A Risk Assessment of the Jaffe vs Enzymatic Method for Creatinine Measurement in an Outpatient Population

Background

The Jaffe and enzymatic methods are the two most common methods for measuring serum creatinine. The Jaffe method is less expensive than the enzymatic method but is also more susceptible to interferences. Interferences can lead to misdiagnosis but interferences may vary by patient population. The overall risk associated with the Jaffe method depends on the probability of misclassification and the consequences of misclassification. This study assessed the risk associated with the Jaffe method in an outpatient population. We analyzed the discordance rate in the estimated glomerular filtration rate based on serum creatinine measurements obtained by the Jaffe and enzymatic method.

Methods

Method comparison and risk analysis. Five hundred twenty-nine eGFRs obtained by the Jaffe and enzymatic method were compared at four clinical decision limits. We determined the probability of discordance and the consequence of misclassification at each decision limit to evaluate the overall risk.

Results

We obtained 529 paired observations. Of these, 29 (5.5%) were discordant with respect to one of the decision limits (i.e. 15, 30, 45 or 60 ml/min/1.73m²). The magnitude of the differences (Jaffe result minus enzymatic result) were significant relative to analytical variation in 21 of the 29 (72%) of the discordant results. The magnitude of the differences were not significant relative to biological variation. The risk associated with misclassification was greatest at the 60 ml/min/1.73m² decision limit because the probability of misclassification and the potential for adverse outcomes were greatest at that decision limit.

Conclusion

The Jaffe method is subject to bias due to interfering substances (loss of analytical specificity). The risk of misclassification is greatest at the 60 ml/min/1.73m² decision limit; however, the risk of misclassification due to bias is much less than the risk of misclassification due to biological variation. The Jaffe method may pose low risk in selected populations if eGFR results near the 60 ml/min/1.73m² decision limit are interpreted with caution.     

A Novel Amplified Enzymatic Assay Method for Hypoxanthine

A sensitive and rapid enzymatic assay for hypoxanthine, using a Clark oxygen electrode as sensor, is proposed. In the presence of sodium sulfite, oxidation of hypoxanthine by milk xanthine oxidase caused very rapid oxygen consumption in excess of the stoichiometric requirement for hypoxanthine oxidation. Hypoxanthine from 0.5 to 10 μM can be assayed within a few minutes by addition of 25 mM sodium sulfite to the reaction mixture. This assay proved to be over 10-times more sensitive and much more rapid than the control method without sulfite.     

A Novel Amplified Enzymatic Assay Method for Hypoxanthine

A sensitive and rapid enzymatic assay for hypoxanthine, using a Clark oxygen electrode as sensor, is proposed. In the presence of sodium sulfite, oxidation of hypoxanthine by milk xanthine oxidase caused very rapid oxygen consumption in excess of the stoichiometric requirement for hypoxanthine oxidation. Hypoxanthine from 0.5 to 10 μM can be assayed within a few minutes by addition of 25 mM sodium sulfite to the reaction mixture. This assay proved to be over 10-times more sensitive and much more rapid than the control method without sulfite.     

A New Enzymatic Reaction: Enzyme Catalyzed Ammonolysis of Carboxylic Esters

A new enzymatic reaction of carboxylic esters and ammonia (ammonolysis) was studied. This reaction provides a synthetically useful and mild alternative for the synthesis of amides. Several lipases and one esterase acted as catalyst. Ammonolysis of esters of chiral carboxylic acids gave higher ee values than hydrolysis under comparable reaction conditions. Furthermore, consecutive enzymatic esterification and ammonolysis provided a convenient one-pot synthesis of carboxylic amides from carboxylic acids.     

