[Acute renal failure secondary to paroxysmal cold hemoglobinuria]. / Insuficiencia renal aguda secundaria a hemoglobinuria paroxística al frío.

Paroxysmal cold hemoglobinuria is a type of hemolytic anemia that mainly affects children. Tubular renal injury induced by the heme pigment in intravascular hemolysis is a rare cause of renal failure. We describe the case of a 5-year-old boy who presented with dehydration and dark urine a few hours after exposure to cold. The child had had an upper respiratory infection the previous week. He developed anemia and acute renal failure. The direct antiglobulin test was positive with anti-C3c and C3d. The diagnosis of paroxysmal cold hemoglobinuria was confirmed by the presence of biphasic antibody in the Donath-Landsteiner test.

Insuficiencia renal aguda secundaria a hemoglobinuria paroxística al frío / Acute renal failure secondary to paroxysmal cold hemoglobinuria

La hemoglobinuria paroxística al frío es una forma de anemia hemolítica que afecta principalmente a niños. La toxicidad tubular renal del pigmento hemo en situaciones de hemólisis intravascular constituye una etiología rara de insuficiencia renal. Se presenta el caso de un paciente de 5 años que presenta deshidratación y orina oscura horas después de exponerse al frío. Una semana antes había un antecedente de infección de las vías aéreas superiores. En su evolución desarrolla anemia e insuficiencia renal aguda. El test de Coombs directo es positivo con anti-C3c y C3d. La presencia de anticuerpos bifásicos queda demostrada con la prueba de Donath-Landsteiner, lo que confirma el diagnóstico de hemoglobinuria paroxística al frío

Paroxysmal cold hemoglobinuria is a type of hemolytic anemia that mainly affects children. Tubular renal injury induced by the heme pigment in intravascular hemolysis is a rare cause of renal failure. We describe the case of a 5-year-old boy who presented with dehydration and dark urine a few hours after exposure to cold. The child had had an upper respiratory infection the previous week. He developed anemia and acute renal failure. The direct antiglobulin test was positive with anti-C3c and C3d. The diagnosis of paroxysmal cold hemoglobinuria was confirmed by the presence of biphasic antibody in the Donath-Landsteiner test

Molecular studies in Portuguese patients with Smith-Lemli-Opitz syndrome and report of three new mutations in DHCR7.

Smith-Lemli-Opitz syndrome (SLO) is an autosomal recessive disorder characterised by craniofacial dysmorphism, mental retardation, multiple congenital anomalies, and increased levels of 7-dehydrocholesterol (7-DHC) in body tissues and fluids. SLO is caused by mutations in the DHCR7 gene which encodes 7-dehydrocholesterol reductase, the last enzyme of cholesterol biosynthesis pathway. In our investigation, we screened 682 dysmorphic/mentally retarded Portuguese patients for abnormal levels of 7-DHC in blood by UV spectrometry. We identified six unrelated patients with SLO (0.87% of total). Mutational analysis of the DHCR7 gene led to the identification of seven distinct mutations, three of which are new (F174S, H301R, and Q98X). The common IVS8-1G > C and T93M variants together with the H301R accounted for 70% of the all SLO alleles in our population. Our findings contribute to the variegate array of pathological changes in the DHCR7 gene among different European populations.

Prenatal diagnosis of 21 cases of metabolic disorder

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Evaluation of a non-invasive fluorescence technique as a marker for diabetic lenses in vivo.

BACKGROUND: A fluorescence spectrometer has been constructed to study in vitro and in vivo fluorescence of human lenses. This instrument can measure fluorescence emission spectra, which can be useful in the characterisation of the lens endogenous fluorophores and evaluation of the feasibility of fluorescence measurement as a non-invasive marker for diabetes. The spectrometer allows determination of the optimum excitation and emission wavelengths, which can be used in simpler instrumentation for monitoring purposes. METHODS: To est the application in such studies a homogeneous group of type II diabetic subjects and normal controls was studied. For each subject the fluorescence emission spectra was measured using a spectrometer prototype consisting of a modified slit lamp coupled to a optical multichannel analyser (OMA). The incorporation of narrow-band filters allows the selection of three different excitation wavelengths: 404 nm, 436 nm and 485 nm. RESULTS: With both in vitro and in vivo measurements, no significant differences were found between diabetic and normal lenses concerning the wavelength of maximum emission of fluorescence. However, the spectra (lambda(exc)=436 nm) between 480 and 550 nm were better defined with diabetic lenses. Using ratios of fluorescence intensity at two different wavelengths (490/610, 510/610, and 550/610) allows for good discrimination between normal controls and diabetic patients. The use of ratios largely removes the effects due to attenuation of excitation light and emitted fluorescence. CONCLUSIONS: The non-invasive evaluation of lens fluorescence is proposed as early indicator of ocular complications associated with diabetes.

