Urinary biomarker of oxidative stress in patients with psoriasis vulgaris and atopic dermatitis.

BACKGROUND: The involvement of oxidative stress in the pathogenesis of various skin disorders has been suggested for decades. However, few clinical studies have assessed oxidative stress in skin diseases. The easiest and least invasive method to assess oxidative stress in patients may be the measurement of oxidation products in urine. OBJECTIVE: This study aims to assess oxidative stress in psoriasis and atopic dermatitis patients. METHODS: Urine samples were collected from 29 psoriasis patients (25 males and 4 females), 21 atopic dermatitis patients (14 males and 7 females) and 20 healthy controls (16 males and 4 females). The severity and extent of psoriasis and atopic dermatitis was assessed by their area and severity index. We measured nitrate as a metabolite of nitric oxide, malondialdehyde as a major lipid oxidation product, and 8-hydroxydeoxyguanosine (8-OHdG) as a DNA oxidation marker. RESULTS: Urinary nitrate and 8-OHdG levels, but not malondialdehyde, were significantly higher in psoriasis patients than those in healthy controls. On the contrary, only urinary nitrate level was significantly higher in atopic dermatitis patients than those in healthy controls. The severity and extent of both psoriasis and atopic dermatitis significantly correlated with urinary nitrate level and malondialdehyde level, but it did not correlate with urinary 8-OHdG level. CONCLUSIONS: Measurement of these three urinary oxidative products is non-invasive. Above all, measurement of urinary nitrate may be most useful in the clinical assessment of oxidative stress in both psoriasis and atopic dermatitis patients. There is a possibility that urinary 8-OHdG level may indicate the different pathogenesis between psoriasis and atopic dermatitis.

Adenosines scavenged hydroxyl radicals and prevented posttraumatic epilepsy.

Intracortical injection of iron ions has been used as a model of posttraumatic epilepsy. Oxidation of lipids in neural membranes by reactive oxygen species, especially hydroxyl radicals (OH), is involved in the mechanisms responsible for iron-induced seizures. We examined the scavenging effects of adenosine (Ado) and 2-chloroadenosine (Cl-Ado) on OH radicals and superoxide (O2.-) using an electron spin resonance (ESR) spectrometer, and the occurrence of epileptic discharges in electrocorticogram (ECoG) induced by FeCl3 injection into the sensorimotor cortex of rats. Though DMPO-O2.- spin adducts generated by the hypoxanthine-xanthine oxidase system were not quenched by Ado or Cl-Ado, 5 mM of each showed a quenching effect on DMPO-OH spin adducts (5.3 x 10(16) spins/ml) generated by the Fenton reagent. In ECoG of rats, spike discharges appeared 15-45 min after FeCl3 injection (500 nmol) into the sensorimotor cortex, and polyspikes and/or ictal patterns were observed 70-90 min after the injection. Cl-Ado (1 mg/kg) or Ado (5 mg/kg) injected intraperitoneally 30 min prior to the FeCl3 injection suppressed or delayed the occurrence of epileptic discharges induced by FeCl3. Cl-Ado and Ado may suppress the occurrence of epileptic discharges by scavenging OH and by their anticonvulsant effect.

Direct detection of circulating free radicals in the rat using electron spin resonance spectrometry.

We developed a new technique for directly observing in vivo free radical formation in the circulating blood of living rats using electron spin resonance (ESR) spectrometry without any labeling or trapping agents. It was found that a doublet peak spectrum was obtained following ferric citrate and ascorbic acid injection. The signals were confirmed in different ways to be due to ascorbic acid radicals. These results provide evidence to support the involvement of free radical intermediates in iron-ascorbic acid reactions, and further confirm the suggested mechanisms of both the adverse and protective effects of ascorbic acid in biological systems. Furthermore, this method of direct observation is a new application of ESR spectrometry to living animals.

Neurochemical changes related to ageing in the senescence-accelerated mouse brain and the effect of chronic administration of nimodipine.

