Validation of a HPLC assay for porphobilinogen synthase in human erythrocytes for use in the clinical laboratory

Abstract

(Uncorrected OCR) Abstract

Porphobilinogen (PBG) synthase condenses two molecules of

aminolaevulinic acid (ALA) to form PBG in heme biosynthesis. The enzyme

activity is sensitive to inhibition by heavy metals such as lead. It can act as a biological indicator of chronic lead POis~r\g to identify the risk group,

especially in children, so that early treatment can be given to prevent possible

permanent damages. A reversed-phase ion-pair HPLC analytical method for

the assay of the PBG synthase activity based on detection of PBG production

has been validated. A Hypersil CN column (150 x 4.6 mm; 5 urn) was

employed together with a mixture of acetonitrile-40 mM phosphate buffer at pH

2.4 with 5 mM 1-heptanesulphonic acid (8:92, v/v). UV detection was

performed at 240 nm. PBG was eluted as a spectrally pure peak resolved from

its impurities in the methanol-inhibited enzyme reaction. The method was

sensitive with a limit of quantitation of 2 ~M. The within-run and between-run

precisions were 8.2% and 13.8% respectively. The recovery was 93.4 �7.1%

(n=6). The preliminary reference range of the PBG synthase activities in the

local pediatric population were from 21.5 to 26.3 ~mol/L RBC/min. Bland and Altman statistical analysis showed that the HPLC assay and the colorimetric assay could not be used interchangeably. The HPLC assay was an alternative way to assess the PBG synthase activities in the human erythrocyte samples.

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