Column-chromatographic separation of isoenzymes of aspartate aminotransferase.

We describe a column-chromatographic method for separating the mitochondrial and cytoplasmic isoenzymes of aspartate aminotransferase in human serum. Bed height of the ion exchanger, pH, and salt concentrations in the eluting buffers are shown to be variables affecting the separation of the isoenzymes. Under the optimized conditions selected for this study, a 30% increase in volume was observed in one fraction, associated with changing the salt concentration of the eluting buffer and attributed to a contraction of the DEAE-Sephadex A-50. Elution profiles (enzyme activity vs. fraction number) were examined with highly purified mitochondrial and cytoplasmic isoenzymes of human origin in bovine serum albumin and human serum. Recovery of the enzyme in the eluted fractions averaged 102% (SD, 2.0%) for specimens prepared from the purified isoenzymes and 104% (SD, 10.7%) for 38 human serum specimens. The separation technique showed linearity to catalytic concentrations in excess of 200 U/liter (reaction temperature 30 degrees C) for each isoenzyme. Additional information is presented regarding among-day precision and the effect of specimen dilution.     

Isoelectric focusing on cellulose acetate membrane: a separation procedure for alkaline phosphatase isoenzymes

An isoelectric focusing technique for the separation of alkaline phosphatase isoenzymes on cellulose acetate membrane is described. Optimal conditions for isoelectric focusing were established by changing ampholine concentration and focusing conditions. Bone, liver, intestinal, and placental isoenzymes can be resolved into vacious sub-bands in a pH range of 4.1 to 5.2. These sub-bands were correlated with the findings of electrophoretic isoenzyme separation. The whole procedure proves very simple to perform and comparatively time saving (4 h). This procedure may help clarify the problems of ALP isoenzyme differention when electrophoretic patterns are unresolved.     

Clinical significance of serum high-molecular-mass alkaline phosphatase,alkaline phosphatase–lipoprotein-X complex,and intestinal variant alkaline phosphatase

This paper is a study to identify the clinical significance of high-molecular-mass alkaline phosphatase (ALP:E:C.3.1.3.1.), ALP–lipoprotein–X complex (LP-X) and intestinal variant ALP. We used cellulose acetate and agarose gels and techniques including wheat germ lectin, cetavlon-diethyl ether, thermostatability, neuraminidase and L -phenylalanine to improve the electrophoretic separation of the alkaline phosphatase isoenzymes. Patients' serum samples were electrophoresed from a diverse group of individuals ill with cholestasis, neoplastic disease metastatic to the liver, hepatocellular carcinoma, cirrhosis, diabetes mellitus, and chronic renal disease. Agarose gels provided better separation of ALP isoenzymes than cellulose acetate gels. The results also indicated that high-molecular mass ALP is present in patient's serum in conditions associated with cholestasis especially caused by hepatic malignancy. High-molecular mass ALP was frequently found to co-exist with the liver isoenzyme and LP–XALP complex. The intestinal variant was identified in patients with malignancy, cirrhosis, chronic renal disease and diabetes mellitus. Intestinal ALP coexisted concomitantly with a variant intestinal ALP. Intestinal variant ALP is most likely composed of intestinal ALP attached to a cellular membrane-binding domain, or may be an artifact produced by neuraminidase incubation © 1994 Wiley-Liss, Inc.     

以N-甲基葡胺为磷酸化受体测定血清碱性磷酸酶的实验探讨

目的评估以N-甲基葡胺作为磷酸化受体缓冲液测定血清碱性磷酸酶(ALP)活性的方法。方法通过对试剂的合理组合和实验条件的优选,建立基于N-甲基葡胺的血清ALP测定方法,并与基于其他缓冲液的推荐方法进行对比。结果精密度,批内:CV=0.62%,批间:CV=2.3%;线性范围至少可达到2 073 U/L;分别与以2-氨基-2-甲基-1-丙醇(AMP,X1)和二乙醇胺(DEA,X2)为磷酸化受体的ALP测定法比较:Y=1.29X1 13.25,r=0.999;Y=0.65X2 19.19,r=0.998。健康成人参考区间为44~133 U/L[(80.33±20.97)U/L]。结论N-甲基葡胺可替代AMP或DEA作为磷酸化受体缓冲液用于临床血清ALP活性的常规测定。     

