Comparison of alkaline phosphatase isoenzymes determined by an inhibition method and by electrophoresis

A chemical inhibition procedure suitable for the routine determination of alkaline phosphatase (AP) isoenzymes in serum has been adapted for use with a fast kinetic analyzer, System Olli 3000. The results of this procedure are compared with the electrophoretic separation of alkaline phosphatase isoenzymes. The comparison of the results obtained indicates that the AP-urea/AP ratio can be used to differentiate between patients with bone and liver disease and that it is possible to estimate the relative bone and liver isoenzyme activities from this ratio quickly using two simple equations.     

Isoelectric focusing of neuraminidase-treated alkaline phosphatase isoenzymes on agarose gel

We attempted to separate bone and liver alkaline phosphatase (EC 3.1.3.1) isoenzymes in human serum by isoelectric focusing on agarose gel. We found that in a pH 3-10 gradient the liver and bone isoenzymes focused into so many bands over a narrow pH range such that the information could not be quantified. However, when the bone isoenzyme in serum was first desialylated at 37 degrees C for a minimum of 6 h, catalyzed by neuraminidase (EC 3.2.1.18) at pH 5.8-6.0, we could detect four distinct bands with pls of 6.7, 6.8, 6.9, and 7.0. Under the same conditions, the liver isoenzyme in human serum focused into one band at pH 7.0. The multiple banding we observed for the desialylated bone isoenzyme has not been previously reported. The method is suited as a qualitative technique for detecting the bone alkaline phosphatase isoenzyme in serum.     

Affinity electrophoresis of alkaline phosphatase using polyacrylamide gels.

A method is described for the separation of liver and bone isoenzymes of alkaline phosphatase in serum using wheat germ lectin affinity electrophoresis in a polyacrylamide gel matrix. The electrophoretic mobilities of liver and intestinal isoenzyme are essentially not affected by lectin, but the bone enzyme is retarded and separated from the liver fraction. Affinity electrophoresis in polyacrylamide gel, combined with agarose gel electrophoresis, and a solid-phase linked antibody precipitation procedure for intestinal alkaline phosphatase allowed the various isoenzyme fractions, biliary, liver, bone and intestinal, to be quantitated.     

Separation and identification of alkaline phosphatase isoenzymes and isoforms in serum of healthy persons by isoelectric focusing

We have developed an isoelectric focusing procedure for resolving alkaline phosphatase (EC 3.1.3.1) isoenzymes and isoforms in serum. We use a thin-layer agarose gel film containing synthetic carrier ampholytes and a "separator" to flatten the pH gradient in the region of the isoenzyme and isoform isoelectric points. Sharp, highly resolved zones of enzyme activity are obtained by limiting diffusion; for this we rapidly couple the released product, 1-naphthol, to a diazonium salt, which forms a colored precipitate at the site of activity. We have resolved and identified 12 zones of alkaline phosphatase activity in the serum of ostensibly healthy persons within a wide age range. Theoretically, three basic isoenzymes are produced from independent gene loci: intestinal, placental, and nonspecific tissue alkaline phosphatase. The other zones of activity may be isoforms.     

琼脂糖等电聚焦法测定碱性磷酸酶的同工酶

本文采用琼脂糖等电聚焦法测定了人体组织和健康人血清中的碱性硝酸酶同工酶，发现小肠中至少有一种碱性磷酸酶同工酶的亚型，胎盘中有四种，肝脏中有两种，实验证明，本法的重复性好，分辨率高，灵敏度高，最低检测限要达２５Ｕ／Ｌ，克服了国外报道中常有的区带扩散，分辨率低等弊病。     

Simultaneous separation of serum creatine kinase and lactate dehydrogenase isoenzymes by ion-exchange column chromatography.

