Human bone marrow stromal cells: the impact of anticoagulants on stem cell properties.

Background: Bone marrow stromal cells (BMSCs) are the source of multipotent stem cells, which are important for regenerative medicine and diagnostic purposes. The isolation of human BMSCs from the bone marrow (BM) cavity using BM aspiration applies the method with collection into tubes containing anticoagulants. Interactions with anticoagulants may affect the characteristics and composition of isolated BMSCs in the culture. Thus, we investigated how anticoagulants in isolation procedures and cultivation affect BMSC molecular characteristics. Methods: BM donors (age: 48-85 years) were recruited from the hematology clinic. BM aspirates were obtained from the iliac crest and divided into tubes coated with ethylenediaminetetraacetic acid (EDTA) or heparin anticoagulants. Isolated BMSCs were analyzed by flow cytometry and RNA-seq analysis. Further cellular and molecular characterizations of BMSCs including CFU, proliferation and differentiation assays, cytometry, bioenergetic assays, metabolomics, immunostaining, and RT-qPCR were performed. Results: The paired samples of isolated BMSCs obtained from the same patient showed increased cellular yield in heparin vs. EDTA samples, accompanied by the increased number of CFU colonies. However, no significant changes in molecular characteristics were found between heparin- and EDTA-isolated BMSCs. On the other hand, RNA-seq analysis revealed an increased expression of genes involved in nucleotide metabolism and cellular metabolism in cultivated vs. non-cultivated BMSCs regardless of the anticoagulant, while genes involved in inflammation and chromatin remodeling were decreased in cultivated vs. non-cultivated BMSCs. Conclusion: The type of anticoagulant in BMSC isolation did not have a significant impact on molecular characteristics and cellular composition, while in vitro cultivation caused the major change in the transcriptomics of BMSCs, which is important for future protocols using BMSCs in regenerative medicine and clinics.

Osteoporosis in adults in clinical practice (1): diagnosis and differential diagnosis.

Osteoporosis is a systemic metabolic disease of the skeleton characterized by low bone strength that results in an increased risk of fracture. Fractures are associated with serious clinical consequences, including pain, disability, loss of independence, and death, as well as high healthcare costs. Early identification and intervention with patients at high risk for fracture is needed to reduce the burden of osteoporotic fractures. The identification of a patient at high risk of fracture should be followed by evaluation for factors contributing to low bone mineral density (BMD) and/or low bone quality, falls, and fractures. Components of the osteological evaluation include an assessment of BMD by dual-energy X-ray absorptiometry, osteoporosis-directed medical history and physical exam, laboratory studies, and possibly skeletal imaging. Disorders other than osteoporosis, requiring other types of treatment, may be found. This overview summarizes the basic procedures for the diagnosis and differential diagnosis of osteoporosis, which are necessary before starting treatment.

Tumor induced osteomalacia - A long way toward correct diagnosis and management.

Tumor-induced osteomalacia (TIO) is an uncommon type of osteomalacia associated with phosphaturic mesenchymal tumors (PMTs). Due to nonspecific symptoms, the diagnosis and appropriate management of the disease is often delayed for many years. Involvement of spine with TIO associated tumors is exceedingly rare. We present a 53-year-old woman with a 10-year history of bone pain, muscle weakness and multiple bone fractures that markedly impaired her quality of life. Biochemical evaluation revealed hypophosphatemia due to renal phosphate wasting and elevated plasma fibroblast growth factor 23 (FGF-23) concentration indicating PMT. It was found using 68Ga DOTA TOC PET/CT scan in the vertebral body L2. The patient underwent surgical resection of the tumor. Postoperatively, there was a significant decrease in phosphaturia, normalization of serum phosphate, 1.25 dihydroxyvitamin D and plasma FGF23 concentration. Thereafter the patient's condition markedly improved concerning her motility and basic daily activities. This case report demonstrates the first known case of TIO in the Slovakia and points to a long way from onset of symptoms toward correct diagnosis and successful surgical management.

Osteoporosis in men: underappreciated and undertreated.

Osteoporotic fractures of the vertebrae and the proximal end of the femur dramatically impair quality of life and increase morbidity and mortality. Although up to 40% of all osteoporotic fractures occur in men, physicians tend to underestimate the osteoporosis in men, and it remains underdiagnosed and undertreated. Though, there is no evidence that current approved osteoporosis medications work any less well in men than in women, insufficient awareness of the risk of fractures, fear of side effects of drugs and other barriers have made management challenging in men at risk for fracture. Our review provides updates on pathophysiology and current options for diagnosis and treatment of male osteoporosis.

