uterine leiomyomas. Humoral hypercalcemia of benignancy is a recently described syndrome characterized by hypercalcemia induced by PTHrP produced by a benign tumor. Clinical cases such as ovarian dermoid cysts, intestinal leiomyoma, mammary hyperplasia, pheochromocytoma, and a patient with a uterine leiomyoma have been reported. The purpose of this study was to investigate the level of PTHrP in the plasma of women with benign gynecological disease (uterine leiomyomas (n=36), endometriosis (n=29), adenomyosis (n=25)) and controls(n=52). Real-time quantitative RT-PCR was also performed from excised leimyoma tissue from the leiomyoma group. In the leimyoma group 17% of patients had an elevated PTHrP level, levels of which correlated with the size of the leimyoma(s). Two women of the leiomyoma group that had relatively high plasma levels of PTHrP had evidence of hypercalcemia. None of the women in the other groups had elevated plasma PTHrP concentration or evidence of hypercalcemia. Real-time quantitative RT-PCR showed significant overexpression of PTHrP in all leimyoma tissue compared to surrounding myometrial tissue. These findings suggest that women that present with uterine leimyomas and symptoms of hypercalcemia should be examined for humoral hypercalcemia of benignancy.
SIGNIFICANCE OF GLUCOCORTICOIDS FOR THE REGULATION OF OSTEOBLAST FUNCTION BY VITAMIN D
M. Eijken*, F. A. A. Weyts, M. Koedam, C. J. Buurman, M. van Driel, H.A.P. Pols, J. P. T. M. van Leeuwen
Department of Internal Medicine, Erasmus MC, Rotterdam, The Netherlands
Glucocorticoids have profound effects on bone formation and, when administered in pharmacological doses, cause osteoporosis, mainly by suppressing bone formation. However, in vitro studies show that glucocorticoids are essential for the differentiation and function of human osteoblasts and are known to modulate hormone effects.
To gain more insight into the action of glucocorticoids in osteoblasts we studied the effects of dexamethasone (Dex) on the human osteoblast cell-line SV-HFO, that has the potency to differentiate during culture, forming a mineralized extracellular matrix in a 3-week-period. Cells were cultured for 23 days in the presence or absence of Dex and 1,25(OH)2D3 (VitD). Throughout culture-time, we studied mRNA expression of the bone matrix proteins osteocalcin (OC), osteopontin (OPN) and collagenIalpha1. Furthermore we studied mRNA expression of the transcription factor Cbfa1, as well as mineral deposition and alkaline phosphatase activity (ALP). To quantify mRNA expression we used real-time PCR.
Both ALP-activity and mineralization were induced by Dex in a dose-dependent manner (10-8-10-6M). VitD stimulated ALP activity and mineralization, however this stimulation was dependent on the presence of 10-6M Dex. Dex also increased collagenIalpha1 and Cbfa1 expression, by 1.6 and 1.5 fold, respectively. While Dex had no effect on basal OC mRNA expression, it reduced vitD-stimulated OC expression from approximately 4.0- to 2.6-fold. This was supported by measurements of OC protein. Dex inhibited basal OPN mRNA expression by 30% and completely blocked vitD-stimulated OPN expression. All effects of Dex and vitD were independent of the stage of SV-HFO differentiation.
In conclusion, these data underscore the complex and multifaceted role of glucocorticoids in bone cell metabolism. Dex stimulated CollagenIalpha1, Cbfa1, and was essential for mineralization and ALP activity, supporting a crucial role for Dex in osteoblast differentiation and function. Moreover, VitD-stimulation of ALP-activity and mineralization were dependent on the presence of Dex. However, Dex markedly reduced basal or vitD-stimulated expression of OC and OPN, which suggests and supports a negative role for OC and OPN in the process of mineralization. Together, glucocorticoids have a positive effect on osteoblast genes and proteins involved bone matrix formation and mineralization while it inhibits the expression of proteins limiting mineralization.
DIETARY ISOFLAVONES MODULATE BONE MARROW OSTEOPROGENITOR CELLS IN FEMALE PIGLETS: IN VIVO AND EX VIVO RESULTS
A. De Wilde1, C. M. Rassi1, M. Lieberherr1*, C. Colin1, H. C. Lacroix1, G. Chaumaz1, V. Coxam2, C. Bennetau-Pelissero3, G. Cournot1, A. Pointillart1
1LNSA, INRA, Jouy en Josas, France
2U3M, INRA, Theix, France
3Unité Micronutriments, Reproduction, Santé, ENITA, Bordeaux, France
Food containing soybeans provide isoflavone phytoestrogens that can preserve bone mass in postmenopausal women, and prevent bone loss in ovariectomized rats. But the effects of dietary isoflavones on bone remain unclear, particularly on bone formation during growth. We investigated the effects of an isoflavone-rich diet in growing female piglets. Two groups of 8 pre-pubertal piglets were fed a basal (controls) or an isoflavone-enriched (S800) diet for six weeks. The S800 diet contained 800 mg SoyLifeTM /kg, providing 2.8 mg isoflavones/kg body weight/day; the control diet contained no isoflavone supplement. Blood samples, genital tracts, and several bones were collected at slaughter. Bone strength and density were evaluated on the main metatarsal and metacarpal bones and tibia. Bone marrow, rich
in osteoblastic and osteoclastic progenitor cells, was collected from humeri for in vitro studies. The plasma concentrations of isoflavones were increased (daidzein was the main circulating molecule) in the pigs given the isoflavone rich diet, but growth rate, body weight, plasma bone markers (alkaline phosphatase, osteocalcin), bone mineral density and strength were unaffected. In contrast, cultured stromal cells from these pigs had more alkaline phosphatase-rich cells and mineralized nodules, secreted more osteocalcin and osteoprotegerin, and expressed more estrogen receptors ER alpha and ER beta than cells from controls. Cultured mononucleated nonadherent bone marrow cells from isoflavone-treated pigs developed fewer tartrate-resistant acid phosphatase mononucleated cells (osteoclast progenitors) when cultured with 1,25(OH)2D3, and resorbed a smaller area of dentine slices than did those of controls. Freshly isolated bone marrow osteoclast progenitors from isoflavone-treated pigs had more caspase-3 cleavage activity than cells from controls. These cells expressed both estrogen receptors, ER alpha and ER beta, and their production was stimulated by the isoflavone-rich diet. The S800 piglets had heavier ovaries with more follicles, but their uterus weight was unaffected. We concluded that dietary isoflavones act on progenitor bone cells in the bone marrow of growing female piglets via ERs, particularly ER beta, before influencing in vivo bone homeostasis.
