Measurement Of Isoenzymes Research Articles

Development of monoclonal antibodies and immunochromatographic lateral flow device for rapid test of alanine aminotransferase isoenzyme 1

BackgroundAlanine aminotransferase (ALT) has been used as a sensitive marker for liver injury in people and in preclinical toxicity studies. But measurement of ALT isoenzymes, ALT1 and ALT2, was reported to be of more diagnostic value. The aim of this study is to develop an ideal pair of anti-ALT1 monoclonal antibodies (MAbs) of high specificity and affinity, and subsequently prepare a Immunochromatographic lateral flow device (LFD) for rapid test of ALT1 in human serums. MethodsThe complete coding sequence of ALT1 gene (1500 bp) was cloned from human hepatoma G2 cells (HepG2) and inserted into the expression vector pET-32a(+). ALT1 recombinant protein was routinely prepared by E. coli BL21 (DE3) expression and Ni2+ affinity purification. Balb/c mice were immunized with purified ALT1 and the splenocytes were fused with Sp2/0 myeloma cells. The positive clones, verified by indirect enzyme-linked immunosorbent assay (ELISA) using purified ALT1, were subcloned to single clones by limiting dilution process. A MAb pair was selected from the obtained MAbs according the sandwich ELISA pairing results and then used for lateral flow device (LFD) production. After evaluation of the sensitivity and specificity, the LFD strips were employed to test human serum samples with known ALT activity levels. ResultsALT1 recombinant protein was expectedly prepared by expression and purification. A total of 8 stable clones that produced antibodies specifically recognizing ALT1 protein were developed. After sandwich ELISA pairing, an ideal pair of anti-ALT1 MAbs, designated as BD7 and DG3, were selected and proved to be of high specificity, titer and affinity. Based on the MAb pair, LFD strips specifically for ALT1 rapid test were subsequently prepared. The detection threshold of the LFD strips was 12 U/L. No cross reaction was found. ConclusionsThe ALT1 LFD with high sensitivity and specificity was successfully developed. It is valuable for testing ALT1 protein in human sera and can be a beneficial complement for traditional ALT test.

The activity of class I, II, III and IV alcohol dehydrogenase isoenzymes and aldehyde dehydrogenase in renal cell carcinoma

ObjectivesEthanol has been considered as a lifestyle risk factor for cancer in humans. While some studies have indicated that alcohol intake has a preventive effect for renal cell cancer, others have not. The metabolism of alcohol in cancer cells may be in many ways different than in healthy tissue and its disturbances could be associated with carcinogenesis. The aim of this study was to compare the metabolism of renal cell cancer cells and normal renal cells by measurement of ADH isoenzymes and ALDH activities in these tissues. Material and methodsThe study material consisted of 43 cancerous renal tissues (14 patients in stage II, 19 in stage III and 10 in stage IV). Class III and IV ADH and total ADH activities were measured by the photometric method and class I and II ADH and ALDH activities by the fluorometric method with class-specific fluorogenic substrates. ResultsThe activity of the class I ADH isoenzyme and the total ADH was significantly higher in every stage of renal cell cancer as compared to healthy tissues. Analysis of ALDH activity did not show statistically significant differences between cancer and healthy cells. ConclusionThe increased activity of total ADH in renal cell cancer, especially the class I isoenzyme and normal activity of ALDH, may be the factor intensifying carcinogenesis because of increasing the ability to highly carcinogenic acetaldehyde formation and causing disorders in metabolism of many biologically important substances.

The activity of class I, II, III and IV alcohol dehydrogenase isoenzymes and aldehyde dehydrogenase in ovarian cancer and ovarian cysts

The metabolism of cancerous cells is in many ways different than in healthy cells. In ovarian cancer, cells exhibit activity of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), which participate in metabolism of many biological substances. The aim of this study was to compare the metabolism of ovarian cancer cells, ovarian cysts and normal ovarian cells by measurement of ADH isoenzymes and ALDH activities. The study material consisted of 36 cancerous ovarian tissues. Class III, IV of ADH and total ADH activity was measured by the photometric method and class I, II ADH and ALDH activity by the fluorometric method with class-specific fluorogenic substrates. The activity of the class I ADH isoenzyme and the total ADH was significantly higher in ovarian cancer as compared to ovarian cysts and healthy tissues but there are no significant differences between ovarian cysts and healthy cells. The other classes of ADH tested, did not show significant differences between activity of cancerous cells and healthy ovary. The increased activity of total ADH in ovarian cancer, especially the class I isoenzyme and normal activity of ALDH, may be the factor for the disturbances in important biological substances metabolism and could increase the concentration of highly carcinogenic acetaldehyde.