Diabetes, Cardiovascular Disorders and 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Body Burden in Czech Patients 50 Years After the Intoxication.

The correlation between 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) intoxication and the parameters of metabolic impairment was examined in the last eight male survivors of 80 workers exposed to TCDD during the production of herbicides in a chemical factory in 1965-1967. Their median TCDD blood level was 112 (46-390) pg/g lipids, and the median TCDD body deposit was 3.9 (0.8-11.7) µg. This puts these patients into the most severely intoxicated group of subjects, according to back-calculated levels of TCDD. The median TCDD blood level in eight controls was 12 pg/g (<0.10 to 22.2 pg/g). Markers of metabolic impairment - diabetes, dyslipidaemia, arterial hypertension, carotid artery plaque, skin microvascular reactivity, eye fundus hypertensive angiopathy and history of coronary heart disease - were assessed and compared to a general male population of comparable age. Measured parameters compared with a population of comparable age were as follows: prevalence of diabetes (62.5% versus 17.6%), arterial hypertension (87.5% versus 71.8%), dyslipidaemia (87.5% versus 88.8%), history of coronary heart disease (62.5% versus 26.0%) and eye fundus hypertension angiopathy (50% versus 14%). All eight patients (100% versus 43%) developed plaques in carotid arteries, six had stenosis >50% and two had a carotid intervention (stenting or endarterectomy). Total cholesterol levels decreased compared to the earlier study this patient group in 2008, most likely due to a more intensive use of lipid-lowering drugs. Several metabolic parameters were higher (diabetes as much as 3.5-fold) in the group of severely TCDD-intoxicated subjects than in a general population of comparable age. This suggests that TCDD plays a role in the development of metabolic impairment and vascular changes.

Effects of teriparatide and risedronate on new fractures in post-menopausal women with severe osteoporosis (VERO): a multicentre, double-blind, double-dummy, randomised controlled trial.

BACKGROUND: No clinical trials have compared osteoporosis drugs with incident fractures as the primary outcome. We compared the anti-fracture efficacy of teriparatide with risedronate in patients with severe osteoporosis. METHODS: In this double-blind, double-dummy trial, we enrolled post-menopausal women with at least two moderate or one severe vertebral fracture and a bone mineral density T score of less than or equal to -1·50. Participants were randomly assigned to receive 20 µg of teriparatide once daily plus oral weekly placebo or 35 mg of oral risedronate once weekly plus daily injections of placebo for 24 months. The primary outcome was new radiographic vertebral fractures. Secondary, gated outcomes included new and worsened radiographic vertebral fractures, clinical fractures (a composite of non-vertebral and symptomatic vertebral), and non-vertebral fractures. This study is registered with ClinicalTrials.gov (NCT01709110) and EudraCT (2012-000123-41). FINDINGS: We enrolled 680 patients in each group. At 24 months, new vertebral fractures occurred in 28 (5·4%) of 680 patients in the teriparatide group and 64 (12·0%) of 680 patients in the risedronate group (risk ratio 0·44, 95% CI 0·29-0·68; p<0·0001). Clinical fractures occurred in 30 (4·8%) of 680 patients in the teriparatide group compared with 61 (9·8%) of 680 in the risedronate group (hazard ratio 0·48, 95% CI 0·32-0·74; p=0·0009). Non-vertebral fragility fractures occurred in 25 (4·0%) patients in the teriparatide group and 38 (6·1%) in the risedronate group (hazard ratio 0·66; 95% CI 0·39-1·10; p=0·10). INTERPRETATION: Among post-menopausal women with severe osteoporosis, the risk of new vertebral and clinical fractures is significantly lower in patients receiving teriparatide than in those receiving risedronate. FUNDING: Lilly.

Prevalence and Risk Factors of Osteoporosis in Postmenopausal Women with Type 2 Diabetes Mellitus.

