Alkaline Picrate Research Articles

Alternative method for measurement of albumin/creatinine ratio using spectrophotometric sequential injection analysis

A simple, automatic and practical system for successive determination of albumin and creatinine has been developed by combining sequential injection analysis (SIA) and highly sensitive dye-binding assays. Albumin detection was based on the increase in the absorbance due to complex formation between albumin and eosin Y in acidic media. The absorbance of the complex was monitored at 547 nm. For the creatinine assay, the concentration of creatinine was measured by reaction with alkaline picrate to form a colored product which absorbs at 500 nm. The influences of experimental variables such as effects of pH, reagent concentration, standard/sample volume and interferences were investigated. Under optimal conditions, the automated method showed linearity up to 20 mg L −1 for albumin and 100 mg L −1 for creatinine. The 3 σ detection limits were 0.6 and 3.5 mg L −1 for albumin and creatinine, respectively, and the relative standard deviations ( n = 10) were 2.49% for 20 mg L −1 albumin, and 3.14% for 20 mg L −1 creatinine. Application of the proposed method to the direct analysis of urinary samples yielded results which agreed with those obtained from the Bradford protein assay and a creatinine enzymatic assay according to a paired t-test. The results obtained should be a step towards developing a fully automated and reliable analytical system for clinical research, which requires direct determination of albumin and creatinine and/or its ratios.

Variation of serum creatinine, cystatin C, and creatinine clearance tests in persons with normal renal function

Background To determine the potential sensitivity of several renal function tests for detecting early changes in renal function, we compared the within-individual (W-I) variation over 5 months of serum creatinine, serum cystatin C, and creatinine clearance. Methods On 31 healthy subjects, blood and timed urine specimens were collected once each month to get 6 collections. Creatinine (enzymatic) in serum and urine and cystatin C (immunonephelometric) in serum were measured and glomerular filtration rate (GFR) by creatinine clearance and the Modification of Diet in Renal Disease (MDRD) equation were calculated. To compare W-I variations between different creatinine methods, we also measured creatinine by both enzymatic and kinetic alkaline picrate methods on 15 sets of frozen samples. Results For the 31 volunteers, the mean W-I variations for serum creatinine (5.8%) and cystatin C (5.4%) were both much lower than the W-I variation of creatinine clearance (18.7%). As expected, the MDRD GFR had a similar W-I variation (6.7%) to that of serum creatinine and its values were markedly different than GFR by creatinine clearance. On the 15 sets of frozen samples, the W-I variation of creatinine measured by the enzymatic method (CV 5.2%) was slightly less than by the picrate method (CV 6.2%). Conclusions The low W-I variation of both serum cystatin C and serum creatinine suggests that serial measurements of either would detect a changes in renal function earlier than would GFR by creatinine clearance or MDRD equation, which allows reporting only for GFRs < 60 ml/min/1.7 m 2. While we measured only creatinine clearance, the large variability, difficulty, and cost of all clearance measurements make them impractical for routine monitoring of patients.

Enzymatic assays for creatinine: time for action

Accurate serum creatinine measurements in glomerular filtration rate estimation (eGFR) using equations are critical to ongoing global public health efforts to improve the diagnosis and treatment of chronic kidney disease. There is now an ongoing activity to promote worldwide standardization of methods to determine creatinine together with the introduction of a revised eGFR equation appropriate for use with standardized creatinine methods. Standardization of calibration, i.e., implementation of calibration traceability to high-order reference measurement procedures and reference materials, does not, however, correct for analytical interferences of field methods (non-specificity bias). To account for the sensitivity of alkaline picrate-based methods to non-creatinine chromogens, some manufacturers have adjusted the calibration to minimize the pseudo-creatinine contribution of plasma proteins, producing results more closely aligned to the reference method (isotope dilution-mass spectrometry), but this strategy makes an assumption that the non-creatinine chromogen interference is a constant among samples, which is an oversimplification. Thus, analytical non-specificity for substances found in individual patient samples can affect the accuracy of eGFR computed from serum creatinine values for any alkaline picrate method, including the so-called "compensated" Jaffé methods. The use of assays that are more specific for serum creatinine determination, such as those based on enzymatic reactions, may provide more reliable eGFR values. Supporting the choice of more specific assays by clinical laboratories represents one of the main tasks of our profession in order to achieve the ultimate clinical goal, which is to routinely report an accurate eGFR in all the pertinent clinical situations.