The activity of class I, II, III and IV alcohol dehydrogenase isoenzymes and aldehyde dehydrogenase in cervical cancer

Objective The aim of this study was to compare the metabolism of cervical cancer cells and normal cervical cells by measurement of ADH isoenzymes and ALDH activities. Methods The study material consisted of 40 cancerous cervical tissues. Class III, IV of ADH and total ADH activity was measured by the photometric method and class I, II ADH and ALDH activity by the fluorometric method with class-specific fluorogenic substrates. Results The activity of the class I ADH isoenzyme and the total ADH were significantly higher in cervical cancer as compared to healthy tissues. Class I of ADH and total ADH activity are significantly higher in every stage of cancer as compared to the control. There are no significant differences between planoepitheliale and adenocarcinoma. Conclusion The increased activity of total ADH in cervical cancer may be the cause of some metabolic disorders in cancer cells, which may intensify carcinogenesis in this organ.

Prognostic Value of Troponin and Creatine Kinase Muscle and Brain Isoenzyme Measurement after Noncardiac Surgery

There is uncertainty regarding the prognostic value of troponin and creatine kinase muscle and brain isoenzyme measurements after noncardiac surgery. The current study undertook a systematic review and meta-analysis. The study used six search strategies and included noncardiac surgery studies that provided data from a multivariable analysis assessing whether a postoperative troponin or creatine kinase muscle and brain isoenzyme measurement was an independent predictor of mortality or a major cardiovascular event. Independent investigators determined study eligibility and abstracted data in duplicate. Fourteen studies, enrolling 3,318 patients and 459 deaths, demonstrated that an increased troponin measurement after surgery was an independent predictor of mortality (odds ratio [OR] 3.4, 95% confidence interval [CI] 2.2-5.2), but there was substantial heterogeneity (I(2) = 56%). The independent prognostic capabilities of an increased troponin value after surgery in the 10 studies that assessed intermediate-term (≤ 12 months) mortality was an OR = 6.7 (95% CI 4.1-10.9, I(2) = 0%) and in the 4 studies that assessed long-term (more than 12 months) mortality was an OR = 1.8 (95% CI 1.4-2.3, I(2) = 0%; P < 0.001 for test of interaction). Four studies, including 1,165 patients and 202 deaths, demonstrated an independent association between an increased creatine kinase muscle and brain isoenzyme measurement after surgery and mortality (OR 2.5, 95% CI 1.5-4.0, I(2) = 4%). An increased troponin measurement after surgery is an independent predictor of mortality, particularly within the first year; limited data suggest an increased creatine kinase muscle and brain isoenzyme measurement also predicts subsequent mortality. Monitoring troponin measurements after noncardiac surgery may allow physicians to better risk stratify and manage their patients.

Pattern of Cerebrospinal Fluid Lactic Dehydrogenase During Treatment of Bacterial Meningitis

Bacterial and aseptic meningitis are characterized by distinctive lactic dehydrogenase isoenzyme patterns. No studies have quantified the dynamics of lactic dehydrogenase isoenzyme distribution during treated bacterial meningitis. We used a retrospective case-series design, and reviewed files of all neonates with bacterial meningitis who attended our pediatric tertiary medical center for 8 years period. We identified neonates in whom a repeated lumbar puncture was indicated. Findings of cerebrospinal fluid analysis, including levels of lactic dehydrogenase isoenzymes, were compared with an age-matched reference group. In two patients with meningitis, lumbar puncture with cerebrospinal fluid analysis was repeated because of inadequate response to treatment or initially obscure etiologic pathogens. Both patients had initially low levels of lactic dehydrogenase-1 and lactic dehydrogenase-2 and high levels of lactic dehydrogenase-4 and lactic dehydrogenase-5, similar to other patients with bacterial meningitis. The distribution pattern of lactic dehydrogenase isoenzyme normalized after adequate antibiotic treatment. In light of the encouraging results in these two patients, further studies are warranted regarding the value of lactic dehydrogenase isoenzyme measurements for follow-up purposes and for evaluations of response to treatment.

