L-cysteine's effect on modulated rat brain enzymes with forced swimming.

BACKGROUND: Forced exercise is associated with oxidative stress, and L-cysteine (L-cys) administration reduces free-radical production. AIM: To investigate whether L-cys (5 mg/kg) intraperitoneal administration can ameliorate modulated total antioxidant status (TAS), protein concentration, and the activities of acetylcholinesterase (AChE), (Na+,K+)-ATPase, and Mg2+-ATPase in rat brain after 2 and 3 hr of forced swimming. METHODS: TAS, protein, and enzyme activities were measured spectrophotometrically before and after 2 and 3 hr of exercise without or with L-cys administration. RESULTS: TAS concentration (55.6 +/- 1.5 vs. 42.1 +/- 1.0 vs. 37.4 +/- 1.2 micromol/L, p < .001), protein concentration (5.68 +/- 0.36 vs. 5.40 +/- 0.18 vs. 4.01 +/- 0.16 mg/ml, p < .01), and AChE activity (0.89 +/- 0.05 vs. 0.61 +/- 0.04 vs. 0.48 +/- 0.03 DeltaOD/min x mg protein, p < .001) were significantly reduced, whereas Na+,K+-ATPase (6.00 +/- 0.36 vs. 10.44 +/- 1.04 vs. 11.90 +/- 1.21 micromol phosphorus inorganic/hr x mg protein, p < .001) and Mg2+-ATPase activity (7.20 +/- 0.65 vs. 10.88 +/- 1.08 vs. 11.55 +/- 1.22 mmol phosphorus inorganic/hr x mg protein, p < .001) were statistically significantly increased after 2 and 3 hr of forced exercise. Post-L-cys administration, AChE activity was decreased (0.90 +/- 0.04 vs. 0.47 +/- 0.02 DeltaOD/min x mg protein, p < .001) and remained unaltered (0.64 +/- 0.04 vs. 0.67 +/- 0.04 DeltaOD/min x mg protein, p > .05) 2 and 3 hr postexercise (0.47 +/- 0.02 vs. 0.54 +/- 0.02 DeltaOD/min x mg protein, p > .05). Na+,K+-ATPase was decreased and remained unchanged (1.85 +/- 0.17 vs. 1.77 +/- 0.19 mumol phosphorus inorganic/hr x mg protein, p > .05) 2 and 3 hr postswimming (1.91 +/- 0.19 vs. 2.06 +/- 0.17 mumol phosphorus inorganic/hr x mg protein, p > .05). Mg2+-ATPase activity was similar with L-cys supplementation pre- vs. postswimming. CONCLUSIONS: L-cys administration might ameliorate modulated rat brain enzyme activities induced by free-radical production during forced swimming.

Evidence for the participation of the stimulated sympathetic nervous system in the regulation of carnitine blood levels of soccer players during a game.

Catecholamines and carnitine blood levels are closely implicated with training. The aim of the study was to investigate the effect of sympathetic nervous system stimulation on carnitine and its fraction levels during training. Blood was obtained from 14 soccer players pregame, at intermission, and postgame. Catecholamines were measured with high-performance liquid chromatography methods; muscle enzymes creatine kinase and lactate dehydrogenase as well as lactate, pyruvate, and total antioxidant status with commercial kits; and carnitine and fraction levels with tandem mass spectrometry. Total antioxidant status (2.97 +/- 0.13 vs 0.96 +/- 0.10 mmol/L, P < .01) as well as free carnitine levels (20.47 +/- 4.0 vs 12.30 +/- 2.8 micromol/L, P < .001) were remarkably decreased especially postgame. Total acylcarnitines (5.20 +/- 1.8 vs 9.42 +/- 3.0 micromol/L, P < .001) and especially total very long-chain acylcarnitines (0.80 +/- 0.01 vs 1.85 +/- 0.03 micromol/L, P < .001) as well as catecholamine levels (adrenaline: 230 +/- 31 vs 890 +/- 110 pmol/L, P < .01; noradrenaline: 1.53 +/- 0.41 vs 3.7 +/- 0.6 nmol/L, P < .01) were significantly increased in players postgame. A statistically significant inverse correlation was found between adrenaline and free carnitine (r = -0.51, P < .01); and a positive correlation was found between adrenaline, total acylcarnitines (r = 0.58, P < .01), and total long-chain acylcarnitine (r = 0.49, P < .01). The significant positive correlation of adrenaline levels with total acylcarnitine and total long-chain acylcarnitine blood levels in athletes as well as the inverse correlation with free carnitine levels may indicate participation of the stimulated sympathetic nervous system in the regulation of some carnitine fraction levels during exercise.

The effect of alpha-Tocopherol supplementation on training-induced elevation of S100B protein in sera of basketball players.

