Use of salicylate with high pressure liquid chromatography and electrochemical detection (LCE) as a sensitive measure of hydroxyl free radicals in adriamycin treated rats

Hydroxyl free radicals react with salicylate to form 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA). Utilizing the technique of high pressure liquid chromatography with electrochemical detection (LCED), it is possible to detect DHBAs at the level of femtomoles. Since salicylate is relatively non-toxic, we have administered it as a trapping agent in a first attempt to examine the use of the LCED method as a sensitive measure of in vivo OH production. Utilizing adriamycin administration as a model to induce oxygen free radical tissue damage, we found that the level of DHBAs present in drug treated rats versus controls was increased 100-fold in heart and muscle, 30-fold in lung, and 3- and 4-fold in brain and blood, respectively. These first observations support the theory that adriamycin induced OH in tissue and indicates that the LCED method may prove to be useful to measure oxygen free radical production in vivo.     

Detection of Free Radical Activity During Transient Global Ischemia and Recirculation: Effects of Intraischemic Brain Temperature Modulation

Abstract: To obtain direct evidence of oxygen radical activity in the course of cerebral ischemia under different intraischemic temperatures, we used a method based on the chemical trapping of hydroxyl radical in the form of the stable adducts 2,3- and 2,5-dihydroxybenzoic acid (DHBA) following salicylate administration. Wistar rats were subjected to 20 min of global forebrain ischemia by two-vessel occlusion plus systemic hypotension (50 mm Hg). Intraischemic striatal temperature was maintained as normothermic (37°C), hypothermic (30°C), or hyperthermic (39°C) but was held at 37°C before and following ischemia. Salicylate was administered either systemically (200 mg/kg, i.p.) or by continuous infusion (5 mM) through a microdialysis probe implanted in the striatum. Striatal extracellular fluid was sampled at regular intervals before, during, and after ischemia, and levels of 2,3- and 2,5-DHBA were assayed by HPLC with electrochemical detection. Following systemic administration of salicylate, stable baseline levels of 2,3- and 2,5-DHBA were observed before ischemia. During 20 min of normothermic ischemia, a 50% reduction in mean levels of both DHBAs was documented, suggesting a baseline level of hydroxyl radical that was diminished during ischemia, presumably owing to oxygen restriction to tissue at that time. During recirculation, 2,3- and 2,5-DHBA levels increased by 2.5- and 2.8-fold, respectively. Levels of 2,3-DHBA remained elevated during 1 h of reperfusion, whereas the increase in 2,5-DHBA levels persisted for 2 h. The increases in 2,3- and 2,5-DHBA levels observed following hyperthermic ischemia were significantly higher (3.8- and fivefold, respectively). In contrast, no significant changes in DHBA levels were observed following hypothermic ischemia. The postischemic changes in DHBA content observed following local administration of salicylate were comparable to the results obtained with systemic administration, thus confirming that the hydroxyl radicals arose within brain parenchyma itself. These results provide evidence that hydroxyl radical levels are increased during postischemic recirculation, and this process is modulated by intraischemic brain temperature. Hence, these data suggest a possible mechanism for the effects of temperature on ischemic outcome and support a key role for free radical-induced injury in the development of ischemic damage.     

Glutamate accumulation and increased hydroxyl free radical formation in the abdominal aorta and heart of gerbil after ischemia/reperfusion insult.

The concentration of glutamate as well as the hydroxylation of salicylate, as an index of hydroxyl free radical formation, has been determined in the abdominal aorta and heart of gerbils undergoing an ischemia/reperfusion insult (IRI) and compared to control sham-operated gerbils. The amount of glutamate and hydroxylated salicylate (dihydroxybenzoic acid, DHBA) was significantly increased in both the aorta and heart of IRI-treated gerbils as compared to the aorta and in the heart of sham-operated gerbils. Vitamin E (90 mg/kg at 24 and 1 h prior to an IRI) pretreatment prevented the increase of both glutamate and DHBA in both the aorta and heart of IRI-lesioned gerbils. The results suggest that increases in glutamate, perhaps due to the decreased activities of glutamine synthetase, are correlated with increased oxygen free radical formation during an ischemia/reperfusion insult to the heart.     

