Effects of uremic solutes on reactive oxygen species in vitro model systems as a possibility of support the renal function management.

BACKGROUND: In view of the prevalence of oxidative stress in chronic kidney disease (CKD) patients, the loss of low-molecular-weight biomolecules by hemodialysis and the antioxidant potential of some uremic solutes that accumulate in CKD, we used in vitro model systems to test the antioxidant potential of the following uremic solutes: uric acid, hippuric acid, p-cresol, phenol, methylguanidine, L-arginine, L-tyrosine, creatinine and urea. METHODS: The in vitro antioxidant efficiencies of the uremic solutes, isolated or in mixtures, were tested with the following assays: i) ABTS radical cation decolorization assay; ii) hypochlorous acid (HOCl/OCl(-)) scavenging activity; iii) superoxide anion radical (O2(•-)) scavenging activity; iv) crocin bleaching assay (capture of peroxyl radical, ROO(•)); v) hydrogen peroxide (H2O2) scavenging activity. RESULTS: Four of the tested uremic solutes (p-cresol, phenol, L-tyrosine, uric acid) were effective antioxidants and their IC50 were found in three model systems: ABTS(•+), HOCl/OCl(-) and crocin bleaching assay. In the 4-solutes mixtures, each one of the solute captured 12.5% for the IC50 of the mixture to ABTS(•+) or HOCl/OCl(-), exhibiting a virtually exact additive effect. In the 2-solutes mixtures, for ROO(•) capture, it was observed the need of more mass of uremic solutes to reach an IC50 value that was higher than the projected IC50, obtained from the IC50 of single solutes (25% of each, in the binary mixtures) in the same assay. In model systems for O2(•-) and H2O2, none of the uremic solutes showed scavenging activity. CONCLUSIONS: The use of the IC50 as an analytical tool to prepare and analyze mixtures allows the determination of their scavenging capacities and may be useful for the assessment of the antioxidant status of biological samples under conditions of altered levels of the endogenous antioxidant network and/or in the employment and monitoring of exogenous antioxidant therapy.

Nicotine-enhanced oxidation of low-density lipoprotein and its components by myeloperoxidase/H2O2/Cl- system.

In this study, the effect of nicotine on the LDL oxidation by the MPO/H2O2/Cl- system and the effect of HOCl on LDL and some of its components, such as methyl linoleate, vitamin E and the amino acid tryptophan were explored. Nicotine, in micromolar concentrations, enhanced the tryptophan oxidation, either present in LDL or free, in solution. Nicotine also decreased the formation of conjugated dienes and oxygen consumption in a methyl linoleate / HOCl system, and there was evidence to suggest an increase in chlorohydrin formation. Acceleration of the vitamin E oxidation by HOCl was also observed in the presence of nicotine. These data show that the interaction of nicotine and HOCl can promote significant biochemical modifications in LDL particle and some of its components involved in the pathogenesis of cardiovascular and other diseases.

Chemical, enzymatic and cellular antioxidant activity studies of Agaricus blazei Murrill.

Mushrooms possess nutritional and medicinal properties that have long been used for human health preservation and that have been considered by researchers as possible sources of free radical scavengers. In this work, the antioxidant properties of water extracts from Agaricus blazei Murill, produced by maceration and decoction, are demonstrated in vitro. Resistance to oxidation is demonstrated through three mechanisms: i) inhibition of enzymatic oxidative process, with 100% inhibition of HRP (horseradish peroxidase) and MPO (myeloperoxidase); ii) inhibition of cellular oxidative stress, with 80% inhibition of the oxidative burst of polymorphonuclear neutrophils (PMNs); and iii) direct action over reactive species, with 62% and 87% suppression of HOCl and superoxide anion radical (O2• -), respectively. From the data, it was concluded that the aqueous extract of A. blazei has significant antioxidant activity, indicating its possible application for nutraceutical and medicinal purposes.

Curcumin-supplemented yoghurt improves physiological and biochemical markers of experimental diabetes.

