Serum creatine kinase MM sub-band determination by isoelectric focusing: a potential method for the monitoring of myocardial infarction

Fresh myocardium homogenates analyzed by thin-layer isoelectric focusing revealed the presence of two prominent creatine kinase (CK; EC 2.7.3.2) sub-bands, MMO (pI 7.10) and MM1 (pI 6.88), in approximately equal proportion. While these forms represented together as much as 85% of the cellular MM fraction, they accounted only for viz. 2.2 and 27.7% of the total serum MM activity when measured 8 h before the CK peak in patients with myocardial infarction. Incubation of the isolated MMO and MM1 with normal human serum demonstrated that the former turned to MM1 within 5 h at 37°C; further changes affecting MM1 gave rise to other sub-bands, MM2 (pI 6.70), MM3 (pI 6.45), and MM4 (pI 6.25). In our patient population, these three forms represented more than 75% of the serum CK-MM activity at the CK peak; hence, soon after the enzyme release, the serum MM isoenzyme mainly consists of degradation products arising from the labile MMO and MMl. Among the two cellular forms, MMO was the best related to the total enzyme activities and the most efficient for differentiating the patients with left ventricular failure from the others during the entire survey period (F = 3.8, p < 0.05). Because its presence in the blood provides evidence for a very recent CK release from the tissues, serum CK-MMO determinations might be proposed for following the extension of the lesion after a myocardial infarct.     

抗氧化剂预防低血糖所致肌肉损伤的研究

目的 探讨低血糖状态下血浆和组织中谷胱甘肽含量的变化以及还原型谷胱甘肽（GSH）对血清酶活性的抑制作用。方法 静脉注射胰岛素诱发低血糖，并持续60min，然后输注葡萄糖解除低血糖。结果发现诱发低血糖后6h，血浆GTSH，氧化型谷胱甘肽（GSSG），谷胱甘肽总量（TGSH）及GSSG/TGSH比值均明显升高，肝脏GSH和TGSH明显减少，心肌和骨骼肌CSSG和GSSG/TGSH比值明显增高，同时伴有血清酶（ALT，AST，LDH，CK）活性升高，预先注射CSH后诱发低血糖组，血浆GSSG和GSSG/TGSH比值明显下降，并抑制了血清酶的活性。结论 低血糖所致的血清酶活性的升高与体内谷胱甘肽氧化还原状态的改变有关。补充抗氧化剂GSH可以预防低血糖所致的肌肉损伤。     

Heterogeneity of serum creatine kinase isoenzyme MM in myocardial infarction: standardization of patterns by use of cord-blood serum

We investigated serum creatine kinase (CK; EC 2.7.3.2) isoenzyme MM in myocardial infarction, using isoelectric focusing in polyacrylamide gels. As many as 14 sub-band species were detected, and sequential sampling revealed a progressive anodal shift in their distribution. A nomenclature for sub-band typing is proposed. It denotes the major sub-bands as 1 (pl 6.91), 2 (pl 6.65), and 3 (pl 6.35); which are usually detected in sera of normal CK range. Abnormal sub-bands (associated with increases in the concentrations of CK) are denoted as a (pl 7.55), b (pl 7.35), c (pl 7.25), d (pl 7.05), e (pl 6.85), f (pl 6.72), g (pl 6.50), h (pl 6.40), i (pl 6.28), j (pl 6.20), and k (pl 6.15). Cord-blood sera gave highly reproducible CK-MM patterns characterized by prominent detection of sub-bands 1-3 and faint detection of c, e, f, g, h, i, and j. It is recommended as a standard for CK-MM sub-band typing.     

