A fluorometric kinetic method for creatine in urine

A fluorometric kinetic method is described for the analysis of creatine in urine. The method is based on the specific enzymatic reaction of creatine kinase on creatine and two coupled indicator reactions. Fluorogenic compounds in urine are removed by activated carbon. The rate of decrease of NADH fluorescence at 460 nm is measured and equated to creatine content. The method is more sensitive than the comparable spectrophotometric procedure.     

常见肌酐测定方法中存在的干扰

目的 探讨目前常见肌酐测定方法中存在干扰。方法 以漂白土（fuller's earth)吸附法为标准，对Jaffe'氏反应速率法、紫外法、比色 和电极法进行方法学评价。结果 在所采用的8种干扰物（丙酮酸钠、α －酮戊二酸、胆红素、血红蛋白、肌酸、多巴胺、抗坏酸、地塞米松）中，Jaffe'氏反应速率法受干扰物影响最大，α－酮戊二酸（≥0.82mmol/L)、丙酮酸钠（≥0.186mmol/L)对该法有正干扰，而胆红素（≥165.5umol/L）则有明显负干扰。紫外法基本不受干扰物影响；比色法不仅受肌酸（≥0.21umol/L）的正干扰，而且也受多巴胺（≥0.28mmol/L)的负干扰；电极法仅受到肌酸（≥0.62 mmol/L）的正干扰。结论 紫外法是目前测定肌酐最好的方法。     

Erythrocyte creatine as a marker of excessive erythrocyte destruction due to hypersplenism in patients with liver cirrhosis

OBJECTIVE: Erythrocyte creatine is a sensitive marker of erythrocyte age, and can be used to detect slight and continuous hemolysis. Excessive blood cell destruction caused by increased spleen function is important evidence of hypersplenism. This study evaluates the usefulness of erythrocyte creatine as a sensitive marker of excessive erythrocyte destruction due to hypersplenism in patients with liver cirrhosis. DESIGN AND METHOD: Erythrocyte creatine was determined by an enzymatic method in 50 patients with postnecrotic liver cirrhosis and 50 healthy controls. The spleen size was measured by ultrasonography and expressed as a spleen index. RESULTS: The patients with splenomegaly showed significantly higher erythrocyte creatine than those without splenomegaly (p < 0.005) and healthy controls (p < 0.001), but there was no significant difference in erythrocyte creatine between healthy controls and those without splenomegaly. Fourteen (93%) of the 15 patients with abnormally high erythrocyte creatine (> 1.8 micromol/g hemoglobin) had splenomegaly. There were no significant differences in reticulocyte count between healthy controls and the patients with and without splenomegaly. Erythrocyte creatine showed good correlations with spleen index (r = 0.67; p < 0.001) and reticulocytes (r = 0.63; p < 0.001). CONCLUSIONS: Erythrocyte creatine can be used for predicting erythropoietic status and estimating hypersplenism in patients with liver cirrhosis.     

Enzymatic erythrocyte creatine determinations as an index for cell age

Creatine concentration in red blood cells was determined after ammoniumsulfate precipitation on a clear hemoglobin-free filtrate with a new enzymatic assay making use of bacterial creatinase. The method described is more specific than Griffiths' method and can easily be mechanised and adapted for use in a routine laboratory using classical automated equipment. By contrast with Griffiths' method no significant interferences of amino acids and creatine-like molecules were found. Reference values for this method were 0.379 +/- 0.076 mmol/l. In patients with high turnover of erythrocytes, e.g. haemodialysis patients (0.529 +/- 0.122 mmol/l), and renal insufficiency patients (0.565 +/- 0.145 mmol/l), significantly increased creatine concentration in erythrocytes were observed. Low erythrocyte creatine concentrations were found in chronic ambulatory dialysis patients (0.311 +/- 0.042 mmol/l).     

Electrophoretic identification of the brain isoenzyme of creatine kinase following treatment with anti-BB antisera

A procedure is described for accurately determining the presence of true creatine kinase isoenzyme BB by fluorescent staining following electrophoresis on either cellulose acetate or agarose. Treatment of sera with anti-BB inhibiting antibody prior to electrophoresis and subsequent enzymatic staining allows clear identification of creatine kinase BB in presence of interfering artifacts. The utilization of an immunological means of identifying BB following electrophoresis avoids the pitfall of associating an artifactual BB isoenzyme with a disease state. Using this technique, presence of creatine kinase BB in patients with carcinoma, cardiac disease and renal failure has been verified.     

