Cancer, pancreatitis, and the detection of the isoenzymes of DNAase, RNAase and amylase.

Substrate films of starch, RNA, and DNA were used to identify the isoenzymes of amylase, RNAase, and DNAase found in human ductal pancreatic juice subjected to isoelectric focusing. The pancreatic secretions from 15 patients were shown to contain as many as four isoenzymes of RNAase; the two major forms had isoelectric points of 7.87 and 7.52, and the two minor forms, of 7.25 and 6.90. Six DNAase bands were detected; the major bands had pI values of 4.86 and 4.79, and sometimes appeared as one band. The minor bands had pI values of 5.08, 5.00, 4.68, and 4.58. Purified bovine DNAase I, analyzed similarly, showed four bands (5.29, 5.19, 5.04 and 4.96). Nine isoenzymes of alpha-amylase were observed in the secretions from 15 patients. The major alpha-amylase isoenzyme had a pI value of 6.84 in 14 patients and of 7.04 in 1 patient. Secondary bands were seen with pI values of 6.23, 6.53 and 6.69. Additional isoenzymes were found with pI values of 7.16, 6.39, 6.00 and 5.78. The amylase isoenzyme with a pI value of 6.39 was found in 7 of the 8 patients with a normal pancreas or carcinoma of the pancreas, and in only 1 of 7 patients with acute or chronic pancreatitis.     

Electrophoretic amylase fractionation as an aid in diagnosis of pancreatic disease.

Six alpha-amylase (EC 3.2.1.1) isoenzymes have been resolved electrophoretically on cellulose acetate membranes in a discontinuous buffer system. The fastest migrating isoenzymes are of salivary origin (S1, S2, S3), the slower ones of pancreatic origin (P1, P2, P3). We determined the amylase isoenzyme distribution in the sera of 240 subjects. A specific pancreatic isoenzyme (P3) was observed in all clinically diagnosed cases of acute or chronic pancreatitis as well as in 15 of 40 renal-transplant patients. Moreover, P3 isoenzyme activity declined during apparent recovery from pancreatitis. The P2 isoenzyme appeared in 95% of all specimens, P1 in only 2%. The pancreatic isoenzymes were preferentially excreted in the urine of both renal-transplant patients and normal individuals. The major salivary isoenzyme, S1, was observed in 95% of all serum and urine samples; however, the S2 and S3 appeared less consistently. Our method is simple and rapid, and quite applicable for use in clinical evaluation of patients with pancreatitis or with certain nonpancreatic dysfunctions.     

Quantitative gel-electrophoretic determination of serum amylase isoenzyme distributions.

I report a direct, sensitive, quantitative method for determining serum amylase isoenzyme activity with commercially available reagents. Day-to-day reproducibility (CV) was 3--4% for the isoenzymes in normal serum; within-run precision was 8, 3, and 2% for low, normal and high isoenzyme activities. Amylase isoenzymes, separated into the pancreatic and salivary types by electrophoresis in polyacrylamide gel, are then quantified by directly incubating the gels in soluble-starch solution, staining with iodine, and densitometry. The proportion of pancreatic isoenzyme (47 normal sera) was 43 +/- 8% (mean +/- SD). Isoenzyme activities as low as 2% of normal can be measured accurately in 10 micro L of serum. The reproducibility, precision, and sensitivity indicate that the method is applicable to differential diagnosis of hyperamylasemia or hypoamylasemia, and is suited for monitoring the subtle changes in serum amylase isoenzyme distribution that may accompany disease progression or therapy.     

Electrophoretic patterns of alkaline phosphatase isoenzymes in human sera with abnormally high activity, and an unusual band observed in sera of patients with pancreatic cancer.

Alkaline phosphatase isoenzymes in sera were resolved by electrophoresis on cellulose acetate membranes into seven different bands (L1, B, Pl, L2, l1, l2, and Pa, in decreasing order of electrophoretic mobility). The slowest moving band (Pa) was observed in the sera of 16 patients--15 with cancer of the pancreas and one with hemochromatosis. Sera of 50 other patients with malignant or benign diseases did not show the Pa band. The Pa band is more heat labile than is the liver isoenzyme (L1). Its behavior toward inhibitors (L-phenylalanine and L-homoarginine) is similar to that of L1. Sera containing the Pa band exhibit a diffuse band in the region where isoenzymes of intestinal origin migrate; however, its heat stability and sterospecific inhibition are different from those of intestinal isoenzymes in sera that show no Pa band.     

