An Automated p-Nitrophenylphosphate Serum Alkaline Phosphatase Procedure for the AutoAnalyzer

1965 ◽  
Vol 11 (9) ◽  
pp. 876-888 ◽  
Author(s):  
Stanley Morgenstern ◽  
Gerald Kessler ◽  
Joseph Auerbach ◽  
Richard V Flor ◽  
Bernard Klein
Keyword(s):  
Alkaline Phosphatase ◽  
Serum Alkaline Phosphatase ◽  
Reaction Rates ◽  
Small Sample ◽  
Automated System ◽  
Enzyme Action ◽  
Linear Reaction ◽  
Simplified Procedure ◽  
Automated Determination

Abstract A new and simplified procedure for the automated determination of serum alkaline phosphatase uses the AutoAnalyzer. The substrate p-nitrophenylphosphate in 2-amino-2-methyl-1-propanol offers the prime advantage of providing directly its own chromogen, p-nitrophenol, following enzyme action. The procedure also permits use of small sample volumes, provides linear reaction rates, and has a simple manifold design. Correlations are presented between the manual procedure and the automated system.

An Automated p-Nitrophenylphosphate Serum Alkaline Phosphatase Procedure for the "Robot Chemist"

1965 ◽  
Vol 11 (9) ◽  
pp. 889-897 ◽  
Author(s):  
Stanley Morgenstern ◽  
Gerald Kessler ◽  
Joseph Auerbach ◽  
Richard V Flor ◽  
Bernard Klein
Keyword(s):  
Alkaline Phosphatase ◽  
Serum Alkaline Phosphatase ◽  
Reaction Rates ◽  
Small Sample ◽  
Enzyme Action ◽  
Nitrophenyl Phosphate ◽  
Linear Reaction ◽  
Accurate Procedure ◽  
Automated Determination

Abstract A new, precise, and accurate procedure is described for the automated determination of serum alkaline phosphatase on the Robot Chemist. The substrate, p-nitrophenyl-phosphate in 2-amino-2-methyl-1-propanol, offers the prime advantage of providing directly its own chromogen, p-nitrophenol, following enzyme action. Other advantages are the small sample volumes required and linear reaction rates. Correlations are presented between the manual procedure and those used with both the Technicon Auto-Analyzer and the Robot Chemist.

An Automated Determination of Alkaline Phosphatase Utilizing p-Nitrophenol Phosphate

1964 ◽  
Vol 10 (12) ◽  
pp. 1112-1116 ◽  
Author(s):  
Rex E Sterling ◽  
Alan A Wilcox ◽  
Arnold G Ware ◽  
Mary K Umehara
Keyword(s):  
Alkaline Phosphatase ◽  
Production Rate ◽  
Serum Alkaline Phosphatase ◽  
Automated Determination

Abstract A technic is described for the automated determination of serum alkaline phosphatase. This method permits a production rate of 60 determinations per hour on 0.2 ml. of serum per determination. Calculations are simplified since no serum blank is required.

Automated Determination of Serum Alkaline Phosphatase Using a Modified Bodansky Technic

1964 ◽  
Vol 10 (1) ◽  
pp. 75-82 ◽  
Author(s):  
H Keay ◽  
J A Trew
Keyword(s):  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Inorganic Phosphate ◽  
Serum Alkaline Phosphatase ◽  
Automated Determination

Abstract An automated procedure for the measurement of alkaline phosphatase activity by a modification of the Bodansky method is described. It has been possible to adapt the Fiske-SubbaRow method for phosphate so that alkaline phosphatase is determined by a second run, immediately following the determination of inorganic phosphate. Studies of the effect of time, pH, and concentration of barbital on the enzyme activity are discussed, and the advantages of the method are listed.