The activity of class I, II, III, and IV alcohol dehyrogenase isoenzymes and aldehyde dehydrogenase in endometrial cancer

The metabolism of cancerous cells is in many ways different than in healthy cells. In endometrial cancer, cells exhibit activity of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), which participate in the metabolism of many biological substances. The aim of this study was to compare the metabolism of endometrial cancer cells and normal endometrial cells by measurement of ADH isoenzymes and ALDH activities in these tissues. The study material consists of cancerous endometrial tissues obtained from 34 patients. Total ADH activity was measured using the photometric method and ALDH activity using the fluorometric method. For the measurement of class I and II ADH isoenzyme activity, we employed the fluorometric method, with class-specific fluorogenic substrates. The activity of class III and IV ADH was measured using the photometric method. The activity of the class I ADH isoenzyme was significantly higher in the endometrial cancer tissues when compared with normal endometrial tissues. The other classes of ADH tested did not show significant differences between activity of cancerous cells and healthy endometrium. The activity of total ADH was also significantly higher in endometrial cancer. The increased activity of total ADH in endometrial cancer, especially the class I isoenzyme and normal activity of ALDH, may be the cause of disorders in metabolic pathways that use these isoenzymes and could increase the concentration of acetaldehyde, which is cancerogenic substance.

Establishment and characterization of a fibroblast line from Simmental cattle

A fibroblast line (named SCF36) from ear marginal tissue of Simmental cattle was established successfully by direct culture of explants and cell cryopreservation techniques. Biological analysis showed that the population doubling time of the thawed cells was 42.8 h. The average viability of the cells was 96.8% before freezing and 91.5% after thawing. Measurements of lactic dehydrogenase and malic dehydrogenase isoenzymes showed no cross-contamination of this cell line with other species. Karyotyping showed that the frequency of cells with chromosome number 2 n = 60 was more than 90%. Tests for bacteria, fungi, viruses and mycoplasmas were negative. The efficiencies of expression of enhanced green, yellow and red fluorescent protein genes (pEGFP-N 3, pEYFP-N 1 and pDsRed1-N 1) were between 11.3% and 28.8% after transfection; fluorescence was well distributed in the cytoplasm and nucleus except for some cryptomeric vesicles. This Simmental cattle fibroblast line not only contains the germline of this important cattle breed, which is preserved at the cellular level, but valuable material has also been provided for genomic, postgenomic and somatic cloning research. Moreover, the establishment of these methods may provide both technical and theoretical support for preserving the genetic resources of other livestock and poultry at the cellular level.

Measurement of alkaline phosphatase isoenzymes in individual mouse bone marrow fibroblast cells based on capillary electrophoresis with on-capillary enzyme-catalyzed reaction and electrochemical detection.

A novel method for the determination of alkaline phosphatase (ALP) isoenzymes in individual fibroblast cells of mouse bone marrow was developed by combining capillary electrophoresis with an on-capillary enzyme-catalyzed reaction and electrochemical detection. In this method, a single an cell, followed by 5.0 x 10(-2) mol/L Na2B4O7- 3.0 x 10(-2) mol/L NaCl (pH 9.8) as cell lysis solution, was injected into the inlet of the separation capillary by electromigration. The cell was lysed by applying a voltage of 2 kV. The ALP isoenzymes in the cell were preseparated at 20 kV for 1 min, and then allowed to react for 30 min with disodium phenyl phosphate as enzyme substrate in the running buffer. ALP converted disodium phenyl phosphate into its product, phenol, at a relatively high reaction rate without consumption, with resultant amplification of the signal on prolonged reaction time, producing an adequate amount of product for final detection. A mass detection limit as low as 3.5 x 10(-21) mol/L (corresponding to 1.5 nU) was achieved. Finally, the two zones of products generated by ALP isoenzymes were detected at the outlet of the capillary by using the end-capillary amperometric detection at a carbon fiber microdisk bundle electrode with a constant potential.

The use of biochemical markers for diagnosis of the acute coronary syndromes

It was recognized very early that the specificity of AST, LDH, and CK was not high for cardiac diseases, as injury to the liver and skeletal muscles and hemolysis produced falsely positive results. Thus the measurement of isoenzymes becomes important to improve the specificity of total enzyme measurements. Assays for CK and LD isoenzymes were initially developed using electrophoresis. Because this assay was time consuming and difficult to automate, imunooinhibition assays were soon developed. The development of immunoassay measurements enabled determination of the enzyme concentration which was not dependent on whether or not the enzyme was active. These "mass assays" also enabled the measurement of protein markers which are themselves not enzymes. This led to the investigation of many other markers for potential use in cardiac diseases. With changing clinical practices, cardiac markers are now needed to detect the presence of minor myocardial infarction in patients with unstable angina. Outcome studies have shown that patients with increased troponin are at high short-term risk for death and AMI. The success of new cardiac markers such as troponin is due to their high cardiac specificity and the existence of assays with low detection limits.