Modifications of plasma enzyme activities after severe head injury; evaluation of prognosis using multivariate methods

The aim of this study was to analyse the plasma variations of four enzymatic activities (lactate dehydrogenase, aldolase, creatine kinase, aspartate aminotransferase) in 134 patients suffering from severe head injury. Enzymatic activities were assayed daily for 3 days after the trauma. Means of the four enzymatic activities were significantly different according to their evolution (death or survival), except for creatine kinase, 48 h after the trauma. Multivariate analysis indicated that lactate dehydrogenase and aldolase levels were useful in order to discriminate between potential survivors and non-survivors. The value of multivariate analysis in head traumatology is discussed.     

Interferences in current methods for measurements of creatinine

We measured the interference of carbonyl compounds, drugs, and other substances in human serum on the determination of creatinine by the two-point, fixed-time kinetic modification of the Jaffé reaction as well as by four enzymatic methods. We added known concentrations of the interfering substances to a solution of creatinine in water. For bilirubin, we used both pooled normal sera with added bilirubin and icteric patient sera. The magnitude of interferences varies widely from method to method. Carbonyl compounds, dopamine, cephalosporines, and bilirubin interfere with the Jaffé reaction. Bilirubin, creatine, dopamine, ascorbic acid, and sarcosine interfere with the enzymatic methods. We conclude that the elimination of interferences in the determination of creatinine has still not been achieved.     

Radioimmunoassay of creatine kinase isoenzymes in human serum: isoenzyme MM

Measurement of the mass concentration of serum enzymes by radioimmunoassay provides direct quantitation of specific isoenzymes and may be less subject to some of the limitations of traditional assay procedures for enzymes. We describe the development of a sensitive and specific radioimmunoassay for the muscle isoenzyme of creatine kinase, CM-MM, in human serum. CK-MM, purified from human skeletal muscle, was used to raise high-titer antisera and for iodination by the Chloramine T method. The radioimmunoassay required 50 microliter of sample, utilized a double-antibody separation method, and was completed in 24 h. Cross reactivity with CK-BB was virtually zero, 3--17% with CK-MB. The mass concentration of CK-MM in the serum of healthy subjects ranged from 36 to 1668 microgram/liter and correlated closely with total CK enzymatic activity. Serum concentrations of CK-MM from casually selected patients correlated less well with total CK enzymatic activity, suggesting the existence of other CK isoenzymes or the presence of inactive forms.     

IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C.

This paper is the eighth in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C and the certification of reference preparations. Other parts deal with: Part 1. The concept of reference procedures for the measurement of catalytic activity concentrations of enzymes; Part 2. Reference procedure for the measurement of catalytic concentration of creatine kinase; Part 3. Reference procedure for the measurement of catalytic concentration of lactate dehydrogenase; Part 4. Reference procedure for the measurement of catalytic concentration of alanine aminotransferase Part 5. Reference procedure for the measurement of catalytic concentration of aspartate aminotransferase Part 6. Reference procedure for the measurement of catalytic concentration of gamma-glutamyltransferase; Part 7. Certification of four reference materials for the determination of enzymatic activity of gamma-glutamyltransferase, lactate dehydrogenase, alanine aminotransferase and creatine kinase at 37 degrees C. The procedure described here is deduced from the previously described 30 degrees C IFCC reference method. Differences are tabulated and commented on.     