We investigated the effects of prolonged treatment of diabetic rats with curcumin-supplemented yoghurt on the physiological and biochemical changes associated with diabetes mellitus. An established metabolic cage model was used to assess these changes in three groups of streptozotocin-diabetic rats which had been administered, by gavage, curcumin blended into yoghurt in the doses of 30, 60 and 90 mg/kg body weight (BW) per d (groups DC30, DC60, DC90) for 31 d. One group of non-diabetic rats was also treated with 90 mg/kg BW per d curcumin (NDC90). Three control groups of diabetic animals received water (DW), yoghurt (DY) and insulin at 27·78 µmol/d by subcutaneous injection (DI). Also, two groups of non-diabetic animals received water (NDW) and yoghurt (NDY). Groups DI and DC90 exhibited significant falls, relative to DW and DY, in food and water intake, urine volume, glycaemia, urinary urea and glucose, proteinuria, serum TAG and activities of aspartate and alanine aminotransferases, and higher hepatic glycogen and BW. These improvements were greater in DI than in DC90. No difference was observed in the serum levels of total cholesterol or HDL-cholesterol, or in the masses of adipose and muscular tissues, between DC90 and DW or DY. Moreover, the improvements in curcumin-treated rats, relative to DW and DY, were significant and dose-dependent. The NDC90 group also showed no difference from the NDW or NDY groups, in any of the markers for diabetes. In conclusion, curcumin mixed into yoghurt at the highest dose tested exhibited anti-diabetic activity, improving significantly most of the markers assessed in this study.

Susceptibility of clinical isolates of Candida to photodynamic effects of curcumin.

BACKGROUND AND OBJECTIVE: The resistance of Candida species to antifungals represents a major challenge for therapeutic and prophylactic strategies. This study evaluated photodynamic therapy (PDT) mediated by Curcumin (CUR) against clinical isolates of C. albicans, C. tropicalis, and C. glabrata, both in planktonic and biofilm forms. STUDY DESIGN/MATERIALS AND METHODS: Suspensions of Candida were treated with three CUR concentrations and exposed to four LED fluences. The protocol that showed the best outcomes for inactivation of the planktonic phase was selected to be evaluated against Candida biofilms. In addition, two higher CUR concentrations were tested. The metabolic activity of biofilms was evaluated by means of XTT reduction assay and the biofilm biomass was evaluated using crystal violet (CV) staining assay. Data were analyzed in a mixed model nested ANOVA, Wilcoxon's nonparametric tests, and the Kruskal-Wallis test (α = 5%). RESULTS: The use of CUR in association with light was able to promote a significant antifungal effect against the planktonic form of the yeasts. When using 40 µM of CUR, the metabolic activity of C. albicans, C. glabrata, and C. tropicalis biofilms was reduced by 85%, 85%, and 73%, respectively, at 18 J/cm(2) . CUR-mediated PDT also decreased the biofilm biomass of all species evaluated. In addition, CV staining showed that C. albicans isolates were strong biofilm-forming strains, when compared with C. glabrata and C. tropicalis isolates. CONCLUSION: The results from the present investigation showed that low CUR concentrations can be highly effective for inactivating Candida isolates when associated with light excitation.

Investigation of the photodynamic effects of curcumin against Candida albicans.

This study describes the association of curcumin with light emitting diode (LED) for the inactivation of Candida albicans. Suspensions of Candida were treated with nine curcumin concentrations and exposed to LED at different fluences. The protocol that showed the best outcomes for Candida inactivation was selected to evaluate the effect of the preirradiation time (PIT) on photodynamic therapy (PDT) effectiveness, the uptake of curcumin by C. albicans cells and the possible involvement of singlet oxygen in the photodynamic action. Curcumin-mediated PDT was also assessed against biofilms. In addition to the microbiological experiments, similar protocols were tested on a macrophage cell line and the effect was evaluated by Methyltetrazolium assay (MTT) and SEM analysis. The optical properties of curcumin were investigated as a function of illumination fluence. When compared with the control group, a statistically significant reduction in C. albicans viability was observed after PDT (P < 0.05), for both planktonic and biofilm cultures. Photodynamic effect was greatly increased with the presence of curcumin in the surrounding media and the PIT of 20 min improved PDT effectiveness against biofilms. Although PDT was phototoxic to macrophages, the therapy was more effective in inactivating the yeast cell than the defense cell. The spectral changes showed a high photobleaching rate of curcumin.