The protection of creatine kinase MM sub-bands by EDTA during storage

We examined the effect of a 5 mmol/1 concentration of EDTA on the stabilization of the five serum creatine kinase MM isoenzymes, resolved by thin-layer isoelectric focusing.In patient sera, total CK and CK-MB activities were stable during storage of the samples for two months at 4° C even in the absence of EDTA. However, EDTA stabilized the labile MM and MM1 sub-bands, which are the first to appear in the blood after the release from the damaged tissue and its addition to blood samples intended for determining the MM sub-band pattern is recommended.The stabilizing effect of EDTA was emphasized at higher temperatures. EDTA protected the CK-MM pattern in myocardium extracts made in normal serum and incubated at 37°C during 40 h, but was unnecessary when myocardium was homogenized in heat-inactivated serum. It is thought that EDTA could act by inhibiting a heat-labile component of human serum.     

Blood glutathione disulfide: in vivo factor or in vitro artifact?

BACKGROUND: The reported mean concentration of glutathione disulfide (GSSG) in human blood/erythrocytes varies widely (1 to >500 micromol/L), as does that of reduced glutathione (GSH) to a lesser extent. We have identified and investigated possible pitfalls in measurement of both GSH and GSSG. METHODS: We measured GSH and GSSG using a spectrophotometer with a modification of the GSH recycling method; the same samples were also measured by reversed-phase HPLC after derivatization of thiols (dithiothreitol was used to reduce disulfides) with monobromobimane. The thiol-bimane adduct was measured by a fluorescence detector. RESULTS: Measured GSH/GSSG concentrations were affected by the following: (a) oxidation of thiols in acidified samples; (b) oxidation after restoring neutral-alkaline pH; (c) oxidation during acid deproteinization; (d) shift in the GSH/GSSG equilibrium because of irreversible blocking of free thiols; and (e) reaction of electrophiles with amino groups. In particular, oxidation during sample deproteinization with acid influenced and produced artifacts (30-150 micromol/L GSSG was produced by this procedure); this phenomenon was directly correlated with the presence of oxygenated hemoglobin, being minimized by both oxygen deprivation and incubation in an atmosphere of 5% carbon monoxide. CONCLUSIONS: GSSG is present in healthy human blood at low concentrations (2-6 micromol/L), and most published data on GSSG may be affected by artifacts.     

Measurement of creatine kinase isoforms by agarose gel electrophoresis in the diagnosis of myocardial infarction.

To evaluate a method to quantitate the isoforms of serum creatine kinase isoenzyme MM (CK-MM) by agarose gel electrophoresis, sera of normal subjects (n = 74) and patients with acute myocardial infarction (n = 21) and other diseases (n = 67) were studied. The within-assay imprecision (CV) for CK-MM1, -MM2, and -MM3 was 1.9%, 0.8%, and 2.2% at the activity of 79, 105, and 64 U/L (30 degrees C, CK-NAC), respectively; while the assay-to-assay imprecision was 4.8%, 3.2% and 3.9%, respectively. The method could detect 5 U/L or more of any CK-MM isoform and was linear with respect to CK activity at values less than 1100 U/L. Sera from healthy subjects (n = 74) contained mainly CK-MM1 (mean = 48.5%), with lesser amounts of CK-MM2 (mean = 30.6%) and CK-MM3 (mean = 20.8%). The central 95-percentile reference range for the ratio of MM3/MM1 was 0.12-1.34 with mean = 0.49. The sensitivity of CK-MM3/MM1 ratio greater than 1.3 in the diagnosis of acute myocardial infarction employing the first available sample was 90% at a specificity of 91%, compared with a sensitivity of 81% and specificity of 87% for the conventional CK-MB assay. At CK-MM3/MM1 ratio of 1.6 or more, the specificity increased to 96% while sensitivity remained unchanged at 90%. This procedure for the quantitation of serum CK-MM isoforms is convenient, practical and suitable for inclusion in the routine panel of cardiac tests.     

