Stylianos Tsakiris

Effect of diet on plasma total antioxidant status in phenylketonuric patients

Background: Phenylketonuria (PKU), an inborn error of phenylalanine (Phe) metabolism, is treated with a low Phe lifelong diet, which is a vegetarian and contains many antioxidants.Aim: The aim of this study was to evaluate the effect of diet on plasma total antioxidant status in our PKU patients.Methods, Results: Twenty-two PKU patients on strict diet (group A), 24 PKU patients who did not accurately follow their dietary control (group B) and 40 healthy children (controls) of comparable age took part in this study. Nutrients, as well as blood levels of lipids, vitamin C, β-carotene and α-tocopherol were evaluated in all groups. Vitamin C intake and its blood levels did not differ between the groups. However, the intake of β-carotene, α-tocopherol (2211±116, 14±1.0u2005mg/24u2005h) and their blood levels (0.7±0.09, 34±0.9u2005µmol/l) in group A were statistically significantly higher (P<0.001) as compared with those of group B (1352±118, 10±1.0u2005mg/24u2005h and 0.49±0.08, 22±0.6u2005µmol/l) and controls (1290±120, 9.0±0.9u2005mg/24u2005h and 0.40±0.09, 24±1.6u2005µmol/l). Lipid intakes and their blood levels were lower in patients on the strict diet. Plasma total antioxidant status was higher in the same group of patients (group A). Additionally, positive correlations were found between the antioxidant vitamin blood levels and the plasma total antioxidant status in the groups, especially in the group A. PKU patients of group A showed significantly higher antioxidant status (1.6±0.2u2005mmol/l) as compared with those of group B (1.0±0.19u2005mmol/l; P<0.001) and controls (1.01±0.2u2005mmol/l).Conclusions: The high plasma antioxidant status in patients with PKU, especially in those with a good compliance with their diet, is possibly due to the amounts of antioxidants which are present in their special low Phe vegetarian diet.

Alpha-tocopherol supplementation prevents the exercise-induced reduction of serum paraoxonase 1/arylesterase activities in healthy individuals

Objective:To investigate PON 1/Aryl activities in basketball players with or without α-T supplementation pre- and post-training. Vitamin E (α-tocopherol, α-T) reduces lipid peroxidation. Paraoxonase 1/arylesterase (PON 1/Aryl) activities are closely related to oxidation and atherogenesis.Subject/Methods:Blood was obtained from 10 players pre- (group A), post-exercise (group B) and after 1 month on α-T (200u2009mg per 24u2009h orally) supplementation pre- (group C) and post-exercise (group D). Lactate, pyruvate, muscle enzyme activities, creatine kinase, lactate dehydrogenase and total antioxidant status (TAS) were measured with commercial kits. Catecholamines and α-T were determined with high-performance liquid chromatography methods and PON 1/Aryl activities spectrophotometrically.Results:Lactate, pyruvate, muscle enzyme activities and catecholamines were increased (P<0.001) in all groups post-training. Alpha-T levels remained unaltered pre- vs post-exercise. TAS was decreased in all the groups post training. PON 1/Aryl activities were significantly decreased post-exercise (group B) (PON1: 65±12u2009Uu2009min−1u2009ml−1, Aryl: 58±14u2009KUu2009min−1u2009ml−1) as compared to those pre-exercise (group A) (PON1: 142±16u2009Uu2009min−1u2009ml−1, Aryl: 114±12u2009KUu2009min−1u2009ml−1, P<0.001). In contrast, the studied enzyme activities remained practically unaltered after α-T supplementation pre- vs post-training. Both enzyme activities positively correlated to TAS (r=0.60, P<0.001).Conclusions:Alpha-T supplementation may result in protection of the enzyme PON 1/Aryl activities from free radical production.

