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.

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.

Serum Alkaline Phosphatase: Total Activity and Isoenzyme Determinations Made by Use of the Centrifugal Fast Analyzer

1972 ◽  
Vol 18 (12) ◽  
pp. 1468-1474 ◽  
Author(s):  
Bernard E Statland ◽  
H Harold Nishi ◽  
D S Young
Keyword(s):  
Alkaline Phosphatase ◽  
Clinical Laboratory ◽  
Serum Alkaline Phosphatase ◽  
Kinetic Method ◽  
Total Activity ◽  
Alkaline Phosphatase Isoenzymes ◽  
Chemical Inhibition ◽  
Routine Clinical Laboratory ◽  
Adult Volunteers ◽  
Total Alkaline

Abstract We present a kinetic method in which the centrifugal analyzer is used to determine total alkaline phosphatase activity and to measure the isoenzymes in bone, liver, and the L-phenylalanine-sensitive fraction (intestine, tumor, and placenta). Measurements were made at 30°C in diethanolamine buffer, with p-nitrophenylphosphate as substrate. The alkaline phosphatase isoenzymes were separated and measured by selective chemical inhibition with 10 mmol/liter L-phenylalanine and 3.3 mol urea per liter. Analyses were performed on sera of a group of healthy pediatric and young adult volunteers and, in addition, on sera from patients with clinically documented osteoblastic disorders and hepatobiliary diseases. The precision and accuracy of the centrifugal analyzer were evaluated, as was the suitability of the instrument for routine clinical laboratory use.

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.

scholarly journals Reaction Rate Retardation as a Method for Serum Alkaline Phosphatase Isoenzyme Measurement

1980 ◽  
Vol 17 (4) ◽  
pp. 192-198 ◽  
Author(s):  
Pamela B Brown ◽  
K O Lewis
Keyword(s):  
Alkaline Phosphatase ◽  
Reaction Rate ◽  
Serum Alkaline Phosphatase ◽  
Enzyme Reaction ◽  
Intestinal Alkaline Phosphatase ◽  
Wide Range ◽  
Alkaline Phosphatase Isoenzymes ◽  
Sensitivity And Selectivity ◽  
Rate Retardation ◽  
Alkaline Phosphatase Isoenzyme

A method for serum alkaline phosphatase isoenzymes using an enzyme reaction rate analyser is described. The complete urea-induced degradation of enzyme activity is monitored, from which individual isoenzyme activities are obtained by calculating the constituent exponential components of the degradation curve. Activities have been measured with adequate sensitivity and selectivity for up to four isoenzyme components in normal and in pathological sera. The identity of each isoenzyme present is assigned from its characteristic degradation half-life, and by this method bone and liver alkaline phosphatase are clearly distinguished and quantitated, and a composite value for placental-intestinal alkaline phosphatase activity is obtained. The approach promises to be applicable to a wide range of isoenzymes, and in analogy with ‘reaction rate’ the term ‘reaction rate retardation’ is suggested for the procedure.

Alkaline phosphatase isoenzymes.

1982 ◽  
Vol 28 (10) ◽  
pp. 2007-2016 ◽  
Author(s):  
D W Moss
Keyword(s):  
Alkaline Phosphatase ◽  
Quantitative Analysis ◽  
Posttranslational Modifications ◽  
Quantitative Methods ◽  
Molecular Forms ◽  
Future Research ◽  
Alkaline Phosphatases ◽  
Isoenzyme Analysis ◽  
Alkaline Phosphatase Isoenzymes ◽  
Alkaline Phosphatase Isoenzyme

Abstract The human alkaline phosphatases constitute a system of multiple molecular forms of enzymes in which heterogeneity is partly due to genetic factors and partly to posttranslational modifications. Recognition of the nature and occurrence of these multiple forms has made a significant contribution both to the understanding of changes in alkaline phosphatase values for serum in disease and to the use of alkaline phosphatase measurements in diagnosis. Many of the diagnostic advantages of alkaline phosphatase isoenzyme analysis can be obtained with the aid of qualitative methods such as zone electrophoresis. However, quantitative methods are needed to take full advantage of the potential benefits of isoenzyme analysis. Selective inactivation methods can be applied successfully to the quantitative analysis of bone and liver alkaline phosphatases in serum. However, the aim of future research should be to remove the limitations at present imposed on quantitative analysis by the close similarities of bone and liver alkaline phosphatases.

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.

"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.

The analysis of alkaline phosphatase isoenzyme using 4-methylumbelliferyl phosphate as substrate on a cellulose acetate membrane

1979 ◽  
Vol 91 (3) ◽  
pp. 273-276 ◽  
Author(s):  
Fukuko Watanabe ◽  
Megumi Takano ◽  
Fumiko Tanaka ◽  
Nobuyuki Amino ◽  
Chozo Hayashi ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Cellulose Acetate ◽  
Cellulose Acetate Membrane ◽  
Alkaline Phosphatase Isoenzyme

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

1975 ◽  
Vol 21 (8) ◽  
pp. 1067-1071 ◽  
Author(s):  
Chung-Ja Mo Cha ◽  
Balduino Mastrofrancesco ◽  
Sungman Cha ◽  
Henry T Randall
Keyword(s):  
Alkaline Phosphatase ◽  
Heat Stability ◽  
Diffuse Band ◽  
Electrophoretic Patterns ◽  
Cancer Of The Pancreas ◽  
Cellulose Acetate Membranes ◽  
Intestinal Origin ◽  
Alkaline Phosphatase Isoenzymes ◽  
Human Sera ◽  
Moving Band

Abstract Alkaline phosphatase isoenzymes in sera were resolved by electrophoresis on cellulose acetate membranes into seven different bands (L1, B, Pl, L2, I1, I2, 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 stereospecific inhibition are different from those of intestinal isoenzymes in sera that show no Pa band.

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