The levels of neurotransmitters and related metabolic enzyme activities in the brain of young-adult (3 months old), aged (11 months old) and nimodipine-administered (11 months old) senescence-accelerated mouse (SAM) were compared. Nimodipine, a calcium antagonist, was administered orally for 5 months. Acetylcholine (ACh), serotonin (5-HT) and dopamine (DA) levels all decreased with age but this decrease was attenuated by nimodipine. Choline acetyltransferase and choline esterase activities increased with age, and nimodipine enhanced their activities. Tryptophan hydroxylase activity was not affected by age or nimodipine administration. Monoamine oxidase-A activity increased with age, and was decreased by nimodipine administration. These results suggest that SAM rapidly undergoes neurochemical changes which are considered to be part of the normal aging process, and these changes were attenuated by chronic administration of nimodipine.

Age-related increase in nitric oxide synthase activity in senescence accelerated mouse brain and the effect of long-term administration of superoxide radical scavenger.

The levels of nitric oxide (NO) and NO synthase (NOS) activities were compared in the brains of young adult (3 months old), aged (11 months old) and TJ-960 administered (11 months old) senescence accelerated mice (SAM), of which the SAMP8 substrain is inferior in acquisition of learning due to the abnormality of glutamatergic neurotransmission in the cerebral cortex. TJ-960, which is based on the Kampo (Japanese traditional herbal medicine) prescription Sho-saiko-to-go-keishi-ka-shakuyaku-to, acts as a superoxide radical scavenger and attenuates the deterioration of neuronal activity associated with aging. We administered TJ-960 orally for 5 months. In the cerebral cortex of aged SAMP8, NOS activity was increased compared with that of young adult SAMP8. Though TJ-960 did not alter the contents of NO in any brain region compared with those in aged SAMP8, it did prevent the increase in NOS activity in the aged cerebral cortex. Our data suggest that NOS activity may increase to compensate for the reduced sensitivity of the NO reaction system in the aging process, and that TJ-960 may normalize this increased NOS activity in the cerebral cortex, although further work is clearly needed to ascertain maintenance in the acquisition of learning.

Structure-activity relationships of arginine analogues on nitric oxide synthase activity in the rat brain.

Nitric oxide (NO) is synthesized by nitric oxide synthase (NOS) from L-arginine (Arg) which has a guanidino group in its molecule. We examined the effect of 23 different Arg analogues on NOS activity in the rat brain. Though homoarginine, epsilon-guanidinocaproic acid and canavanine act as substrates of NOS, production of NO from them was lower than that from Arg. alpha-Guanidinoglutaric acid (2-GGA) and arcaine inhibited NOS activity at levels equal to NG-monomethyl-L-arginine (MeArg), a well known NOS inhibitor. Though almost all previously reported NOS inhibitors were synthesized by substituting the guanidino nitrogen of Arg, the guanidino nitrogens of arcaine and 2-GGA were not substituted. Furthermore, 2-GGA is a known endogenous convulsant in mammals, and arcaine, which was isolated from a marine mollusc, is also a convulsive substance. Hence, 2-GGA and arcaine will be excellent drugs to investigate not only the chemical nature of NOS but also the physiologic function of NO.

Application of automated flow injection analysis to determine nitrite and nitrate in mouse brain.

Nitric oxide level in the mouse brain was estimated by determination of nitrite and nitrate using an automated flow injection analyser for NOx. Different experimental conditions were examined to determine which produced reproducible results. After pretreatment of tissue specimens by the ZnSO4-NaOH method for deproteinization, reproducible and constant values were obtained. The values were more accurate immediately after sectioning without freezing than after 24 h with freezing. Two sacrifice methods, decapitation and microwave irradiation of the head, were investigated, but there was no significant difference between the two. No substances in the mouse brain exerted a positive or negative influence on the results. These results show that our method is indeed applicable to the brain tissue.

Inhibitory effect of arcaine on nitric oxide synthase in the rat brain.

