Non-creatinine Chromogens Research Articles

Comparison of 150mmol/L and 250mmol/L concentration of sodium hydroxide in Jaffe's two point method for serum creatinine measurement with respect to linearity of assay

Objectives: Objective of the study is to perform linearity check for Creatinine measurement with Jaffe's two point method using CLSI EP-6 guideline for 150 mmol/L, 250mmol/L NaOH concentration in final reagent mixture. Material & Method: Leftover serum of samples received by the laboratory, devoid of patient identification, was used to make pool of serum. Serum creatinine reagents have NaOH of 150 mmol/L, 250 mmol/L were prepared. Serial dilution of pooled serum spiked with creatinine powder was prepared. Creatinines were measured in series of dilution. Linear regression analysis of each dilution series to find linearity limit was performed. esult & Discussion: This study evaluated Linearity difference plot using CLIA criteria (15% or 0.3 mg/dl whichever is higher) for creatinine at 95% confidence limit.7.5% of result or 0.15 mg/dl were used for plotting Linearity Difference plot. If any the two conditions are satisfied at a given point in the Linearity Difference Plot, the point is considered within linearity. 250 mmol/L NaOH in final reagent mixture gives limit of linearity 23.77mg/dl and 150 mmol/L NaOH in final reagent mixture gives limit of linearity 31.16 mg/dl. Conclusion: 150 mmol/L final reagent concentration of NaOH gives higher linearity of 31.16 mg/dl, low slop (a=29.23) indicating high sensitivity of the reagent, low y-intercept (b=-0.4578) indicating minimum effect of non-creatinine chromogens; i.e. high specificity than 250mmol/L final reagent concentration of NaOH.

Serum creatinine: advantages and pitfalls

Serum creatinine (sCr) is today the most commonly used marker to estimate glomerular filtration rate (GFR). However, measuring and interpreting creatinine is not so simple. Some physiological reasons, particularly its dependence on muscle mass makes sCr an imperfect biomarker of GFR. However, there are also analytical reasons that could be further improved. The implementation of ID-MS traceable creatinine assays (enzymatic and compensated Jaffe) improved estimation of GFR by reducing bias. Enzymatic methods which are more precise and less susceptible to interfere with non-creatinine chromogens than compensated Jaffe methods, provide more reliable estimations of GFR.

Creatinine Estimation and Interference

Sir, Creatinine is one of the most common analyte used as the indicator of glomerular filtration rate (GFR) and kidney function. In 1886, Jaffe reported a reaction of creatinine and picric acid in an alkaline medium forming complex having absorbance maxima at 520 nm. The reaction has been brought to use for the measurement of creatinine by Folin and Wu in 1919. Since then, Jaffe’s reaction has been used for estimation of creatinine in serum and urine sample. For more than 90 years, this method is in use for the measurement of creatinine concentration due to its simplicity and low cost. Creatinine is produced as the byproduct of creatine metabolism and is transported through bloodstream to the kidneys for excretion. Malfunctioning of kidney is reflected by lowering of the amount of creatinine in urine and rise of its level in blood. Creatinine levels in blood and urine also indicate the creatinine clearance, accounting for GFR. Creatinine readings indicate the total kidney GFR which is the sum of filtration rates of all functional nephrons. Early structural damage to the renal cells involving reduction in functional nephron number may not affect an individual’s total GFR as remaining renal units may perform compensatory function, enabling the kidneys to maintain kidney function temporarily even after the loss of functional tissue. Thus, creatinine/GFR may not indicate the early damage to kidney tissue. Jaffe also elaborated the interference in alkaline picrate reaction by number of organic compounds (e.g. acetone, glucose) which has been described as pseudochromogens. Still, creatinine remains the gold standard for measuring the glomerular filtration rate and kidney functioning. And Jaffe method remains the default method for estimation of creatinine. In last century, many caveats in this method were reported which we have indicated in brief in this article. Recently we have reported an interference by streptomycin (an aminoglycoside) in high doses which act as false positive in Jaffe reaction [1]. It is known from literature that aminoglycosides can be nephrotoxic and may potentially affect kidney function. If it is so, then, nephrotoxicity due to high dose of aminoglycosides (say streptomycin) can never be correctly estimated as defect in excreting out creatinine would be masked by the false positive Jaffe reaction given by streptomycin. Antibiotics like cephapirin, cefazolin, cefamandole, cephalothin, and cefoxitin have been shown to interfere with creatinine determination at various concentrations [1, 2]. Phenacemide and cephalothin may interfere either positively or negatively with the determination of creatinine by the Jaffe reaction [3] . Cephalosporin antibiotics have been shown to form non-creatinine chromogen with alkaline picrate reagent and produce false positive results for creatinine. Serum creatinine and creatinine clearance values are positively interfered by cefoxitin in the Jaffe reaction, the routine laboratory method of creatinine measurement. Both invitro and invivo analysis suggest the same [4]. Many biomolecules like bilirubin, glucose, acetone etc. are also known to interfere with the Jaffe method of creatinine estimation and therefore the method has gone through modifications [5]. To overcome the effect of interference specific enzymatic method of creatinine estimation has been also developed [6]. But in-spite of various modification of the Jaffe method and development of the specific enzymatic method for creatinine estimation, the classical alkaline picrate method remains popular in India.

