Improved agarose electrophoretic method for separating alkaline phosphatase isoenzymes in serum.

1988 ◽  
Vol 34 (9) ◽  
pp. 1857-1862 ◽  
Author(s):  
V O Van Hoof ◽  
L G Lepoutre ◽  
M F Hoylaerts ◽  
R Chevigné ◽  
M E De Broe
Keyword(s):  
Alkaline Phosphatase ◽  
Monoclonal Antibodies ◽  
Polyclonal Antibody ◽  
High Molecular Mass ◽  
High Reproducibility ◽  
Neuraminidase Treatment ◽  
Electrophoretic Method ◽  
Electrophoretic Mobilities ◽  
Before And After ◽  
Alkaline Phosphatase Isoenzymes

Abstract 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.

Electrophoretic Method for Assessing the Normal and Pathological Distribution of Alkaline Phosphatase Isoenzymes in Serum

1975 ◽  
Vol 21 (8) ◽  
pp. 1128-1135 ◽  
Author(s):  
Lilian M V Lee ◽  
Margaret A Kenny
Keyword(s):  
Alkaline Phosphatase ◽  
Distribution Patterns ◽  
Bone Diseases ◽  
Discontinuous System ◽  
Electrophoretic Method ◽  
Electrophoretic Mobilities ◽  
Alkaline Phosphatase Isoenzymes ◽  
Rf Values ◽  
Per Se ◽  
Tissue Of Origin

Abstract We describe a simple, reproducible, discontinuous system for polyacrylamide disc gel-electrophoresis, with which the alkaline phosphatase isoenzymes in human serum can be fractionated. No sample preparation is needed. The isoenzymes are classified according to their electrophoretic mobilities (RF values) and quantitated by peak area measurements from spectrophotometric scans. The four alkaline phosphatase isoenzymes usually present in normal sera, in order of descending mobilities (and designated according to principal tissue of origin) are: "fast" liver, "slow" liver, bone, and intestine. Sera of diseased patients show a greater variety of isoenzyme distribution patterns, but the most frequently observed patterns are the same as normal patterns. We conclude that the finding of "fast" liver only is not pathognomonic, as previously reported by others, and that information on relative distributions per se is not diagnostically useful, although information on specific increases in activity is useful. With this system, hepatobiliary disorders can be differentiated from other forms of liver and bone diseases.

Electrophoretic Separation on Agarose Thin Film of Isoenzymes of Alkaline Phosphatase from Human Serum and Tissue

1971 ◽  
Vol 17 (4) ◽  
pp. 290-295 ◽  
Author(s):  
J A Demetriou ◽  
J M Beattie
Keyword(s):  
Thin Film ◽  
Alkaline Phosphatase ◽  
Plasma Proteins ◽  
Enzymic Activity ◽  
Electrophoretic Method ◽  
Electrophoretic Mobilities ◽  
Alkaline Phosphatase Isoenzymes ◽  
Human Sera ◽  
Barbital Buffer ◽  
Isoenzyme Patterns

Abstract An electrophoretic method is presented for separating alkaline phosphatase isoenzymes on agarose gel by using barbital buffer. α-Naphthyl AS-MX phosphate was converted to the highly fluorescent α-naphthol AS-MX, to detect the zones of enzymic activity on the thin film. The fluorescent zones on the electropherograms were scanned with a recording fluorometer and the electrophoretic mobilities of the enzymes were compared with those of the plasma proteins. This method was used to assess the isoenzyme patterns in human sera and tissues (liver, bone, kidney, spleen, and placenta).

Cellulose Acetate Electrophoresis of Alkaline Phosphatase Isoenzymes in Human Serum and Tissue

1972 ◽  
Vol 18 (5) ◽  
pp. 417-421 ◽  
Author(s):  
H A Fritsche ◽  
H R Adams-Park
Keyword(s):  
Alkaline Phosphatase ◽  
Human Serum ◽  
Cellulose Acetate ◽  
High Sensitivity ◽  
Heat Inactivation ◽  
Cellulose Acetate Electrophoresis ◽  
Electrophoretic Method ◽  
Large Numbers ◽  
Alkaline Phosphatase Isoenzymes ◽  
Isoenzyme Patterns

Abstract We describe a new electrophoretic method for the characterization of human serum and tissue alkaline phosphatases on cellulose acetate plates. Enzymes are localized fluorometrically with the substrate α-naphthol AS-MX phosphate or colorimetrically by coupling the reaction product with Fast Blue RR. Both localization techniques are sensitive enough to demonstrate isoenzyme patterns in micro-scale samples of normal sera. Our electrophoretic studies indicate that sera of children and adults normally contain isoenzymes originating from both liver and bone. The high sensitivity of the method allows the use of normal sera as markers rather than tissue extracts, and isoenzyme patterns may be visually assessed after heat inactivation and chemical inhibition. The method is suitable for the electrophoretic fractionation of alkaline phosphatase in large numbers of sera, with equipment and technique familiar to many laboratories.