Creatine kinase and lactate dehydrogenase isoenzymes in serum and tissues of patients with stomach adenocarcinoma

Total activities of creatine kinase (EC 2.7.3.2; CK) and lactate dehydrogenase (EC 1.1.1.27; LD) and their isoenzymes were estimated in serum and tissue samples from patients with stomach adenocarcinomas who were to undergo gastric resection. Total CK activity (U/g protein) appeared to be markedly decreased in neoplastic stomach tissue. CK-BB was the predominant isoenzyme in both neoplastic and normal stomach tissues; however, the CK-BB/total CK ratio was increased in adenocarcinoma tissue. Macro CK type 1 was found in two neoplastic tissues and macro CK type 2 in 11. LD4 and LD5 isoenzymes were predominant in gastric tissues, but LD5 and the LD5/LD1 ratio were higher in adenocarcinoma tissue. At 24 h before surgery, CK-BB was demonstrated in sera of all patients and CK-MB in 69%. The CK-BB probably originated from neoplastic stomach tissue, which contains high CK activity, with BB isoenzyme predominating. After gastrectomy, CK and LD isoenzymes in sera were markedly increased by 24 h postsurgery. These alterations were attributed to release from damaged tissue during gastric resection.     

Alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) activity in the sera of patients with liver cancer

The principal enzymes catalyzing the conversion of ethanol to acetate are alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). The activities of these enzymes are elevated in the serum during the course of alcoholism or cirrhosis. In previous investigations we have found elevated levels of ADH, ALDH, and class I ADH activity in liver cancer cells. It can suggest that these changes may be reflected by enzyme activity in the serum. In this work, the activity of ADH isoenzymes, and ALDH in the sera of patients with liver cancer was measured. Serum samples were taken from 64 patients (28 drinkers, 36 nondrinkers), with liver cancer. 25 patients had primary and 39 metastatic liver tumors. Total ADH activity was measured by photometric method with p-nitrosodimethylaniline (NDMA) as a substrate and ALDH activity by the fluorimetric method with 6-methoxy-2-naphtaldehyde as a substrate. For the measurement of the activity of class I and II isoenzymes we employed the fluorimetric methods, with class-specific fluorogenic substrates. The activity of class III ADH was measured by the photometric method with formaldehyde and class IV with m-nitrobenzaldehyde as a substrate. A statistically significant increase of class I ADH isoenzymes was found in the sera of cancer patients. The median activity of this class isoenzyme in the total cancer group increased about 51% (2.94 mU/L) in the comparison to the control level (1.43 mU/L). The activity of the class I ADH isoenzyme was significantly higher in the sera of patients with metastatic tumors than with primary cancers. The activity of this class in the sera of drinkers and group of moderate drinkers was significantly higher in comparison to the control group and higher in the sera of heavy drinkers when compared with moderate drinking patients. The total ADH activity was significantly higher (44%) among patients with cancer than healthy ones. The activity of class I ADH isoenzymes was elevated only in the serum of patients with metastatic liver cancer. This increase of activity seems to be caused by the enzyme released from liver cancer cells and primary tumors originating in other organs.     

Quantitative fractionation of alkaline phosphatase isoenzymes according to their thermostability.

Continuous monitoring of heat denaturation of a mixture of alkaline phosphatase isoenzymes at 60 degrees C and pH 7.5 permits the simultaneous direct identification and quantitation of three isoenzymes: the placental isoenzyme, the L-phenylalanine-sensitive intestinal isoenzyme, and the liver isoenzyme (hepatocytic). The isoenzyme that is principally of bone origin cannot be identified as such without the help of other diagnostic aids and the patient's medical history. All human tissues contain alkaline phosphatase, many organs more than one of the isoenzymes. Liver alkaline phosphatase, which constitutes 40-50% of normal serum alkaline phosphatase activity, was measured in the serum of persons with various liver diseases. Its activity exceeded normal in all types of liver disease; in 80% of cases this increase was accompanied by increased gamma-glutamyl-transferase activity, but the quantitative correlationship (r = 0.54) was not as good as expected if both enzymes come from the same source and are indices of liver dieases. Liver alkaline phosphatase activity increases in the blood early in liver disease, before most liver tests show abnormalities. The other major isoenzyme of normal serum probably represents a mixture of isoenzymes from bone and reticulo-endothelial and vascular tissues, which all contain the same "very heat-labile" alkaline phosphatase. Cord blood and children's sera contain mostly this very heat-labile isoenzyme.     