Inhibition of peroxidase activity and scavenging of reactive oxygen species by astilbin isolated from Dimorphandra mollis (Fabaceae, Caesalpinioideae).

Astilbin (5,7,3',4'-tetrahydroxy-2,3-dihydroflavonol-3-ß-o-rhamnoside), a flavonoid with a large range of biological activities, was isolated from Dimorphandra mollis, a shrub common to the Brazilian Cerrado. The purpose of this study is to verify the effects of astilbin on myeloperoxidase (MPO) and horseradish peroxidase (HRP), and its antioxidant activity against hypochlorous acid (HOCl) and total antioxidant activity (TAC) by the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS•+). Astilbin inhibited MPO and HRP activities in a concentration-dependent relationship and effectively scavenged HOCl. The TAC by ABTS•+ of astilbin (IC50 ~ 20 mM) was higher than that of uric acid, which was used as a positive control. These data demonstrate that astilbin is a potent antioxidant and that it inhibits MPO and HRP activities efficiently.

Inhibition of peroxidase activity and scavenging of reactive oxygen species by astilbin isolated from Dimorphandra mollis (Fabaceae, Caesalpinioideae)

Astilbin (5,7,3’,4’-tetrahydroxy-2,3-dihydroflavonol-3-ß-o-rhamnoside), a flavonoid with a large range of biological activities, was isolated from Dimorphandra mollis, a shrub common to the Brazilian Cerrado. The purpose of this study is to verify the effects of astilbin on myeloperoxidase (MPO) and horseradish peroxidase (HRP), and its antioxidant activity against hypochlorous acid (HOCl) and total antioxidant activity (TAC) by the 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS•+). Astilbin inhibited MPO and HRP activities in a concentration-dependent relationship and effectively scavenged HOCl. The TAC by ABTS•+ of astilbin (IC50 ~ 20 mM) was higher than that of uric acid, which was used as a positive control. These data demonstrate that astilbin is a potent antioxidant and that it inhibits MPO and HRP activities efficiently.

Tratamento crônico com extrato alcoólico de Pterogyne nitens não melhora parâmetros clássicos do diabetes experimental / Continuous treatment with alcohol extract from Pterogyne nitens leaves does not alter typical variables of experimental diabetes

Tem sido atribuído ao flavonóide kaempferitrina e ao alcalóide galegina efeito hipoglicêmico. Folha de Pterogyne nitens, por conter tais compostos, poderia ser antidiabética. Assim, avaliamos o efeito do tratamento com Pterogyne nitens a ratos diabéticos sobre níveis glicêmicos e parâmetros fisiológicos. Ratos diabéticos (50 mg estreptozotocina/Kg peso) foram tratados durante 32 dias, 2 vezes ao dia, por gavagem com extrato etanólico de folhas de Pterogyne nitens (76 mg/0,5 mL glicerina 10 por cento por rato) (DTPn). Grupos diabéticos controles foram tratados com: glicerina 10 por cento (0,5 mL) (DTG), insulina (2,5 U/0,3 mL) (DTI) e água (0,5 mL) (DTA). Semanalmente determinamos: peso corporal, ingestão hídrica e alimentar, volume urinário e nível glicêmico. Os resultados dos grupos DTPn, DTG e DTA foram diferentes do DTI para todos os parâmetros, ocorrendo ganho de peso corporal e redução dos demais parâmetros no DTI. O grupo DTPn apresentou resultados semelhantes aos DTG e DTA. Através dos resultados apresentados no grupo DTI, constatamos que o modelo de estudo foi adequado. Também concluímos que o extrato vegetal e a glicerina não melhoraram e nem exacerbaram o quadro diabético. Resta a possibilidade da planta promover melhoria do diabetes com diferente: dose do extrato, via de administração ou severidade do diabetes induzido.