Cholesterol oxides accumulate in human cataracts.

Human lens membranes contain the highest cholesterol content of any known biological membrane. Although cholesterol is prone to oxidation, the presence of its oxidation products in human cataract has not been shown before. This study was designed to investigate the presence of cholesterol oxides in human cataractous lenses. Human clear lenses (n = 48) were obtained from Coimbra University Hospital Eye Bank. Human cataracts (n = 54) were obtained by routine extracapsular surgery. Cholesterol oxides were isolated by solid-phase extraction on a C18 cartridge and quantified as TMS-ether derivatives by gas chromatography. The extraction procedure allows for an efficient recovery of the major cholesterol oxides, while retaining virtually all cholesterol. Exposure of membranes isolated from transparent human lenses to the free radical generator 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH) produced 7 alpha-hydroxycholesterol (6%), 7 beta-hydroxycholesterol (19%), 5 alpha, 6 alpha-epoxycholestanol (1%) and 7-ketocholesterol (74%) as major oxidation products. Cataractous lenses contained quantifiable amounts of 7 beta-hydroxycholesterol (7.3 +/- 0.74 mmol mol-1 cholesterol), 7-ketocholesterol (4.2 +/- 0.32 mmol mol-1 cholesterol), 5 alpha, 6 alpha-epoxycholestanol (0.9 +/- 0.16 mmol mol-1 cholesterol), 20 alpha-hydroxycholesterol (0.6 +/- 0.13 mmol mol-1 cholesterol) and 25-hydroxycholesterol (0.1 +/- 0.02 mmol mol-1 cholesterol), whereas clear lenses contained no detectable amounts of cholesterol oxides. We have shown, for the first time, that oxysterols accumulate in human cataracts. Although the total amount of oxidized cholesterol in cataracts is not likely to be high it may account for much of the membrane damage associated with cataract formation.

Crystallin composition of human cataractous lens may be modulated by protein glycation.

BACKGROUND: This study was designed to establish whether increased glycation of human crystallins could be related to an increased susceptibility to aggregation and insolubilization. The study was focused particularly on the glycation levels and composition of low-molecular-weight (LMW) peptides present in human cataractous lenses. METHODS: Lens crystallins from the water-soluble fraction were separated on a preparative scale by gel filtration. Each crystallin was purified and its glycation level evaluated as furosine content. The peptides were further purified by reverse-phase chromatography. The amino acid composition of each of these peptides was also determined by RP-HPLC using PITC pre-column derivatization. RESULTS: The high-molecular-weight (HMW), alpha L-crystallin and LMW crystallins from diabetic patients present high furosine content. LMW peptides were shown to constitute a heterogeneous population of three major peptides with a lysine content similar to that observed for native crystallins. These peptides were shown to present glycation levels ten times higher than those observed for the crystallins. Glycated proteins from insoluble fraction were found to be mostly urea soluble and were present at higher concentration in diabetic cataracts. CONCLUSIONS: LMW peptides are suggested to play a major role in protein aggregation and insolubilization, probably via a mechanism involving protein glycation. This process seems to be particularly relevant to diabetic cataract development.

Role of glycation in human lens protein structure change.