Simultaneous determination of ipratropium and salbutamol in rat plasma by LC-MS/MS and its application to a pharmacokinetic study

A novel, sensitive and specific LC-MS/MS method with silica-based solid-phase extraction was developed for simultaneous determination of ipratropium (IPR) and salbutamol (SAL) in rat plasma. Chromatographic separation was achieved on a Shiseido Capcell Pak CR column (SCX:C(18)=1:4, 150 mm × 2.0 mm, 5 μm) with a mobile phase consisting of methanol/water (85:15, v/v) containing 20 mmol/L ammonium formate and 0.1% formic acid at a flow rate of 0.3 mL/min. A tandem mass spectrometric detection with an electrospray ionization (ESI) interface was conducted via multiple reaction monitoring (MRM) under positive ionization mode. This method was validated in terms of specificity, linearity, accuracy (within ±115.4%), intra- and inter-day precision (<11.4%) over the concentration range of 8-1612 pg/mL for IPR and 50-10,000 pg/mL for SAL. In addition, stability and matrix effects of IPR and SAL in plasma were evaluated. This method has been successfully applied to the pharmacokinetic study of compound ipratropium bromide aerosol mainly containing ipratropium bromide (IB) and salbutamol sulphate (SS) after inhalation in rats.     

Melatonin scavenges hydroxyl radical and protects isolated rat hearts from ischemic reperfusion injury

During postischemic reperfusion, free radicals are produced and have deleterious effects in isolated rat hearts. We investigated whether melatonin (MEL) reduces the production of hydroxyl radical (*OH) in the effluent and aids in recovery of left ventricular (LV) function. Hearts were subjected to 30 min of ischemia followed by 30 min of reperfusion. Salicylic acid (SAL) was used as the probe for *OH, and its derivatives 2,5- and 2,3-dihydroxybenzoic acid (DHBA) were quantified using HPLC. In addition, thiobarbituric acid reactive substances (TBARS) in the myocardium was measured. Plateaus in the measurement of 2,5- and 2,3-DHBA were seen from 3 to 8 min after reperfusion in each group. The group that received 100 microM MEL+ SAL had significantly reduced amounts of 2,5- and 2,3-DHBA by multiple folds, compared to the SAL group. TBARS was significantly decreased in the 100 microM MEL group (1.20+/-0.36 vs 1.85+/-0.10 micromol/g of drug-free group, p<0.001). More importantly, the 100 microM MEL group significantly recovered in LV function (LV developed pressure, +dp/dt, and -dp/dt; 63.0%, 60.3%, and 59.4% in the 100 microM MEL group; 30.2%, 29.7%, and 31.5% in the drug-free group, respectively; p<0.05). Duration of ventricular tachycardia or ventricular fibrillation significantly decreased in the 100 microM MEL group (100 microM MEL, 159+/-67 sec; drug-free, 1244+/-233 sec; p<0.05). As a result of scavenging *OH and reducing the extent of lipid peroxidation, MEL is an effective agent for protection against postischemic reperfusion injury.     

Resolution of catecholic tetrahydroisoquinoline enantiomers and the determination of R- and S-salsolinol in biological samples by gas chromatography-mass spectrometry

Tetrahydroisoquinolines (TIQs) might be formed endogenously and can act centrally to promote a mechanism governing alcohol drinking behaviour. The possibility that biosynthesis occurs through a stereospecific enzymatic reaction is considered. Several TIQs were transformed into diastereomers by a two-step derivatization with N-methyl-N-trimethylsilyltrifluoracetamide and R-(−)-2-phenylbutyrylic acid and were analyzed by gas chromatography-mass spectrometry (GC-MS). High resolution of the TIQ enantiomers was achieved. This method was applied to the quantification of the enantiomers of salsolinol (SAL) in urine and plasma of healthy humans. Deuterated SAL was used as the internal standard. SAL was extracted from biological material using phenulboronic Deuterated SAL was used as the internal standard. SAL was extracted from biological material using phenylboronic phase cartridges and transformed into diastereomers. The sensitivity and specificity of the assay permit the determination of the enantiomeric composition of SAL in plasma and urine. The limit of quantification was found to be 100 pg/ml for each enantiomer. The described method has the advantage that optimal resolution of the SAL enantiomers without peak overlapping between analyte and other compounds can be achieved. Contrary to other findings, our GC-MS studies have demonstrated that endogenously formed SAL is racemic in plasma as well as in urine of healthy subjects.     

