Determination of LD-Ig complex with counter immunoelectrophoresis using terylene cellulose acetate membrane as supporting media.

OBJECTIVE: In order to determine abnormal LD isoenzyme bands in serum, we established a method of counter immunoelectrophoresis using terylene cellulose acetate as supporting media. SETTING: Department of Clinical Laboratory, Southwestern Hospital, Chongqing 400038 China. PRACTICE DESCRIPTION: LD isoenzymes were electrophoretically fractionated with a Paragon electrophoretic system. LD anomaly was identified by counter immunoelectrophoresis using terylene cellulose acetate as supporting media. MAIN OUTCOME MEASUREMENTS: Identification of the LD-Ig complex using a simple and practical method. RESULTS: An abnormal LD-4 band and an extra band on the cathodic side of LD-5 were observed in the serum of a post-burn patient and proved to be an LD-IgG complex. CONCLUSION: We have established a useful and efficient protocol that uses both isoenzyme electrophoresis and counter immunoelectrophoresis to identify abnormal isoenzyme bands. The method of counter immunoelectrophoresis we describe, using terylene cellulose acetate as supporting media, is simple and suitable for routine clinical use.     

The properties and clinical significance of some electrophoretically slow forms of alkaline phosphatase.

Sera from 8 patients with a marked slow-moving alkaline phosphatase band on electrophoresis were investigated. Inhibitor studies and treatment with neuraminidase showed that all the patients had slow bands with alkaline phosphatase properties resembling those of the liver or bone isoenzyme. In no case did the slow band resemble the intestinal isoenzyme. Immunoelectrophoretic and molecular weight studies indicated that the slow band consisted of an IgG-alkaline phosphatase complex of molecular weight 540 000. Serum from a patient with the slow band was able to bind liver or bone, but not intestinal, alkaline phosphatase from other patients to form the slow band. Serum from patients with the slow band probably contains an abnormal IgG molecule which can bind alkaline phosphatase in the ratio 2:1. No clinical condition was common to all 8 patients although most of them had either intestinal or lung disease.     

Incubation with neuraminidase and affinity electrophoresis with wheat-germ lectin compared for separating and quantifying alkaline phosphatase isoenzymes in plasma

Of 98 patients' specimens examined for alkaline phosphatase (EC 3.1.3.1) isoenzymes by electrophoresis on cellulose acetate membrane after incubation with neuraminidase, 50 showed only a single liver or bone isoenzyme staining band; in 15 of these, the tissue origin of the fraction could not be accurately identified from its electrophoretic location. In the remaining 48 specimens, both liver and bone fractions were identifiable, but in only 25 of these was the electrophoretic resolution sufficient to yield separate peaks on densitometry. In contrast, both liver and bone alkaline phosphatase isoenzymes were identified in 95 of the 98 specimens by affinity electrophoresis involving wheat-germ lectin, the detection of both fractions being in agreement with the results of sequential heat inactivation. The tissue origin of the enzyme bands was readily ascertainable from their consistent electrophoretic location in this medium, and in 89 of the specimens the isoenzyme fractions could be resolved into separate peaks on densitometry. We conclude that resolution of liver and bone alkaline phosphatase by incubation with neuraminidase followed by cellulose acetate electrophoresis is greatly inferior to that obtained by wheat-germ lectin affinity electrophoresis.     

Lysosomal acid phosphatase: activity and isoenzymes in separated normal human blood cells

The present study was devised to investigate the activity and isoenzymes of lysosomal acid phosphatase in individual normal human blood cells, including the T- and B-population of lymphocytes, with the aim to contribute to the classification of haematopoietic neoplasias on the basis of cell specific isoenzyme patterns. Platelets, erythrocytes, granulocytes, monocytes and T-lymphocytes were isolated from blood by gradient centrifugation or immune adsorption. B-lymphocytes were obtained from human tonsils. After purity control and isolation of lysosomes the concentration of acid phosphatase was assayed using the conventional spectrophotometric method. Isoenzymes were separated by isoelectric focusing on polyacrylamide thin layer slabs. Monocytes revealed the highest activity with 14 mU/10(7) cells, about three times more than granulocytes. T-lymphocytes showed an activity of 2.85 mU/10(7) cells and B-lymphocytes of 1.83 mU/10(7) CELLS. The lowest activity was found in platelets with 0.08 mU/10(7) cells. Granulocytes showed 12 isoenzyme bands, whilst the number for monocyte, B-lymphocytes, T-lymphocytes and platelets were respectively 11, 12, 1 and 4 isoenzyme bands. Thus it became evident that the different blood cell populations can be distinguished on the basis of their acid phosphatase isoenzyme pattern.     