Improved agarose electrophoretic method for separating alkaline phosphatase isoenzymes in serum

A modified agarose electrophoretic system for the separation of alkaline phosphatase (ALP, EC 3.1.3.1) isoenzymes is described. Bone, liver, high-molecular-mass, and intestinal ALP are separated with high reproducibility. The sensitivity of the agarose system is superior to cellulose acetate in detecting high-Mr ALP. Correlation is good between bone ALP fractions scanned before and after treatment with neuraminidase. Immunoglobulin-bound ALPs, the ALP-lipoprotein-X complex, and the additional ALP fraction observed in transient hyperphosphatasemia in children are detected by their peculiar electrophoretic mobility in the proposed system. Approximately 25% of the samples contained an additional fraction of intestinal-type ALP, as evidenced by neuraminidase treatment and use of polyclonal and monoclonal antibodies. Because the electrophoretic mobilities of this "intestinal variant" and of some immunoglobulin-bound ALP fractions are identical to those of bone and intestinal ALP, respectively, treatment of the samples with a polyclonal antibody that reacts with intestinal ALP is advised.     

Alkaline phosphatase isoenzymes of liver and bone origin are incompletely resolved by wheat-germ-lectin affinity chromatography

We used wheat-germ-lectin affinity chromatography as a tool to investigate the structure of alkaline phosphatase (ALP, EC 3.1.3.1) and to obtain fractions enriched in either bone or liver ALP activity. Liver and bone isoenzymes in serum samples were incompletely resolved except that the activity in the nonretained fraction (fraction 1) always represented pure liver isoenzyme and constituted a larger percentage of total activity in pooled sera with increased liver ALP activity than in pooled sera with increased bone activity. In contrast, a more avidly retained ALP activity, presumably with high glycosylation, was found in human serum with high activity of bone ALP. Using a solid-phase immunoassay, we examined the fractions obtained from the wheat-germ-lectin-Sepharose 4B column to determine whether the isoenzyme preference of the monoclonal antibody was markedly influenced by the degree of glycosylation. Whether samples contained high proportions of liver or of bone isoenzyme activity, the nonretained fraction contained a higher percentage of liver ALP, whereas the more strongly bound fraction contained a higher percentage of bone ALP. Except for eluted fractions that either contained no detectable N-acetylglucosamine or the highest percentage of it, the avidity of the liver-isoenzyme-specific monoclonal antibody for ALP seemed to be independent of the degree of glycosylation, suggesting that the epitope for monoclonal antibody may be expressed in some structure other than the carbohydrate moieties.     

Sialidase from different sources compared for electrophoretically separating serum alkaline phosphatase fractions from liver and bone

We compared sialidase (neuraminidase; EC 3.2.1.18) from Vibrio cholerae, Clostridium perfringens, and Arthrobacter ureafaciens, seeking to improve the electrophoretic separation of the liver and bone isoenzymes of alkaline phosphatase (EC 3.1.3.1) on cellulose acetate membranes. Resolution is decisively determined by the type and activity of sialidase used in the preincubation of serum sample. Sialidase from Arthrobacter ureafaciens is not suited for this method. For optimal separation of the two isoenzymes we recommend the use of sialidase from Vibrio cholerae, determination of its activity with a standard procedure such as described here (mucin or sialyl lactose as substrates), and a final concentration of sialidase activity of 2.0 or 2.9 U/L (measured with mucin or sialyl lactose) in the incubation mixture.     