ESTROGEN STIMULATES OSTEOPROTEGERIN SYNTHESIS AND SUPPRESSES RANKL EXPRESSION IN MATURING MEGAKARYOCYTES
S. Bord1*, E. Frith1,2, D. C. Ireland1, M. A. Scott2, J. I. O. Craig2, J. E. Compston1
1University of Cambridge, Department of Medicine, UK
2University of Cambridge, Department of Haematology, UK
High-dose estrogen (E) has been shown to produce anabolic skeletal effects in postmenopausal women and is associated with increased megakaryocyte (MK) population in the bone marrow. In vitro E stimulates megakaryocytopoiesis with altered estrogen receptor expression. To investigate further mechanisms by which MKs may influence bone remodelling, CD34+ cells were isolated from cord blood by magnetic bead technology (MACS) and cultured for 6, 9 and 12 days in a collagen- based system plus or minus 100nM 17beta estradiol. Collagen films were dried and cells immunolocalised for osteoprotegerin (OPG), RANKL and CD61, a marker of early megakaryocyte maturation, using an indirect immunoperoxidase technique. Specific protein expression was measured quantitatively by image analysis. Fluorescence-based immunocytochemistry was used to co-localise OPG and RANKL with CD61.
At 6 days, when only very immature MKs were evident OPG expression was suppressed 3-fold (p<0.01) in the E-treated cultures whilst RANKL remained at basal levels compared to untreated cells. However by 9 days in the E-treated cultures the maturing MKs, demonstrated by a 2-fold induction of CD61 (p<0.001), showed a 2.5- fold (p<0.01) increase in OPG expression. RANKL levels were reduced 0.3-fold (p<0.02) in cells cultured in the presence of E at this time point. Maximal OPG expression was seen at 12 days with a 3-fold induction of expression (p<0.001), whilst RANKL levels were further suppressed by 0.5-fold compared to controls (p<0.01). Co-localisation of CD61 with OPG and RANKL at 12 days confirmed expression of these proteins by MKs. OPG staining in the MKs was markedly more intense in the E- treated cultures, whilst there was no difference in RANKL staining between the E- treated and untreated cultures.
We have demonstrated that in vitro, E stimulates the colony forming potential of CD 34+ cells to a more megakaryocytic phenotype. These maturing MKs show increased OPG and suppressed RANKL expression with E treatment. Our results provide further evidence that megakaryocytes may play a role in bone remodelling and in E-induced changes in osteoclastogenesis and bone resorption.
LOW 25-HYDROXY-VITAMIN D CONCENTRATION ASSOCIATED WITH ELEVATED PARATHYROID HORMONE AND TARTRATE- RESISTANT ACID PHOSPHATASE 5B CONCENTRATIONS DURING WINTER IN PREPUBERTAL FINNISH GIRLS
S. Cheng1,2*, A. Lyytikäinen1, H. Kröger2, J. Halleen3, M. Alen4, C. Lamberg- Allardt5
1University of Jyväskylä, Jyväskylä, Finland
2University of Kuopio, Kuopio, Finland
3University of Turku, Turku, Finland
4PEURUNKA-Medical Rehabilitation Center, Laukaa, Finland
5University of Helsinki, Helsinki, Finland
The aim of this investigation was to study the association of serum 25- hydroxyvitamin D (25-OHD) with intact parathyroid hormone (iPTH) and tartrate- resistant acid phosphatase 5b (TRAP 5b) concentrations during winter in prepubertal girls. The subjects were 10-12 year-old girls (n=193) at Tanner stages I-II. Blood samples were taken in the morning between 7:30 and 9:00 after an overnight fast during period December 3rd to 17th (G1, n=99) and January 10th to February 5th (G2, n=94). The 25-OHD concentration was measured by radioimmunoassay. The iPTH concentration was measured using an immunoradiometric method and TRAP 5b was determined by immunoassay. We found that using the cut-off point of 25 nmol/l for serum 25-OHD, 32% of the girls were deficient in vitamin D and 46% of the girls had concentrations deemed to be insufficient (26 and < 40 nmol/l). The girls in the vitamin D-deficient group had significantly higher iPTH (43.9±15.7 pg/l) and TRAP 5B (12.2 ± 2.9 U/l) concentrations than the insufficient (38.6 ± 11.2 pg/l, p=0.049, 11.0 ± 2.8 U/l, p=0.009) and sufficient (32.7 ± 12.1 pg/l, p<0.001, 10.9 ± 1.9 U/l, p=0.006) groups. When taking into account the difference of blood sample collection time, we found that 25-OHD concentration was significantly lower in G2 compared to G1 (28.5 ± 10.9 vs. 36.4 ± 10.7
nmol/l, p<0.001). This low concentration of 25-OHD was associated with elevated concentrations of iPTH (35.9 ± 12.4 vs. 41.7 ± 14.1 pgl/l, p=0.003) and TRAP 5b (10.4 ± 1.2 vs. 12.2 ± 3.4 U/l, p<0.001). In our sample 87% of the girls reported sub- optimum vitamin D intake (<5mg). Our results show that limited exposure to sunlight and low dietary vitamin D intake are the main reasons for vitamin D deficiency during winter. Vitamin D deficiency with a higher concentration of iPTH is consistent with secondary hyperparathyroidism. A low vitamin D concentration accompanied by high bone resorption (TRAP 5b) may limit the accretion of bone mass. Our results suggest vitamin D supplement on an individual basis or through the food supply is indicated for prepubertal children.
SEXUAL HORMONE BINDING GLOBULIN IS RELATED WITH FEMORAL BONE MINERAL DENSITY IN MALE CARDIAC TRANSPLANT RECIPIENTS
G. Höfle*, G. Tautermann, H. Holzmüller, H. Drexel
Department of Internal Medicine and VIVIT, LKH Feldkirch, a teaching hospital affiliated with the University of Innsbruck, Feldkirch, Austria
Osteoporosis and increased incidence of fractures, particularly vertebral fractures (vfx), are a common problem after cardiac transplantation (CTX). Fractures occur even before Osteodensitometry (DXA) detects osteoporosis.
We performed a cross-sectional analysis of male CTX patients in a late post- transplantation period (4.2 years ±SD 2.6 after CTX, n=21). We measured bone mineral density (BMD) by Osteodensitometry (DXA, Lunar) as well as by quantitative heel ultrasound (Sahara, Hologic). We studied the endocrine characteristics associated with the presence/absence of prevalent vfx in cardiac transplant recipients.