Enzymatic assays for creatinine: Time for action

Estimation of glomerular filtration rate (eGFR) on the basis of serum creatinine concentration measurements using equations is critical to ongoing global public health efforts to improve the diagnosis and treatment of chronic kidney disease. There is now ongoing activity to promote world‐wide standardization of methods to measure creatinine concentrations, together with the introduction of a revised eGFR equation appropriate for use with standardized creatinine methods. Standardization of calibration, i.e. implementation of calibration traceable to higher‐order reference measurement procedures and reference materials, does not, however, correct for analytical interferences of field methods (non‐specificity bias). To account for the sensitivity of alkaline picrate‐based methods to non‐creatinine chromogens, some manufacturers have adjusted the calibration to minimize the pseudo‐creatinine contribution of plasma proteins, thereby producing results more closely aligned with the reference method (isotope dilution‐mass spectrometry), but this strategy makes the assumption that the non‐creatinine chromogen interference is constant among samples, which is an oversimplification. Thus, analytical non‐specificity for substances found in individual patient samples affects the accuracy of eGFR computed from serum creatinine concentrations for any alkaline picrate method, including the so‐called “compensated” Jaffe methods. Using assays that are more specific for serum creatinine, such as those based on enzymatic reactions, may provide more reliable eGFR values. Supporting the choice of more specific assays by clinical laboratories is one of the main tasks of our profession in achieving the ultimate clinical goal, which is to routinely report an accurate eGFR in all pertinent clinical situations.

Calibration of Serum Creatinine in the National Health and Nutrition Examination Surveys (NHANES) 1988-1994, 1999-2004

The calibration of serum creatinine values to standardized creatinine and the commutability of serum creatinine across surveys are essential to the correct use of National Health and Nutrition Examination Survey (NHANES) data for kidney function and for generating estimates of the burden of kidney disease in the United States. Calibration study of serum creatinine in NHANES III (1988-1994) and NHANES 1999-2000, 2001-2002, and 2003-2004 to directly compare creatinine measurements from the original surveys with standard creatinine measured using an assay traceable to known gold-standard methods. We also assessed predictors of differences between methods (potential interferences) in this general population. The NHANES are ongoing cross-sectional surveys of the civilian noninstitutionalized population of the United States. We selected random samples of approximately 200 stored specimens from persons aged 60 years or older from each survey (NHANES III, 1999-2000, 2001-2002, and 2003-2004). Stored serum specimens from the 4 NHANES surveys were analyzed for serum creatinine by using a Roche enzymatic assay implemented at the Cleveland Clinic Research Laboratory (CCRL). The Roche assay is traceable to gold-standard reference methods. The original NHANES serum creatinine values were obtained using the Jaffé method (kinetic alkaline picrate) implemented in several different laboratories. Overall agreement between the original NHANES values (Jaffé method) and CCRL measurements (Roche enzymatic) was high, but substantial biases were observed in NHANES III and 1999-2000. No bias was observed in NHANES 2001-2002 and 2003-2004. Final calibration equations to correct serum creatinine values in the relevant surveys are provided. Assay differences were independent of sex, race/ethnicity, and bilirubin and triglyceride levels, but weakly related to age and glucose concentration. We were not able to examine drift in measurements over time within each survey or directly evaluate freeze-thaw effects. The magnitude of differences in serum creatinine measurements in NHANES III and 1999-2000 from standard creatinine would result in large differences in estimates of kidney function (10% to 20%). Thus, correction of original creatinine values in NHANES III and 1999-2000 is essential, but no correction is needed for NHANES 2001-2002 or 2003-2004.

Effects of Falciparum Malaria Infection on Some Hematological Indices and Renal functions

Parameters including some haematological and renal function indices were evaluated in 30 middle aged patients hospitalised for Plasmodium falciparum falciparum malaria at the Federal Medical Centre (FMC), Owerri. Also 30 apparently healthy age-matched controls were evaluated for the same parameters. Haematological parameters including haemoglobin (Hb) level, total WBC, differential leucocyte and platelet counts were performed using appropriate techniques. Blood urea was estimated using Diacetylmonoxime method and creatinine level was determined using alkaline picrate (Jaffe's reaction). The mean blood urea and creatinine values of P. falciparum malaria patients were significantly high (P