OBJECTIVE: To investigate the effect of alpha-Tocopherol (alpha-T) supplementation on S100B elevated serum levels in basketball players' training. DESIGN: Blood was obtained from 10 basketball players pre-exercise (group A), post-exercise (group B) and after 30 days on alpha-T (200 mg/24 h orally) supplementation pre- (group C) and post-training (group D). Blood samples were taken for the evaluation of total antioxidant status (TAS), alpha-T and catecholamines in plasma and S100B and muscle enzyme levels in serum. METHODS: TAS, muscle enzymes: creatine kinase (CK), lactate dehydrogenase (LDH), and S100B protein levels were measured with commercial kits, whereas alpha-T and catecholamine levels with HPLC methods. RESULTS: TAS was found higher in the groups with alpha-T addition (groups C and D) than in the other ones. On the contrary, CK, LDH and S100B were remarkably lower (116.8+9.5 U/L, 427+22 U/L, 0.18+0.04 microg/L, respectively) in group D than those in group B (286+12 U/L, 688+26 U/L, 0.28+0.06 microg/L, p<0.001, respectively). S100B levels were negatively correlated with TAS (r=-0.64, p<0.001) and positively with CK levels (r=0.58, p<0.001). CONCLUSIONS: alpha-T supplementation may reduce S100B increased release from muscle and nerves induced by training. S100B serum evaluation may be a useful biomarker for the effect of training on the participation of the neuromuscular system.

An in vivo and in vitro study of erythrocyte membrane acetylcholinesterase, (Na+, K+)-ATPase and Mg2+-ATPase activities in basketball players on alpha-tocopherol supplementation. The role of L-carnitine.

BACKGROUND & AIMS: Vitamin E (alpha-tocopherol, alpha-Te) and carnitine reduce lipid peroxidation. THE AIM WAS TO: To investigate the erythrocyte membrane acetylcholinesterase (AChE), Na+, K+-ATPase and Mg2+-ATPase activities in basketball players with or without alpha-Te supplementation, before and after training. In vitro, we aimed to find out any additional effect of L-carnitine (L-C) on the modulated enzyme activities. SUBJECTS AND METHODS: Blood was obtained from 10 players before (group A), after exercise (group B) and after 1 month on alpha-Te (200 mg/24 h orally) supplementation before (group C) and after the game (group D). Lactate, pyruvate, muscle enzyme activities and total antioxidant status (TAS) were measured with commercial kits. Catecholamines and alpha-Te were determined with HPLC methods and membrane enzyme activities spectrophotometrically. RESULTS: Lactate, pyruvate, muscle enzymes and catecholamine levels were increased (P<0.001) in all groups after training. Alpha-Te levels and Mg2+-ATPase activity remained unaltered before and after exercise. TAS was decreased in the groups after the game. AChE activity was increased in group B (P<0.01) and decreased in group D (P<0.01). After the exercise, Na+, K+-ATPase activity was increased in group B and remained unaltered in group D. In vitro incubation of membranes from group D with L-C resulted in a partially restoration of the membrane AChE activity, whereas Na+, K+-ATPase and Mg2+-ATPase activities were found unchanged. CONCLUSIONS: Alpha-Te supplementation in basketball players results in an increase of TAS and AChE activity, whereas the other enzyme activities were found unchanged. L-C addition may restore AChE activity, which was modulated by training in players on alpha-Te.

alpha-Tocopherol supplementation restores the reduction of erythrocyte glucose-6-phosphate dehydrogenase activity induced by forced training.

BACKGROUND: Erythrocyte glucose-6-phosphate dehydrogenase (G6PD) activity is closely related to free radical production. alpha-Tocopherol (alpha-T) is implicated with the reduction of lipid peroxidation. AIM: To investigate the effect of training and alpha-T supplementation on the erythrocyte G6PD activity. METHODS: Blood was obtained from 10 basketball players pre-game (group A), post-game (group B) and after 30 days on alpha-T (dl-alpha-tocopheryl-acetate, 200mg 24h(-1) orally) supplementation pre- (group C) and post-training (group D). alpha-T and catecholamines were evaluated with HPLC methods and creatine kinase, lactate dehydrogenase, total antioxidant status (TAS) and G6PD activity with commercial kits. RESULTS: TAS was increased in the groups with alpha-T addition (groups C and D). Post-exercise, TAS and G6PD activity were remarkably higher (2.10+/-0.13mmoll(-1), 7.92+/-1.5Ug(-1)Hb, respectively) in group D than those in group B (0.92+/-0.10mmoll(-1), 4.8+/-1.4Ug(-1)Hb, p<0.01, respectively). G6PD activity positively correlated with TAS (r=0.64, p<0.001) in all the studied groups. CONCLUSIONS: Supplementation with alpha-T may protect G6PD activity from reduction induced by forced training.

alpha-tocopherol supplementation reduces the elevated 8-hydroxy-2-deoxyguanosine blood levels induced by training in basketball players.