Kaempferitrin (a flavonoid) and galegin (an alkaloid) have been indicated as hypoglycemic agents. Leaves of Pterogyne nitens, which contain both compounds, might be antidiabetic. We therefore treated diabetic rats with these leaves to observe the effects on their glycemia and physiological variables. Streptozotocin-diabetic rats were given ethanolic extract of the leaves (76 mg in 0.5 mL 10 percent glycerol) (DTPn), twice a day by gavage for 32 days. Diabetic controls were given 0.5 mL 10 percent glycerol (DTG), insulin (2.5 U in 0.3 mL) (DTI) or 0.5 mL water (DTA). During this treatment, we measured level of glycemia, the body weight, daily food and water intake and urine volume, once each week. The results for the DTPn, DTG and DTA groups all differed significantly from these for the DTI group. The latter exhibited greater body weights and lower physiological variables and glycemia than the groups DTPn, DTG and DTA, all of which gave similar results. From the data for DTI rats, we conclude that the study model was appropriate. Therefore, the plant extract (plus glycerol) neither improved nor worsened the diabetic state of the rats. It is possible that this plant might ameliorate diabetes experimental if the dose of extract, treatment route or severity of induced diabetes were altered.

Free radical scavenging profile and myeloperoxidase inhibition of extracts from antidiabetic plants: Bauhinia forficata and Cissus sicyoides.

There is abundant evidence that reactive oxygen species are implicated in several physiological and pathological processes. To protect biological targets from oxidative damage, antioxidants must react with radicals and other reactive species faster than biological substrates do. The aim of the present study was to determine the in vitro antioxidant activity of aqueous extracts from leaves of Bauhinia forficata Link (Fabaceae-Caesalpinioideae) and Cissus sicyoides L. (Vitaceae) (two medicinal plants used popularly in the control of diabetes mellitus), using several different assay systems, namely, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) decolorization, superoxide anion radical (O2(.-)) scavenging and myeloperoxidase (MPO) activity. In the ABTS assay for total antioxidant activity, B. forficata showed IC50 = 8.00+/-0.07 microg/mL, while C. sicyoides showed IC50 = 13.0+/-0.2 microg/mL. However, the extract of C. sicyoides had a stronger effect on O2(.-) (IC50 = 60.0+/-2.3 microg/mL) than the extract of B. forficata (IC50 = 90.0+/-4.4 microg/mL). B. forficata also had a stronger inhibitory effect on MPO activity, as measured by guaiacol oxidation, than C. sicyoides. These results indicate that aqueous extracts of leaves of B. forficata and C. sicyoides are a potential source of natural antioxidants and may be helpful in the prevention of diabetic complications associated with oxidative stress.

Estudo fitoquímico de goiaba (Psidium guajava L. ) com potencial antioxidante para o desenvolvimento de formulação fitocosmética / Phytochemical study of guava (Psidium guajava L. ) with potential antioxidant activity aiming at developing a phytocosmetic formulation

Considerando-se a qualidade dos fitoterápicos, é importante salientar que a preocupação com esta questão inclui rigoroso acompanhamento das diferentes etapas do desenvolvimento e produção destes produtos, desde a coleta do vegetal até a disponibilidade do produto final. Neste trabalho, foram realizados o controle da qualidade, o potencial antioxidante como também ensaios biológicos in vitro do fruto da goiabeira (Psidium guajava L.), para o desenvolvimento de uma formulação fitocosmética. Os resultados obtidos mostraram que o fruto apresenta taninos e flavonóides, bem como atividades antioxidante e antimicrobiana. A análise microbiológica não apresentou crescimento de patógenos na formulação desenvolvida entre os outros testes realizados. Destaca-se a importância do estabelecimento do controle da qualidade para as plantas, a fim de que sejam utilizadas para o desenvolvimento de uma formulação fitocosmética segura, eficaz e com qualidade.