Osteopenia (lumbar and/or femoral neck DXA T-Score < -1 SD) was common (10 out of 21 patients, 47.6%) and 7 patients (33.3 %) had osteoporosis as defined by lumbar and/or femoral neck DXA T-Score < -2,5 SD. Only 4 patients had a BMD result within the reference range (19.1%). Vfx were present in 10 of 21 patients (47.6 %; n=37 vfx). They were observed in 1 out of 4 patients with normal DXA results, 5 out of 10 osteopenic and 4 out of 7 osteoporotic cardiac transplant recipients.
A significant negative correlation was observed between sexual hormone binding globuline (SHBG) and femoral neck BMD (r = -0.699; p value = 0.001) and BMD at Ward's triangle (r = -0.439; p value = 0.046). Although quantitative heel ultrasound parameters and lumbar spine BMD correlated well with each other, there was no significant relationship with SHBG. We did not find an association of higher SHBG levels with a lower body mass index (BMI). Prevalent vfx showed a significant correlation with femoral neck bone mineral density (r = 0.486; p value = 0.03). However, SHBG, total and free testosterone as well as estradiol and serum ß- CrossLaps did not exhibit significant associations with vfx in our patients.
The association of high SHBG levels with low femoral neck BMD is demonstrated in a cohort of male cardiac transplant recipients. Our data support a pathogenic role of SHBG in post-transplantation bone disease, although - possibly due to the small number of patients - we could not proof an interrelation of SHBG with prevalent vfx.
DYNAMIC REGULATION OF RANKL AND OPG EXPRESSION DURING HUMAN OSTEOBLAST DIFFERENTIATION: RELATION WITH MINERALIZATION
H. Jahr1,2*, M. Eiken1, B. C. J. van der Eerden1, H. Weinans2, J. A. N. Verhaar2, H. A. P. Pols1, J. P. T. M. van Leeuwen1
1Department of Internal Medicine, Erasmus MC, Rotterdam, The Netherlands
2Department of Orthopedics, Erasmus MC, Rotterdam, The Netherlands
Osteoblasts play a pivotal role in bone metabolism. Besides bone formation they also control osteoclastogenesis. This is mediated through receptor activator of NF kappa B (RANK) localized on the osteoclast, its osteoblast-bound ligand RANKL, and osteoprotegerin (OPG), a natural inhibitory decoy receptor for RANKL secreted by osteoblasts. Actually little is known about at what functional stage osteoblasts stimulate or inhibit osteoclast formation.
The current study was setup to assess RANKL and OPG expression in relation to osteoblast differentiation and function. For this we used a human osteoblast differentiation model that forms and mineralizes an extracellular matrix in the presence of dexamethasone (+DEX). The expression was analyzed in the +DEX, - DEX (non-mineralizing) condition and in the absence/presence of vitamin D by real- time RT-PCR at three time points during culture: 1) early proliferative, matrix synthesis (day 7); 2) peak alkaline phosphatase activity, onset of mineralization (day 14), and 3) mineralization (day 21).
Both OPG and RANKL expression increase during differentiation with the strongest increase starting at day 14. This increase in RANKL expression was very strong in the +DEX condition but virtually absent in the -DEX condition. For OPG expression no difference between these conditions was observed. Comparison of RANKL and OPG expression showed that OPG is more abundantly expressed but that the increase in expression was more pronounced for RANKL than for OPG, i.e. the ratio of RANKL/OPG increases during mineralization. Independent of the condition (DEX or no DEX) vitamin D did not change RANKL expression. In the +DEX condition vitamin D didn't change OPG expression, however, in the absence of DEX vitamin D strongly induced the expression of OPG.
In conclusion, RANKL and OPG expression is dynamically regulated during human osteoblast differentiation. Dexamethasone and vitamin D alone or in interaction regulate the transcription of these genes. The expression of RANKL but not OPG is dependent on whether osteoblasts are in a mineralized surrounding. This is logical from a physiological point of view: induction of osteoclastogenesis is unwanted in a non-mineralizing situation. Moreover, the strong induction of OPG expression by vitamin D in the non-mineralizing condition is in line with this.
CLINICAL USEFULNESS OF A NEW ASSAY FOR PARATHYROID HORMONE IN POSTMENOPAUSAL PATIENTS WITH PRIMARY HYPERPARATHYROIDISM
S. Dionisi1*, E. Romagnoli1, I. Nofroni2, J. Pepe1, S. De Geronimo1, F. Paglia1, G. Tonnarini1, S. Minisola1
1Department of Clinical Sciences, University 'La Sapienza', Rome, Italy
2Department of Experimental Medicine and Pathology, University 'La Sapienza', Rome, Italy
To our knowledge, there are no studies directly comparing first and second generation IRMA for parathyroid hormone (PTH) in patients with primary hyperparathyroidism (PHPT).
We evaluated serum levels of PTH in 39 postmenopausal patients with PHPT (mean age ±SD = 63.9 ±8.2 years) using a classical IRMA (PTH S: N-tact PTH SP; DiaSorin Inc., Stillwater, MN, USA; antibodies directed against epitopes 1-34 and 39- 84) and a new method (Duo PTH; Scantibodies Lab. Inc. Santee, CA, USA; PTH W: antibodies directed against epitopes 1-4 and 39-84; PTH T: antibodies against epitopes 7-34 and 39-84). The values obtained have been compared with those of 70 healthy postmenopausal females of similar mean age (65.6 ±10.9 years), renal function and vitamin D status (PTH S = 37.1 ±12.3picog/ml; PTH W = 24.4 ±9.9picog/ml; PTH T = 37.5 ±13.8picog/ml). Furthermore, we assayed serum PTH levels every 24 hours for 5 days after successful surgery in 10 PHPT patients.
We found very high significant linear correlations among the assays employed in PHPT patients (r=0.91-0.92). Serum PTH levels were above the 2 SD of control population in the following percentage: PTH S 59%, PTH W 77%, PTH T 82%. However in the ROC-curve analysis none of the areas under the curve of the 3 PTH assays significantly differed from each other. Considering the PTH W/PTH T x 100 ratio, we observed a gaussian distribution and similar mean values in both normal subjects and patients (64.5 ±9.5% and 63.4 ±13.3%, respectively).
Our results indicate that the assays we employed have a similar diagnostic sensitivity, even though the number of patients with PTH W and T values above 2 SD of control subjects is higher. The finding of a similar distribution of the PTH W/PTH T ratio suggests that the disease doesn't influence the percentage of biologically inactive fragments derived from central secretion or peripheral metabolism. The similar post-operative curves seem to be at variance to the hypothesis of a possible effect of hypocalcemia in modulating the central secretion of hormonal fragments, at least in this particular acute condition.
