Effect of smoking and smoking cessation on bone mass, bone remodeling, vitamin D, PTH and sex hormones.

OBJECTIVE: To assess the effect of smoking and smoking cessation on bone density, bone remodeling markers, sex hormones, and vitamin D-PTH axis in healthy young subjects. MATERIALS AND METHODS: We studied 74 healthy people (31 men, 43 women; mean age 32.2 (7) years) divided into 52 never smokers and 22 smokers, 15 of which stopped smoking for one month. RESULTS: Male smokers compared with never smokers showed lower BMD (0.971 (0.11) g/cm(2) vs. 1.069 (0.09) g/cm(2), P=0.042); higher plasma estrone levels (32.37 (10.13) pg/mL vs. 20.91 (5.46) pg/mL, P=0.001); and lower serum iPTH levels (16.2 (3.5) pg/mL vs. 28.8 (2.0) pg/mL, P=0.008). In women, BMD values were similar in smokers than in never smokers, but 25-hydroxyvitamin D levels were lower in smokers (31.9 (15.1) ng/mL vs. 16.8 (9.9) ng/mL, P=0.002). After adjusting by age and coffee consumption, female smokers had higher urinary-NTX levels than never smokers. After smoking cessation, statistically significant decreases of 25-hydroxyvitamin D and SHBG plasma levels were observed in men and women, respectively. CONCLUSIONS: Tobacco increases bone resorption and affects bone mass by some alterations in sex hormone metabolism, but also importantly by alterations on the vitamin D-PTH axis.

Osteoblasts in HIV-infected patients: HIV-1 infection and cell function.

BACKGROUND: HIV-infected patients have been shown to have a severe alteration in osteoblast function that appears to be related to the infection. OBJECTIVE: To determine whether normal human osteoblasts express CD4, whether osteoblasts from patients with HIV infection are infected by HIV-1 and whether osteoblast dysfunction observed in vivo also occurs in vitro. METHODS: Osteoblast cultures from bone marrow biopsies of HIV-infected patients (n = 14) and control patients (n = 10) were used in a cross-sectional study and a case-control prospective study. Expression of CD4 was analysed using flow cytometry and reverse transcriptase polymerase chain reaction; the presence of HIV-1 particles was determined by measuring p24 antigen in the supernatants of osteoblast cultures and viral DNA or RNA in the osteoblasts using the polymerase chain reaction. Osteoblast function was assessed by measuring cell proliferation, type I collagen and osteocalcin synthesis. RESULTS: In human osteoblasts, CD4 expression could not be determined using flow cytometry, although low levels of mRNA coding for CD4 were detected. HIV infection was not observed in osteoblast cultures from HIV-infected patients nor was there any alteration in replication and synthesis of type I collagen, although osteocalcin synthesis was increased. CONCLUSIONS: It is unlikely that HIV-1 infects human osteoblasts in vivo; therefore, the hypothesis that these cells could act as local HIV-1 reservoirs should be reconsidered.

DNA image cytometry. An alternative method in osteoblast proliferation assays.

OBJECTIVE: To assess whether DNA image cytometry can be used as an alternative method to tritiated thymidine uptake quantification in osteoblast proliferation assays. STUD DESIGN: Proliferation of normal human osteoblasts incubated with normal human serum at 0%, 2.5%, 5%, 10%, 20% and 40% was quantified by tritiated thymidine uptake quantification and DNA image cytometry. RESULTS: Tritiated thymidine uptake quantification showed that normal human serum stimulated the proliferation of normal human osteoblasts and that the degree of stimulation was directly related to the concentration of serum in the culture medium. Similar results were obtained when the DNA image cytometry assay was used. A highly significant linear relationship between the ranks of both methods was found (Spearman's r = 1.00, P = .0253). CONCLUSION: DNA image cytometry may be a valuable alternative when the use of radioactive material is not desired and/or subsequent morphologic or immunocytochemical characterization of cells under study is required.

In vitro synthesis of type I collagen: quantification of carboxyterminal propeptide of procollagen type I versus tritiated proline incorporation.