OBJECTIVE: Patients with type 2 diabetes (T2DM) are at increased risk of fractures. The aim of this study is to analyze the prevalence and risk factors of osteoporosis and osteoporosis related fractures in postmenopausal women with T2DM. METHODS: A total of 112 postmenopausal women with T2DM and 171 control nondiabetic women received a standardized questionnaire on osteoporosis risk factors, and were evaluated for bone mineral density (BMD, by using a dual energy X-ray absorptiometry), biochemical markers of bone and glucose metabolism, soluble receptor for advanced glycation end products (sRAGE) and its gene polymorphisms (rs1800625 or rs2070600). RESULTS: In T2DM patients the prevalence of osteoporosis was 25% and low trauma vertebral (Vfx) and non-vertebral fractures were found in 8% and 19% women, respectively. When compared between subjects with and without fractures, there were no significant differences in BMD at any site between the groups, except for distal radius, which was significantly lower in T2DM women with Vfx (p<0.05 vs.non-fractured without osteoporosis). We found no associations between bone and glucose metabolism variables, sRAGE and BMD. No significant differences were observed in sRAGE levels according to their rs1800625, rs 2070600 genotype or fracture prevalence. Serum osteocalcin was significantly lower in T2DM women (p<0.01 vs. controls) and in T2DM women with Vfx (p<0.05) vs. non-fractured without osteoporosis. T2DM women with low daily walking activity (< 2 h daily) had significantly higher serum sclerostin levels (p<0.05 vs. those who were walking > 2 h daily). CONCLUSION: Diabetes-specific parameters as well as RAGE polymorphisms did not associate with BMD or fractures in T2DM postmenopausal women. Lower levels of osteocalcin, namely in those with Vfx and higher sclerostin levels in those with low daily walking activity suggest lower bone remodeling and/or decreased bone quality in T2DM.

High Prevalence of Hypovitaminosis D in Postmenopausal Women with Type 2 Diabetes Mellitus.

The link between vitamin D and type 2 diabetes mellitus (T2DM) is intensively studied. This study aims to define the serum concentration of 25-hydroxyvitamin D (25-OH D) and to investigate the relationship between 25-OH D status, glycated hemoglobin (HbA1c) and body composition in postmenopausal women with T2DM and in non-diabetic controls. In this cross-sectional study, 75 women with T2DM and 32 control subjects were selected. Serum 25-OH D, intact parathyroid hormone (PTH), calcium, fasting glucose and HbA1c, were measured. The mean 25-OH D level was 21.4±11.4 ng/ml (range 4.1-50.7 ng/ml) in diabetic women and 30.3±9.4 ng/ml (range 10.8-54.2 ng/ml) in control group (p<0.001). The prevalence of hypovitaminosis D (<30 ng/ml) was higher in vitamin D3 non-supplemented T2DM women (89% vs. 63% controls); the difference diminished in vitamin D3 (500-1000 IU per day) supplemented subgroups (45% diabetics vs. 42% controls). In T2DM women, 25-OH D levels were not associated to HbA1c, duration of diabetes, fasting glucose and PTH levels, however, 25-OH D levels negatively associated with body mass index (p=0.011), total body fat mass (p=0.005) and total body lean mass (p=0.004). The prevalence of hypovitaminosis D is higher in non-supplemented postmenopausal women with T2DM than in non-diabetic controls (89% vs. 63%). Obesity is a risk factor for vitamin D insufficiency in T2DM postmenopausal women. Further studies evaluating relationships between fat, muscle, bone and vitamin D metabolism in T2DM patients are warranted.

Increased oxidative/nitrosative stress markers measured non- invasively in patients with high 2,3,7,8-tetrachloro-dibenzo-p-dioxin plasma level.

