Isolation from neutrophil membranes of a complex containing active NADPH oxidase and cytochrome b-245.

NADPH-dependent O2- forming activity was extracted with deoxycholate from subcellular particles of guinea-pig neutrophils following stimulation with phorbol myristate acetate. The solubilized enzyme was purified by chromatography on Ultrogel AcA22, by isopycnic glycerol density gradient centrifugation and by treatment with 0.4 M NaCl. This procedure yielded a high-molecular-weight complex containing phospholipids, cytochrome b-245 and NADPH oxidase activity. Cytochrome b was found to be purified to the same extent as NADPH oxidase activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the various purification fractions showed a progressive enrichment of a band whose molecular weight is 3.2 X 10(4). The enrichment of this protein band paralleled those of NADPH oxidase activity and of cytochrome b, indicating that it is a component of the oxidase system. The possibility that this band corresponds to either cytochrome b or a flavoprotein/cytochrome b complex is considered.

The cytochrome b and flavin content and properties of the O2- -forming NADPH oxidase solubilized from activated neutrophils.

NADPH-dependent O2- -generating activity was extracted and partially purified from guinea pig polymorphonuclear leukocytes. The most active preparation generated 202.8 nmol O2- min/min per mg protein. This activity was 30-fold higher than that of extracts from resting cells, indicating that the activated state of the oxidase was retained after solubilization. The solubilization and purification of the enzyme activity were followed by a parallel solubilization and purification of cytochrome b. Spectroscopic studies showed that solubilized cytochrome b has an Em of -245 mV and binds CO to about 30%. Cytochrome b was reduced by NADPH in anaerobiosis at a low rate and was rapidly reoxidized by air. A correlation was found between the inhibition of O2- formation caused by the SH reagent p-chloromercuribenzoate and the alterations induced by this compound on the Em of cytochrome b. These observations strongly support the participation of cytochrome b in the catalytic activity of the solubilized NADPH oxidase. The enzyme preparations contained FAD, which was found to be associated both with NADPH oxidase and with diaphorase activities. The fraction with the highest O2- forming activity contained FAD and cytochrome b in a ratio of about 0.5:1. The participation of FAD in the electron transport from NADPH to O2 is supported also by the inhibitory effect exerted by quinacrine on O2- formation.

Determination of lipid hydroperoxides in native low-density lipoprotein by a chemiluminescent flow-injection assay.

Lipid hydroperoxides have been implicated in the pathogenesis of atherosclerosis. This work was therefore set up to obtain a fast and specific chemiluminescent assay for measuring hydroperoxides in native low-density lipoprotein (LDL). The apparatus was a complete HPLC system including two pumps, an autosampler, a computer and a chemiluminescent detector with a T-mixing coil in the place of the column. Samples were injected from the autosampler and mixed with luminescent reagent (3 microM luminol and 1 microM microperoxidase in 0.1 M carbonate buffer (pH 10)) in the T-piece. To generate a calibration curve, linoleic acid hydroperoxide was obtained by incubating soybean lipoxygenase with linoleic acid. The calculated conjugated diene concentration was in good agreement with the nominal linoleic acid hydroperoxide concentration. The chemiluminescence was linear with the amount of linoleic acid hydroperoxide injected and the detection limit was about 3 pmol linoleic acid hydroperoxide. The chemiluminescence induced by copper-oxidized LDL was linear with concentration; the detection limit, when compared with linoleic acid hydroperoxide, was similar. The reproducibility of the linoleic acid hydroperoxide and of oxidized LDL hydroperoxide was examined in single pools. The coefficient of variation on the triplicates of each pool was about 3%. The titre of the linoleic acid hydroperoxide and oxidized LDL peroxides was quite stable for at least 10 days when stored under argon at 4 degrees C in the presence of EDTA. The mean value of the LDL hydroperoxides in 16 control subjects was 145.20 +/- 98.81 pmol/mg LDL protein. In conclusion, the microperoxidase-luminol-dependent chemiluminescence flow-injection assay is a rapid, sensitive and selective method for measuring lipid hydroperoxides in native LDL.

