Hypoxanthine-guanine phosphoribosyl transferase: assay using high performance liquid chromatography

Area of Science:

• Biochemistry
• Enzymology
• Analytical Chemistry

Background:

• Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a crucial enzyme in purine metabolism.
• HGPRT deficiency leads to Lesch-Nyhan syndrome, a severe genetic disorder.
• Accurate and sensitive assays are needed for diagnosing HGPRT deficiency.

Purpose of the Study:

• To develop and validate a high-performance liquid chromatography (HPLC) method for quantifying HGPRT activity.
• To establish a reliable assay for screening red cell lysates for HGPRT deficiency.

Main Methods:

• HPLC separation of inosine monophosphate (IMP) using a C18 reversed-phase silica column.
• Detection of IMP by absorbance at 254 nm.
• Simultaneous measurement of hypoxanthine consumption.

Main Results:

• The assay accurately quantifies HGPRT activity, with results expressed as nmol IMP . h-1 . mg-1 haemoglobin.
• A normal range of 94 +/- 15 nmol IMP . h-1 . mg-1 haemoglobin was established.
• The method can accurately assay HGPRT activities down to 1% of normal levels.

Conclusions:

• The developed HPLC method provides a sensitive and accurate means for estimating HGPRT activity.
• This assay is suitable for routine screening of red cell lysates to identify HGPRT deficiency.
• The method facilitates early diagnosis and management of conditions related to HGPRT deficiency.