Simultaneous automated measurement of serum total CK and CK-MM lsoform ratio in serum

We automated a two-step kinetic procedure for determining serum CK-MM isoform ratio using an immunoinhibition method. By measuring the total CK activity and the residual CK activity (serum CK-MM isoform) remaining after the inhibition by tissue CK-MM isoform specific monoclonal antibody reagent (CK-M01) the CKMM isoform ratio is calculated using the difference between total CK and residual CK activities divided by the residual CK activity. Linearities of total CK and residual CK assays were?7750 U/L and 2,500 U/L, respectively; within-run CVs of isoform ratio (N = 10) were 2.8 and 7.0% (mean 0.14 and 0.60), respectively. The MM³/MM¹ isoform ratio obtained with the proposed method (X) correlated well with the results of electrophoretic method (Y) according to the equation: Y = 0.98X ?0.3, r = 0.988. The normal reference range of isoform ratios obtained by assaying 1,222 serum samples from healthy subjects was 0.09–0.75. The isoform ratio increased after onset of chest pain, peaking at 2–6 hr thereafter. A mean isoform ratio of 1.86 was obtained with serum sample from 86 patients diagnosed as having an acute myocardial infarction (AMI). This method is accurate and highly sensitive, as the detection and early diagnosis of AMI can be completed in 10 min. © 1994 Wiley-Liss, Inc.     

Peripheral oxidative stress in relapsing-remitting multiple sclerosis

ObjectivesTo evaluate levels of oxidative stress in blood samples in patients with relapsing–remitting MS (RR-MS).Design and methodsPeripheral blood samples were collected from 24 RR-MS patients and 15 healthy controls. Levels of the following were measured: carbonylated proteins, 8-hydroxy-2′deoxyguanosine (8OHdG), total glutathione, reduced glutathione (GSH), oxidized glutathione (GSSG), GSH/GSSG ratio, superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GRd), glutathione-S-transferase (GST), myeloperoxidase (MPO), antioxidant gap, total antioxidant capacity (PAO), global oxidative stress (GOS), serum vascular cell adhesion molecule-1 (sVCAM-1) and serum inter-cellular adhesion molecule 1 (sICAM-1).ResultsValues for carbonylated proteins, 8OHdG, total glutathione, GSH, GSH/GSSG ratio, SOD, GRd and GOS were significantly higher in RR-MS patients than in healthy controls. By contrast, PAO, GSSG, GPx and GST were lower in RR-MS patients.ConclusionOxidative stress plays a major role in MS, and is observed prior to relapse.     

Monoclonal antibody inhibiting creatine kinase MM3 but not isoform MM1

Monoclonal antibody CKM-G01 inhibited greater than 99% of the activity of porcine and human creatine kinase(CK)-MM isoenzyme purified from muscle. However, it inhibited only 54% of CK-MM in human serum. Chromatofocusing of serum CK-MM showed that CKM-G01 inhibited 100% of MM3 but not isoform MM1. CKM-G01 inhibited CK-MM2 by 57%. CKM-G01 specifically inhibited only the original CK-M subunit and not the subunit modified by removal of C-terminal lysine by carboxypeptidase N. CKM-G01 can be used for assay of CK isoforms. We devised a new diagnostic reagent involving it, which requires no analytical separation of isoforms, based on the immunoinhibition method, and applied it to early diagnosis of acute myocardial infarction. The "inhibition index," (inhibited CK activity/total CK activity) x 100, increased more rapidly than did total CK and CK-MB. Evidently this diagnostic reagent can be used for easy, early diagnosis of acute myocardial infarction.     

Dramatic reduction of erythrocyte glucose-6-phosphate dehydrogenase activity in athletes participating in the ultradistance foot race "Spartathlon"

OBJECTIVE: To investigate the effect of a long-distance endurance exercise "Spartathlon" on erythrocyte glucose-6-phosphate dehydrogenase (G(6)PD) activity. MATERIAL AND METHODS: The study comprised 15 male runners, median age 36.5 years. Blood samples were obtained in the 15 min before the race and again within 15 min after the end of the race. Erythrocyte glutathione (GSH and GSSG) and plasma malonyldialdehyde were measured with HPLC methods, and total antioxidant capacity (TAC), total hyperoxides and G(6)PD activity with commercial kits. Lipids, uric acid and total bilirubin were determined with a clinical chemistry analyser. RESULTS: Total hyperoxides were found statistically reduced, whereas total bilirubin was measured elevated post-race. Interestingly, GSSG levels were found increased (167.3+/-12.0 versus 219.5+/-20.3 micromol/L; p<0.005) as well as GSSG/GSH ratio (16.0+/-1.3 versus 20.60+/-1.65; p<0.05) post-race. In contrast, G(6)PD activity was found remarkably decreased (8.72+/-3.10 versus 3.8+/-2.5 U/g Hb; p<0.0001) pre versus post the event. CONCLUSION: Red blood cell G(6)PD activity in athletes may be reduced post-race as a consequence of the modulation of NADP/NADPH levels and elevation of the erythrocyte GSSG, and especially GSSG/GSH ratio, resulting in an impairment of the hexose monophosphate shunt.     