The salicylate metabolite gentisic acid, but not the parent drug, inhibits glucose autoxidation-mediated atherogenic modification of low density lipoprotein

Oxidation of low density lipoprotein (LDL) by glucose-derived radicals may play a role in the aetiology of atherosclerosis in diabetes. Salicylate was shown to scavenge certain radicals. In the present study, aspirin, salicylate and its metabolites 2,5- and 2, 3-dihydroxybenzoic acid (DHBA) were tested for their ability to impair LDL oxidation by glucose. Only the DHBA derivatives, when present during LDL modification, inhibited LDL oxidation and the increase in endothelial tissue factor synthesis induced by glucose oxidised LDL. The LDL glycation reaction was not affected by DHBA. The antioxidative action of DHBA may be attributed to free radical scavenging and/or chelation of transition metal ions catalysing glucose autoxidation.     

Hydroxyl radicals detected via brain microdialysis in rats breathing air and during hyperbaric oxygen convulsions

Microdialysis was done on 300-400 g, awake, male rats with microdialysis probes inserted through guide cannulas into the striatum (Bregma co-ordinates A 0.5, L 2.9, D -4.0 for guide cannulas implanted 5 days previously). Rats were exposed to hyperbaric oxygen (HBO; 6 atm absolute, 5 atm gauge pressure of oxygen with carbon dioxide absorbed by soda lime). Artificial cerebrospinal fluid (CSF) containing 5 mM sodium salicylate was perfused at 1 microl/min and collected over sequential 10 min intervals with rats breathing air, then HBO, and after decompression. Times to convulsions and duration and severity of convulsions were observed and recorded. CSF samples were analyzed for 2,3- and 2,5-dihydroxybenzoic acid (DHBA), reaction products of hydroxyl radicals with salicylate, by HPLC and compared to authentic standards. Recovery of DHBAs was 48% from fluid surrounding microdialysis probes, based on in vitro tests. The average time to the first convulsion was 21 min and rats convulsed an average of 4 times during 40 min in HBO. There were no significant differences in hydroxyl radical production by this protocol during any of the 10 min collection periods in air or HBO (average in pmoles for 10 microl of all samples: 2,3-DHBA = 7.0 +/- 2.5 and 2,5-DHBA = 11.3 +/- 4.1). The failure to detect an increase in hydroxyl radicals in HBO prior to or during convulsions appears valid since each rat served as its own control.     

Hydroxyl radicals detected via brain microdialysis in rats breathing air and during hyperbaric oxygen convulsions

Abstract

Microdialysis was done on 300–400 g, awake, male rats with microdialysis probes inserted through guide cannulas into the striatum (Bregma co-ordinates A 0.5, L 2.9, D –4.0 for guide cannulas implanted 5 days previously). Rats were exposed to hyperbaric oxygen (HBO; 6 atm absolute, 5 atm gauge pressure of oxygen with carbon dioxide absorbed by soda lime). Artificial cerebrospinal fluid (CSF) containing 5 mM sodium salicylate was perfused at 1 µl/min and collected over sequential 10 min intervals with rats breathing air, then HBO, and after decompression. Times to convulsions and duration and severity of convulsions were observed and recorded. CSF samples were analyzed for 2,3- and 2,5-dihydroxybenzoic acid (DHBA), reaction products of hydroxyl radicals with salicylate, by HPLC and compared to authentic standards. Recovery of DHBAs was 48% from fluid surrounding microdialysis probes, based on in vitro tests. The average time to the first convulsion was 21 min and rats convulsed an average of 4 times during 40 min in HBO. There were no significant differences in hydroxyl radical production by this protocol during any of the 10 min collection periods in air or HBO (average in pmoles for 10 µl of all samples: 2,3-DHBA = 7.0 ± 2.5 and 2,5-DHBA = 11.3 ± 4.1). The failure to detect an increase in hydroxyl radicals in HBO prior to or during convulsions appears valid since each rat served as its own control.     