Troglitazone reduces LDL oxidation and lowers plasma E-selectin concentration in NIDDM patients.

Troglitazone, an oral antidiabetic agent with antioxidant properties, has previously been shown to increase the resistance of LDL to oxidation in vitro and in vivo in healthy volunteers. In a randomized, placebo-controlled, parallel-group study in 29 patients with NIDDM, we tested the effect of troglitazone (200 mg once daily) on the resistance of LDL to oxidation and on circulating levels of preformed lipid hydroperoxides and the adhesion molecule E-selectin. Resistance of LDL to oxidation was assessed by measuring 1) fluorescence development induced by copper treatment (lag phase), and 2) amount of thiobarbituric acid-reactive substances (TBARS) generated by incubation with umbilical vein endothelial cells. At 8 weeks, the lag phase was increased by 23% (P < 0.01 by analysis of covariance [ANCOVA]) in the patients receiving troglitazone (n = 18) compared with the group receiving placebo (n = 11). At the same time, TBARS were 3.63 +/- 0.10 nmol/l (vs. 5.32 +/- 0.10 nmol/l in the placebo group, P = 0.009), LDL hydroperoxide concentration was reduced from 1.48 +/- 0.03 to 1.19 +/- 0.03 ng/mg (no change in the placebo group, P < 0.01), and plasma E-selectin levels decreased from 56.5 +/- 2.33 to 43.7 +/- 1.77 microg/l (no change in the placebo group, P < 0.01). In NIDDM, troglitazone may slow down the development of atherosclerosis by modifying LDL-related atherogenic events.

Antioxidants inhibit the expression of intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1 induced by oxidized LDL on human umbilical vein endothelial cells.

The oxidative modification of low density lipoprotein (LDL) and the endothelial expression of adhesion molecules are key events in the pathogenesis of atherosclerosis. In this study we evaluated the effect of oxidized LDL on the expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin on human umbilical vein endothelial cells (HUVECs). The hypothesis that oxidized LDL functions as a prooxidant signal was also evaluated, by studying the effect of different radical-scavenging antioxidants on expression of adhesion molecules. LDL was oxidized by using Cu2+, HUVECs or phospholipase A2 (PLA2)/ soybean lipoxygenase (SLO), the degree of oxidation being measured as thiobarbituric acid-reactive substances (TBARS) and conjugated dienes (CD). Exposure of 200 micrograms/ml of native LDL to 1 microns Cu2+, HUVECs and to PLA2/ SLO resulted in four- to fivefold higher levels of TBARS and CD than in native LDL. Cu(2+)-(1 microM), HUVEC-, and PLA2/SLO-oxidized LDL caused a dose-dependent, significant increase of ICAM-1 and VCAM-1 (p < .01). The expression of E-selectin did not change. LDL oxidized with a 2.5 and 5 microM Cu2+ did not increase ICAM-1 and VCAM-1 significantly. Both the Cu(2+)- and HUVEC-oxidized LDL, subjected to dialysis and ultrafiltration, induced ICAM-1 and VCAM-1 expression. After incubation with the ultrafiltrate, the expression of ICAM-1 and VCAM-1 was not significantly different from that obtained with native LDL. LDL pretreated with different antioxidants (vitamin E and probucol) and subjected to oxidation by Cu2+ and HUVECs induced a significantly lower expression of ICAM-1 and VCAM-1 than nonloaded LDL (p < .01). The pretreatment of HUVECs with vitamin E and probucol significantly reduced the expression of VCAM-1 on HUVECs induced by oxidized LDL (p < .01); the effect on ICAM-1 was much less evident. In conclusion, oxidized LDL can induce the expression of different adhesion molecules on HUVECs; this induction can be prevented by pretreating either the LDL or the cells with radical-scavenging antioxidant.

