Metabolomics reveals elevated urinary excretion of collagen degradation and epithelial cell turnover products in irritable bowel syndrome patients.

INTRODUCTION: Irritable bowel syndrome (IBS), the most commonly diagnosed functional gastrointestinal (GI) disorder in developed countries, is characterized by chronic abdominal pain, and altered bowel habits. OBJECTIVES: Accurate and timely diagnosis is challenging as it relies on symptoms and an evolving set of exclusion criteria to distinguish it from other related GI disorders reflecting a complex etiology that remains poorly understood. Herein, nontargeted metabolite profiling of repeat urine specimens collected from a cohort of IBS patients (n = 42) was compared to healthy controls (n = 20) to gain insights into the underlying pathophysiology. METHODS: An integrated data workflow for characterization of the urine metabolome with stringent quality control was developed to authenticate reliably measured (CV < 30%) and frequently detected (> 75%) metabolites using multisegment injection-capillary electrophoresis-mass spectrometry. Complementary statistical methods were then used to rank differentially excreted urinary metabolites after normalization to osmolality that were subsequently identified by high resolution tandem mass spectrometry and their electrophoretic migration behavior. RESULTS: Our work revealed ten consistently elevated urinary metabolites in repeat samples collected from IBS patients at two different time points (q < 0.05 after age and Benjamini-Hochberg/FDR adjustment), which were associated with greater collagen degradation and intestinal mucosal turn-over processes likely due to low-grade inflammation. IBS-specific metabolites identified in urine included a series of hydroxylysine metabolites (O-glycosylgalactosyl-hydroxylysine, O-galactosyl-hydroxylysine, lysine), mannopyranosy-L-tryptophan, imidazole propionate, glutamine, serine, ornithine, dimethylglycine and dimethylguanosine. A major limitation in this retrospective case-control study was significant co-morbidity of IBS patients with other illnesses, including depression and prescribed medications as compared to healthy controls. CONCLUSION: This work provides new mechanistic insights into the pathophysiology of IBS while also offering a convenient way to monitor patient disease progression and treatment responses to therapy based on a panel of urinary metabolites that avoids invasive blood sampling, colonoscopy and/or tissue biopsies.

Calcium intake in winter pregnancy attenuates impact of vitamin D inadequacy on urine NTX, a marker of bone resorption.

PURPOSE: Pregnancy is characterised by increased bone turnover, but high bone turnover with resorption exceeding formation may lead to negative maternal bone remodelling. Recent studies are conflicting regarding the effect of calcium on skeletal health in pregnancy. The aim of this study was to examine the seasonal effect of serum 25-hydroxyvitamin D (25OHD) and dietary calcium on a marker of bone resorption. METHODS: This was prospective study of 205 pregnant women [two cohorts; early pregnancy at 13 weeks (n = 96), and late pregnancy at 28 weeks (n = 109)]. Serum 25OHD and urine cross-linked N-telopeptides of type I collagen (uNTX) were measured at both time points. Intakes of vitamin D and calcium were recorded using 3-day food diaries at each trimester. RESULTS: Compared to summer pregnancies, winter pregnancies had significantly lower 25OHD and significantly higher uNTX. Higher calcium intakes were negatively correlated with uNTX in winter, but not summer. In late pregnancy, compared to those reporting calcium intakes ≥1000 mg/day, intakes of <1000 mg/day were associated with a greater increase in uNTX in winter pregnancies than in summer (41.8 vs. 0.9%). Increasing calcium intake in winter by 200 mg/day predicted a 13.3% reduction in late pregnancy uNTX. CONCLUSIONS: In late pregnancy, during winter months when 25OHD is inadequate, intakes of dietary calcium <1000 mg/day were associated with significantly increased bone resorption (uNTX). Additional dietary calcium is associated with reduced bone resorption in late pregnancy, with greater effect observed in winter. Further research regarding optimal dietary calcium and 25OHD in pregnancy is required, particularly for women gestating through winter.

Diagnostic and prognostic role of urinary collagens in primary human bladder cancer.