THE EXPRESSION OF 25-HYDROXYVITAMIND 24- HYDROXYLASE AND 25-HYDROXYVITAMIND 1 ALPHA- HYDROXYLASE IN HUMAN OSTEOBLASTS
G. J. Atkins1, P. H. Anderson2, H. A. Morris2, A. C. W. Zannettino3, P. Kostakis1,3, D. M. Findlay1*
1Dept. of Orthopaedics and Trauma, University of Adelaide, Adelaide, South Australia 5000, Australia
2Division of Endocrinology, Institute of Medical and Veterinary Science, Adelaide, South Australia 5000, Australia
3Division of Haematology, Institute of Medical and Veterinary Science, Adelaide, South Australia 5000, Australia
1,25-dihydroxyvitaminD3 (vitD3) is a key regulator of calcium homeostasis through its actions on the intestine, kidney, and bone. Although a major role for vitD3 in bone formation in vivo is still uncertain, the vitD3 receptor, VDR, is known to be expressed by osteoblasts. Ligation of vitD3 with VDR induces the expression in osteoblasts of a variety of osteogenic genes, such as osteocalcin and osteopontin. In addition, vitD3 appears to play a direct role in the osteoclastogenic response by the induced expression in osteoblasts of RANKL, and also has effects on the differentiation of human osteoblasts (see our accompanying abstract, Atkins GJ et al). In this study, we used real-time RT-PCR, to examine the expression in normal human osteoblasts (NHBC) of mRNA encoding VDR, the 25-hydroxyvitaminD 24- hydroxylase (CYP24), and the 25-hydroxyvitaminD 1 alpha-hydroxylase (CYP27B1). We show that NHBC express abundant mRNA for VDR, and the expression of CYP24 mRNA increases dramatically in response to treatment with vitD3. Furthermore, the level of CYP24 mRNA induction correlated with the level of VDR mRNA. Our results suggest that human osteoblasts are capable of responding to vitD3, regulating the response through the induction of CYP24, and together with the expression of CYP27B1 mRNA, perhaps also synthesising biologically active vitD3. Therefore vitD3 may play a more complex and intrinsic role in human osteoblast biology than has been hitherto recognised.
LUTEINIZING HORMONE RECEPTOR KNOCKOUT (LURKO) MICE AND TRANSGENIC HUMAN CHORIONIC GONADOTROPIN OVEREXPRESSING MICE (HCG AB+) HAVE BONE PHENOTYPES
S. J. Yarram1*, M. J. Perry2, T. J. Christopher2, K. Westby1, N. Brown1, S. B. Rulli3, F-P. Zhang3, I. Huhtaniemi3, J. R. Sandy1, J. P. Mansell1
1Division of Child Dental Health, University of Bristol, Bristol, UK
2Department of Anatomy, University of Bristol, Bristol, UK
3Department of Physiology, University of Turku, Turku, Finland
Considerable attention has been paid to the role of sex steroids during periods of major skeletal turnover but the interaction of the gonadotropic hormones, which include LH, FSH and hCG, within bone tissue have been overlooked. The question is pertinent due to the recent detection of extragonadal expression of gonadotropin receptors. To this end we determined if primary human osteoblasts (hOB's) and the human osteoblast cell line, MG63, expressed functional LH receptors. Further, we examined bone mineral density (BMD) and bone histomorphometry in the following models: (1) an LH receptor null mutant (LuRKO) mouse, (2) a transgenic mouse overexpressing hCG (hCG a-b+) and (3) an ovariectomized (OVX) hCG a-b+ model. A combination of Western blotting, immunolocalization, nested RT-PCR and subsequent Southern blotting supported the presence of osteoblast LH receptors. However treatment of these cells with hCG failed to generate cAMP.
Male LuRKO mice showed a decrease in BMD after 5 months, apparently secondary to suppressed gonadal steroid production. Similarly, 9-10 week old female LuRKO mice exhibited decreases in histomorphometric parameters tested. The data indicate that loss of LH signaling results in a reduction in bone formation. By contrast there were significant increases in BMD and histomorphometric indices for female, but not male, hCG ab+ mice indicating that chronic exposure to hCG results in bone formation. However, OVX of the hCG ab+ mice resulted in a significant reduction in BMD comparable to OVX wild type controls. The profound increase in BMD for hCG ab+ mice may be attributed to the raised estrogen in this model. To explore this possibility an assessment of BMD in overexpressing mice treated with tamoxifen is ongoing. Although gonadotropin levels are tightly linked to sex steroid titers it appears that their effects on the skeleton are indirect.
DOES LEPTIN HAVE AN EFFECT ON BONE MINERAL DENSITY IN PERIMENOPAUSAL WOMEN?
M. D. Kontogianni1*, U. Dafni2, J. Routsias1, F. N. Skopouli1
1Department of Dietetics and Nutritional Science, Harokopio University, Athens, Greece
2Laboratory of Biostatistics, School of Nursing, University of Athens, Greece
Introduction: Fat mass is an important determinant of bone density, but the mechanism of this relationship is uncertain. Leptin, as a circulating peptide of adipocyte origin, is a potential contributor to this relationship. We investigated the role of leptin in mediating fat mass effects on the skeleton of perimenopausal women.
Subjects and Methods: Twenty-five premenopausal and 55 postmenopausal, healthy women (age 52.4 ± 5.7, BMI 29.3 ± 4.5) participated in our study. BMD was measured with Dual X-ray absorptiometry, leptin levels with ELISA, body composition with the method of bioelectrical impedance analysis and other factors that are known to affect BMD were also assessed. Backward multiple regression analysis was performed using BMD, expressed with T-score at the lumbar spine, and total body bone mineral content, as the dependent variables and leptin, oestrogen, free testosterone, physical activity, calcium intake, smoking habits, body fat and fat free mass, BMI, age and years since menopause (YSM) as the independent variables.
Results: The only variables that had a relation with BMD were YSM (â= -0.0113, p=0.003) and BMI (â= 0.0101, p=0.016), (R2= 0.205). Only when insulin was added to the independent variables, leptin (â= -0.0039, p=0.061) as well as insulin (â= 0.0168, P=0.002) (R2=0.394) were shown to have a strong relationship to BMD. The same results were demonstrated using ANOVA between the normal and osteopenic/osteoporotic women.
In a subgroup of 42 women, in which the total body bone mineral content was also measured, the regression model (R2= 0.739) demonstrated that YSM (â= -19.229, p=0.022), fat free mass (â= 63.375, p=0.000), body fat (â= 19.621, p=0.05) as well as insulin (â= 26.477, p=0.014) and leptin (â= -12.730, p=0.013) had a strong relationship with the bone mineral content.