Lipoic acid ameliorates oxidative stress and renal injury in alloxan diabetic rabbits

The therapeutic potential of lipoic acid (LA) in diabetes and diabetic nephropathy treatment was elucidated. Alloxan diabetic rabbits were treated daily for three weeks with either 10 or 50 mg of LA per kg body weight (i.p.). The following parameters were measured: 1) serum glucose, urea, creatinine and hydroxyl free radical (HFR) levels; 2) blood glutathione redox state; 3) urine albumin concentration; 4) hepatic and renal HFR levels, GSH/GSSG ratios, cysteine contents and the activities of the enzymes of glutathione metabolism; and 5) the activity of renal NADPH oxidase. Histological studies of kidneys were also performed. The treatment of diabetic rabbits with 50 mg of LA resulted in lethal hypoglycaemia in 50% of animals studied. Although the low dose of LA did not change serum glucose concentration, it decreased serum urea and creatinine concentrations, attenuated diabetes-induced decline in GSH/GSSG ratio and abolished hydroxyl free radicals accumulation in serum, liver and kidney cortex. LA did not change the activities of the enzymes of glutathione metabolism, but it elevated hepatic content of cysteine, which limits the rate of glutathione biosynthesis. Moreover, LA lowered urine albumin concentration and attenuated glomerulopathy characteristic of diabetes. However, it did not affect diabetes-stimulated activity of renal NADPH oxidase. In view of these data, it is concluded that low doses of LA might be useful for the therapy of diabetes and diabetic nephropathy. Beneficial action of LA seems to result mainly from direct scavenging of HFR and restoring glutathione redox state due to elevation of intracellular cysteine levels.     

Determinations of catechols in small volumes of plasma using ion-pair reversed phase liquid chromatography/electrochemistry

A method with improved sensitivity for detection of catechols (CA) in small volumes of plasma using an ion-pair reversed phase HPLC system with electromechemical detection is presented. Fast isocratic separations were obtained by using 7.5 cm x 4.6 mm (i.d.) reversed phase columns with 3C18 3 micron silica particles. The CA:s L-DOPA, Noradrenaline (NA), Adrenaline (A), Dihydroxybenzylamine (DHBA, i.s.), DOPAC and Dopamine (DA) were separated in less than 4 min. The performance of three different electrochemical cells was compared with respect to hydrodynamic voltammogram, band broadening effect, linearity and detection limit. The sample preparation procedure using alumina extraction of CA:s, was modified to improve recoveries and decrease dilution factors. A modified carbon paste cell (CP-O) gave a response 4-8 times higher than what is previously reported for GC cells. Detection limits were: L-DOPA 80, NA 1.25, A 1.25, DHBA 0.4, DOPAC 1.25 and DA 0.6 pg/injection. Application to plasma from rat and fish (cod) under rest, exercise and stress is reported. The method allows determination of CA:s in small volumes of plasma (less than 500 microliter) obtained several times a day from the same animal even if it is small (less than 1/2 kg), is under rest and parts of the plasma sample are to be used for analysis of other parameters than CA:s.     

Brain Hydroxyl Radical Generation in Acute Experimental Head Injury

Abstract: The time course and intensity of brain hydroxyl radical (^?OH) generation were examined in male CF-1 mice during the first hour after moderate or severe concussive head injury. Hydroxyl radical production was measured using the salicylate trapping method in which the production of 2,3- and/or 2,5-dihydroxybenzoic acid (DHBA) in brain 15 min after salicylate administration was used as an index of ^?OH formation. In mice injured with a concussion of moderate severity as defined by the 1-h posttraumatic neurologic recovery (grip score), a 60% increase in 2,5-DHBA formation was observed by 1 min after injury compared with that observed in uninjured mice. The peak in DHBA formation occurred at 15 min after injury (+67.5%; p < 0.02, compared with uninjured). At 30 min, the increase in DHBA lost significance, indicating that the posttraumatic increase in brain ^?OH formation is a transient phenomenon. In severely injured mice, the peak increase in DHBA (both 2,3- and 2,5-) was observed at 30 min after injury, but also fell off thereafter as with the moderate injury severity. Preinjury dosing of the mice with SKF-525A (50 mg/kg i.p.), an inhibitor of microsomal drug oxidations, did not blunt the posttraumatic increase in salicylate-derived 2,5-DHBA, thus showing that it is not due to increased metabolic hydroxylation. Neither injury nor SKF-525A administration affected the DHBA plasma levels. However, saline perfusion of the injured mice to remove the intravascular blood before brain removal eliminated the injury-induced increase in 2,5-DHBA, but did not affect the baseline levels seen in uninjured mice. This implies that the source of the increased DHBA in the injured mice is the microvasculature, probably the endothelium. The administration of the 21-aminosteroid lipid antioxidant, tirilazad mesylate, which possesses ^?OH scavenging properties, also attenuated the posttraumatic increase in DHBA, further supporting that it reflects an increase in ^?OH radical formation. These results are the first direct demonstration of the occurrence and time course of increased ^?OH production in injured brain.     