OBJECTIVES: 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) is a highly toxic persistent environmental contaminant, classified as a human carcinogen affecting any target organ. The mechanism of carcinogenesis by TCDD is unclear as TCDD shows a lack of direct genotoxicity. Experimental studies also support the role of oxidative stress in TCDD neurotoxicity and vascular dysfunction. The aim was to investigate markers of oxidative/nitrosative stress and inflammation using non-invasive methods in subjects who got ill due to severe occupational exposure to TCDD in the years 1965-1968. METHODS: In 11 TCDD-exposed patients, and 16 controls, the analysis of following oxidative products of lipids, proteins and nucleic acids in plasma, urine and exhaled breath condensate (EBC) was performed: 8-iso-prostaglandin F2α (8-isoprostane), 4-hydroxy-trans-2-nonenale (HNE), malondialdehyde (MDA), o-tyrosine (o-Tyr), 8-hydroxyguanosine (8-OHG), 8-hydroxy-2´-deoxy-guanosine (8-OHdG), 5-hydroxymethyluracil (5-OHMeU). In addition, nitric-oxide-tyrosine (NO-Tyr) and leukotriene (LT) B4, C4, D4, and E4 were detected by liquid chromatography-mass spectrometry/mass spectrometry (LC-ESI-MS/MS). TCDD was measured by HRGC/HRMS, body lipid content by densitometry. Single-photon emission spectrometry (SPECT) of the brain was performed and compared with the findings of the patients in 2008. RESULTS: Mean TCDD plasma level in 2010 was 175 ± 162 pg/g lipids (population level about 2 pg/g), total TCDD content in the body 5.16 ± 4.62 mg. Reduction of cerebral blood flow in SPECT progressed in 8 patients, finding was stable in 2 subjects, and improvement occurred in 1 patient. In the EBC, 10 from 12 markers (all except LT D4 and LT E4), were significantly increased in the patients (p<0.05). In the urine, 7 markers were significantly higher than in the controls (p<0.05): 8-isoprostane, MDA, HNE, LT C4, LT E4, o-Tyr and NO-Tyr. In plasma, only NO-Tyr and 8-OHG were elevated (p<0.05). CONCLUSION: NO-Tyr was increased in all matrices in dioxin-exposed patients. EBC is not limited to lung disorders as the markers of oxidative stress and inflammation were elevated in EBC of patients with normal lung functions. TCDD-induced oxidative stress and inflammation markers can be detected non-invasively in the EBC and urine in the follow-up of the highly-exposed patients. Their prognostic value, however, needs to be elucidated.

Bone health in patients with multiple sclerosis.

Multiple sclerosis (MS) is a gait disorder characterized by acute episodes of neurological defects leading to progressive disability. Patients with MS have multiple risk factors for osteoporotic fractures, such as progressive immobilization, long-term glucocorticoids (GCs) treatment or vitamin D deficiency. The duration of motor disability appears to be a major contributor to the reduction of bone strength. The long term immobilization causes a marked imbalance between bone formation and resorption with depressed bone formation and a marked disruption of mechanosensory network of tightly connected osteocytes due to increase of osteocyte apoptosis. Patients with higher level of disability have also higher risk of falls that combined with a bone loss increases the frequency of bone fractures. There are currently no recommendations how to best prevent and treat osteoporosis in patients with MS. However, devastating effect of immobilization on the skeleton in patients with MS underscores the importance of adequate mechanical stimuli for maintaining the bone structure and its mechanical competence. The physical as well as pharmacological interventions which can counteract the bone remodeling imbalance, particularly osteocyte apoptosis, will be promising for prevention and treatment of osteoporosis in patients with MS.

The effect of timing of teriparatide treatment on the circadian rhythm of bone turnover in postmenopausal osteoporosis.

BACKGROUND: We hypothesized that with the administration of teriparatide (TPTD) treatment at different times, we would be able to modify the physiological circadian rhythm of bone turnover. METHODS: The concentration of serum C-terminal telopeptide of collagen type I (ßCTX), serum N-terminal propeptide of procollagen type I (P1NP), serum ionized calcium (iCa), and plasma PTH were measured every 3 h over a 24 h period in 14 postmenopausal osteoporotic women (aged 72.4±9.3 years) treated with 20 µg TPTD for long term, given at different times of the day. General linear model-repeated measurements (GLM RM) were performed to analyze the circadian rhythms as well as intergroup comparisons. RESULTS: GLM-RM for both related groups showed a significant influence of time of day on all measured variables except P1NP. The analysis for each group separately provided a powerful model for ßCTX (P<0.001, η(2)=0.496), serum iCa (P<0.001, η(2)=0.423), plasma PTH (P<0.001, η(2)=0.283), and serum PINP (P<0.001, η(2)=0.248). While the evening TPTD treatment showed a marked circadian rhythm for serum ßCTX, the morning TPTD treatment rather suggested circasemidian rhythm. The P1NP rhythm followed a much smaller amplitude of the rhythm than ßCTX. Changes in serum iCa were positively related to changes in serum ßCTX (P<0.001) and negatively related to changes in PTH (P<0.001). CONCLUSION: Timing of TPTD administration may significantly change the 24 h variation in bone turnover markers as well as calcium-parathyroid axis in postmenopausal osteoporotic women.

Association of macrophage inhibitory cytokine-1 with nutritional status, body composition and bone mineral density in patients with anorexia nervosa: the influence of partial realimentation.