Effect of L-carnitine administration on the modulated rat brain protein concentration, acetylcholinesterase, Na+K+-ATPase and Mg2+-ATPase activities induced by forced swimming

Background: Forced exercise produces free radicals and L-carnitine (L-C) administration reduces oxidative stress. Aim: To investigate whether short (2 hours) or prolonged (3 hours) forced swimming could modulate total antioxidant status (TAS), protein concentration and activities of acetylcholinesterase (AChE), Na+K+-ATPase and Mg2+-ATPase in rat brain following intraperitonal administration of L-C (300 mg/kg). Methods: TAS, protein and enzyme activities were measured spectrophotometrically. Results: TAS, protein concentration and AChE activity were reduced, whereas Na+K+-ATPase and Mg2+-ATPase were significantly increased after either 2 or 3 hours of training. L-C administration resulted in a profound restoration of TAS and protein concentration whereas AChE and Na+K+-ATPase were increased before exercise, followed by AChE restoration and Na+K+-ATPase reduction after exercise. Mg2+-ATPase remained unchanged. An in vitro study using L-C incubation of brain homogenates previously treated with L-C resulted in complete restoration of the modulated enzymes, whereas the enzyme activities from untreated animals remained unaltered. Conclusions: Short or prolonged swimming in rats may result in a reduction of brain TAS, protein concentration and AChE activity, and an activation of Na+K+-ATPase and Mg2+-ATPase. L-C administration may prevent reduction in TAS and protein concentration, and a decrease in AChE and Na+K+-ATPase activity; the latter reached pre-exercise values after L-C incubation.

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 (G6PD) activity. Material and methods. The study comprised 15 male runners, median age 36.5 years. Blood samples were obtained in the 15u2005min before the race and again within 15u2005min 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 G6PD 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±20.3u2005µmol/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, G6PD activity was found remarkably decreased (8.72±3.10 versus 3.8±2.5u2005U/g Hb; p<0.0001) pre versus post the event. Conclusion. Red blood cell G6PD 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.

Serum paraoxonase/arylesterase activities in phenylketonuric patients on diet.

Aim:To compare serum paraoxonase/arylesterase (PON-aryl) activities in phenylketonuric (PKU) patients with high and low phenylalanine (Phe) blood concentration.Patients and methods:Seventeen poorly controlled PKU children (off diet) underwent clinical and laboratory examinations before and after 30 days adhering to their special diet (on diet), whereas controls (N=24) were examined once. Lipid, lipoprotein levels and paraoxonase (PON 1) activities were measured with the Bayer Advia 1650 Clinical Chemistry System. Apolipoprotein AI (Apo AI) levels were determined by the Dade Behring BN ProSpec nephelometer, whereas total antioxidant capacity (TAC), PON-aryl and Phe levels were measured spectrophotometrically.Results:Phe significantly differed among the groups. Lipids and lipoproteins, except high-density-lipoprotein-cholesterol (HDL-C) and Apo AI, were higher when off diet than those on diet. HDL-C and Apo AI were similar in patients and controls. TAC (0.99±0.19u2009mmol/l) was significantly lower when the patients were off diet than when they adhered to diet and controls (1.71±0.20 and 1.81±0.20u2009mmol/l P<0.001 respectively). PON 1 and PON-aryl activities (68±2u2009U/min/ml, 88±26u2009KU (min/ml) in children with high Phe were reduced as compared with those with low blood Phe levels (152±41u2009U/min/ml, 107±23u2009KU/min/ml P<0.001) and controls (146±43u2009U/min/ml, 109±41u2009KU/min/ml P<0.001). The enzyme activities positively correlated with HDL-C and Apo AI when PKU patients were on diet and controls as well as with TAC in all the groups, whereas negatively correlated with Phe levels.Conclusions:PON-aryl activities are strongly related to the dietary control of PKU patients.

The effect of L -cysteine and glutathione on inhibition of Na + , K + -ATPase activity by aspartame metabolites in human erythrocyte membrane

