Ruth Ellen Simoni

Uric acid changes in urine and plasma: An effective tool in screening for purine inborn errors of metabolism and other pathological conditions

SummaryPurine inborn errors of metabolism (IEM) are serious hereditary disorders, which should be suspected in any case of neonatal fitting, failure to thrive, recurrent infections, neurological deficit, renal disease, self-mutilation and other manifestations. Investigation usually starts with uric acid (UA) determination in urine and plasma. UA, the final product of purine metabolism in humans, may be altered not only in purine IEM, but also in other related pathologies and clinical conditions. However, data and information about abnormal UA levels are scattered in the literature, often being controversial and confusing. A comprehensive overview has been elaborated, according to abnormal UA levels in urine and plasma, which associates these alterations with purine IEM. Other possible diseases, clinical conditions, diet and drug intake, related to the metabolism of uric acid, are also presented. The article includes tables that classify the disorders according to different patterns of UA alterations, with pertinent enzymes, clinical symptoms, inheritance and comments. Additionally, summarized pathophysiological mechanisms of important disorders are described. The overview is intended to assist in the interpretation of the results of UA analyses. It demonstrates that variation of UA concentrations in urine and plasma may constitute an effective tool in screening for purine IEM and other related pathological conditions.

Fumaric aciduria: an overview and the first Brazilian case report

Fumaric aciduria is a rare metabolic disease, with 40 cases reported so far. Fumarase deficiency leads mainly to brain abnormalities, developmental delay, and great accumulation of fumaric acid in urine. This work presents the first case of fumaric aciduria described in Brazil, which presented with some interesting clinical and biochemical findings such as colpocephaly, hepatic alterations, and marked metabolic acidosis since birth. Common findings were ventriculomegaly, hypotonia, and microcephaly. Biochemically, besides the high urinary fumaric acid excretion, atypical elevation of plasma citrulline, tyrosine and methionine levels were also observed. In order to show all features and variants of fumaric aciduria, literature data of 40 patients was reviewed and compared with the case reported here. Findings in all these patients demonstrate that this disorder does not yet have its phenotype completely defined; it is important that more patients be described.

Monitoring of a one-month ascorbic acid therapy in an alcaptonuric child; determinations by HPLC

No successful treatment has been devised so far for alcaptonuria (McKusick 203500) and therapeutic measures aim only at prevention and correction of the complications of the disease: darkening of urine, owing to excretion of homogentisic acid (HA) and ochronotic arthritis, in adulthood. Several groups have investigated the effect of ascorbic acid (AA) on the urinary concentration of HA. Most of these studies were performed in the early years, with short AA intake periods and titration procedures complicated by the similar properties of HA and AA. Some reported ineffectiveness. More recently, conflicting results were found in studies using HPLC and urine samples collected before and after the therapy. We now report on monitoring of a one-month AA supplementation, with daily urine collections (before, during and after the therapy), determinations by HPLC, and statistical analyses of the results. Our patient is a 3-year-old Brazilian girl, previously diagnosed and described at the age of 10 months (Simoni et al 1993). During a period of 30 days, 1 g AA was administered daily, in two 500 mg oral doses. The childs diet was kept normal and constant. Two urine samples per day were collected, during two periods of 10 consecutive days. These consisted of 5 days before and 5 days after the beginning and the end of the treatment. An HPLC system (with UV detection) developed by the authors, based on the method described by Bory et al (1990), allowed the simultaneous determination of urinary HA, AA and creatinine. The results (mg/mg creatinine) were analysed statistically with Bayesian dynamic models (West and Harrison 1989) and revealed a reduction in urinary HA concentration during the treatment. Three distinct levels of HA concentration were demonstrated, corresponding to the period before (I), during (II) and after (III) AA intake. The reduction, shown by level II, started at the beginning of AA supplementation and remained until the end of the treatment, when an increase was observed. Comparison of the levels by t-test confirmed a significant difference between periods I and II (p = 0.05). Example HA concentrations (mg/mg creatinine) for the two daily samples were 9.07 and 8.49 on the 5th day before, 9.42 and 6.17 on the 5th day after the supplementation; and during the treatment, 4.66 and 5.97, 6.52 and 6.54, 8.18 and 5.81, 3.49 and 6.60, on the 2nd, 5th, 26th and 29th days, respectively. The observed reduction is not yet understood. It might be a consequence of two or more different effects caused by AA intake. Besides being a powerful reducing

Screening for hydroxyethyl starch (HES) doping in sport.

The artificial colloid hydroxyethyl starch (HES) is among the most frequently used plasma volume expanders in the medical field. However, in 1998, its misuse by the athletic community was officially reported and since 2000, HES is prohibited by the International Olympic Committee (IOC). Therefore, several methods enabling the detection of HES in urine were developed, most based on gas chromatography-mass spectrometry (GC-MS). In the present work, a simple and low-cost screening method, intended to reduce the number of samples to be analysed by GC-MS, was developed. The method is based on the acid hydrolysis of HES and detection of the resulting glucose and hydroxyethyl glucose derivatives by Benedicts reaction (reduction of copper sulfate to brick-red cuprous oxide by glucose and/or derivatives). Samples considered suspect were submitted to GC-MS analysis for identification of HES. The method was successfully applied for screening of HES in 2627 urine samples from 1346 Brazilian soccer players and 1281 athletes from the Pan-American Games (Rio de Janeiro, 2007); 71 (2.7%) samples, considered suspect, were submitted to GC-MS, but no positive results were found. Moreover, a thin layer chromatography (TLC) method was adapted for visualisation of the characteristic band pattern of HES hydrolysis products. The results indicate that the methods are efficient and useful for the screening of HES in urine.