Collagen type 4 alpha 1 (COL4A1) and collagen type 13 alpha 1 (COL13A1) produced by urothelial cancer cells support the vital oncogenic property of tumor invasion. We investigated the diagnostic and prognostic capability of COL4A1 and COL13A1 in voided urine and compared the observed values with those of fragments of cytokeratin-19 (CYFRA21-1), nuclear matrix protein 22 (NMP-22), and voided urine cytology in bladder cancer (BCa). We collected voided urine samples from 154 patients newly diagnosed with BCa, before surgery and from 61 control subjects. Protein levels of COL4A1, COL13A1, CYFRA21-1, and NMP-22 in urine supernatants were measured using enzyme-linked immunosorbent assays. Diagnostic performance and optimal cut-off values were determined by receiver operating characteristic analysis. Urine levels of COL4A1, COL13A1, the combined values of COL4A1 and COL13A1 (COL4A1 + COL13A1), and CYFRA21-1 were significantly elevated in urine from patients with BCa compared to the controls. Among these biomarkers, the optimal cut-off value of COL4A1 + COL13A1 at 1.33 ng/mL resulted in 57.4%, 83.7%, 56.1%, 80.7%, and 91.7% sensitivity for low-grade tumors, high-grade tumors, Ta, T1, and muscle invasive disease, respectively. We evaluated the prognostic value of preoperative urine levels in 130 non-muscle invasive BCa samples after the initial transurethral surgery. A high urinary COL4A1 + COL13A1 was found to be an independent risk factor for intravesical recurrence. Although these data need to be externally validated, urinary COL4A1 and COL13A1 could be a potential diagnostic and prognostic biomarker for BCa. This easy-to-use urinary signature identifies a subgroup of patients with a high probability of recurrence and progression in non-muscle invasive and muscle invasive BCa.

Alterations in urinary collagen peptides in lupus nephritis subjects correlate with renal dysfunction and renal histopathology.

BACKGROUND: The excessive accumulation of extracellular matrix (ECM) in the renal tubulointerstitium is a key component of chronic renal damage in lupus nephritis (LN) and a critical determinant of the disease progression to renal failure. Detection of fibrosis requires renal biopsy and is therefore limited by high risks associated with an invasive procedure. This study explores whether a unique LN urinary peptidome can be identified and whether LN-specific alteration reflects the underlying fibrogenic process of altered ECM turnover. METHOD: Urinary peptides were analyzed for 36 LN and 35 nonrenal systemic lupus erythematosus (SLE) subjects and 58 healthy volunteers (HVs). RESULTS: In total, 70 collagen and 230 noncollagen peptides were significantly changed between LN and nonrenal SLE and between LN and HV and defined as 'LN peptides'; 14 proteases associated with observed LN collagen peptides were identified and activities in 9 proteases were significantly different between LN and nonrenal SLE; 28 collagen peptides were correlated with at least one parameter of clinical renal dysfunction or histolopathology. CONCLUSION: Urinary peptidomic alterations likely reflect pathogenic pathways involving ECM turnover in LN kidneys and potentially could be developed as biomarkers to monitor renal disease progression.

Monoclonal antibody against macrophage colony-stimulating factor suppresses circulating monocytes and tissue macrophage function but does not alter cell infiltration/activation in cutaneous lesions or clinical outcomes in patients with cutaneous lupus erythematosus.

This study's objective was to assess the effects of PD-0360324, a fully human immunoglobulin G2 monoclonal antibody against macrophage colony-stimulating factor in cutaneous lupus erythematosus (CLE). Patients with active subacute CLE or discoid lupus erythematosus were randomized to receive 100 or 150 mg PD-0360324 or placebo via intravenous infusion every 2 weeks for 3 months. Blood and urine samples were obtained pre- and post-treatment to analyse pharmacokinetics and pharmacodynamic changes in CD14(+) CD16(+) monocytes, urinary N-terminal telopeptide (uNTX), alanine/aspartate aminotransferases (ALT/AST) and creatine kinase (CK); tissue biopsy samples were taken to evaluate macrophage populations and T cells using immunohistochemistry. Clinical efficacy assessments included the Cutaneous Lupus Erythematosus Disease Area and Severity Index (CLASI). Among 28 randomized/analysed patients, peak/trough plasma concentrations increased in a greater-than-dose-proportional manner with dose increases from 100 to 150 mg. Statistically significant differences were observed between active treatment and placebo groups in changes from baseline in CD14(+) CD16(+) cells, uNTX, ALT, AST and CK levels at most time-points. The numbers, density and activation states of tissue macrophages and T cells did not change from baseline to treatment end. No between-group differences were seen in CLASI. Patients receiving PD-0360324 reported significantly more adverse events than those receiving placebo, but no serious adverse events. In patients with CLE, 100 and 150 mg PD-0360324 every 2 weeks for 3 months suppressed a subset of circulating monocytes and altered activity of some tissue macrophages without affecting cell populations in CLE skin lesions or improving clinical end-points.