肌酸激酶同工酶检测在急性心肌梗死中的临床价值

目的 探讨肌酸激酶（CK）MB和MM同工酶（CK-MB和CK-MM）亚型在急性心肌梗死（AMI）患者中的变化规律与其预后的关系,评价CK同工酶的亚型检测在AMI心肌早期再灌注、梗死延迟或再梗死诊断中的临床价值。方法 采用琼脂糖凝胶电泳系统将血清CK同工酶亚型分离为CK-MM3、CK-MM2、CK-MM1、CK-MB2和CK-MB1,并分析比较21例AMI患者血清CK同工酶亚型在发病后0～6小时、24小时和72小时的动态变化。结果 AMI患者血清CK-MB和CK-MM在发病后6h开始升高,其中以CK-MB2和CK-MM3升高为主,MB2／MB1〉1．36,MM3／MM1〉0．7;12-24小时达峰值,CK-MB／CK〉30％。15例早期再灌注的AMI患者血清CK、CK-MB和CK-MM在72小时下降至正常,但6例无早期再灌注患者仍处于较高水平,其中MB2／MB1〉1．29,MM3／MM1〉0．65。结论 CK同工酶的亚型检测能反映AMI患者心肌组织损伤的动态过程,可作为一项较灵敏的生化指标,有助于诊断AMI心肌早期再灌注、梗死延迟或再梗死。     

Changes in glutathione concentration in hypothermically perfused dog kidneys

A loss of glutathione from the kidney can cause increased sensitivity to oxygen free radical-induced injury. In this study we investigated the effects of kidney preservation on glutathione and how various glutathione precursors affect glutathione concentration in the dog kidney. During 5-day continuous machine perfusion of the kidney at 5 degrees C, a loss of glutathione from the cortex tissue was seen (24% +/- 1% glutathione remained after 5 days). Perfusion with reduced glutathione (GSH, 3 mmol/L) suppressed this loss (77% +/- 11% of glutathione remained after 5 days). Oxidized glutathione (GSSG) did not prevent the loss of glutathione. The addition of the three amino acids that make up glutathione (glycine, glutamic acid, and cysteine, 3 mmol/L each) also suppressed the loss of glutathione (82% +/- 13% remained at 5 days). The glutathione precursor, thioproline, a cysteine delivery compound, in combination with glycine and glutamic acid (3 mmol/L each), stimulated the synthesis of glutathione in the kidney during hypothermic perfusion (137% +/- 23% of control values at 5 days). The increase in tissue glutathione stimulated by GSH or other precursors was sensitive to the glutathione synthetase inhibitor, buthionine sulfoximine. This indicated the existence of active glutathione metabolism even at 5 degrees C in perfused kidneys. This study showed that in kidney preservation a loss of glutathione occurred that could be suppressed by the addition of various precursors for glutathione synthesis. The loss of glutathione from preserved kidneys may be one cause of posttransplant renal injury that could be prevented by use of the appropriate glutathione precursors.     