Streptomycin interference in Jaffe reaction — Possible false positive creatinine estimation in excessive dose exposure

ObjectivesTo study the potential of commonly used aminoglycoside antibiotics to form non-creatinine chromogen with alkaline picrate reagent. Design and methodsWe studied the non-creatinine chromogen formation of various concentrations of streptomycin, amikacin, kanamycin, netilmicin, gentamicin and tobramycin added to known creatinine concentrations by the Jaffe reaction based creatinine estimation. ResultsOnly streptomycin above therapeutic concentrations of 10mg/mL interfered in the Jaffe reaction and acted as non-creatinine chromogen. ConclusionsTherapeutic doses of the aminoglycosides do not form non-creatinine chromogens.

Point-of-Care Measurement of Serum Creatinine in the Intensive Care Unit

Objective: To test the precision and limits of agreement of point-of-care testing (POCT)-based measurement of serum creatinine (Cr) in critically ill patients. Methods: We studied 250 paired blood samples from 82 critically ill patients from a general intensive care unit by simultaneous POCT and central laboratory testing (Jaffé method). Correlation, precision, bias, and limits of agreement were assessed. Possible confounders for interference of noncreatinine chromogens were evaluated by multivariate linear regression analysis. Results: The mean difference in serum Cr measured by central laboratory and POCT was +9.6 μmol/L (95% limits of agreement: −11.2 to +30.4 μmol/L). The mean percentage difference between the two techniques was 8.7% (95% limits of agreement −7.8% to +25.1%). On multivariate regression, the difference in serum Cr was increased with greater hemoglobin and lactate levels but decreased with greater bilirubin, albumin, and calcium levels. Conclusions: Compared with the central laboratory testing, POCT-based measurement of serum Cr in critically ill patients carried a small negative bias. This difference appeared affected by the blood levels of biochemical variables known to affect the Jaffé method. POCT-based Cr measurement appears sufficiently accurate for clinical use.

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.

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.

Does the ID-MS traceable MDRD equation work and is it suitable for use with compensated Jaffé and enzymatic creatinine assays?

International recommendations suggest that measurement of serum creatinine should be supplemented with an estimate of glomerular filtration rate (GFR) using the Modification of Diet in Renal Disease (MDRD) study equation. One problem has been the lack of standardization of commercially available creatinine assays resulting in varying estimates of GFR. A revision of the MDRD equation offers traceability to a reference method. This study evaluates the use of isotope dilution mass spectrometry (ID-MS), the compensated Jaffe and enzymatic creatinine methods compared with the Beckman CX3 Jaffe assay used to derive the MDRD equation and investigates their impact on GFR estimation using both the original and ID-MS-traceable MDRD equations. Serum creatinine was measured in 277 patients by (i) ID-MS, (ii) a Roche enzymatic assay, (iii) a Roche compensated kinetic Jaffe assay and (iv) a Beckman CX3 kinetic Jaffe assay. Estimated GFR was calculated using the MDRD equations. The ID-MS (-7.5%), Roche enzymatic (-8.6%) and compensated kinetic Jaffe (-11.9%) assays were all negatively biased (P < 0.0001) compared with the Beckman CX3 assay, causing predictable, clinically significant, overestimation of GFR when the original MDRD equation is used. This positive bias was reduced (ID-MS 6.7 to 0.4%; enzymatic 8.8 to 3.4%; compensated kinetic Jaffe 13.7 to 7.1%) when GFRs were calculated using the ID-MS-traceable MDRD equation. Compensated assays that account for non-creatinine chromogen interference produce significantly higher estimates of GFR when using the original MDRD equation. Use of the ID-MS-traceable MDRD equation ameliorates this effect. There is good agreement between estimated GFR derived from the original MDRD equation using Beckman Astra CX3 data and estimated GFR derived from the new ID-MS-traceable MDRD equation using a local ID-MS creatinine assay. This suggests that the ID-MS-traceable MDRD equation may be reliably used with both ID-MS and true ID-MS-traceable creatinine assays without the requirement for standardization to the MDRD laboratory.

