The use of an immobilized enzyme nylon tube reactor incorporating a four enzyme system for creatinine analysis

A single immobilized enzyme nylon tube reactor was produced incorporating a four enzyme system for the analysis of creatinine. The enzyme activity ratios in the coupling solution used to prepare the reactor were found to be of extreme importance in governing the activity of the latter. The reactor was incorporated into a continuous flow analysis system used to assay creatinine in urine samples and the results were correlated with a manual technique employing the same enzyme system in solution. The precision, correlation, high specificity, simplicity, and speed of the analysis were concluded to be factors in favor of the method's suitability for urine creatinine determinations.     

Qualitative and quantitative analysis of uric acid,creatine and creatinine together with carbohydrates in biological material by HPTLC

Summary In clinical diagnosis creatine, creatinine and uric acid are important parameters for the evaluation of renal diseases, and are partially responsible for gout and the formation of renal calculi. The determination of various carbohydrates, especially glucose, in urine and serum serves as an indicator of diabetes and other carbohydrate anomalies. A simple, rapid and economic method for the simultaneous screening and quantitation of these compounds is presented. No sample preparation is necessary for the determination in urine or in serum. The proposed method consists of separations on an amino modified HPTLC precoated plate. The detection of all relevant substances is reproducibly performed by simply heating the chromatogram to give stable fluorescent derivatives. The detection limits in all cases lie significantly below the physiological range.     

Enzymatic Assay by Flow Injection Analysis with Detection by Chemiluminescence: Determination of Glucose,Creatinine, Free Cholesterol and Lactic Acid Using an Integrated Fia Microconduit

Abstract

A miniaturized flow injection system for the determination of D-glucose, L-lactic acid, creatinine and free cholesterol is described. All substrates are degraded enzymatically by means of oxidases which, along with ancillary coenzymes (creatinine assay), are immobilized on controlled porosity glass and incorporated into small PVC column reactors. The hydrogen peroxide generated by the individual oxidases is determined by chemiluminescence with an alkaline reagent containing luminol and hexacyanofer rate (III). The injection valve, flow channels, enzyme reactor and light detector are integrated into a FIA microconduit. The detection limits were 0.03 mg glucose/dl, 0.03 mg lactate/dl, 0.3 mM creatinine and 0.5 mg cholesterol/dl. The enzyme reactors all showed little change in activity over a 3 months period of operation and were found fully compatible with serum samples.     

高效毛细管区带电泳测定人尿液中尿酸、肌酐和伪尿核苷的含量

报道了采用高效毛细管区带电泳技术直接将人尿液注入毛细管进行尿液中肌酐、尿酸及伪尿核苷含量测定的新方法。试验表明,以磷酸盐（ｐＨ６．１）作缓冲液,对人体尿液中肌酐、尿酸及伪尿核苷进行直接分析具有较高的灵敏度和较好的重复性。     

Towards a definitive assay of creatinine in serum and in urine: separation by high-performance liquid chromatography

A fast and sensitive method for the separation of serum and urinary creatinine is described. For the preliminary purification of serum and urine, a cation-exchange column is used to remove protein, anions and neutral compounds prior to isolation of creatinine by high-performance liquid chromatography. A reversed-phase system with 0.01 M ammonium acetate solution as the mobile phase can separate creatinine in 7.5 min at a flow-rate of 1 ml/min. The purity of the separated creatinine is proved by derivatization using trifluoracetic anhydride, followed by gas chromatography and mass spectrometry. Although this method of purification was designed for incorporation into a definitive assay, the ease and speed of analysis makes is very attractive for routine clinical use.     

