Direct non-radioactive assay of galactose-1-phosphate:uridyltransferase activity using high performance liquid chromatography

Martijn Lindhout; M Estela Rubio-Gozalbo; Jaap A. Bakker; Jorgen Bierau

doi:10.1016/j.cca.2010.03.032

Direct non-radioactive assay of galactose-1-phosphate:uridyltransferase activity using high performance liquid chromatography

Martijn Lindhout, M Estela Rubio-Gozalbo, Jaap A. Bakker, Jorgen Bierau^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

Background Galactose-1-phosphate uridyltransferase (GALT) catalyses the conversion of galactose-1-phosphate (Gal-1-P) and UDP-glucose (UDP-Glc) into glucose-6-phosphate and UDP-galactose (UDP-Gal) Complete, or near complete, deficiency of GALT causes classic galactosaemia. The diagnosis is confirmed by measuring GALT activity in erythrocytes The most commonly used assays require radio labelled substrates or indirect coupled assays Methods: GALT activity was measured in erythrocyte lysates using optimal concentrations of the substrates galactose-1-phosphate and UDP-Glc UDP-Gal and UDP-Glc were separated using reversed-phase high performance liquid chromatography with UV detection Clinical validity was assessed using blood samples from galactosaemic patients Results UDP-Gal and UDP-Glc were separated with HPLC. The assay was linear with incubation times up 80 min and between 0 and 425 nmol haemoglobin Within-day and between-day imprecision at 50, 75 and 100% enzyme activity was

Original language	English
Pages (from-to)	980-983
Journal	Clinica Chimica Acta
Volume	411
Issue number	13-14
DOIs	https://doi.org/10.1016/j.cca.2010.03.032
Publication status	Published - 4 Jul 2010

Keywords

Galactosaemia
Galactose-1-phosphate uridyltransferase

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10.1016/j.cca.2010.03.032

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title = "Direct non-radioactive assay of galactose-1-phosphate:uridyltransferase activity using high performance liquid chromatography",

abstract = "Background Galactose-1-phosphate uridyltransferase (GALT) catalyses the conversion of galactose-1-phosphate (Gal-1-P) and UDP-glucose (UDP-Glc) into glucose-6-phosphate and UDP-galactose (UDP-Gal) Complete, or near complete, deficiency of GALT causes classic galactosaemia. The diagnosis is confirmed by measuring GALT activity in erythrocytes The most commonly used assays require radio labelled substrates or indirect coupled assays Methods: GALT activity was measured in erythrocyte lysates using optimal concentrations of the substrates galactose-1-phosphate and UDP-Glc UDP-Gal and UDP-Glc were separated using reversed-phase high performance liquid chromatography with UV detection Clinical validity was assessed using blood samples from galactosaemic patients Results UDP-Gal and UDP-Glc were separated with HPLC. The assay was linear with incubation times up 80 min and between 0 and 425 nmol haemoglobin Within-day and between-day imprecision at 50, 75 and 100% enzyme activity was ",

keywords = "Galactosaemia, Galactose-1-phosphate uridyltransferase",

author = "Martijn Lindhout and Rubio-Gozalbo, {M Estela} and Bakker, {Jaap A.} and Jorgen Bierau",

year = "2010",

month = jul,

day = "4",

doi = "10.1016/j.cca.2010.03.032",

language = "English",

volume = "411",

pages = "980--983",

journal = "Clinica Chimica Acta",

issn = "0009-8981",

publisher = "Elsevier B.V.",

number = "13-14",

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TY - JOUR

T1 - Direct non-radioactive assay of galactose-1-phosphate:uridyltransferase activity using high performance liquid chromatography

AU - Lindhout, Martijn

AU - Rubio-Gozalbo, M Estela

AU - Bakker, Jaap A.

AU - Bierau, Jorgen

PY - 2010/7/4

Y1 - 2010/7/4

N2 - Background Galactose-1-phosphate uridyltransferase (GALT) catalyses the conversion of galactose-1-phosphate (Gal-1-P) and UDP-glucose (UDP-Glc) into glucose-6-phosphate and UDP-galactose (UDP-Gal) Complete, or near complete, deficiency of GALT causes classic galactosaemia. The diagnosis is confirmed by measuring GALT activity in erythrocytes The most commonly used assays require radio labelled substrates or indirect coupled assays Methods: GALT activity was measured in erythrocyte lysates using optimal concentrations of the substrates galactose-1-phosphate and UDP-Glc UDP-Gal and UDP-Glc were separated using reversed-phase high performance liquid chromatography with UV detection Clinical validity was assessed using blood samples from galactosaemic patients Results UDP-Gal and UDP-Glc were separated with HPLC. The assay was linear with incubation times up 80 min and between 0 and 425 nmol haemoglobin Within-day and between-day imprecision at 50, 75 and 100% enzyme activity was

AB - Background Galactose-1-phosphate uridyltransferase (GALT) catalyses the conversion of galactose-1-phosphate (Gal-1-P) and UDP-glucose (UDP-Glc) into glucose-6-phosphate and UDP-galactose (UDP-Gal) Complete, or near complete, deficiency of GALT causes classic galactosaemia. The diagnosis is confirmed by measuring GALT activity in erythrocytes The most commonly used assays require radio labelled substrates or indirect coupled assays Methods: GALT activity was measured in erythrocyte lysates using optimal concentrations of the substrates galactose-1-phosphate and UDP-Glc UDP-Gal and UDP-Glc were separated using reversed-phase high performance liquid chromatography with UV detection Clinical validity was assessed using blood samples from galactosaemic patients Results UDP-Gal and UDP-Glc were separated with HPLC. The assay was linear with incubation times up 80 min and between 0 and 425 nmol haemoglobin Within-day and between-day imprecision at 50, 75 and 100% enzyme activity was

KW - Galactosaemia

KW - Galactose-1-phosphate uridyltransferase

U2 - 10.1016/j.cca.2010.03.032

DO - 10.1016/j.cca.2010.03.032

M3 - Article

C2 - 20359473

SN - 0009-8981

VL - 411

SP - 980

EP - 983

JO - Clinica Chimica Acta

JF - Clinica Chimica Acta

IS - 13-14

ER -

Direct non-radioactive assay of galactose-1-phosphate:uridyltransferase activity using high performance liquid chromatography

Abstract

Keywords

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