Oxidized forms of glutathione in peripheral blood as biomarkers of oxidative stress

BACKGROUND: Reduced glutathione (GSH) and its redox forms, glutathione disulfide (GSSG) and glutathionylated proteins (PSSG), are biomarkers of oxidative stress, but methodologic artifacts can interfere with their measurement. We evaluated the importance of correct sample handling during the preanalytical phase for GSH, GSSG, and PSSG measurement. METHODS: We used human blood for in vitro experiments with oxidants [tert-butylhydroperoxide (t-BOOH), diamide, and menadione]. For in vivo experiments, we used rats in which we cannulated the jugular and femoral veins for both oxidant administration and blood collection. We measured GSH, GSSG, and PSSG with HPLC with or without sample pretreatment with N-ethylmaleimide (NEM) to prevent artifacts. We also measured malondialdehyde (MDA) with HPLC, and protein carbonyls (PCO) with spectrophotometric procedures. RESULTS: When methodologic artifacts were prevented by pretreatment with NEM, GSSG results increased up to 3-fold over the basal concentrations, even in the presence of 5 micromol/L t-BOOH or diamide and 20 micromol/L menadione. PSSG increased by approximately 50% at 20 micromol/L t-BOOH or diamide and at 50 micromol/L menadione. PCO and MDA remained unchanged. In vivo oxidation treatments elicited immediate and significant increases in GSSG and PSSG over basal values (up to 200-fold), whereas PCO and MDA showed only slight variation 120 or 180 min after treatment. CONCLUSIONS: With the use of artifact-free measurement methods, GSH, GSSG, and PSSG are potentially powerful and reliable biomarkers of oxidative stress status and can be used to evaluate whether, and to what extent, oxidative stress may be involved in various diseases.     

Biliary excretion of glutathione and glutathione disulfide in the rat. Regulation and response to oxidative stress

Regulation of the biliary excretion of reduced glutathione (GSH) and glutathione disulfide (GSSG) and responses to selected model toxins were examined in male Sprague-Dawley rats. In control and phenobarbital-pretreated rats in which the intrahepatic concentration of GSH was modulated by the administration of diethyl maleate or acetaminophen, the biliary concentration of GSH was consistently lower than, but directly proportional to, the intrahepatic concentration of GSH. Furthermore, increments in bile flow produced by the infusion of sulfobromophthalein (BSP)-glutathione were associated with proportional increases in the biliary excretion of GSH, suggesting that GSH passes into bile passively along a concentration gradient. In contrast, GSSG appears to be secreted into bile against a steep concentration gradient. An increased hepatic production and biliary excretion of GSSG resulted from the administration of t-butyl hydroperoxide. Measurement of biliary GSSG and BSP during a constant infusion of the GSH adduct of BSP indicated that GSSG shares a common excretory mechanism with GSH adducts. Diquat, nitrofurantoin, and paraquat also markedly stimulated the biliary excretion of GSSG. On a molar basis, these compounds generated much more GSSG than a direct substrate for glutathione peroxidase such as t-butyl hydroperoxide, indicating that the compounds undergo redox-cycling with concomitant production of hydrogen peroxide. Aminopyrine (0.8 mmol/kg) also significantly increased biliary GSSG. This increase, however, was associated with a proportional increase in bile flow and in the biliary excretion of GSH such that the GSSG/GSH ratio in bile did not change. Acetaminophen and chloroform, two compounds generating electrophilic metabolites that deplete intrahepatic GSH, led to a progressive decrease in the biliary excretion of GSH and GSSG. Furosemide and dimethylnitrosamine, the electrophilic metabolites of which do not deplete hepatic GSH, minimally altered biliary GSH and GSSG. Similarly, carbon tetrachloride and iproniazid, which yield organic radical metabolites that can peroxidize membrane lipids, did not increase the biliary excretion of GSSG. This finding indicates that membrane-bound lipid hydroperoxides may not be good substrates for glutathione peroxidases. The measurement of the biliary excretion of GSSG and of the GSSG/GSH ratio in bile is a sensitive index of oxidative stress in vivo and thus complements other in vivo parameters for the study of reactive intermediates of xenobiotics such as the determination of covalent binding, the formation of lipid hydroxy acids, and the depletion of intracellular GSH.     

