Establishment of a highly sensitive sandwich ELISA for the N-terminal fragment of titin in urine.

Muscle damage and loss of muscle mass are triggered by immobilization, loss of appetite, dystrophies and chronic wasting diseases. In addition, physical exercise causes muscle damage. In damaged muscle, the N-terminal and C-terminal regions of titin, a giant sarcomere protein, are cleaved by calpain-3, and the resulting fragments are excreted into the urine via glomerular filtration. Therefore, we considered titin fragments as promising candidates for reliable and non-invasive biomarkers of muscle injury. Here, we established a sandwich ELISA that can measure the titin N-terminal fragment over a biologically relevant range of concentrations, including those in urine samples from older, non-ambulatory Duchenne muscular dystrophy patients and from healthy donors under everyday life conditions and after exercise. Our results indicate that the established ELISA could be a useful tool for the screening of muscular dystrophies and also for monitoring the progression of muscle disease, evaluating the efficacy of therapeutic approaches, and investigating exercise-related sarcomeric disruption and repair processes.

Comparative Tissue Proteomics of Microdissected Specimens Reveals Novel Candidate Biomarkers of Bladder Cancer.

More than 380,000 new cases of bladder cancer are diagnosed worldwide, accounting for ∼150,200 deaths each year. To discover potential biomarkers of bladder cancer, we employed a strategy combining laser microdissection, isobaric tags for relative and absolute quantitation labeling, and liquid chromatography-tandem MS (LC-MS/MS) analysis to profile proteomic changes in fresh-frozen bladder tumor specimens. Cellular proteins from four pairs of surgically resected primary bladder cancer tumor and adjacent nontumorous tissue were extracted for use in two batches of isobaric tags for relative and absolute quantitation experiments, which identified a total of 3220 proteins. A DAVID (database for annotation, visualization and integrated discovery) analysis of dysregulated proteins revealed that the three top-ranking biological processes were extracellular matrix organization, extracellular structure organization, and oxidation-reduction. Biological processes including response to organic substances, response to metal ions, and response to inorganic substances were highlighted by up-expressed proteins in bladder cancer. Seven differentially expressed proteins were selected as potential bladder cancer biomarkers for further verification. Immunohistochemical analyses showed significantly elevated levels of three proteins-SLC3A2, STMN1, and TAGLN2-in tumor cells compared with noncancerous bladder epithelial cells, and suggested that TAGLN2 could be a useful tumor tissue marker for diagnosis (AUC = 0.999) and evaluating lymph node metastasis in bladder cancer patients. ELISA results revealed significantly increased urinary levels of both STMN1 and TAGLN2 in bladder cancer subgroups compared with control groups. In comparisons with age-matched hernia urine specimens, urinary TAGLN2 in bladder cancer samples showed the largest fold change (7.13-fold), with an area-under-the-curve value of 0.70 (p < 0.001, n = 205). Overall, TAGLN2 showed the most significant overexpression in individual bladder cancer tissues and urine specimens, and thus represents a potential biomarker for noninvasive screening for bladder cancer. Our findings highlight the value of bladder tissue proteome in providing valuable information for future validation studies of potential biomarkers in urothelial carcinoma.

ß-Hydroxy-ß-methylbutyrate free acid reduces markers of exercise-induced muscle damage and improves recovery in resistance-trained men.

The purpose of the present study was to determine the effects of short-term supplementation with the free acid form of b-hydroxyb-methylbutyrate (HMB-FA) on indices of muscle damage, protein breakdown, recovery and hormone status following a high-volume resistance training session in trained athletes. A total of twenty resistance-trained males were recruited to participate in a high-volume resistance training session centred on full squats, bench presses and dead lifts. Subjects were randomly assigned to receive either 3 g/d of HMB-FA or a placebo. Immediately before the exercise session and 48 h post-exercise, serum creatine kinase (CK), urinary 3-methylhistadine (3-MH), testosterone, cortisol and perceived recovery status (PRS) scale measurements were taken. The results showed that CK increased to a greater extent in the placebo (329%) than in the HMB-FA group (104%) (P»0·004, d » 1·6). There was also a significant change for PRS, which decreased to a greater extent in the placebo (9·1 (SEM 0·4) to 4·6 (SEM 0·5)) than in the HMB-FA group (9·1 (SEM 0·3) to 6·3 (SEM 0·3)) (P»0·005, d » 20·48). Muscle protein breakdown, measured by 3-MH analysis, numerically decreased with HMB-FA supplementation and approached significance (P»0·08, d » 0·12). There were no acute changes in plasma total or free testosterone, cortisol or C-reactive protein. In conclusion, these results suggest that an HMB-FA supplement given to trained athletes before exercise can blunt increases in muscle damage and prevent declines in perceived readiness to train following a high-volume, muscle-damaging resistance-training session.

Mass spectrometric detection of candidate protein biomarkers of cancer cachexia in human urine.