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.     

The purification of human creatine kinase BB with high specific activity

This paper describes the purification of human creatine kinase BB with high specific activity (1,122 U/mg). The procedure used resulted in a protein yield of 5.4 mg (21% recovery) from 150 g of brain tissue. Two-dimensional electrophoresis and PAGE studies indicated that purified CK-BB might exist as native isoenzyme along with structural aggregates since the multi-banded appearance was reduced to a single band with sodium dodecyl sulfate treatment but not with 2-mercaptoethanol. Investigators are cautioned not to store brain tissue for prolonged periods of time before isolation of the isoenzyme as this may lead to protein redistribution with additional bands becoming evident on PAGE.     

Abnormal serum lactate dehydrogenase isoenzyme in a case of laryngeal carcinoma and thyrotoxicosis.

An abnormal lactate dehydrogenase (LDH) isoenzyme was found in the serum of a patient with laryngeal carcinoma and hyperthyroidism. On electrophoresis it migrated as an additional band between LDH-1 and LDH-2. Follow-up studies suggested that this higher molecular weight, rather thermostable LDH isoenzyme, might have originated from the cancer tissue, though a possible relationship with the thyrotoxic state cannot be excluded.     

Immunochemical Evidence for Protein Abnormalities in Platelets from Patients with Glanzmann''s Thrombasthenia and Bernard-Soulier Syndrome

Crossed immunoelectrophoresis of Triton X-100 solubilized proteins from normal and abnormal platelets was performed with rabbit antibodies raised against normal platelets. In Bernard-Soulier platelets protein 13 was not detected, and neither the amphiphilic (probably GP Ib) nor the hydrophilic (glycocalicin) glycocalicin-related proteins were seen when monospecific antiglycocalicin antiserum was used. The most prominent precipitate, 16, and platelet fibrinogen, 24 were not detected in platelets of two patients with type I thrombasthenia, whereas in one patient with type II thrombasthenia fibrinogen was clearly detected, but the amount of protein 16 remained severely reduced. Protein 16 was heavily labeled after lactoperoxidase-catalyzed ¹²⁵I iodination of normal platelets, and was precipitated by IgG-L, an alloantibody from a polytransfused thrombasthenic patient. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or protein 16 cut out from immunoplates showed two ¹²⁵I-labeled glycoprotein bands, which migrate as GP IIb and GP IIIa. SDS-PAGE of ¹²⁵I-labeled type I thrombasthenic platelets showed no periodic acid-Schiff bands or peaks of radioactivity in the GP IIb and GP IIIa regions, whereas in the GP I region both the periodic acid-Schiff band intensity and the radiolabeling were within the normal range. Autoradiography after crossed immunoelectrophoresis of iodinated thrombasthenic platelets showed that the bulk of radioactivity was bound to protein 17. This glycoprotein, which was also present in normal and Bernard-Soulier platelets, migrates in the GP I region on SDS-PAGE. Thus, the bulk of radioactivity observed in the GP I region after SDS-PAGE is associated with protein 17 and not with glycocalicin.     

Stability and electrophoretic characteristics of creatine kinase BB extracted from human brain and intestine

Creatine kinase (CK; EC 2.7.3.2) isoenzyme BB extracted from brains of rats reportedly undergoes modification at 37 degrees C, leaving an electrophoretic variant that accounts for most of the residual CK activity. This variant, called CK-BB', migrates on electrophoresis similarly to creatine kinase isoenzyme MB. Using electrophoresis and immunoinhibition with antiserum to creatine kinase isoenzyme MM, we found CK-BB to be the only identifiable cytoplasmic isoenzyme in surgical samples from human brain and intestine. In contrast, we found that some samples of brain obtained at autopsy contain CK-BB'. We also found that CK-BB extracted from human brain was converted to CK-BB' upon incubation in serum or plasma at 37 degrees C. We found a similar development of CK-BB' in incubation mixtures of serum or plasma containing CK-BB obtained from surgical samples of human intestine. The development of CK-BB' during infarction of the gastrointestinal system may thus be a source of false-positive CK-MB in the laboratory verification of myocardial infarction when electrophoresis is used as the only method to identify CK isoenzymes.     

Hemopoietic origin of factor XIII A subunits in platelets, monocytes, and plasma. Evidence from bone marrow transplantation studies.