Conclusion: In our sample of perimenopausal women, leptin had a significant negative renationship with lumbar BMD as well as with total body bone mineral content only when insulin levels were taken into account.
SERUM LEVELS OF TESTOSTERONE AND ESTRADIOL ARE ASSOCIATED WITH BONE MINERAL DENSITY AND RISK OF FRACTURES IN OLDER MEN AND WOMEN
S. M. F. Pluijm1*, J. H. Smit2, M. A. Blankenstein3, P. Lips4
1Institute for Research in Extramural Medicine (EMGO Institute), VU University Medical Centre (Vumc), Amsterdam, The Netherlands
2Department of Sociology and Social Gerontology, Vrije Universiteit, Amsterdam, The Netherlands
3Department of Clinical Chemistry, VU University Medical Centre (Vumc), Amsterdam, The Netherlands
4Department of Endocrinology, VU University Medical Center (Vumc), Amsterdam, The Netherlands
The aim of this study was to examine whether serum levels of estradiol and testosterone are associated with bone mineral density (BMD) and quantitative ultrasound measurements (QUS), and can predict the risk of fracture in a population- based sample of older men and women.
Morning serum levels of estradiol and sex hormone-binding globulin (SHBG) were determined in 636 men and 663 women of 65 years and older who were all participants of the Longitudinal Aging Study Amsterdam (LASA). Serum levels of testosterone were analysed only in men. In the total sample, QUS measurements were assessed and a three year prospective study on fractures was performed. In a subsample (N=514), total body bone mineral content (TBBMC) and BMD of the hip and lumbar spine were measured with DXA. In addition, prevalent vertebral deformities were identified on lateral radiographs of the thoracic and lumbar spine using a semiquantitative method. Data were analysed using multivariate regression analyses, adjusted for age, body mass index, chronic diseases, mobility, smoking and depressive symptoms. All analyses were stratified by sex.
In women, median [IQR] serum levels of estradiol (E) and free estradiol index (fE=E/SHBG) were 28.9 [13.6] pmol/l and 0.60 [0.59]. In men, mean (SD) serum levels of E, fE, testosterone (T) and free testosterone index (fT= T/SHBG) were 77.3 (25.4) pmol/l, 2.01 (0.96), 15.5 (4.9) nmol/l and 0.39 (0.13), respectively. In women, both lower E and fE serum levels were associated with lower QUS values, lower TBBMC, and lower BMD of the hip, and lumbar spine. In men, lower serum levels of E and T were associated with lower QUS values, lower TBBMC, and lower BMD of the total hip and lumbar spine, whereas fE and fT were only significantly associated with QUS. In women, a serum level of E in the lowest quartile resulted in an adjusted relative risk of fractures of 2.32 (95% CI 1.17-4.63). In men, T was linearly inversely associated with fracture risk (RR: 0.92; 95%CI: 0.84-0.99).
These data indicate that decreased serum levels of estradiol and testosterone are independent risk factors for low BMD, QUS and increased risk of fractures in older men and women.
THERAPY IN OSTEOPOROSIS: ESTROGEN AND PHYTOESTROGENS
U. W. Wehr1*, E. Lochmüller2, S. Krammer3, P. Weber3, C. Riegger3, M. Schlachter3, W. A. Rambeck1
1Institute for Animal Physiology, Department of Veterinary Medicine, University of Munich, Germany
2Department of Medicine, University of Munich, Germany
3Hoffmann-LaRoche, Basel, Switzerland
To this day therapy and prevention of the osteoporosis is still unsatisfactory. In this connection a new group of substances came into focus: the phytestrogens. They indicate structural similarity to oestradiol and thereby unfold their oestrogen-like effects on bone. Phytoestrogens seem to be able to have a protective effect on bone without the negative side effects of estrogenes (i.e. possible increase of the breast cancer risk).
The aim of our study was to investigate the bone protective effect of the soy constituent genistein, during a marginal supply of vitamin D and K, which can be observed in patients with osteoporosis.
50 female fischer 344-rats were either sham-operated (SHAM, n=10) or ovariectomized. The ovariectomized animals were either assigned to subcutaneously injected estrogen or to different concentrations of genistein (5 or 15 mg/kg body weight) given orally.
The vitamin D and K supply amounted to 500 IU, respectively to 20 mg per kilogram body weight during the entire duration of test.
After 28 days the cocentration of the marker of bone resorption Pyrodinolin (PYD in nmol/mmol Crea) in the urine and the marker of bone formation Osteocalcin (Oc in ng/ml) in the serum were assessed. After 84 days bone mineral density (right tibia) was measured by Dual X-Ray Absorptiometry (DXA). As a control, the weight of the uterus was also determined.
In the groups supplemented with phytestrogens, the increased bone resorption caused by the ovariectomy was effectively reduced. In rats, given higher doses of genistein even bone loss due to ovariectomy could be prevented. In addition there could not be determined any influence on female sexual organs (weight of uterus).
The protective effect of genistein on osteoporotic bone seems to be a result of the reduction of osteoclast activity.
REPAIR OF SKELETAL DEFECTS WITH A SELECTIVE PROSTAGLANDIN E2 (PGE2) EP-2 RECEPTOR AGONIST
F. Borovecki1*, V. M. Paralkar2, S. Vukicevic1, D. D. Thompson2
1Department of Anatomy, School of Medicine, University of Zagreb, Zagreb, Croatia
2Pfizer Global Research and Development, Groton, CT, USA
Prostaglandin E2 (PGE2) causes significant increases in bone mass when administered systemically or locally to the skeleton and mitigates its tissue-specific pharmacological activity via four different G-protein coupled receptor subtypes, EP1, EP2, EP3 and EP4. We identified a known acyclic sulfonamide as a selective EP2 agonist and following chemical modifications, discovered a more potent and highly selective pyridyl sulfonamide, CP-533,536. It bound with high affinity to the rat EP2 receptor (IC50 = 50 nM) and is approximately 250-fold more selective for binding to the EP2 receptor subtype compared to the EP4 receptor subtype (IC50 = 3200 nM). The addition of CP-533,536 to cells transfected with the EP2 receptor resulted in an equivalent increase in intracellular cAMP with an IC50 of 5 nM. In a model of local rat bone marrow injection, it was observed that CP-533,536 dose-dependently induced both trabecular and cortical local bone formation in a robust manner at the injection site. Next, the ability of CP-533,536 to heal tibial osteotomy defects in a canine model was tested. A single application induced full rebridgement of the defect in 75% of dogs (n=8) treated with 5mg or 25 mg of CP-533,536 dissolved in 0,5 ml of PLGH matrix. The newly formed bone rebridged the tibial osteotomy defect by week 8. Dogs treated with the vehicle (PLGH) alone did not show any significant healing as assessed by radiography and histology. The potent bone anabolic activity of CP- 533,536 offers a new chemical alternative for the enhancement of fracture treatment in patients.
