L-alpha-glycerol-phosphate oxidase

This was a cross-sectional study involving participants from the ongoing Cape Town Vascular and Metabolic Health (VMH) study. VMH is an extension of the Cape Town Bellville South study, which has been described in details previously [42 (link), 43 (link)]. Data collection was based on a standardised questionnaire available in the electronic form on a password-protected personal digital assistant (PDA). Upon completion of the assessment of each participant, data were automatically encrypted and transmitted via mobile internet connection to a dedicated server, from which they were checked for completion, downloaded and stored for future use. Physical examination involved data collection on blood pressure (BP) which was measured according to the World Health Organisation (WHO) guidelines [44 (link)], using a semi-automatic digital blood pressure monitor (Omron M6 comfort-preformed cuff BP Monitor) on the right arm in sitting position and at rest for at least 10 minutes. Body weight (to the nearest 0.1 kg) was measured with the subject in light clothing and without shoes, using an Omron body fat meter HBF-511digital bathroom scale, which was calibrated and standardized using a weight of known mass. Height to the nearest centimetre was measured with a stadiometer, with subjects standing on a flat surface. Body Mass Index (BMI) was calculated as weight per square meter (kg/m²). Waist circumference was measured with a non-elastic tape at the level of the narrowest part of the torso, as seen from the anterior view. BP and anthropometric measurements were performed three times and their average used for analysis.
All participants underwent a 75 g oral glucose tolerance test (OGTT) as recommended by the WHO [20 (link)]. Further, the following biochemical parameters were analyzed at an ISO 15189 accredited Pathology practice (PathCare, Reference Laboratory, Cape Town, South Africa): glycated haemoglobin (HbA1c) by High Perfomance Liquid Chromotography (Biorad Variant Turbo, BioRad, South Africa); serum insulin by a paramagnetic particle chemiluminescence assay (Beckman DXI, Beckman Coulter, South Africa); serum cotinine by Competitive Chemiluminescent (Immulite 2000, Siemens, South Africa); plasma glucose by enzymatic hexokinase method (Beckman AU, Beckman Coulter, South Africa); total cholesterol (TC); high density lipoprotein cholesterol (HDL-c) by enzymatic immunoinhibition – End Point (Beckman AU, Beckman Coulter, South Africa);; triglycerides (TG) by glycerol phosphate oxidase-peroxidase, End Point (Beckman AU, Beckman Coulter, South Africa); low density lipoprotein cholesterol (LDL) by enzymatic selective protection – End Point (Beckman AU, Beckman Coulter, South Africa); and ultrasensitive C-reactive protein (CRP) by Latex Particle immunoturbidimetry (Beckman AU, Beckman Coulter, South Africa). In addition, blood sample were collected in a Tempus RNA tube (Applied Biosystems) and stored at -80 degrees for RNA extraction and analysis. Participants selected for this analysis were 12 individuals with screen-detected diabetes, 12 with prediabetes and 12 with normal glucose tolerance, matched for age, blood pressure, smoking and body mass index.

Cultures were monitored every day to check for contamination by plating on BHI agar media and ziehl neelsen stain. Cultures from chemostat were regularly sampled for measuring OD and colony forming units (Beste et al, 2011 (link)). Carbon‐di‐oxide production from the cultures was monitored using Gas analyzer. The supernatant was collected, filtered using 0.22 μm unit filters and used for substrate consumption and product excretion analyses. To measure the glycerol uptake, the amounts of glycerol in the supernatant and fresh medium was measured using glycerol assay kit by a coupled enzyme assay involving glycerol kinase and glycerol phosphate oxidase, resulting in a colorimetric (570 nm) product, proportional to the glycerol present. To measure NH₄Cl uptake, the amounts of NH₄Cl were measured using ammonia assay kit by reaction of ammonia present in the samples involving L‐glutamate dehydrogenase activity. Dry weight of the cells was measured by centrifuging cultures, drying the cell pellet using freeze dryer and weighing the cells. The dried pellet was used for protein analysis using Bicinchoninic Acid Kit for Protein Determination.

Serum concentrations of total triglyceride and cholesterol were directly determined by commercial assay kits based on glycerol phosphate oxidase-p-aminophenazone method. According to the instruction, the low-density lipoprotein cholesterol level was determined by commercial assay kits. Enzyme linked immunosorbent assay kits was applied to quantify concentrations of very low-density lipoprotein cholesterol, growth hormone, insulin, and leptin and insulin-like growth factors-1. All kits were supplied by Nanjing Jiancheng Bioengineering Institute (Nanjing, China).