Troponin I as a Diagnostic Marker of a Perioperative Myocardial Infarction in the Orthopedic Population

Study Objective: To assess the utility of troponin I, the only molecular marker of myocardial injury not expressed in regenerating muscle, in diagnosing perioperative myocardial infarction (MI) in the setting of orthopedic surgery where false elevations in creatine kinase MB isoenzymes (CKMB) are known to occur. Design: Prospective study. Setting: University-affiliated hospital. Patients: 85 patients with risk factors for coronary artery disease (CAD) who were scheduled for orthopedic surgery, including total knee arthroplasty, 34; total hip arthroplasty, 36; posterior spine fusion, 7; and other orthopedic operations, 8. Interventions: Patients were observed in the postanesthesia care unit for at least 24 hours where they had an electrocardiogram (ECG) performed, and blood drawn to rule out MI. Measurements: Blood samples for measurement of creatine kinase MB isoenzymes (CKMB) and troponin I were drawn at 8-hour intervals for up to 24 hours. Main results: Five (5/85) patients had elevated levels of both CKMB and troponin I postoperatively. New ECG abnormalities were present in all but one patient who had an old anterolateral MI. Troponin I peaked within 16 hours except in one patient where it continued to increase. That female patient developed cardiogenic pulmonary edema. All the others did well clinically. Six patients (6/85) had a positive CKMB index, and a negative troponin I level. None had ECG changes, except for one in whom subsequent cardiac catheterization showed insignificant CAD. They all did well clinically. All patients with an elevated troponin I level had a positive CKMB index. Conclusions: Troponin I is as sensitive a marker of MI as CKMB in the orthopedic population, but it has a higher specificity in the perioperative setting. Troponin I can be helpful in properly identifying the source of CKMB elevation postoperatively when this elevation is questionable.

Increased mortality after coronary artery bypass graft surgery is associated with increased levels of postoperative creatine kinase-myocardial band isoenzyme release: Results from the GUARDIAN trial

OBJECTIVESWe sought to determine if elevated cardiac serum biomarkers after coronary artery bypass graft surgery (CABG) are associated with increased medium-term mortality and to identify patients that may benefit from better postoperative myocardial protection.BACKGROUNDThe relationship between the magnitude of cardiac serum protein elevation and subsequent mortality after CABG is not well defined, partly because of the lack of large, prospectively studied patient cohorts in whom postoperative elevations of cardiac serum markers have been correlated to medium- and long-term mortality.METHODSThe GUARD during Ischemia Against Necrosis (GUARDIAN) study enrolled 2,918 patients assigned to the entry category of CABG and considered as high risk for myocardial necrosis. Creatine kinase-myocardial band (CK-MB) isoenzyme measurements were obtained at baseline and at 8, 12, 16 and 24 h after CABG.RESULTSThe unadjusted six-month mortality rates were 3.4%, 5.8%, 7.8% and 20.2% for patients with a postoperative peak CK-MB ratio (peak CK-MB value/upper limits of normal [ULN] for laboratory test) of <5, ≥5 to <10, ≥10 to <20 and ≥20 ULN, respectively (p < 0.0001). The relationship remained statistically significant after adjustment for ejection fraction, congestive heart failure, cerebrovascular disease, peripheral vascular disease, cardiac arrhythmias and the method of cardioplegia delivery. Receiver operating characteristic curve analysis revealed an area under the curve of 0.648 (p < 0.001); the optimal cut-point to predict six-month mortality ranged from 5 to 10 ULN.CONCLUSIONSProgressive elevation of the CK-MB ratio in clinically high-risk patients is associated with significant elevations of medium-term mortality after CABG. Strategies to afford myocardial protection both during CABG and in the postoperative phase may serve to improve the clinical outcome.

Isolation and measurement of carbonic anhydrase isoenzymes in erythrocytes of dogs.