PURPOSE: Protein glycation may be involved in cataract development, by altering protein structure, particularly amino acid composition, and formation of fluorophores through a Maillard reaction. This study was designed to evaluate major changes in early and advanced (fluorescent) glycation products, with special emphasis on glycation-induced changes in amino acid composition of lens proteins. METHODS: We analyzed 50 human cataractous lenses (25 diabetic and 25 non-diabetic). Glycated proteins were isolated by affinity chromatography. Glycated and non-glycated proteins were separated by molecular sieve chromatography and further analyzed by RP-HPLC to establish the amino acid content. Early glycation levels were determined as furosine content and advanced glycation products were quantified by the characteristic fluorescence. RESULTS: Specific lens fractions (HMW and LMW) present significant differences in fluorescence levels between glycated and non-glycated proteins, specially in cataractous lenses from diabetic patients in which all proteins analyzed presented higher glycation levels than in non-diabetic patients. The amino and analysis of glycated proteins also revealed some important differences in specific basic residues (namely Lys, Arg and His) compared to the non-glycated fraction. CONCLUSIONS: The results suggest that protein glycation may be involved in changes in amino acid composition and fluorophore formation. This process may well account for the increased risk factor that diabetes represents for cataract development.

An experimental model for the evaluation of lipid peroxidation in lens membranes.

Lipid peroxidation has been associated with a number of specific manifestations related both to lens aging and cataract development. The assessment of the effect of various naturally occurring prooxidants as well as the development of antioxidant strategies has often been limited by the lack of appropriate and simple experimental models. In this study we discuss the adaptation of a method based on the incorporation of a fluorescent probe (parinaric acid) into biological membranes to monitor early stages of lipid peroxidation. After establishing the appropriate conditions, the method can be successfully applied to study peroxidation in bovine lens membranes, allowing for the evaluation of the effect of several free radical generating systems, including the following metal-dependent initiators: ascorbate/ iron, hydrogen peroxide/copper and cumene hydroperoxide/ copper. The inhibitory effect of the chelating agent diethylene-triaminepenta-acetic acid and the competitive hydroxyl radical scavenger sorbitol, was consistently observed on parinaric acid degradation, on hydroxyl radical yield and on the amount of thiobarbituric acid reactive material produced. It could be shown that oxidative degradation of the probe gives direct information on lens membrane susceptibility to a specific peroxidation system. Parinaric acid can therefore be used as an efficient oxidation probe to evaluate oxidative damage inflicted to lens membranes by different systems, allowing also the evaluation of the antioxidant effect of various drugs including those with potential anticataractogenic effect.

A technical approach to the evaluation of glucose oxidation: implications for diabetic cataract.

Oxidative stress has recently been involved in a number of diseases including development of diabetic cataract. If hyperglycemia is the relevant factor in diabetes, then it is reasonable to assume that under physiological conditions glucose may be toxic. The mechanisms involved in such a type of glucose 'toxicity' are still poorly understood but may involve glucose autoxidation. In this study we discuss a new methodological approach to the evaluation of glucose-induced oxidative damage to bovine lens membranes. The method is based on the incorporation of a fluorescent probe (parinaric acid) into lens membranes. The oxidative degradation of the probe is evaluated by monitoring its fluorescence decrease. It was possible to show that glucose may induce oxidative damage in the presence of trace amounts of transition metals. Furthermore, the data obtained by monitoring oxidative degradation of parinaric acid could be related to the amount of thiobarbituric acid-reactive substances formed under identical periods of time. The technique was shown to be reproducible, straightforward and highly sensitive as compared to other classical methods. Moreover, this methodological approach allows not only the evaluation of the extension of oxidative stress inflicted upon lens membranes but also the evaluation of the antioxidant effect of various compounds including some drugs with a potential anticataractogenic effect.

Oxidative damage to lens membranes induced by metal-catalyzed systems.

The oxidative effect of hydrogen peroxide, ascorbic acid and glucose in the presence of transition metals was studied. Incubation of bovine lens membranes with hydrogen peroxide/metal, ascorbic acid/metal and glucose/metal systems resulted in a significant augmentation of thiobarbituric acid-reactive substance content in the membranes. Presence of alpha-tocopherol decreased the extent of oxidative damage. This oxidation was found to be mediated by hydroxyl radicals. Dependence of hydroxyl radical generation upon the buffer used has been observed. In all experiments Cu(II) ions exhibited a higher efficiency compared to Fe(II) ions.

Bendazac decreases in vitro glycation of human lens crystallins. Decrease of in vitro protein glycation by bendazac.