Radioimmunoassay of the carboxyterminal propeptide of human type I procollagen has been recently introduced to measure in vitro synthesis of type I collagen by osteoblasts and fibroblasts. However, it has not been assessed whether the equivalent results are obtained with this new assay and with tritiated proline incorporation to collagen protein. To this purpose, both methods were used to quantify synthesis of type I collagen in normal human osteoblast cultures to which fetal calf serum and human serum were added in order to stimulate protein synthesis. A positive correlation in the results obtained by both methods was obtained (r = 0.95, P = 0.0001). Given the technical advantages of detection of levels to carboxyterminal propeptide of type I procollagen, we consider that this is the technique of choice for the quantification of in vitro synthesis of type I collagen by normal human osteoblasts.

99mTc-labelled red blood cell scintigraphy for localization of gastrointestinal bleeding in chronic renal failure.

Angiodysplasia (AD) may be the source of bleeding in patients with gastrointestinal hemorrhage, with special occurrence in the elderly population and in patients with chronic renal failure (CRF). Although several techniques have been tested for its diagnosis, the gold standard is not well defined yet. We analyze the usefulness of 99mTc-labelled red blood cell (99mTc RBC) scintigraphy in the localization of bleeding from AD lesions in a cohort of 21 patients. Other investigative methods include fibrocolonoscopy examination, angiography, or diagnostic laparotomy. Group A (AD and CRF): 11 patients. Group B (AD without CRF): 10 patients. 99mTc RBC scintigraphy showed 88.9% sensitivity and specificity in group A, while in group B it had 100% sensitivity and specificity. Arteriography showed 100% sensitivity and specificity. On the contrary, fibrocolonoscopy had a very low sensitivity (30%). Our results suggest that 99mTc RBC scintigraphy may be the preferred diagnostic tool for AD, especially in patients with CRF, in whom arteriography may accelerate the decline of renal function.

[Screening for bone disease risk with clinical factors in women after physiologic menopause]. / Cribado de riesgo óseo mediante factores clínicos en mujeres tras menopausia fisiológica.

BACKGROUND: Densitometric screening for osteoporosis in postmenopausal women has not been demonstrated cost-effective. We have tried to identify clinical factors for screening previous to densitometry avoiding unnecessary explorations. SETTING: outpatient clinics of a menopausal unit in a 450-bed general hospital. Cross-sectional study, in two steps, of two groups of 140 and 284 women attending for physiological menopause. A clinical questionnaire, physical data and lumbar densitometry (Hologic QDR 1000) were obtained classifying the cases as "normal" or "low bone mass" (osteopenia or osteoporosis) according with the WHO criteria. In the first group a logistic regression analysis was done to identify predictive factors for abnormal densitometry, then validated in the second group. Sensitivity, specificity, predictive values (PV) and classification ability of clinical factors were analyzed. RESULTS: Four factors were independent predictors of abnormal densitometry: age > 51 (odds ratio [OR] = 6.64; 95% CI, 2.36-18.7); body weight < 70 kg (OR = 4.32; 95% CI, 1.71-10.09); years of fertility < 32 (OR = 3.77; 95% CI, 1.36-10.04), and number of live births > 2 (OR = 3.47; 95% CI, 1.27-9.53). Presence of one factor offers: sensitivity 91.9%; specificity 15%; positive PV 66.6%, and negative PV 50%, whereas the presence of two factors offers: sensitivity 62.7%; specificity 70%; positive PV 79.9%, and negative PV 50.3%. Clinical screening allows, when two factors are present, to avoid a 35.5% of densitometries and the false-negative cases represent 18%. CONCLUSIONS: Detection of bone-risk clinical factors (abnormal densitometry) yields a screening, previous to densitometry, that avoids at least one third of explorations in women with physiological menopause, improving the efficiency of the test.

Effect of alendronate on cultured normal human osteoblasts.