Effect of plasma on the degradation of hydroperoxides of unesterified linoleic acid and copper-peroxidized LDL.

The determination of lipid hydroperoxides in plasma and lipoproteins recently reached a clinical relevance in disorders such as atherosclerosis, where oxidative reactions have been suggested to play a fundamental pathogenetic role. The peroxide content of lipoproteins is usually measured after ultracentrifugation and extraction. During this procedure, some peroxides might decompose causing a too low recovery. To screen this possibility, the disappearance, in the presence of human plasma, of hydroperoxides of linoleic acid and Cu-oxidized low density lipoprotein (LDL) have been investigated, using both a iodometric titration and an enzymatic assay. While only in the presence of GSH plasma decomposes linoleic acid hydroperoxides quite rapidly, peroxides in Cu-oxidized LDL were stable both in presence as well as in absence of GSH. This indicated that lipid hydroperoxides are stable in plasma and that peroxides of Cu-oxidized LDL are not substrate for the glutathione-dependent peroxidase activity in plasma. The relevant decrease of the iodometric titre of LDL peroxides observed in the presence of elevated amounts of plasma was shown to be artifactual, since some compounds extracted from plasma do react with iodine generated by peroxides. Whole plasma itself, indeed, has been shown to reduce back to I- appreciable amount of free iodine.

Effects of guar gum on plasma lipoproteins and apolipoproteins C-II and C-III in patients affected by familial combined hyperlipoproteinemia.

In recent years, guar gum has been shown to be a potent hypocholesterolemic agent. The effects of this fiber on triglycerides are less clear. In order to evaluate the influence of guar supplementation on plasma lipoproteins and apolipoprotein C-II (apoC-II) and apolipoprotein C-III (apoC-III) isoforms (apoC-III2, apoC-III1, apoC-III0), 16 g/day of guar gum were administered to 12 outpatients affected by familial combined hyperlipoproteinemia for a period of 60 days. Mean total cholesterol and triglyceride levels significantly decreased after 15 days of treatment and persisted reduced at the 30th and 60th day of guar supplementation. While low-density lipoprotein cholesterol paralleled the reduction of total plasma cholesterol, we did not observe any change in the cholesterol content of high-density lipoprotein (HDL) subfractions (HDL2 and HDL3) during the study. A redistribution of the relative content of very low-density lipoprotein apoC-III isoforms with a significant increase of apoC-III1 and a decrease of apoC-III0 was observed after 15, 30, and 60 days of guar gum administration. The results show that guar gum reduces not only cholesterol but also triglyceride levels in patients affected by familial combined hyperlipoproteinemia. Further studies are needed to confirm the suggestion that the different distribution of very low-density lipoprotein apoC-III isoforms induced by guar supplementation may influence the behavior of plasma triglycerides.

Long-term effect of a low-fat, high-carbohydrate diet on plasma lipids of patients affected by familial endogenous hypertriglyceridemia.

We evaluated the effect of a low-fat, high carbohydrate (LFHC) diet on plasma lipids in 10 patients affected by familial endogenous hypertriglyceridemia. All the patients studied underwent a base-line period of 4 wk, a 12-wk intervention period, and an 8-wk switch-back period. During the control periods patients consumed approximately 45% of energy as fat and approximately 40% as carbohydrate. During the intervention period they consumed an isocaloric diet containing approximately 25% of energy as fat and approximately 60% as carbohydrate. Total plasma triglyceride and cholesterol levels decreased significantly after 45 and 90 d of treatment (p less than 0.01). The reduction of plasma cholesterol was associated mostly with the decrease in VLDL cholesterol (p less than 0.01) while LDL cholesterol increased at days 45 and 90 of the LFHC diet (p less than 0.01). Finally, we observed a significant increase in HDL cholesterol both at days 45 and 90 of the LFHC diet (p less than 0.01). The LFHC diet we used may be an useful tool in the management of hypertriglyceridemia.

Predisposition to LDL oxidation in patients with and without angiographically established coronary artery disease.

