Measurement of serum bone-specific alkaline phosphatase activity in cynomolgus macaques

Serum levels of bone-specific alkaline phosphatase activity (B-ALP) in cynomolgus monkeys were evaluated as an index of elevated bone turnover following ovariectomy. The enzyme immunoassay 96-well microtiter plate B-ALP assay, developed by Metra Biosystems (Mountain View, CA) for human use, was employed and compared with a standard automated assay measuring total serum levels of alkaline phosphatase activity (T-ALP). The B-ALP assay was first validated for use in these monkeys. Ovariectomy led to increased bone turnover as indicated by approximately 2-fold higher activity in both assays and this elevation was inhibited by daily estradiol administration. Although both assays provided generally similar results, several monkeys were observed to have greatly elevated values of T-ALP but not B-ALP. This discrepancy is believed to result from high levels of the liver isoform of alkaline phosphatase in monkeys with hepatic dysfunction, which are not detected by the B-ALP assay.     

Depression of activity of intestinal mucosal alkaline phosphatase with gastrointestinal microorganisms.

Depression of intestinal alkaline phosphatase activity can be caused by the formation of the microbial ecosystem with several kinds of viable microorganisms in rats.     

Measurement of serum alkaline phosphatase

Alkaline phosphatase is a commonly requested enzyme test in clinical chemistry. However, the enzyme is not particularly substrate specific, which has led to a proliferation of methods for its analysis. It can exhibit a variable instability effect depending on the techniques required for its storage or analysis. Methods can also be highly dependent on sample isoenzyme distribution and reagent purity, leading to problems in the quality control of its analysis and in the comparison of results obtained from different methods. Alkaline phosphatase is not tissue specific and this may on occasion lead to uncertainty in the interpretation of its measured activity in blood serum. In recent years there has been a number of attempts to standardize methodology for this and other enzymes. Perhaps an alternative approach to the measurement of alkaline phosphatase activity will alleviate some of the problems encountered.     

Correlation between serum alkaline phosphatase activity and blood glucose levels

Fasting blood glucose levels and serum alkaline phosphatase activity of age-matched Libyan diabetic men (168) and women (168) were determined. The mean levels of blood glucose of men and women were 227 +/- 6 and 237 +/- 5 mg/dl, respectively. The respective values of serum alkaline phosphatase were 179 +/- 5 and 199 +/- 6 IU/l. The mean serum phosphatase activity of women was significantly higher (p less than 0.001) than that of their male counterparts. A statistically significant positive correlation was found between serum alkaline phosphatase and blood glucose levels of these diabetic patients (r = 0.35; p less than 0.001).     

Activation of thiol-dependent antioxidant activity of human serum albumin by alkaline pH is due to the B-like conformational change

Antioxidant activity of human serum albumin (HSA) increased steeply as the reaction mixture was shifted from neutral to alkaline pH. The antioxidant activity was also remarkably increased by Ca(2+) or a cationic detergent (cetyltrimethylammonium chloride). Carboxyl group modification of HSA resulted in about 40-fold increase of the antioxidant activity. The chemical modification study indicated that in addition to functional cysteine(s), cationic amino acid residues such as histidine, arginine and lysine appeared to involve in the antioxidant reaction. HSA also exhibited alkaline-pH dependent peroxidase activity to remove fatty acid hydroperoxide. At neutral pH, only two thiols of Cys-289 and free Cys-34 of HSA were modified by a thiol-specific modification reagent, 5-((((2-iodoacetyl)amino)ethy)amino)naphthalene-1-sulfonic acid (I14), regardless of the presence or absence of dithiothreitol (DTT), and the resultant antioxidant activity was not decreased, suggesting that Cys-289 and Cys-34 did not participate in the antioxidant reaction. At alkaline pH, I14 modified several additional HSA thiols in the presence, but did not in the absence of DTT. The antioxidant activity of the modified HSA was remarkably decreased to as much as 30% of the antioxidant activity given by the unmodified HSA in the absence of DTT. The HPLC pattern for tryptic peptides containing modified cysteine(s) derived from the I14-treated c-HSA (carboxyl group-modified HSA) at pH 7.0 with DTT was very similar to that of the I14-modified HSA at pH 8.0 with DTT. Taken together, these results suggest that activation of thiol-dependent antioxidant activity of HSA at alkaline pH is due to the conformational change favorable for the functional cysteine(s)-mediated catalysis.     

Phosphoprotein-phosphatase activity associated with human placental alkaline phosphatase.

Human placental alkaline phosphatase, a marker protein for some nontrophoblastic neoplasms, was found to have phosphoprotein phosphatase activity. This was demonstrated by the dephosphorylation of ³²P-labeled histones, protamine, glycogen synthetase, casein, and phosvitin at various pH values. Unlike the general phosphoprotein phosphatase, the placental alkaline phosphatase does not have phosphorylase $\text{a}$ phosphatase activity.     

