New enzymatic assay for calcium in serum

Abstract

We established a simple and rapid kinetic assay for measurement of calcium in serum by using urea amidolyase (EC 3.5.1.45) from yeast species. The method is based on inhibition of the enzyme by calcium. In the assay, we eliminated endogenous ammonium ion by use of glutamate dehydrogenase (GLDH; EC 1.4.1.4); then in the presence of urea amidolyase, urea, ATP, bicarbonate, magnesium, and potassium ions, ammonium ion production was inversely proportional to calcium ion concentration in serum. The concentration of ammonium ion formed was determined by adding GLDH to produce NADP+ in the presence of 2-oxoglutarate and NADPH; we then monitored the change of absorbance at 340 nm. The within-run CVs of this method were 1.7-3.2% (n = 10) at 1.53-3.08 mmol/L, respectively. Day-to-day (total) CVs were 2.8-4.1%. Analytical recovery was 92-112%. The presence of other ions, ascorbic acid, reduced glutathione, bilirubin, hemoglobin, citrate, lipemic material, or human serum albumin did not affect this assay system. The correlation between values obtained with our method (y) and o-cresolphthalein complexone method (CPC) (x) was: y = 1.001x + 0.077 mmol/L (r = 0.949, Sy[symbol: see text]x = 0.079, n = 100); with the other enzymatic method (x) it was: y = 0.952x + 0.021 mmol/L (r = 0.955, Sy[symbol: see text]x = 0.074, n = 100). The SEs for each method were: 0.025 mmol/L, our method; 0.023 mmol/L, CPC method; and 0.025 mmol/L, the other enzymatic method.

Identification of differentially expressed genes and pathways for intramuscular fat deposition in pectoralis major tissues of fast-and slow-growing chickens

“…Samples of the right pectoralis major muscle were homogenized using the method of Folch [ 53 ]. The contents of triglyceride (TG) and phospholipids (PLIP) in the solvent phase, after centrifugation, were analyzed with TG [ 54 , 55 ] and PLIP [ 56 , 57 ] kits (Deliman Biochemical technology Co., LTD, Beijing, China).…”

Validation of a point-of-care handheld blood total calcium analyzer in postpartum dairy cows

“…Frozen plasma was thawed at room temperature and tested with the CM, whereas frozen serum was sent to the Animal Health Laboratory (University of Guelph, ON, Canada) for measurement of tCa using a Cobas Calcium Gen 2 kit (Roche Diagnostics, Indianapolis, IN), which was considered the reference test (RT). Although atomic absorption spectrophotometry is considered the gold standard to measure calcium concentration, photometric methods are commonly used in clinical practice (Kimura et al, 1996;Bazydlo et al, 2014). These validated methods are available at a lower cost (Kimura et al, 1996;Bourguignon et al, 2014).…”

“…Although atomic absorption spectrophotometry is considered the gold standard to measure calcium concentration, photometric methods are commonly used in clinical practice (Kimura et al, 1996;Bazydlo et al, 2014). These validated methods are available at a lower cost (Kimura et al, 1996;Bourguignon et al, 2014).…”