Aplicação da espectrometria de absorção atômica de alta resolução e fonte contínua com forno de grafite e amostragem direta de sólidos na determinação de cobre hepático para o auxílio do diagnóstico da doença de Wilson

Abstract

Copper is an important cofactor for many enzymes, but it is toxic when free. Most of the serum copper is transported bound to ceruloplasmin, a part of the albumin and transcuprein complexes. Wilson's disease is an autosomal recessive disease caused by a P-type ATPase mutation that prevents copper from binding to ceruloplasmin. Therefore, there is copper deposition that occurs in hepatocytes, in the brain, in the periphery of the cornea and in the kidneys. The diagnosis of Wilson's disease is a challenge, especially in the absence of neurological alterations and Kayser – Fleisher rings. Low serum concentrations of ceruloplasmin are observed, as there is no incorporation of copper in apoceruloplasmin for its formation, but some patients have normal concentrations. Furthermore, 2% of the heterozygous population for P-type ATPase mutations have low concentrations of copper and ceruloplasmin, so it is not a good reference for trying to diagnose a patient. There are tests that can be used to investigate patients with Wilson's disease, such as hepatic copper. In the present study, an analytical method was developed for the direct determination of copper by solid liver sampling using Solid Sample High Resolution Continuum Source Graphite Furnace Atomic Absorption Spectrometry (SS HR CS GF AAS). The method was validated using DOLT-2 certified reference material and lyophilized livers from laboratory animals, which were also analyzed by ICP-MS for comparison. Due to the large range of copper mass that can be found in the liver, three different absorption lines (217,894 nm, 249,2146 nm and 244,164 nm) were used in order to obtain a greater dynamic range of concentration. The detection and quantification limits obtained, in mg kg-1, for these lines were, respectively: 0.082 and 0.272; 10.8 and 35.8; and 49.3 and 164 for copper. A liver biopsy sample from a patient suspected of having Wilson's Disease was analyzed and the results indicated that the patient is a carrier of the disease, due to having a concentration greater than 250 mg kg-1 of Cu in liver dry mass, value higher than the cutoff concentration established in the literature.