Automated Alkaline Phosphatase Determination

1967 ◽  
Vol 13 (4) ◽  
pp. 290-298 ◽  
Author(s):  
Bernard Klein ◽  
James H Kaufman
Keyword(s):  
Alkaline Phosphatase ◽  
Excellent Agreement ◽  
Enzyme Activities ◽  
Present Method ◽  
Serum Alkaline Phosphatase ◽  
Control Analysis

Abstract An automated procedure for the determination of serum alkaline phosphatase uses phenolphthalein monophosphate as the substrate. Important assets of this substrate are: (1) A chromogenic product is enzymically produced and can be measured directly; (2) Bilirubin does not interfere, thus eliminating the need for a control analysis. Excellent agreement is obtained when the enzyme activities determined by the present method and by the automated p-nitrophenylphosphate procedure are compared.

Automated Determination of Serum Hexosaminidase A by pH Inactivation for Detection of Tay-Sachs Disease Heterozygotes

1974 ◽  
Vol 20 (5) ◽  
pp. 538-543 ◽  
Author(s):  
Abraham Saifer ◽  
Guta Perle
Keyword(s):  
Automated System ◽  
Ashkenazi Jewish Population ◽  
Tay Sachs Disease ◽  
Hexosaminidase A ◽  
Ph Inactivation ◽  
The Mean ◽  
Manual Test ◽  
Normal Adults ◽  
Automated Determination

Abstract We have automated a manual test for detection of heterozygotes of Tay-Sachs disease by assay of hexosaminidase A in serum, based on pH inactivation [Clin. Chim. Acta 43, 417 (1973)]. The same manifold is used both for the total hexosaminidase and pH-inactivation (hexosaminidase B) procedures. Automation expedites mass screening of the Ashkenazi Jewish population for carriers of the Tay-Sachs gene (prevalence rate, 1:30), because 100 or more tests can be performed daily. The mean percentage value and range (±2 SD) of hexosaminidase A for normal adults is 68.6 (58-79) and for carriers is 48.8 (39-59) with the automated pH-inactivation procedure. "Presumed carriers" (<53% hexosaminidase A) and individuals in the uncertain range (53 to 58%) should be retested by using leukocytes, to avoid the effect of certain physical ailments, before being labeled as carriers. The same automated system used for this assay can also be used to detect carriers of at least seven other sphingolipidoses for which artificial fluorogenic substrates are available.

An Automated Alkaline Phosphatase Assay with Phenolphthalein Monophosphate as Substrate

1967 ◽  
Vol 13 (4) ◽  
pp. 281-289 ◽  
Author(s):  
Kirsten Hviid
Keyword(s):  
Alkaline Phosphatase ◽  
Normal Values ◽  
Serum Alkaline Phosphatase ◽  
Colorimetric Determination ◽  
Alkaline Phosphatase Assay ◽  
Phosphatase Assay ◽  
Hydrolysis Of

Abstract The manual procedure of Babson et al. (1) for the determination of serum alkaline phosphatase has been automated. The assay is based on the colorimetric determination of phenolphthalein formed on hydrolysis of phenolphthalein monophosphate. The procedure utilizes 0.16 ml. of serum without dialysis. Blanks are required only for turbid sera. Results are compared with those obtained by the manual procedure, and data relating to sample interaction, precision, blank values, and normal values are presented.

BIOASSAY OF PARATHYROID HORMONE IN RATS BY DETERMINATION OF PLASMA CALCIUM, URINARY 32P EXCRETION AND SERUM ALKALINE PHOSPHATASE

1966 ◽  
Vol 35 (3) ◽  
pp. 229-238 ◽  
Author(s):  
R. J. TREACHER
Keyword(s):  
Alkaline Phosphatase ◽  
Parathyroid Hormone ◽  
Serum Calcium ◽  
Calcium Concentration ◽  
Serum Alkaline Phosphatase ◽  
Plasma Calcium ◽  
Significant Alteration ◽  
Good Precision ◽  
Serum Calcium Concentration

SUMMARY Methods for assay of parathyroid hormone based on an increase in serum calcium concentration, urinary 32P excretion and serum alkaline phosphatase elevation in parathyroidectomized rats have been compared and modifications introduced to improve sensitivity, precision, speed and ease of manipulation. Both the serum calcium and urinary 32P assay gave good precision (mean λ = 0·23 and 0·29, respectively) but by the serum calcium method less than 10 USP units of parathyroid hormone could not be detected, whereas the phosphaturic assay detects as little as 0·5 USP unit. Both assays are simple to perform and each requires only 2 days to complete. They can be combined in a single design using the same animals. Assays based on serum alkaline phosphatase levels in parathyroidectomized rats were not successful since it was impossible to produce a significant alteration in serum alkaline phosphatase by the administration of parathyroid hormone.