Increased membrane permeability and myofibrillar protein breakdown are established features of cancer cachexia. Proteins released from cachectic muscle may be excreted in urine to act as biomarkers of the cachectic process. One-dimensional SDS polyacrylamide gel electrophoresis followed by matrix-assisted laser desorption/ionisation or liquid chromatography tandem mass spectrometry was used to compare the protein content of urine from cachectic (>10% weight loss) (n=8) and weight-stable (n=8) gastro-oesophageal cancer patients and healthy controls (n=8). Plasma creatine kinase concentration was used as a marker of gross muscle breakdown. The number of protein species identified in cachectic samples (median 42; range 28-61; total 199) was greater than that identified in weight-stable cancer (median 15; range 9-28; total 79) and control samples (median 12.5; range 5-18; total 49) (P<0.001). Many of the proteins identified have not been reported previously in the urine of cancer patients. Proteins identified specifically in cachectic samples included muscle (myosin species), cytoskeletal (alpha-spectrin; nischarin) and microtubule-associated proteins (microtubule-actin crosslinking factor; microtubule-associated protein-1B; bullous pemphigoid antigen 1), whereas immunoglobulin kappa-light chain and zinc alpha-2 glycoprotein appeared to represent markers of cancer. The presence of myosin in urine (without an increase in plasma creatine kinase) is consistent with a specific loss of myosin as part of the cachectic process. Urinary proteomics using mass spectrometry can identify muscle-specific and non-muscle-specific candidate biomarkers of cancer cachexia.

Creatine monohydrate supplementation does not increase muscle strength, lean body mass, or muscle phosphocreatine in patients with myotonic dystrophy type 1.

Creatine monohydrate (CrM) supplementation may increase strength in some types of muscular dystrophy. A recent study in myotonic muscular dystrophy type 1 (DM1) did not find a significant treatment effect, but measurements of muscle phosphocreatine (PCr) were not performed. We completed a randomized, double-blind, cross-over trial using 34 genetically confirmed adult DM1 patients without significant cognitive impairment. Participants received CrM (5 g, approximately 0.074 g/kg daily) and a placebo for each 4-month phase with a 6-week wash-out. Spirometry, manual muscle testing, quantitative isometric strength testing of handgrip, foot dorsiflexion, and knee extension, handgrip and foot dorsiflexion endurance, functional tasks, activity of daily living scales, body composition (total, bone, and fat-free mass), serum creatine kinase activity, serum creatinine concentration and clearance, and liver function tests were completed before and after each intervention, and muscle PCr/beta-adenosine triphosphate (ATP) ratios of the forearm flexor muscles were completed at the end of each phase. CrM supplementation did not increase any of the outcome measurements except for plasma creatinine concentration (but not creatinine clearance). Thus, CrM supplementation at 5 g daily does not have any effects on muscle strength, body composition, or activities of daily living in patients with DM1, perhaps because of a failure of the supplementation to increase muscle PCr/beta-ATP content.

Solitary plasmacytoma of bone.

From 1978 to 1992, 19 patients were diagnosed with solitary plasmacytoma of bone (SPB) accounting for 5.5% of plasma cell neoplasms in Chang Gung Memorial Hospital. Fourteen were male and five were female, with ages ranging from 21 to 71 years (median, 53 years). M-protein was detected at diagnosis in five patients, and during follow-up in three. The most common sites of SPB were the vertebral bodies, with seven involving the thoracic, four involving the lumbar and two involving the cervical spine. The remaining sites included the skull in four, and femur and pelvis in one each. Fifteen of the patients received local radiotherapy, local control was achieved in all; nine patients were alive with no evidence of disease for seven to 77 months, two patients remained in apparent remission at 14 and 92 months, two patients progressed to multiple myeloma at 12 and 84 months, one patient developed extramedullary plasmacytoma at three months, and one patient died of intercurrent disease at four months. The four patients who were not treated with radiation therapy ultimately developed local recurrence or dissemination within four years. In our series, local recurrence or dissemination was always associated with the appearance of, or an increase in, the M-component; thus, measurement of M-protein is helpful in assessing tumor control or progression. Based on our experience and a review of the literature, it is recommended that irradiation of the primary sites is the treatment of choice. Chemotherapy is indicated in those patients with disease progression, recurrence, or dissemination.

Muscle protein degradation in premature human infants.

1. Myofibrillar protein degradation has been measured by the rate of 3-methylhistidine excretion in premature infants weighing between 635 g and 1295 g. Analyses were made in conjunction with 1--3 day nitrogen balance studies. 2. In 56 balance studies in 36 infants, total muscle protein breakdown varied between 0.70 and 2.58 (mean 1.05) g day-1 kg-1 body weight while the percentage of total muscle protein degraded each day was between 3.3 and 8.3 (mean 4.8). 3. Both total and fractional rates of protein breakdown showed highly significant negative correlations with nitrogen retention but no relationship to total energy input. 4. Protein degradation was higher than average in infants who were losing weight at the time of the balance study, lower in infants who were gaining weight and higher in those who died within 2 weeks of the analysis. 5. Myofibrillar protein breakdown was not different between infants fed orally and those receiving total parenteral nutrition. 6. Generally the effects of nitrogen and evergy status on muscle protein degradation in the premature infants are different from changes reported in adult human beings or adult rats. We suggest that this difference may be a consequence of the very limited energy reserves of the premature infant.