Determination of creatinine and creatine by capillary electrophoresis : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Chemistry at Massey University

The assessment of creatinine and creatine in biological fluids is important in the evaluation of renal and muscular functions. For routine creatinine determinations in the clinical laboratory, the most frequently used method is the spectrophotometric one based on the Jaffé reaction. However, this reaction is not specific for creatinine. For this reason, several methods have been proposed, but the elimination of interferences in the determination of creatinine has still not been achieved in some of these methods; others solved this problem either with expensive equipment that does not suit routine analysis or necessitates time-waste procedures. In this thesis capillary electrophoresis was the new tool investigated. It was applied in an attempt to achieve both the separation of creatinine from the non-creatinine 'Jaffé- reacting' chromogens and the determination of creatine in serum. Capillary zone electrophoresis was performed with detection at wavelength 480 nm to separate creatinine from the non-creatinine 'Jaffé-reacting' chromogens in urine. The principle was based upon the different migration times due to the different molecule weights, molecular sizes and charges under the applied high voltage. The picric acid was employed as part of the running buffer to allow reaction of creatinine and picrate to take place after the sample injection. This procedure eliminated the negative influence of the reaction time that is controlled manually in the common Jaffé reaction method. Therefore, compared to the Jaffé reaction method, the new method achieved more accuracy and precision in the determination of creatinine. Determination of creatinine in serum and urine were studied at a new wavelength 417 nm, which gave a higher sensitivity of detection than at 480 nm. This wavelength shift made the determination of creatinine in serum possible by capillary zone electrophoresis without the non-creatinine 'Jaffé-reacting' chromogens interfering. In this method, serum only needed a simple filtration before the analysis. Creatine was discovered to have absorption at 417 nm in alkaline medium. Moreover, specific sample stacking was introduced in this method. The sample was dissolved in a mixture of two-volumes acetonitrile and one-volume 3 % ammonium chloride to give a 10-fold enhancement of detection sensitivity

The Comparison of Creatinine and Cystatin C Value in Preeclampsia Severity and Neonatal Outcome

Objectives: to compare the levels of creatinine and cystatin C with the severity of preeclampsia, and assess neonatal outcomes.Materials and Methods: Creatinine, cystatin C, and neonatal outcomes were assesed in 17 normotensive samples, 17 samples of mild preeclampsia and 17 samples of severe preeclampsia. Analysis of data with statistical tests of ANOVA and t test differences between 2 proportions.Results: The mean levels of creatinine in the normotensive group, mild preeclampsia, severe preeclampsia are 0.56 mg/dL, 0.67 mg/ dL, and 0.75 mg/dL, p=0.138; While on cystatin C are 0.82 mg/L, 1.03 mg/L and 1.32 mg/L, p=0.000. The adverse neonatal out-come wasn't found in the normotensive group. In mild pre-eclampsia obtained 1 preterm birth and 1 intrauterine fetal death (IUFD), whereas in severe preeclampsia obtained 3 babies born preterm, 1 IUFD, and 1 intrauterine growth restriction (IUGR).Conclusion: levels of cystatin C was increased significantly in line with increased severity of preeclampsia, whereas creatinine was not increased significantly. Cystatin C is better than crea-tinine as a marker of renal dysfunction in preeclampsia patients. There was an increase in adverse neonatal outcomes in the group of preeclampsia

Urinary Neutrophil Gelatinase-associated Lipocalin as a Marker for Identification of Acute Kidney Injury and Recovery in Dogs with Gentamicin-induced Nephrotoxicity.

BackgroundAcute kidney injury (AKI) is associated with high mortality rates in dogs, which may be a consequence of late recognition using traditional diagnostic tests. Neutrophil gelatinase-associated lipocalin (NGAL) is a protein-induced during kidney injury that may identify AKI earlier than traditional tests.Objectives/hypothesisTo evaluate urinary NGAL (uNGAL) and uNGAL-to-urinary creatinine ratio (UNCR) as early markers of kidney injury and recovery in an AKI model in dogs. It was hypothesized that these markers would document AKI earlier than serum creatinine concentration.AnimalsFive purpose-bred dogs.MethodsProspective study. Acute kidney injury, defined as a > 50% increase in serum creatinine concentration above baseline, was induced in dogs by gentamicin administration (8-10 mg/kg SC q8h). Blood and urine collected for biochemical analyses and uNGAL and urinary creatinine concentrations, respectively, during AKI induction and recovery.ResultsAcute kidney injury was diagnosed significantly earlier based on a 7-fold increase in UNCR compared to a > 50% increase in serum creatinine concentration (day 8; range, 2-10 mg/dl vs day 16; range, 14-19 mg/dl; P = .009). During recovery, the initial decrease in UNCR preceded the decrease in serum creatinine concentration by a median of 2 days. The uNGAL changes paralleled UNCR changes, but the increase in uNGAL was triphasic; the initial peak occurred earlier than UNCR (median, day 11 versus median, day 19).Conclusions and clinical importanceThe UNCR was early marker of gentamicin-induced AKI and its decrease documented onset of renal recovery. Additional studies are needed to validate this marker in dogs with naturally occurring renal injury