血液贮存时间与红细胞内肌酸含量的关系

目的应用红细胞内肌酸的酶测定方法，观察人红细胞内肌酸在血液贮存期间的变化量及其与无偿献血次数的关系。方法分组测定不同无偿献血次数者及库存不同时间血液的红细胞内肌酸的含量。结果血液库存期间，红细胞内肌酸的变化范围为：0％～72．5％（4℃库存0～42d）、0％～51.8％（室温储存0～28d）。不同无偿献血次数的献血者红细胞内肌酸测定结果经方差分析，各组之间差异无显著性（P〉0．05）。结论红细胞内肌酸随血液库存时间延长红细胞衰老而降低，降低幅度与红细胞衰老程度呈正比，通过红细胞内肌酸降低的程度，可估计红细胞在库存期间的衰老变化程度；不同无偿献血次数的献血者红细胞内肌酸含量无明显差异，科学合理献血对献血者红细胞无影响。     

A new immunoassay for the measurement of myoglobin in serum

Because the concentration of serum myoglobin (Mb) increases within 2 to 4 hours after the first sign of acute myocardial infarction, it has been proposed as an early marker of the condition. Our aim was to evaluate a new assay that provides a rapid, quantitative determination of Mb (Baxter Stratus Myoglobin) based on the radial partition technique. We compared the results obtained by this technique with those from nephelometric and radioimmunoassay methods. A significant agreement was observed, the correlation coefficients (r) being 0.999 and 0.996, respectively. The method evaluated provided good reproducibility with CVs between 3.14% and 4.87%, and its linearity and analytical sensitivity were satisfactory. The clinical evaluation of this assay demonstrates that Mb increases in serum of patients with acute myocardial infarction before total creatine kinase and creatine kinase MB isoenzyme. Mb concentration shows an early peak and earlier return to normal values after the necrosis compared to enzymatic activities. Moreover the assay is rapid and fully automated. The method is therefore considered appropriate for contributing to the early diagnosis of AMI in clinical laboratories. © 1994 Wiley-Liss, Inc.     

血清肌酐二步酶法检测新技术研究

目的探讨二步酶法测定肌酐的实验方法。方法以肌酐酰氨基水解酶为试剂Ⅱ,以N-(2-羟基-3-磺丙基)-3,5-二甲氧基苯胺钠盐(HDAOS)和4-氨基安替比林(4-AAP)色原物作为试剂Ⅰ,建立二步酶法测定血清肌酐。通过内空白法消除内源性肌酸和改变光谱吸收方法消除脂血、溶血、黄疸干扰。采用二步酶法测定36例患者血清肌酐,并与高效液相色谱(HPLC)法和一步酶法比较;同时测定200名健康体检者血清肌酐,以建立参考范围。结果患者组肌酐二步酶法结果[(84.2±26.6)μmol/L]明显低于一步酶法[(116.6±29.6)μmol/L,t=32.12,P<0.01];与HPLC法测定结果[(83.9±26.8)μmol/L]差异无统计学意义(t=0.541 6,P>0.05),且呈良好相关性(Y二步酶法=1.042XHPLC法-0.182 1,r2=0.982 4)。二步酶法测定血清肌酐的线性范围达4 500μmol/L,平均回收率为100.8%,批内和批间变异系数(CV)分别为2.91%～4.20%和3.20%～4.60%,应用二步酶法每天测定室内质控定值血清[低(78.0μmol/L)、中(206.0μmol/L)、高(900.0μmol/L)],共测定180 d,其日间总CV分别为4.46%、5.27%、7.24%。二步酶法试剂至少能稳定180 d。健康人群血清肌酐的参考范围男性为56～132μmol/L,女性为41～109μmol/L。结论以肌酐酰氨基水解酶为试剂Ⅱ和以HDAOS、4-AAP为色原物(试剂Ⅰ)的二步酶法可以消除肌酸和脂血、溶血、黄疸血的干扰,可用于临床肌酐常规测定。     

An investigation of factors influencing the measurement of creatine phosphokinase activity in serum using coupled enzymatic methods.

1. The factors influencing the measurement of creatine phosphokinase (CPK) activity in serum by coupled enzymatic methods were investigated to establish optimum conditions for this type of assay. Such a study was indicated following observations by the authors of poor performance of commerically produced reagent kits together with the failure of most of the established an well accepted methods to operate under true optimum zero order kinetics in the reaction phase state. 2. The factors invested were the effects of pH, substrate concentrations (creatine phosphate, glucose and NADP+), added auxiliary (hexokinase) and indicator (glucose-6-phosphate dehydrogenase) enzymes, dithiothreitol (DTT) as an activator and conditions of storage of substrate stability. DTT was found to be a suitable activator but not a reactivator of the reaction. The optimum concentrations of creatine phosphate, glucose and NADP+ were found to be 20.0, 20.0 and 2.0 mmol/litre, respectively. Optimum activieies of the enzymes, glucose-6-phosphate dehydrosenase and hexokinase were 1000 and 2000 units/litre, respectively. 3. The between-day precision of the method for measuring serum at pH 6.8 and 30 degrees C at three activity levels under the optimum conditions developed was excellent yielding coefficients of variation ranging from 2.0 to 2.7%.     

