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.     

Resolution of alkaline phosphatase isoenzymes in serum by isoelectric focusing in immobilized pH gradients

This new method for fractionation of serum alkaline phosphatase isoenzymes is based on isoelectric focusing on a mixed-type polyacrylamide support containing an immobilized pH gradient with a superimposed carrier-ampholyte gradient. All known forms of alkaline phosphatase are separated in an Immobiline pH 3.5-6.0 gradient, the sample being applied into pockets cast on a pH 8.0 plateau. Sharp zymogram bands are obtained by substituting alkaline-stable 5-bromo-4-chloro-3-indoxyl phosphate and tetrazolium salts for the standard 1- and 2-naphthyl phosphate-diazonium salt combinations. After hydrolysis of the phosphate group by the alkaline phosphatase the indoxyl moieties reduce tetrazolium salts to nearly insoluble and nondiffusible formazan precipitates. Normal sera show an array of about 10 isobands isoelectric between pH 3.9 and pH 4.79. In Paget's disease, two sharp isobands with pls of 4.97 and 5.09 are seen. Placental alkaline phosphatase overlaps with the higher pl bands of normal serum; however, upon heat destruction of the latter, it shows four sharp bands with the following pl's: 4.59, 4.62, 4.67 and 4.73.     

Serum alpha-amylase isozymes in mumps: estimation of salivary and pancreatic isozymes by isoelectric focusing

Serum alpha-amylase isozymes were separated into three major isozymes by thin-layer gel isoelectric focusing and detected by a starch-iodine zymogram procedure. Of the three groups of isozymes, one (S isozyme) corresponded to a salivary specific form, one (P isozyme) to a pancreatic specific form and the third (SP isozyme) to isozymes of similar isoelectric point common to both secretions. The levels of total alpha-amylase and of these three isozymes were estimated in the sera of 54 patients with mumps. Total alpha-amylase and salivary isozyme concentrations were greatly increased in the sera of all patients compared with controls. Pancreatic isozyme concentrations however, were only slightly increased and did not correlate with clinical pancreatitis. Indeed, in patients with mumps associated with pancreatitis, meningoencephalitis or orchitis, levels of total serum amylase, although higher than controls, were lower than levels in patients who presented solely with mumps sialadenitis.     

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.     

Heterogeneity of monoclonal immunoglobulin-G proteins studied by isoelectric focusing

Heterogeneity of myelomatous and nonmyelomatous monoclonal IgG₁ proteins was investigated by isoelectric focusing experiments in thin-layer polyacrylamide gels.It appears from this study that nonmyelomatous monoclonal IgG₁ proteins possess the same individuality and limited heterogeneity as myelomatous monoclonal IgG₁ proteins.     

Mathematical treatment of data for calculating activities of alpha-amylase isoenzymes

Methods for determining alpha-amylase isoenzymes by selective inhibition with a wheat-germ protein are practical and easy, and give accurate, precise results. The incomplete specificity of the inhibitory action is not a major drawback but does necessitate mathematical treatment of the data (i.e., enzymic activities measured before and after preincubation with the inhibitor) to ascertain the amount of the different isoamylases. Such an algorithm is quite simple and straightforward, because the isoenzymes can be calculated either arithmetically or geometrically, by using a linear standard curve, empirically obtained, that relates the fraction of activity remaining after inhibition (R/T) to the pancreatic isoenzyme fraction or to the percentage of total alpha-amylase (P/T or 100 X P/T). An alternative method, plotting R/T against the ratio of pancreatic to salivary isoenzyme (P/S), is inconvenient, necessarily yields a nonlinear curve, needlessly complicates the calculations, and has been a persistent source of confusion in many articles dealing with the differentiation of isoamylases.     

Apolipoprotein E phenotyping from plasma by isoelectric focusing and immunoblotting

A method for apolipoprotein (apo) E phenotyping directly from plasma by isoelectric focusing (IEF) and immunoblotting was confirmed. Ten microliters plasma were delipidated. IEF in 5% polyacrylamide flat gel with 6.4 mol/l urea and 2.8% pharmalyte (PH 4-6.5) was carried out at 3,000 V for 1 hr. Seventeen samples were applied per one flat gel, and IEF of two flat gels was made. Then, Western blotting on nitrocellulose membrane was done at 75 V for 3 hr. Immunostaining was performed using goat-anti-human apo E as first antibody and biotinylated anti-goat IgG as second antibody, and 4-chlorodel-1-naphthol as a substrate. In approximately 5% of the samples, we had difficulty in discriminating between homozygotes and heterozygotes (i.e., apo E3/3 and apo E3/2, or apo E4/4 and apo E4/3) because of equally strong sialated band, but this problem was solved by sialidase treatment of plasma before delipidation. As a result, six apo E phenotypes were clearly demonstrated. Apo E phenotyping of 34 samples could be made simultaneously in 2 days. It is concluded that the polyacrylamide gel IEF and immunoblotting method is useful for apo E phenotyping if it is made up for by sialidase treatment.     