Renal and Hepatic Dysfunction in Malaria Patients in Minna, North Central Nigeria

Information on kidney and liver involvement in malaria in Africa is still very scanty. Kidney and liver functions were assessed in 70 malaria patients using serum levels of creatinine and urea and urinary protein levels as test indicators of kidney function and serum levels of bilirubin, aspartate aminotranferase (AST or SGOT), alanine aminotransferase (ALT or SGPT), and alkaline phosphatase (ALP) as indicators of liver function. Descriptive analysis of results obtained showed that 67.14% of patients had creatinine level above the 126µmole/L which is considered the upper limit of the normal range. Three cases (4.29%) had creatinine levels well above 265µmoles/L. The serum concentrations of creatinine, urea, protein, conjugated and total bilirubin, AST, ALT, and ALP in malaria patients were significantly higher (p<0.05) than those of malaria free individuals. We conclude that renal dysfunction, acute renal failure and liver dysfunction are clinical features of malaria in Minna, North Central Nigeri

Effect of strategies to reduce exposure of infants to environmental tobacco smoke in the home : cross sectional survey

Objective To examine parents' reported knowledge and use of harm reduction strategies to protect their infants from exposure to tobacco smoke in the home, and the relation between reported use of strategies and urinary cotinine to creatinine ratios in the infants. Design Cross sectional survey. Settings Coventry and Birmingham. Main outcome measures Parents’ reported knowledge and use of harm reduction strategies and urinary cotinine to creatinine ratios in their infants. Participants 314 smoking households with infants. Results 86% of parents (264/307) believed that environmental tobacco smoke is harmful, 90% (281/314) believed that infants can be protected from it in the home, and 10% (32/314) were either unaware of measures or reported using none. 65% of parents (205/314) reported using two or more measures, but only 18% (58/314) reported not allowing smoking in the home. No difference was found in mean log e transformed urinary cotinine to creatinine ratio in infants from households that used no measures compared with households that used less strict measures. Mean log cotinine to creatinine ratios were significantly different in households banning smoking in the home compared with those using less strict or no measures. Banning smoking in the home was independently associated with a significant reduction in urinary cotinine to creatinine ratio by a factor of 2.6 (1.6 to 4.2) after adjustment for average household cigarette consumption, tenure, and overcrowding. Conclusions Less than a fifth of parents in smoking households ban smoking in the home. Banning smoking was associated with a small but significant reduction in urinary cotinine to creatinine ratio in infants, whereas less strict measures compared with no measures had no effect on the infants’ exposure to environmental tobacco smoke

Creatinine, diet, micronutrients, and arsenic methylation in West Bengal, India.

BackgroundIngested inorganic arsenic (InAs) is methylated to monomethylated (MMA) and dimethylated metabolites (DMA). Methylation may have an important role in arsenic toxicity, because the monomethylated trivalent metabolite [MMA(III)] is highly toxic.ObjectivesWe assessed the relationship of creatinine and nutrition--using dietary intake and blood concentrations of micronutrients--with arsenic metabolism, as reflected in the proportions of InAS, MMA, and DMA in urine, in the first study that incorporated both dietary and micronutrient data.MethodsWe studied methylation patterns and nutritional factors in 405 persons who were selected from a cross-sectional survey of 7,638 people in an arsenic-exposed population in West Bengal, India. We assessed associations of urine creatinine and nutritional factors (19 dietary intake variables and 16 blood micronutrients) with arsenic metabolites in urine.ResultsUrinary creatinine had the strongest relationship with overall arsenic methylation to DMA. Those with the highest urinary creatinine concentrations had 7.2% more arsenic as DMA compared with those with low creatinine (p &lt; 0.001). Animal fat intake had the strongest relationship with MMA% (highest tertile animal fat intake had 2.3% more arsenic as MMA, p &lt; 0.001). Low serum selenium and low folate were also associated with increased MMA%.ConclusionsUrine creatinine concentration was the strongest biological marker of arsenic methylation efficiency, and therefore should not be used to adjust for urine concentration in arsenic studies. The new finding that animal fat intake has a positive relationship with MMA% warrants further assessment in other studies. Increased MMA% was also associated, to a lesser extent, with low serum selenium and folate