To purify canine carbonic anhydrase (CA) isoenzymes CA-I and CA-II and to determine concentrations of CA-I and CA-II in erythrocytes of Beagles and dogs native to Japan. Blood samples from 116 Beagles, including 24 pregnant Beagles, and blood samples from 29 dogs native to Japan. Canine CA-I and CA-II were purified by use of column chromatography. Concentrations of CA-I and CA-II in erythrocytes of dogs were determined, using an ELISA. Mean (+/- SD) concentrations of CA-I and CA-II in erythrocytes of Beagles were 3.21+/-0.86 and 1.63+/-0.39 mg/g of Hb, respectively. Mean concentration of CA-I was greater in male Beagles than female Beagles. In contrast, mean concentration of CA-II was greater in female Beagles than male Beagles. Furthermore, concentration of CA-II was greater in pregnant female Beagles than male or nonpregnant female Beagles. Mean concentrations of CA-I and CA-II in erythrocytes of dogs native to Japan were 11.03+/-4.39 and 3.29+/-0.91 mg/g of Hb, respectively. Mean concentration of CA-I was greater in male dogs from Japan than female dogs from Japan. The ELISA used in this study proved to be precise and sensitive for determining CA-I and CA-II concentrations in dogs. The ELISA may enable study of changes in isoenzymes associated with hereditary or metabolic disorders of blood or other body fluids, using only a small sample. Measurement of the concentrations of CA isoenzymes in dogs may be of diagnostic value.

Standardizing utilization of biomarkers in diagnosis and management of acute cardiac syndromes

Publications on the development and use of myocardial markers have exploded in the decade of the 1990s. According to subscriptions to proficiency testing surveys, enzymatic measurement of creatine kinase and lactate dehydrogenase isoenzymes have largely been replaced by CK-MB mass immunoassays. Laboratories reporting use of myoglobin and cardiac troponin have increased tremendously over the past few years. In this field of medicine where there have been dramatic changes, development of consensus guidelines can be helpful to provide assistance to clinicians and laboratorians as to how they can make the best use of new cardiac markers for clinical practice.

Role of cardiac troponin I in the evaluation of myocardial injury

Cardiac troponin I (cTnl) is highly specific for cardiac muscle. In this study, we compared the utility of CK and CK-MB index versus cTnl in the assessment of myocardial infarction in 155 patients being evaluated for myocardial damage. As a cardiac marker for MI, Troponin I seems to be superior to CK-MB. In the subset of patients with renal disease, cTnl has definite advantages over CK-MB. In addition, the use of cTnl has the potential to replace the measurement of lactate dehydrogenase isoenzymes.

Cardiac Troponin T in Serum as Marker for Myocardial Injury in Newborns

In neonates, acute perinatal asphyxia may lead to ischemic myocardial damage (1). In some cases, subendocardial infarction has been documented (2). Generally, diagnosis of the myocardial injury (MI) is based on clinical findings, suggestive electrocardiographic and echocardiographic patterns (3), decrease in myocardial uptake of thallium (4), and classical creatine kinase (CK)-MB isoenzyme measurement (5). However, CK-MB in serum cannot be regarded as a cardiac-specific marker in the neonate, and extreme caution should be used in the interpretation of increased CK-MB activity during this period (6). Cardiac troponin T (cTnT), the structural protein that binds the troponin complex to the tropomyosin molecular strand, has recently been proposed as a more specific biochemical marker for diagnosis of myocardial infarction in the adult population (7). Here we evaluated the use of cTnT measurement in serum in the diagnosis of MI in newborns, as well as that of the determination of CK-MB mass concentration by a sensitive and specific monoclonal anti-CK-MB antibody-based immunoassay. Three groups of infants were studied. Group I consisted of 27 preterm infants (gestational age ranging from 28 to 36 weeks) without major respiratory and cardiovascular dysfunctions. Group II was 27 healthy full-term newborns (15 born by vaginal delivery and 12 by cesarean section) with a mean gestational age of 39.7 weeks. Group III was composed of seven infants (four preterm and three term) who demonstrated, during the first 3 days after birth, clinical, electrocardiographic, and echocardiographic signs of MI. In particular, in electrocardiogram (ECG) evaluation, MI was considered to be present when inversion of T waves or ST-segment depression ≥1 mm in more than two precordial leads was noted. Groups I and II underwent a clinical examination, ECG, echocardiogram, and blood collection for the measurement of total CK, CK-MB, and cTnT on day 2 after birth. Group III was evaluated …

Hyperamylasemia after cardiac surgery in infants and children.