Inhibition of Fatty Acid Amidohydrolase,the Enzyme Responsible for the Metabolism of the Endocannabinoid Anandamide,by Analogues of Arachidonoyl-serotonin

Arachidonoyl-serotonin inhibits in a mixed-type manner the metabolism of the endocannabinoid anandamide by the enzyme fatty acid amidohydrolase. In the present study, compounds related to arachidonoyl-serotonin have been synthesised and investigated for their ability to inhibit anandamide hydrolysis by this enzyme in rat brain homogenates. Removal of the 5-hydroxy from the serotonin head group of arachidonoyl-serotonin produced a compound (N-arachidonoyltryptamine) that was a 2.3-fold weaker inhibitor of anandamide hydrolysis, but which also produced its inhibition by a mixed-type manner (K_i(slope) 1.3 µM; K_i(intercept) 44 µM). Replacement of the amide linkage in this compound by an ester group further reduced the potency. In contrast, replacement of the arachidonoyl side chain by a linolenoyl side chain did not affect the observed potency. N-(Fur-3-ylmethyl) arachidonamide (UCM707), N-(fur-3-ylmethyl)linolenamide and N-(fur-3-ylmethyl)oleamide inhibited anandamide hydrolysis with pI50 values of 4.53, 5.36 and 5.25, respectively. The linolenamide derivative was also found to be a mixed-type inhibitor. It is concluded that the 5-hydroxy group of arachidonoyl-serotonin contributes to, but is not essential for, inhibitory potency at fatty acid amidohydrolase.     

Gibbs-Ensemble Molecular Dynamics: A New Method for Simulations Involving Particle Exchange

We discuss a novel simulation method suitable for simulating phenomena involving particle exchange. The method is a molecular dynamics version of the Gibbs-Ensemble Monte Carlo technique, which has been developed some years ago for the direct simulation of phase equilibria in fluid systems. The idea is to have two separate simulation boxes, which can exchange particles or molecules in a thermodynamically consistent fashion. We discuss the general idea of the Gibbs-Ensemble Molecular Dynamics technique and present examples for different simple atomic and molecular fluids. Specifically we will discuss Gibbs-Ensemble Molecular Dynamics simulations of gas-liquid and liquid-solid equilibria in Lennard-Jones systems and in hexane as well as an application of the method to adsorption.     

A Similarity-Based Method for Predicting Enzymatic Functions in Yeast Uncovers a New AMP Hydrolase

Despite decades of research and the availability of the full genomic sequence of the baker’s yeast Saccharomyces cerevisiae, still a large fraction of its genome is not functionally annotated. This hinders our ability to fully understand cellular activity and suggests that many additional processes await discovery. The recent years have shown an explosion of high-quality genomic and structural data from multiple organisms, ranging from bacteria to mammals. New computational methods now allow us to integrate these data and extract meaningful insights into the functional identity of uncharacterized proteins in yeast. Here, we created a database of sensitive sequence similarity predictions for all yeast proteins. We use this information to identify candidate enzymes for known biochemical reactions whose enzymes are unidentified, and show how this provides a powerful basis for experimental validation. Using one pathway as a test case we pair a new function for the previously uncharacterized enzyme Yhr202w, as an extra-cellular AMP hydrolase in the NAD degradation pathway. Yhr202w, which we now term Smn1 for Scavenger MonoNucleotidase 1, is a highly conserved protein that is similar to the human protein E5NT/CD73, which is associated with multiple cancers. Hence, our new methodology provides a paradigm, that can be adopted to other organisms, for uncovering new enzymatic functions of uncharacterized proteins.     

Endo-Xylogalacturonan Hydrolase, a Novel Pectinolytic Enzyme

We screened an Aspergillus tubingensis expression library constructed in the yeast Kluyveromyces lactis for xylogalacturonan-hydrolyzing activity in microwell plates by using a bicinchoninic acid assay. This assay detects reducing carbohydrate groups when they are released from a carbohydrate by enzymatic activity. Two K. lactis recombinants exhibiting xylogalacturonan-hydrolyzing activity were found among the 3,400 colonies tested. The cDNA insert of these recombinants encoded a 406-amino-acid protein, designated XghA, which was encoded by a single-copy gene, xghA. A multiple-sequence alignment revealed that XghA was similar to both polygalacturonases (PGs) and rhamnogalacturonases. A detailed examination of conserved regions in the sequences of these enzymes revealed that XghA resembled PGs more. High-performance liquid chromatography and matrix-assisted laser desorption ionization–time of flight mass spectrometry of the products of degradation of xylogalacturonan and saponified modified hairy regions of apple pectin by XghA demonstrated that this enzyme uses an endo type of mechanism. XghA activity appeared to be specific for a xylose-substituted galacturonic acid backbone.     