Oxidative modification of low density lipoprotein (LDL) may play an important role in the mechanism of atherosclerotic damage to blood vessels. In the present study the LDL isolated from the plasmas of 73 coronary artery disease (CAD) patients, 28 valvular heart disease (VHD) patients, 59 subjects affected by type IIa hyperlipoproteinemia and 71 controls was oxidatively modified by incubation with copper ions. In 15 CAD and 15 Type IIa patients and 15 controls the LDL chemical composition and polyunsaturated fatty acid (PUFA) content were also measured. Differences in the LDL susceptibilities to lipid peroxidation were studied by measuring the changes of fluorescence intensity. The lag phase in the CAD patients was found to be significantly lower than in the VHD and controls (P < 0.001). The lag phase in the type IIa patients was significantly higher than in the CAD patients (P < 0.01), and significantly lower than the VHD and controls (P < 0.01). The LDL isolated from the type IIa patients had an increase in the relative content of free and esterified cholesterol (P < 0.05), while the CAD patients had a decrease in the relative content of free cholesterol (P < 0.05), and an increase in the relative content of protein (P < 0.05). The lowest value of the LDL cholesterol to protein ratio and LDL size, was found in the CAD patients (P < 0.05). When expressed in micrograms/mg LDL cholesterol, the concentration of the LDL PUFAs was significantly higher in the CAD group than in the others (P < 0.05). The LDL alpha-tocopherol concentration was quite similar in the different groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Lacidipine inhibits the activation of the transcription factor NF-kappaB and the expression of adhesion molecules induced by pro-oxidant signals on endothelial cells.

OBJECTIVE: The adhesion of monocytes to endothelium, an early event in atherosclerosis is mediated by cell adhesion molecules. Signal-transduction pathways for these binding molecules include the translocation of the transcription factor NF-kappaB; moreover, intracellularly generated oxygen-derived free radicals play a major role in this process. In this study we evaluated the extent to which lacidipine, a calcium antagonist with antioxidant properties, affects the expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin on human umbilical vein endothelial cells, induced by different pro-oxidant signals such as oxidized low density lipoprotein (LDL) and tumor necrosis factor-alpha (TNF-alpha). METHODS: We incubated 5 micromol/l Cu2+-oxidized LDL and TNF-alpha (2 ng/ml) with human umbilical vein endothelial cells for 48 and 6 h, respectively. ICAM-1, VCAM-1 and E-selectin were measured by flow cytometry. NF-kappaB was evaluated by electrophoretic mobility shift assay. RESULTS: The incubation of 5 micromol/l Cu2+-oxidized LDL not only caused a dose-dependent increase in ICAM-1, VCAM-1 and E-selectin (P < 0.001), but also synergically increased their TNF-alpha-induced expression (P < 0.001). The addition of lacidipine to human umbilical vein endothelial cells significantly reduced the expression of ICAM-1, VCAM-1 and E-selectin induced by TNF-alpha alone or with oxidized LDL (P < 0.001). The reduction in adhesion molecule expression caused by lacidipine was paralleled by a significant fall in NF-kappaB translocation. CONCLUSIONS: The results suggest that lacidipine may have prevented NF-kappaB-mediated adhesion molecule expression by exerting its effects on oxygen-derived free radicals. The results support previous observations that lacidipine may have therapeutic effects in atherosclerosis.

Oxidized low-density lipoprotein increases the production of intracellular reactive oxygen species in endothelial cells: inhibitory effect of lacidipine.