Changes in mass and catalytic activity concentrations of aspartate aminotransferase isoenzymes in serum after a myocardial infarction

We used an RIA and inhibition of enzyme activity to monitor the changes in mass and catalytic concentrations of the aspartate aminotransferase (EC 2.6.1.1;AST) isoenzymes in serum after myocardial infarction. Cytosolic (c-AST) and mitochondrial (m-AST) forms of AST were present in sera from all 38 of our patients. Although the immunological and catalytic concentrations of both isoenzymes correlated well with the size of the infarct, c-AST gave a better measure than did m-AST. About 20% of the total enzyme activity at peak activity was from the mitochondrial isoenzyme. Both isoenzyme activities peak at very nearly the same time, but m-AST has the longer half-life. Immunological evidence of the mitochondrial isoenzyme can be detected in serum for at least eight days after the infarct. The presence of left ventricular failure produces greater serum isoenzyme activities than in those without failure.     

碱性磷酸酶同工酶对原发性胆汁性肝硬化的诊断价值初探

目的　初步探讨血清碱性磷酸酶(ALP)同工酶对原发性胆汁性肝硬化(PBC)的辅助诊断价值。方法 　用Sebia琼脂糖凝胶电泳分离30名正常人、23例PBC患者和25例其他肝胆疾病患者的血清ALP同工酶,经 光密度扫描仪扫描后,分析各型同工酶的相对含量。结果　血清ALP同工酶经电泳后可分为肝型、胆汁型、肠 型和骨型4个区带。PBC患者的肝型ALP的相对含量最高,约为56%～70%,显著高于正常对照及乙肝后肝硬 化患者(P<0.05),而其胆汁型ALP的相对含量显著低于肝外阻塞性黄疸患者(P<0.05)。结论　ALP同工酶 分析可辅助诊断PBC。     

Separation and analysis of arylsulfatase isoenzymes in body fluids of man.

Soluble arylsulfatase (EC 3.1.6.1) is present in the body fluids of man in the form of two isoenzymes, arylsulfatase A and B, which reportedly are useful biochemical markers for certain types of malignancy. However, rapid assay of the individual isoenzymes is extremely difficult; procedures based on differential inhibition or activation of the isoenzymes in a mixture yield only semiquantitative results. A feature of these isoenzymes is their inhibition by some common anions (notably phosphate) at physiologic concentrations. The isoenzymes can be separated by anion-exchange chromatography, the B isoenzyme being eluted in the void volume and the A isoenzyme and the anionic inhibitors retarded. Lead is used to sequester phosphate, enabling measurement of A in the salt-eluted fraction. Using this technique, we have found significant elevations of B in the sera of patients with colorectal cancer. The potential of rapid, chromatographic separation coupled with continuous monitoring for arylsulfatase activity is discussed.     

Serum type-2 macro-creatine kinase isoenzyme is not a useful marker of severe liver diseases or neoplasia

We studied the creatine kinase (CK) isoenzyme pattern in sera from 332 patients affected by hepatic cirrhosis and several neoplastic diseases (102 cirrhosis, 36 hepatocarcinoma, 16 metastatic liver tumor, 40 breast cancer, 18 other neoplastic diseases and 120 cases of leukemia or lymphoma) to evaluate both its diagnostic utility for cancer diagnosis and its power as a prognostic index. Type-2 macro CK (mitochondrial creatine kinase) was detected, with no statistical difference in cirrhosis (14%), hepatocarcinoma (16%), metastatic liver tumor (31%), breast cancer (5%) and other tumors (6%). It was not detected in any patient with leukemia or lymphoma. The presence of type-2 macro CK was unrelated to the stage of either cirrhosis or hepatocarcinoma, according to Child and Okuda, respectively, nor was it correlated to serum cytolytic enzyme levels or to gamma-globulin levels. In cirrhotics, type-2 macro CK was not linked to serum levels of the following tumor markers: alpha-fetoprotein, pseudouridine and gamma-glutamyltransferase isoenzymes complexed to low-density lipoprotein. In addition, the atypical band persisted in several patients with cirrhosis monitored for six months who did not show any evidence of evolution toward hepatocarcinoma. Thus, type-2 macro CK has poor diagnostic sensitivity for neoplastic diseases, and lacks prognostic value both in cirrhosis and neoplastic diseases.     