Evaluation of "true" creatinine clearance in rats reveals extensive renal secretion.

The renal clearance of endogenous creatinine is widely used to assess glomerular filtration rate (GFR) and renal function in animal investigations. The objective of the present investigation was to evaluate the extent of renal secretion of endogenous creatinine in rats and the effect of probenecid, the classical inhibitor of organic anion transport, on creatinine clearance. Ten female Lewis rats received 3H-inulin (5-muCi i.v. bolus followed by 5 muCi/hr) throughout a 6-hr period. Three hours after initiation of the inulin infusion, probenecid was administered (92.4-mg/kg i.v. bolus followed by 0.59 mg/min/kg). Steady-state serum concentrations of about 500 micrograms/ml probenecid were achieved. Renal clearance was assessed between 1 and 3 hr (control) and between 4 and 6 hr (probenecid treatment). A preliminary study in seven rats demonstrated no time-dependent change in inulin or creatinine clearance between these two study intervals. Creatinine clearances were determined by an alkaline picrate assay which incorporated Fuller's earth (Lloyd reagent) to remove interfering noncreatinine chromogens from serum samples and these values were compared with those using a nonspecific picrate assay. "True" clearance ratios of creatinine to inulin (Clcr/CLin) were greater than unity (2.33 +/- 0.83, mean +/- SD) and were significantly decreased after probenecid treatment (1.26 +/- 0.28, P less than 0.01). Probenecid had no effect on GFR, as assessed by inulin clearance. Using the nonspecific picrate assay, CLcr/CLin was 1.12 +/- 0.41, which was not significantly different from unity and which decreased to 0.53 +/- 0.12 after probenecid treatment. Therefore, creatinine undergoes extensive renal secretion in female Lewis rats.

Determination of excretion of inulin, creatinine, sodium sulfanilate, and phenolsulfonphthalein to assess renal function in goats

SUMMARY Excretion of creatinine, sodium sulfanilate (ss), and phenolsulfonphthalein (psp) was studied in healthy goats. In conscious goats, mean (± sem) inulin clearance was 2.26 ± 0.08 ml/min/kg of body weight. Endogenous creatinine clearance, 1.97 ± 0.09 ml/min/kg, underestimated inulin clearance (P &lt; 0.01), probably because of the presence of noncreatinine chromogens in caprine plasma. The estimated renal clearance of psp was 6.88 ± 0.39 ml/min/kg, whereas the estimated renal clearance of ss was 3.71 ± 0.39 ml/min/kg. Both exceeded inulin clearance (P &lt; 0.01), confirming renal tubular secretion of both compounds. In 6 anesthetized goats, exogenous creatinine clearance and ss clearance exceeded inulin clearance (P &lt; 0.05). Results of stop-flow experiments documented secretion of creatinine and ss by the proximal portion of the caprine nephron. Plasma half-life of psp in uninephrectomized goats exceeded that in intact goats (20.2 ± 1.5 min vs 11.9 ± 0.7 min; P &lt; 0.01). Similarly, plasma half-life of ss was greater in goats after uninephrectomy (58.2 ± 6.2 min vs 30.4 ± 1.2 min; P &lt; 0.01).

Specific creatinine determination in laboratory animals using the new enzymatic test kit "Creatinine-PAP".

The new Creatinine-PAP test kit from Boehringer Mannheim GmbH was evaluated for the determination of creatinine in dogs, rabbits, guinea pigs, rats, and mice. In comparison with methods based on the reaction with alkaline picrate, the enzymatic method gave the lowest serum creatinine values. The differences are caused by serum substances (non-creatinine chromogens) reacting with picric acid. Noticeable differences between the standard inulin clearance and creatinine clearance (e. g., in rats) arise because the non-creatinine chromogens interfere differently in the picric acid methods, and serum and urine contain different concentrations of interfering substances. The concentrations of non-creatinine chromogens are different in the various laboratory animals. Since these substances cannot be entirely removed (e. g., in rats, dogs) by the adsorption procedure with Fuller's earth, we recommend the peroxidase-coupled enzymatic test as a practical and specific method for routine measurements of creatinine in laboratory animals.

