Detection of cardiac-specific creatine kinase isoenzyme in sera with normal or slightly increased total creatine kinase activity.

We describe a spectrophotometric kinetic assay for detecting creatine kinase MB isoenzyme activity in the 1 to 10 U/liter range. The MB isoenzyme was isolated [Clin. Chem. 20, 36 (1974)] and assayed (Rosalki method) with an Abbott ABA-100. Good reproducibility was demonstrated for MB isoenzyme activities near 1 U/liter (CV = 2.6%). Sera with normal or slightly increased total creatine kinase activity were evaluated. Sera of 14 patients with acute myocardial infarction contained, per liter, 84 to 236 U of total creatine kinase activity and 4.6 to 28.0 U of isoenzyme MB activity; corresponding ranges for sera from healthy lab technicians and patients with noncardiac disease were 36 to 277 and 0 to 2.6 U. MB isoenzyme activity for infarction patients rose and fell sharply within three days after the infarction. Atypical time-course patterns, MB isoenzyme activity remaining abnormally great for five days, were observed in serum from patients with prolonged atrial fibrillation and congestive heart failure or cardiomyopathy; the BB isoenzyme (1 to 5 U/liter) was also detected in sera of such patients but was absent in sera from infarcation patients. Quantification of column-isolated MB by the assay described is rapid, easy, specific, and extremely sensitive for measuring MB in the 1 to 10 U/liter range.     

New manual and automated method for determining activity of creatine kinase isoenzyme MB, by use of dithiothreitol: clinical applications.

The method is based on the selective activating capacity of dithiothreitol on creatine kinase isoenzyme MB, after isoenzyme MM is activated by glutathione. Isolated isoenzymes MM and MB of human and canine origin were assayed individually and in mixtures of known activities. When glutathione was present in the assay medium the activity of each isoenzyme could be measured individually, but glutathione did not activate isoenzyme MB if it was present in a mixture with MM. Dithiothreitol, added to the serum before assay, activated the isoenzyme MB in the mixture. Values for MB activities obtained for isolated isoenzyme MB and for the isoenzyme mixture after dithiothreitol was added averaged 110 and 111 U/liter, respectively (r = 0.998; y = 1.007 x + 0.298; n = 10). In the serum of 40 patients with documented acute transmural myocardial infarction, the mean proportion of isoenzyme MB activity measured in this way was 5.5% (coefficient of variation, 7.7%). Isoenzyme MB activities measured by use of dithiothreitol compared well with those obtained by conventional electrophoresis/spectrophotometry (r = 0.998; y = 1.09x -0.65) and spectrofluorometry (r = 0.996; y = 1.10 x + 0.80). The assay of MB activity by the dithiothreitol method was automated, by use of an Abbott Bichromatic Analyser and a Calbiochem Super-Stat Pack Kit. In 60 isoenzyme MB determinations the manual and automated method correlated well (r = 0.990; y = 1.0x -1.36). The simplicity of isoenzyme MB determination by use of dithiothreitol and its ease of automation allow routine monitoring of the isoenzyme activity in patients with ischemic heart disease.     

Alkaline phosphatase activity from human osteosarcoma cell line SaOS-2: an isoenzyme standard for quantifying skeletal alkaline phosphatase activity in serum

Earlier we described a kinetic assay for quantifying skeletal alkaline phosphatase (ALP) isoenzyme activity in serum. The precision of the assay depends on including ALP standards for the skeletal, hepatic, intestinal, and placental isoenzymes. We wondered whether human osteosarcoma cells could provide an efficient alternative to human bone or Pagetic serum as a source of the skeletal ALP standard. ALP activities prepared from five human osteosarcoma cell lines were compared with a bone-derived ALP standard with respect to heat stability and sensitivity to chemical effectors. Two of the cell lines (SaOS-2 and TE-85) contained ALP activities that resembled the bone-derived standard. We selected SaOS-2 cells for additional evaluation (as a potential source of isoenzyme standard), because they contained 40-50 times more ALP activity than did the TE-85 cells. To include the SaOS-2 cell-derived ALP activity in the quantitative isoenzyme assay, we diluted the enzyme in a solution containing heat-inactivated (i.e., ALP-negative) human serum. Surprisingly, this dilution caused a 60-125% increase in maximum enzyme activity. In the quantitative assay of ALP isoenzyme in serum, the SaOS-2 derived ALP was indistinguishable from the serum skeletal ALP standard, with respect to the above criteria and assay variations. Evidently ALP from SaOS-2 cells is suited as a standard for measuring skeletal ALP activity in this assay.     