Quantitation of creatine kinase isoenzymes in human tissues and sera by an immunological method

Antisera against the crystallized creatine kinase isoenzymes from human skeletal muscle (MM) and from human brain (BB) were produced in rabbits. Both the MM and BB isoenzymes were precipitated quantitatively by their homologous antisera. No cross-reaction was observed. The hybrid MB from human heart muscle could not be precipitated completely by either of the two antisera. In artifical mixtures the concentrations of individual creatine kinase isoenzymes were determined from the percentage of non-precipitable activity in the supernatant after reaction with each of the antisera.This immunotitration assay was applied to study the quantitative distribution of creatine kinase isoenzymes in extracts of human tissues. The isoenzyme patterns obtained were compared with those determined by electrophoretic analysis.In sera of patients with myocardial infarction, the immunotitration assay allowed the sensitive and rapid quantitation of creatine kinase isoenzymes, especially of the “infarct-specific” hybrid MB, even in sera with low total activity. This indicates that the method is of diagnostic value.     

Alteration in isoenzyme patterns of serum galactosyltransferase activity in ovarian cancer patients: preliminary results

Isoelectric focusing on agarose gel was used to separate the isoenzymes of serum galactosyltransferase (uridine diphosphogalactose: N-acetylglucosaminyl galactosyltransferase, EC 2.4.1.22) from 8 healthy women, and 11 ovarian cancer patients of whom 4 were in clinical remission. In all cases, we found 7 major peaks with isoelectric points ranging from 4.0-5.4. The most acidic peaks were preferentially elevated in the tumor-bearing patients, particularly the peak with pI 4.44.     

Spurious brain creatine kinase in serum from patients with renal disease.

Creatine kinase isoenzyme I(BB) is generally not detectable in normal serum, and its occurrence in serum has been documented in only a few disease states. In particular, increased activity of this isoenzyme has been reported in association with chronic renal failure, hemodialysis, and renal transplantation. The present study demonstrates that the apparent creatine kinase observed in the serum of such renal patients is an artifact, observed as a result of measuring creatine kinase isoenzymes by fluorescence. Our observations resemble those of McKenzie et al. [Clin. Chim. Acta 70, 333(1976)] concerning an artifact in the fluorometric determination of lactate dehydrogenase isoenzymes in the sera of patients with end-stage renal failure. The artifact binds to albumin, is not a protein, and occurs in some normal sera at very low concentrations. This artifact can be mistakenly identified as isoenzyme I in renal-disease patients if CK isoenzymes are determined fluorometrically.     

Clinical relevance of alkaline phosphatase isoenzyme determinations by high performance liquid chromatography

A new method for the separation of alkaline phosphatase isoenzymes by means of high performance liquid chromatography (HPLC) is presented. One isoenzyme was identified in homogenate of small intestine, two were identified in bone, and two in liver, and fragment and biliary isoenzymes were identified in bile. Sera from 32 patients with different diseases of the skeletal system or the liver were analysed. High activities of the bone isoenzymes were detected in bone diseases, of the second liver isoenzyme in acute hepatitis and of the first liver and biliary isoenzymes in biliary obstruction. There are indications that the first liver isoenzyme is derived from the cell membrane and the second liver isoenzyme from the cytosol. The biliary isoenzyme is considered to be a highly sensitive and specific indicator for cholestasis.     

The origin of the elevated activities of creatine kinase and other enzymes in the sera of patients with myxoedema.

An attempt has been made to establish the origin of the elevated serum creatine kinase which occurs in most patients with myxoedema. Parallel determinations of a number of other serum enzymes were made but the incidence of elevated values was appreciably less than in the case of creatine kinase. Rather surprisingly, the serum amylase activity was found to be increased in more than 50% of the patients studied. Creatine kinase isoenzymes were separated by starch-gel electrophoresis of the sera of 26 patients with myxoedema. In 25 the MM isoenzyme only could be identified while the remaining serum also contained a trace of the MB fraction. Similar isoenzyme studies were made with the sera of normal and thyroidectomized rats, all of which are shown to contain all three isoenzymes. (MM, MB and BB) irrespective of thyroid functional status. No consistent difference was apparent between the patterns exhibited by the thyroidectomized and control groups, and it was concluded that thyroidectomized rats cannot be regarded as a suitable experimental model for the study of this aspect of human hypothyroidism. It is suggested that enzyme release in myxoedema is a non-specific effect, possibly die to diminution in the ATP content of tissues generally. The greater incidence of creatine kinase elevation is probably due to the relatively high concentrations of this enzyme in skeletal muscle, the mass of which is much greater than that of any other tissue.     