Preoperative Urodynamic Parameters (Valsalva Leak Point Pressure and Maximum Urethral Closure Pressure), Urinary Collagen and Plasma Vitamin D Levels as Predictors of Mid Urethral Sling Surgery Outcome.

PURPOSE: To determine the best predictor of the mid urethral sling outcome we calculated the AUC of ROC curves of preoperative parameters, including Valsalva leak point pressure, maximum urethral closure pressure, urinary NTx (N-telopeptide of crosslinked type I collagen) and plasma vitamin D values (D2, D3 and D2 plus D3). MATERIALS AND METHODS: This was an ancillary study of TOMUS (Trial of Mid-urethral Slings) and the ValUE (Value of Urodynamics Evaluation) trial in which subjects underwent mid urethral sling surgery for stress urinary incontinence. Valsalva leak point pressure and maximum urethral closure pressure were measured in 427 subjects, whereas NTx, vitamin D2, vitamin D3 and vitamin D2 plus D3 levels were obtained from 150, 116, 115 and 116 subjects respectively. Outcome success was defined using identical outcome (subjective and objective) variables for all subjects. ROC curves with corresponding AUC values were compared. RESULTS: TOMUS and ValUE subjects were significantly different in age, body mass index, UDI (Urogenital Distress Inventory) scores. TOMUS subjects had a lower surgical success rate compared to ValUE subjects (66.3% vs 76.0%, p = 0.03). The AUC values of Valsalva leak point pressure, maximum urethral closure pressure, NTx, and vitamins D2, D3 and D2 plus D3 were 0.542, 0.561, 0.702, 0.627, 0.645 and 0.640, respectively. The AUC of NTx was significantly higher than the AUCs of Valsalva leak point pressure and maximum urethral closure pressure (p = 0.02 and 0.03, respectively). CONCLUSIONS: Urinary NTx was the best predictor of the mid urethral sling outcome. This test is not only noninvasive, it is also modifiable. Finding ideal modifiable risk factors prior to mid urethral sling surgery should be subject to future investigations.

Stage-specific quantitative changes in renal and urinary proteome during the progression and development of streptozotocin-induced diabetic nephropathy in rats.

Diabetic nephropathy (DN) is a microvascular complication associated with diabetes causing slow deterioration of kidneys leading to end-stage renal disease. Timely intervention and diagnosis are crucial in order to ameliorate and halt the progression of DN. Current diagnosis of DN consists of urine assays for detection of microalbuminuria, which have inadequate specificity and sensitivity. Hence, there arises a need to discover stage-specific biomarkers which can aid in the early detection of DN and also in identifying the mechanisms underlying pathogenesis of DN. Therefore the present study was undertaken to identify the differentially expressed proteins in the urine and to examine the pattern of proteomic changes occurring in the rat kidneys during the course of progression of streptozotocin-induced model of DN in rats. Two-dimensional gel electrophoresis coupled to MALDI-TOF mass spectrometry was employed to identify the differentially expressed proteins under diabetic conditions. Among the identified proteins Calgranulin A and Calgranulin B appeared in the urinary proteome at the fourth week of induction of diabetes while we recorded a time-dependent decrease in the expression of major urinary protein (alpha 2u globulin) in the urine as well as kidneys of diabetic rats. Parallel monitoring of targeted proteomic changes in the renal proteome revealed an increase in histone H2B phosphorylation at serine14 along with a gradual decrease in Bcl-2 and MMP-13 expression during the course of progression and development of streptozotocin-induced DN.

Effects of dietary protein and glycaemic index on biomarkers of bone turnover in children.