Simultaneous measurement of monoamines, their metabolites and 2,3- and 2,5-dihydroxybenzoates by high-performance liquid chromatography with electrochemical detection. Application to rat brain dialysates

A reversed-phase chromatographic method with electrochemical detection was developed for the simultaneous determination of 2,3- and 2,5-dihydroxybenzoates, indicators of in vivo hydroxyl free radical formation, monoamines (NE, DA, 5-HT) and their metabolites (MHPG, DOPAC, HVA, 3MT, 5-HIAA). Linearity was observed from 10 pg to 10 ng injected. Reproducibility is correct (C.V. about 9%) except for 3MT and 5-HT. The limit of detection for almost all products was about 20 pg injected on the column. An application of this method in the study of the neurotoxicity of high pressure oxygen in rat is described. The limit of quantification for all compounds was 5 ng/ml except for HVA (10 ng/ml). Some basal levels DA, 5-HT, 5-HIAA, HVA, DOPAC, 3MT, 2,5-DHBA and 2,3-DHBA in microdialysates coming from striatum of normoxic restrained rats are given.     

Involvement of Free Radicals in Excitotoxicity In Vivo

Abstract: Recent evidence has linked excitotoxicity with the generation of free radicals. We examined whether free radical spin traps can attenuate excitotoxic lesions in vivo. Pretreatment with N-tert -butyl-α-(2-sulfophenyl)-nitrone (S-PBN) significantly attenuated striatal excitotoxic lesions in rats produced by N -methyl- d -aspartate (NMDA), kainic acid, and α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA). In a similar manner, striatal lesions produced by 1-methyl-4-phenylpyridinium (MPP⁺), malonate, and 3-acetylpyridine were significantly attenuated by either S-PBN or α-phenyl- N-tert -butylnitrone (PBN) treatment. Administration of S-PBN in combination with the NMDA antagonist MK-801 produced additive effects against malonate and 3-acetylpyridine toxicity. Malonate injections resulted in increased production of hydroxyl free radicals (^•OH) as assessed by the conversion of salicylate to 2,3- and 2,5-dihydroxybenzoic acid (DHBA). This increase was significantly attenuated by S-PBN, consistent with a free radical scavenging effect. S-PBN had no effects on malonate-induced ATP depletions and had no significant effect on spontaneous striatal electrophysiologic activity. These results provide the first direct in vivo evidence for the involvement of free radicals in excitotoxicity and suggest that antioxidants may be useful in treating neurologic illnesses in which excitotoxic mechanisms have been implicated.     

Effect of imipramine on 1-methyl-4-phenylpyridinium ion-induced hydroxyl radical generation in rat striatum

We examined the effect of imipramine (a tricyclic antidepressant drug) on hydroxyl radical (.OH) generation induced by 1-methyl-4-phenylpyridinium ion (MPP(+)) in extracellular fluid of rat striatum, using a microdialysis technique. Imipramine enhanced the formation of.OH trapped as 2,3-dihydroxybenzoic acid (DHBA) induced by MPP(+) (5 mM). Introduction of imipramine (0.1, 0.5 and 1.0 mM) dose-dependently increased the level of dopamine (DA) release. Concomitantly, imipramine enhanced DA efflux and the level of DHBA induced by MPP(+), as compared with MPP(+)-treated control. When corresponding experiments were performed with reserpinized rats, there were small increases in the levels of DA and nonsignificant increase in the formation of DHBA. When iron (II) was administered to imipramine (1 mM)-treated animals, a marked elevation of DHBA was observed, compared with MPP(+)-only treated animals. A positive linear correlation was observed between iron (II) and DHBA (R(2)=0.985) in the dialysate. These results indicate that imipramine enhances generation of.OH induced by MPP(+) during enhanced DA overflow.     