Factor XIII A subunit (FXIIIA) is found in plasma, platelets, and monocytes. The hemopoietic contributions to FXIIIA in these components were studied in patients transplanted with marrows from donors with different FXIIIA phenotypes. In three patients with successful engraftment (by DNA genotyping, red cell phenotyping, and cytogenetic studies) platelet and monocyte FXIIIA changed to donor phenotypes with hematologic recovery. Thus, FXIIIA in platelets and monocytes is synthesized de novo and/or from their progenitor cells. Plasma FXIIIA phenotype change after transplantation was more complex. Patient I changed from phenotype 1-1 (one electrophoretically fast band) to 1-2 (three bands) in 115 d; patients 2 and 3 did not change completely from phenotype 1-2 to 1-1 in up to 458 d, but did show enrichment of the fastest band. Thus, while there is a definite contribution of donor hemopoiesis to plasma FXIIIA, another source of recipient FXIIIA appears to be present to delay or prevent the phenotype change.     

Partial characterization of an abnormal lactate dehydrogenase isoenzyme, LDH-1ex, in serum from a patient with hepatocellular carcinoma

Serum from a patient with hepatocellular carcinoma contained an abnormal isoenzyme of lactate dehydrogenase (LDH; EC 1.1.1.27), LDH-1ex, that on electrophoresis on 10-g/L agarose gel migrated anodally to the LDH-1 band. This isoenzyme was partly purified by ultrafiltration and preparative electrophoresis. Gel chromatography and sodium dodecyl sulfate/polyacrylamide gel electrophoresis studies of the resulting LDH-1ex preparation suggested that this isoenzyme is probably a tetramer made up of four single polypeptide chains (monomers), each having a molecular mass of about 32,000 Da. LDH-1ex was heat stable and reacted more readily with 2-hydroxybutyrate than did the slower migrating LDH-4 and LDH-5 isoenzymes. LDH-1ex showed no activity when lactate was omitted from the substrate solution or replaced by ethanol.     

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.     

碱性磷酸酶同工酶在老年女性骨质疏松诊断中的应用

目的 探讨碱性磷酸酶（ALP）同工酶在老年女性骨质疏松诊断中的应用。方法 采用神经氨酸苷酶,用琼脂糖电泳法对体检正常者50例（对照组）和老年女性骨质疏松患者55例（骨质疏松组）血清中的ALP同工酶进行分离分析。结果 经神经氨酸苷酶处理后,琼脂糖电泳法可以分离肝和骨ALP;两组肝型ALP比较,差异无显著性意义（P〉0.05）;骨型ALP比较,差异有显著性意义（P〈0.05）;热失活法和电泳法检测骨ALP具有显著的相关关系（r=0.92,P〈0.05）。结论 骨型ALP可为临床诊断老年女性骨质疏松提供可靠的依据。     

Binding of urokinase to plasma proteinase inhibitors

125I-labelled urokinase was incubated with plasma and plasminogen free plasma, and the incubation mixtures were analyzed by agarose gel electrophoresis. Autoradiography demonstrated that non-reacted urokinase remained around the slit and that complex-formation with inhibitors altered the migration and resulted in two bands, a major one and a minor one. Crossed immunoelectrophoresis combined with autoradiography showed that the major band contained a complex between alpha 2-antiplasmin and urokinase. The minor band contained a complex between alpha 2-macroglobulin and urokinase. Also di-isopropylfluorophosphate-inactivated urokinase was bound to alpha 2-macroglobulin but not to alpha 2-antiplasmin. Thus, an intact active site of urokinase is not necessary for complex formation with alpha 2-macroglobulin.     

A simplified method for the estimation of urinary total hydroxyproline.

An oxidative activity has been demonstrated in different tissues of various animals species (man, rabbit, hen, rat, mouse) measuring it by means of adrenaline, by starch gel electrophoresis and acrylamide gel electrofocusing, followed by incubation of these gels with DOPA.Kidney, liver and erythrocytes of rabbit, rat, mouse and hen, human erythrocytes and platelets display a high oxidative activity. This activity, which is not completely inhibited by KCN, is found in one band on starch gel electrophoresis and in two distinct bands on electrofocusing in the case of platelets and erythrocytes of man, rabbit and rat.The starch gel electrophoretic migration and the electrofocusing pattern are tissue and species dependent.     

Comparison of acid phosphatase isoenzymes of human seminal fluid, prostate, and leukocytes.