Alkaline phosphatase isoenzymes resolved by electrophoresis on lectin-containing agarose gel.

1986 ◽  
Vol 32 (8) ◽  
pp. 1570-1573 ◽  
Author(s):  
W E Schreiber ◽  
L Whitta
Keyword(s):  
Heat Treatment ◽  
Alkaline Phosphatase ◽  
Cellulose Acetate ◽  
Wheat Germ ◽  
Agarose Gel ◽  
Electrophoretic Method ◽  
Activity Staining ◽  
Agarose Gels ◽  
Alkaline Phosphatase Isoenzymes ◽  
Wheat Germ Lectin

Abstract With this electrophoretic method the liver, biliary, and bone isoenzymes of alkaline phosphatase are clearly separated on agarose gels. Wheat-germ lectin, incorporated in the gel matrix, binds the bone isoenzyme selectively, forming a precipitate near the origin. Neither liver nor biliary isoenzyme is affected. Activity staining with an indigogenic dye substrate reveals the liver isoenzyme migrating nearest the anode, followed by the biliary and bone isoenzymes. Results are generally similar to those of electrophoresis on cellulose acetate. However, the lectin-agarose gels better resolve the liver and bone isoenzymes, and heat treatment of samples is not required before electrophoresis.

Electrophoretic variants of alanine aminopeptidase, alkaline phosphatase, and gamma-glutamyltransferase in urine.

1984 ◽  
Vol 30 (6) ◽  
pp. 856-859 ◽  
Author(s):  
K Jung ◽  
U W Wischke
Keyword(s):  
Alkaline Phosphatase ◽  
Electrophoretic Mobility ◽  
Brush Border ◽  
Neuraminidase Treatment ◽  
Electrophoretic Variants ◽  
Brush Border Enzymes ◽  
Gamma Glutamyltransferase ◽  
Electrophoretic Mobilities ◽  
Alanine Aminopeptidase ◽  
Triton X

Abstract The three brush-border enzymes--alanine aminopeptidase (EC 3.4.11.2), alkaline phosphatase (EC 3.1.3.1), and gamma-glutamyltransferase (EC 2.3.2.2)--are present in the urine of healthy persons in two variants, a particulate form and a soluble one. They can be separated by electrophoresis in agarose gel and by ultracentrifugation. The particulate forms exhibit similar electrophoretic mobility, but the soluble forms of these brush-border enzymes differ in their electrophoretic mobilities. The enzyme components of the particulate activity can be mobilized by Triton X-100 and trypsin. The electrophoretic mobility of the soluble forms of alanine aminopeptidase and gamma-glutamyltransferase is slowed by neuraminidase treatment. Both forms of gamma-glutamyltransferase are influenced in their electrophoretic mobility by treatment with n-butanol/diisopropyl ether, showing their lipid dependence. These findings enhance our knowledge of the biochemical nature of brush-border enzymes in urine.

An improved electrophoretic method for alkaline phosphatase isoenzymes determination

1975 ◽  
Vol 8 (1-6) ◽  
pp. 415-418
Author(s):  
Samuel Y. Chu ◽  
V.E. Turkington ◽  
C. Vea ◽  
P. Cheung
Keyword(s):  
Alkaline Phosphatase ◽  
Electrophoretic Method ◽  
Alkaline Phosphatase Isoenzymes

A Study of Various Electrophoretic and Inhibition Techniques for Separating Serum Alkaline Phosphatase Isoenzymes

1972 ◽  
Vol 18 (2) ◽  
pp. 110-115 ◽  
Author(s):  
Roy B Johnson ◽  
Karen Ellingboe ◽  
Phyllis Gibbs
Keyword(s):  
Alkaline Phosphatase ◽  
Serum Alkaline Phosphatase ◽  
Bone Alkaline Phosphatase ◽  
New Technique ◽  
Optimal Conditions ◽  
Before And After ◽  
Alkaline Phosphatase Isoenzymes ◽  
A New Technique ◽  
Serum Alkaline Phosphatase Isoenzymes

Abstract Optimal conditions for the identification and semiquantitation of the isoenzymes of serum alkaline phosphatase are reviewed and a new technique is presented. In the recommended scheme, specific disc electrophoretic conditions are used to produce good separations, which are improved by comparing the alkaline phosphatase patterns of sera before and after treatment with heat or urea (3 mol/liter). This method is compared with other electrophoretic and inhibition techniques. Interpretation is aided by (a) reference to the relative mobilities of each isozyme, and (b) inactivation of bone alkaline phosphatase by heat or urea. The isozymes originating from bile, intestine, placenta, bone, and liver can be independently resolved as separate bands with untreated sera except when bone alkaline phosphatase is grossly increased. The proposed method permits identification and semiquantitation of all the recognized alkaline phosphatase isozymes in most sera, as an aid to diagnosis.

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