Bioanalytical studies of porphyric disorders using HPLC with fluorescence detection

Bioanalytical studies of porphyric disorders using HPLC with fluorescence detection

Author Cypriani, Michele Gotelipe de Souza Corrêa Autor UNIFESP Google Scholar
Alves, Atecla Nunciata Lopes Autor UNIFESP Google Scholar
Bechara, Etelvino José Henriques Autor UNIFESP Google Scholar
Assunção, Nilson Antonio Autor UNIFESP Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Universidade de São Paulo (USP)
Abstract We describe here the development, validation, quantification and application of a method for determination of heme porphyrin precursors in the urine of porphyric patients. The isomers coproporphyrinogen I and III (COPRO I and III), uroporphyrinogen I (URO I), heptacarboxylporphyrinogen I (HEPTA I), pentacarboxylporphyrinogen (PENTA I), and hexacarboxylporphyrinogen I (HEXA I) were analyzed. These six urinary heme precursors were determined in urine samples collected from 24 patients by high-performance liquid chromatography (HPLC) equipped with a fluorescence detector. The inter- and intra-day precision (coefficient of variation < 5%) and accuracy (95-99%) were evaluated. The limits of detection and of quantification of the porphyrins, expressed in nmol L-1, were as follows: URO I, 0.62 and 2.05; HEPTA I, 0.59 and 1.96; HEXA I, 0.54 and 1.81; PENTA I, 0.52 and 1.73; COPRO I, 2.03 and 6.77; and COPRO III, 0.43 and 1.44. The method described here satisfactorily results in an acceptable cost-benefit ratio, precision and speed for determining the concentrations of heme precursors in the urine of latent or symptomatic acute intermittent porphyria individuals or porphyria cutanea tarda carriers. Since it was analytically validated, this method may be used for accurate and reliable diagnostic reports to follow-up the onset of acute crisis in porphyria carriers to adopt preventive pharmacological treatment.

Neste artigo, desenvolvemos, validamos e aplicamos um método para separação e quantificação de porfirinas precursoras do grupo heme na urina de portadores de porfirias. Os isômeros coproporfirinogenio I e III (COPRO I e III), uroporfirinogenio I (URO I), heptacarboxilporfirinogenio I (HEPTA I), pentacarboxilporfirinogenio I (PENTA I) e hexacarboxilporfirinogenio (HEXA I) foram determinados em amostras coletadas de 24 pacientes de porfiria aguda intermitente e de porfiria cutânea tarda. Utilizou-se cromatografia líquida de alta eficiência (HPLC) e detector de fluorescência. As concentrações de porfirinas foram determinadas com precisão inter e intra-dias (< 5%) e exatidão dentro da faixa 95-99%. Os limites de detecção e quantificação das porfirinas, expressos em nmol L-1, foram os seguintes: URO I, 0,62 e 2,05; HEPTA I, 0,59 e 1,96; HEXA I, 0,54 e 1,81; PENTA I, 0,52 e 1,73; COPRO I, 2,03 e 6,77; e COPRO III, 0,43 e 1,44. O método descrito aqui obedece a parâmetros analíticos satisfatórios, com excelente relação custo-benefício, e foi aplicado a amostras de urina de portadores assintomáticos e pacientes de porfirias. Este método foi validado analiticamente e mostrou potencial para diagnóstico de portadores de diferentes tipos de porfirias, imediatamente antes ou durante crises, e até mesmo para monitorar um tratamento farmacológico.
Keywords porphyrins
heme biosynthesis
acute intermittent porphyria
porphyria cutanea tarda
porphyria diagnosis
HPLC analysis of porphyrinogens
Language English
Sponsor Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
INCT Redoxoma Redox Processes in Biomedicine
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Grant number 2006/60245-3 e 2006/56530-4
Date 2014-10-01
Published in Journal of the Brazilian Chemical Society. Sociedade Brasileira de Química, v. 25, n. 10, p. 1918-1926, 2014.
ISSN 0103-5053 (Sherpa/Romeo, impact factor)
1678-4790 (Sherpa/Romeo, impact factor)
Publisher Sociedade Brasileira de Química
Extent 1918-1926
Origin http://dx.doi.org/10.5935/0103-5053.20140230
Access rights Open access Open Access
Type Article
Web of Science ID WOS:000344490900018
SciELO ID S0103-50532014001000018 (statistics in SciELO)
URI http://repositorio.unifesp.br/handle/11600/8616

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