For decades, it has been debated whether high protein intake compromises bone mineralisation, but no long-term randomised trial has investigated this in children. In the family-based, randomised controlled trial DiOGenes (Diet, Obesity and Genes), we examined the effects of dietary protein and glycaemic index (GI) on biomarkers of bone turnover and height in children aged 5-18 years. In two study centres, families with overweight parents were randomly assigned to one of five ad libitum-energy, low-fat (25-30% energy (E%)) diets for 6 months: low protein/low GI; low protein/high GI; high protein/low GI; high protein/high GI; control. They received dietary instructions and were provided all foods for free. Children, who were eligible and willing to participate, were included in the study. In the present analyses, we included children with data on plasma osteocalcin or urinary N-terminal telopeptide of collagen type I (U-NTx) from baseline and at least one later visit (month 1 or month 6) (n 191 in total, n 67 with data on osteocalcin and n 180 with data on U-NTx). The level of osteocalcin was lower (29.1 ng/ml) in the high-protein/high-GI dietary group than in the low-protein/high-GI dietary group after 6 months of intervention (95% CI 2.2, 56.1 ng/ml, P=0.034). The dietary intervention did not affect U-NTx (P=0.96) or height (P=0.80). Baseline levels of U-NTx and osteocalcin correlated with changes in height at month 6 across the dietary groups (P<0.001 and P=0.001, respectively). The present study does not show any effect of increased protein intake on height or bone resorption in children. However, the difference in the change in the level of osteocalcin between the high-protein/high-GI group and the low-protein/high-GI group warrants further investigation and should be confirmed in other studies.

Urinary collagen peptides: Source of markers for bone metabolic processes in kidney transplant recipients.

INTRODUCTION: Kidney transplant recipients (KTRs) are at an increased risk of fractures. Total urinary hydroxyproline excretion served as marker for bone resorption (BR) but was replaced by ß-CrossLaps (CTX), a C-terminal collagen α-1(I) chain (COL1A1) telopeptide. We investigated the low-molecular-weight urinary proteome for peptides associated with changes in bone metabolism after kidney transplantation. METHODS: Clinical and laboratory data including serum levels of CTX in 96 KTR from two nephrology centers were correlated with signal intensities of urinary peptides identified by capillary electrophoresis mass spectrometry. RESULTS: Eighty-two urinary peptides were significantly correlated with serum CTX levels. COL1A1 was the predominant peptide source. Oral bisphosphonates were administered for decreased bone density in an independent group of 11 KTR and their effect was evaluated on the aforementioned peptides. Study of the peptides cleavage sites revealed a signature of Cathepsin K and MMP9. Seventeen of these peptides were significantly associated with bisphosphonate treatment, all showing a marked reduction in their excretion levels compared to baseline. DISCUSSION: This study provides strong evidence for the presence of collagen peptides in the urine of KTR that are associated with BR and that are sensitive to bisphosphonate treatment. Their assessment might become a valuable tool to monitor bone status in KTR.

Increased Collagen Turnover Is a Feature of Fibromuscular Dysplasia and Associated With Hypertrophic Radial Remodeling: A Pilot, Urine Proteomic Study.

Fibromuscular dysplasia (FMD), a nonatherosclerotic, noninflammatory disease of medium-sized arteries, is an underdiagnosed disease. We investigated the urinary proteome and developed a classifier for discrimination of FMD from healthy controls and other diseases. We further hypothesized that urinary proteomics biomarkers may be associated with alterations in medium-sized, but not large artery geometry and mechanics. The study included 33 patients with mostly multifocal, renal FMD who underwent in depth arterial exploration using ultra-high frequency ultrasound. The cohort was separated in a training set of 23 patients with FMD from Belgium and an independent test set of 10 patients with FMD from Italy. For each set, controls matched 2:1 were selected from the Human Urinary Proteome Database. The specificity of the classifier was tested in 700 additional controls from general population studies, patients with chronic kidney disease (n=66) and coronary artery disease (n=31). Three hundred thirty-five urinary peptides, mostly related to collagen turnover, were identified in the training cohort and combined into a classifier. When applying in the test cohort, the area under the receiver operating characteristic curve was 1.00, 100% specificity at 100% sensitivity. The classifier maintained a high specificity in additional controls (98.3%), patients with chronic kidney (90.9%) and coronary artery (96.8%) diseases. Furthermore, in patients with FMD, the proteomic score was positively associated with radial wall thickness and wall cross-sectional area. In conclusion, a proteomic score has the potential to discriminate between patients with FMD and controls. If confirmed in a wider and more diverse cohort, these findings may pave the way for a noninvasive diagnostic test of FMD.

