imProving Outcomes after STroke clinical pilot trial protocol.

RATIONALE: One in three patients experience depression after stroke and this risk is consistent over time. A strategy to prevent depression that could be economically delivered to most stroke patients and ideally which also has a low likelihood of adverse events needs to be developed and evaluated. Aims POST aims to determine whether a simple intervention (postcards) prevents depression (Hospital Anxiety and Depression rating Scale, HADS depression subscale score > or =8) in patients with a recent stroke. Secondary end-points include reduced anxiety (HADS anxiety subscale score > or =8) and improved health-related quality of life in patients with a recent stroke. DESIGN: A single-centre randomised, double-blind, pilot trial to prevent depression in patients with a recent (within 8 weeks) stroke presenting to hospital. Patients will be enrolled over 12 months and randomised to receive three trial-specific assessments (baseline, 3- and 6-month assessments of mood, HRQoL and social functioning), or three trial-specific assessments plus a postcard sent centrally in a sealed envelope at 1, 2, 3, 4 and 5 months after discharge from hospital. Blinded follow-up telephone assessments will be conducted for both groups. STUDY OUTCOMES AND SAMPLE SIZE: For the primary end-point the POST trial will have 80% power to detect a relative risk of 0.4 given an incidence of depression of 30%. For the secondary aims POST has 90% power to detect a difference of 3 points on the HADS depression subscale (assuming a standard deviation of 6 points) between randomised groups. This includes an inflation factor of 15% to account for patients lost to follow-up. DISCUSSION: Evidence of efficacy will determine whether a multi-centre, international trial is warranted.

Effect of pH and ionic strength on mu- and m-calpain inhibition by calpastatin.

The objectives of this study were to determine the extent to which pH and ionic strength influence mu- and m-calpain activity and the inhibition of calpains by calpastatin. Calpastatin, mu-calpain, and m-calpain were purified from at-death porcine semimembranosus. Mu-calpain or m-calpain (0.45 U) were incubated with the calpain substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methyl coumarin in the presence of calpastatin (0, 0.15, or 0.30 U of calpain inhibitory activity) under the following pH and ionic strength conditions: pH 7.5 and 165 mM NaCl or 295 mM NaCl; pH 6.5 and 165 mM NaCl or 295 mM NaCl; and pH 6.0 and 165 mM NaCl or 295 mM NaCl. The reactions were initiated with addition of 100 microM (mu-calpain) or 1 mM CaCl2 (m-calpain), and calpain activity was recorded at 30 and 60 min. Mu-calpain had the greatest (P < 0.01) activity at pH 6.5 at each ionic strength. Higher ionic strength decreased mu-calpain activity (P < 0.01) at all pH conditions. Inhibition percent of mu-calpain by calpastatin was not affected by pH; however, it was influenced by ionic strength. Inhibition of mu-calpain by calpastatin was higher (P < 0.01) at 295 mM NaCl than at 165 mM NaCl when 0.3 units of calpastatin were included in the assay. Activity of m-calpain was greater (P < 0.01) at pH 7.5 than at pH 6.5. m-Calpain activity was not detected at pH 6.0. Inhibition of m-calpain was greater (P < 0.01) when 0.15 and 0.3 U calpastatin were added at pH 6.5 than 7.5 at 165 mM NaCl, whereas percentage inhibition of m-calpain was greater (P < 0.01) at 295 mM than 165 mM NaCl at pH 7.5 and 6.5. These observations provide new evidence that defines further the influence of pH decline and increased ionic strength on mu-calpain, m-calpain, and calpastatin activity, thereby helping to more accurately define a role for these enzymes in the process of postmortem tenderization.

Oxidative environments decrease tenderization of beef steaks through inactivation of mu-calpain.

This study was designed to test the hypothesis that oxidative conditions in postmortem (PM) tissue decrease calpain activity and proteolysis, subsequently minimizing the extent of tenderization. To achieve different levels of oxidation, the diets of beef cattle were supplemented with vitamin E for the last 126 d on feed, and beef steaks were irradiated early PM. Ten steers were fed a finishing diet with the inclusion of vitamin E at 1,000 IU per steer daily (VITE). Another 10 beef steers were fed the same finishing diet without added vitamin E (CON). At 22 to 24 h PM, strip loins from each carcass were cut into 2.54-cm-thick steaks and individually vacuum packaged. Within 26 h PM, steaks were irradiated at 0 or 6.4 kGy and then aged at 4 degrees C for 0, 1, 3, 7, and 14 d postirradiation. Steaks from each time point were used to determine Warner-Bratzler shear force (WBSF) and calpain activity, and for western blotting of sarcoplasmic proteins and myofibrillar proteins. Calpastatin activity was determined at 0, 3, and 14 d postirradiation. At 1, 3, 7, and 14 d postirradiation, WBSF values of irradiated steaks were higher (P < 0.03) than for nonirradiated steaks. Western blots of troponin-T and desmin showed decreased proteolysis in irradiated samples compared with nonirradiated samples. At 2 d PM, troponin-T degradation products were more evident (P < 0.03) in nonirradiated steaks supplemented with VITE than nonirradiated steaks from the CON diet. Similarly, VITE treatment resulted in steaks with lower (P < 0.05) calpastatin activity at 1 d PM than in steaks from steers fed the CON diet. Irradiation diminished the rate of calpastatin inactivation. Irradiated samples, regardless of diet, had no detectable levels of intact titin or nebulin. Irradiation decreased mu-calpain activity and autolysis, whereas mu-calpain activity was not affected by diet or irradiation. Inactivation of mu-calpain by oxidation during early times PM decreased the amount of myofibrillar proteolysis, thereby decreasing the extent of tenderization of beef steaks.