Background:Reports have implicated Aspartame (N-L-a-aspartyl-L-phenylalanine methyl ester, ASP) in neurological problems.Aim:To evaluate Na+, K+-ATPase activities in human erythrocyte membranes after incubation with the ASP metabolites, phenylalanine (Phe), methanol (MeOH) and aspartic acid (Asp).Methods:Erythrocyte membranes were obtained from 12 healthy individuals and were incubated at 37°C for 1u2009h with the sum or each of the ASP metabolites separately, which are commonly measured in blood after ASP ingestion. Na+, K+-ATPase and Mg2+-ATPase activities were measured spectrophotometrically.Results:Membrane Mg2+-ATPase activity was not altered. The sum of ASP metabolite concentrations corresponding to 34, 150 or 200u2009mg/kg of the sweetener ingestion resulted in an inhibition of the membrane Na+, K+-ATPase by −30, −40, −48%, respectively. MeOH concentrations of 0.14, 0.60 or 0.80u2009mM decreased the enzyme activity by −25, −38, −43%, respectively. Asp concentrations of 2.80, 7.60 or 10.0u2009mM inhibited membrane Na+, K+-ATPase by −26, −40, −46%, respectively. Phe concentrations of 0.14, 0.35 or 0.50u2009mM reduced the enzyme activity by −24, −44, −48%, respectively. Preincubation with L-cysteine or reduced glutathione (GSH) completely or partially restored the inhibited membrane Na+, K+-ATPase activity by high or toxic ASP metabolite concentrations.Conclusions:Low concentrations of ASP metabolites had no effect on Na+, K+-ATPase activity. High or abuse concentrations of ASP hydrolysis products significantly decreased the membrane enzyme activity, which was completely or partially prevented by L-cysteine or reduced GSH.

The effect of the mode of delivery on the maternal-neonatal erythrocyte membrane acetylcholinesterase activity.

UNLABELLEDnFree radical production and high catecholamine levels are implicated with the modulation of acetylcholinesterase (AChE) activity.nnnOBJECTIVEnTo investigate the effect of the mode of delivery on maternal-neonatal erythrocyte membrane AChE activity.nnnSUBJECTS AND METHODSnSome women with normal pregnancy (N = 30) were divided into two groups: group A (N = 16) with normal labour and vaginal delivery and group B (N = 14) with scheduled Cesarean section, twenty non-pregnant women were the controls. Blood was obtained from controls and from mothers pre- vs post-delivery as well as from the umbilical cord (CB). Total antioxidant status (TAS), membrane AChE activities and catecholamine blood levels were measured with a commercial kit, spectrophotometrically and HPLC methods, respectively.nnnRESULTSnTAS and catecholamine levels as well as membrane AChE activities were similar in the two groups of mothers pre-delivery and in controls. TAS levels were reduced whereas AChE activities and catecholamine levels were increased post-delivery in mothers of group A and unaltered in group B at the same times of study. AChE activity was similarly lower in the CB of neonates than those of their mothers pre-delivery.nnnCONCLUSIONSnDuring a normal delivery process, the low TAS, the increased levels of catecholamines and the increased AChE activity, post-delivery, may be due to the increased stress due to the participation of uterus and skeletal muscles as during endurance exercise. The low AChE activity in newborns may be related to perinatal immaturity.

L-Cysteine and glutathione restore the reduction of rat hippocampal Na+, K+-ATPase activity induced by aspartame metabolites.

Studies have implicated aspartame (ASP) ingestion in neurological problems. The aim of this study was to evaluate hippocampal Na(+),K(+)-ATPase and Mg(2+)-ATPase activities after incubation with ASP or each of ASP metabolites, phenylalanine (Phe), methanol (MeOH) and aspartic acid (asp) separately. Suckling rat hippocampal homogenates or pure Na(+),K(+)-ATPase were incubated with ASP metabolites. Na(+),K(+)-ATPase and Mg(2+)-ATPase activities were measured spectrophotometrically. Incubation of hippocampal or pure Na(+),K(+)-ATPase with ASP concentrations (expected in the cerebrospinal fluid (CSF)) after ASP consumption of 34, 150 or 200mg/kg resulted in hippocampal enzyme activity reduction of 26%, 50% or 59%, respectively, whereas pure enzyme was remarkably stimulated. Moreover, incubation with hippocampal homogenate of each one of the corresponding in the CSF ASP metabolites related to the intake of common, high/abuse doses of the sweetener, inhibited Na(+),K(+)-ATPase, while pure enzyme was activated. Hippocampal Mg(2+)-ATPase remained unaltered. Addition of l-cysteine (cys) or reduced glutathione (GSH) in ASP mixtures, related with high/toxic doses of the sweetener, completely or partially restored the inactivated membrane Na(+),K(+)-ATPase, whereas the activated pure enzyme activity returned to normal. CSF concentrations of ASP metabolites related to common, abuse/toxic doses of the additive significantly reduced rat hippocampal Na(+),K(+)-ATPase activity, whereas pure enzyme was activated. Cys or GSH completely or partially restored both enzyme activities.