Cyanide Content of Parts of Pride of Barbados (&lt;i&gt;Caesalpina pulcherima&lt;/i&gt;) Grown in Nigeria

Cyanide contents of some parts of Pride of Barbados (Caesalpina pulcherima), grown in Nigeria, were studied. Analyses were conducted on intact seed, extra –cotyledonous deposit, cotyledon, flower, pod (with seeds removed), apical and pre-apical leaves and stalks, basal and apical stems, root and testa of Pride of Barbados. Alkaline picrate method was used in cyanide determination. Results obtained showed that mean cyanide contents of parts of Pride of Barbados were in the following ranges: 0.6 – 3.3; 0.4 – 3.1; 0.4 – 4.0 and 0.8 – 4.1mgkg-1 dry weight for samples from west (Ekpoma), east (Enugu), south (Port Harcourt) and north (Jos) of Nigeria respectively. The amount of cyanide determined in the edible extra-cotyledonous deposit of this legume was minimal and equally very low in all other parts studied (p>0.05). Pride of Barbados is presumably among the legumes with the lowest cyanide content. Keywords: Cyanide content, Pride of Barbados. Journal of Medical Laboratory Science Vol. 13 (2) 2004: pp. 29-32

Expressing the Modification of Diet in Renal Disease Study Equation for Estimating Glomerular Filtration Rate with Standardized Serum Creatinine Values

We sought to reexpress the 4-variable Modification of Diet in Renal Disease (MDRD) Study equation for estimation of glomerular filtration rate (GFR) using serum creatinine (S(cr)) standardized to reference methods. Serum specimens included creatinine reference materials prepared by the College of American Pathologists (CAP), traceable to primary reference material at the NIST, with assigned values traceable to isotope dilution mass spectrometry (IDMS), a calibration panel prepared by the Cleveland Clinic Research Laboratory (CCRL), and frozen samples from the MDRD Study. Split specimens were measured at the CCRL using the Roche enzymatic and Beckman CX3 kinetic alkaline picrate assays. Roche enzymatic assay results on CAP samples were comparable to IDMS-assigned values. Beckman CX3 assay results in 2004-2005 were significantly higher than but highly correlated with simultaneous Roche enzymatic assay results (r(2) = 0.9994 on 40 CCRL samples) and showed minimal but significant upward drift from Beckman CX3 assay results during the MDRD Study in 1989-1991 (r(2) = 0.9987 in 253 samples). Combining these factors, standardized S(cr) = 0.95 x original MDRD Study S(cr). The reexpressed 4-variable MDRD Study equation for S(cr) (mg/dL) is GFR = 175 x standardized S(cr)(-1.154) x age(-0.203) x 1.212 (if black) x 0.742 (if female), and for S(cr) (micromol/L) is GFR = 30849 x standardized S(cr)(-1.154) x age(-0.203) x 1.212 (if black) x 0.742 (if female) [GFR in mL x min(-1) x (1.73 m(2))(-1)]. When the calibration of S(cr) methods is traceable to the S(cr) reference system, GFR should be estimated using the MDRD Study equation that has been reexpressed for standardized S(cr).

Does Bilirubin Cause Interference in Roche Creatinine Methods?

Bilirubin interference in kinetic alkaline picrate (Jaffe) creatinine assays has been well documented (1)(2). The exact mechanism of interference is still unclear, however, and both conjugated and unconjugated bilirubin have been implicated. Enzymatic assays, which reportedly suffer less interference, are an alternative, but the extra reagent cost has restricted their use. Historical data continue to be used to estimate the extent of bilirubin interference, although modifications to these assays or instruments may have taken place since the original interference studies were performed. We used liquid chromatography tandem mass spectrometry (LC-MS/MS) as the reference method to investigate the extent of bilirubin interference in 2 automated creatinine assays. We have previously shown that this LC-MS/MS assay compares well to the 2 automated methods at creatinine concentrations of 100 samples with bilirubin concentrations within reference intervals. We added varying amounts of creatinine (Sigma) to phosphate buffered saline (PBS) pH 7.4, containing 40 g/L bovine serum albumin, to give concentrations of 37.5–1000 μmol/L. One liter of PBS contained 8 g sodium chloride, 0.2 …

Estimated GFR: Are There Limits to Its Utility?: Monitoring Kidney Function in Type 2 Diabetic Patients with Incipient and Overt Diabetic Nephropathy. Diabetes Care 29: 1024-1030, 2006.