A modified radioimmunoassay for arylsulfatase A in human serum and urine

A radioimmunoassay was developed for the determination of arylsulfatase A (EC 3.1.6.1) in human serum and urine. An isoenzyme of arylsulfatase A purified from human urine was used as a standard antigen. The enzyme was radioiodinated with 125I using the Chloramine T method and was stable for about 4 wk. Antibody-bound enzyme was separated from free enzyme by means of a double antibody technique in the presence of polyethylene glycol (PEG). The working range of the method was 0.15-5.0 ng of arylsulfatase A per assay. The within-assay CV was about 8% for both biological fluids and the between-assay CV for serum was 14.1%. Analytical recoveries were 93.2 +/- 9.1% and 97.8 +/- 5.5% for serum and urine, respectively, and the sensitivity was 0.040 ng of arylsulfatase per assay. Serum samples of 50 healthy blood donors were assayed to establish the normal serum level of immunoreactive enzyme, which was found to be 8.3 ng/ml +/- 1.8 ng/ml of serum. Storage of frozen serum was shown to have no significant effect on results obtained using this RIA.     

急性心肌梗死患者血浆B型钠尿肽及血清心肌梗死指标变化的临床分析

目的探讨急性心肌梗死(AMI)患者血浆B型钠尿肽(BNP)与其他AMI生化指标之间有无相关性。方法采用免疫荧光法定量检测40例健康体检者及47例AMI患者的BNP,并同时检测其他几项AMI生化指标,进行比较分析及相关分析。结果 (1)AMI组血浆BNP、肌酸激酶同工酶(CK-MB)、肌钙蛋白I(TnI)及肌红蛋白(MYO)检测结果分别为(856.8±295.7)ng/L、(41.71±13.25)μg/L、(0.520±0.180)μg/L、(6.8±16.8)μg/L,健康对照组分别为(165.4±40.6)ng/L、(3.27±1.48)μg/L、(0.010±0.005)μg/L、(22.2±8.7)μg/L,两组比较差异均有统计学意义(P<0.05)。(2)AMI患者血浆BNP与其他AMI指标的相关性分析结果表明,血浆BNP与CK-MB、TnI和MYO之间呈正相关。结论血浆BNP可作为临床预测冠心病患者发生AMI危险性的指标,并可与CK-MB、TnI及MYO一起作为AMI的监测及疗效观察的指标。     

Reference intervals for amylase isoenzymes in serum and plasma of infants and children

Measurement of pancreatic (P) and salivary-like (S) amylase isoenzyme activity in serum of adults is useful as an indirect indicator of pancreatic and salivary gland exocrine dysfunction. To extend the use of this assay to the pediatric population, we measured amylase isoenzymes in 546 serum and plasma samples and defined normal reference intervals for the P and S isoenzymes as a function of age in newborns, infants, and children. The mean activity of P isoenzyme in newborns is 3% of that of adults, begins to increase at seven to eight months, and reaches adult values by five years. The mean activity of S isoenzyme in serum is 32% of the adult mean at birth, begins to increase by three to four months, and reaches adult values by 19 months; children five to 12 years old have slightly higher values than adults. These changes with age underscore the importance of the use of age-matched reference intervals when serum amylase isoenzyme activities are measured as diagnostic indicators.     

Time-dependent changes in bone, placental, intestinal, and hepatic alkaline phosphatase activities in serum during human pregnancy

To measure changes in bone alkaline phosphatase (EC 3.1.3.1) activity in serum as a function of duration of pregnancy, we adapted our existing alkaline phosphatase (ALP) isoenzyme assay (which has been used to measure bone, hepatic, and intestinal ALP activities in serum, in the absence of placental ALP) to allow quantification of individual ALP isoenzyme activities in the presence of placental ALP. The resulting CV for repeat measurements of bone ALP activity in artificial isoenzyme mixtures ranged from 23% for samples in which the bone isoenzyme represented 7% of total ALP activity to 11% for samples in which bone ALP accounted for 48% of total ALP activity. Values for repeat determinations of bone ALP activity in human serum samples (i.e., including samples obtained from pregnant women and from nonpregnant controls) varied by an average of 18%. We find, in initial applications of this method, that (a) the amount of bone ALP activity in serum is increased during pregnancy (P less than .001), and remains increased at six weeks postpartum, in non-lactating women (P less than .001), and (b) bone ALP activity at term was not significantly different in pregnant women with pre-eclampsia, diabetes, premature rupture of membranes, or premature labor, compared with normal pregnancies at term. Our data support the hypothesis that maternal bone formation may be increased during pregnancy.     