Development of a creatinine enzyme-based bar-code-style lateral-flow assay

A lateral-flow, enzyme-based, bar-code assay for creatinine employing the concept of combination of diffusion and kinetics controlled has been developed. Unlike the traditional bar-code version of immunochromatographic assay, which depends on the stepwise capture of colorimetric tracer-labeled antibody–antigen complex by the immobilized antibody on each successive line, the principle of our proposed assay is based on the delay in TMB release and its diffusion in combination with horseradish peroxidase kinetics. Hydrogen peroxide (H₂O₂) produced from enzymatic reactions acts as a limiting factor, which controls the rate of conversion of TMB to blue color complex. The assay takes advantage of giving ladder bar result therefore without the need of any reading device. Depending on the amount of enzymes used, the assay can be one (9 min) or two steps (19 min). The strip assay semiquantitatively measures creatinine concentrations ranging from 0 to 400 μM. Thirty urine samples and thirty serum samples were tested, and the assay showed 90.0% and 86.7% agreement compared with conventional Jaffé method, respectively. This assay provides a tool for quick identification of creatinine for patients without the requirement of any instrument. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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 3σ 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.     

Simultaneous determination of uric acid and creatinine in biological fluids by column-switching liquid chromatography with ultraviolet detection

A column-switching liquid chromatographic method for the simultaneous determination of uric acid and creatinine in human serum and urine was developed. Creatinine and uric acid were separated by size-exclusion chromatography on a hydrophilic gel column (C1) and creatinine eluted from Cl was separated from proteins by filtration through a longer hydrophilic gel column (C2). The creatinine fraction eluted from C2 was transferred to a weakly acidic cation-exchange column (C3) and then to a strongly acidic cation-exchange column (C4). Uric acid eluted from Cl after creatinine was transferred to an anion-exchange column (C5) and then to a hydrophilic gel column (C6). The mobile phase was a mixed buffer of pH 5.1 (propionic acid-succinic acid-NaOH, 60:15:60 mmol/1 in water). Diluted serum and urine could be injected onto C1, and Cl was backflushed after the transfer of uric acid from Cl to C5.

Creatinine and uric acid in the eluate were determined by measuring their ultraviolet absorption at 234 and 290 nm, respectively. The recovery of uric acid and creatinine added to diluted serum (20-fold dilution, concentration 20 and 5 μmol/1, respectively) was 98.9±0.56% and 100.9±1.29%, respectively. The recovery of uric acid and creatinine added to diluted urine (100-fold dilution, concentration 50 and 100 μmol/l, respectively) was 99.4±0.72% and 98.7±1.45%, respectively (mean±R.S.D., n=6).     

大鼠尿液中游离皮质酮昼夜节律的分析研究

为探讨大鼠尿液游离皮质酮昼夜节律性变化,采集24 h内8个时间段大鼠尿液,应用ELISA法检测了尿游离皮质酮含量,生化法检测了尿肌酐含量,以皮质酮与尿肌酐比值反映尿游离皮质酮水平,对各个时间段尿游离皮质酮水平的生物时间序列进行了宏观分析与比较.结果表明,大鼠尿液游离皮质酮与尿肌酐比值呈现昼夜波动性;尿液游离皮质酮的检测...     

A rapid urine creatinine assay by capillary zone electrophoresis.

Using capillary zone electrophoresis, the urine creatinine (uCr) assay was validated in extemporaneous diluted urine, both in healthy subjects and athletes, with the uCr concentration as a reference value to compare excretion rates of other metabolites in the same samples. The electrokinetic sample injection was carried out at 10 kV per 10 s; UV absorbance detection was at 254 nm. Using standard samples, the creatinine migration mean time in 100 mmol/L acetate buffer, pH 4.4, was 3.3+/-0.2 min; the repeatability for absolute migration mean time was 0.6% and peak height repeatability was 2.9%. The correlation coefficient of the standard curve was r = 0.999 and the detection limit was 23.1 micromol/L. Intra- and interassay coefficients of variation (CV) were 3.0 and 3.6%, respectively; recovery was 99+/-3% and linearity was r= 0.98. Normal urine samples were diluted 1:80 in run buffer. The present CE urine creatinine assay showed a good correlation with HPLC and with Jaffe methods (r = 0.98 and r = 0.97, respectively; p < 0.0001). The uCr in the morning urine samples of 34 healthy males (M), 38 healthy females (F), and 83 male athletes (A) was 10.4+/-6.1 mmol/L, 10.8+/-8.1 mmol/L and 13.2+/-6.5 mmol/L, respectively. The uCr difference (p < 0.02) between M and A and a correlation (p < 0.05) with age in A were observed.     