Estimated GFR: Are There Limits to Its Utility?: Monitoring Kidney Function in Type 2 Diabetic Patients with Incipient and Overt Diabetic Nephropathy. Diabetes Care 29: 1024-1030, 2006.

Studies on Residual Hydrocyanic Acid (HCN) in Garri Flour Made from Cassava (Manihot Spp.)

Garri flour was produced from mashed cassava (Manihot spp) roots, which had been subjected to fermentation for time periods of between 6 and 72 h. The hydrocyanic acid content of each batch was estimated by spectrophotometric alkaline picrate method. White and yellow garri flour samples procured from markets in Abia State, Nigeria were also analyzed for their HCN contents using the above-mentioned method. The HCN content decreased significantly with the length of fermentation period of the grated cassava mash. Levels of residual HCN were also significantly influenced by the source and type of garri flour (whether white or yellow). The white garri flour had higher HCN levels than the yellow.

Creatinine concentration in plasma from dog, rat, and mouse: a comparison of 3 different methods

There are numerous methods for analyzing creatinine concentration in plasma, including the Jaffé alkaline picrate method in various modifications, enzymatic tests, and chromatographic methods. The purpose of this study was to evaluate whether an enzymatic method could replace a Jaffé method for routine creatinine measurements in plasma from dogs, rats, and mice. The enzymatic method and a compensated Jaffé method were tested against a high-pressure liquid chromatography (HPLC) method, regarded as the gold standard for creatinine measurement. Heparinized plasma samples were obtained from 20 beagle dogs, 20 Wistar rats, and 20 CD1-strain mice. The 2 test kits (Roche Diagnostics), Creatinine Jaffé Compensated and the enzymatic Creatinine Plus Version 2 reagent, were used on a Cobas Integra 400. The Jaffé compensated method used a calibration adjustment of 18 micromol/L to correct for the protein matrix in serum and plasma. The HPLC method was an isocratic method using a weak cation-exchange column following protein precipitation. Creatinine concentrations obtained using the enzymatic and the Jaffé methods differed significantly from the results obtained by the HPLC method. For dog plasma, mean values of 61.2, 61.8, and 67.8 micromol/L were obtained by the compensated Jaffé, enzymatic and HPLC methods, respectively. In the rat, respective mean values were 26.7, 21.9, and 23.0 micromol/L, and in the mouse, respective mean values were 14.2, 5.4, and 9.2 micromol/L. The enzymatic method can replace the Jaffé method for plasma creatinine determination in dogs, rats, and mice because results from the enzymatic method were closer to HPLC values than were those of the Jaffé method.

Estimation of Renal Function in Subjects With Normal Serum Creatinine Levels: Influence of Age and Body Mass Index

The Cockcroft-Gault (CG) and simplified Modification of Diet in Renal Disease (MDRD) formulas are the most widely used estimates of renal function. The influence of age and body mass index (BMI) on the performance of these equations was analyzed in 850 subjects with serum creatinine levels less than 1.5 mg/dL (<133 micromol/L). Glomerular filtration rate (GFR) was measured as urinary clearance of continuously infused technetium Tc 99m-labeled diethylene triaminopentaacetic acid. Performance was assessed as bias, precision, and accuracy. In the total population, the CG and MDRD calculations based on enzymatic measurement of serum creatinine (which is constantly less than that obtained by using the alkaline picrate [Jaffé] method) significantly underestimated GFR by 4.9 and 12.4 mL/min/1.73 m2 (0.08 and 0.21 mL/s/1.73 m2), respectively. In patients 65 years and older, underestimation by means of the CG formula was enhanced, whereas that by means of the MDRD formula was blunted, compared with the group younger than 65 years (-11.3 versus -3.7 mL/min/1.73 m2 [-0.19 versus -0.06 mL/s/1.73 m2] for CG and -3.7 versus -14.0 mL/min/1.73 m2 [-0.06 versus -0.23 mL/s/1.73 m2] for MDRD). GFR was underestimated to a large extent by means of the MDRD equation irrespective of BMI. Conversely, the underestimation by means of the CG formula found in lean people (-13.0 mL/min/1.73 m2 [-0.22 mL/s/1.73 m2]) was blunted in overweight people (BMI, 25 to 30 kg/m2) and reversed to overestimation (+10.1 mL/min/1.73 m2 [+0.17 mL/s/1.73 m2]) in obese subjects (BMI > 30 kg/m2). As suggested by estimations obtained using enzymatic serum creatinine measurement, the MDRD equation may be the estimation of choice in elderly patients, whereas the CG estimate is preferable in subjects younger than 65 years. Nevertheless, when obesity is present, no reliable estimation can be obtained by using the CG or MDRD formula.