Bendazac has been used as an anti-cataractogenic drug. It has been reported that this acts by preventing protein denaturation. In this study the ability of bendazac to inhibit in vitro glycation of human lens crystallins was evaluated. Possible effects of bendazac were detected by incubation of WS crystallins with the reducing sugars glucose and fructose. The efficiency of bendazac was evaluated by means of selected parameters including: browning, glycation (measured as tyrosine content) and specific NTP-fluorescence. The results showed clearly that bendazac (bendazac L-lysine and sodium) inhibits the early stages of protein glycation, as well as the formation of fluorescent advanced glycation products. Bendazac lysine (20 mM) proved to be more effective in inhibiting fluorescence development (67% inhibition) that the corresponding sodium salt (35% inhibition). No significant differences were found with respect to furosine levels; about 40% inhibition was produced with either bendazac lysine or sodium salt bendazac clearly inhibits glycation of human lens crystallins, as can be efficiently monitored by following specific changes in lens protein fluorescence. These results may constitute a new and relevant therapeutic approach to monitoring cataract development.

Age-related changes in normal and cataractous human lens crystallins, separated by fast-performance liquid chromatography.

The water-soluble crystallins from normal human lenses (n = 32), cataractous lenses of diabetic patients (n = 9) and cataractous lenses of nondiabetic patients (n = 9) were analyzed with fast-performance liquid chromatography. Six different crystallin classes were separated reproducibly by chromatography on Superose 6. The fractions were identified as alpha H, alpha L, beta H, beta L1, beta L2 and low-molecular-weight (LMW) crystallins by their elution order and molecular mass. The results obtained show that during lens aging there is a progressive increase in alpha H-crystallin and a decrease in alpha L-crystallin content, while no significant age-related changes were observed in the LMW fraction. Analysis of changes in crystallin content in human cataractous lenses showed that apparently cataractogenesis can be described as an acceleration of the normal aging process. Important differences were found between the chromatographic profiles of cataracts from diabetic and nondiabetic patients mainly in the LMW fraction, suggesting that in cataract formation of diabetics alternative mechanisms may be superimposed on the normal aging process.

Protein glycation and in vivo distribution of human lens fluorescence.

Glycated proteins formed by the Maillard reaction were measured by furosine determination in human normal lenses and in senile and diabetic cataracts. Furosine, an hydrolysis product of fructose-lysine adduct formed in the early stages of the Maillard reaction, was measured by high performance liquid chromatography (HPLC). Furosine levels in diabetic cataracts were found to be 3 to 4 times higher than those observed for senile cataracts. The increased glycation levels both in cortex and nucleus were related to the increase of fluorescence determined in vitro by fluorometry and in vivo by Scheimpflug photography. Lens proteins were incubated with glucose and it has been demonstrated that protein glycation occurred parallel with the increase in concentration of fluorescent chromophores that present similar characteristics as those observed in vivo. The results indicate that protein insolubilization seemed to involve preferentially glycated proteins and at least in diabetic cataracts, the process seems to be initiated in the cortical region.

Monitoring in vivo lens changes. A comparative study with biochemical analysis of protein aggregation.

In this study the AA attempted to evaluate the relationship between lens optical density and lens fluorescence determined in vivo, with some specific (in vitro) biochemical changes occurring during cataract development. Special attention has been given to the comparison between diabetic and non diabetic cataracts. Prior to surgery all lenses were analysed by Scheimpflug photography to evaluate the topography of opacities and fluorescence distribution. Individual lenses were separated into cortex and nucleus and the amount of high molecular weight (HMW) protein aggregates was determined by FPLC (Fast Performance Liquid Chromatography). The results found in this study have shown that, as it would be expected, diabetic cataractous lenses present higher fluorescence levels than senile cataracts. It has also been shown that the increase in lens optical density, determined by Scheimpflug photography is clearly related to the increase in the amount of HMW-aggregates. Furthermore, in diabetic cataracts, a good correlation between protein aggregation and lens fluorescence determined in vivo has been found. Thus, it seems that in diabetic cataracts chemical or metabolic mechanisms leading to the production of fluorescent chromophores may be related to protein aggregation and therefore to the major processes involved in cataract development.

Spectrophotometric analysis of sodium fluorescein aqueous solutions. Determination of molar absorption coefficient.