OBJECTIVE: The mechanisms by which oxidized low-density lipoprotein (ox-LDL) induces the expression of adhesion molecules on endothelial cells (HUVECs) are still not clear. The signal transduction pathways for these binding molecules include the translocation of the transcription factor NF-kB and the intracellular reactive oxygen species (ROS) are said to play a key role in this process. Aim of this study was (1) to evaluate the effect of ox-LDL on intracellular production of ROS in culture of HUVECs; (2) to evaluate if the intracellular increase of ROS induced by ox-LDL is mediated by the binding to a specific endothelial receptor; (3) to ascertain if lacidipine can decrease ox-LDL-induced ROS production in HUVECs. METHODS: Five microM Cu2+ ox-LDL were incubated with HUVECs for 5 min. 2',7'-Dichlorofluorescein (DCF) as an expression of intracellular ROS production, was measured by flow cytometry. RESULTS: ox-LDL induced a significant dose-dependent increase in DCF production (P < 0.001) through the binding to a specific receptor. The preincubation of HUVECs with radical scavengers compounds and lacidipine significantly reduced (P < 0.001) the ox-LDL-induced DCF production. CONCLUSIONS: ox-LDL increased the intracellular formation of ROS through the ligation to a specific endothelial receptor. Preincubation of HUVECs with lacidipine, a calcium antagonist with antioxidant properties, significantly reduced the intracellular ROS formation induced by ox-LDL. We propose that the effect of lacidipine on adhesion molecule expression and on NF-kB activation can be explained by its effect on intracellular ROS formation.

Comparative effects of different dihydropyridines on the expression of adhesion molecules induced by TNF-alpha on endothelial cells.

OBJECTIVE: Lacidipine has already been demonstrated to reduce the expression of some adhesion molecules induced by pro-oxidant signals on endothelial cells. In order to verify if this effect is a peculiarity of this molecule, or belongs to other dihydropyridinic compounds (DHPs), the activity of lacidipine was compared with that of lercanidipine, amlodipine, nimodipine and nifedipine. DESIGN AND METHODS: The compounds were incorporated in human umbilical vein endothelial cells (HUVECs) using native low-density lipoprotein as a carrier. The drug concentrations in HUVECs were measured by mass spectrometry. Human recombinant tumour necrosis factor-alpha was then incubated with HUVECs for 7 h at 37 degrees C for adhesion molecule expression. RESULTS: The cellular amount of lacidipine, lercanidipine and amlodipine was similar, while nimodipine and nifedipine were almost undetectable or undetectable, respectively. Lacidipine, at any concentration, determined a dose-dependent significant decrease of the expression of intercellular adhesion molecule-1 (ICAM-1) ICAM-1, vascular cell adhesion molecule-1 (VCAM-1) VCAM-1 and E-selectin (P < 0.01). Lercanidipine and amlodipine determined variable decreases of adhesion molecules at the intermediate and highest concentrations. Nimodipine and nifedipine determined no effect on ICAM-1, VCAM-1 and E-selectin. The lowest IC50, i.e. the concentration determining the 50% reduction of ICAM-1, VCAM-1 and E-selectin expression was obtained with lacidipine for all the adhesion molecules considered (P < 0.01). CONCLUSIONS: It is concluded that the effect of the DHPs used in this study on adhesion molecule expression is determined first by their lipophilicity and then by their intrinsic antioxidant activity.

Predisposition to LDL oxidation during copper-catalyzed oxidative modification and its relation to alpha-tocopherol content in humans.

The predisposition to LDL oxidation during copper-catalyzed oxidative modification and its relationship with LDL alpha-tocopherol concentration was studied in 41 control subjects. The results show that the predisposition of LDL to oxidation expressed as duration of the inhibition period and rate of the propagation period varied greatly in the controls, but did not correlate with the values of LDL alpha-tocopherol. On the contrary the experiments with alpha-tocopherol incorporated in LDL demonstrate that even small increases of incorporated alpha-tocopherol, under circumstances where other variables were probably largely unaffected, increased proportionally the length of the inhibition period and reduced the rate of the propagation period. The values of LDL alpha-tocopherol achieved after the enrichment turned out to be positively correlated with the duration of the inhibition period and negatively with the rate of the propagation period. Finally the results of this study also show that there was a variability in the LDL alpha-tocopherol decay of different subjects under the same oxidative stress. In our conditions however, the time in which alpha-tocopherol contributed to the LDL protection was much shorter than the mean length of the inhibition period. The results demonstrate that the variability in the predisposition to LDL oxidation during copper-catalyzed oxidative modification is not determined only by the concentration of alpha-tocopherol in LDL and that therefore its value as a sole indicator of antioxidant status is probably inadequate.