Progesterone receptor measurement by isoelectric focusing: a potential microassay

Sixty-two selected breast cancers were used to compare the conventional dextran-coated charcoal (DCC) assay with a new method of separating progesterone receptor by isoelectric focusing in flat beds of agarose gel. Ninety assays were performed. Isoelectric focusing indicated correctly the presence of receptor in 92% and the absence of receptor in 86% of assays, when compared with the DCC assay. The relationship between the results of the two methods was linear. Isoelectric focusing underestimated receptor to a variable extent, finding relatively less receptor at higher absolute levels of binding than at lower levels. The lower limit of sensitivity of isoelectric focusing was 30 fmol/ml cytosol. The protein concentrations of cytosols prepared from 46 needle biopsy samples (mean weight 25 mg) ranged from 0.5 to 30 g/l (median 4 g/l, 10th percentile 0.75 g/l). Isoelectric focusing is a satisfactory method of progesterone receptor measurement and can be applied to samples too small for conventional techniques.     

Characterization of human beta2-microglobulin by isoelectric focusing.

b2-Microglobulin (B2M) isolated from the urine of normal subjects and patients with cadaveric renal transplantation, showed 2 homologues by isoelectric focusing, one with a pI 5.3, the other with a pI 5.7. These proteins show identical molecular weights by dextran gel filtration and sodium dodecyl sulfate acrylamide gel electrophoresis. The immunogenic reactivity demonstrates partial identity using antiserum to human B2M from 2 different sources.     

Heterogeneity of human red cell autoantibodies assessed by isoelectric focusing

Human red cell (RBC) autoantibodies may be the products of a single lymphocyte clone or of a restricted number of clones. For insight into the clonal distribution of human RBC autoantibodies, serum fractions from 28 individuals with various forms of autoimmune hemolytic anemia (AHA) and two nonanemic individuals with positive direct antiglobulin tests were separated by isoelectric focusing (IEF), and RBC binding in each fraction was quantitated with a solid-phase radioimmunoassay. IEF fractions of serum from normal volunteers and patients with nonimmune hemolytic anemia served as controls. These studies indicate that RBC antibodies are found in a restricted number of IEF fractions in sera from some patients with immune hemolytic anemia. IEF fractions containing RBC-binding activity vary among patients with idiopathic AHA, and distinct patterns of binding activity are found in serum from some patients with AHA associated with alphamethyldopa and procainamide or with B-cell immunoproliferative diseases. These findings suggest that the mechanism leading to autoantibody production may differ among patients with the various forms of immune hemolytic anemia.     

Separation of alpha-amylase isoenzymes using cellulose acetate electrophoresis

alpha-Amylase isozymes were separated by electrophoresis in cellulose acetate to detect the isoforms of the chromogenic substrate manufactured by Lachema, Czechoslovakia. In a group of 20 normal subjects aged 25 to 45, alpha-amylase pancreatic isozyme activity prevailed. Patients with acute myocardial infarction developed, during 36 hours after the onset of the anginal attack, a reduction in the ratio of pancreatic amylase/salivary amylase activities in both increased and normal total alpha-amylase activities. The suggested modified technique of electrophoretic separation of alpha-amylase isozymes may become an effective method for differential diagnosis. Use of this method will help locate the source of hyperamylasemia in various diseases and will thus specify the diagnosis, ruling out the useless therapy for pancreatitis.     

Specific immunoassay of alpha-amylase isoenzymes in human serum

A monoclonal antibody (66C7) was prepared that specifically binds human salivary amylase (EC 3.2.1.1); it cross reacts with human pancreatic amylase by less than 1%. Two procedures are described for determination of isoamylases in human serum with this antibody: an enzyme immunoassay for determining amylase of salivary origin, and a routine method in which this amylase is immunoprecipitated and the remaining (pancreatic) amylase activity is assayed. Results by the two methods correlate well.     