The usefulness of sodium fluorescein (SF) and related physical parameters were analysed. Two factors that may affect the molar absorption coefficient (epsilon) of this compound were the presence of impurities and the pH of the solution. As discrepant values can be found in the literature for that coefficient, a purification technique was used and SF quantification was performed according to sodium concentration determined by atomic absorption spectrophotometry. The molar absorption coefficient of the SF solution in phosphate buffer pH 7.4 was determined. To study the influence of pH on epsilon determination, absorption spectra at pH1, 3, 5, and 10 were also analysed.

Effect of calcium dobesilate on the blood-retinal barrier in early diabetic retinopathy.

The effect of calcium dobesilate on the alteration of the blood-retinal barrier was studied in 41 adult-onset, non-insulin dependent diabetic patients with minimal or no retinopathy, randomly assigned to receive either oral calcium dobesilate (1000 mg twice daily) or a placebo for 12 months. The posterior vitreous value and the penetration ratio, determined by vitreous fluorophotometry, reflected stabilisation of blood-retinal barrier permeability in the calcium dobesilate patients and deterioration of blood retinal barrier in those given placebo. During the relatively short period of the study, one year, no significant change in microaneurysm and capillary closure gradings was observed. No side effects were associated with calcium dobesilate.

[Functional evaluation of the corneal endothelium using anterior segment fluorophotometry]. / Evaluation fonctionnelle de l'endothélium cornéen par la fluorophotométrie du segment antérieur.

The AA have studied the permeability of the corneal endothelium by anterior segment fluorophotometry using instillation of fluorescein. The Fluorotron Master TM has been modified with the introduction of a new slit (58.4 microns) and of the voltage output (6.5 v) has also been increased. The resolution has been improved from 2.39 mm to 0.59 mm. Of this study, we have examined 20 patients submitted to extracapsular cataract extraction with intraocular lens implantation (ECCE + IOL). The patients have been divided in two groups, according to the surgical technic used: "can-open" and intracapsular. Each patient have been submitted before surgery and 1 week after to an ophthalmologic examination, paquimetry and anterior segment fluorophotometry. The permeability coefficient (PAC) increases with the duration of surgery and when using "can-opener" instead of endocapsular.

[Use of aminoglycoside antibiotics in a general hospital: kinetics consequences in those patients whose blood levels are not monitored]. / Utilización de antibióticos aminoglucósidos en un hospital general: consecuencias cinéticas en aquellos pacientes en los que no se monitorizan sus niveles plasmáticos.

A surprise monitoring of plasma aminoglycoside level was carried out in our Hospital in 55 patients. Basal samples and samples one hour after i.m. or i.v. administration were obtained. A protocol sheet with personal, clinical and therapeutic data was filled for each patient. The FPIA analysis technique (CV less than 5%) was used. Only the data related to gentamicin were evaluated, because the number of patients receiving other aminoglycosides was very low (5 cases). The most usual dose and interval were 80 mg (91%) and 12 hours (69%), respectively. The administration routes were i.v. (52%) and i.m. (48%). Blood urea and/or creatinine levels were not measured in 22% of patients. The baseline gentamicin levels were subtherapeutic in 30%, therapeutical in 57%, and toxic in 13%, and 50%, 46% and 4%, respectively, for the levels after one hour. In patients with abnormal renal function a good correlation could be demonstrated (r = 0.96; p less than 0.001) between creatinine level and gentamicin 1/2 time. This correlation was absent in patients with normal renal function. When the dose of the antibiotic was adjusted to the weight of the patient, the frequency of therapeutic levels was higher (47%; p less than 0.03); in these cases, the most common administration interval was 8 hours (p less than 0.01). We emphasize the lack of control of the renal function in 22% of patients, stressing that the lack of monitoring systematically results in potentially subtherapeutic levels in more than one half of the investigated patients (57%).

[Quantification of the foveolar zone in normal and diabetic patients]. / Quantification de la zone avasculaire centrale chez les sujets normaux et diabétiques.

The AA studied the foveolar avascular zone (FAZ) in diabetic patients using digital image processing (512 x 521) and comparing different methods of measurements. The results obtained from eighteen diabetic patients and twelve healthy nondiabetic controls were compared. Each patient was submitted to fluorescein angiography. The foveal avascular zone was quantified both, digitalized or by manual procedure. The different methods of analysis are compared and the results presented.