l-Cysteine and glutathione restore the modulation of rat frontal cortex Na+, K+-ATPase activity induced by aspartame metabolites

BACKGROUNDnStudies have suggested that aspartame (ASP) ingestion is implicated in neurological problems.nnnAIMnThe aim of this study was to evaluate rat frontal cortex Na+, K+ -ATPase and Mg2+ -ATPase activities after incubation with ASP or each of its metabolites, phenylalanine (Phe), methanol (MeOH) and aspartic acid (asp) separately.nnnMETHODnSuckling rat frontal cortex homogenates or pure Na+, K+ -ATPase were incubated with ASP metabolites. Na+, K+ -ATPase and Mg2+ -ATPase activities were measured spectrophotometrically.nnnRESULTSnIncubation of frontal cortex homogenate or pure Na+, K+ -ATPase with various ASP concentrations as expected in the cerebrospinal fluid (CSF) after ASP consumption of 34, 150 or 200mg/kg, decreased the frontal cortex enzyme activity by 33%, 53% or 57%, respectively, whereas pure enzyme was remarkably stimulated. Moreover, incubation of frontal cortex homogenate with each one of the expected ASP metabolites in the CSF, except MeOH, which are related to the intake of the above mentioned doses of the sweetener, resulted in an activation of the membrane Na+, K+ -ATPase, as well as pure enzyme. Frontal cortex Mg2+-ATPase remained unaltered. Addition of l-cysteine (cys) or reduced glutathione (GSH) to ASP metabolites mixtures, corresponding to 150 or 200mg/kg doses of the sweetener, completely or partially restored to normal the modulated membrane and pure Na+, K+ -ATPase activities.nnnCONCLUSIONnCSF concentrations of the sum of ASP metabolites corresponding to the intake of common, abuse or toxic doses (34 or 150 or 200mg/kg, respectively) of the additive significantly increased rat frontal cortex Na+, K+ -ATPase and pure enzyme activities. Cys or GSH completely or partially restored to normal both enzyme activities, possibly due to amelioration of the cellular GSH reduction from the action of MeOH, a metabolite of the sweetener and/or by their scavenging effect.

Maternal–neonatal erythrocyte membrane Na+, K+-ATPase and Mg2+-ATPase activities in relation to the mode of delivery

Free radical production and high catecholamine levels are implicated in the modulation of Na+, K+-ATPase, and Mg2+-ATPase activities. The aim of this study was to investigate the effect of the mode of delivery on the above-mentioned enzyme activities in maternal–neonatal erythrocyte membrane. Women with normal pregnancy (Nxa0=xa030) were divided into two groups: Group A (Nxa0=xa016) with normal labor and vaginal delivery, and Group B (Nxa0=xa014) with scheduled cesarean section; 20 non-pregnant women were the controls. Blood was obtained from controls and mothers, pre- versus post-delivery, and from the umbilical cord (CB). Total antioxidant status (TAS), membrane enzyme activities, and catecholamine blood levels were measured with a commercial kit, spectrophotometrically, and by HPLC methods, respectively. The results showed that: TAS levels, catecholamine, and the membrane enzyme activities were similar in the two groups of mothers pre-delivery, whereas both enzyme activities were lower than those of controls. TAS levels were reduced whereas Na+, K+-ATPase activities (0.35xa0±xa00.03 vs. 0.65xa0±xa00.06xa0μmolxa0Pi/hxa0×xa0mg protein, Pxa0<xa00.001), and catecholamine levels were increased post-delivery in mothers of Group A and unaltered in Group B (0.38xa0±xa00.02 vs. 0.40xa0±xa00.03xa0μmolxa0Pi/hxa0×xa0mg protein, Pxa0>xa00.05), at the same times of study. Mg2+-ATPase activities remained unaltered in both groups of mothers and newborns. Na+, K+-ATPase activity was similarly lower in the CB of neonates than those of their mothers, pre-delivery. Our results suggest that: (a) during a normal vaginal delivery process, the low TAS and the increased levels of catecholamines may increase Na+, K+-ATPase activity, post-delivery; (b) the low enzyme activities evaluated in mothers pre-delivery may be due to the high estrogen levels and those in newborns due to perinatal immaturity.