Determination of Creatinine in Human Urine with Flow Injection Tandem Mass Spectrometry

Background/Aims: Excretion of urinary compounds in spot urine is often estimated relative to creatinine. For the growing number of liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays of urine-excreted molecules, a fast and accurate method for determination of creatinine is needed. Methods: A high-throughput flow injection tandem mass spectrometry method for exact quantitation of creatinine in urine has been developed and validated. Sample preparation used only two-step dilution for protein precipitation and matrix dilution. Flow injection analysis without chromatographic separation allowed for total run times of 1 min per sample. Creatinine concentrations were quantitated using stable isotope dilution tandem mass spectrometry. Selectivity and coelution-free quantitation were assured by qualifier ion monitoring. Results: Method validation revealed excellent injection repeatability of 1.0% coefficient of variation (CV), intraday precision of 1.2% CV and interday precision of 2.4% CV. Accuracy determined from standard addition experiments was 106.1 +/- 3.8%. The linear calibration range was adapted to physiological creatinine concentrations. Comparison of quantitation results with a routinely used method (Jaffe colorimetric assay) proved high agreement (R-2 = 0.9102). Conclusions: The new method is a valuable addition to the toolbox of LC-MS/MS laboratories where excretion of urinary compounds is studied. The `dilute and shoot' approach to isotope dilution tandem mass spectrometry makes the new method highly accurate as well as cost-and time-efficient. Copyright (C) 2012 S. Karger AG, Base

Effects of dietary long-chain polyunsaturated fatty acids on plasma amino acids and indices of protein metabolism in infants: Results from a randomized clinical trial

Background/Aim: Previous studies in vitro and in animals in vivo found that alpha-linolenic acid (C18:3 omega 3) may enhance oxidative damage of essential amino acids. We investigated whether the addition of the long-chain polyunsaturated fatty acids (LCPUFA) arachidonate (C20:4 omega-6; AA) and docosahexaenoate (C22:6-omega 3; DHA) in the form of egg phospholipids to infant formula affects plasma amino acid concentrations and indices of protein metabolism in term infants. Methods: In a double-blind, randomized clinical trial, healthy infants were fed from day 5 of life formula with or without preformed LCPUFA (n = 10 and 12, respectively). At the age of 5 days and 1, 2, 3 and 4 months, blood samples were obtained and analyzed for plasma amino acids by high-performance liquid chromatography and for plasma phospholipid fatty acid composition by gas chromatography. Results: At the age of 3 months, plasma threonine concentrations were significantly lower in infants receiving dietary LCPUFA than in controls (124 +/- 16 vs. 216 +/- 28 mu mol/l, p < 0.05). Values of other plasma essential amino acids, total protein, albumin, creatinine and urea nitrogen did not differ between the two feeding groups throughout the study. At the age of 5 days, plasma phospholipid AA and DHA concentrations were inversely correlated with histidine concentrations (AA: r = -0.60, p = 0.01; DHA: r = -0.53, p < 0.05). At the age of 3 months, DHA concentrations were inversely related to plasma histidine, methionine and threonine concentrations (r = -0.66, -0.62, and -0.64, respectively, p < 0.05). Conclusions: The dietary LCPUFA supplementation of infant formula used in this study has no adverse effects on infant plasma amino acid concentrations and indicators of protein metabolism. Nonetheless, the apparent interaction of LCPUFA with some amino acids in formula-fed infants warrants further investigation