This study was conducted to clarify the incidence of hyperamylasemia after cardiac surgery in infants and children. 186 infants and children operated on at Children's Hospital. Helsinki, during an 11-month period were enrolled in the study. Serum samples were taken before and on 3 consecutive days after cardiac surgery at the intensive care unit and before discharge from the hospital. We measured serum total amylase and serum pancreatic amylase with two different assays (1) reduction of salivary amylase from total amylase activity and (2) measurement of mass concentration with monoclonal antibodies. Preoperative values for both total amylase and pancreatic isoenzymes were strongly age-related. At least one of the three tests showed postoperative hyperamylasemia (> +/- 2 SD above starting values of the age group and maximal value > 3 times the individual starting value) in 64/186 (34%) patients. 22/186 (12%) patients had abnormal results in all assays. A more than tenfold rise in pancreatic amylase, suggesting pancreatitis, was found in 14 patients (8%). Mortality was 21% in this subgroup, but 5% in the rest of the patients. Hyperamylasemia was more common after 1 year of age, and after open-heart surgery, especially homograft implantation or cardiac transplantation. Hyperamylasemia is a common finding after cardiac surgery in pediatric patients. Amylase isoenzyme measurements are needed for clinical decision making. Age-group-related reference values are mandatory for the right interpretation of amylase values.

Bone alkaline phosphatase isoenzyme in renal osteodystrophy.

Serum total alkaline phosphatase is the most commonly used biochemical marker of bone disease in renal patients, but alkaline phosphatase originates from different organs and sometimes lacks specificity. Bone isoenzyme measurement is considered superior to total alkaline phosphatase for the assessment of bone metabolism. We have studied the value of bone isoenzyme, determined by a new. IRMA (Tandem-R-Ostase), in haemodialysis patients with secondary hyperparathyroidism and renal osteodystrophy. Fifty-six haemodialysis patients were studied. Intact parathyroid hormone (PTH), osteocalcin, total alkaline phosphatase and bone alkaline phosphatase were determined. A transiliac bone biopsy was performed in 20 of the 56 patients after double tetracycline labelling. There was a significant correlation between bone alkaline phosphatase and PTH (r = 0.79, P < 0.001) and between bone and total alkaline phosphatase (r = 0.84, P < 0.001) in all patients. The patients who underwent a bone biopsy showed osteitis fibrosa in 17, mixed lesion in one, adynamic bone disease in one and normal bone in one. Bone alkaline phosphatase showed a significant correlation with static and dynamic histomorphometric indices similar to that obtained with PTH and better than those of total alkaline phosphatase and osteocalcin. It is concluded that bone alkaline phosphatase (ostase) seems to be a useful non-invasive marker of bone metabolism in patients on haemodialysis with high turnover bone disease. More studies are necessary to know its value in low turnover bone disease.

Alteration of superoxide dismutase activity in the anterior horn in motoneuron disease patients

Studies on anterior horn dissected from MND patients have demonstrated that MnSOD activity is increased when expressed as a percentage of total SOD activity when compared to controls. Total SOD activity (mean ± SD) in controls = 414 ± 105 U mg −1 tissue and in MND patients = 373 ± 115 U mg −1 tissue, whereas CuZnSOD activity was higher in controls (290 ± 91 U mg −1 tissue) than in MND patients (197 ± 81 U mg −1 tissue). The reduction in CuZnSOD was compensated for by a concomitant elevation of MnSOD activity (control = 123 ± 40.5 U mg −1 tissue, MND patients = 175 ± 47 U mg −1 tissue). These data not only show an important deficit in anterior horn cell body protection against free radical-mediated cell damage, but are also are the first measurements of SOD isoenzymes in anterior horn neurones.

Automated measurement of alpha-amylase isoenzymes with 6(3)-deoxymaltotriose as selective amylase inhibitor

We developed an automated method for measurement of alpha-amylase isoenzymes in serum by a single kinetic assay (SKA) and a double kinetic assay (DKA) with 2-chloro-4-nitrophenyl-6(5)-azido-6(5)-deoxy-beta-maltopentaoside as a substrate and 6(3)-deoxymaltotriose (DOG3) as a novel selective amylase inhibitor. DOG3 showed a large difference in inhibitory activity between human pancreatic alpha-amylase (HPA; 86.9% inhibition) and salivary alpha-amylase (32.1% inhibition) at 0.33 mmol/L. Constant inhibition was obtained immediately after addition of DOG3. The inhibitory effect did not change with variation in concentrations of amylase up to approximately 3000 U/L. The results obtained by SKA correlated well with those obtained by three methods: monoclonal antibody (r = 0.988), wheat germ inhibitor (r = 0.989), and DKA (r = 0.995). The within-run and between-run CVs for HPA were 0.63-2.32% on SKA, 0.69-1.81% on DKA. No significant interferences by endogenous serum compounds were observed with the proposed methods.