Influence of early postmortem protein oxidation on beef quality.

The objective of this study was to examine the effect of early postmortem protein oxidation on the color and tenderness of beef steaks. To obtain a range of oxidation levels, the longissimus lumborum muscles (LM) from both strip loins of 20 steers fed either a finishing diet with vitamin E (1,000 IU per steer daily, minimum of 126 d [VITE]; n = 10 steers) or fed the same finishing diet without vitamin E (CON; n = 10 steers) were used. Within 24 h after slaughter, the LM muscle from each carcass was cut into 2.54-cm-thick steaks and individually vacuum packaged. Steaks from each steer were assigned to a nonirradiated group or an irradiated group. Steaks were irradiated within 26 h postmortem, and were aged at 4 degrees C for 0, 1, 3, 7, and 14 d after irradiation. Steaks from each diet/irradiation/aging time treatment were used to determine color, shear force, and degree of protein oxidation (carbonyl content). Steaks from steers fed the VITE diet had higher (P < 0.01) alpha-tocopherol contents than steaks from steers fed the CON diet. Immediately following irradiation, steaks that had been irradiated had lower (P < 0.05) L* values regardless of diet. Irradiated steaks, regardless of diet, had lower a* (P < 0.05) and b* (P < 0.01) values than nonirradiated steaks at all aging times. Carbonyl concentration was higher (P < 0.05) in proteins from irradiated steaks compared to nonirradiated steaks at 0, 1, 3, and 7 d postirradiation. Immunoblot analysis showed that vitamin E supplementation decreased the number and extent of oxidized sarcoplasmic proteins. Protein carbonyl content was positively correlated with Warner-Bratzler shear force values. These results indicate that increased oxidation of muscle proteins early postmortem could have negative effects on fresh meat color and tenderness.

Creatine monohydrate supplemented in swine finishing diets and fresh pork quality: III. Evaluating the cumulative effect of creatine monohydrate and alpha-lipoic acid.

The objective of this study was to evaluate short-duration supplementation of alpha-lipoic acid (ALA) and creatine monohydrate (CMH) to improve fresh pork quality. Forty-eight commercial hybrid barrows were blocked by BW and randomly allotted to one of four treatments: 1) no CMH or ALA; 2) supplementation of 24 g of CMH(-1) x pig(-1) x d(-1); 3) supplementation of 600 mg ALA(-1) x pig(-1) x d(-1); or 4) combined CMH and ALA supplements. Twelve pigs per treatment were individually penned with ad libitum access to water and a finishing diet. Treatments were hand-fed to individual pigs daily (divided into three equal doses) for 5 d before slaughter at 113 kg BW in two separate groups of 24 pigs each. Intramuscular pH was recorded at 45 min postmortem and again at 24 h in the ham semimembranosus (SM) and the longissimus muscle (LM) between the 10th and 11th rib. A Meatcheck (SFK Technology, Peosta, IA) conductivity probe was inserted in the same anatomical locations as pH measurement, providing an index value (PY) from 0 to 100 (a higher index value indicates more intact muscle cells and higher water-holding capacity). Color (L, a, b values) measurements were obtained at 24 h postmortem on the ham gluteus medius (GM), SM, and LM. Two 2.54-cm-thick loin chops were removed from the loin for determination of Warner-Bratzler shear force and glycolytic potential. The intact SM and the posterior portion of the boneless loin were vacuum-packaged and stored for 7 d to determine purge loss. Creatine-supplemented pigs had a higher (P = 0.03) PY value in the SM (66.67) at 45 min postmortem than either ALA, singularly (63.50), or in the combined CMH/ALA (62.27) treatments. (A higher PY index indicates superior water-holding capacity.) Lipoic acid supplementation resulted in the highest pH at 45 min (P = 0.029). These results justify further evaluation of the potential positive influence of supplementing alpha-lipoic acid to improve pork quality.