Nitric oxide is synthesized by nitric oxide synthase (NOS) from L-Arg, which contains a guanidino group. Arcaine is the diguanidino compound and a derivative of Arg. This study was conducted to investigate the effects of arcaine on rat brain NOS activity, using nitrite and nitrate as indicators. Arcaine inhibited NOS activity in a linear mixed manner (K1 = 18.68 microM). Almost all previously reported NOS inhibitors were synthesized by substituting the guanidino nitrogen of Arg, but the guanidino nitrogens of arcaine were not substituted. Arcaine was also reported to be a competitive antagonist of the polyamine site on the N-methyl-D-aspartic acid (NMDA) receptor. Arcaine appears to be an excellent drug to investigate not only the chemical nature of NOS but also the functional and structural relationship between NOS and NMDA receptors.

Nitric oxide synthase inhibited by audouine in the rat brain.

Nitric oxide synthase (NOS) synthesizes nitric oxide (NO) from L-arginine (Arg) which has a guanidino group in its molecule. Audouine, a derivative of Arg, is the diguanidino compound. In this study, the effects of audouine on rat brain NOS activity were investigated by measuring nitrite and nitrate formation. Audouine inhibited NOS activity in a competitive (Ki = 2.10 microM) and partially uncompetitive (Ki = 49.7 microM) manner. Audouine is not substituted at the guanidino nitrogen, in contrast to most previously reported NOS inhibitors which were synthesized by substituting the guanidino nitrogen of Arg. Audouine is a novel inhibitor of NOS and should be useful for investigating the chemical nature of NOS and the roles of NO in the central nervous system.

Effects of glutamic acid relatives on the electrical activity of an identified molluscan giant neurone (Achatina fulica Férussac).

Effects of glutamic acid and its relatives were examined on the electrical activity of two kinds of neurones (the PON, periodically oscillating neurone, and the TAN, tonically autoactive neurone) identified in the suboesophageal ganglia of an African giant snail, Achatina fulica Férussac. L- and D-Glu, L-Asp, Gly and beta-Ala did not show any effect on the two neurones. However, beta-hydroxyglutamic acid (BHGA) showed a remarkable inhibitory effect on the PON. Erythro-L-BHGA had the strongest effect of these stereoisomers, and the critical concentration of this substance to produce the effect was 10(-6)-3 X 10(-5) g/ml (6-18 muM) when administered in the bath application. We confirmed by the microdrop application that erythro-L-BHGA directly hyperpolarized the PON neuromembrane. When the curve of current-voltage relationships (I-V curve) of the PON neuromembrane measured under erythro-L-BHGA at 10(-5) g/ml (61 muM) was superimposed on the curve of the normal state using the firing level as the common standard, these two curves showed concordance in a wide range of membrane polarization level. This concordance implies that the membrane resistance was maintained normally under erythro-L-BHGA at this concentration. A higher concentration of 10(-4) g/ml (0.61 mM) of this substance caused a decrease of PON membrane resistance and a remarkable elevation of its firing level. The TAN was not sensitive to BHGA, but sensitive to GABA and its derivatives.

Determination of neurotransmitter release into the caudate nucleus during convulsions induced by pentylenetetrazole using in vivo differential pulse voltammetry.

In vivo differential pulse voltammetry using an electrochemically treated carbon fiber electrode was applied to the investigation of the in vivo release of indoleamine and catecholamine within the caudate nucleus of freely moving and immobilized rats during convulsions induced by pentylenetetrazole (PTZ). Two distinct oxidation peaks, on at 130 mV (3,4-dihydroxyphenylacetic acid (DOPAC] and the other at 300 mV (5-hydroxyindoleacetic acid), were observed in voltammograms obtained from the caudate nucleus. In the caudate nucleus of freely moving rats, the in vivo oxidation current that peaked at 300 mV showed almost no change during and after tonic convulsions induced by 60 mg/kg of PTZ i.p. During tonic convulsions, the amplitude of the DOPAC oxidation peak significantly decreased to 75% of the peak height recorded prior to the injection of PTZ, and the minimum lasted for about 30 min; then the mean curve slowly recovered to the control level within 60 min. These results suggest that the release of dopamine (DA) in the caudate nucleus of freely moving rats decreased during tonic convulsions induced by PTZ. In another experiment, the EEGs of immobilized rats were recorded simultaneously, and the changes in the EEG pattern were used as an index of convulsion. In voltammograms from the caudate nucleus of immobilized rat, the peak height of the 130-mV oxidation potential significantly increased during ictal seizures. The increase lasted for 3-6 min after the ictal seizures. The severe electrical activity of the brain during ictal seizures interfered with the recording of some voltammograms.(ABSTRACT TRUNCATED AT 250 WORDS)