Use of fluorescence HPLC for determining extrarenal creatinine clearance in rats

In order to clarify whether plasma creatinine concentration is overestimated due to the presence of non-creatinine chromogens when determined by the Jaff/'e method and also to elucidate the significance of tubular secretion in relation to creatinine clearance, the creatinine clearance in rats was measured by the use of a fluorescence HPLC method for the specific determination of creatinine. When creatinine was determined by the Jaffé method, plasma creatinine concentration was always overestimated. When creatinine was determined by the fluorescence HPLC method, the clearance of endogenous creatinine was found to be hardly influenced by tubular secretion, and it appears to be a reliable index of GFR in rats with normal renal function. The clearances for endogenous and exogenous creatinine were well correlated to inulin clearance, although the clearance of exogenous creatinine at plasma concentrations ranging from 22.6 to 289.9 μg/ml was slightly higher than the inulin clearance. The phenomenon of extrarenal clearance of exogenous creatinine was studied carefully in rats with normal renal function under steady-state conditions and after a single injection. The contribution of extrarenal clearance to overall clearance was 37.9% in the former case and 21.1% in the latter case. Although there is about a 2-fold difference between these values, extrarenal clearance appears to be significant in rats with normal renal function, and plasma clearance of exogenous creatinine cannot be necessarily regarded as a valid index of GFR.

The use of an enzymatic kit to measure plasma creatinine in the mouse and three other species

1. 1. Plasma creatinine concentrations were determined in mice, rats, dogs and humans using a kinetic alkaline picrate method and an enzymatic method with a centrifugal analyser. 2. 2. Lower creatinine values were obtained for all four species using the enzymatic method, but the differences between methods were greater for mice and rats. 3. 3. Removal of creatininase from the enzymatic method reagents gave proportionally higher values for non-creatinine chromogens in mice and rats. A single enzymatic method was not specific for creatinine when used for these species.

Evaluation of serum creatinine measurement by the direct acidification method for errors contributed by non-creatinine chromogens

Serum creatinine determination by the direct acidification method was found to be non-specific. Chromogens other than creatinine could react with alkaline picrate and their developed color was also destroyed by acid. Only elevated levels of bilirubin and acetoacetate caused a significant interfering effect. The degree of this interference depended on: (1) the amount of the interfering substance; (2) the reaction time allowed for color development; and (3) the final acidity for decolorization.

PLASMA CREATININE DETERMINATION IN THE NEONATE: USE OF A KINETICS METHOD

Plasma creatinine in newborns is elevated at birth and has been reported to stabilize at lower values only after several weeks. These elevated values resulting from renal and nonrenal phenomena have severely limited the use of creatinine in the neonate as an assessment of GFR. A kinetics method of creatinine analysis that minimizes the effect of noncreatinine chromogens was used to measure plasma creatinine in 38 newborns (66 determinations) during the first 4 weeks of life. Diagnoses included prematurity, transient hyperbilirubinemia, moderate RDS, and intrauterine growth retardation. Average creatinine concentrations (umol/1) during the 1st and 2nd days of life were 96 and 73, with ranges of 59-188 and 18-185, respectively. All creatinine values (n=29) observed after 5 days of life except 1 (71) were below 48. The mean plasma creatinine observed in infants aged 5-30 days was 34, the range being 14-71. A transient, but significant increase in plasma creatinine (up to values of 101) was seen in 3 patients receiving gentamicin, a potentially nephrotoxic agent. We conclude that the kinetics method of creatinine determination, which requires only 50 ul of plasma, provides a valid index of renal function in neonates as early as the first days of life.

Evaluation of the Beckman Creatinine Analyzer 2

The Beckman Creatinine Analyzer 2 was evaluated in our laboratory. This new instrument determines creatinine by a Jaffe rate method and utilizes an optical detection system which measures the rate of change in absorbance at 520 nm 25.6 seconds after sample introduction. The linearity of the instrument was verified up to creatinine concentrations of 24 mg/dl. Recovery averaged 92% from serum and 97% from urine. Comparison studies using serum specimens were made against a manual, non-kinetic Jaffe method and a Technicon SMA 12/60. Urine comparisons were made against the manual method. All comparisons indicate the Beckman analyzer yields lower creatinine values due to the minimization of interferences from non-creatinine chromogens. No significant change in these correlations was observed with elevated bilirubin concentrations (greater than 10 mg/dl) or with lipemic specimens (triglycerides up to 3390 mg/dl). Both within-day and day-to-day precision were shown to be within the stated manufacturer's specifications for both sera and urine specimens.