Comparison of Heat Fractionation and Gel Electrophoresis Methods for the Quantitative Determination of Alkaline Phosphatase Isoenzymes

2020 ◽  
Vol 154 (Supplement_1) ◽  
pp. S8-S8
Author(s):  
Kayode Balogun ◽  
Megan Lee ◽  
Kelly Doyle
Keyword(s):  
Alkaline Phosphatase ◽  
Quantitative Determination ◽  
Electrophoretic Mobility ◽  
Gel Electrophoresis ◽  
Heat Stability ◽  
Small Sample ◽  
Placental Alkaline Phosphatase ◽  
Heat Stable ◽  
Alkaline Phosphatase Isoenzymes

Abstract Introduction Alkaline phosphatase (ALP) is important in the diagnostic work-up for hepatobiliary and bone diseases. ALP isoenzymes are expressed in the bone, liver, kidney, placenta, and intestine, and vary in heat stability and electrophoretic mobility. Distinguishing the different ALP isoenzymes is clinically important for the diagnosis of pathologies associated with elevated ALP activity. Current modalities available to measure ALP isoenzymes utilize the heat stability, electrophoretic mobility, and immunochemical properties of the isoenzymes. The differences inherent in these methods allow for unique benefits of each method in identifying ALP isoenzymes. The objective of this study was to compare bone, liver, and placental ALP isoenzyme results determined by heat fractionation and gel electrophoresis and to characterize the heat-stable non-liver fraction (t1/2 >11 min), reported by heat fractionation, using gel electrophoresis. Methods A total of 72 de-identified serum samples that span a wide range of known ALP isoenzyme concentrations and disease states were used to measure ALP using gel electrophoresis and heat fractionation. Heat fractionation was achieved by selective inactivation of the isoenzymes at 56 °C in 10, 15, and 20-minute intervals. Log-percent activity of the total and heat-inactivated fractions at each time point was plotted against time in minutes. The linear activity decay between 10 and 20 minutes determined the relative amount of liver isoenzyme activity and the slope of the line determined the half-lives of ALP isoenzymes. Electrophoresis was performed according to the manufacturer’s protocol using the Hydragel ISO-PAL gel to resolve ALP isoenzymes based on their electrophoretic mobility and interaction with lectin. ALP isoenzymes were quantified by densitometry. Results Our results show a significant correlation coefficient (r) of 0.98, Deming regression slope of 1.1, and bias of -1.2% for the liver isoenzyme (n=43). However, liver fractions are not distinguishable by heat fractionation when heat-stable isoforms are present. The bone fraction (n=43) showed a coefficient of correlation of 0.86, slope of 0.55, and bias of -31%. Although, with a small sample size (n=6), the placental isoenzyme showed a significant agreement between the two methods: r = 0.999, slope = 0.98, and a -3.5% bias. Of the non-liver fractions reported by heat fractionation (n=13, ALP >100 U/L) eleven (85%) showed distinct qualitative bands in the intestinal lane on gel electrophoresis; however, quantitative values did not correlate between the two methods. Conclusion Our data support an agreement between the heat fractionation and gel electrophoresis methods for the quantitative determination of liver and placental alkaline phosphatase isoenzymes. Although there is an association between the two methods, the activity of the bone isoenzyme was underestimated by the gel electrophoresis method, likely due to saturation of the gel and densitometry scan because of elevated protein concentrations. The non-liver fractions were qualitatively identified as intestinal isoenzyme.

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