Increasing the efficiency of in-capillary electrophoretically mediated microanalysis reactions via rapid polarity switching.

We report herein a new approach to enhance the sensitivity or speed of CE-based methods that involve in-line reactions. Rapid polarity switching (RPS) is used as a novel means for in-line mixing of two reactant solutions via rapid (1-5 s) and sequential switching of the applied potential field. By employing the RPS approach with a model chemical reaction, that between creatinine and alkaline picrate, significant enhancement in sensitivity (or a decrease in analysis time) is realized. Both increased convection and electrophoretic stacking of the ionic reagent appear to contribute to the rise in apparent reaction rate. When coupled with in-line chemistry of the Jaffe method for creatinine, the RPS methodology allows for 3-fold faster determination of creatinine in the concentration range needed for the analysis of clinical blood serum specimens. The new approach also allows the analysis to be performed without the need for the cumbersome and problematic enhanced sensitivity cell.

Are urea and creatinine values reliable indicators of azotaemia in canine babesiosis?

Serum urea and creatinine are extensively used as parameters to screen for azotaemia. Their reciprocal plots roughly correlate with glomerular filtration rate (GFR). They are, however, subject to influence by non-renal factors and to increase their specificity they are often tested concurrently. In renal disease they are expected to behave similarly, with both parameters increasing as GFR decreases. Haemolysis, as it occurs in canine babesiosis, may cause non-renal elevations in serum urea, possibly due to ammonia loading. Furthermore, haemolysis with its related elevations in serum bilirubin and serum haemoglobin, may negatively bias the measurement of serum creatinine due to interference of these substances with the chemical analysis of serum creatinine. This negative bias occurs when the alkaline picrate method, or when direct enzymatic methods based on the measurement of hydrogen peroxide, are used. In order to investigate the significance of these perturbations in canine babesiosis, paired values of serum urea and serum creatinine from Babesia canis-negative, non-haemolysis dogs (Group 1), were used to establish a relationship between urea and creatinine over a range of azotaemia by linear regression analysis. This relationship was then used to predict serum creatinine values from actual serum urea values in B. canis-positive dogs (Group 2). The mean of the predicted serum creatinine values for Group 2 (237.03 micromol/l) was then compared with the mean of the actual serum creatinine values for Group 2 (131.31 micromol/l). For Group 2, the mean actual serum creatinine demonstrated a significant negative bias relative to the mean predicted creatinine value. There was also a higher correlation between serum urea and serum creatinine in Group 1 than in Group 2. These findings may have been caused by either nonrenal elevations of serum urea values or by interference with the measurement of serum creatinine. Therefore, although it is possible that some Group 2 dogs with B. canis with high serum urea and normal, low, or zero values for serum creatinine were not azotaemic, it is also possible that other Group 2 dogs with these biochemical findings did in fact have azotaemia. This study concluded that urea and creatinine do not behave in a similar and predictable manner over a range of azotaemia in canine babesiosis and are therefore not ideally suited for the detection of renal disease in this clinical setting.

Utility of endogenous creatinine clearance as a measure of renal function in mice