Considering the quality of the phytotherapic products, it is important to point out that the concern with this question includes rigorous accompaniment of the different stages of the development and production of these products, since the collection of the vegetable until the availability of the end product. In this work the quality control, the antioxidant potential were performed as also biological assays in vitro of guava fruit (P. guajava L.), for the development of a phytocosmetic formulation. The results had shown that the fruit presents tannins and flavonoids, as well as antioxidant and antimicrobial activities. The microbiological analysis did not present growth of pathogens. The importance of the establishment of the quality control for the plants is relevant, so that they are used for the development of a phytocosmetic formulation considered safe, efficient and with quality.

Free radical scavenging profile and myeloperoxidase inhibition of extracts from antidiabetic plants: Bauhinia forficata and Cissus sicyoides

There is abundant evidence that reactive oxygen species are implicated in several physiological and pathological processes. To protect biological targets from oxidative damage, antioxidants must react with radicáis and other reactive species faster than biological substrates do. The aim of the present study was to determine the in vitro antioxidant activity of aqueous extracts from leaves of Bauhinia forficata Link (Fabaceae - Caesalpinioideae) and Cissus sicyoides L. (Vitaceae) (two medicinal plants used popularly in the control of diabetes mellitus), using several different assay systems, namely, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) decolorization, superoxide anión radical (0(2)•-) scavenging and myeloperoxidase (MPO) activity. In the ABTS assay for total antioxidant activity, B. forficata showed IC50 = 8.00±0.07 μg/mL, while C. sicyoides showed IC50 = 13.0±0.2 μg/mL. However, the extract of C. sicyoides had a stronger effect on 0(2)•- (IC50 = 60.0±2.3 μg/mL) than the extract of B. forficata (IC50 = 90.0±4.4 μg/ mL). B. forficata also had a stronger inhibitory effect on MPO activity, as measured by guaiacol oxidation, than C. sicyoides. These results indicate that aqueous extracts of leaves of B. forficata and C. sicyoides are a potential source of natural antioxidants and may be helpful in the prevention of diabetic complications associated with oxidative stress.

The effect of rifampicin and pyrazinamide on isoniazid pharmacokinetics in rats.

Tuberculosis chemotherapy involves combination of the drugs isoniazid (INH), rifampicin (RMP) and pyrazinamide (PYR) for a 6-month period. The present work investigated the influence of RMP and PYR on the pharmacokinetic parameters of INH when groups of rats were pre-treated for 21 days with INH alone or in combination with RMP and/or PYR, in the following amounts per kg body weight: INH 100 mg; INH 100 mg+RMP 100 mg; INH 100 mg+PYR 350 mg; INH 100 mg+PYR 350 mg+RMP 100 mg. It was found that the co-administration of PYR caused an increase in the INH distribution volume (V(d)/F), half-life of elimination (t(1/2beta)) and clearance (Cl(T)/F), and a decrease in the area under curve 0 to 24 h (AUC). Co-administration of RMP caused an increase in the Cl(T)/F and a decrease in the AUC. The combination INH+PYR+RMP caused an increase in the Cl(T)/F and a decrease in the AUC. These significant pharmacokinetic interactions between the tuberculostatic drugs might be related to differences in the therapeutic and toxic effects.

The effect of pyrazinamide and rifampicin on isoniazid metabolism in rats.

Hepatotoxicity is the main concern during tuberculosis chemotherapy with the first-line drugs isoniazid (INH), rifampicin (RMP) and pyrazinamide (PYR). Since these hepatotoxic events have been associated with INH metabolites, the study aimed to measure the area under curve (AUC) parameter for INH and its metabolites acetylisoniazid (AcINH), hydrazine (Hz) and acetylhydrazine (AcHz), when groups of rats were pre-treated for 21 days with INH alone or in combination with RMP and/or PYR, in the following amounts per kg body weight: INH 100 mg; INH 100 mg + RMP 100 mg; INH 100 mg + PYR 350 mg; INH 100 mg + PYR 350 mg + RMP 100 mg. It was found that co-administration of RMP, PYR and RMP + PYR caused a significant decrease in the AUC for INH. Co-administration of PYR was the only treatment that caused a significant increase in the AUC for Hz and a decrease in the AUC for its acetylated product AcHz. The AUC for AcINH was not significantly altered in any experimental group. In conclusion, the increased metabolism of INH in all the drug combinations and the significantly higher production of Hz in the group INH + PYR might be linked with exacerbated hepatotoxic effects of these drug associations.

