Ultra fast and sensitive liquid chromatography tandem mass spectrometry based assay for galactose-1-phosphate uridylyltransferase and galactokinase deficiencies

Abstract

The diagnosis of transferase and galactokinase deficiency galactosemia usually involves the measurement of erythrocyte galactose-1-phosphate uridylyltransferase (GALT) and galactokinase (GALK) enzyme activity, respectively. The current gold standard assays for these enzymes are radioactive assays, which are laborious and/or incapable of measuring low enzyme activities. To further our knowledge of genotype-phenotype relationships, we had developed an assay for GALT activity alone using LC-MS/MS. In this study we generated a robust and sensitive LC-MS/MS based GALT and GALK assay using a novel normal phase chromatographic condition. We improved upon our earlier assay by drastically reducing the instrument run time and eliminating the use of an ion pairing reagent. Stable isotope labeled substrates were utilized in the GALT and GALK assays. The enzymatic products ([13C6]-uridine diphosphate galactose in GALT assay and [13C6]-galactose-1-phosphate in GALK assay) were quantified in a 3 min LC-MS/MS run. The assays were sensitive enough to allow for the quantification of enzyme activities as low as 0.2% and 0.3% of normal control values in the GALT and GALK assays, respectively. Thirty-three samples from non-galactosemic patients were assayed to have erythrocyte GALT activity of 23.4 ± 4.2 and GALK activity of 1.8 ± 0.47 (mean ± SD) µmol·(g Hgb) −1·hr−1. Erythrocyte GALT activities in a cohort of 16 patients with classic galactosemia were measured: 4 patients had GALT activity less than 1% of normal control values and the remaining 12 had no detectable GALT activity. No GALK activity was detected in a GALK deficient sample we analzyed. Lastly, we tested the feasibility of adapting this LC-MS/MS based GALT/GALK assay as a newborn screening (NBS) test.

Subfertility and growth restriction in a new galactose-1 phosphate uridylyltransferase (GALT) - deficient mouse model

“…Using the LC-MS/MS procedure published by Li et al, 26 we confirmed the total absence of GALT activity in the homozygous GalT genetrapped mice (Figure 1b). Also, the homozygous GalT gene-trapped mice accumulated up to 60.8±27.3 mM of gal-1P in RBC even when they were unchallenged (ie, fed with normal chow; Figure 1c).…”

“…8 GALT activity was determined according to procedure published by Li et al, 26 which used a direct method with LC-MS/MS detection of the product of the reaction. Waters XBridge BEH amide column (2.1 mm  100 mm, 3.5 mm) and AB Sciex API 4000 system (AB Sciex IS, Framingham, MA, USA) equipped with Shimadzu LC units was used in this study.…”

“…Organs harvested immediately from killed animals were fixed in 10% formalin for at least 48 h before sending off to the histology research core at ARUP Laboratories (Salt Lake City, UT, USA) for tissue processing and slide Red blood cell GALT activity was measured in 10-day-old mice with various GALT genotypes by a LC-MS/MS method described by Li et al 26 (c) Gal-1P contents in RBC lysate of mice fed with normal chow/high-galactose diet were measured by enzymatic method previously described. 8 Mouse model of GALT deficiency M Tang et al preparations.…”

Laboratory diagnosis of galactosemia: a technical standard and guideline of the American College of Medical Genetics and Genomics (ACMG)

“…43,44 These methods can be multiplexed and allow the simultaneous determination of enzyme activity for all three galactosemia types with minimal sample volume. This represents a significant improvement for diagnostic testing.…”

“…Recently, LC-MS/MS methods have been developed to measure GALK and GALE activity in RBCs and/or DBS using 13 C-labeled substrate. [43][44][45] Metabolite assays…”