Lactate dehydrogenase isoenzymes were partially separated by use of a previously described column technique for creatine kinase [Clin. Chem. 20, 36 (1974)]. Extracts of lactate dehydrogenase-rich tissues were used to evaluate column resolution. Samples layered on mini-columns containing DEAE-Sephadex were eluted with Tris-buffered sodium chloride (100 and 200 mmol/liter). Lactate dehydrogenase activity in column effluents was measured by the Wacker method, and their isoenzyme content was assessed by electrophoresis on polyacrylamide gel. Dehydrogenase isoenzymes 3, 4, and 5 were separated from isoenzymes 1 and 2, and the separation was tissue-specific and reproducible. The electrophoretic technique for isoenzymes 3, 4, and 5 gave values about 20% lower than did the column technique. Sera from 15 healthy laboratory technicians contained total lactate dehydrogenase, isoenzymes 1 and 2, and isoenzymes 3, 4, and 5 in the ranges 94 to 152, 34 to 64, and 38 to 75 U/liter, respectively. Activities of sera from 15 patients with acute myocardial infarction (total lactate dehydrogenase) ranged from 212 to 800 U/liter and lactate dehydrogenase isoenzymes 1 and 2 ranged from 138 to 628 U/liter. Lactate dehydrogenase and creatine kinase isoenzymes were rapidly and easily measured after being simultaneously separated. The procedure is specific and sensitive for following the post-infarct time course of changes in isoenzyme activities.     

Clinical significance of acidic pI isoenzyme of ribonuclease in human serum

Employing isoelectric focusing with an Ampholine column, the presence of three major pI isoenzymes (basic, neutral and acidic) of human ribonuclease (RNase) is demonstrated. The acidic pI isoenzyme was observed in pancreatic and hepatic cancer cells and in fetal pancreas and liver tissues, and was frequently detected in the sera of patients with pancreatic and hepatic cancer. This isoenzyme was not, however, detected in the sera of non-cancerous patients. In pancreatic cancer tissues, other pI isoenzymes, except for the acidic one, were markedly suppressed and the isoelectrophoretic patterns of the cancerous tissues closely resembled those obtained from fetal pancreatic tissue. These results suggest that the acidic pI isoenzyme could represent one of the carcinofetal proteins and that the detection of this pI isoenzyme in the patient's serum may be useful for the diagnosis of pancreatic and hepatic cancer.     

Importance of assay conditions in visualization and quantitation of serum alkaline phosphatase isoenzymes separated by electrophoresis

The importance of separation and identification of serum alkaline phosphatase (ALP; E.C. 3.1.3.1) fractions/isoenzymes has been frequently reported. Each serum ALP fraction/isoenzyme quantitation has both practical and theoretical importance. In the present work, serum was collected from Wistar rats and, in identical experimental conditions, total serum ALP activity and serum ALP electrophoretic fractions/isoenzymes activities were quantified. Different results for both kinds of ALP activity were obtained when different buffers or mixture of these buffers (carbonate/bicarbonate; 2-amino-2-methyl-1-propanol/HCl; Veronal, sodium diethylbarbiturate/HCl), pH conditions (9.4 and 10.4) and substrates (alpha- and beta-naphthyl phosphates) were used. Higher total serum ALP activity was always observed with beta-naphthyl phosphate, independently of the buffer (or mixture of buffers) and pH used. Electrophoresis allowed the separation of two serum ALP fractions. Activity of both serum ALP electrophoretic fractions was always higher with beta-naphthyl phosphate, except with carbonate/bicarbonate pH 10.4. The effect of a change in pH was buffer- (or mixture of buffers) and substrate-dependent; the addition of a second buffer (to that previously used) was not always accompanied by an increase or decrease (of the same magnitude) in our results. The results obtained with different buffers (or mixture of buffers) were not identical with substrates and pH values. It is concluded that (i) from the same electrophoretic separation of serum ALP fractions/isoenzymes, different values for its activity can be obtained by changing the assay conditions used for ALP visualization (revelation, staining); (ii) the same assay conditions for quantitation of total serum ALP and serum ALP electrophoretic fractions/isoenzymes should be used; (iii) the choice of assay conditions should take into account the biochemical problem being studied in each case.     

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.     

