Liquid Chromatography-Tandem Mass Spectrometry Assay of Leukocyte Acid α-Glucosidase for Post-Newborn Screening Evaluation of Pompe Disease.

BACKGROUND: Pompe disease (PD) is the first lysosomal storage disorder to be added to the Recommended Uniform Screening Panel for newborn screening. This condition has a broad phenotypic spectrum, ranging from an infantile form (IOPD), with severe morbidity and mortality in infancy, to a late-onset form (LOPD) with variable onset and progressive weakness and respiratory failure. Because the prognosis and treatment options are different for IOPD and LOPD, it is important to accurately determine an individual's phenotype. To date, no enzyme assay of acid α-glucosidase (GAA) has been described that can differentiate IOPD vs LOPD using blood samples. METHODS: We incubated 10 µL leukocyte lysate and 25 µL GAA substrate and internal standard (IS) assay cocktail for 1 h. The reaction was purified by a liquid-liquid extraction. The extracts were evaporated and reconstituted in 200 µL methanol and analyzed by LC-MS/MS for GAA activity. RESULTS: A 700-fold higher analytical range was observed with the LC-MS/MS assay compared to the fluorometric method. When GAA-null and GAA-containing fibroblast lysates were mixed, GAA activity could be measured accurately even in the range of 0%-1% of normal. The leukocyte GAA activity in IOPD (n = 4) and LOPD (n = 19) was 0.44-1.75 nmol · h-1 · mg-1 and 2.0-6.5 nmol · h-1 · mg-1, respectively, with no overlap. The GAA activity of pseudodeficiency patients ranged from 3.0-28.1 nmol · h-1 · mg-1, showing substantial but incomplete separation from the LOPD group. CONCLUSIONS: This assay allows determination of low residual GAA activity in leukocytes. IOPD, LOPD, and pseudodeficiency patients can be partially differentiated by measuring GAA using blood samples.

Sulfatide Analysis by Mass Spectrometry for Screening of Metachromatic Leukodystrophy in Dried Blood and Urine Samples.

BACKGROUND: Metachromatic leukodystrophy (MLD) is an autosomal recessive disorder caused by deficiency in arylsulfatase A activity, leading to accumulation of sulfatide substrates. Diagnostic and monitoring procedures include demonstration of reduced arylsulfatase A activity in peripheral blood leukocytes or detection of sulfatides in urine. However, the development of a screening test is challenging because of instability of the enzyme in dried blood spots (DBS), the widespread occurrence of pseudodeficiency alleles, and the lack of available urine samples from newborn screening programs. METHODS: We measured individual sulfatide profiles in DBS and dried urine spots (DUS) from MLD patients with LC-MS/MS to identify markers with the discriminatory power to differentiate affected individuals from controls. We also developed a method for converting all sulfatide molecular species into a single species, allowing quantification in positive-ion mode upon derivatization. RESULTS: In DBS from MLD patients, we found up to 23.2-fold and 5.1-fold differences in total sulfatide concentrations for early- and late-onset MLD, respectively, compared with controls and pseudodeficiencies. Corresponding DUS revealed up to 164-fold and 78-fold differences for early- and late-onset MLD patient samples compared with controls. The use of sulfatides converted to a single species simplified the analysis and increased detection sensitivity in positive-ion mode, providing a second option for sulfatide analysis. CONCLUSIONS: This study of sulfatides in DBS and DUS suggests the feasibility of the mass spectrometry method for newborn screening of MLD and sets the stage for a larger-scale newborn screening pilot study.

HIV-1 integration sites are flanked by potential MARs that alone can act as promoters.

Matrix attachment regions (MARs) are cis regulatory elements that modulate gene expression in a tissue and cell stage specific manner. Recent reports show that viral integration within the genome takes place at nonrandom active genes. We have checked for the presence of MARs in the vicinity of the reported 524 HIV-1 integration sites. Our studies show that in 92.5% cases, MARs flank the integration sites. Similarly, for adeno-associated virus, two potential MARs were present next to the integration site on the human chromosome. Earlier we have shown that short MAR sequences present upstream of HIV-1 LTR promote processive transcription at a distance. Here, using a well-studied IgH-MAR and another potential MAR from p53 promoter, we demonstrate that MARs alone can act as promoters. Thus, we propose that MAR elements near the HIV-1 integration sites can act as potential promoters, which may facilitate proviral integration and transcription.

Modulation of stress induced by isometric handgrip test in hypertensive patients following yogic relaxation training.

13 essential hypertensive patients aged 41 to 60 years were given yoga training for 60 min daily, Monday through Saturday, for a total duration of 4 weeks. Blood pressure and heart rate (HR) were measured with non-invasive semi-automatic blood pressure monitor. Measurements were recorded before the training and at weekly intervals during the 4 week training period. Results of our study show a significant (P<0.001) reduction in resting HR and rate-pressure-product (RPP) after 2 weeks of yoga training. Systolic pressure (SP), diastolic pressure (DP) (P<0.001) and mean pressure (MP) (P<0.05) showed a significant reduction at 3 weeks of training period. After 4 weeks of training, there was further fall in SP, DP, pulse pressure (PP) (P<0.05), MP (P<0.001), HR and RPP. Isometric handgrip test before yoga training produced a significant rise in SP and MP and insignificant rise in DP, HR and RPP. After yoga training, there was a significant rise in all these parameters. Our results show that yoga training optimises the sympathetic response to stressful stimuli like isometric handgrip test and restores the autonomic regulatory reflex mechanisms in hypertensive patients.