Alendronate is an aminobisphosphonate with a potent anti-reabsorptive action that does not appear to interfere with bone mineralization, and is even able to increase bone mineral density in osteoporotic postmenopausal women through a still not fully understood mechanism. This study was conducted to assess the direct effect of alendronate on diverse aspects of normal human osteoblast physiology. For that purpose, the in vitro effect of a wide range of concentrations [from 10(-1) to 10(-12) mol/L] of alendronate on cell viability, proliferation, collagen synthesis, and the mineral-depositing capacity of normal human osteoblasts was tested. Alendronate effects were examined at 48 and 96 h of culture in the presence or absence of fetal calf serum. In vitro alendronate affected osteoblast viability at concentrations equal to or higher than 10(-4) mol/L. At concentrations equal to or higher than 10(-3) mol/L, no viable cells were observed in cultures. In vitro alendronate at concentrations between 10(-5) and 10(-12) mol/L did not have any effect on the proliferative capacity of normal human osteoblasts determined by two different techniques: (1) tritiated thymidine incorporation to DNA and (2) cell counting. Collagen synthesis by normal human osteoblasts showed a tendency to decrease following incubation with alendronate supplemented with fetal calf serum. This decrease was only statistically significant after 96 h of culture; however, a dose-response effect could not be documented. Finally, no effect of alendronate was observed on calcium deposition in vitro by normal human osteoblasts at concentrations equal to or lower than 10(-5) mol/L. In conclusion, the present study shows that alendronate in vitro does not affect viability, proliferation, and mineral deposit capacity of normal human osteoblasts at the concentration at which it inhibits by 50% the resorptive capacity of osteoclasts that for this drug has been reported as 2 x 10(-9) mol/L.

Vitamin D treatment in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are a group of clonal disturbances with defective cellular differentiation. Vitamin D3 (VD) analogues can act on the differentiation and maturity of different cell lines. We studied the effects of VD on a series of patients with MDS in an open-design trial. Nineteen patients, 12 men and seven women, with MDS were included. Patients were 74.8 +/- 5.6 years (mean +/- SD), seven had refractory anaemia with ringed sideroblasts, five had refractory anaemia, one had refractory anaemia with excess of blasts and six had chronic myelomonocytic leukaemia. All the patients were in a low to intermediate risk group. Mean follow-up period was 26.21 months, range 9-75. Responders were defined as follows: granulocyte or platelet count increase by 50%, or haemoglobin increase of 1.5 g/dl or transfusion needs decrease by 50%. The first five patients received 266 microg of calcifediol three times a week and the other 14 received calcitriol (0.25-0.75 microg/d). Response was observed in 11 patients. In the calcifediol-treated group, one case responded, three were nonresponders, and one showed progression. In the calcitriol group, 10 were responders (two with major response), and four were non-responders. No correlation was observed between baseline levels of vitamin D metabolites and the presence of response. No hypercalcaemia was observed. Treatment with vitamin D3 metabolites could induce a long-standing response of the haematological disturbance in some low-intermediate risk MDS patients without inducing hypercalcaemia.

Immunoreactive osteocalcin forms in conditioned media from human osteoblast culture and in sera from healthy adult control subjects and patients with bone pathologies.

BACKGROUND: The aim of this work was to study the immunoreactive forms of bone Gla protein (BGP) present in conditioned media of human osteoblast cultures (BGP released from osteoblast) and in the sera of healthy adult control subjects and patients with bone pathologies (chronic renal failure on haemodialysis, Paget's disease of bone and post-menopausal osteoporosis). METHODS: The technical procedure used was a combination of high-performance liquid chromatography (HPLC) and different BGP assays with several specificities to analyse BGP levels in the different HPLC fractions. Aliquots of conditioned media or sera were purified through a Sephadex G-50m column and by HPLC (C4 reverse-phase column) in a 25-40% acetonitrile gradient. Two-minute fractions were collected and divided into three aliquots in order to determine osteocalcin content using three different assays: (a) ELSA-OST-NAT IRMA, which only detects intact osteocalcin; (b) ELSA-OSTEO IRMA, which detects intact osteocalcin and N-terminal fragments; and (c) OSCA Test RIA, which detects intact osteocalcin, C-terminal and other fragments. RESULTS: We found different immunoreactive forms of osteocalcin in the culture medium of human osteoblasts and in sera from control subjects and patients for the bone pathologies studied. We did not find great qualitative differences between the immunoreactive osteocalcin profile found in the culture medium from human osteoblasts and the sera from healthy control subjects. However, the different bone pathologies show different characteristic patterns of immunoreactive forms of osteocalcin. CONCLUSIONS: An interesting finding has been the detection, both in sera and in osteoblast culture media, of several immunoreactive forms of intact osteocalcin that eluted from HPLC at different acetonitrile percentages, and therefore correspond to different molecular forms.