MM subisoenzymes of creatine kinase as an index of disease activity in polymyositis

Creatine kinase (CK; EC 2.7.3.2), although the most commonly measured enzyme for assessing disease activity in polymyositis, is not always a reliable indicator of the extent and severity of myositis. Recently, the CK-MM isoenzyme has been found to undergo post-synthetic modifications upon release into the serum, such that electrophoretically identifiable sub-bands or subisoenzymes--MM1, MM2, and MM3--are produced. To determine the diagnostic and discriminative value of these subisoenzymes in polymyositis, we analyzed CK and its MM subisoenzyme forms in serum samples from 22 patients with myositis and from 23 controls. In the presence of inflammatory myositis and increased total CK activity, MM-patterns correlated with the clinical trend, often more accurately than did measurements of total CK. MM1 proportions greater than 30% of total CK-MM or ratios of MM3 to MM1 less than 1 were associated with an improving or stable condition, whereas MM1 activity less than 30% or MM3/MM1 greater than 1 reflected a deteriorating course of disease. Patients whose disease was assessed to be clinically deteriorating were clearly distinguished from patients with improving disease by their subisoenzyme patterns (p less than 0.01). Thus these patterns add significantly to the information obtainable by routine blood analysis.     

Effect of creatine kinase-MM subtype composition on a CK-MB immunoinhibition assay

Immunoinhibition (INH) by use of polyclonal anti-human CK-M antibody may be used to measure CK-MB in serum. Previous studies have shown that inhibiting antibodies prepared against purified muscle extracts may inhibit CK-MM by greater than 99%. Using patients' sera and muscle homogenates incubated with human serum, we studied the effect of CK-MM subtype composition on an INH assay. We found that with increasing time from the CK-releasing event, e.g., myocardial infarction, or with longer in vitro incubation, the proportion of CK-MM1 increased and the proportion of uninhibited CK-MM increased from 0.2% to 0.7-0.8%. As a consequence, CK-MB activity may be overestimated by as much as 1.6% of total CK when uncorrected INH results are used. Inhibition was maximal in samples containing 100% CK-MM3, the tissue subtype. Because of the time-dependent change in CK-MM subtypes, published results for INH from studies in which CK-MM purified from muscle was used may not be directly applicable to clinical specimens.     

An immunologic pathway for intravascular catabolism of creatine kinase subform MM3.

In vitro incubation of the MM3 subform of human creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2, CK) with fresh human serum resulted in the formation of a complex of high relative molecular mass (Mr 320 kDa). The formed complex (macro CK-MM3) consists of both CK-MM3 and immunoglobulin A (IgA), and its amount of the formed complex was proportional to CK-MM3 activity and IgA concentration. Two molecules of CK-MM3 combined with one molecule of IgA, and the immunoglobulin inhibited the enzyme activity. As IgA does not form complexes with other subforms (CK-MM2 and CK-MM1) or CK-MB, the antigen specificity of IgA to CK-MM3 is definitely exacting. The circumstantial evidence suggests that macro CK-MM3 is a specific antigen-antibody complex. Macro CK-MM3 was detected in all of the examined sera of adult patients with more than 2001 U/1 CK activity (the positive percentage of macro CK-MM3 in all adult patients was 73%), but not detected in sera of patients who were younger than 12 months old. No relationship was observed between macro CK-MM3 and the patients' underlying diseases. Macro CK-MM3 formation suggested to be an immunologic pathway for intravascular catabolism of CK-MM3 when its activity increases.     

Oxygen radical generation of neutrophils: a reason for oxidative stress during marathon running?