Spondylocheiro dysplastic form of the Ehlers-Danlos syndrome--an autosomal-recessive entity caused by mutations in the zinc transporter gene SLC39A13.

We present clinical, radiological, biochemical, and genetic findings on six patients from two consanguineous families that show EDS-like features and radiological findings of a mild skeletal dysplasia. The EDS-like findings comprise hyperelastic, thin, and bruisable skin, hypermobility of the small joints with a tendency to contractures, protuberant eyes with bluish sclerae, hands with finely wrinkled palms, atrophy of the thenar muscles, and tapering fingers. The skeletal dysplasia comprises platyspondyly with moderate short stature, osteopenia, and widened metaphyses. Patients have an increased ratio of total urinary pyridinolines, lysyl pyridinoline/hydroxylysyl pyridinoline (LP/HP), of approximately 1 as opposed to approximately 6 in EDS VI or approximately 0.2 in controls. Lysyl and prolyl residues of collagens were underhydroxylated despite normal lysyl hydroxylase and prolyl 4-hydroxylase activities; underhydroxylation was a generalized process as shown by mass spectrometry of the alpha1(I)- and alpha2(I)-chain-derived peptides of collagen type I and involved at least collagen types I and II. A genome-wide SNP scan and sequence analyses identified in all patients a homozygous c.483_491 del9 SLC39A13 mutation that encodes for a membrane-bound zinc transporter SLC39A13. We hypothesize that an increased Zn(2+) content inside the endoplasmic reticulum competes with Fe(2+), a cofactor that is necessary for hydroxylation of lysyl and prolyl residues, and thus explains the biochemical findings. These data suggest an entity that we have designated "spondylocheiro dysplastic form of EDS (SCD-EDS)" to indicate a generalized skeletal dysplasia involving mainly the spine (spondylo) and striking clinical abnormalities of the hands (cheiro) in addition to the EDS-like features.

Deletion of soluble epoxide hydrolase gene improves renal endothelial function and reduces renal inflammation and injury in streptozotocin-induced type 1 diabetes.

Studies suggest that soluble epoxide hydrolase (sEH) inhibition reduces end-organ damage in cardiovascular diseases. We hypothesize that sEH gene (Ephx2) knockout (KO) improves endothelial function and reduces renal injury in streptozotocin-induced diabetes. After 6 wk of diabetes, afferent arteriolar relaxation to acetylcholine was impaired in diabetic wild-type (WT) mice, as the maximum relaxation was 72% of baseline diameter in the WT but only 31% in the diabetic mice. Ephx2 KO improved afferent arteriolar relaxation to acetylcholine in diabetes as maximum relaxation was 58%. Urinary monocyte chemoattractant protein-1 (MCP-1) excretion significantly increased in diabetic WT mice compared with control (868 ± 195 vs. 31.5 ± 7 pg/day), and this increase was attenuated in diabetic Ephx2 KO mice (420 ± 98 pg/day). The renal phospho-IKK-to-IKK ratio and nuclear factor-κB were significantly decreased, and hemeoxygenase-1 (HO-1) expression increased in diabetic Ephx2 KO compared with diabetic WT mice. Renal NADPH oxidase and urinary thiobarbituric acid reactive substances excretion were reduced in diabetic Ephx2 KO compared with diabetic WT mice. Albuminuria was also elevated in diabetic WT mice compared with control (170 ± 43 vs. 37 ± 13 µg/day), and Ephx2 KO reduced this elevation (50 ± 15 µg/day). Inhibition of sEH using trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (tAUCB) also reduced renal inflammation and injury in diabetic WT mice. Furthermore, inhibition of HO with stannous mesoporphyrin negated the reno-protective effects of tAUCB or Ephx2 KO during diabetes. These data demonstrate that Ephx2 KO improves endothelial function and reduces renal injury during diabetes. Additionally, our data also suggest that activation of HO-1 contributes to improved renal injury in diabetic Ephx2 KO mice.