BACKGROUND: Macrophage inhibitory cytokine-1 (MIC-1) is a key inducer of cancer-related anorexia and weight loss. However, its possible role in the etiopathogenesis of nutritional disorders of other etiology such as anorexia nervosa (AN) is currently unknown. METHODS: We measured fasting serum concentrations of MIC-1 in patients with AN before and after 2-month nutritional treatment and explored its relationship with nutritional status, metabolic and biochemical parameters. Sixteen previously untreated women with AN and twenty-five normal-weight age-matched control women participated in the study. We measured serum concentrations of MIC-1 and leptin by ELISA, free fatty acids by enzymatic colorimetric assay, and biochemical parameters by standard laboratory methods; determined resting energy expenditure by indirect calorimetry; and assessed bone mineral density and body fat content by dual-energy X-ray absorptiometry. ANOVA, unpaired t-test or Mann-Whitney test were used for groups comparison as appropriate. The comparisons of serum MIC-1 levels and other studied parameters in patients with AN before and after partial realimentation were assessed by paired t-test or Wilcoxon Signed Rank Test as appropriate. RESULTS: At baseline, fasting serum MIC-1 concentrations were significantly higher in patients with AN relative to controls. Partial realimentation significantly reduced serum MIC-1 concentrations in patients with AN but it still remained significantly higher compared to control group. In AN group, serum MIC-1 was inversely related to Buzby nutritional risk index, serum insulin-like growth factor-1, serum glucose, serum total protein, serum albumin, and lumbar bone mineral density and it significantly positively correlated with the duration of AN and age. CONCLUSIONS: MIC-1 concentrations in AN patients are significantly higher relative to healthy women. Partial realimentation significantly decreased MIC-1 concentration in AN group. Clinical significance of these findings needs to be further clarified.

Marked reduction of bone turnover by alendronate attenuates the acute response of bone resorption marker to endogenous parathyroid hormone.

The aim of this study was to assess the effects of the antiresorptive treatments of alendronate (ALN), risedronate (RIS) and raloxifene (RLX) on the response of bone to endogenous parathyroid hormone (PTH) induced by acute hypocalcemia. Forty women (age, 55-80 years) with postmenopausal osteoporosis (treated with ALN, RIS and RLX or untreated-control group) were given infusions of sodium ethylenediaminetetraacetic acid (EDTA; 10 mg/kg of body weight). Serum ionized calcium (iCa), plasma intact PTH and marker of bone resorption, serum beta C-terminal telopeptide of type I collagen (beta-CTX; beta CrossLaps) were followed for 180 min. In all women, decrease in serum iCa following the EDTA load resulted in an acute increase in serum PTH. Between 60 and 180 min, plasma PTH in the ALN and RIS treated women remained significantly higher than in the control group. The integrated beta-CTX responses (area under curves, AUCs) to peaks of PTH were significantly lower in the ALN treated women than in those treated with RIS, RLX or control group. There was no significant difference in beta-CTX AUC response to PTH between RIS, RLX and control women. Taken together, these findings suggest that in women with postmenopausal osteoporosis treated with ALN, a substantial reduction of bone turnover blunts the acute bone resorbing effect of endogenous PTH.

Marker of Bone Resorption in Acute Response to Exogenous or Endogenous Parathyroid Hormone.

Parathyroid hormone (PTH) changes morphology of osteoclasts within minutes after its systemic administration. The aim of our study was to test in healthy men whether both exogenous and endogenous PTH could change acutely (minutes to hours) the serum cross-linked C-telopeptide of type I collagen (beta CTX), which is released during osteoclastic resorption of bone. Twelve healthy men (age range 24-34 yr) were each studied during 180 min on a control period, after a single subcutaneous injection of teriparatide, and after 30 min EDTA infusion to stimulate endogenous PTH secretion. The tests were started after overnight fast, 3 h after a standard calcium load. The EDTA infusion induced a significant decrease in serum ionized calcium (by 8.5% at 33 min) and a significant increase in plasma PTH (by 305% at 33 min). Both the EDTA and teriparatide resulted in a significant increase in beta CTX (p < 0.001) with maximum increases of 64% and 80%, respectively. A mild, but significant decrease in beta CTX was observed during the control test period. In conclusion, single-dose teriparatide injection as well as a stimulation of endogenous PTH in healthy men results in an acute increase of the bone resorption marker.

Markers of bone remodeling predict rate of bone loss in multiple sclerosis patients treated with low dose glucocorticoids.