一株新的豆豉纤溶酶产生菌的研究

从全国各地采集豆豉样品,经富集培养并利用纤维蛋白平板法获得一株形态与现存产纤溶酶微生物差异较大的菌株HS9.通过传统方法、化学方法以及16S rRNA序列分析对HS9进行分类鉴定,属于Pseudomonas aeruqinosa,是未见报道的产豆豉纤溶酶菌株.发酵培养HS9获得粗酶,经20%～70%硫酸铵梯度盐析、Sephadex G-75凝胶过滤以及CM-Sepharose Fast Flow阳离子交换层析分离纯化后,得到了电泳纯酶.通过SDS-PAGE了解该酶分子量约为34 kD,pH 8.0～8.5时酶活性最高,最适作用温度48℃,作用方式为直接水解纤维蛋白,胃蛋白酶抑制剂在工作浓度1μmol/L时能完全抑制其活性,推测该酶为天冬氨酸蛋白酶,是一种新型的豆豉纤溶酶.     

用质粒作底物测定核糖体失活蛋白酶活性的新方法

植物核糖体失活蛋白(ribosome-inactivating protein,RIP)是一类能作用于核糖体最大RNA的独特蛋白质.它是研究蛋白质生物合成中核糖体RNA结构与功能的有力工具.利用RIP能在DNA中脱去一些腺嘌呤碱基使超螺旋DNA解旋的特点,分别以常用的质粒PUC18、PUC19和PBR322 DNA为底物,建立了测定RIP酶活性的一种新方法,其灵敏度是50ng(天花粉蛋白)和5ng(还原型的辛纳毒蛋白),酶催化反应的时间是60min.这个新方法具有方便、快捷、灵敏的特点,避免了常用方法中制备核糖体、提取RNA的仪器和技术条件的限制,检测的时间由原来的几天缩短到约120min,大大地降低了检测的费用,为广泛和深入地研究RIP提供了有利的条件.     

A New Enzyme,d-Fructose Dehydrogenase

Genetic relatedness of 14 yeast strains and 2 mold strains was studied by the DNA-DNA hybridization method. The hybridization was performed between mitochondrial-DNA-free, ³²p-labeled DNA of Saccharomyces cerevisiae IAM 4009 and cold DNA of other strains. The DNA homology indices deviated considerably even among S. cerevisiae strains having similar GC contents, but, in general, yeast strains known to be able to mate with S. cerevisiae, showed high homology indices (35∽70%). Other species of Saccharomycetaceae and 6 asporogenous yeast strains exhibited values of 10∽20%. The relatedness suggested from these results was confirmed by the competition experiments and also by the hybridization with ³²P-DNA of Candida pulcherrima IFO 0561. DNA’s of Aspergillus oryzae I and Neurospora crassa IFO 6067 also exhibited low but appreciable homology indices (5∽7%). These results were discussed from the aspects of phylogenetics and also of gene conservation in microorganisms.     

Rat Brain Free Glucose and Lactate Measurement by a Novel Method Using Bisecting Decapitation-Extrusion and Enzyme Denaturation at Five Seconds

This study investigates a novel method as a means for animal decapitation with rapid brain removal and enzyme denaturation. Briefly, the rat head is simultaneously decapitated and bisected. Either half of the in situ brain is aspirated under -250 mm Hg pressure into a modified small plastic syringe and then extruded through a needle as a fine strand into a relatively large volume of 2 M urea at 95 degrees C. After cooling, sonication, and centrifugation of the brain homogenate, the supernatant is measured enzymatically for brain free glucose and lactate concentration. Enzyme denaturation is effected within 4-6 s. The results are in good agreement with published values for glucose and lactate using other rapid enzyme inactivation techniques.     