Delta-guanidinovaleric acid as an endogenous and specific GABA-receptor antagonist: electroencephalographic study.

Sporadic spike discharges recorded on EEGs from epidural electrodes appeared 5-10 min after topical application of 0.3 nmol delta-guanidinovaleric acid (DGVA) on the pia mater of the sensorimotor cortex, on the same side as the application. Spike discharges induced by DGVA were completely suppressed within 10 min of supplementary application of GABA (50 nmol), (3R)-(-)-4-amino-3-hydroxybutanoic acid (L-GABOB) (5 nmols) or muscimol (5 nmols) on the pia mater, but the discharges were not affected by supplementing with 500 nmol of alpha-amino-DGVA, i.e., arginine (Arg). Whereas spike discharges were not induced by DGVA together with L-GABOB or muscimol, DGVA applied together with Arg induced spike discharges. Neither phenobarbital (PB) (20 mg/kg, i.m.), diazepam (DZ) (10 mg/kg, i.p.), sodium valproate (200 mg/kg, i.p.) nor diphenylhydantoin (20 mg/kg, i.p.) showed any suppressive effects on spike discharges induced by DGVA. DGVA induced spike discharges 20 min after pre-injection of PB or DZ. These electroencephalographic findings suggest that DGVA, which has one more carbon in its chain than N-amidino-GABA, might act directly on the GABA-receptor to induce spike discharges and might be a specific GABA-receptor antagonist.

Reduction in nitric oxide synthase activity with development of an epileptogenic focus induced by ferric chloride in the rat brain.

Intracortical injection of iron ion has been shown to induce recurrent seizures and epileptic discharges in electrocorticograms. The importance of the effects of NO on seizure control systems and their regulation is suggested. In this paper, we examined the changes in nitric oxide synthase (NOS) activity in the epileptogenic focus induced by intracortical injection of iron ion at 5 min, 10 min, 1 h, 3 h and 3 days. Iron ion significantly decreased NOS activity in the cortex at the injection site 5 min, 3 h and 3 days after injection. These results suggest that the formation of an epileptic focus induced by iron ion is accompanied by decreased NOS activity.

The biosynthesis of 2-guanidinoethanol in intact mice and isolated perfused rabbit kidneys.

The metabolic pathway for the synthesis of 2-guanidinoethanol (GEt) was studied in intact mice and isolated perfused rabbit kidneys. GEt excretions in 24-hr urine increased after the intraperitoneal injection of ethanolamine (EA) into mice. Perfusion of isolated rabbit kidneys with EA and L-arginine (Arg) enhanced the GEt excretion from the ureter. This enhancement was observed in an EA concentration-dependent manner under the presence of Arg. When glycine (Gly) was added to the perfusion medium together with EA and Arg, the enhancement of GEt excretion was inhibited, whereas, guanidinoacetic acid excretion was increased to the same extent as during the perfusion with Gly and Arg. These results indicate that GEt is synthesized from Arg and EA in the kidney and that this synthesis is catalyzed by Arg:Gly amidinotransferase (EC 2.1.4.1.). We also described the guanidino compound excretion levels, including levels of GEt, in the rabbit, mouse, rat, and cat. The levels varied considerably with mammalian species.