Screening for inborn errors of metabolism in high-risk children from Rio de Janeiro, Brazil

From 1988 to 1995, our laboratory at the Institute of Chemistry of the Federal University of Rio de Janeiro, in Rio de Janeiro, screened 2650 samples from 2000 high-risk patients (mostly children) for Inborn Errors of Metabolism (IEM). Chemical tests, various chromatographic techniques and enzyme assays were performed on urine, plasma and in some cases, cerebrospinal fluid (CSF). A total of 145 cases of IEM (7.2%) was identified. These were related to: the metabolism of amino acids (41) and carbohydrates (17), organic acids (7), lysosomal enzymes (61), membrane transport system (16), metals (2), intestinal disaccharidases (1) and porphyrin metabolism (3). Furthermore, a relevant number of patients with abnormal findings is still under investigation. Biochemical results and clinical symptoms are presented and the importance of reference laboratories for the detection of IEM is stressed.

Plasma volume expanders: use in medicine and detecting misuse in sports

Plasma volume expanders comprise a heterogeneous group of substances used in medicine that are intravenously administered in cases of great blood loss owing to surgery or medical emergency. These substances, however, can also be used to artificially enhance performance of healthy athletes in sport activities, and to mask the presence of others substances. These practices are considered doping, and are therefore prohibited by the International Olympic Committee and the World Antidoping Agency. Consequently, drug testing procedures are essential. The present work provides an overview of plasma volume expanders, assembling pertinent data such as chemical characteristics, physiological aspects, adverse effects, doping and analytical detection methods, which are currently dispersed in the literature.

Hyperargininaemia : A late-diagnosed brazilian case with increased urinary excretion of homocystine

Hyperargininaemia: A late-diagnosed Brazilian case with increased urinary excretion of homocystine R. E. Simoni1, C. P. Honório de Oliveira1, M. J. Braga1, C. R. Gayer de Menezes1, J. C. Llerena Jr2, P. S. Correia2, A. A. Santa Rosa2, D. G. Horovitz2, C. R. M. Chaves2, M. L. Corta de Oliveira1* 1Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Cidade Universitária, Rio de Janerio, 2Instituto Fernandes Figueira, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil *Correspondence: Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Cidade Universitária, Ilha do Fundão, Centro de Tecnologia, Bloco A, 21941-900, Rio de Janerio, Brazil

Systematic analysis of glycerol: colourimetric screening and gas chromatography–mass spectrometric confirmation

Glycerol is a naturally occurring polyol in the human body, essential for several metabolic processes. It is widely used in the food, pharmaceutical, and medical industries and in clinical practice as a plasma volume expander (PVE). Athletes, however, may use glycerol to mask the presence of forbidden substances or to enhance performance, inclusively through hyperhydration achieved by glycerol ingestion with added fluid. These practices are considered doping, and are prohibited by the World Anti-Doping Agency (WADA). Therefore, glycerol was introduced in the prohibited list. Doping through glycerol ingestion can readily be identified by detection of elevated glycerol concentrations in urine. In this paper, a protocol for the fast detection of glycerol in urine is proposed. It consists of a previous visual colourimetric screening, followed by a quantitative/qualitative confirmation analysis by mass spectrometry. The screening procedure involves a reaction in which polyhydric alcohols are oxidized by periodate to formic acid and formaldehyde, which is detected by the addition of a fuchsin solution. For the subsequent qualitative/quantitative confirmation analysis, a gas chromatography-mass spectrometry based approach with a non-deuterated internal standard and a drying step of only 10 min is proposed. The linear correlation was demonstrated within WADA´s threshold range. The calculated RSD were 2.1% for within-day precision and 2.8% for between-day precision. The uncertainty estimation was calculated, and a value of 2.7% was obtained. The procedure may also be used for the analysis of other polyols in urine, as for example the PVE mannitol.

A Survey on Inborn Errors of Metabolism in 323 Neonates from the State of Rio de Janeiro, Brazil

Inborn errors of metabolism (IEM) are a relevant cause of morbidity and death among children, and neonates in particular. However, little is known about the prevalence of these disorders in Brazilian newborns. Our laboratory of IEM (LABEIM) at the Department of Biochemistry, Institute of Chemistry, Federal University of Rio de Janeiro (UFRJ), has been working on the diagnosis of IEM since 1988. Out of 3,300 patients (90% children), screened and evaluated from 1989 to 2,000 because of a high clinical suspicion of having an IEM, 323 (9.8%) were neonates. Patients came from different regions of the state of Rio de Janeiro, in which lives approximately 8.5% of the total Brazilian population. Chemical tests, various chromatographic techniques and enzyme assays were performed in urine, plasma and in some cases, cerebrospinal fluid (CSF). This study describes our laboratory and the experience with the 323 investigated neonates, among which 28 cases (8.7%) of IEM were identified and 18 (5.6%), strongly suspected. All these cases were related mainly to the metabolism of amino acids, organic acids, lysosomal enzymes and carbohydrates. Furthermore, data on population, community and health services are presented.