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Postgraduate Thesis: Validation of a HPLC assay for porphobilinogen synthase in human erythrocytes for use in the clinical laboratory
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TitleValidation of a HPLC assay for porphobilinogen synthase in human erythrocytes for use in the clinical laboratory
 
AuthorsSuen, Kin-wah
孫建華
 
Issue Date2004
 
PublisherThe 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
 
DegreeMaster of Medical Sciences
 
SubjectHigh performance liquid chromatography.
Porphyrins.
Erythrocytes.
Microbiological assay.
 
Dept/ProgramMedical Sciences
 
DC FieldValue
dc.contributor.authorSuen, Kin-wah
 
dc.contributor.author孫建華
 
dc.date.issued2004
 
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.natureabstract
 
dc.description.naturetoc
 
dc.description.thesisdisciplineMedical Sciences
 
dc.description.thesislevelmaster's
 
dc.description.thesisnameMaster of Medical Sciences
 
dc.identifier.hkulb2962489
 
dc.languageeng
 
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)
 
dc.relation.ispartofHKU Theses Online (HKUTO)
 
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.
 
dc.source.urihttp://hub.hku.hk/bib/B29624897
 
dc.subject.lcshHigh performance liquid chromatography.
 
dc.subject.lcshPorphyrins.
 
dc.subject.lcshErythrocytes.
 
dc.subject.lcshMicrobiological assay.
 
dc.titleValidation of a HPLC assay for porphobilinogen synthase in human erythrocytes for use in the clinical laboratory
 
dc.typePG_Thesis
 
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<item><contributor.author>Suen, Kin-wah</contributor.author>
<contributor.author>&#23403;&#24314;&#33775;</contributor.author>
<date.issued>2004</date.issued>
<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 &#65533;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</description.abstract>
<language>eng</language>
<publisher>The University of Hong Kong (Pokfulam, Hong Kong)</publisher>
<relation.ispartof>HKU Theses Online (HKUTO)</relation.ispartof>
<rights>The author retains all proprietary rights, (such as patent rights) and the right to use in future works.</rights>
<source.uri>http://hub.hku.hk/bib/B29624897</source.uri>
<subject.lcsh>High performance liquid chromatography.</subject.lcsh>
<subject.lcsh>Porphyrins.</subject.lcsh>
<subject.lcsh>Erythrocytes.</subject.lcsh>
<subject.lcsh>Microbiological assay.</subject.lcsh>
<title>Validation of a HPLC assay for porphobilinogen synthase in human erythrocytes for use in the clinical laboratory</title>
<type>PG_Thesis</type>
<identifier.hkul>b2962489</identifier.hkul>
<description.thesisname>Master of Medical Sciences</description.thesisname>
<description.thesislevel>master&apos;s</description.thesislevel>
<description.thesisdiscipline>Medical Sciences</description.thesisdiscipline>
<description.nature>abstract</description.nature>
<description.nature>toc</description.nature>
<bitstream.url>http://hub.hku.hk/bitstream/10722/30769/5/Abstract.pdf</bitstream.url>
<bitstream.url>http://hub.hku.hk/bitstream/10722/30769/6/Table%20of%20Contents.pdf</bitstream.url>
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