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.     

Bone isoenzyme of serum alkaline phosphatase in acromegaly.

In 37 patients with active acromegaly and in 15 patients with inactive acromegaly, activity of bone isoenzyme of serum alkaline phosphatase correlated (P less than 0.001) with serum concentration of immunoreactive growth hormone. By using stepwise regression analysis, the predication of serum growth hormone values based on serum levels of bone isoenzyme of serum alkaline phosphatase, gamma-glutamyl transferase and calcium in these patients with acromegaly was within 1 S.D. range in 37 patients and in only 2 patients was it out of 2 S.D. range. By using discriminant analysis, based on bone and liver isoenzymes of serum alaline phosphatase and urinary hydroxyproline excretion, 87%, 60% and 97% of the classification of patients with active and inactive acromegaly and healthy adults, respectively, was correct. The multivariate approach offers a quantitative appraisal of the biochemical parameters of peripheral growth hormone action used as an indicator of growth hormone concentration in patients with acromegaly.     

Creatine kinase isoenzyme MB determination on the ACA: dependence on serum matrix and other effectors

Creatine kinase isoenzyme MB catalytic activities in human serum, determined by ACA ion exchange chromatography and immunoinhibition, differ significantly, the correlation coefficient being 0.88. The reasons for this variation are interference of antibodies with the creatine kinase B subunit in the immunoinhibition assay, nonreproducible elution of creatine kinase isoenzyme MB from the ion exchange resin in the ACA pack, due to varying protein concentrations in the serum samples and increasing elution of creatine kinase isoenzyme MM from the ion exchange column caused by a preceding partial inactivation of creatine kinase isoenzyme MM. Pretreatment of serum samples with a solution containing magnesium sulphate, maleate and 2-oxoglutarate (solution A) prior to determination of creatine kinase isoenzyme MB catalytic activities on the ACA significantly improves the sensitivity and specificity of the method; the correlation coefficient for the values from the ACA and immunoinhibition then becomes 0.92. Dilution of serum samples with bovine serum albumin solution is now practicable.     

beta-N-acetyl-D-glucosaminidase isoenzymes by chromatofocusing from serum and skin in diabetes

beta-N-Acetyl-D-glucosaminidase and its kinetic characteristics were determined in serum and skin from both diabetes mellitus and impaired glucose tolerance patients and controls. Mean total activity was reduced (p less than 0.05) in the skin of both patient groups. beta-N-Acetyl-D-glucosaminidase isoenzyme expression was investigated using chromatofocusing on PBE-94 coupled with automated enzyme assay. The isoenzyme profiles from serum showed two major forms (A and B) whose ratio varied from 2:1 in controls to 3:1 in diabetics. Moreover, diabetes mellitus and impaired glucose tolerance subjects displayed an intermediate (I) form. The A/B isoenzyme ratio was completely reversed in skin.     

Hepatic infarction: biochemical study of a case

Hepatic infarction was observed post mortem in a 27-year-old man who died of aortic dissection. Blood had been sampled at admission and 12 and 19 hours later. Values for aspartate aminotransferase and alanine aminotransferase in serum were markedly above normal, whereas those for alkaline phosphatase and gamma-glutamyltransferase were only marginally increased. A threefold-increased creatine kinase was ascribable solely to isoenzyme CK-3, suggesting muscle breakdown. Moreover, total lactate dehydrogenase activity was increased threefold, accounted for by a ninefold increase in LD-5 isoenzyme. Those enzyme activities in serum that evidently are associated with acute hepatocellular necrosis increase quickly in hepatic infarction, and CK isoenzyme assay is a useful adjunct if LD-5 increases are significant.     