Monoamine release in the rat striatum is induced by delta-guanidinovaleric acid and inhibited by GABA agonists.

delta-Guanidinovaleric acid (GVA) is an endogenous convulsant and is thought to be a specific gamma-aminobutyric acid (GABA) antagonist. In this study, we examined the effects of GVA and GABA agonists, GABA, muscimol and baclofen, on the release of dopamine (DA) and serotonin (5-HT) in the rat striatum using a brain dialysis technique. GVA produced a significant increase in the amount of DA and 5-HT released compared with controls. Both GABA (10mM) and muscimol (10mM) inhibited the GVA-induced release of DA and 5-HT. Muscimol was a more potent inhibitor of 5-HT release than DA release. Baclofen (10mM) inhibited only the GVA-induced DA release. These results suggest that the activation of GABA receptors inhibits the release of DA and 5-HT in the striatum, and that the dopaminergic system regulates GABA-B receptors and the serotonergic system mainly regulates GABA-A receptors.

Identification of delta-guanidinovaleric acid in human urine.

delta-Guanidinovaleric acid (DGVA) was identified in human urine using thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS). In the TLC, all Rfs of sample from urine developed by 6 solvent systems were identical to that of authentic DGVA. In the GC/MS, the mass spectrum of the sample was identical to the trifluoroacetylated dimethylpyrimidyl derivative of DGVA butylester (M+ = 375). In the HPLC analysis, the DGVA peak was observed just before 15 min in either chromatogram obtained by analysis of human urine or authentic DGVA, and the content of DGVA in pooled human urine was calculated at 2.4 nmol/ml.

Synthesis of neuroactive guanidino compounds by rat kidney L-arginine: glycine amidinotransferase.

Several neuroactive guanidino compounds have been reported to be synthesized in mammals by transamidination reactions. The enzyme(s) responsible for their synthesis and their location in the body has not been well established. The purpose of this investigation was to determine if purified homogeneous rat kidney alpha- and beta-L-arginine : glycine amidinotransferase (transamidinase) would catalyze the synthesis of certain neuroactive guanidino compounds, and if so, to determine if any catalytic specificity existed between the two forms of the enzymes. L-Arginine (Arg) was used as the amidino group donor and the following compounds were investigated for their ability to accept the amidino group: ethanolamine; 4-aminobutyric acid; lysine; 5-aminovaleric acid; 3-aminopropionic acid; taurine; L-glutamic acid (Glu); L-aspartic acid (Asp); and histidine (His). All of the above listed compounds served as amidino group acceptors for the enzyme except Glu, Asp and His. No differences were found between the alpha- and beta-transamidinase in any of the experiments reported, and the synthesis of 2-guanidinoethanol by the enzyme was by a sequential mechanism with a Km for Arg and ethanolamine of 14mM and 163mM, respectively. The possibility that the site of synthesis of the neuroactive guanidino compounds in the kidney and perhaps pancreas is discussed.

Formation of 2-guanidinoethanol by a transamidination reaction from arginine and ethanolamine by the rat kidney and pancreas.

The formation of 2-guanidinoethanol (GEt) from L-arginine (Arg) and ethanolamine (EA) was studied using rat kidney homogenates. Maximum GEt formation was observed between pH 8.7 and 9.1, and the enzyme catalyzing the GEt synthesis was stable between pH 5.6 and 9.1. The rate of GEt formation from Arg and EA by rat kidney homogenates obeyed simple Michaelis-Menten type kinetics. L-Ornithine and glycine inhibited GEt formation by rat kidneys. Both of them inhibited GEt formation in a linear mixed-type inhibitory manner when Arg concentrations were varied at a fixed concentration of EA, while they showed competitive inhibition when EA concentrations were varied at a fixed concentration of Arg. L-Canavanine and guanidinoacetic acid as well as Arg acted as an amidine donor for GEt formation, but L-homoarginine, 3-guanidinopropionic acid and 4-guanidinobutyric acid did not. GEt synthesis was also observed in the rat pancreas. It had almost half of the activity of rat kidney to form GEt. This ratio of kidney to pancreas was approximately equal to that of L-arginine:glycine amidinotransferase (transamidinase, EC 2.1.4.1) in kidney and pancreas. These results suggest that GEt may be synthesized from Arg and EA by a transamidinase catalyzing reaction.