The susceptibility of low-density lipoprotein to in vitro oxidation is increased in hypercholesterolemic patients.

It has been suggested that the oxidative modification of low-density lipoprotein (LDL) plays a major role in atherogenesis. We evaluated the oxidative resistance to copper-induced oxidative changes of LDL derived from patients affected by type IIa hyperlipoproteinemia compared with healthy subjects and faced the question of the importance of the antioxidants and polyunsaturated fatty acids (PUFAs) contained in LDL in determining its variability. LDL isolated from the plasmas of 25 subjects affected by familial hypercholesterolemia and 15 control subjects was oxidatively modified with Cu2+ in vitro, and the differences in LDL susceptibilities (lag and propagation phases) to lipid peroxidation were studied by measuring the changes in fluorescence intensity. LDL alpha-tocopherol and PUFAs were also measured. The lag phase was significantly lower and the propagation phase significantly higher in the type IIa patients than in control subjects (p < 0.01). The linoleic and arachidonic acids, expressed as percentage of total LDL fatty acids, were significantly higher in type IIa patients than in the control subjects (p < 0.01). There was a positive significant correlation between the LDL cholesterol and the linoleic and arachidonic acids as percentage of total LDL fatty acids (p < 0.01). Both linoleic and arachidonic acids turned out to be negatively correlated with the lag phase and positively with the propagation phase (p < 0.01). The concentration of LDL alpha-tocopherol was similar in the two groups. Therefore, type IIa patients have a greater susceptibility to LDL oxidation than control subjects. This may be due to a relative higher concentration of linoleic and arachidonic acids in LDL derived from patients with familial hypercholesterolemia.

Increased susceptibility of LDL to in vitro oxidation in patients on maintenance hemodialysis: effects of fish oil and vitamin E administration.

Oxidized low-density lipoproteins (LDL) play an important role in the pathogenesis of atherosclerosis. An increased sensitivity of red blood cell membranes to lipid peroxidation has been previously demonstrated in patients with chronic renal failure, suggesting that the antioxidant defence of lipoproteins might be impaired. Fish oil supplementation has been proposed in dialysis patients, but it is still unclear if the positive effects of fish oil depend only on its polyunsaturated fatty acid content or on other factors, such as the usually added antioxidants. Moreover, the increased concentration of highly peroxidable n-3 polyunsaturated fatty acids induced by fish oil in LDL particles could favour LDL oxidation and possibly the development of atherosclerosis. The present study was designed to evaluate the susceptibility of LDL to in vitro oxidation (lag phase) and the rate of lipid peroxidation (propagation phase) by fluorescence development during copper exposure in 14 hemodialysis patients. A further aim was to compare the effects on lipid metabolism and LDL oxidation of fish oil supplementation (20 ml containing vitamin E 20 IU as antioxidant) for 30 days and of vitamin E administration (50 IU) for another 30 days. The length of the lag phase and vitamin E concentration were significantly reduced (p < 0.01) in hemodialysis patients and increased significantly (p < 0.01) after administration of both fish oil and vitamin E. Fish oil supplementation also reduced plasma lipids significantly (p < 0.01) and increased the propagation phase (p < 0.01). Our results demonstrate that the susceptibility of LDL to oxidation is enhanced in hemodialysis patients, suggesting a possible relationship between excessive LDL peroxidation and accelerated atherosclerosis. The increased susceptibility of LDL to in vitro oxidation can be explained, at least partially, by a reduced LDL vitamin E concentration. Since fish oil increased the lag phase to the same extent as vitamin E supplementation, the positive effect of fish oil could be partly explained by its antioxidant content.

