"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.

Hypophosphatasia (adult form): quantitation of serum alkaline phosphatase isoenzyme activity in a large kindred.

1980 ◽  
Vol 26 (7) ◽  
pp. 840-845 ◽  
Author(s):  
J L Millán ◽  
M P Whyte ◽  
L V Avioli ◽  
W H Fishman
Keyword(s):  
Alkaline Phosphatase ◽  
Serum Alkaline Phosphatase ◽  
Hepatic Metabolism ◽  
Total Activity ◽  
Heat Inactivation ◽  
Mean Values ◽  
Adult Form ◽  
Cellulose Acetate Membranes ◽  
Alkaline Phosphatase Isoenzymes ◽  
Alkaline Phosphatase Isoenzyme

Abstract We used heat inactivation, L-phenylalanine inhibition, and electrophoresis on polyacrylamide gel and cellulose acetate membranes--with and without use of specific antisera against the liver-bone, intestinal, and placental isoenzymes--to distinguish and quantitate the different alkaline phosphatase isoenzymes in sera from 23 adult members of a kindred affected by the adult form of hypophosphatasia. Nine subjects had values for total activity more than two standard deviations below the mean values for age- and sex-matched normal persons. Bone isoenzyme was diminished in all nine, whereas liver isoenzyme was subnormal in only four. Phosphoethanolamine and phosphoserine in the urine of eight hypophosphatasemic individuals correlated inversely with both total and liver alkaline phosphatase activity in their serum, but not with the activity of the bone isoenzyme. Total activity in the serum of adult kindred members correlated best with the circulating liver isoenzyme activity. The findings suggest that altered hepatic metabolism is responsible for the increased urinary excretion of phosphoethanolamine, and perhaps phosphoserine, in hypophosphatasia.

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.

Hypophosphatasia (adult form): quantitation of serum alkaline phosphatase isoenzyme activity in a large kindred.

1980 ◽  
Vol 26 (7) ◽  
pp. 840-845
Author(s):  
J L Millán ◽  
M P Whyte ◽  
L V Avioli ◽  
W H Fishman
Keyword(s):  
Alkaline Phosphatase ◽  
Serum Alkaline Phosphatase ◽  
Hepatic Metabolism ◽  
Total Activity ◽  
Heat Inactivation ◽  
Mean Values ◽  
Adult Form ◽  
Cellulose Acetate Membranes ◽  
Alkaline Phosphatase Isoenzymes ◽  
Alkaline Phosphatase Isoenzyme

Abstract We used heat inactivation, L-phenylalanine inhibition, and electrophoresis on polyacrylamide gel and cellulose acetate membranes--with and without use of specific antisera against the liver-bone, intestinal, and placental isoenzymes--to distinguish and quantitate the different alkaline phosphatase isoenzymes in sera from 23 adult members of a kindred affected by the adult form of hypophosphatasia. Nine subjects had values for total activity more than two standard deviations below the mean values for age- and sex-matched normal persons. Bone isoenzyme was diminished in all nine, whereas liver isoenzyme was subnormal in only four. Phosphoethanolamine and phosphoserine in the urine of eight hypophosphatasemic individuals correlated inversely with both total and liver alkaline phosphatase activity in their serum, but not with the activity of the bone isoenzyme. Total activity in the serum of adult kindred members correlated best with the circulating liver isoenzyme activity. The findings suggest that altered hepatic metabolism is responsible for the increased urinary excretion of phosphoethanolamine, and perhaps phosphoserine, in hypophosphatasia.

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.

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.

Characterization of placental isoenzyme of alkaline phosphatase in human pregnancy serum

1969 ◽  
Vol 47 (2) ◽  
pp. 147-155 ◽  
Author(s):  
N. K. Ghosh ◽  
W. H. Fishman
Keyword(s):  
Alkaline Phosphatase ◽  
Serum Alkaline Phosphatase ◽  
Heat Stability ◽  
Biochemical Properties ◽  
Heat Inactivation ◽  
Total Serum ◽  
Placental Alkaline Phosphatase ◽  
Human Placental Alkaline Phosphatase ◽  
Alkaline Phosphatase Isoenzyme ◽  
In Pregnancy

Human placental alkaline phosphatase isoenzyme has been characterized in pregnancy serum by several biochemical criteria. The total serum alkaline phosphatase, its L-phenylalanine-sensitive moiety, heat inactivation, and the ratio of enzyme activity at pH 10.7 versus 9.8 (10.7/9.8 R) were measured during parturition and 59 weeks of pre- and post-natal periods. The extent of L-phenylalanine inhibition, heat stability, and 10.7/9.8 R of serum alkaline phosphatase progressively increased during gestation attaining maximum values during the delivery, after which they gradually declined. The electrophoretic behaviors of alkaline phosphatase isoenzymes of pregnancy sera were followed by starch- and Sephadex-gel electrophoreses. Alkaline phosphatase has been purified 300-fold from the placenta of the subject whose serum enzyme was investigated. The biochemical properties, including the electrophoretic behavior and neuraminidase sensitivity of heat-stable alkaline phosphastase in pregnancy sera at term, were comparable to those of purified placental alkaline phosphatase. The values for 10.7/9.8 R of the pregnancy sera were statistically different from those of sera from normal nonpregnant women. The results obtained in this study suggest that the enhanced level of pregnancy serum alkaline phosphatase is due to the enrichment of the circulation with an isoenzyme of placental origin.

Atypical alkaline phosphatase isoenzyme in serum from a patient with Hodgkin's disease.

1984 ◽  
Vol 30 (5) ◽  
pp. 800-802 ◽  
Author(s):  
J P Beilby ◽  
P Garcia-Webb ◽  
C I Bhagat ◽  
A Prins
Keyword(s):  
Alkaline Phosphatase ◽  
Molecular Mass ◽  
Hodgkin's Disease ◽  
Serum Alkaline Phosphatase ◽  
Hodgkin’S Disease ◽  
Heat Inactivation ◽  
Relative Molecular Mass ◽  
Agarose Gel Electrophoresis ◽  
Post Translational Modification ◽  
Alkaline Phosphatase Isoenzyme

Abstract An alkaline phosphatase isoenzyme that did not move from the origin in agarose gel electrophoresis was detected in serum from a 51-year-old woman with Hodgkin's disease. Inhibitor and heat-inactivation studies of the patient's serum alkaline phosphatase showed properties resembling those of both liver and bone isoenzymes. No immunoglobulin or high-molecular-mass complexes with the alkaline phosphatase isoenzyme were detected. The relative molecular mass (Mr) of the atypical alkaline phosphatase isoenzyme was 182 000, that of the liver alkaline phosphatase isoenzyme control 170 000. Treatment of both of these isoenzymes with neuraminidase gave a product with an Mr of 140 000. We propose that a post-translational modification increased the carbohydrate content of the liver alkaline phosphatase isoenzyme, thus changing the charge characteristics of the enzyme and decreasing its electrophoretic mobility. We believe this to be the first report of a post-translational modification in a heat-sensitive isoenzyme of alkaline phosphatase.

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

1989 ◽  
Vol 35 (9) ◽  
pp. 1955-1957 ◽  
Author(s):  
K Jung ◽  
M Pergande ◽  
S Klotzek
Keyword(s):  
Alkaline Phosphatase ◽  
Vibrio Cholerae ◽  
Serum Alkaline Phosphatase ◽  
Standard Procedure ◽  
Final Concentration ◽  
Sialidase Activity ◽  
Optimal Separation ◽  
Cellulose Acetate Membranes ◽  
Different Sources

Abstract 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.

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.

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