Effects of cardiac surgery on renal function in children

In order to determine whether cardiac surgery with cardiopulmonary bypass (CPB) affects renal function in children, glomerular filtration rate (GFR and renal plasma flow (RPF) were determined preoperatively and on the second postoperative day by a single-injection, urineless technique in a randomly selected group of 10 children. The GFR was 126.7 +/- 6.8 ml. per minute per 1.73 sq. M. (mean +/- S.E.M.) before the operation and 127.4 +/- 16.2 on the second postoperative day (p = 0.95). The RPF was 541.7 +/- 80.6 ml. per minute per 1.73 sq. M. preoperatively and 536.0 +/- 82.1 on the second postoperative day (p = 0.8 to 0.9). The results in this group of 10 children indicate that renal function is not markedly altered 2 days after cardiac surgery with CPB and deep hypothermia with circulatory arrest. The study also revealed a falsely decreased creatinine clearance. This was due to an increase in postoperative plasma creatinine values, probably related to an increase in plasma noncreatinine chromogens.

New Method for Direct Determination of "True" Creatinine

Abstract Determination of serum creatinine by the Jaffé reaction may give erroneous results because of interfering noncreatinine chromogens. Studies of the Jaffé reaction in alkaline picrate media of various pH demonstrated that creatinine, proteins, and certain chromogens have characteristic properties as regards the appearance and intensity of the color reaction. Thus a method for determining "true" creatinine has been developed in which simple spectrophotometric measurements are made at 500 nm in two alkaline picrate reagents, buffered at pH 9.65 and 11.50. Results agree well with those obtained by the use of an ion-exchange method. The proposed method is easy and rapid and offers the advantages of increased specificity and direct analysis of serum and urine.

Renal handling of creatinine in nephrotic and non-nephrotic patients.

1. The endogenous creatinine/GFR (inulin or free [57Co]cyanocobalamin or [125I]iothalamate) clearance ratios were determined in ninety-nine non-nephrotic patients and subjects and in sixteen nephrotic patients. The clearance ratios of the nephrotic patients were not significantly different from those of the non-nephrotic patients and normal subjects. 2. The clearance ratios increased as the GFR fell from 176 to below 15 ml/min, then decreased toward unity at lower GFR values. 3. In an attempt to explain this biphasic relationship, two further studies were performed. Endogenous creatinine/inulin and [14C]creatinine/inulin clearance ratios were simultaneously determined in seventeen additional patients. The [14C]creatinine/inulin clearance ratio was the larger of the two in all patients with a GFR greater than 15 ml/min. In another group of eight patients with unequal-sized kidneys, who were studied during bilateral ureteral catheterization, the endogenous creatinine/inulin clearance ratios were determined and found not to differ significantly. 4. The three studies suggest that there is significant tubular secretion of creatinine at all levels of renal function. The increasing clearance ratio of endogenous creatinine/GFR as the GFR decreases is not due to increased tubular secretion of creatinine nor a result of a difference in creatinine handling by diseased kidneys, but rather a reflection of the decreasing fraction of non-creatinine chromogen to the total creatinine chromogen. The smaller clearance ratio at a very low GFR would be expected if the maximal tubular secretory rate of creatinine was exceeded.

Endogenous creatinine clearance by rats.

Thirty-one white male albino rats of weights ranging from 200 to 450 g were used to study 24-hour, undisturbed creatinine clearances. Determinations were made using apparent creatinine, true serum and apparent urinary creatinine and true serum and urinary creatinine. The resting creatinine clearance was 0.709 ml/min/100 g body weight, using true serum creatinine and apparent urinary creatinine, and compared closely with the values obtained by Muntwyler and Griffin of 0.73 ml/min/100 g body weight. The urinary non-creatinine chromogen averaged 7.3% of the total chromogen; under some circumstances this value is sufficiently large to be significant. Correlations of creatinine clearance with body weight, kidney weight, and surface area were approximately the same. Therefore, in this weight range it seems most practical to use body weight as the unit for relating clearances.