Improvement of an HPLC method to determine urinary δ-aminolevulinic acid / Aprimoramento de um método de HPLC para determinar ácido δ-aminolevulínico urinário

OBJECTIVE: The purpose of this study was to evaluate and improve a high performance liquid chromatography (HPLC) methodology to determine urinary δ-aminolevulinic acid (ALA-U) with small volumes of sample. METHOD: The method was based on the formation of a fluorescent compound and subsequent 15-minute chromatographic run. RESULTS: The method shows suitable linearity, precision and recovery. Urine samples showed 1.2 ± 0.9 mg/l (media ± standard deviation) of ALA-U. CONCLUSION: The method was considered suitable for the routine analysis of ALA-U.

INTRODUÇÃO E OBJETIVO: A proposta deste estudo foi avaliar e aprimorar uma metodologia de cromatografia líquida de alta eficiência (CLAE)(11, 12), a fim de determinar o ácido δ-aminolevulínico urinário (ALA-U) utilizando volumes reduzidos de amostra. MÉTODO: O método baseia-se na formação de um composto fluorescente e posterior corrida cromatográfica de 15 minutos. RESULTADOS: O método apresentou linearidade, precisão e recuperação adequadas. Os resultados para as amostras de urina testadas foram 1,2 ± 0,9 mg/l (média ± desvio padrão) de ALA-U. CONCLUSÃO: O método foi considerado adequado para análises de rotina de ALA-U.

Warfarin sensitivity genotyping: a review of the literature and summary of patient experience.

The antithrombotic benefits of warfarin are countered by a narrow therapeutic index that contributes to excessive bleeding or cerebrovascular clotting and stroke in some patients. This article reviews the current literature describing warfarin sensitivity genotyping and compares the results of that review to the findings of our study in 189 patients at Mayo Clinic conducted between June 2001 and April 2003. For the review of the literature, we identified relevant peer-reviewed articles by searching the Web of Knowledge using key word warfarin-related adverse event. For the 189 Mayo Clinic patients initiating warfarin therapy to achieve a target international normalized ratio (INR) in the range of 2.0 to 3.5, we analyzed the CYP2C9 (cytochrome P450 2C9) and VKORC1 (vitamin K epoxide reductase complex, subunit 1) genetic loci to study the relationship among the initial warfarin dose, steady-state dose, time to achieve steady-state dose, variations in INR, and allelic variance. Results were compared with those previously reported in the literature for 637 patients. The relationships between allelic variants and warfarin sensitivity found in our study of Mayo Clinic patients are fundamentally the same as in those reported by others. The Mayo Clinic population is predominantly white and shows considerable allelic variability in CYP2C9 and VKORC1. Certain of these alleles are associated with increased sensitivity to warfarin. Polymorphisms in CYP2C9 and VKORC1 have a considerable effect on warfarin dose in white people. A correlation between steady-state warfarin dose and allelic variants of CYP2C9 and VKORC1 has been demonstrated by many previous reports and is reconfirmed in this report. The allelic variants found to most affect warfarin sensitivity are CYP2C9*1*1-VKORC1BB (less warfarin sensitivity than typical); CYP2C9*1*1-VKORC1AA (considerable variance in INR throughout initiation); CYP2C9*1*2-VKORC1AB (more sensitivity to warfarin than typical); CYP2C9*1*3-VKORC1AB (much more sensitivity to warfarin than typical); CYP2C9*1*2-VKORC1AB (much more sensitivity to warfarin than typical); CYP2C9*1*3-VKORC1AA (much more sensitivity to warfarin than typical); and CYP2C9*2*2-VKORC1AB (much more sensitivity to warfarin than typical). Although we were unable to show an association between allelic variants and initial warfarin dose or dose escalation, an association was seen between allelic variant and steady-state warfarin dose. White people show considerable variance in CYP2C9 allele types, whereas people of Asian or African descent infrequently carry CYP2C9 allelic variants. The VKORC1AA allele associated with high warfarin sensitivity predominates in those of Asian descent, whereas white people and those of African descent show diversity, carrying either the VKORC1BB, an allele associated with low warfarin sensitivity, or VKORC1AB or VKORC1AA, alleles associated with moderate and high warfarin sensitivity, respectively.

Monitoração terapêutica da azatioprina: uma revisão: [revisão] / Therapeutic drug monitoring of azathioprine: a review: [review]

Os nucleotídeos de tioguanina (6-TGN), metabólitos ativos da azatioprina (AZA) e da 6-mercaptopurina (6-MP), atuam como antagonistas das purinas, inibindo as sínteses de DNA, RNA e a protéica, e induzindo à citotoxicidade/imunossupressão. A enzima geneticamente determinada, tiopurina metiltransferase (TPMT), está envolvida no metabolismo desses agentes e, hipoteticamente, determina a resposta clínica às tiopurinas. A baixa atividade dessa enzima diminui a metilação das tiopurinas, resultando em potencial sobredose, enquanto altos níveis de TPMT levam à superprodução do metabólito tóxico 6-metilmercaptopurina (6-MMP) e à não-efetividade terapêutica da AZA e da 6-MP. Várias mutações no gene da TPMT têm sido identificadas e correlacionadas com fenótipos de baixa atividade. Neste artigo, também se discute a monitoração terapêutica desses fármacos por meio da medida dos níveis de 6-TGN intra-eritrocitários, os quais se correlacionam com imunossupressão e mielotoxicidade. Já a 6-MMP está diretamente relacionada com hepatotoxicidade. Esses ensaios estão associados ao uso de doses adequadas dessa droga, resultando num melhor controle da doença e menor uso de corticosteróides.

Thioguanine nucleotides (6-TGN), active metabolites of azathioprine (AZA) and 6-mercaptopurine (6-MP), act as purine antagonists, inhibiting DNA, RNA, and protein synthesis and inducing cytotoxicity and immunosuppression. The genetically determined thiopurine methyltransferase enzyme (TPMT) is involved in the metabolism of these agents and, theoretically, determines the clinical response to thiopurines. Low activity of this enzyme decreases the methylation of thiopurines, what results in potential overdosing, whereas high TPMT status leads to overproduction of toxic metabolite 6-methilmercaptopurine (6-MMP) and ineffectiveness of AZA and 6-MP. Several mutations in the TPMT gene have been identified and correlated with low activity phenotypes. In this study, we also discuss the therapeutic monitoring of these drugs by means of red blood cell 6-TGN levels, which correlate with immunosuppression and mielotoxicity. 6-MMP is directly connected with hepatotoxicity. These metabolites assays are associated with the use of appropriate doses of this drug, what results in a better control of the disease and a decreased use of corticosteroids.

Cerebrospinal fluid oligoclonal bands in the diagnosis of multiple sclerosis. Isoelectric focusing with IgG immunoblotting compared with high-resolution agarose gel electrophoresis and cerebrospinal fluid IgG index.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. We compared the diagnostic performance of isoelectric focusing (IEF) combined with IgG immunoblotting to high-resolution agarose gel electrophoresis for the detection of intrathecal synthesis of IgG due to the MS disease process. In 20 patients with definite MS, IEF and high-resolution agarose gel electrophoresis had sensitivities of 90% and 60%, respectively. In the 51 patients with no evidence of MS, the methods had specificities of 94% and 96%, respectively. With a prevalence of 15% in this test population, IEF and high-resolution agarose gel electrophoresis had positive predictive values of 73% and 73% and negative predictive values of 98% and 93%, respectively. The IEF method improves the sensitivity and the negative predictive value of the oligoclonal-banding assay, the IEF gels are easier to interpret, and the IEF assay requires a smaller cerebrospinal fluid volume.