Investigation of alkaline phosphatase activity in the serum of pregnant rats

An investigation was undertaken to determine if the placental alkaline phosphatase of the rat enters the maternal circulation and to study some of its characteristics. Unlike human, rat placental alkaline phosphatase was found to be heat labile and the alkaline phosphatase activity in the serum of both pregnant and non-pregnant rats was also found to be heat labile. Also unlike the human, the alkaline phosphatase activity in rat serum does not increase as pregnancy progresses to term. In an endeavour to establish if the rat placental enzyme is present in the serum of the pregnant rat, the characteristics of the enzyme in both placental extracts and serum of non-pregnant and 1-, 2- and 3-week pregnant rats were studied using the techniques of heat stability at 56°, gel filtration through Sephadex columns, disc gel electrophoresis, and L-phenylalanine inhibition. The presence of rat placental alkaline phosphatase in maternal serum could not be positively demonstrated by any of these procedures, suggesting that rat placental alkaline phosphatase does not enter the maternal serum.     

Intracellular alkaline phosphatase activity in cultured human cancer cells

Summary The effect of saponin treatment in demonstrating intracellular portion of alkaline phosphatase activity in human cancer cell lines was evaluated. Previous reports using standard lead-salt techniques visualized enzyme almost exclusively on the plasma membrane and sometimes in the lysosomes. However, by treating cells with saponin before or during the cytochemical incubation, intracellular alkaline phosphatase became demonstrable at the endoplasmic reticulum, Golgi apparatus, Golgi-derived vesicles and mitochondria as well as lysosomes and plasma membrane. These intracellular catalytic activities were significantly inhibited by the specific amino acid inhibitors characteristic for each cell line, and this suggested that intracellular alkaline phosphatase is the same isoenzyme as that present in the plasma membrane. The results of our current and previous studies therefore indicate that saponin reveals latent intracellular alkaline phosphatase activity by changing the membrane's physical state; thereby increasing the availability of both catalytic and antigenic sites of the enzyme to substrate and to antibody respectively.This work was supported by National Institutes of Health Grant No. CA 21967     

Stabilization of human serum alkaline phosphatase to histidine-induced heat inactivation by tryptic digestion.

1. Serum alkaline phosphatase [EC 3.1.3.1] was strongly inactivated by histidine during incubation at pH 8.0 and 45degrees; however, tryptic digestion of the serum strongly protected the enzyme against inactivation by histidine. In the absence of histidine, however, neither heat inactivation of the phosphatase nor the effect of trypsin [EC 3.4.21.4] was observed. Factors affecting the alkaline phosphatase inactivation were studied further. 2. The effect of trypsin on the histidine-induced heat inactivation differed considerably according to the tissue source of the enzyme, which suggests a possible method for distinguishing alkaline phosphatase isoenzymes.     

Differences in phosphatidate hydrolytic activity of human alkaline phosphatase isozymes

Hydrolytic activities of human alkaline phosphatase isozymes were investigated using phosphatidases with various fatty acyl chains (egg phosphatidate and dioleoyl, distearoyl, dipalmitoyl, dimyristoyl and dilauroyl phosphatidates). In the presence of sodium deoxycholate, purified human placental and intestinal alkaline phosphatases hydrolyzed all the phosphatidates examined. The hydrolytic activity was maximal in the presence of 10 g/l sodium deoxycholate. Of the phosphatidates, dilauroyl phosphatidate was the best substrate. Using the same unit of the enzyme, the phosphatidate hydrolytic activity of placental alkaline phosphatase was 2- to 3-times higher than that of the intestinal enzyme. In contrast, liver alkaline phosphatase did not hydrolyze phosphatidates with long fatty acyl chains (C16-18) even in the presence of sodium deoxycholate. The liver enzyme hydrolyzed dimyristoyl and dilauroyl phosphatidates very slowly. These results show that the phosphatidates with long fatty acyl chains were useful to differentiate placental and intestinal alkaline phosphatases from the liver enzyme, and suggest that the former enzymes play a different physiological role from the liver enzyme.     

Bone-specific alkaline phosphatase activity is inhibited by bisphosphonates

Bisphosphonates (BPs) are drugs widely used in the treatment of various bone diseases. BPs localize to bone mineral, and their concentration in resorption lacunae could reach almost milimolar levels. Bone alkaline phosphatase (ALP) is a membrane-bound exoenzyme that has been implicated in bone formation and mineralization. In this study, we investigated the possible direct effect of three N-containing BPs (alendronate, pamidronate, and zoledronate) on the specific activity of bone ALP obtained from an extract of UMR106 rat osteosarcoma cells. Enzymatic activity was measured by spectrophotometric detection of p-nitrophenol product and by in situ visualization of ALP bands after an electrophoresis on cellulose acetate gels. Because ALP is a metalloprotein that contains Zn²⁺ and Mg²⁺, both of which are necessary for catalytic function, we also evaluated the participation of these divalent cations in the possible effect of BPs on enzymatic activity. All BPs tested were found to dose-dependently inhibit spectrophotometrically measured ALP activity (93–42% of basal) at concentrations of BPs between 10⁻⁵ M and 10⁻⁴ M, the order of potency being zoledronate ≊ alendronate > pamidronate. However, coincubation with excess Zn²⁺ or Mg²⁺ completely abolished this inhibitory effect. Electrophoretic analysis rendered very similar results: namely a decrease in the enzymatic activity of the bone-ALP band by BPs and a reversion of this inhibition by divalent cations. This study shows that N-containing BPs directly inhibit bone-ALP activity, in a concentration range to which this exoenzyme is probably exposed in vivo. In addition, this inhibitory effect is most possibly the result of the chelation of Zn²⁺ and Mg²⁺ ions by BPs.