VITAMIN D3 AT 90 OR 700 MCG WEEKLY FOR 1 YEAR: RESPONSES OF 25-HYDROXYVITAMIND, PTH, URINE AND PLASMA CALCIUM
R. Vieth1*, M. Dogan1,3, D. E. C. Cole1,5, L. A. Rubin2,4, I. L. Bromberg1,3, R. Josse2,4, P. G. Walfish2,3
1Dept of Laboratory Medicine and Pathobiology, Canada
2Dept of Medicine, University of Toronto, Canada
3Mount Sinai Hospital
4St Michael's Hospital
5Sunnybrook and Women's College Hospital
One teaspoon of cod-liver oil provides 9.4 microg (375 IU) of vitamin D3, a daily dose appropriate for the skeletal development of an infant or child. Because of body size, adults may require ten times the infant amount. Here, we report on 114 healthy adults, of whom 59 have been followed to 1-yr in an ongoing study comparing the double-blind, randomized use of vitamin D3, at 90 or 700 microg once weekly (equivalent to 15 or 100 microg/day; 4 200 or 28 000 IU/wk). Subjects were healthy women and men, mean age 49 y. Before taking the vitamin D, mean plasma 25(OH)D was 60.0 nmol/L. Blood and urine were tested every 4th month on vitamin D3. In subjects given 90 microg/wk, plasma 25(OH)D reached a plateau mean value of 82 nmol/L by 4 months. In subjects given 700 microg/wk, plasma 25(OH)D increased throughout the year: at the 4th, 8th, and 12th months, it was, 105, 119, and 141 nmol/L respectively. By 12 months, the average change in 25(OH)D for each 1 microg/day of vitamin D3 dose was 1.5 nmol/L with 95 microg/wk, and 0.82 nmol/L with 700 microg/wk. There were no changes in blood ionized calcium in either group. In subjects given 700 microg/wk only, average urine calcium/creatinine ratio was 0.72, i.e. 44% higher than in the lower-dose group (p<0.04). At baseline, parathyroid hormone (PTH) concentration averaged 5.7 pmol/L for all subjects; at 12 months, it was 4.27 in the 90 microg/wk group, and 3.71 in the 700 microg/wk group (difference
between dose groups not significant). Thus, both vitamin D3 doses lowered PTH without affecting plasma calcium. With the higher dose, 25(OH)D took longer to reach its plateau. This study supports the 1-yr safety of vitamin D consumption at 700 microg weekly.
GENE EXPRESSION PROFILING REVEALS NEW FUNCTIONS OF VITAMIN D
F. Graedler1*, U. Zeitz1,2, G. Moeller1, R. G. Erben2, J. Adamski1
1GSF National Research Institute for Health and Environment, Institute of Experimental Genetics, Ingolstaedter Landstr. 1, 85764 Neuherberg, Germany
2Institute of Animal Physiology, University of Munich, Veterinaerstrasse 13, 80539 Munich, Germany
The hormone 1,25-dihydroxyvitamin D(3) [1,25-(OH)2D3], the biologically active form of vitamin D, plays an essential role in bone mineralization through its regulatory function on calcium resorption, transport and bone formation. Functional inactivation of the vitamin D receptor (VDR) in transgenic mice results in a phenotype charcterized by growth retardation, impaired bone formation, alopecia and secondary hyperparathyroidism, symptoms similar to those of patients with hereditary vitamin D resistant rickets.
In this study we used cDNA microarray technology to compare gene expression profiles of various tissues from vitamin D receptor knockout (VDRKO) mice to the wild type (wt). Using a commercially available clone set of 20.000 sequence-verified mouse cDNAs and ESTs from Lion Biosciences Inc., we produced microarrays covering a large part of the mouse genome. Differentially expressed genes were detected as intensity ratios (VDR-KO / wt) in dual-colour hybridisations.
In kidney a number of genes involved in mineral metabolism were found to be regulated. A prominent example of these is calbindin D9K, which has a putative role in transcellular calcium transport in the renal distal nephron facilitating the process of renal calcium reabsorbtion. In agreement with previous studies it was found to be strongly downregulated in kidneys from VDRKO mice and can not be normalized by feeding the VDRKO mice with a diet rich in calcium and phosphorous. Upregulated were in VDRKO kidney genes involved in lipidogenesis like fatty acid synthase. Activation of lipidogenesis with high systemic vitamin D levels can be due to increased levels of cytosolic calcium, which in turn indicates the presence of a membrane calcium channel, responsive to vitamin D, that has been postulated recently.
In femur upregulation of bone matrix proteins and metalloproteinases correlate with results from histological studies indicating an elevated rate of bone remodelling and build-up of unmineralized matrix in bone from VDRKO mice.
Selected data were confirmed by quantitative PCR. In short, the performance of gene expression profiling using microarray technology could give a profound insight in the effects of VDR gene ablation on a physiological level.
VITAMIN-D DEFICIENCY IS PREVALENT IN CHILDREN OF IMMIGRANTS FROM THE MIDDLE EAST AND AFRICA LIVING IN DENMARK
K. Kramme1*, N. Wedderkopp2, L. Heickendorff3, L. B. Andersen4, B. Abrahamsen1, K. Froberg2, K. Brixen1
1Dept.of Endocrinology, Odense University Hospital, Denmark
2Institute of Sport Science and Clinical Biomechanics, University of Southern Denmark, Denmark
3Department of Clinical Chemistry, Aarhus University Hospital, Denmark
4Institute of Sport Science, Copenhagen University, Denmark
Introduction: In Denmark, food is not fortified with vitamin-D and the main source of vitamin-D is production in the skin when exposed to UV-light. This process only takes place during the 3 summer months due to the location at 56 degree northern latitude, and the production is lower in persons with pigmented skin and, furthermore, decreases with age. A number of studies have demonstrated that vitamin-D deficiency is prevalent in adult immigrants from e.g. Arabic countries to Northern Europe. Data on children, however, are scarce. Clarification of this is important in the light of the possible detrimental effect of vitamin-D deficiency on bonemetabolism, musclefunction and mood.
Aim: To compare serum levels of 25-OH-vitamin-D in children of immigrants and of Danish descent, and estimate the prevalence of hypo-vitaminosis-D and marginal vitamin-D deficiency.