BACKGROUND: The aim of this study was to evaluate the clinical value of markers of bone remodeling in assessment of rate of bone loss in patients with multiple sclerosis (MS) long term treated with low dose glucocorticoids. METHODS: The study involved 70 patients with MS. Motor function of the patients was evaluated using the Kurtzke Expanded Disability Status Scale (KEDSS). Bone mineral density (BMD) was determined at the lumbar spine and proximal femur at baseline and after 1.8 +/- 0.8 years. Bone remodeling was assessed using circulating concentrations of type 1 collagen cross-linked C-telopeptide (beta CTX), aminoterminal propeptide of type I procollagen, and N-MID osteocalcin (OC). A control group of 140 age-matched healthy subjects was used to compare bone-turnover markers. RESULTS: The plasma CTX concentration was the most significant parameter of bone remodeling which correlated with the rate of bone loss and with the KEDSS. The rate of bone loss at the proximal femur was not significantly different between tertiles of plasma OC concentrations. CONCLUSION: In physically active patients with MS treated with low-dose GC, the bone-turnover markers were not different from controls. Patients having plasma CTX but markers of bone formation higher as compared to controls were confirmed 2 years later as bone losers.

Calcitonin load test to assess the efficacy of salmon calcitonin.

BACKGROUND: Monitoring treatments of osteoporosis is required to identify patients not responding to the treatment in a way that reflects mechanism of action of the antiresorption drug on bone. Neither bone mineral measurement nor the available biochemical markers of bone remodeling can be used to monitor efficacy of treatment with nasal spray salmon calcitonin (sCT) since the changes in individual patients are modest and do not exceed the least significant change. METHOD: The novel calcitonin load test (CLT) was developed to assess the biological response to sCT in postmenopausal osteoporotic women. The CLT is based on the time course and an extent of suppression of serum C-terminal telopeptide of types I collagen (CTX) after the intranasal and subcutaneous administration of sCT. The CLT was conducted in 30 untreated postmenopausal osteoporotic women (control group, mean age, 67.7+/-8.4 years), and in 120 postmenopausal osteoporotic women (mean age, 68.5+/-8.1 years) treated with 200 IU of sCT (Miacalcic Nasal, Novartis, Switzerland), for up to 8.4 years (mean, 3.5+/-2.1 years). RESULTS: After 90 min from the intranasal administration of 400 IU of sCT, a decrease (p<0.01) in serum CTX by 58+/-11% was found in the control group, and by 60+/-11% in 74% of the treated patients. In the remaining treated patients, the decrease in CTX did not exceed the least significant change. The number of patients not responding to the CLT increased with duration of the treatment up to 34% in patients treated for over 4 years. Of the non-responders to the nasal spray sCT, 63% failed to respond to the subcutaneous administration of 10 IU of sCT. In the treated group, a significant negative correlation has been found between the percentual changes in CTX from its baseline levels detected during the CLT, and a rate of changes in the femoral neck BMD (p<0.01). CONCLUSION: The CLT can be used as a tool to identify patients that respond to administration of CT, and will profit from a continued treatment with sCT.

Plasma type 1 collagen cross-linked C-telopeptide: a sensitive marker of acute effects of salmon calcitonin on bone resorption.

BACKGROUND: The aim of the study was to determine the acute effects of salmon calcitonin (sCT) on bone resorption as assessed by plasma type 1 collagen cross-linked C-telopeptide (CTX). In addition, the plasma aminoterminal propeptide of type I procollagen (PINP) and osteocalcin (OC) were evaluated as markers of bone formation. METHODS: In six healthy young women, after overnight fasting, single subcutaneous doses of 2, 10 and 50 IU as well as a nasal dose of 200 IU of sCT were administered consecutively with a 1-week washout period. During the control period, no drug was given. Blood samples were drawn at 0700 a.m. (baseline) and throughout the 9-h fasting period. RESULTS: Both intranasal and subcutaneous sCT resulted in a significant reduction in plasma CTX by 50-60% as early as within 1 h. The plasma CTX showed a dose-dependent decrease over a dosage range of 2, 10 and 50 IU after subcutaneous administration of sCT. No significant difference was observed between the areas under curves (AUC) for plasma CTX following intranasal and subcutaneous administration of 200 and 2 IU of sCT, respectively. Synthesis of types I collagen remained unaffected by the dose of 50 IU of subcutaneously administered sCT when the acute effects were considered. CONCLUSIONS: Plasma CTX is a sensitive marker in detecting acute changes of bone resorption after sCT administration.