Hematological parameters and blood markers that indicate oxidative stress, such as lipid peroxides (LPO), reduced and oxidized glutathione (GSH, GSSG), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), were measured in 18 marathon runners before, immediately after the race, and after 8 days of rest. In parallel, the oxygen radical generation of neutrophils (PMN) was measured by chemiluminescence in six randomly selected runners. After the race, a 4.4-fold enhanced PMN count and a 1.4-fold increased capacity to generate oxygen radicals of the PMN (2.20+/-0.38 vs. 3.12+/-0.69 arb. unit/10(6) cells) were found. Consequently, a 6.25-fold increased capacity to generate oxygen radicals of the post-run blood (7.26+/-1.3 vs. 45.40+/-10.3 arb. unit/ml blood) was calculated. This points to PMN as an important oxygen radical source established in the runners' blood, which could contribute to the oxidative stress indicated in the post-run blood by increased LPO (11.46+/-3.09 vs. 13.09+/-3.14 micromol/l plasma), GSSG (0.038+/-0.003 vs. 0.045+/-0. 005 mmol/l blood) and GSSG/GSH ratio (3.8+/-0.5 vs. 4.1+/-0.6%) and by decreased SOD (15.63+/-1.78 vs. 14.58+/-1.51 10(3)U/mmol Hb) and GSH-Px (485.1+/-107.1 vs. 434.9+/-101.7 U/mmol Hb). Despite the decline of the oxygen radical source during rest, the oxidative stress in the blood did not decrease in all runners.     

Differences in glutathione status and lipid peroxidation of red and white muscles: alterations following ischemia and reperfusion

Glutathione status and products from lipid peroxidation [measured as thiobarbituric acid reactive substances (TBARS)] were determined in red and white muscle tissue of the rat. Marked differences between both muscle types were found in reduced glutathione (GSH) and oxidized glutathione (GSSG) content, exhibiting 163% and 183%, respectively, higher levels in red than in white muscle tissue, while the ratio of GSSG/GSH showed no differences. These characteristics may be due to an adaptive mechanism related to the 48% higher baseline level of TBARS in red muscle tissue. Immediately after 4 h of tourniquet-ischemia GSH, GSSG, and TBARS were increased (16%, 32%, 45% in white muscle; 19%, 49%, and 42% in red muscle, respectively), whereas the GSSG/GSH ratio remained unchanged. During the subsequent reperfusion period, GSH decreased within 2 h by 39% in white and 89% in red muscle to a minimal level of 5 mmol/g protein in both types of muscle. No recovery from the depletion was observed up to 12 h of reperfusion. The GSH decrease was parallelled by a marked increase of the GSSG/GSH ratio (150% in white and 450% in red muscle) and followed by about 150% increase in TBARS in both muscle types. This suggests that the increase in damaging TBARS is a secondary event after depletion of cellular antioxidants. Treatment of the animals during the reperfusion period with methyl-prednisolone, deferoxamine, or superoxide dismutase and catalase did not prevent the GSH decrease, but were effective in reducing the GSSG/GSH ratio to near normal and reducing the TBARS increase by about 50%.     

Differences in glutathione status and lipid peroxidation of red and white muscles: alterations following ischemia and reperfusion

Glutathione status and products from lipid peroxidation [measured as thiobarbituric acid reactive substances (TBARS)] were determined in red and white muscle tissue of the rat. Marked differences between both muscle types were found in reduced glutathione (GSH) and oxidized glutathione (GSSG) content, exhibiting 163% and 183%, respectively, higher levels in red than in white muscle tissue, while the ratio of GSSG/GSH showed no differences. These characteristics may be due to an adaptive mechanism related to the 48% higher baseline level of TBARS in red muscle tissue. Immediately after 4 h of tourniquet-ischemia GSH, GSSG, and TBARS were increased (16%, 32%, 45% in white muscle; 19%, 49%, and 42% in red muscle, respectively), whereas the GSSG/GSH ratio remained unchanged. During the subsequent reperfusion period, GSH decreased within 2 h by 39% in white and 89% in red muscle to a minimal level of 5 mmol/g protein in both types of muscle. No recovery from the depletion was observed up to 12 h of reperfusion. The GSH decrease was parallelled by a marked increase of the GSSG/GSH ratio (150% in white and 450% in red muscle) and followed by about 150% increase in TBARS in both muscle types. This suggests that the increase in damaging TBARS is a secondary event after depletion of cellular antioxidants. Treatment of the animals during the reperfusion period with methyl-prednisolone, deferoxamine, or superoxide dismutase and catalase did not prevent the GSH decrease, but were effective in reducing the GSSG/GSH ratio to near normal and reducing the TBARS increase by about 50%.