Participants and methods: In 1999 a cross-sectional survey of 1020 children and adolescents aged 8-10 and 14-16 years (3rd and 9th grade) living in Odense, Denmark, was started . These participants in the European Youth Heart Study were recruited through two-stage cluster sampling from schools stratified according to school type, location and socio-economic character of uptake area. In a subgroup of 81 girls and 51 boys information on ethnicity and dietary and clothing habits were collected by a questionnaire, and serum levels of 25-OH-vitamin-D, PTH, albumin- corrected calcium, and alkaline phosphatase were determined.
Results: Serum levels of 25-OH-vitamin-D were significantly lower in children descending from immigrants from Middle East or Africa compared with those of Danish descent (25 ± 4 vs. 65 ± 3 nmol/l, p<0.001) while serum PTH was significantly higher (7.4 ± 1.2 vs. 4.8 ± 0.2 pmol/l, p<0.05). No difference in serum 25-OH-vitamin-D or PTH was seen between boys or girls of Danish descent. Girls of Middle East descent, however, had significantly lower serum 25-OH-vitamin-D compared with boys (p<0.05). In the Middle East group, 74% of the children had 25- OH-vitamin-D levels below 25 nmol/l (hypo-vitaminosis-D) compared with 5% in the Danish group. Using a threshold of 50 nmol/l (marginal deficiency) 87% of ethnic children was below this as compared with 35% of Danish descent.
Conclusion: In Denmark, hypo-vitaminosis-D, as defined by serum 25-OH- vitamin-D levels below 25 nmol/l, is highly prevalent in children of immigrants from the Middle East and Africa. Efforts to prevent this are highly warranted.
HYPOESTROGENISM AND HYPOANDROGENISM AND BONE MASS IN MALES OVER 40 YEARS OF AGE
M-R. Mascarenhas1*, S. Guerra1, J. Camolas1, J. Garcia-e-Costa1, F. Almeida2, D. Santos-Pinto3, A. Galvão-Teles1
1Endocrinology, Diabetes and Metabolism Unit, Faculty of Medicine of Lisbon, Santa Maria University Hospital, Lisboa, Portugal
2Osteoporosis Unit, Endlab, Lda., Lisboa, Portugal
3Laboratorio Medico Dr. David Santos Pinto, Lda., Lisboa, Portugal
In adult males the sex steroids as well as the bone mineral density (BMD) seem to decline slowly with the ageing process. The impact of the androgen levels on the alterations to the bone mass density in the male is still controversial.
AIM Contribution to the study of the BMD difference dependence on 17beta- estradiol (E2), total and free testosterone (T and FT) plasma concentrations, age, height, weight and body mass index (BMI) in men over 40 years of age.
MATERIALS AND METHODS A group of men 40 years old or over was studied (n = 273). Age, height, weight and body mass index (BMI) were evaluated. Fast blood was collected for E2, T and FT measurements. This group was subdivided according to the E2, the T and T>4 ng/mL) and FT plasma levels. Andropause was considered for T less than or equal to 2,0 ng/mL (n=25) or FT less than or equal than 10 pg/mL (n=28) and hypoestrogenism for E2 less than or equal to 20 pg/mL (n=111).
The BMD at the lumbar spine (L1-L4), at the hip (total) and at the distal forearm (total) and at the whole body, the total body fat and lean masses were accessed by DEXA with the Hologic-QDR-4500-Acclaim densitometer.
The multiple regression analysis was used to differentiate the several parameters (statistical significance=P < 0.05).
RESULTS The mean age was similar among the androgen and E2 subgroups, the mean stature was low in the FT less than or equal to 10 pg/mL subgroup, the mean weight was elevated in the T less than or equal to 2 ng/mL subgroup and the mean BMD were higher in the andropause subgroups.
The mean BMD at the lumbar spine, at the hip and at the whole body were reduced in the hypoandogenism and hypoestrogenism subgroups; the BMD at the forearm was diminished in the E2 less than or equal to 20 pg/mL and FT less than or equal to 10 pg/mL subgroups.
Significant E2/BMD relationships at every skeletal regions were detected.
CONCLUSIONS Men 40 years old or over with lower T, FT and E2 concentrations may present an increased risk to osteoporosis complications - higher morbility and mortality - due to the reduced BMD. As in the post-menopausal women, BMD is also reduced in males over 40 years of age and may be influenced partly on estrogen plasma levels.
BONE MASS IS REDUCED IN THE TREATED HYPOGONADOTROPIC HYPOGONADAL MALES - BUT NOT IN THE TREATED HYPERGONADOTROPIC HYPOGONADISM
M-R. Mascarenhas1*, S. Guerra1, J. Garcia-e-Costa1, J-M. Aragüès1, L. Oliveira- Lopes1, F. Almeida2, A. Galvão-Teles1
1Endocrinology, Diabetes and Metabolism Unit: Faculty of Medicine of Lisbon, Santa Maria University Hospital, Lisboa, Portugal
2Osteoporosis Unit, Endlab, Lda., Lisboa, Portugal
Hypogonadism in males is considered a risk for osteoporosis, nevertheless it remains not apparent if the hypogonadotropic or hypergonadotropic hypogonadism treatment reduces the risk for a low bone mass.
Bone mineral density (BMD) evaluation in males, during the treatment of hypogonadotropic and hypergonadotropic hypogonadism.
MATERIALS AND METHODS
The BMD at the lumbar spine, at the hip, at the distal forearm and at the whole body were accessed by using the QDR 4500 Acclaim densitometer (Hologic Inc.) in adult and adolescents males under medical therapeutics. A group of hypergonadotropic hypogonadal (n = 21) males was under androgen replacement (mean duration of 7.25 years) and a group of hypogonadotropic hypogonadism (n =
21) patients were treated with testosterone and/or bromocriptine (mean duration of 7.67 years). The BMD of the 42 males and the 2 subgroups were compared with those of normal males (control group, n=156).
Multiple regression analyses were used to compare the BMD at several skeletal regions between groups and duration of therapy correlations (statistical significance: P<0.05).
The trabecular and cortical BMD were reduced in the treated hypogonadotropic group, if compared with the treated hypergonadotropic subgroup.
The BMD at several skeletal sites of the treated hypergonadotropic hypogonadal subgroup and the control group were identical.
The detected BMD/duration of therapy relationships in the hypogonadism groups were not significant.
A severe decrease in the BMD was detected in the hypogonadotropic hypogonadal males, under medical therapy more than 7 years. However, the treated hypergonadotropic hypogonadal group reached a BMD similar to the normal men group.