Amylase in serum of healthy subjects. The relationship between catalytic concentration and substance concentration is not effected by the isoenzyme pattern

Catalytic activity concentrations and substance concentrations of immunoreactive amylase were measured in serum samples from 140 healthy subjects with various electrophoretic isoenzyme patterns. The relationship between catalytic activity concentrations and corresponding substance concentrations of immunoreactive amylase was independent of the actual amylase isoenzyme pattern.     

Creatine kinase isoenzyme BB in serum of healthy adults and children

The activity of the creatine kinase isoenzyme BB was determined in the serum of 26 healthy adults and 31 children. The isoenzyme BB could be proved as a normal component in the human serum. In the adults examined, an activity of $\text{0.56 ± 0.16}\text{I.U./l}\text{(}\text{x}\text{±}\text{S.D.}\text{)}$ was determined. The activity of creatine kinase isoenzyme BB in the serum does not depend on sex but is subject, however, to a strong age dependence. Only at an age of more than 18 years, isoenzyme BB activities adjust to those of adults.     

Tay-Sachs disease: one-step assay of beta-N-acetylhexosaminidase in serum with a sulphated chromogenic substrate

A sulphated chromogenic compound, p-nitrophenyl-6-sulpho-2-acetamido-2-deoxy-beta-D-glucopyranoside, which can be hydrolysed enzymatically to p-nitrophenol and the sulphated amino sugar, was used as a substrate for the determination of activity of beta-N-acetylhexosaminidase isoenzymes in human serum. The sera of six Tay-Sachs patients lacking isoenzyme A and heat-inactivated control serum exhibited 6% of the mean normal enzyme activity of 1.32 U/l (1-s range = 1.07-1.57 U/l). In 10 obligate carriers of the Tay-Sachs gene the enzyme activity was 52% (1-s range = 45-60%) of the mean normal value. Therefore, by using the sulphated chromogenic substrate Tay-Sachs disease can be diagnosed enzymatically in a simple one-step procedure, but the 2-s activity ranges of heterozygotes and normals overlap. The assay is not absolutely specific for isoenzyme A of beta-N-acetylhexosaminidase, because the substrate can be hydrolysed to a certain extent by beta-N-acetylhexosaminidase I.     

Evaluation of a commercial immunoenzymometric assay kit for creatine kinase MB isoenzyme determination using monoclonal antibodies

A simultaneous two-site immunoenzymometric assay for creatine kinase MB determination (Hybritech Tandem-E CK-MB) using monoclonal antibodies was evaluated and compared with cellulose acetate electrophoresis using fluorometric scanning densitometry. The assay has satisfactory precision (between-day analysis gives a coefficient of variation between 2.1 and 9.4%) and is not susceptible to interference by concentrations of creatine kinase MM up to 5000 micrograms/l (3400 U/l) and creatine kinase BB up to 1000 micrograms/l (1085 U/l). The upper limit of MB isoenzyme concentration in 250 apparently healthy people was 5.5 micrograms/l. Comparison between the immunoenzymometric assay (y) and electrophoresis (x) yielded the following linear regression equation: y = 0.37x + 1.9, with a correlation coefficient of 0.828. The characteristics of the temporal kinetics of MB isoenzyme, calculated by two methods, in 49 patients with acute myocardial infarction, were nearly identical in terms of the rate of creatine kinase MB release and the time at which the peak value is obtained, but not in terms of the rate of elimination of the isoenzyme. The fractional disappearance rate of MB isoenzyme from the circulation was significantly higher if calculated with Tandem-E results rather than with electrophoresis results (-0.035 vs -0.028, p less than 0.001). Whereas in the first day after infarction immunoenzymometric assay and electrophoresis had the same clinical sensitivity for identifying patients with acute myocardial infarction, in specimens collected more than 24 hours after the onset of the chest pain, the clinical sensitivity of the immunoenzymometric method was lower. Our results show that it is still premature to draw definitive clinical conclusions from the immunoassay results.     