The use of endogenous plasma creatinine levels and creatinine clearance as a tool to evaluate renal function in mice has come under scrutiny as prior studies have reported that the Jaffé alkaline picrate method grossly overestimates true plasma creatinine in mice. As members of the NIDDK Animal Models of Diabetic Complications Consortium (AMDCC), we evaluated the performance and feasibility of an alternative high-performance liquid chromatography (HPLC)-based method for standard determination of plasma creatinine and creatinine clearance in mice. Our purpose was to develop a simple method that provides a reliable, reproducible, and sensitive assay for small volumes (<25 microL) of mouse plasma and sera. We compared creatinine clearance measured by HPLC with the Jaffé method and HPLC creatinine clearance with inulin clearance [fluoroscein isothiocyanate (FITC) inulin in an osmotic pump implanted in mouse] in C57BL/6J mice. Different groups of mice underwent either one of two protocols. Protocol A included dietary intervention with normal, low salt plus enalapril, or high salt. Protocol B induced diabetes using streptozotocin. First, mean plasma creatinine levels were significantly lower (P < 0.0001) by HPLC (0.128 +/- 0.026 mg/dL) vs. Jaffé (0.4 +/- 0.12 mg/dL) for mice on a normal diet. Urine creatinine concentrations measured by HPLC were 10% lower than by Jaffé (P < 0.01). Second, mean creatinine clearance by HPLC for mice on a normal diet was 255 +/- 68 microL/min. Mice on low salt diet plus enalapril had reduced creatinine clearance (72.8 +/- 24.2 microL/min) while mice on high salt diet had an elevated creatinine clearance (355 +/- 105 microL/min). Third, diabetic mice (19 to 24 weeks of diabetes) exhibited hyperfiltration as creatinine clearance was 524 +/- 214 microL/min whereas nondiabetic age/gender-matched mice showed a mean creatinine clearance of 206 +/- 41 microL/min. Finally, significant correlation was demonstrated for creatinine clearance by HPLC vs. inulin clearance (R= 0.643; P < 0.001). HPLC is highly accurate, much more sensitive and specific than the Jaffé method for plasma creatinine measurements in mice. Creatinine clearance in mice measured by HPLC reflects changes in renal function induced by diet and diabetes.

A simplified method for HPLC determination of creatinine in mouse serum.

Mouse models are frequently used to study renal function. However, mouse serum contains chromagens that interfere with standard picric acid-based assays for serum creatinine. Several alternative methods exist for serum creatinine measurements, including assay by high-performance liquid chromatography (HPLC), but only one has been adapted to mouse serum. Creatinine was measured in serum by acetonitrile deproteinization, followed by isocratic, cation exchange HPLC. The HPLC method was compared with a standard alkaline picrate colorimetric assay, using serum from animals with low-to-moderate renal injury. Acidification of acetonitrile with HCl in the deproteinization step produced variable results, including an extra peak that interfered with integration of the creatinine peak or loss of the creatinine peak. Deproteinizing with acetonitrile alone resulted in a more reliable measurement of serum creatinine, which was validated by a series of known additions of creatinine standard. The HPLC assay was reproducible with coefficients of variation from 1.6 to 5.1%. The picric acid assay overestimated serum creatinine, when directly compared with the HPLC assay. The extent of overestimation, up to sixfold, was greatest at normal (0.1 to 0.2 mg/dl) to moderately elevated (0.5 mg/dl) serum creatinine levels. Mouse serum contains substances that interfere with standard picric acid assays for creatinine. Our new HPLC assay can accurately detect creatinine from 5 microl of mouse serum. These results support the widespread adoption of HPLC to accurately measure serum creatinine in mouse models of renal injury.

Impact of serum creatinine measurement error on dose adjustment in renal failure.

Doses of renally eliminated drugs should be adjusted according to kidney function to prevent adverse drug events and cost. Dose adjustment can be based on serum creatinine level, subsequent creatinine clearance estimation, and dosage calculation with consideration of the renal elimination properties of the respective compound. Our objective was to quantify the impact and relevance of serum creatinine measurement error on dose adjustment in renal failure. We analyzed 27914 measurements from external quality assessment surveys of 1878 German laboratories that used a kinetic alkaline picrate (69% of results) or an enzymatic method (25%) for creatinine determination. Linear models were fit for both methods combined and separately. On the basis of 95% confidence intervals (CIs) for creatinine values, 95% CIs for drug dosing were calculated. The 95% CI for a measured serum creatinine value was 0.80. Measured value < Reference method value < 1.28. Measured value for the kinetic alkaline picrate method and 0.87. Measured value < Reference method value < 1.21. Measured value for the enzymatic method. Applied to a data set of 6.5 million simulated patients with all possible combinations of characteristics relevant for drug dosing, the dosing error caused by serum creatinine measurement error did not exceed 25% in patients with creatinine clearance estimates lower than 50 mL/min according to the Cockcroft-Gault equation. For drugs completely eliminated by the kidneys in active form, the dosing error was up to 6-fold smaller than that which would occur if doses were not adjusted. The serum creatinine measurement error of current laboratory methods is small and is comparable to other errors influencing dose adjustment.