Acute effects of ethanol on mineral metabolism and trabecular bone in Sprague-Dawley rats.

In order to assess the effects of acute ethanol intoxication on bone, 45 female Sprague-Dawley rats were studied. Five rats were sacrificed at baseline. The remainder received either ethanol (2 g/kg of body weight) intraperitoneally or isotonic saline. Rats were sacrificed in groups of 10 (5 intoxicated and 5 placebo) at 1, 4, 8, and 24 hours after injection. At the time of sacrifice, a blood sample was obtained and the 4th vertebra was excised for histomorphometric analysis of undecalcified bone. Effect of ethanol was assessed by an analysis of variance test using a Scheffé procedure. In ethanol-treated rats we observed (mean +/- SD, ethanol versus controls, maximum difference point, P value) a significant decrease in osteiod surface with osteoblasts (42.86 +/- 15.61% versus 64.57 +/- 6.24%, P < 0.05); osteoclast number (0.05 +/- 0.02 n/mm2 versus 0.17 +/- 0.09 n/nm2, P < 0.05), and osteocalcin (36.9 +/- 2.21 ng/ml versus 45.8 +/- 5.1 ng/ml, P < 0.05). Osteoclast surface was initially reduced (0.129 +/- 0.09% versus 0.425 +/- 0. 26%, P < 0.01) but showed a subsequent increase (0.765 +/- 0.24% versus 0.226 +/- 0.17%, P < 0.01) attributable to alcohol. There was also a significant decrease in serum Ca (8.51 +/- 0.23 mg/dl versus 9.10 +/- 0.29 mg/dl, P < 0.01) and parathyroid hormone values (23.51 +/- 5.72 pg/ml versus 76.39 +/- 11.66 pg/ml, P < 0.001). We conclude that acute alcohol intoxication in rats induces early striking changes in bone histology and analytical parameters, not completely reversed after 24 hours. These data are consistent with a toxic effect induced by alcohol on bone.

Osteoblastic proliferation in bone biopsies from patients with end-stage chronic renal failure.

Osteoblasts have traditionally been considered to be terminally differentiated cells and therefore unable to divide. Data in recent years, however, indicate that cellular differentiation does not usually preclude preservation of proliferative ability and that most differentiated cells are able to divide under adequate stimuli. The aim of this study was to assess whether cubic osteoblasts undergo proliferation during the formation phase of the remodeling cycle under a stimulus that increased bone turnover. For that purpose, the osteoblastic proliferation index (OPI) was analyzed by DNA image cytometry in transiliac bone biopsies from 33 patients with chronic renal failure (23 men, 10 women; mean age 50.4 +/- 15.1 years) who have been classified into low (n = 13), normal (n = 15), and high (n = 15) bone turnover according to activation frequency (Ac.f). OPI was significantly higher (p < 0.002) in the high bone turnover group (13.90 +/- 4.72%) compared with the low (2.38 +/- 4.13%) and normal turnover groups (2.84 +/- 4.04%). There was a positive correlation between OPI and the following histomorphometric parameters: bone formation rate, surface referent (r = 0.76, p = 0.00001), activation frequency (r = 0.73, p = 0.00001), mineral apposition rate (r = 0.73, p = 0.00001), bone formation rate, volume referent (r = 0.71, p = 0.00001), and mineralizing surface (r = 0.62, p = 0.0001). This study shows that a rise in bone turnover is associated with a marked increase of bone-forming cell proliferation in patients with end-stage chronic renal failure. From this finding, it may be concluded that cubic osteoblasts do not behave as "terminally differentiated" cells in vivo, because a high proportion of them are still able to divide.