We used ion-exchange column chromatography and electrophoresis on polyacrylamide gel to compare the acid phosphatase isoenzymes of prostate and leukocytes. The major isoenzyme of the prostate is band 2A; only a trace of band 2B was observed. However, the major isoenzymes of leukocytes are band 4 and band 2B, and only a small amount of band 2A was observed. The three isoenzymes isolated from leukocytes or prostate gland react to the antiserum prepared against the aicd phosphatase isoenzyme of seminal fluid. Acid phosphatases of leukocytes other than the three isoenzymes mentioned above did not interact with the antiserum.     

Association of high-molecular-mass and electrophoretically atypical alkaline phosphatases

Polyacrylamide gel electrophoresis of alkaline phosphatase may yield abnormally migrating fractions; these include high-molecular-mass alkaline phosphatase, which remains at the gel origin, and immunoglobulin-alkaline phosphatase complexes, which have a mobility approximately 1/3 that of liver isoenzyme. We performed a retrospective study of 19 patients whose sera exhibited atypical alkaline phosphatase fractions, defined as bands whose mobility was slower than bone, liver, or intestinal alkaline phosphatase; 17 had a mobility approximately 1/3 that of liver isoenzyme and 16 also exhibited gel origin enzyme activity or high-molecular-mass bands. The strong association of the atypical and high-molecular-mass alkaline phosphatases suggests that they may be structurally related, both consisting of either immunoglobulin-enzyme complexes or membrane-alkaline phosphatase complexes. This hypothesis is supported by (1) one serum available for investigation containing alkaline phosphatase-immunoglobulin complexes in both abnormally migrating fractions, but on detergent treatment showing no evidence of membrane-bound enzyme; (2) detergent treatment of serum from patients with only high-molecular-mass alkaline phosphatase creating bands with a mobility of approximately 1/3 that of the liver isoenzyme.     

Lactate dehydrogenase isoenzyme pattern in sera of patients with malignant diseases

Total lactate dehydrogenase (LDH; EC 1.1.1.27) activity and the percentage distribution of LDH isoenzymes were determined in 127 patients with malignant diseases. A shift in the isoenzyme patterns was observed toward the M-type, with an increase in the percentage of LDH-4 and LDH-5 isoenzymes and a slight increase in total LDH activity of all patients. Serum samples from 68 of the patients contained an abnormal isoenzyme of LDH, "LDH-1 ex," that, on agarose gel electrophoresis at pH 8.6, migrated between albumin and LDH-1 isoenzyme. Chemotherapy, radiotherapy, or surgical removal of the tumor was accompanied by disappearance of this abnormal isoenzyme. The heat stability of LDH-1 ex isoenzyme appears to be similar to that of LDH-1 but greater than that of the other LDH isoenzymes. Statistical analysis of these data demonstrated a significant correlation between malignancy and the appearance of LDH-1 ex isoenzyme (P less than 0.001). In contrast, the relationship between LDH-1 ex isoenzyme and metastasis or anatomical location of the malignancy is not statistically important (P less than 0.1).     

Enolase isoenzymes as tumour markers

alpha- and gamma-enolase isoenzyme substance concentrations were measured in serum and plasma from healthy subjects and from 174 patients with different solid tumours. While alpha-enolase was found to be increased in the plasma of patients with tumours of quite different origin, gamma-enolase apparently reflected malignancies of the neuroendocrine system. Before the beginning of the cytotoxic therapy gamma-enolase was increased above the upper limit of the reference range (10 micrograms/l) in 27/27 patients (100%) suffering from small cell lung cancer. Most patients with squamous cell carcinoma of the lung or with prostatic cancer exhibited normal gamma-enolase, while both tumour types produced high plasma substance concentrations of the alpha-isoenzymes of enolase.     

Aberrant multimeric structure of von Willebrand factor in a new variant of von Willebrand''s disease (type IIC).

A variant of von Willebrand's disease has been identified in which sodium dodecyl sulfate agarose electrophoresis provides evidence that the von Willebrand factor present is structurally abnormal. Rather than the repeating triplet seen in normal subjects and in patients with the IIA and IIB variants, a repeating doublet was present in the propositus. None of the bands had the same mobility as bands in normal subjects or previously described von Willebrand's disease patients. The larger multimers of von Willebrand factor were lacking both from plasma and platelets, and did not appear in the circulation after infusion of 1-deamino-[8-D-arginine]-vasopressin. There was a marked increase in the concentration of the smallest multimer in the propositus and his phenotypically normal children, indicating that this abnormality of von Willebrand factor is inherited in an autosomal-recessive manner.