Continuous-flow enzymatic determination of creatinine with improved on-line removal of endogenous ammonia

A new continuous-flow automated enzymatic method suitable for the direct determination of creatinine in physiological samples is described. The proposed system utilizes an on-line gas predialysis unit in conjuction with a flow-through enzyme reactor coil and a potentiometric ammonia detector. The enzyme reactor contains immobilized creatinine iminohydrolase (EC 3.5.4.21) which converts creatinine to ammonia and N-methylhydantoin. Ammonia liberated from this reaction is detected downstream with the membrane electrode-based detector. The novel gas predialysis unit effectively removes >99.8% of endogenous ammonia (up to 1 mM) present in the sample. Thus, final peak potentials recorded by the electrode detector are directly proportional to the logarithm of creatinine concentrations present. The method is shown to be precise (<3%), selective, and capable of accurately determining creatinine in serum and urine samples containing abnormally high endogenous ammonia levels. Determinations of creatinine in serum samples (n = 30) using this new method correlate well with an existing Technicon AutoAnalyzer colorimetric method (r = 0.996).     

Application of multisegment injection on quantification of creatinine and standard addition analysis of urinary 5-hydroxyindoleacetic acid simultaneously with creatinine normalization

In this paper, the development of a simple dilute-and-shoot method for quantifying urinary creatinine by CE–ESI–MS was described. The creatinine analysis time was about 7 min/sample by conventional single injection (SI) method and can be significantly reduced to less than 2 min/sample with multi-segment injection (MSI). In addition, the standard addition analysis of 5-hydroxyindole-3-acetic acid (5-HIAA) and creatinine normalization was performed within one run by the MSI technique, and the total analysis time was 14-min faster compared to the SI method for analyzing the same set of samples. The uses of isotopic and non-isotopic internal standards (ISs) were compared. Creatinine-(methyl-¹³C) and 5-hydroxyindole-4,6,7-D₃-3-acetic-D₂ acid (5-HIAA-D₅) used as isotopic ISs can provide both accurate and precise results. In contrast, 1,5,5-trimethylhydantoin (1,5,5-TH) used as the non-isotopic IS for creatinine may cause a bias of over 13% in SI method and even worse when the MSI technique was used. Another compound, 2-methyl-3-indoleacetic acid (2-MIAA), was determined not suitable for MSI analysis of 5-HIAA due to endogenous interferences despite its acceptable performance in conventional methods of analysis.     

The micro water/1,2-dichloroethane interface as a transducer for creatinine assay

Facilitated ammonium ion transfer reactions, using the crown ether dibenzo-18-crown-6, at a single microhole supported water/1,2-dichloroethane interface were used as the transducer to detect the biomolecule creatinine. The ammonium ion was produced during the enzymatic action of creatinine deiminase on creatinine. A prototype sensing device incorporating a Teflon gas permeable membrane, to exclude possible interferents, yielded a linear amperometric response to added creatinine from 20 M to 1 mM. This range is sufficient for that required for the determination of creatinine in serum.     