Bone remodeling alterations in myelodysplastic syndrome.

There is a close relationship between hematopoietic bone marrow and bone cells. Thus, the profound derangement of hematopoiesis in myelodysplastic syndromes (MDS) might be expected to affect bone cell function. We studied the dynamic histomorphometric changes in bone in 22 MDS patients to examine this relationship and analyze the influence of hematological disease on bone remodeling. Bone-regulating hormones and histomorphometry of undecalcified transiliac bone biopsies, after double tetracycline labeling, were studied. Serum calcium, phosphorus, creatinine, alkaline phophatase, osteocalcin, iPTH, 25(OH)D3, 1,25(OH)2D3, hydroxyprolinuria, and calcium/creatinine ratio in urine were normal compared with controls. Histomorphometry showed a significant decrease in osteoblast surface (Ob.S/BS) (0.30 +/- 0.40 vs. 0.8 +/- 1.1, p = 0.031), wall thickness (W.Th), (22.03 +/- 5.5 vs. 31.8 +/- 5.8, p < 0.005), osteoclast number (N.Oc/T.Ar) (0.004 +/- 0.01 vs. 0.017 +/- 0.01, p = 0.03), mineral apposition rate (MAR) (0.16 +/- 0.15 vs. 0.53 +/- 0.19, p < 0.005), bone formation rate, surface referent (BFR/BS) (0.004 +/- 0.10 vs. 0.016 +/- 0.016, p = 0.009), and activation frequency (Ac.f) (0.06 +/- 0.07 vs. 0.21 +/- 0.23, p = 0.008). An increase in mineralization lag time (MLT) (119.2 +/- 78.6 vs. 29.6 +/- 77, p < 0.005), (mean +/- SD, unpaired Student t-test) was observed. Bone volume (BV/ TV), eroded surfaces (ES/BS), and osteoid thickness (O.Th) remained unchanged. This picture of adynamic bone with decreased mineral apposition rate and markedly decreased osteoclast number is a characteristic finding in MDS patients. Thus, bone histomorphometric finding in MDS patients show the relationships and interactions between hematopoietic and bone cells.

Bone growth and modeling changes induced by periosteal stripping in the rat.

In this study, the changes in longitudinal bone growth and metaphyseal modeling induced by middiaphyseal periosteal stripping in the rat femur were analyzed by means of histomorphometrical techniques. One hundred forty-four male 30-day-old Sprague-Dawley albino rats distributed in 4 groups of 36 were studied: a control group, a sham group, a group with middiaphyseal right femoral periosteal stripping, and a group with a polyethylene ring wrapped around the stripped zone. The animals were euthanized at 1, 2, or 4 weeks from the start of the experiment, after double tetracycline labeling. A statistically significant, albeit small, longitudinal overgrowth of stripped femurs was observed after a latency period of 2 to 4 weeks. The metaphyseal diameters were greater in stripped femurs than nonstripped femurs. This finding was associated with a lower osteoclastic index in the external metaphyseal surface and with a lower bone formation rate in the internal surface of the metaphyseal cortex. These latter findings have not been reported previously in the literature and may support the role of the periosteum in controlling metaphyseal modeling.

Bone remodelling in human immunodeficiency virus-1-infected patients. A histomorphometric study.

The aim of this study was to identify and describe possible alterations of bone histomorphometry in patients with human immunodeficiency virus (HIV-1) infection and to assess the relation between these alterations and disease severity. Forty-four HIV-1-infected patients seen successively at our hospital were evaluated for the study. In an attempt to avoid confounding factors as far as possible, we excluded patients who fulfilled any of the following criteria: age less than 18 or greater than 40 years; recent history of extended bed rest; previous diagnosis of metabolic bone disease, renal insufficiency, or hepatic failure; clinical or echographic signs of liver cirrhosis; diabetes mellitus or previous diagnosis of other endocrine diseases; drug therapy that could act on bone metabolism; and/or moderate to severe nutritional alteration. Twenty-two patients (13 men, 9 women; age: 27.9 +/- 4.1 years, mean +/- standard deviation) were included in the study. Plasma and urine biochemistry and calcium-regulating hormones were determined. Bone mineral content was measured on vertebrae L2 to L4 and on the neck and intertrochanteric areas of the femur by dual-photon absorptiometry. A transiliac bone biopsy was performed after double-tetracycline labelling, with histomorphometric study of undecalcified bone. Serum osteocalcin was found to be lower in patients who, according to the Centers for Disease Control (CDC) classification, had greater disease severity, and showed a positive correlation with the number of CD4+ T lymphocytes. No alterations in bone densitometry were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of cyclosporine A on normal human osteoblasts in vitro.