Determination of alkylresorcinol metabolites in human urine by gas chromatography–mass spectrometry

Alkylresorcinols (ARs) are phenolic lipids present at high concentrations in the outer parts of rye and wheat kernels and have been proposed as biomarkers for intake of whole grain and bran products of these cereals. AR are absorbed in the small intestine and after hepatic metabolism two major metabolites, 3,5-dihydroxybenzoic acid (DHBA) and 3-(3,5-dihydroxyphenyl)-1-propanoic acid (DHPPA), are excreted in urine either as such or as conjugates. Urine samples from nine individuals were incubated with different enzymes to assess type and extent of conjugates. In comparison with DHBA, which was mostly found in the free form, the less polar DHPPA was conjugated to a greater extent and the major conjugates were glucuronides. In this method, urine samples were hydrolyzed using β-glucuronidase from Helix pomatia and syringic acid was used as internal standard. Samples, silylated with BSTFA, were analyzed by GC–MS utilizing a BP-5 fused silica capillary column and single ion monitoring of molecular ions (m/z 370 [DHBA], m/z 398 [DHPPA]). Recoveries of DHBA and DHPPA were estimated to be 94% and 93%, respectively. The average intra-assay/inter-assay coefficients of variation were 4.9/5.7% for DHBA and 7.6/9.3% for DHPPA.     

Protective effect of histidine on para-nonylphenol-enhanced hydroxyl free radical generation induced by 1-methyl-4-phenylpyridinium ion (MPP+) in rat striatum.

The present study examined the antioxidant effect of histidine, a singlet oxygen ((1)O(2)) scavenger, on para-nonylphenol (an environmental estrogen-like chemical)-enhanced hydroxyl radical (.OH) generation induced by 1-methyl-4-phenylpyridinium ion (MPP+) in extracellular fluid of rat striatum. Rats were anesthetized, and sodium salicylate in Ringer's solution (0.5 nmol/microl/min) was infused through a microdialysis probe to detect the generation of.OH as reflected by the non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) in the striatum. Introduction of para-nonylphenol (10 microM) significantly enhanced MPP+ -induced.OH generation. Histidine (25 mM) decreased the para-nonylphenol-enhanced.OH formation. Although the level of MPP+ -induced.OH formation trapped as DHBA after para-nonylphenol treatment increased, para-nonylphenol failed to increase either the level of dopamine and DHBA formation in the reserpinized animals. These results indicate that para-nonylphenol and MPP+ -enhanced.OH generation was based on 1O(2) production, and histidine may have a preventive effect on para-nonylphenol and MPP+ -induced.OH generation in rat striatum.     

Determination of hydroxyl free radical formation in human platelets using high-performance liquid chromatography with electrochemical detection

The formation of the hydroxyl free radical (HFR) can be quantified indirectly, by measuring two products of the hydroxylation of salicylic acid, 2,3-dihydroxybenzoate (2,3-DHB) and 2,5-dihydroxybenzoate (2,5-DHB). In this study, we used reversed-phase high-performance liquid chromatography with electrochemical (coulometric) detection to measure 2,3- and 2,5-DHB levels in human platelets. The limits of detection of the method were 10 and 5 fmol on column for 2,3-DHB and 2,5-DHB, respectively. We tested the technique by measuring increases in dihydroxybenzoate levels after exposure of platelets to experimentally induced oxidative stress. Then, we measured platelet levels of 2,3- and 2,5-DHB in patients with Parkinson’s disease, under therapy with l-DOPA, and in normal subjects. We also measured platelet concentrations of l-DOPA and its major metabolite, 3-O-methyldopa (3-OMD). Parkinsonian patients showed increased levels of both 2,3- and 2,5-DHB. Platelet levels of 2,3-DHB were positively correlated with platelet levels of l-DOPA and 3-OMD. The technique we describe proved simple and extremely sensitive and may represent a useful tool for the study of oxidative stress in humans.     