A new enzymatic serum creatinine measurement based on an endogenous creatine-eliminating system

We describe a new colorimetric determination of serum creatinine which does not require a blank to correct for endogenous creatine. In the first reaction, creatinase (creatinase amidinohydrolase EC 3.5.3.3) and sarcosine oxidase (sarcosine: oxygen oxidoreductase (demethylating) EC 1.5.3.1) were used in the enzymatic hydrolysis of endogenous creatine to produce hydrogen peroxide. In the presence of horseradish peroxidase, 2,4-dichlorophenolsulfonate (2,4-DCPS) was converted to a colorless polymer by hydrogen peroxide. In the second reaction, creatininase (creatinine amidohydrolase EC 3.5.2.10) and 4-aminoantipyrine (4-AA) were added, and only the creatine generated from creatinine by creatininase was hydrolyzed sequentially by creatinase and sarcosine oxidase to produce hydrogen peroxide. This newly-formed hydrogen peroxide was measured at 510 nm in a coupled reaction catalyzed by peroxidase, with 2,4-DCPS/4-AA as a chromogen. The standard curve was linear up to 20 mg/dl; 40 microliter of serum and 20 min were required for determination. Analytical recovery of creatinine added to either normal or abnormal sera averaged 98.5%. Within-day and day-to-day studies gave CV values of less than or equal to 2.9% and less than or equal to 4.8%, respectively. No significant interferences were observed with the proposed method. The results obtained by the present method correlated well with those obtained by the Jaffé procedure.     

The isoelectric focusing of creatine kinase variants: II. The heterogeneity of creatine kinase in human serum with normal and elevated catalytic concentrations

An effective and reliable method for the quantitative estimation of creatine kinase-MB, creatine kinase-MM variants and mitochondrial forms of creatine kinase in serum is presented. The high resolving power of isoelectric focusing allows the use of tetrazolium salts and meldola blue for the quantitative measurement without interfering non-specific reduction. The addition of thiol compounds to the agarose medium increases the sensitivity of the method, due to the inhibition of sulfhydryl group oxidation, and prevents enzyme degradation, which is a possible cause of an artificial heterogeneity. Depending upon the type of muscle and the degree of cell damage, we found 3-4 creatine kinase-MM sub-bands in sera with activities below 80 U/l. At elevated creatine kinase activities 3-11 creatine kinase-MM sub-bands were found. The appearance of creatine kinase-MB in serum indicates that damage has occurred to certain organs, especially the cardiac muscle. An organ with moderate or massive cell damage could release, in addition to the sarcoplasmatic creatine kinase variants, other forms with more alkaline isoelectric points (mitochondrial creatine kinase). The presence of such bands in serum of patients correlates with poor prognosis. Besides the separation of creatine kinase-MM sub-bands, creatine kinase-MB, creatine kinase-BB and of macroforms 1 and 2, the advantage of this method is the detection of mitochondrial creatine kinase forms, which in cellulose acetate electrophoresis migrate with creatine kinase-MM.     

Continuous-flow enzymic determination of creatine in urine

A new enzymic method is described for the determination of creatine in urine by continuous-flow analysis. The measurement is accomplished by transforming creatine to formaldehyde in reactions catalyzed by creatinase (creatine amidinohydrolase) and sarcosine dehydrogenase. The formaldehyde is reacted with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole to form a purple product, which is measured colorimetrically. The method gives a linear standard curve for creatine concentrations up to 100 mg/L. Precision and analytical recovery are excellent, and results correlate well with those by the more-difficult Folin method, which currently is most often used for this analysis.     

Distribution of serum concentrations of creatine kinase MM and BB isoenzymes measured by radioimmunoassay.