A novel automated assay for malate dehydrogenase isoenzymes

We have developed a new automated method for the determination of malate dehydrogenase (MDH) isoenzymes in serum employing guanidine hydrochloride. Our proposed method showed good reproducibility; within-run precision coefficient of variations (CVs) were less than 2.5 (mean 13.6–42.9 U/L) for total MDH (T-MDH) and less than 6.7% (mean 6.3–23.6 U/L) for mitochondrial MDH (m-MDH) (n = 10). The upper detection limit of the proposed method exhibited good linearity up to 1,000 U/L for both T-MDH and m-MDH. In the proposed m-MDH reagent, the presence of up to 2,000 U/L of cytosolic MDH(c-MDH) activity had no effect on the outcome of m-MDH assay. Results of our proposed method (y) correlated well with those of the electrophoretic method (x) giving the regression equation: y = 1.46 x + 6.87 (N = 30); r = 0.99. Normal concentrations of various anticoagulants and bilirubin did not affect the assay results. Both ascorbic acid and glucose exhibited a slight positive interference with the proposed assay. Clinically, we found that m-MDH activity in serum had greater diagnostic predictive value than T-MDH activity for judging successful outcome of reperfusion therapy; the prognosis was poor when the m-MDH/T-MDH ratio was greater than 20%. © 1996 Wiley-Liss, Inc.     

Behavior of fetal hemoglobin during isoelectric focusing

The results of isoelectrofusing applied to haemolysates of newborns, a case of beta thalassaemia and a case of hereditary persistence of fetal haemoglobin show that fetal haemoglobin F(alpha 2 gamma 2) can be oxidized into two products. The first corresponds to fetal ferrihaemoglobin (alpha +2 gamma +2), the oxidation product of the four Fe atoms of the molecule. The second, which is called fetal intermedial methaemoglobin, and has the same pH as adult ferrihaemoglobin A1, represents probably a product of partial oxydation of fetal haemoglobin.     

Genetic polymorphism of mouse immunoglobulin light chains revealed by isoelectric focusing

Light chains isolated from normal immunoglobulin of unimmunized mice were analyzed by gel isoelectric focusing. Examination of the focusing patterns of light chains from nine inbred mouse strains showed that six of the strains (SWR/J, C3H/HeJ, DBA/1J, A/J, CBA/J, and C57BL/6J) possessed a virtually identical spectrum of focusing bands, while the remaining three strains (RF/J, AKR/J, and C58/J) showed clear differences involving several bands. Analysis of the light chains of individual SWR/J, C58/J, and F1 hybrid mice indicated that the differences in focusing pattern were inherited in a simple codominant fashion. A new procedure was developed for the rapid analysis of light chains from small quantities of serum.     

Human serum inhibitors of collagenase as revealed by preparative isoelectric focusing

Single step separation of pooled normal human serum by means of preparative isoelectric focusing in the range from pH 3.5--9.8 revealed more heterogeneous inhibition of collagenolytic activity than previously reported. Essentially three inhibition zones were resolved. According to their electrophoretic behaviour the respective serum fractions displaying inhibitory activity were designated alpha-, beta- and gamma-collagenase inhibitors. The main component responsible for collagenase inhibition in the alpha-zone was found to be alpha 2-macroglobulin. In the beta-zone inhibitory activity focused around pH 6.3. In the gamma-range a non-dialysable cationic component focusing at pH 9.2 was also able to decrease collagenolytic activity derived from rheumatoid arthritis synovial culture supernatant. These findings were supported by single step separation of serum on DEAE-anion exchange chromatography.     

Human globin chain separation by isoelectric focusing in ultrathin polyacrylamide gels

We describe three basic modifications of our previous method for thalassemia screening by isoelectric focusing of heme-free globin chains: the gel thickness is reduced from 2 mm to 240 μm; the level of the detergent Nonidet P-40 in the gels is decreased from 3% to 0.5%; the polyacrylamide slab is covalently fixed to the supporting glass plate by treatment with silane A-174. By the present method the entire focusing process, starting from gel moulding up to gel destaining and drying, is completed within 4 h, a fraction of the time needed in our previous technique. More than a 100 samples can be analyzed per working day. The present technique also affords increased sensitivity: less than l μg protein/band is detected and less than 200 picomol of hemoglobin are needed for each analysis. Band sharpness and resolution in our ultrathin gels is also considerably increased.     

Oligoclonal bands in cerebrospinal fluid detected by PhastSystem isoelectric focusing.

Pharmacia's "PhastSystem" for semi-automated isoelectric focusing (IEF) in thin precast polyacrylamide gels (PAGE) was found to be as sensitive as high-resolution protein electrophoresis (HRPE) in agarose gels and conventional PAGE-IEF for detection of oligoclonal banding (OB) in concentrated cerebrospinal fluid (CSF) samples. Both PhastSystem IEF and HRPE revealed OB in CSF from eight of nine multiple sclerosis patients and four of 10 patients with various types of infection of the central nervous system as opposed to only two of 70 patients with miscellaneous neuropsychiatric disorders. The PhastSystem also frequently detected OB in silver-stained, unconcentrated CSF from patients with multiple sclerosis.