气相色谱-质谱联用法同时测定乳及乳制品中的三聚氰胺及肌酐

建立了同时测定乳制品中三聚氰胺及肌酐的气相色谱-质谱联用方法。样品经1%三氯乙酸溶液萃取,混合型阳离子交换固相萃取净化,提取液用氮气吹干后加入N,O-双(三甲基硅基)三氟乙酰胺-三甲基氯硅烷(BSTFA-TMCS)硅烷化试剂,于75 ℃下衍生60 min,最后采用选择离子模式下的气相色谱-质谱测定。三聚氰胺和肌酐的定量限分别为0.10 mg/kg和0.20 mg/kg;在0.1～50 mg/L范围内的线性相关系数均大于0.99。实际样品中,肌酐在10～100 mg/kg和三聚氰胺在0.1～5.0 mg/kg添加范围内的回收率分别为80.7%～116.8%和77.6%～107.5%,相对标准偏差分别小于9.4%和8.5%。该方法能有效除去干扰,灵敏度高,回收率较好,可用于乳制品中三聚氰胺和肌酐的同时测定。     

Determination of uric acid and creatinine in human urine using hydrophilic interaction chromatography

Uric acid is the end-product of purine metabolism and a major antioxidant in humans. The concentrations of uric acid in plasma and urine are associated with various diseases and routinely measured in clinical and biomedical laboratories using enzymatic conversion and colorimetric measurement. In this study a hydrophilic interaction chromatographic (HILIC) method was developed for simultaneous determination of uric acid and creatinine, a biomarker of urine dilution and renal function, in human urine. Urine samples were pretreated by dilution, protein precipitation, centrifugation and filtration. Uric acid and creatinine were separated from other components in urine samples and quantified using HILIC chromatography. A linear relationship between the ratio of the peak area of the standards to that of the internal standard and the concentration of the standards was obtained for both uric acid and creatinine with the square of correlation coefficients >0.999 for both analytes. The detection limits were 0.04 μg/mL for creatinine and 0.06 μg/mL for uric acid. The described HILIC method has proved to be simple, accurate, robust and reliable.     

Capillary electrophoresis and ultra-high-performance liquid chromatography methods in clinical monitoring of creatinine in human urine: A comparative study

Creatinine is an important diagnostic marker and is also used as a standardization tool for the quantitative evaluation of exogenous/endogenous substances in urine. This study aimed at evaluating and comparing three analytical approaches, based on hyphenations of different separation [two-dimensional capillary isotachophoresis (CITP–CITP), capillary zone electrophoresis (CZE), ultra-high-performance liquid chromatography (UHPLC)] and detection [conductivity (CD), ultraviolet (UV), tandem mass spectrometry (MS/MS)] techniques, for their ability to provide reliable clinical data along with their suitability for the routine clinical use (cost, simplicity, sample throughput). The developed UHPLC–MS/MS, CITP–CITP–CD, and CZE–UV methods were characterized by favorable performance parameters, such as linearity (r ˃ 0.99), precision (relative standard deviation, 0.22–2.97% for the creatinine position in analytical profiles), and recovery (87.1–115.1%). Clinical data, obtained from the analysis of 24 human urine samples by a reference enzymatic method, were comparable with those obtained by the tested methods (Passing–Bablok regression and Bland–Altman analysis), approving their usefulness for the routine clinical use. In this context, the UHPLC–MS/MS method provides benefits of enhanced orthogonality/accuracy and high sample throughput (threefold shorter total analysis times than the CE methods), whereas advantages of the CE methods for routine labs are simplicity and low cost of both the instrumentation and measurements.     

毛细管电泳多次进样法快速测定尿肌酐

采用毛细管电泳多次进样技术快速测定了尿肌酐。采用３６ｃｍ×５０μｍｉ．ｄ涂层柱,０．１ｍｍｏｌ／ＬｐＨ２．５的磷酸缓冲液,在２００ｎｍ处检测。连续进样５个样品总分析时间为１２ｍｉｎ,比单次进样节省２０ｍｉｎ左右。日内、日间变异系数均小于７．０％,用吡啶作内标,在肌酐浓度为５～８０ｍｇ／Ｌ范围内,肌酐浓度与肌酐同吡啶的峰高比值的线性关系很好（ｒ＝０．９９９６）。测定１２个样品同生化分析仪测定的结果相关性好（ｒ＝０．９７７３）,且分析时间节省了约３０ｍｉｎ。     

Amperometric biosensor based on reductive H2O2 detection using pentacyanoferrate-bound polymer for creatinine determination