Human, rabbit, bovine, and porcine creatine kinase isoenzymes are glycoproteins

Human, rabbit, bovine, and porcine creatine kinase (CK) isoenzyme preparations were extensively purified by isoelectric focusing and high-performance liquid chromatography and tested for the presence of carbohydrate. In this study, all the CK isoenzymes demonstrated positive periodic acid-Schiff (PAS) reactions, indicating the presence of carbohydrate. It is concluded that CK is a glycoprotein as a carbohydrate, associated with the M subunit of human, rabbit, bovine, and porcine isoenzymes. Also, carbohydrate is an integral part of the B subunit of both rabbit brain and porcine heart CK. Loss of carbohydrate may be important in enzyme catabolism, yielding a pool of circulating modified CK protein that, to date, has remained undetected by traditional methods.     

The value of combined determination of high molecular mass API and LP-X in the differential diagnosis of intrahepatic and extrahepatic obstruction

Combined determination of serum lipoprotein-X and electrophoretic separation of high molecular mass (HMr) alkaline phosphatase has been proposed as a marker for the differential diagnosis between intrahepatic cholestasis and extrahepatic obstructive jaundice. Of 32 patients who were known to be lipoprotein-X positive and in whom a definitive diagnosis had been made, 13 had intrahepatic cholestasis and 17 extrahepatic obstruction, and 2 had both intrahepatic and extrahepatic obstruction. The detection of HMr alkaline phosphatase isoenzyme proved to be a sensitive and specific test for detecting liver disease, particularly obstructive liver disease. The diagnostic significance of the combined determination of serum lipoprotein-X to demonstrate or exclude cholestasis and electrophoretic separation of HMr alkaline phosphatase isoenzymes to allow differentiation between intrahepatic cholestasis and extrahepatic obstruction was investigated.     

Heterogeneity of serum creatine kinase isoenzyme MM in myocardial infarction: standardization of patterns by use of cord-blood serum

We investigated serum creatine kinase (CK; EC 2.7.3.2) isoenzyme MM in myocardial infarction, using isoelectric focusing in polyacrylamide gels. As many as 14 sub-band species were detected, and sequential sampling revealed a progressive anodal shift in their distribution. A nomenclature for sub-band typing is proposed. It denotes the major sub-bands as 1 (pl 6.91), 2 (pl 6.65), and 3 (pl 6.35); which are usually detected in sera of normal CK range. Abnormal sub-bands (associated with increases in the concentrations of CK) are denoted as a (pl 7.55), b (pl 7.35), c (pl 7.25), d (pl 7.05), e (pl 6.85), f (pl 6.72), g (pl 6.50), h (pl 6.40), i (pl 6.28), j (pl 6.20), and k (pl 6.15). Cord-blood sera gave highly reproducible CK-MM patterns characterized by prominent detection of sub-bands 1-3 and faint detection of c, e, f, g, h, i, and j. It is recommended as a standard for CK-MM sub-band typing.     

Interference by platelets in the electrophoretic determination of enolase isoenzymes in plasma

Serum and plasma gave different electrophoretic patterns when enolase isoenzymes were evaluated by electrophoresis on cellulose acetate. Plasma isoenzyme bands were more intense, and there was an additional one (band P) that was not present in serum. We show that, under the conditions of electrophoresis, some of the residual platelets in the plasma are ruptured, releasing intracellular enolases and consequently leading to intensification of the isoenzyme bands. The band P originated from the remaining unruptured platelets. Thus plasma samples must be platelet-free for determination of enolase isoenzyme to be reliable.     

Erum-induced changes in electrophoretic mobility of creatine phosphokinase isoenzymes?

BB isoenzyme of creatine phosphokinase (CK-BB) obtained from human brain-extract changes its electrophoretic mobility after incubation in human serum at 37° C. No change of electrophoretic mobility of CK-BB is observed after incubation in isotonic saline. We have shown by means of immunoprecipitation with specific antibodies that the structure of CK-BB is not changed. These findings are supported by other authors and make the diagnostic value of electrophoretic separation of CK isoenzymes doubtful as after a 3-h incubation CK-BB migrates similarly to CK-MB and consequently may be misinterpreted.     