5'-Nucleotide phosphodiesterase isoenzymes in human serum: quantitative measurement and some biochemical properties

A method based on native PAGE is used for the quantitative measurement of 5'-nucleotide phosphodiesterase isoenzymes (5'-NPD; EC 3.1.4.1) in human serum. In contrast to other techniques this method works with a commercially available substrate. In sera of healthy donors four isozymes could be separated, designated as 5'-NPD-I, 5'-NPD-II, 5'-NPD-III and 5'-NPD-IV in the reverse order of their electrophoretic mobility. When low amounts of serum were applied to the gel, the separation between all four activities was sufficient enough to allow their quantitation. Higher amounts of serum impaired the separation between the isoenzymes I and II. However, even when using high amounts of serum, the quantitation of these activities was possible when taking advantage of some of their biochemical properties which are described herein.     

Microheterogeneity of serum alkaline phosphatase isoenzymes as revealed by isoelectric focusing

The microheterogeneity of human serum alkaline phosphatase (ALP) was investigated by means of isoelectric focusing. Liver and bone isoenzymes focused in a similar pattern, with about 10 bands located between pH 3.7 and 4.9, but differed in the relative intensity of the various bands. Intestinal ALP exhibited 7 to 8 bands at pH 4.9-5.1, and the placental enzyme showed 2 to 3 bands at pH 4.9. Mild digestion with neuraminidase revealed that the banding of liver and bone isoenzymes was at least partly due to differences in the sialic acid content of the various fractions. Extensively desialylated liver and bone isoenzymes showed apparently identical patterns with 6 to 7 bands focused at pH 6.2-6.7. Isoelectric focusing is a useful method for characterizing the microheterogeneity of alkaline phosphatase isoenzymes. The clinical value of this method seems to be limited, however, since it did not distinguish between liver and bone isoenzymes and failed to detect 'specific' isoelectric fractions correlated to various diseases.     

Suitability of commercial control sera for the quality control of activity determination of alkaline phosphatase.

The suitability of thirteen commercially available control sera for measuring alkaline phosphatase (EC 3.1.3.1; orthophosphoric acid monoester phosphohydrolase, ALP) activity in human serum was tested. Apart from differences in ALP activity observed in some reconstituted commercial sera, the behaviour of control materials towards experimental variables such as the nature and concentration of the substrate, pH and type of buffer (or PO4-acceptor) together with the composition of the isoenzymes present in human serum highlights the problems and difficulties if commercial materials are to be used as control sera. The half-saturation constants in control sera were in all cases smaller than those of ALP isoenzymes from bone and liver. The shape of substrate activity curves and the pH optimum in most of control sera differed from that of human serum. The discrepant kinetic data of control materials and human serum may mask or suggest changes relevant to commercial quality control serum but not to samples of human serum.     

Physiologic concentrations of bile salts inhibit rat hepatic alkaline phosphatase but not the intestinal isoenzyme

Objective: The effect of bile salts on alkaline phosphatase (EC 3.1.3.1) activity from Wistar rat liver, duodenum, jejunum, and serum was investigated.

Design and results: For concentrations higher than 1 mM conjugated bile salts (glycocholate, glycochenodeoxycholate, taurocholate, taurodeoxycholate, and taurochenodeoxycholate) inhibited hepatic ALP but, up to concentrations of 10 mM, had no effect on intestinal ALP. Also cholate, deoxycholate, and chenodeoxycholate, within the same concentration range, did not have any effect on intestinal ALP. ALP inhibition induced by conjugated bile salts was significantly higher in serum of starved rats than in serum of fed animals, what is in good agreement with the known higher proportion of hepatic ALP and lower proportion of intestinal ALP in serum of starved rats.

Conclusions: Bile salts can, thus, be used to help discriminating between tissue-nonspecific and intestinal ALP isoenzymes and identifying pathologic conditions where the relative quantities of these isoenzymes are altered in serum. Inhibition of hepatic ALP by physiologic concentrations of bile salts may bear some relation to the bile salts effects on their own enterohepatic circulation and/or biosynthesis.     