Alkaline phosphatase isoenzymes in serum determined by high-performance anion-exchange liquid chromatography with detection by enzyme reaction

This rapid, reproducible method for separating and determining individual alkaline phosphatase (EC 3.1.3.1) isoenzymes in serum is based on high-performance liquid chromatography with a weak anion-exchange column (SynChropak AX 300). The isoenzymes so resolved are detected by using an on-line enzyme reaction followed by spectrophotometric monitoring at 405 nm of the 4-nitrophenol formed. Complete diagnostic profiles of the various isoenzymes present in normal and pathological sera are obtained within 20 min. The mean (and SD) normal concentrations of the bone B1 and intestinal isoenzymes in serum of adults were 3.7 (4.3) and 4.5 (3.9) U/L, respectively (n = 14), and of the bone isoenzyme B2 and liver isoenzymes L1 and L2, 5.8 (8.6), 33.0 (10.6), and 12.0 (4.8) U/L, respectively (n = 17). Concentrations of the B2 and L1 isoenzymes in adults over age 40 years differed significantly from those in adults younger than 40 years, that of bone isoenzyme being lower (P less than 0.05) and that of the liver isoenzyme being higher (P less than 0.001) in the younger adults.     

Cobalamin malabsorption due to nondegradation of R proteins in the human intestine. Inhibited cobalamin absorption in exocrine pancreatic dysfunction.

In vivo studies demonstrate that the pancreatic enzymes and the ionic environment in the upper gastrointestinal tract are essential determining factors for transport and absorption of cobalamin in man. Jejunal fluid was aspirated from healthy human volunteers after administration of cyano[57Co]cobalamin preparations. Immunochemical analysis of the aspirates demonstrated that all isotopic vitamin was transferred to a protein that is identical to the gastric intrinsic factor in terms of molecular mass (57,500), ionic nature (mean pI, 5.09), and reactivity with anti-intrinsic factor sera. However, in the aspirates from patients with exocrine pancreatic dysfunction the vitamin was found to be coupled > 60% to a protein identical to R proteins in terms of molecular mass (125,000), ionic nature (mean pI, 3.51), and reactivity with anti-R protein and anti-intrinsic factor sera. The preferential transfer of cobalamin to R proteins in the patients and to intrinsic factor in healthy subjects was associated, respectively, with low and normal levels of pancreatic enzymes in the intestine and these in turn were paralleled respectively by impaired and normal ileal absorption of cobalamin. These findings confirm the suggestion that the formation of unabsorbable cobalamin complexes may be the reason of impaired vitamin absorption in exocrine pancreatic insufficiency. Observations made with other selected patients demonstrate: (a) that decreased enzyme activity and nondegradation of R proteins may also be due to nonactivation of pancreatic zymogens in an acidic pH of the intestinal juice the vitamin transported to the jejunum couples to intrinsic factor when pancreatic function is normal, and to intrinsic factor and R protein in exocrine pancreatic insufficiency. The observations made with these selected patients may explain why not all patients with exocrine pancreatic insufficiency develop imparied cobalamin absorption, and also why the malabsorption is corrected by the administration of bicarbonate in certain patients.     

Serum adenosine deaminase: isoenzymes and diagnostic application.

Human adenosine deaminase (ADA; EC 3.5.4.4) consists of three isoenzymes: ADA1, ADA1+CP, and ADA2. We developed an electrophoretic technique to distinguish between these three isoenzymes. The isoenzyme pattern was studied in tissue and cell homogenates, as well as in serum from normal subjects and from patients with increased serum ADA who had either hepatitis, infectious mononucleosis, tuberculosis, pneumonia, rheumatoid arthritis, or acute lymphoblastic leukemia (ALL). The highest ADA activity was found in lymphocytes and monocytes. ADA2 could be detected only in monocytes (18% of total ADA activity). It was also the predominant isoenzyme in the sera of controls and all disease groups, except for ALL--the only condition evaluated that is not of an inflammatory nature. We conclude that serum ADA reflects monocyte/macrophage activity or turnover in most diseases studied. The exception is ALL, where serum ADA most probably originates from lymphocyte precursors.     

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

Objective: 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. Methods: 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. Results: 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. Conclusion: 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. J. Clin. Lab. Anal. 24:334–339, 2010. © 2010 Wiley‐Liss, Inc.