UNLABELLED: Cyclosporin-A (CsA) inhibits in vitro proliferation of non-human tumour-cloned osteoblasts. Our aims were to study the direct effect of CsA on proliferation of normal human osteoblast (NHOb) cultures and to ascertain whether CsA-treated patients' sera (CsATPS) may exert effects on the osteoblast which differ from the direct effects of CsA. We studied tritiated thymidine ([3H]thymidine) incorporation in NHOb cultures incubated with (a) increasing CsA concentrations (1.2 to 4800 ng/ml), (b) the same concentrations as in the previous experiment but with the addition of 20% fetal calf serum (FCS) or 20% normal human serum (NHS), (c) 40% NHS or 40% CsATPS. Results at 96 h in (a) CsA inhibited uptake from 300 ng/ml, in (b) CsA inhibited [3H]thymidine uptake from 2400 ng/ml for cultures with FCS and 4800 ng/ml for cultures with NHS, in (c) CsATPS produced [3H]thymidine uptake inhibition compared with NHS. CONCLUSION: CsA alone inhibited [3H]thymidine incorporation in NHOb from concentrations similar to therapeutic concentrations. With FCS or NHS, inhibition was produced at higher concentrations. CsATPS inhibited at CsA concentrations lower than those of the two previous experiments.

Alcohol-induced bone disease in the absence of severe chronic liver damage.

To define and identify metabolic bone disease and mineral alterations induced by chronic heavy alcoholism in patients without severe liver damage, we studied a prospective series of unselected patients admitted to a 300-bed general hospital in Barcelona (Spain). A total of 26 chronic heavy drinkers of more than 150 g/day for at least 3 years were included. A general analytic and hormonal study, including liver biopsy in cases with any abnormality in liver function tests, and plasma and urine biochemistry with calcium regulating hormones and osteocalcin levels were determined. A transiliac bone biopsy after double-tetracycline labeling, with histomorphometric study of undecalcified bone, was performed. Statistical analysis was adjusted by age and sex by means of logistic regression. A total of 26 (20 men and 6 women) chronic alcohol abusers were studied. After adjustment for age and sex, alcoholic patients showed slight but significantly increased concentrations of plasma calcium (9.56 +/- 0.56; OR = 17.93; 95% CI 3.17-101.48) and decreased cPTH (0.36 +/- 0.11; OR = 0.097; 95% CI 0.018-0.528) compared with controls. Osteocalcin values were low (1.49 +/- 0.89, normal range 1.8-6.6). There was a significant decrease in bone volume, BV/TV (12.56 +/- 5.29; OR = 0.06; 95% CI 0.01-0.34), with increased resorption surfaces, ES/BS (4.28 +/- 2.43; OR = 9.86; 95% CI 2.16-45.07), and increased osteoclast number, N.Oc/TA (0.21 +/- 0.37; OR = 6.41; 95% CI 1.27-32.25).(ABSTRACT TRUNCATED AT 250 WORDS)

Long-standing remission after 25-OH D3 treatment in a case of chronic myelomonocytic leukaemia.

This report describes a case of chronic myelomonocytic leukaemia (CMML) in whom a complete remission was achieved and sustained 15 months after treatment with 25-OH vitamin D3. No side-effects were observed. Although vitamin D1 has been used in the treatment of myelodysplastic syndromes, to our knowledge this is the first case of long-standing remission in a patient with CMML. This 'differentiation therapy' might be considered as a possible alternative in the management of this disease.