BACKGROUND: The aim of the present study was to investigate the effect of alpha-tocopherol (alpha-Te) supplementation on DNA oxidative damage induced by heavy training in basketball players. METHODS: Blood was obtained from 10 players before (group A) and after training (group B) and after 1 month on alpha-Te (200 mg/day, orally) supplementation, before (group C) and after training (group D). Total antioxidant status (TAS), muscle enzyme activities and the biomarker of DNA oxidation, 8-hydroxy-2-deoxyguanosine (8-OHdG), were measured using commercial kits. alpha-Te and catecholamine blood levels were determined using HPLC methods. RESULTS: TAS was higher in the groups with alpha-Te (groups C and D). Levels of 8-OHdG and muscle creatine kinase (CK) and lactate dehydrogenase (LDH) were remarkably lower (0.20+/-0.03 ng/mL, 120+/-15 U/L and 430+/-90 U/L, respectively) in the group with alpha-Te (group D) than in group B (0.42+/-0.05 ng/mL, 286+/-12 U/L and 688+/-88 U/L, respectively; p<0.001). 8-OHdG levels were negatively correlated to TAS and positively to CK levels. CONCLUSIONS: alpha-Te supplementation may reduce DNA oxidation induced by training by protecting muscle cell "death" from glutamate entry and/or by elevation of TAS via amelioration of lipid peroxidation.

The effect of in vitro homocystinuria on the suckling rat hippocampal acetylcholinesterase.

UNLABELLED: Homocystinuria is due to enzymatic deficiencies resulting in elevated blood levels of homocysteine (Hcy), homocystine (Hci), and/or methionine (Met) and the clinical presentation of mental retardation, seizures, and cardiovascular disease. Since these symptoms may be closely implicated with acetylcholinesterase (AChE) activity, we aimed to investigate whether this metabolic disorder affects the hippocampal AChE activity in 21 days suckling Wistar rat hippocampus. Various concentrations of Hcy, Hci (0.05-0.5 mM), or Met (0.05-2 mM) as well as Mixture A (Mix A) (0.3 mM (Hcy)+0.2 mM (Hci)+1.0 mM (Met) = in vitro cystathionine beta-synthase deficiency homocystinuria), Mix B1 (Hcy 0.3 mM + Hci 0.2 mM=in vitro severe methylenetetrahydrofolate reductase deficiency homocystinuria) or Mix B2 (Hcy 0.1 mM+Hci 0.05 mM=in vitro mild methylenetetrahydrofolate reductase deficiency homocystinuria) were preincubated with homogenized hippocampii or with eel Electrophorus electricus pure AChE. AChE was evaluated spectrophotometrically. Hcy or Met stimulated hippocampal AChE by 50% (p < 0.001) at low concentrations of the amino acids (up to 0.3-0.5 mM), whereas Hci inhibited the enzyme by 40% (p < 0.001). Mix A, Mix B1, or Mix B2 activated hippocampal AChE by 40, 30, (p < 0.001), and 12% (p < 0.01), respectively. In contrast, the S-containing amino acids, Mix A, Mix B1, Mix B2 failed to affect the pure AChE activity. CONCLUSIONS: a) The presence of -SH group in Hcy and Met may result in hippocampal AChE stimulation and the redox isomer Hci in the inhibition of the enzyme, probably by producing free radicals, and b) The SH-amino acids seem to affect the hippocampal enzyme indirectly, possibly by lipid(s)-protein modifications(s) and Hci by inducing oxidative stress, since no effect was observed on pure AChE activity.

The beneficial effect of L-cysteine supplementation on DNA oxidation induced by forced training.

BACKGROUND: Forced training is closely implicated with free radicals production and indication of tissue damage as well as DNA oxidation. AIM: To investigate the effect of L-cysteine (L-cys) supplementation on DNA oxidative damage found in basketball players after forced training. SUBJECTS AND METHODS: Blood was obtained from 10 players pre-game (group A), post-game (group B) and after 1 month L-cys (0.5 g 24 h(-1), orally) supplementation, pre- (group C) and post-training (group D). Total antioxidant status (TAS) and the biomarker of DNA oxidative damage 8-hydroxy-2-deoxyguanosine (8-OHdG) as well as creatine kinase (CK) and lactate dehydrogenase (LDH) were measured with commercial kits. RESULTS: TAS was increased in the groups with L-cys (group C and group D). Post-exercise 8-OHdG levels, CK and LDH were remarkably lower (0.16+/-0.03 ng ml(-1), 115+/-15 U l(-1), 417+/-90 U l(-1), respectively) in group D than those in group B (0.36+/-0.05 ng ml(-1), 286+/-12 U l(-1), 688+/-88U l(-1), p<0.001, respectively). 8-OH dG levels were negatively correlated with TAS(r=-0.718, p<0.01) and positively with CK levels(r=0.590, p<0.01). CONCLUSIONS: L-cys supplementation in basketball players may reduce DNA damage induced by training. The sulfur-containing amino acid may protect muscle cells "death" by increasing TAS and the cellular defense against oxidative stress.