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.     

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.     

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.     

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.     

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.     

IgA-CK-BB complex with CK-MB electrophoretic mobility can lead to erroneous diagnosis of acute myocardial infarction

This patient, on admission, presented with a tentative diagnosis of myocardial infarction: the electrocardiogram showed a nonspecific ST-segment and T-wave abnormalities, and total creatine kinase (CK; EC 2.7.3.2) activity was slightly increased (238 U/L). However, a high electrophoretic value for CK-MB (50% of total CK activity) and the electrophoretic pattern of lactate dehydrogenase (EC 1.1.1.27) isoenzymes ruled out myocardial infarction. The isoenzyme migrating as CK-MB was found later to contain no immunologically normal CK-M subunits, and it was bound to IgA. A mixture of the patient's serum and a human serum control containing all CK isoenzymes showed altered electrophoretic mobility only for CK-BB, indicating that the patient's serum contained antibodies to the B unit of CK. Elution from a Sephadex G-200 column showed that the peak at which most of the anodic CK was eluted corresponded to a molecular mass of approximately 200 kDa. Evidently this atypical isoenzyme was an IgA-CK-BB complex. Because this macro CK type 1 can mimic CK-MB, it may therefore be a source of confusion.     

Simultaneous separation of creatine kinase and lactate dehydrogenase isoenzymes using an electrophoresis method]

A specific and sensitive rapid method for simultaneous electrophoretic separation of the blood serum creatine kinase (CK) and lactate dehydrogenase (LDH) isoenzymes is described, fit for rapid diagnosis. A common electrophoresis scheme was used for the separation of both enzymes on the same cellulose acetate plate. Then the isoenzymes were separately detected by their enzymic activities. To detect the enzymic activities at the sites of isoenzymes' localization, the samples were incubated with substrate gel. Optimal conditions for the detection of CK and LDH activities were defined. To illustrate the diagnostic value of the method, the blood serum CK and LDH isoenzymic activities were measured in the patients with acute myocardial infarction and in those operated on for aortocoronary shunting.     

Optimized determination and properties of NADPH-dependent glutathione reductase in serum. Studies on serum glutathione reductase, I. (author's transl)]

Reaction conditions were optimized for the determination of serum glutathione reductase, which has not yet been investigated systematically. Imidazole was found to be the most suitable buffer material; the highest glutathione reductase activity in serum was always obtained with imidazole/HCl buffer, which, in contrast to all other tested buffers, also resulted in the maximal enzyme activity without preincubation. In imidazole buffer, the pH-activity curve of serum glutathione reductase shows a broad optimum between pH 6.5 and 6.9. A GSSG concentration of 2 mmol/l and a NADPH concentration of 0.43 mmol/l gave maximal enzyme activity and a linear reaction over 10 min up to 20 U/l test solution. An investigation of serum glutathione reductase activity from 100 clinically healthy probands gave values between 20 and 50 U/l. In the optimized assay system the glutathione reductase in the serum reacts specifically with GSSG and NADPH.     

改良琼脂糖电泳法分离碱性磷酸酶同工酶及其在肾脏疾病诊断中的应用

目的　用改良琼脂糖电泳法分离碱性磷酸酶同工酶并探讨在肾脏疾病诊断中的应用。方法　用改良琼脂糖电泳法对正常体检者和 16 2例肾脏疾病患者碱性磷酸酶 (ALP)同工酶进行分离和定量分析。结果　发现在慢性肾衰患者血清中存在胆汁型ALP ;肾小球肾炎和肾盂肾炎的ALP同工酶活性与正常者无显著性差异 ,慢性肾衰的ALP同工酶活性则显著高于正常体检者。在 6 9例慢性肾衰患者中 ,有 2 4例出现胆汁型ALP ,阳性率为 34 8%。结论　改良琼脂糖电泳法能定量分析肝、骨、胆汁等ALP同工酶 ,适合常规实验室应用