Predictors of serum creatinine in haemodialysis patients: a cross-sectional analysis.

C-reactive protein (CRP) levels, an acute phase response index, predict cardiovascular outcome and are inversely related to visceral proteins, including albuminaemia in haemodialysis patients. Less definite is the relationship between inflammation and markers of somatic proteins such as serum creatinine in such patients. To explore these questions, a cross-sectional analysis of potential predictors of serum creatinine was performed. One hundred and seventy-nine prevalent haemodialysis patients as of June 2001 were included in the cohort. Midweek pre-dialysis blood samples were collected during the months of June, September through to December 2001 inclusive, and determinations of serum urea (urease method), creatinine (alkaline picrate method) and CRP levels by means of a high sensitivity immunonephelometric method were performed. Furthermore, pre- and post-dialysis body weights were recorded and 2 min post-dialysis serum urea levels were determined three times. They were utilized for the calculation of single pool Kt/V and of normalized protein catabolic rate (nPCR). Each of the data represents the mean of three determinations made every 3 months in the study period. The analysis of multivariate linear regression was able to validate our model characterized by a dependent variable, serum creatinine and four independent variables (age, CRP, Kt/V and nPCR) (R(2)=0.60; F=24.10; P<0.00001; SE=1.94). Age (-0.08 mg/dl decrease in serum creatinine per 1-year increase in age), Kt/V (-0.25 mg/dl decrease in serum creatinine per 0.1 increase in Kt/V) and nPCR (0.10 mg/dl increase in serum creatinine per 0.1 g protein/kg/day increase in nPCR) were independently predictive of serum creatinine (P<0.00001). CRP and dialysis vintage did not predict serum creatinine. Stratifying the patients for the effects of CRP, only CRP values </=4 mg/l were directly predictive of serum creatinine (P<0.00001), whereas CRP values >4 mg/l were not. A further insight was given by the stratification of the patients for the effects of the interquartile ranges of CRP: it showed a progressive and statistically significant reduction of beta-coefficient inversely related to the increasing CRP values (P=0.003). Thus, the nature of the correlation between CRP and serum creatinine changes with increasing CRP values: from being a direct one, it shows a trend towards a transformation into an indirect one with beta=0 at a CRP value of approximately 9 mg/l. However, this indirect relationship does not reach statistical significance. The present cross-sectional study suggests that the activation of acute phase response does not influence creatinine metabolism in haemodialysis patients; in contrast, age, Kt/V and nPCR predict serum creatinine levels. Larger prospective trials are needed to achieve a definitive answer about the relationship between somatic proteins, acute phase response activation and nutrition in dialysis patients.

Comparison of electrochemical, electrophoretic and spectrophotometric methods for creatinine determination in biological fluids

Quantitation of creatinine in biological fluids was achieved using both a biosensor based electrochemical flow cell and a micellar electrokinetic chromatography (MEKC) separation method. The results obtained from each method were compared with the widely recognised clinical method based on the Jaffé reaction. This is a standard spectrophotometric assay based on the Jaffé reaction of creatinine with alkaline picrate. The biosensor for creatinine was constructed by co-immobilising the enzymes creatininase, creatinase and sarcosine oxidase (SO) using a bovine serum albumin (BSA)–glutaraldehyde (GA) procedure at a platinum electrode. Detection of the hydrogen peroxide product of the third enzyme-catalysed reaction (SO) allowed the measurement of creatinine at +0.7 V versus Ag/AgCl. The biosensor was incorporated into a flow cell arrangement which allowed rapid determination of creatinine concentrations in urine samples with a limit of detection of 4.5 μM, a response time of 1 min, a linear range of 0–500 μM and a precision of 1.14% ( n=10). The results for five urine samples were compared with the Jaffé reaction method and based on this number of samples results from the spectrophotometric method were 36.7% higher than those determined using the flow cell method. A MEKC method for creatinine determination in serum samples was also developed and employed for serum analysis, with only a simple filtration step as sample preparation. The method showed a limit of detection of 11.5 μM, a linear range of 0–4.4 mM and a peak area precision of 2.7% ( n=10). The method was applied to creatinine determination in serum samples ( n=13) from patients undergoing renal dialysis and the results compared to the Jaffé reaction procedure. The Jaffé method generated values on average 26.7% greater than the corresponding MEKC results.