An early hypogonadotropic hypogonadism diagnosis and therapy may be important, in order to increase the bone mass, as these data strongly suggest that it may signify a risk factor for the progress of a low bone mass in the male.
THE EFFECTS OF ESTROGEN, PROGESTAGEN AND TAMOXIFEN ON RAT VERTEBRAL COLUMN AND FEMURAL BONE MINERAL DENSITY
E. I. Hinoue1, J. M. Soares Jr1, A. M. Massad Costa1, M. G. Nunes1, M. A. Haidar1, M. J. Simoes2, R. S. Simoes2, E. L. A. Motta1, G. Rodrigues de Lima1, E. C. Baracat1*
1Department of Gynecology, Federal University of São Paulo/Escola Paulista de Medicina, Brazil
2Department of Histology, Federal University of São Paulo/Escola Paulista de Medicina, Brazil
The aim of this work was to evaluate the effect of conjugated equine estrogens (CEE), medroxyprogesterone acetate (MPA) and Tamoxifen (TAM)on the bone tissue. Hundred-twenty animals was used for this experiment. All animals were submitted to the bone mineral density (BMD) analyses of spine (L1-L4) and femur (baseline) using a densitometer (HOLOGIC 4500-A) then all animals were ovariectomizes. After 60 days, the rats were randomly divided into six treament groups: GI - control (propyleneglycol); GII - CEE (50 microg/kg); GIII - MPA (2mg/kg); GIV - CEE plus MPA continuously; GV - CEE plus MPA every 48 hs, GVI - TAM (250 microg/kg). The drugs were administered using oral route (gavage) for 60 consecutive days. At the end of the experiment, all animals were submitted to a new densitometric measurement. Our results showed that bilateral ovariectomy led to significant bone loss in the vetebral column and femur. All drugs increased the BMD on column and femur, however the GIV, GV and GVI presented more BMD on the rat bone after the treatment than other groups. Our data suggested that hormonal replacement improved the rat BMD and TAM and the opposed estrogen to MPA affected more positively the BMD than unopposed estrogen or MPA alone.
THE EFFECTS OF ESTROGEN, MEDROXIPROGESTERONE AND TESTOSTERONE ON THE OVARIECTOMIZED CHICKEN BONE
J. M. Soares Jr1*, P. F. R. Silva1, R. Mosquette2, M. J. Simões2, R. S. Simões2, M. A. Haidar1, M. G. Nunes1, A. M. Carvalho2, G. Rodrigues de Lima1, E. C. Baracat1
1Gynecology Department, Federal University of São Paulo/Escola Paulista de Medicina, Brazil
2Histology Department, Federal University of São Paulo/Escola Paulista de Medicina, Brazil
The aim of this study was to evaluate the effects of medroxiprogesterone acetate (MPA), conjugated equine estrogens (CEE) and testosterone (T) on the ovariectomized chicken bone. Forty animals were randomly divided into four treatment groups: GI - control (vehicle); GII - CEE (50microg/kg); GIII - MPA (2mg/kg); GIV - T (100 microg/kg). All drugs were administered using the oral route (gavage) for 60 days. After this period, all animals were sacrificed and the femur was removed for histological study. Our results showed that MPA increased more the number of osteoclast than vehicle, estrogen and testosterone. In GII, the bone wall was thinner than GIII and GIV but thicker than GI. GII presented a large medullar cavity. In GIII and GIV, the medullar cavity was reduced when compared to GI and GII. Our data suggested that progesterone might increased the bone absorption related to increased number of osteoclast. The estrogen and testosterone might have a beneficial effect on the chicken bone with an increase in the bone wall mass.
THE EFFECT OF ESTROGEN OPPOSED TO PROGESTAGENS ON THE BONE MINERAL DENSITY OF POSTMENOPAUSAL WOMEN
F. A. S. Lima1, J. M. Soares Jr1, A. M. Massad Costa1, M. J. Simões2, E. Hinoue1, M. A. Haidar1*, M. G. Nunes1, E. L. A. Motta1, G. Rodrigues de Lima1, E. C. Baracat1
1Gynecology Department, Federal University of São Paulo/Escola Paulista de Medicina, Brazil
2Histology Department, Federal University of São Paulo/Escola Paulista de Medicina, Brazil
The aim of this study was to evaluate the bone mineral density (BMD) before and after hormone replacement therapy on the menopausal women during one year. Also, we analysed the demographic and clinic features of this patients. Hundred women were evaluated using a densitometer before (baseline) and after (treatment) conjugated equine estrogens (CEE) and medroxiprogesterone acetate (MPA). The patients monthly received oral unopposed CEE for 25 days then opposed CEE with MPA for 5 days during one year. Our results showed that vertebral column showed an improvement of 3.5% compared to baseline and the femural BMD increased 2.1% compared to baseline. The treatment was superior in patients with body mass index < 25. The vertebral column BMD showed the best improvement in patients aged over 50 years or with 10 years of menopause. No statistical difference was identified in the femural BMD related to age or time of menopause. Our data suggested that estrogen opposed with MPA is not only beneficial after menopause, but it may have a benefit for women aged over 50 years and with 10 years of menopause, reducing the risk for osteoporosis.
HISTOMORPHOMETRIC STUDY OF TRABECULAR BONE OF OVARIECTOMIZED RATS AFTER ESTROGEN, MEDROXIPROGESTERONE AND TAMOXIFEN
F. A. S. Lima1, J. M. Soares Jr1, A. M. Massad Costa1, M. J. Simões2, E. Hinoue1, M. A. Haidar1*, M. G. Nunes1, E. L. A. Motta1, G. Rodrigues de Lima1, E. C. Baracat1
1Gynecology Department, Federal University of São Paulo/Escola Paulista de Medicina, Brazil
2Histology Department, Federal University of São Paulo/Escola Paulista de Medicina, Brazil
The aim of this study was to evaluate the trabecular bone of ovariectomized rat after conjugated equine estrogen (CEE), medroxiprogesterone acetate (MPA) and tamoxifen (TAM). Forty rats were divided into four treatment groups: GI - vehicle; GII - CEE (50 microg/kg); GIII - TAM (250 microg/kg); GIV - CEE (50microg/kg) plus MPA (2mg/kg). All drugs were administered using the oral route (gavage) for 60 days and the tibial bone was removed for morphohistometric study. Our results showed that GII, GIII and GIV presented a decrease in the bone remodeling process due to a reduction in osteoblastic and osteoclastic surface, eroded bone and medullar cavity compared to GI. We conclued that MPA did not change the estrogen effect and TAM was similar to estrogen on the tibial bone.