Bone resorption in syndromes of the Ras/MAPK pathway.

Disorders of the Ras/mitogen-activated protein kinase (MAPK) pathway have an overlapping skeletal phenotype (e.g. scoliosis, osteopenia). The Ras proteins regulate cell proliferation and differentiation and neurofibromatosis type 1 (NF1) individuals have osteoclast hyperactivity and increased bone resorption as measured by urine pyridinium crosslinks [pyridinoline (Pyd) and deoxypyridinoline (Dpd)]. Pyd and Dpd are hydroxylysine-derived crosslinks of collagen found in bone and cartilage and excreted in the urine. Dpd is most abundant in bone. The aim of this study was to evaluate if other syndromes of the Ras/MAPK pathway have increased bone resorption, which may impact the skeletal phenotype. Participants were individuals with Noonan syndrome (n = 14), Costello syndrome (n = 21), and cardiofaciocutaneous (CFC) syndrome (n = 14). Pyridinium crosslinks from two consecutive first morning urines were extracted after acid hydrolysis and analyzed by high performance liquid chromatography. Three separate analyses of covariance were performed to compare Pyd, Dpd, and Dpd/Pyd ratio of each group to controls after controlling for age. Data were compared to 99 healthy controls. The Dpd and the Dpd/Pyd ratio were elevated (p < 0.0001) in all three conditions compared to controls suggesting that collagen degradation was predominantly from bone. The data suggest that the Ras/MAPK signal transduction pathway is important in bone homeostasis.

Menopausal bone changes and incident fractures in diabetic women: a cohort study.

SUMMARY: The purpose of this study was to evaluate the rate of bone loss and incident fractures in women with diabetes mellitus (DM) across menopause. During menopause, DM women experienced bone mineral density (BMD) loss that was faster at hip and slower at spine and had a higher risk of fractures, perhaps because of their earlier menopause. The increasing DM epidemic will contribute to higher fracture burden. INTRODUCTION: Women with DM have a higher risk of fractures independent of age, body mass index (BMI), and BMD. Our objective is to evaluate if women with DM experience greater bone loss and more fractures across menopause. METHODS: Two thousand one hundred seventy one women, aged 42 to 52 years at baseline (1996), enrolled in the Study of Women's Health Across the Nation (SWAN), a prospective study, with 8 years of annual follow up. One thousand three hundred forty six (62%) completed annual visit 7 (2004). Women with baseline fasting blood glucose level of ≥126 mg/dl and those being treated for diabetes were designated as DM. Annual assessment of menopausal stage, BMD, and urinary N-telopeptide (NTx) were carried out. Rate of change in BMD across menopause and annual self-report data for risk of incident fractures by DM status were determined. RESULTS: Despite higher baseline BMD at hip (p = <0.001), and lumbar spine (p = <0.001), rate of decline in BMD was faster at hip (ß = -0.45 vs. -0.11 gm/cm(2)/year, p = <0.001) for DM women, compared to non-DM. However, lumbar spine bone loss was slower in women with DM as compared to non-DM women (ß = 0.04 vs. -0.25 gm/cm(2)/year, p = 0.004). DM women experienced menopause 3 years earlier than non-DM women (p = 0.002), and age adjusted incident fractures were two fold higher in women with DM compared to non-DM (RR = 2.20, 95% CI: 1.26-3.85, p = <0.006). CONCLUSIONS: BMD loss is greater in hip and slower at spine in DM women during menopausal transition. Women with DM have a higher risk of fractures, perhaps because of their earlier menopause.

Socioeconomic status and bone health in community-dwelling older men: the CHAMP Study.