Erythrocyte membrane acetylcholinesterase activity in subjects with MTHFR 677C-->T genotype.

BACKGROUND: Increased homocysteine (Hcy) blood levels are correlated with vascular and neurological problems. AIM: The aim of the present study was to investigate erythrocyte membrane acetylcholinesterase (AChE) activity in subjects with the MTHFR C677T genotype in relation to Hcy. METHODS: Blood was obtained from 22 individuals with the MTHFR C677T genotype before and after folic acid supplementation and once from controls (n = 30). Plasma folate, vitamin B(12) and total antioxidant status (TAS) were measured with commercial kits, Hcy by a HPLC method and membrane enzyme activity spectrophotometrically. RESULTS: In MTHFR C677T carriers, AChE activity was significantly higher (4.20 +/- 0.12 x mg protein) and decreased to normal levels (3.14 +/- 0.10 x mg protein; p < 0.001) after therapy. TAS differed slightly before and after treatment. Hcy levels were significantly higher before (22.4 +/- 2.8 microM) compared to after (12.1 +/- 2.0 microM; p < 0.001) therapy and compared to controls (10.5 +/- 2.5 micromol/L; p < 0.001). In an in vitro study, incubation of Hcy-activated membrane AChE from controls with phenylalanine resulted in restoration of activity, but failed to reverse the stimulated enzyme from hyperhomocysteinaemic MTHFR C677T subjects before therapy. Alanine incubation protected the enzyme from Hcy activation in controls. CONCLUSIONS: Increased membrane AChE activity may be due to high Hcy levels. In vitro, phenylalanine reversed the Hcy activation of the membrane enzyme from controls and alanine protected it from Hcy action.

The effect of diet on total antioxidant status, erythrocyte membrane Na+,K+-ATPase and Mg2+-ATPase activities in patients with classical galactosaemia.

OBJECTIVE: Classical galactosaemia is characterized by high levels of galactose-1-phosphate (Gal-1-P), galactose and galactitol. In vitro studies have shown modulation of the rat brain Na+,K+-ATPase and Mg2+-ATPase activities by Gal-1-P. The aim of this study was to evaluate the erythrocyte membrane Na+,K+-ATPase and Mg2+-ATPase activities in galactosaemic patients and to correlate them to Gal-1-P, total antioxidant status (TAS) and membrane protein content (PC). PATIENTS AND METHODS: Nine patients (N=9) originally on "loose diet" (group B) were requested to follow their diet strictly (group A). Twelve healthy children were the controls (group C). The activities of the enzymes, TAS and Gal-1-P in blood were determined spectrophotometrically. In the in vitro study, erythrocyte membranes from controls were preincubated with Gal-1-P (300 microM), and then with l-cysteine (0.83 mM) or reduced glutathione (0.83 mM) whereas these from the patients with the antioxidants only. RESULTS: Na+,K+-ATPase, Mg2+-ATPase, TAS and PC were significantly (P<0.001) reduced (0.31+/-0.03, 1.7+/-0.2 micromol Pi/hxmg protein, 0.89+/-0.02 mmol/l, 36.8+/-2.0 g/l, respectively) in group B as compared with those of group A (0.58+/-0.06, 2.5+/-0.2 micromol Pi/hxmg protein, 1.41+/-0.11 mmol/l, 51.5+/-3.1g/l, respectively) and controls (0.67+/-0.05, 3.2+/-0.2 micromol Pi/hxmg protein, 1.65+/-0.12 mmol/l, 64.0+/-3.5 g/l, respectively). Gal-1-P levels in group B was significantly higher than those in group A and controls. Positive correlation coefficients were found between the enzyme activities, PC and TAS whereas Gal-1-P inversely correlated to the enzyme activities. Incubation of the erythrocyte membranes from the patients with the antioxidants failed to restore the activities of inhibited enzymes, whereas the inhibition by Gal-1-P in controls was reversed. CONCLUSIONS: High blood Gal-1-P concentrations resulted in low TAS and PC. The inhibition of Na+,K+-ATPase and Mg2+-ATPase may be due to the presence of free radicals and/or the elevated Gal-1-P.