Selective enrichment of NADPH oxidase activity in phagosomes from guinea pig polymorphonuclear leukocytes.

Recent studies have demonstrated that the activated NADPH oxidase, the enzyme responsible for the stimulation of O2 consumption with O2 formation during phagocytosis, is located in the plasma membrane of leukocytes. The present work deals with whether the activation induced by phagocytosis involves the enzyme of the entire membrane or only that of the portion of the membrane that interacts with the phagocytosable particle and forms the phagosome. The results presented show that the activity of the NADPH oxidase of phagosomal membrane, isolated by centrifugation of homogenates on discontinuous sucrose gradients, is increased 12.6-fold with respect that of homogenate. In contrast, the activities of 5'-nucleotidase and of acid p-nitrophenyl phosphatase, enzyme markers of the plasma membrane not activated during phagocytosis and uniformly distributed on the entire membrane, are increased only about three-fold with respect to that of homogenate. These results indicate that during phagocytosis and activation of NADPH oxidase is a segmentary response that involves only the enzyme that forms the phagocytic vacuole. This fact is relevant for the function of toxic intermediates of oxygen reduction that are discharged in direct contact with the engulfed agent.

[Prevalence of reported child abuse by parents in a pediatric out-patient population]. / Prevalência de violência física relatada contra crianças em uma população de ambulatório pediátrico.

A cross-sectional study was carried out to estimate prevalence of child abuse with battering among patients under age 14 for alleged disciplinary purposes. The reference population were children attending pediatric out-patient services at the University Hospital in Campinas, State of São Paulo, Brazil, a city of one million inhabitants with universal coverage in terms of accessing health care in the unit under study. Interviews were carried out with 130 parents or guardians in a randomly selected group according to order of attendance, and the prevalence of child abuse was estimated at 10.8%, considering as positive cases those where parents referred child battering under age one year or where there was evidence of bodily lesion over age one year. A validation study for the experimental questionnaire is proposed in order to allow for future estimation of population prevalence or incidence. The use of qualitative methods is also recommended as a means of gathering detailed data on socio-psychological determination for the supposedly disciplinary aggression with child abuse.

[Shaken baby syndrome. Report of a case]. / Shaken baby syndrome (síndrome do bebê chacoalhado). Relato de caso]

The authors report the case of a 50-day-old baby who was brought to the Pediatric Emergency Room of the "Hospital de Clinicas da UNICAMP" with seizures. At the examination she presented without external signs of trauma, hypoactive and with generalized seizures. Numerous hemorrhages were found in the ocular fundi. CT scan showed interhemisferic hemorrhage and brain swelling. The child was admitted to the Pediatric Intensive Care Unit, kept under controlled ventilation and hydantal. Although these measures, she died three days later. Since the beginning there was suspicion of child abuse. One day after the admission the father told that he had shaken the baby because she was crying too much. The present paper discuss social and epidemiological aspects, and about the difficulties in the diagnosis of this syndrome that sometimes may be fatal, as in this case.

[Use of alcoholic beverages by children: a not yet investigated phenomenon]. / Uso de bebidas alcoólicas por crianças: fenômeno a ser investigado.

The essay discourses on the ethanol prescription to children, their dependence and the scientific data related to the social abuse. The toxic effects, social and economical perspectives are well exemplified on the follow-up of a child of three years old in a university hospital during eighteen months. At the end, the prevalence of such practice is highlighted as essential for preventive approach on public health.

[Domestic violence against children (First Part)]. / Violência doméstica contra a criança (Parte I).

The authors interviewed 76.2% of the pediatricians from the Pediatric Department-School of Medical Sciences-Campinas University-UNICAMP, to analyse the difficulties of this professional group in the evaluation of the domestic violence against children. From the group of professionals, 86.7% had already attended a situation involving child abuse; the therapeutic aspects were referred in 44.4% as most important, and in 46%, the difficulties in the diagnostic aspects were referred. They conclude that, despite the experiences with the theme, a lot more could be done and studied.