限制酶介导整合技术：一种克隆新基因的新策略

限制酶介导整合（REMI）技术是近年发展起来的一种研究新基因的方法,已被用于盘基网柄菌、酵母和丝状真菌等微生物细胞与发育过程的研究。结合质粒拯救技术,可快速分离质粒两侧未知的基因组DNA序列,进而对该序列做进一步分析和研究。我们简要介绍了REMI技术的基本原理、影响因素、缺点及其应用。     

A Novel Catechol Oxidase Enzyme Electrode for the Specific Determination of Catechol

An enzyme electrode for the specific determination of catechol was developed by using catechol oxidase (EC 1.10.3.1) from eggplant (Solanum melangena L.) in combination with a dissolved oxygen probe. Optimization studies of the prepared catechol oxidase enzyme electrode established a phosphate buffer 50 mM at pH 7.0 and 35°C to provide the optimum conditions for affirmative electrode response. The enzyme electrode response depended linearly on a catechol concentration range of 5?10^-7-30?10^-5 M with a response time of 25 sec and substrate specificity of the catechol oxidase electrode of 100%. The biosensor retained its enzyme activity for at least 70 days.     

Novel Microbial Inhibitors of Angiotensin-converting Enzyme,Aspergillomarasmines A and B

Brush border membrane vesicles (BBMV) from the midgut epithelial cells of silkworm larvae were prepared. ATP hydrolyzing activity (ATPase activity) was associated with the BBMV. ATPase activity without Mg^{2 +} was not observed at pH 7 but substantial ATP hydrolyzing activity was observed at pH 7 with Mg^{2 +}. The enzyme required Mn^{2 +}, Mg^{2 +}, or Ca²⁺ ions. The enzyme also hydrolyzed ITP and GTP but not p-NPP, ADP, or AMP. KNO₃ and NEM strongly inhibited the ATPase activity. Behaviours of the ATPase against inhibitors suggested that it resembled vacuolar type ATPase.     

Acetyl Coenzyme A: Deacetylvindoline O-Acetyltransferase,A Novel Enzyme from Catharanthus

A new enzyme, Acetyl Coenzyme A: deacetylvindoline 0-acetyl transferase (EC 2.3.1. -) which catalyses the synthesis of vindoline from acetyl coenzyme A and deacetylvindoline was isolated from the soluble protein extract of Catharanthus roseus leaves and purified approximately 365-fold. The enzyme had an apparent pI of 4.6 upon chromatofocusing, an apparent molecular weight of 45,000 daltons and a pH optimum between 8.0 to 9.0. Dithiothreitol was essential to maintain enzyme activity.Substrate saturation studies of this enzyme resulted in Michaelis Menton kinetics giving Km values of 5.4 and 0.7µM respectively for acetyl coenzyme A and deacetylvindoline. Studies of the forward reaction demonstrated an absolute requirement for acetyl coenzyme A and deacetylvindoline derivatives containing a double bond at positions 6, 7, whereas the reverse reaction occurred only in the presence of free coenzyme A and vindoline derivatives containing the same double bond. The forward reaction was subject to product inhibition by coenzyme A with an apparent Ki of 8 µM, but was not inhibited by up to 2 mM vindoline. The rate of reaction could therefore be regulated by the level of free coenzyme A in the cell, unaffected by the accumulation of indole alkaloid product.It was suggested that this enzyme catalyses a late step in the biosynthesis of vindoline.     

鞘氨醇-1-磷酸的定量测定——一种新的竞争性结合方法

鞘氨醇-1-磷酸（SPP）是重要的细胞第二信使,影响细胞的生长和死亡.通过培养和收集转染SPP受体-EDG-1的HEK293细胞,与标记及非标记SPP共孵育,利用它们与HEK293细胞的竞争性结合,测定细胞、血清和组织中SPP含量.该法无需特殊仪器,可以测到皮摩尔水平的低含量,批间差异小于15%（6次）.