The reactivity of ortho-methoxy-substituted catechol radicals with sulfhydryl groups: contribution for the comprehension of the mechanism of inhibition of NADPH oxidase by apocynin.

Redox processes are involved in the mechanism of action of NADPH oxidase inhibitors such as diphenyleneiodonium and apocynin. Here, we studied the structure-activity relationship for apocynin and analogous ortho-methoxy-substituted catechols as inhibitors of the NADPH oxidase in neutrophils and their reactivity with peroxidase. Aiming to alter the reduction potential, the ortho-methoxy-catechol moiety was kept constant and the substituents at para position related to the hydroxyl group were varied. Two series of compounds were employed: methoxy-catechols bearing electron-withdrawing groups (MC-W) such as apocynin, vanillin, 4-nitroguaiacol, 4-cyanoguaiacol, and methoxy-catechol bearing electron-donating groups (MC-D) such as 4-methylguaiacol and 4-ethylguaiacol. We found that MC-D were weaker inhibitors compared to MD-W. Furthermore, the radicals generated by oxidation of MC-W via MPO/H(2)O(2), but not for MC-D, were able to oxidize glutathione (GSH) as verified by the formation of thiyl radicals, depletion of GSH, and recycling of the ortho-methoxy-catechols during their oxidations. The capacity of oxidizing sulfhydryl (SH) groups was also verified when ovalbumin was incubated with MC-W, but not for MC-D. Since the effect of apocynin has been correlated with inactivation of the cytosolic fractions of the NADPH oxidase complex and its oxidation during the inhibitory process develops a special role in this process, we suggest that the close relationship between the reactivity of the radicals of MC-W compounds with thiol groups and their efficacy as NADPH oxidase inhibitor could be the chemical pathway behind the mechanism of action of apocynin and should be taken into account in the design of new and specific NADPH oxidase inhibitors.

Chemiluminescent determination of leukocyte alkaline phosphatase: an advantageous alternative to the cytochemical assay.

The determination of leukocyte alkaline phosphatase (LAP) is used as an aid to diagnose many diseases in the laboratory. For example, it can be used to distinguish chronic myeloid leukemia (CML) from other myeloproliferative disorders (particularly myelofibrosis and polycythemia) and leukemoid reactions (LR). Traditionally, this test is performed with the use of subjective cytochemical assays that assign a score to the level of LAP. Here we present a nonsubjective, quantitative, sensitive, and inexpensive chemiluminescent technique that determines LAP based on the commercial reagent Immulite (AMPPD). To validate this methodology, intact leukocytes obtained from 32 healthy subjects, nine CML patients, and nine LR patients were submitted to the optimized protocol. By measuring the light emission elicited by four concentrations of neutrophils, we were able to estimate the activity of LAP per cell (the slope of the curve obtained by linear regression). A high linear correlation was found between the chemiluminescent result (slope) and the cytochemical score. The slope for healthy individuals ranged between 0.61 and 8.49 (10(-5) mV.s/cell), with a median of 2.04 (10(-5) mV.s/cell). These results were statistically different from those of CML patients (range=0.07-1.75, median=0.79) and LR patients (range= 3.84-47.24, median=9.58; P<0.05).