SUMMARY: The association between socioeconomic status (SES) and bone health, specifically in men, is unclear. Based upon data from the large prospective Concord Health in Ageing Men Project (CHAMP) Study of community-dwelling men aged 70 years or over, we found that specific sub-characteristics of SES, namely, marital status, living circumstances, and acculturation, reflected bone health in older Australian men. INTRODUCTION: Previous studies reported conflicting results regarding the relationship between SES and bone health, specifically in men. The main objective of this study was to investigate associations of SES with bone health in community-dwelling men aged 70 years or over who participated in the baseline phase of the CHAMP Study in Sydney, Australia. METHODS: The Australian Socioeconomic Index 2006 (AUSEI06) based on the Australian and New Zealand Standard Classification of Occupations was used to determine SES in 1,705 men. Bone mineral density and bone mineral content (BMC) were determined by dual-energy X-ray absorptiometry. Bone-related biochemical and hormonal parameters, including markers of bone turnover, parathyroid hormone, and vitamin D, were measured in all men. RESULTS: General linear models adjusted for age, weight, height, and bone area revealed no significant differences across crude AUSEI06 score quintiles for BMC at any skeletal site or for any of the bone-related biochemical measures. However, multivariate regression models revealed that in Australian-born men, marital status was a predictor of higher lumbar BMC (ß = 0.07, p = 0.002), higher total body BMC (ß = 0.05, p = 0.03), and lower urinary NTX-I levels (ß=-0.08, p = 0.03), while living alone was associated with lower BMC at the lumbar spine (ß=-0.05, p = 0.04) and higher urinary NTX-I levels (ß=0.07, p = 0.04). Marital status was also a predictor of higher total body BMC (ß = 0.14, p = 0.003) in immigrants from Eastern and South Eastern Europe. However, in immigrants from Southern Europe, living alone and acculturation were predictors of higher femoral neck BMC (ß = 0.11, p = 0.03) and lumbar spine BMC (ß = 0.10, p = 0.008), respectively. CONCLUSIONS: Although crude occupation-based SES scores were not significantly associated with bone health in older Australian men, specific sub-characteristics of SES, namely, marital status, living circumstances, and acculturation, were predictors of bone health in both Australia-born men and European immigrants.

Urinary protein profiles in a rat model for diabetic complications.

Diabetes mellitus is estimated to affect approximately 24 million people in the United States and more than 150 million people worldwide. There are numerous end organ complications of diabetes, the onset of which can be delayed by early diagnosis and treatment. Although assays for diabetes are well founded, tests for its complications lack sufficient specificity and sensitivity to adequately guide these treatment options. In our study, we employed a streptozotocin-induced rat model of diabetes to determine changes in urinary protein profiles that occur during the initial response to the attendant hyperglycemia (e.g. the first two months) with the goal of developing a reliable and reproducible method of analyzing multiple urine samples as well as providing clues to early markers of disease progression. After filtration and buffer exchange, urinary proteins were digested with a specific protease, and the relative amounts of several thousand peptides were compared across rat urine samples representing various times after administration of drug or sham control. Extensive data analysis, including imputation of missing values and normalization of all data was followed by ANOVA analysis to discover peptides that were significantly changing as a function of time, treatment and interaction of the two variables. The data demonstrated significant differences in protein abundance in urine before observable pathophysiological changes occur in this animal model and as function of the measured variables. These included decreases in relative abundance of major urinary protein precursor and increases in pro-alpha collagen, the expression of which is known to be regulated by circulating levels of insulin and/or glucose. Peptides from these proteins represent potential biomarkers, which can be used to stage urogenital complications from diabetes. The expression changes of a pro-alpha 1 collagen peptide was also confirmed via selected reaction monitoring.

Therapeutic effect of Saraca asoca (Roxb.) Wilde on lysosomal enzymes and collagen metabolism in adjuvant induced arthritis.

Rheumatoid arthritis is a chronic, progressive and systemic inflammatory disorder mainly affecting the synovial joints. In the present study, we evaluated the anti-arthritic effect of the methanol extract of Saraca asoca (Roxb.) Wilde., (Fabaceae) on adjuvant induced arthritis by assessing paw swelling, body weight, the levels of lysosomal enzymes, protein bound carbohydrates, serum cytokines, urinary collagen and histopathology of joints. It was found that S. asoca methanol extract at doses of 50, 100 and 200 mg/kg reduced the paw thickness and elevated the mean body weight of arthritic rats. The treatment of S. asoca showed a significant reduction in the levels of both plasma and liver lysosomal enzymes. The protein bound carbohydrates and urinary collagen contents were also decreased at a significant level by the treatment of S. asoca methanol extract. The histopathological study of the joints showed the anti-arthritic property of S. asoca which nearly normalized the histological architecture of the joints. Further, we established the anti-arthritic activity of S. asoca methanol extract by measuring the levels of cytokines in both arthritic and treated rats. The treatment of S. asoca reduced the levels of pro-inflammatory cytokines. In conclusion, S. asoca methanol extract was capable of ameliorating the conditions of arthritis in adjuvant induced arthritic rats.