Hypoglycaemic, antioxidative and nephroprotective effects of taurine in alloxan diabetic rabbits

The therapeutic potential of taurine was investigated under diabetic conditions. Alloxan diabetic rabbits were treated daily for three weeks with 1% taurine in drinking water. The following parameters were measured: 1) serum glucose, urea, creatinine and hydroxyl free radical (HFR) levels; 2) blood glutathione redox state; 3) urine albumin concentration; 4) hepatic and renal HFR levels, GSH/GSSG ratios and the activities of catalase, superoxide dismutase and the enzymes of glutathione metabolism; 5) renal NADPH oxidase activity; 6) the rates of renal and hepatic gluconeogenesis. Histological studies of kidneys were also performed. Taurine administration to diabetic rabbits resulted in 30% decrease in serum glucose level and the normalisation of diabetes-elevated rate of renal gluconeogenesis. It also decreased serum urea and creatinine concentrations, attenuated diabetes-evoked decline in GSH/GSSG ratio and abolished hydroxyl free radicals accumulation in serum, liver and kidney cortex. Animals treated with taurine exhibited elevated activities of hepatic gamma-glutamylcysteine syntetase and renal glutathione reductase and catalase. Moreover, taurine treatment evoked the normalisation of diabetes-stimulated activity of renal NADPH oxidase and attenuated both albuminuria and glomerulopathy characteristic of diabetes. In view of these data, it is concluded that: 1) diminished rate of renal gluconeogenesis seems to contribute to hypoglycaemic effect of taurine; 2) taurine-induced increase in the activities of catalase and the enzymes of glutathione metabolism is of importance for antioxidative action of this amino acid and 3) taurine nephroprotective properties might result from diminished renal NADPH oxidase activity. Thus, taurine seems to be beneficial for the therapy of both diabetes and diabetic nephropathy.     

Binding of fatty acids facilitates oxidation of cysteine-34 and converts copper-albumin complexes from antioxidants to prooxidants

As a transition metal capable of undergoing one-electron oxidation-reduction conversions, copper (Cu) is essential for life and fulfills important catalytic functions. Paradoxically, the same redox properties of copper can make it extremely dangerous because it can catalyze production of free radical intermediates from molecular oxygen. Factors involved in regulation of redox activity of albumin-bound copper have not been well characterized. In the present study, effects of modification of the albumin cysteine-34 (Cys-34) and binding of nonesterified fatty acids on the redox-cycling activity of the complex of copper with human serum albumin (Cu/HSA) were studied. Because ascorbate is the most abundant natural reductant/scavenger of free radicals in blood plasma, the electron paramagnetic resonance assay of ascorbate radical formation was used as a method to monitor Cu/HSA redox-cycling activity. At Cu/HSA ratios below 1:1, the bound Cu was virtually redox inactive, as long as Cys-34 was in reduced state (Cu/HSA-SH). Alkylation, nitrosylation, or oxidation of Cu/HSA resulted in the appearance of redox-cycling activity. Experiments with ultrafiltration of Cu/HSA alkylated with N-ethylmaleimide (Cu/HSA-NEM) showed that at Cu/HSA-NEM ratios below 1:1, the ascorbate radicals were produced by Cu tightly bound to HSA rather than by Cu released in solution. The rate of ascorbate radical production in HSA-NEM and S-nitrosylated HSA (HSA-NO) was, however, more than one order of magnitude lower than that in a solution containing equivalent concentration of free copper ions. While Cu/HSA-SH was redox inactive, binding of oleic or linoleic acids induced Cu-dependent redox-cycling with maximal activity reached at a fatty acid to protein molar ratio of 3:1 for oleic acid and 2:1 for linoleic acid. Binding of fatty acids caused profound conformational changes and facilitated oxidation of the Cys-34 SH-group at essentially the same ratios as those that caused redox-cycling activity of Cu/HSA. We conclude that fatty acids regulate anti-/prooxidant properties of Cu-albumin via controlling redox status of Cys-34.