Quantification of skeletal alkaline phosphatase in osteoporotic serum by wheat germ agglutinin precipitation, heat inactivation, and a two-site immunoradiometric assay

Abstract Three methods for quantifying skeletal alkaline phosphatase (ALP; EC 3.1.3.1) activity/immunoactivity in serum--heat inactivation, wheat germ agglutinin (WGA) precipitation, and an immunoradiometric assay--were tested for recovery and specificity and applied to 81 sera collected from 14 postmenopausal osteoporotic subjects. The heat-inactivation and WGA precipitation assays showed relative recoveries of 91-100% and 16-32%, respectively, for skeletal ALP with complete specificity (no cross-reactivity with hepatic or intestinal ALP); the IRMA showed a relative recovery of 86-100% and 11-14% cross-reactivity with hepatic ALP. There was a closer correlation between the heat-inactivation assay and IRMA (r = 0.833) than between the WGA precipitation assay and IRMA (r = 0.673) or between the heat-inactivation and WGA-precipitation assays (r = 0.568). The WGA precipitation assay failed to detect skeletal ALP in three serum samples that contained significant amounts as determined by the heat-inactivation assay and the IRMA.

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Wheat-germ agglutinin method for measuring bone and liver isoenzymes of alkaline phosphatase assessed in postmenopausal osteoporosis.

Clinical Chemistry ◽

10.1093/clinchem/34.8.1636 ◽

1988 ◽

Vol 34 (8) ◽

pp. 1636-1640 ◽

Cited By ~ 13

Author(s):

S Sørensen

Keyword(s):

Alkaline Phosphatase ◽

Phosphatase Activity ◽

Alkaline Phosphatase Activity ◽

Wheat Germ Agglutinin ◽

Wheat Germ ◽

Hormone Replacement ◽

Heat Inactivation ◽

Precipitation Pattern ◽

Serum Samples ◽

Total Alkaline

Abstract In the method of Rosalki and Foo (Clin Chem 1984;30:1182-6) bone and liver isoenzymes of alkaline phosphatase (EC 3.1.3.1) are quantified by using wheat-germ agglutinin (WGA). I suggest standardizing the procedure by using a WGA concentration that precipitates half of the alkaline phosphatase activity of serum pooled from an equal number of healthy women and men. By applying knowledge of the precipitation pattern in serum samples containing predominantly or exclusively bone or liver sources of alkaline phosphatase, I obtained results for the isoenzymes in healthy subjects that agreed with those by the heat-inactivation methods, as reported earlier in the literature. I then assessed the utility of the standardized procedure in a clinical study of prevention of postmenopausal bone loss. In patients receiving hormone replacement therapy, which is known to decrease bone turnover, the decrease in total alkaline phosphatase activity in serum was entirely ascribable to decreases in the bone isoenzyme activity, probably reflecting reduced bone formation, whereas the activity concentration of liver alkaline phosphatase remained unchanged.

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Reference standards for quantification of skeletal alkaline phosphatase activity in serum by heat inactivation and lectin precipitation

Clinical Chemistry ◽

10.1093/clinchem/39.9.1878 ◽

1993 ◽

Vol 39 (9) ◽

pp. 1878-1884 ◽

Cited By ~ 14

Author(s):

J R Farley ◽

S L Hall ◽

S Herring ◽

C Libanati ◽

J E Wergedal

Keyword(s):

Alkaline Phosphatase ◽

Alkaline Phosphatase Activity ◽

Concanavalin A ◽

Wheat Germ ◽

Bone Cells ◽

Heat Inactivation ◽

Osteosarcoma Cells ◽

Con A ◽

Alp Activity ◽

Skeletal Alkaline Phosphatase

Abstract Putative standards of skeletal alkaline phosphatase (ALP) (from bone, bone cells, osteosarcoma cells, and Pagetic serum) and hepatic ALP (from cholestatic serum and bile) were used to compare three methods for quantifying skeletal ALP activity in serum: heat inactivation, precipitation with wheat germ agglutinin (WGA), and precipitation with concanavalin A (Con A). All the skeletal ALP standards were similarly sensitive to heat inactivation, as were the hepatic ALP standards. Heat inactivation separated skeletal from hepatic ALP by a 50% difference in remaining ALP activities (e.g., 23% and 74% remaining skeletal and hepatic ALP activities after 30 min at 52 degrees C). Differential precipitations with WGA and with Con A were less efficient at separating skeletal from hepatic ALP (maximum differences of < 30% remaining ALP activity). Although both types of hepatic ALP standard (cholestatic serum and bile) were precipitated with similar efficiencies by WGA and Con A, the skeletal ALP standards were not (e.g., at 2.7 g/L, WGA precipitated 78-86% of the ALP activity in Pagetic serum, but only 49% of the ALP activity in extracts of human bone). These data suggest that heat inactivation is preferable to precipitation with WGA or Con A for quantifying skeletal ALP activity in serum: it better separates skeletal from hepatic ALP activity and is not sensitive to glycosyl heterogeneity.

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Immunoadsorption assay for bone alkaline phosphatase compared with wheat germ agglutinin precipitation assay in serum from (pre)pubertal girls

Clinical Chemistry ◽

10.1093/clinchem/42.12.1970 ◽

1996 ◽

Vol 42 (12) ◽

pp. 1970-1974 ◽

Cited By ~ 3

Author(s):

A A Bouman ◽

C M de Ridder ◽

J H Nijhof ◽

J C Netelenbos ◽

H A Delemarre-vd Waal

Keyword(s):

Alkaline Phosphatase ◽

Correlation Coefficient ◽

Wheat Germ Agglutinin ◽

Wheat Germ ◽

Bone Alkaline Phosphatase ◽

Cross Reactivity ◽

Reference Ranges ◽

Correlation Studies ◽

Deming Regression ◽

The Mean

Abstract The performance characteristics of two bone alkaline phosphatase (ALP; EC 3.1.3.1) assays, a wheat germ agglutinin (WGA) precipitation assay and a new immunoadsorption assay (IAA), were compared. The within- and between-run imprecision of the IAA (3.6-4.2% and 3.6-7.7%) was comparable with that of the WGA assay. The mean cross-reactivity with liver ALP appeared to be 4% in the WGA assay and 11% in the IAA. The reference ranges in a group of 155 healthy Caucasian (pre)pubertal schoolgirls were: 149-401 U/L (total ALP, 30 degrees C), 105-349 U/L (bone ALP, 30 degrees C, WGA assay), and 58-205 U/L (bone ALP, 25 degrees C, IAA). Comparison of the WGA assay (x) with the IAA (y) demonstrated a correlation coefficient of 0.95 [Deming regression equation: y = (0.56 +/- 0.01)x + (2.0 +/- 1.5); Sy[symbol: see text]x = 5.3 U/L]. Correlation studies of the WGA assay and the IAA results with total ALP demonstrated r = 0.98 and 0.96, respectively.

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