Serum isoenzyme pattern of creatine kinase and lactate dehydrogenase in various animal species

The creatine kinase and lactate dehydrogenase isoenzyme pattern were determined in the serum of normal and untreated rats, rabbits, dogs, monkeys and pigs. The relative distribution of all isoenzymes in the serum and an electrophoretic pattern for each animal species are presented. The isoenzyme serum pattern showed a great variation between the species. The diagnostic value of serum creatine kinase isoenzyme MB and lactate dehydrogenase isoenzymes 1 and 2 in predicting cardiac lesions in different animal species is briefly discussed.     

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.     

Analysis for alpha-amylase isoenzymes by automated isoelectric focusing

We describe the fractionation of alpha-amylase (EC 3.2.1.1) isoenzymes by use of the Pharmacia "Phastsystem" electrophoresis apparatus. The separation is done by isoelectric focusing on "Phastgel IEF 5-8." A complete run, including staining, takes 4 h and can easily resolve eight isoenzymes. Samples can be analyzed in a routine laboratory with use of conventional reagents.     

heterogeneity of creatine kinase isoenzyme MM in serum in myocardial infarction: interconversion of the "normal" and "abnormal" sub-bands by glutathione

We used isoelectric focusing (IEF) in polyacrylamide gels to investigate the effects of glutathione on the sub-bands of serum creatine kinase (CK; EC 2.7.3.2) isoenzyme MM in acute myocardial infarction. The intensity of the "abnormal" sub-bands c (pI 7.25), e (pI 6.85), and g (pI 6.50) increased, and that of the "normal" sub-bands 1 (pI 6.91), 2 (pI 6.65), and 3 (pI 6.35) decreased, following serum incubation with reduced glutathione (GSH, final concentration 1.25 mmol/L). Further incubation with oxidized glutathione (GSSG, final concentration 5 mmol/L) reversed this change and restored the original pattern, whereas GSSG at 7.5 mmol/L caused sub-bands c, e, and g to disappear and sub-bands 1, 2, and 3 to be enhanced. Sequential incubation of serum with 2.5 mmol of GSSG and 7.5 mmol of GSH per liter produced the opposite sequence of events; i.e., the "abnormal" sub-bands disappeared then reappeared (and GSH at 10 mmol/L enhanced their reappearance). At higher concentrations, glutathione (GSH or GSSG) impaired the detection of the CK-MM sub-bands after IEF, an effect that was "quenched" by heat-inactivated serum of low CK activity. Likewise, the intensity of tissue CK-MM (corresponding to myocardium extracted into 100 mmol/L Tris HCl buffer, pH 7.4) was greatly enhanced by adding heat-inactivated serum to the tissue extract before IEF. We discuss the significance of these findings for the diagnosis of myocardial infarction.     

Rapid electrophoretic determination of neuron-specific enolase isoenzymes in serum

This assay procedure for each of the two neuron-specific enolases (alpha gamma and gamma gamma) and the non-neuronal enolase (alpha alpha) in serum involves two steps: electrophoretic separation of the three isoenzymes--alpha alpha, alpha gamma, and gamma gamma--on cellulose acetate, and bioluminescence measurement of total enolase activity. From these data, the activity concentrations (U/L) of the three isoenzymes in serum are calculated. Both measurement steps are based on the enzymatic activity of enolase and thus differ from the immunological methods currently in use, which require the availability of specific antibodies. The method is rapid (approximately 30 min for both steps) and requires only 10 microL of serum for the complete analysis. Studies of normal children and adults, and of patients suffering from neuroblastoma and small-cell lung cancer, show that it is suitable for clinical use. Furthermore, the fact that both neuron-specific isoenzymes of enolase can be systematically separated is an advantage over immunological techniques in determining isoenzyme patterns for pathological samples.     

An alternative protein precipitation method for antipyrine estimation in plasma

The isoenzymes of aspartate transaminase differ in their kinetic properties in that the cytoplasmic isoenzyme is more readily inhibited by adipate and by 2-oxoglutarate (substrate) at low pH. A differential kinetic assay based on this phenomenon has been optimised for use in assays of serum samples. The new method agrees well with an immune absorption procedure. Methods based on Chromatographic separation of the isoenzymes fail in the presence of serum.