Baseline atherosclerosis parameter could assess the risk of bone loss during pioglitazone treatment in type 2 diabetes mellitus.

SUMMARY: We found that serum osteocalcin, femoral bone mineral density (F-BMD), and 1/3R-BMD were decreased during pioglitazone treatment in patients with type 2 diabetes. Moreover, baseline atherosclerosis parameter, serum insulin-like growth factor-I (IGF-I), and urinary N-terminal cross-linked telopeptide of type I collagen (uNTX) values were associated with changes in bone mineral density (BMD). Therefore, these parameters could assess the risk of BMD reduction in patients treated with pioglitazone. INTRODUCTION: The aim of this study was to investigate the effects of pioglitazone or metformin on bone mass and atherosclerosis in patients with type 2 diabetes. METHODS: A total of 55 Japanese patients were enrolled in this 1-year open-label study and randomized to either pioglitazone (n = 22, 15-30 mg/day) or metformin (n = 23, 500-750 mg/day) groups. BMD at the lumbar spine, femoral neck (F), and one third of the radius (1/3R), bone markers, and atherosclerosis parameters were measured. RESULTS: In the pioglitazone group, serum osteocalcin significantly decreased at 6 months (p < 0.05), although it almost recovered to baseline level at 12 months. F-BMD significantly decreased at 6 months (p < 0.05), and 1/3R-BMD significantly decreased at 6 and 12 months (p < 0.05), while bone markers or BMD at any site were not changed in the metformin group. Although atherosclerosis parameters were not changed in the pioglitazone group, intima-media thickness (IMT)-mean significantly increased at 6 months (p < 0.05) and plaque score significantly increased at 6 and 12 months (p < 0.01) in the metformin group. In the pioglitazone group, %changes in F-BMD were significantly and negatively correlated with baseline IMT-Max, IMT-mean, and plaque scores (r = -0.61, p < 0.01; r = -0.71, p < 0.01; and r = -0.68, p < 0.01, respectively), and %changes in 1/3R-BMD were significantly and negatively correlated with baseline uNTX and IMT-Max (r = -0.57, p < 0.01 and r = -0.48, p < 0.05, respectively) and positively with IGF-I (r = 0.45, p < 0.05). CONCLUSIONS: Baseline IMT, uNTX, and IGF-I could assess the risk of BMD reduction in diabetic patients treated with pioglitazone.

Effect of alendronate on bone mineral density and bone turnover markers in post-gastrectomy osteoporotic patients.

Alendronate decreases the urinary levels of cross-linked N-terminal telopeptides of type I collagen (NTX; about 45% at 3 months) and serum levels of alkaline phosphatase (ALP; about 27% at 24 months), leading to an increase in lumbar spine bone mineral density (BMD; about 9% at 24 months) in postmenopausal Japanese women with osteoporosis. However, the effectiveness of oral bisphosphonates on osteoporosis remains to be established in patients who have undergone a gastrectomy. The objective of the present case series study was to examine the effect of alendronate on BMD and bone turnover markers in post-gastrectomy osteoporotic patients. Sixteen patients (3 men and 13 postmenopausal women) with osteoporosis, who had undergone a gastrectomy (mean age: 69.1 years), were recruited in our outpatient clinic. All the patients were treated with alendronate (5 mg daily or 35 mg weekly) for 24 months. The effects of alendronate on lumbar spine (women) or total hip (men) BMD and urinary NTX and serum ALP levels were examined. A total or partial gastrectomy had been performed for eight patients each. The mean duration after surgery was 16.0 years. With alendronate therapy, urinary NTX levels significantly decreased at 3 months (-27.0%). Serum ALP levels decreased (-12.1%) and lumbar spine BMD increased (+5.2%), but total hip BMD did not significantly change (+0.6%) at 24 months. No severe adverse events were observed, and alendronate therapy was well tolerated. These results suggest that alendronate mildly increases lumbar spine BMD by mildly reducing bone turnover in osteoporotic patients after a gastrectomy.