Smith-Lemli-Opitz syndrome: clinical and biochemical findings in Brazilian patients

Smith-Lemli-Opitz syndrome (SLOS) or RSH syndrome comprises multiple congenital anomalies and mental retardation. The underlying defect is a deficiency in the activity of delta7-sterol reductase, which decreases cholesterol and increases 7-dehydrocholesterol (7-DHC) levels. Our aim was to identify and evaluate the frequency of SLOS manifestations in a group of Brazilian patients. Based on our own data and those reported previously, we present a simple method that allows the estimation of probabilities favoring the diagnosis of SLOS. We evaluated 30 patients clinically and determined their plasma levels of cholesterol and 7-dehydrocholesterol. In 11 patients, the diagnosis was confirmed by ultraviolet spectrophotometry (UV). Of 19 patients with normal laboratory results, 17 showed a high probability favoring the diagnosis of SLOS. The most significant signs and symptoms observed in over 2/3 of the biochemically confirmed cases were mental retardation (10/11), delayed neuropsychomotor development (10/11), syndactyly of 2nd/3rd toes (10/11), and craniofacial anomalies including microcephaly (11/11), incompletely rotated ears (8/11), palpebral ptosis (10/11), anteverted nostrils (10/11), and micrognathia (9/11). Genital anomalies were found in all male patients (6/6).

Temporal response pattern of biochemical analytes in experimental diabetes.

The activities of the enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LD), creatine kinase (CK), amylase (AMS) and angiotensin converting enzyme (ACE) have been used to assess the toxic effects of xenobiotics that have hypoglycaemic action in hepatic, pancreatic, renal and muscle tissue. Using a validated experimental model of diabetes mellitus in rats, we ascertained whether this syndrome itself affected the serum activities of these enzymes over a 53-day period. Levels of hepatic enzymes AST, ALT and ALP were higher in the streptozotocin (STZ)-diabetic rats (group D), but were controlled by insulin therapy (group DI). AMS was reduced in group D and unchanged in group DI rats. Proteinuria was detected 1 day after STZ administration and partially controlled by insulin (group DI); its early presence in group D rats, and the lack of any change in serum ACE in this group, indicates that proteinuria is the better marker for microangiopathy. Microscopic examination of liver, kidney, heart and skeletal muscles (soleus and extensor digitorum longus) revealed various alterations in group D rat tissues, which were less pronounced in group DI. The liver, pancreas and kidney tissue-damage was consistent with the altered serum levels of AST, ALT, ALP and AMS and proteinuria. We conclude that: (i) rigorous control is required when these serum-enzyme levels are used as indicators of tissue toxicity in experimental diabetes, and (ii) LD, CK and bilirubin serum levels, which are unaffected by diabetes, can be used when testing effects of xenobiotics on tissues.

Assessment of monocytic component in acute myelomonocytic and monocytic/monoblastic leukemias by a chemiluminescent assay.

INTRODUCTION: Classically, the monocytic component of acute myelomonocytic (FAB-M4) and acute monocytic/monoblastic (FAB-M5) leukemias is demonstrated by nonspecific esterase positivity in cytochemical stainings. We have previously demonstrated that non-specific esterases from normal monocytes can be determined by a chemiluminescent method. In the present study, we investigated whether this assay can also determine the monocytic component of FAB-M4 and FAB-M5 and distinguish these acute myeloid leukemia (AML) categories. MATERIALS AND METHODS: Bone marrow samples were obtained from 66 patients with AML (M0, two cases; M1, 12 cases; M2, 13 cases; M3, 10 cases; M4, 11 cases; M5, 12 cases; M6, two cases; M7, four cases). Cells were incubated with a standard reaction mixture and chemiluminescence was measured for 10 min. Two parameters were assessed, the peak (PLE) and the integrated light emission (ILE). RESULTS: Both PLE and ILE were higher in FAB-M4 and FAB-M5 subtypes compared to other AML subtypes (P<0.001). In addition, the classification of AML cases into FAB-M4, FAB-M5 and nonmonocytic subtypes based on ILE analysis was concordant with alpha-naphthyl acetate esterase (ANAE) in 97% of cases (kappa coefficient 0.94, P<0.001). CONCLUSIONS: These findings indicate that this chemiluminescent assay was able to determine the monocytic component of FAB-M4 and FAB-M5 cells, and the classification of AML subtypes based on chemiluminescent analysis strongly agreed with the cytochemical ANAE-staining. In conclusion, this chemiluminescent assay is a simple, fast and objective method, which may be useful as an alternative tool in the differential diagnosis of AML subtypes.