Postgraduate Thesis: Validation of a HPLC assay for porphobilinogen synthase in human erythrocytes for use in the clinical laboratory
| Title | Validation of a HPLC assay for porphobilinogen synthase in human erythrocytes for use in the clinical laboratory |
|---|---|
| Authors | Suen, Kin-wah 孫建華 |
| Issue Date | 2004 |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| 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. IV |
| Degree | Master of Medical Sciences |
| Subject | High performance liquid chromatography. Porphyrins. Erythrocytes. Microbiological assay. |
| Dept/Program | Medical Sciences |
| dc.contributor.author | Suen, Kin-wah |
|---|---|
| dc.contributor.author | 孫建華 |
| dc.date.issued | 2004 |
| dc.description.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. IV |
| dc.description.nature | abstract |
| dc.description.nature | toc |
| dc.description.thesisdiscipline | Medical Sciences |
| dc.description.thesislevel | master's |
| dc.description.thesisname | Master of Medical Sciences |
| dc.identifier.hkul | b2962489 |
| dc.language | eng |
| dc.publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| dc.relation.ispartof | HKU Theses Online (HKUTO) |
| dc.rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works. |
| dc.source.uri | http://hub.hku.hk/bib/B29624897 |
| dc.subject.lcsh | High performance liquid chromatography. |
| dc.subject.lcsh | Porphyrins. |
| dc.subject.lcsh | Erythrocytes. |
| dc.subject.lcsh | Microbiological assay. |
| dc.title | Validation of a HPLC assay for porphobilinogen synthase in human erythrocytes for use in the clinical laboratory |
| dc.type | PG_Thesis |