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.

Detection of Serum Alkaline Phosphatase Isoenzymes with Phenolphthalein Monophosphate Following Cellulose Acetate Electrophoresis

1968 ◽  
Vol 14 (1) ◽  
pp. 47-57 ◽  
Author(s):  
William C Romel ◽  
S J LaMancusa ◽  
John K DuFrene
Keyword(s):  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Cellulose Acetate ◽  
Serum Alkaline Phosphatase ◽  
Cellulose Acetate Electrophoresis ◽  
Total Enzyme Activity ◽  
Cellulose Acetate Membranes ◽  
Alkaline Phosphatase Isoenzymes ◽  
Serum Alkaline Phosphatase Isoenzymes ◽  
Total Enzyme

Abstract Serum containing normal and abnormal levels of alkaline phosphatase activity were assayed for total enzyme activity, then fractionated by electrophoresis on cellulose acetate membranes for 20 min. The new substrate, phenolphthalein monophosphate, was employed to locate the isoenzymes on the cellulose acetate membranes and to measure their activity by eluting and scanning procedures. The sensitivity and precision of both technics are presented.

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

1985 ◽  
Vol 31 (7) ◽  
pp. 1198-1200 ◽  
Author(s):  
S B Rosalki ◽  
A Y Foo
Keyword(s):  
Alkaline Phosphatase ◽  
Cellulose Acetate ◽  
Wheat Germ ◽  
Bone Alkaline Phosphatase ◽  
Heat Inactivation ◽  
Cellulose Acetate Membrane ◽  
Affinity Electrophoresis ◽  
Alkaline Phosphatase Isoenzymes ◽  
Wheat Germ Lectin ◽  
Tissue Origin

Abstract 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.

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).

Avian trypanosomes in Simulium and sparrowhawks(Accipiter nisus)

Parasitology ◽  
1990 ◽  
Vol 101 (2) ◽  
pp. 243-247 ◽  
Author(s):  
M. F. Dirie ◽  
R. W. Ashford ◽  
L. M. Mungomba ◽  
D. H. Molyneux ◽  
E. E. Green
Keyword(s):  
Cellulose Acetate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Defined Medium ◽  
Polyacrylamide Gel ◽  
Cellulose Acetate Electrophoresis ◽  
Accipiter Nisus ◽  
Large Numbers ◽  
Isoenzyme Patterns ◽  
Infected Prey

Isolates of avian trypanosomes from nestling sparrowhawks (Accipiter nisus) and from Simulium latipes were compared by isoenzyme electrophoresis with the previously described avian trypanosomes Trypanosoma corvi and T. everetti. Simulium isolates developed into trypomastigotes in semi-defined medium at 37 °C confirming that they belong to the genus Trypanosoma. Cellulose acetate electrophoresis (CAE) and polyacrylamide gel electrophoresis (PAGE) methods were employed. A total of 11 enzymes was examined, of which 8 gave satisfactory results for all lysates (MDH, ICD, PGM, 6PGD, SOD, PEP I, PEP II and PEP D). The zymograms of 3 isolates from Simulium and 4 from A. nisus were similar indicating that they were the same organism and the isoenzyme patterns coincided with those of T. corvi. It is concluded that A. nisus and S. latipes are infected with T. corvi. Flagellate infections were found in the midgut, hindgut and rectum of Simulium where large numbers of epimastigotes were found in the lumen and attached to the cuticular intima of the rectal ampullae by hemidesmosomes at the distal end of an expanded flagellum. Out of 285 nestling sparrowhawks 13 (5%) were infected with trypanosomes. In view of their age and inability to preen themselves it is suggested they became infected via infected prey.

Quantitative alkaline phosphatase isoenzyme determination by electrophoresis on cellulose acetate membranes.

1977 ◽  
Vol 23 (1) ◽  
pp. 28-34 ◽  
Author(s):  
W H Siede ◽  
U B Seiffert
Keyword(s):  
Alkaline Phosphatase ◽  
Cellulose Acetate ◽  
Clinical Laboratory ◽  
Kinetic Assay ◽  
Specific Staining ◽  
Cellulose Acetate Membranes ◽  
Alkaline Phosphatase Isoenzymes ◽  
Elution Method ◽  
Routine Clinical Laboratory ◽  
Alkaline Phosphatase Isoenzyme

Abstract We present a new method for quantitative determination of alkaline phosphatase isoenzymes. This method consists of electrophoretic separation on cellulose acetate membranes, special fixation technique to avoid elution and diffusion of enzyme protein during incubation, specific staining, and quantitative evaluation by densitometric measurement. We highly recommend the precedure for routine clinical laboratory use. In all normal individuals we observe two isoenzymes of hepatic origin and one isoenzyme each of osseous, intestinal, and biliary origin. Quantitative normal values are presented. Precision of the method is calculated, the CV being less than 10%. The exactness of densitometric quantification is proved by comparison with kinetic assay of alkaline phosphatase isoenzymes by use of an elution method. Clinical implications of alkaline phosphatase isoenzymograms are reported and discussed in detail.

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.

"Fragmented" isoenzymes of alkaline phosphatase in the diagnosis of transient hyperphosphatasemia.

1986 ◽  
Vol 32 (12) ◽  
pp. 2211-2213 ◽  
Author(s):  
E Schoenau ◽  
K H Herzog ◽  
H J Boehles
Keyword(s):  
Alkaline Phosphatase ◽  
Liquid Chromatography ◽  
Cellulose Acetate ◽  
Serum Alkaline Phosphatase ◽  
Heat Inactivation ◽  
Similar Increase ◽  
Cellulose Acetate Membranes

Abstract We describe the appearance of "fragmented" isoenzymes of serum alkaline phosphatase (EC 3.1.3.1) in two cases of transient hyperphosphatasemia. We determined the isoenzymes by liquid chromatography, then characterized them by heat inactivation, inhibition with 5 mmol/L L-phenylalanine solution, and electrophoresis on cellulose acetate membranes. We suspect that a virus-induced decrease in clearance of the enzyme from serum is responsible for a similar increase of bone and liver isoenzyme activities and for the presence of these fragmented isoenzymes in transient hyperphosphatasemia.

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