Because of the recent interest in measuring serum enzyme concentration as opposed to catalytic activity, we measured the serum concentration of creatine kinase isoenzymes BB and MM by radioimmunoassay and the total creatine kinase enzymatic activity in healthy adults. For sex-race subgroups we report mean values and the 2.5, 5, 25, 50, 75, 90, 95 and 97.5 percentiles. Differences among average values of the subgroups were highly significant. Results within subgroups frequently departed from a gaussian distribution.     

Determination of creatine kinase isoenzyme MB activity in serum using immunological inhibition of creatine kinase M subunit activity. Activity kinetics and diagnostic significance in myocardial infarction.

This is a new method for the determination of creatine kinase isoenzyme MB activity in serum. The method uses direct activity measurement of creatine kinase B subunit activity after blocking of CK-M subunit activity by inhibiting antibodies. The test takes no longer than 15 min. The method yields an intra-serial C.V. of 2.0-12.9%, and a C.V. from day to day of 5.5%. The detection limit is 3.4 U/l creatine kinase MB. In the 95 cases with proven myocardial infarction several types of creatine kinase MB activity kinetics could be determined. The percentage of creatine kinase MB of peak CK-total is 6-25%, with a mean of 11.1%. The amount of creatine kinase MB with respect to total CK activity after reinfarction is higher than the amount after initial infarction.     

Guanidinoacetate methyltransferase (GAMT) deficiency: non-invasive enzymatic diagnosis of a newly recognized inborn error of metabolism

Guanidinoacetate methyltransferase deficiency is a newly recognized inborn error of creatine biosynthesis. Manifestation of neurologic symptoms occurs in infancy and is partly reversible upon oral substitution of creatine. In the first two index patients, enzymatic diagnosis was established in a liver biopsy, and the underlying molecular defect in the GAMT gene has been identified. In order to provide non-invasive biochemical diagnosis, we have developed an enzyme assay based on the formation of radiolabeled creatine from ¹⁴C guanidinoacetate and S-adenosylmethionine in concentrated and dialyzed extracts from cultivated skin fibroblasts, Epstein–Barr virus transformed lymphoblasts, and cultivated amniotic cells. Cells were investigated from controls, from 1 index patient with proven GAMT deficiency and from 3 additional patients with clinical and biochemical signs of GAMT deficiency. Separation of ¹⁴C guanidinoacetate from ¹⁴C creatine in the reaction mixture was accomplished by HPLC on Hypersil ODS column and radioactivity was determined in fractions according to respective UV signals. GAMT activities in control fibroblasts (n=7), lymphoblasts (n=8) and in amniotic cells (n=2) were 0.38–0.56, 0.61–0.84 and 0.38–0.56 nmol/h/mg protein. Apparent Km values were 9.5–14.8 μM for guanidinoacetate and 68–78 μM for S-adenosylmethionine. In the index patient and in the three additional patients at risk, GAMT activity was <0.1 nmol/h/mg protein. The assay described here allows non-invasive diagnosis of GAMT deficiency in patients at risk.     

Identification of the carboxypeptidase responsible for the post-synthetic modification of creatine kinase in human serum

The enzyme responsible for the post-translational modification of creatine kinase-MM isoenzyme was purified from human plasma. The enzymatic activity of this enzyme (modifying protein) on the synthetic substrates hippuryl-L-arginine, hippuryl-L-lysine, 3-(2-furylacryloyl)-L-arginine and 3-(2-furylacryloyl)-L-alanyl-L-lysine and the ratio of activities on these substrates are in good agreement with the enzymatic activity of the human serum carboxypeptidase N. The effect of metal ions, chelating agents, proteolytic inhibitors and carboxypeptidase N inhibitor could not differentiate the modifying protein from human serum carboxypeptidase N. Affinity chromatography on Concanavalin-A-Sepharose demonstrated the glycoprotein nature of the modifying protein. The difference in molecular weight observed between modifying protein and carboxypeptidase N can be explained by known instability characteristics and the influence of proteolytic enzymes during purification. Double immunodiffusion analysis with purified antiserum to human carboxypeptidase N confirmed the identity of the modifying protein and carboxypeptidase N.