Pentacyanoferrate-bound poly(1-vinylimidazole) (PVI[Fe(CN)₅]) was selected as a mediator for amperometric creatinine determination based on the reductive H₂O₂ detection. Creatinine amidohydrolase (CNH), creatine amidohydrolase (CRH), sarcosine oxidase (SOD), peroxidase (POD), and PVI[Fe(CN)₅] were crosslinked with poly(ethylene glycol) diglycidyl ether (PEGDGE) on a glassy carbon (GC) electrode for a creatinine biosensor fabrication. Reduction current was monitored at −0.1 V in the presence of creatinine and O₂. It is revealed that PVI[Fe(CN)₅] is suitable as a mediator for a bioelectrocatalytic reaction of POD, since PVI[Fe(CN)₅] neither reacts with reactants nor works as an electron acceptor of SOD. The amounts of PVI[Fe(CN)₅], PEGDGE, and enzymes were optimized toward creatinine detection. Nafion as a protecting film successfully prevented the enzyme layer from interferences. The detection limit and linear range in creatinine determination were 12 μM and 12–500 μM (R² = 0.993), respectively, and the sensitivity was 11 mA cm⁻² M⁻¹, which is applicable for urine creatinine tests. The results of the creatinine determination for four urine samples measured with this proposed method were compared with Jaffe method, and a good correlation was obtained between the results.     

Simultaneous quantification of urinary purines and creatinine by ultra high performance liquid chromatography with ultraviolet spectroscopy and quadrupole time‐of‐flight mass spectrometry: Method development,validation, and application to gout study

We present an ultra high performance liquid chromatography with ultraviolet spectroscopy and quadrupole time‐of‐flight mass spectrometry method for the simultaneous quantification of ten purines (adenine, hypoxanthine, guanine, xanthine, deoxyadenosine, adenosine, inosine, guanosine, xanthosine, and uric acid) and creatinine in human urine. After chromatographic separation on an ACE Excel 2 AQ column, high abundant creatinine and uric acid and the other low abundant purines were sequentially detected by ultraviolet and quadrupole time‐of‐flight mass spectrometry within a single run. Method validations including specificity (improved by accurate mass measurement), linearity (correlation coefficients ≥0.9944), limit of quantification (0.002–9.756 µg/mL), intra‐ and interday precision (relative standard deviations ≤9.1 and 14.0%, respectively), accuracy (relative errors ≤13.1%), extraction recovery (between 90.3 and 109.6%), matrix effect (between 85.3 and 110.5%), and stability (relative errors ≤14.3%) were fully evaluated. This approach was applied to characterize the disordered purine metabolism in acute and chronic gout as an example. Quantitative results (normalized by creatinine) showed that an overproduction of urinary purine precursors might be involved in the gout process. The developed method represents a useful tool to investigate the purine disturbances in gout and other relevant diseases.     

Assay for the simultaneous determination of guanidinoacetic acid, creatinine and creatine in plasma and urine by capillary electrophoresis UV-detection

Guanidinoacetic acid (GAA) measurement has recently become of great interest for the diagnosis of creatine (Cn) metabolism disorders, and research calls for rapid and inexpensive methods for its detection in plasma and urine in order to assess a large number of patients. We propose a new assay for the measurement of GAA by a simple CZE UV-detection without previous sample derivatization. Plasma samples were filtered by Microcon-10 microconcentrators and directly injected into the capillary, while for urine specimens a simple water dilution before injection was needed. A baseline separation was obtained in less than 8 min using a 60.2 cm x 75 microm uncoated silica capillary, 75 mmol/L Tris-phosphate buffer pH 2.25 at 15 degrees C. The performance of the developed method was assessed by measuring plasma creatinine and Cn in 32 normal subjects and comparing the data obtained by the new method with those found with the previous CE assay. Our new method seems to be an inexpensive, fast and specific tool to assess a large number of patients both in clinical and in research laboratories.