Increasing the sensitivity of the Jaffe reaction for creatinine

Study of analytical procedure has revealed that linearity of creatinine calibration curve can be extended by using 0.03 molar picric acid solution made up in 70 percent ethanol instead of water. Three to five times more creatinine concentration can be encompassed within linear portion of calibration curve

Do acute elevations of serum creatinine in primary care engender an increased mortality risk?

Background: The significant impact Acute Kidney Injury (AKI) has on patient morbidity and mortality emphasizes the need for early recognition and effective treatment. AKI presenting to or occurring during hospitalisation has been widely studied but little is known about the incidence and outcomes of patients experiencing acute elevations in serum creatinine in the primary care setting where people are not subsequently admitted to hospital. The aim of this study was to define this incidence and explore its impact on mortality. Methods: The study cohort was identified by using hospital data bases over a six month period. Inclusion criteria: People with a serum creatinine request during the study period, 18 or over and not on renal replacement therapy. The patients were stratified by a rise in serum creatinine corresponding to the Acute Kidney Injury Network (AKIN) criteria for comparison purposes. Descriptive and survival data were then analysed. Ethical approval was granted from National Research Ethics Service (NRES) Committee South East Coast and from the National Information Governance Board. Results: The total study population was 61,432. 57,300 subjects with ‘no AKI’, mean age 64.The number (mean age) of acute serum creatinine rises overall were, ‘AKI 1’ 3,798 (72), ‘AKI 2’ 232 (73), and ‘AKI 3’ 102 (68) which equates to an overall incidence of 14,192 pmp/year (adult). Unadjusted 30 day survival was 99.9% in subjects with ‘no AKI’, compared to 98.6%, 90.1% and 82.3% in those with ‘AKI 1’, ‘AKI 2’ and ‘AKI 3’ respectively. After multivariable analysis adjusting for age, gender, baseline kidney function and co-morbidity the odds ratio of 30 day mortality was 5.3 (95% CI 3.6, 7.7), 36.8 (95% CI 21.6, 62.7) and 123 (95% CI 64.8, 235) respectively, compared to those without acute serum creatinine rises as defined. Conclusions: People who develop acute elevations of serum creatinine in primary care without being admitted to hospital have significantly worse outcomes than those with stable kidney function

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

The Contribution of Blood Serum Biomarkers to the Prediction of Cognitive Decline by fMRI and Apolipoprotein-E in Healthy Older Adults

Biomarkers are a promising approach to the prediction and early intervention of Alzheimer\u27s disease. We demonstrated that cortical functional MRI (fMRI) activation during a semantic memory task and apolipoprotein-E ?4 allele inheritance (APOE?4) effectively predicted cognitive decline after 18-months in healthy, asymptomatic elders. Hippocampal volume added modest prediction, while AD family history and demographics were ineffective. Previous studies have linked plasma homocysteine (tHcy), vitamin B12 and creatinine values to cognitive funcitoning, cortical atrophy, hippocampal atrophy and neuropathology, and vascular integrity. Here we incorporated total plasma homocysteine (tHcy), B12 creatinine values into our previous predictive models. Of 78 healthy elders, 27 (34.6%) exhibited significant cognitive decline after 18-months. tHcy, but not B12 or creatinine, was marginally positively correlated with cortical semantic memory fMRI activation, particularly in stable participants. Logistic regression showed that tHcy, when added to APOE?4 and cortical fMRI, was a significant predictor of outcome and strengthed the already significant model (p = .007; C = .80 and R2 = .37). However, control for B12 and creatinine covariates diminished tHcy as a predictor (p = .084), though the model was still stronger than without this factor (C = .78 and R = 31). tHcy did not significantly interact with APOE?4, as has previously been reported. Neither B12 nor creatinine was similarly effective as a predictor. These results suggest that commonly investigated blood serum biomarkers are at best weakly associated with predicting age- and dementia-related cognitive decline in healthy, asymptomatic elders. fMRI and APOE?4 presently provided the best predictive model