急性高血压脑出血患者血清h-FABP水平变化及其临床应用价值

目的 通过观察急性高血压脑出血患者血清心脏型脂肪酸结合蛋白(h-FABP)水平变化,探讨其临床检测价值.方法 血清h-FABP采用双抗体夹心酶联免疫一步法定量检测;cTnI采用固相酶联免疫吸附实验(ELISA);CK-MB采用免疫抑制法测定HT5"H〗结果 急性高血压脑出血患者组血清h-FABP水平显著高于对照组(P<0.01),大量出血者及意识不清者血清h-FABP水平分别显著高于小量出血者及意识清醒者(P<0.01);血清h-FABP阳性率显著高于血清cTnI、CK-MB及心电图的阳性率(P<0.01);死亡率为25.5%(28/110),血清h-FABP、cTnI、CK-MB及心电图异常组的死亡率显著高于正常组(P<0.01);血清h-FABP预测发生死亡具有高的敏感性和阴性预测值,特异性和准确度较低,但心电图具有较高的特异性(64.5%)和准确度(69.1%).结论 血清h-FABP定量测定可作为判断急性高血压脑出血病情轻重及评价发生意外的1项客观指标,联合心电图监测临床价值更高.     

血清心肌肌钙蛋白T(cTnT)检测在不稳定性心绞痛中的临床价值

目的:探讨血清心肌肌钙蛋白T(cTnT)对不稳定性心绞痛(UAP)的诊断及预后判断等方面的价值。方法:用ELISA一步夹心法检测UAP患者血清cTnT的浓度,用酶偶联测定法和抗体免疫抑制法同步检测患者血清CK-MB浓度,并与血清cTnT比较。另检测了40例正常人血清cTnT、CK-MB,供作参比对照。结果:40例对照组血清cTnT浓度0.00-0.04ug/L,CK-MB为0-15U/L,均呈偏态分布。血清cTnT对UAP患者发生AMI的阳性预测值为20％,阴性预测值为96.6％。结论:血清cTnT比CK-MB在反映缺血性心肌损伤方面有更高的灵敏性。血清cTnT对UAP的诊断和预后判断有重要价值。     

Precipitation method for separating and quantifying bone and liver alkaline phosphatase isoenzymes.

We evaluated a method for quantifying bone isoenzyme of alkaline phosphatase (ALP) which utilizes wheat-germ lectin to precipitate this fraction. In precision studies, CVs ranged from 3.2 to 11.4% (within-day) and from 3.7 to 11.5% (day-to-day). The assay procedure was linear to 1100 U/L and was easily adapted to automated kinetic measurement. Comparison of the precipitation method with an affinity electrophoretic method, which utilizes cellulose acetate as a support, demonstrated a satisfactory coefficient of correlation (r = 0.886). The reference range was determined in sera from 188 healthy adult subjects. The distribution of bone ALP values was also studied in 73 healthy children and in 30 healthy adolescents. To evaluate the clinical applicability of the method, the bone isoenzyme was determined in samples from several groups of subjects (pregnant women, patients with hepatobiliary diseases, patients with hepatocellular carcinoma without skeletal involvement, and patients with bone, liver or lymph node metastases). We found the method suitable for routine determination of bone alkaline phosphatase and for the screening of bone metastases. Because of its technical simplicity and satisfactory analytical performance, it can be used instead of the heat-inactivation procedure.     

Production of recombinant human creatine kinase (r-hCK) isozymes by tandem repeat expression of M and B genes and characterization of r-hCK-MB

BACKGROUND: Serum creatine kinase-MB isoenzyme (CK-MB) is widely used as a marker of myocardial injury. We prepared recombinant human CK (r-hCK) MB isoenzyme and examined its potential for use as a control material for assay of CK-MB in serum. METHODS: cDNAs encoding CK-M and CK-B subunits were inserted into the same plasmid vector, followed by transformation of Escherichia coli. The resulting three types of CK isoenzymes were purified by conventional chromatography. RESULTS: The ratio of MB to MM to BB was 50:40:10 on the basis of CK activity. Highly purified CK-MB with a specific activity of 533 U/mg was produced in a yield of 5.7 mg/g of packed cells. Purified r-hCK-MB had the isoelectric point (pI 5.3) and molecular size (46 kDa for the subunit) of native CK-MB. Its immunoreactivity in an ELISA using antibody against native heart enzyme was similar to that of cardiac CK-MB. The r-hCK-MB retained >90% activity for at least 4 months at 11 degrees C in a delipidated serum matrix in a liquid form at a concentration of 118 U/L. CONCLUSIONS: r-hCK-MB shows key properties of the native cardiac isoenzyme and may be useful as a control and calibrator for serum assays of CK-MB.