Subcellular fractionation of frozen skeletal muscle samples.

Cell fractionation can be used to determine the localization and trafficking of proteins between cellular compartments such as the cytosol, mitochondria, and nuclei. Subcellular fractionation is usually performed immediately after tissue dissection because freezing may fragment cell membranes and induce organellar cross-contamination. Mitochondria are especially sensitive to freezing/thawing and mechanical homogenization. We proposed a protocol to improve the retention of soluble proteins in the mitochondrial fraction obtained from small amounts of frozen skeletal muscle. Fifty milligrams of the red portion of gastrocnemius muscle from Wistar rats were immediately processed or frozen in liquid nitrogen and stored at -80 °C for further processing. We compared the enrichment of subcellular fractions from frozen/fresh samples obtained with the modified protocol with those obtained by standard fractionation. Western blot analyses of marker proteins for cytosolic (alpha-tubulin), mitochondrial (VDAC1), and nuclear (histone-H3) fractions indicated that all of the procedures resulted in enriched subcellular fractions with minimal organellar cross-contamination. Notably, the activity of the soluble protein citrate synthase was higher in the mitochondrial fractions obtained with the modified protocol from frozen/fresh samples compared with the standard protocol. Therefore, our protocol improved the retention of soluble proteins in the mitochondrial fraction and may be suitable for subcellular fractionation of small amounts of frozen skeletal muscle samples.

Proteomic profiling of skeletal muscle in an animal model of overtraining.

Excessive training (i.e. overtraining, OT) may result in underperformance, which can be characterized by the time needed to re-establish performance (i.e. functional overreaching (FOR), nonfunctional overreaching, OT syndrome). The present study is an initial screening for proteins presenting altered abundance in the red (RG) and white (WG) portions of the gastrocnemius muscle from rats submitted to an OT protocol that induced FOR. In the RG, compared to the nontrained control, FOR demonstrated an increased abundance of proteins normally related to adaptation to endurance training (e.g. proteins of oxidative phosphorylation complexes, proteins related to lipid metabolism, antioxidants, and chaperones). In the WG, spots identified as mitochondrial aconitase and a component of the succinate dehydrogenase complex were downregulated in FOR, as were proteins related to myofibril stabilization; these latter were upregulated in the RG. This initial study shows that skeletal muscles with different fiber-type compositions respond differently to an OT period. Also, it is likely that actin-interacting proteins have an important role in muscle adaptation to endurance exercise.

Electrocatalytic determination of reduced glutathione in human erythrocytes.

The determination of reduced glutathione (GSH) in human erythrocytes using a simple, fast and sensitive method employing a glassy carbon electrode modified with cobalt tetrasulfonated phthalocyanine (CoTSPc) immobilized in poly(L: -lysine) (PLL) film was investigated. This modified electrode showed very efficient electrocatalytic activity for anodic oxidation of GSH, decreasing substantially the anodic overpotentials for 0.2 V versus Ag/AgCl. The modified electrode presented better performance in 0.1 mol l(-1) piperazine-N,N'-bis(2-ethanesulfonic acid) buffer at pH 7.4. The other experimental parameters, such as the concentration of CoTSPc and PLL in the membrane preparation, pH, type of buffer solution and applied potential, were optimized. Under optimized operational conditions, a linear response from 50 to 2,160 nmol l(-1) was obtained with a high sensitivity of 1.5 nA l nmol(-1) cm(-2). The detection limit for GSH determination was 15 nmol l(-1). The proposed sensor presented good repeatability, evaluated in terms of the relative standard deviation (1.5%) for n = 10. The modified electrode was applied for determination of GSH in erythrocyte samples and the results were in agreement with those obtained by a comparative method described in the literature The average recovery for these fortified samples was 100 +/- 1)%. Applying a paired Student's-t test to compare these methods, we could observe that, at the 95% confidence level, there was no statistical difference between the reference and the proposed methods.

Adequacies of skin puncture for evaluating biochemical and hematological blood parameters in athletes.

OBJECTIVE: The authors tested the effect of blood sampling (skin versus venous puncture) on some biochemical and hematological blood parameters in athletes to answer whether skin puncture could be used as a substitute for venous puncture. DESIGN: Comparative study of 2 methods of blood samples collection. SETTING: The blood was collected in the same athletes at 3 different moments of the preparatory training phase. PARTICIPANTS: Fourteen male indoor soccer players (22 +/- 1 years old) and 7 female handball players (18 +/- 1 years old) participated. MAIN OUTCOME MEASUREMENT: Blood was collected in heparin and K3EDTA by Vacutainer BD or Microvette Sarstedt system for biochemical and hematological analyses, respectively. RESULTS: There were no significant statistical differences between the 2 methods for the values of creatine kinase, urea, creatinine, lymphocytes, and platelets. The other hematological analyzes and uric acid exhibited significant higher values in skin blood, although they were all within the normal expected range. A high degree of correlation was observed between the 2 techniques for all parameters. CONCLUSIONS: Skin puncture is a reliable, easy, accurate, and less invasive sampling method for assessing hematological and some biochemical parameters in athletes, respecting that blood samples should always be obtained from the same site, especially in follow-up studies.