Placental Isoenzyme Research Articles

Comparison of Heat Fractionation and Gel Electrophoresis Methods for the Quantitative Determination of 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.

Interferon-α gene therapy prevents aflatoxin and carbon tetrachloride promoted hepatic carcinogenesis in rats

Retrovirus-mediated interferon alpha (IFN-alpha) gene transfer was evaluated with regard to its possible protective effects against aflatoxin B1 (AFB1)-initiated and carbon tetrachloride (CCl4)-promoted hepatic carcinogenesis in rats. To our knowledge, this is the first time an experimental in vivo gene therapy trial was conducted in Egypt. Two genes were examined in liver tissue by RT-PCR: the first was glutathione-S-transferase placental (GST-P) isoenzyme, as an early marker to detect hepatic malignancy; the second was IFN-alpha gene expression to detect the efficiency of gene uptake and its persistence after transduction. Forty male rats, divided equally into 4 groups, were included in the study: the first group was the control; the second group received CCl4 0.2 ml subcutaneously twice weekly for 12 weeks and AFB1 0.25 mg/kg body wt intraperitoneally twice weekly for 6 weeks; the third group received IFN-alpha (10(8) pfu) intravenously in the tail vein prior to the start of CCl4 and AFB1 injections; and the fourth group received IFN-alpha (10(8) pfu) by intrahepatic injection under ultrasonography guide after termination of the CCl4 and AFB1 injection schedule. The results showed that IFN-alpha has a marked and significant protective effect against hepatic fibrogenesis as well as hepatic carcinogenesis. Pathological examination of liver tissue proved that IFN-alpha minimized both fibrotic and cirrhotic processes. The amount of fibrosis was less in both groups receiving IFN-alpha, with more protection in the group that received IFN-alpha intravenously prior to CCl4 and AFB1. The results of RT-PCR showed that the IFN-alpha gene was significantly expressed in both groups receiving IFN-alpha, with a more intense expression in the group that received IFN-alpha by intrahepatic injection after termination of CCl4 and AFB1 injections. The IFN-alpha gene was detected after three months of gene transduction in rats receiving IFN-alpha intravenously prior to CCl4 and AFB1 and after one month of gene transduction in the post CCl4 and AFB1 rats. IFN-alpha gene was not expressed in the two groups that did not undergo gene transfer. Histopathological signs of premalignant macronodules were evident in the group receiving CCl4 and AFB1, but not IFN-alpha as well as in the group that received IFN-alpha at the end of the experiment. GST-P gene expression was also detected in these two groups, confirming early malignant transformation. In conclusion, IFN-alpha exerts significant protective effects, but more so when the gene is administered before fibrogenic and carcinogenic induction in hepatic tissues. IFN-alpha gene therapy may be justified in clinical trials for high-risk candidates with hepatic carcinogenesis.

Lower alkaline phosphatase activity and occurrence of an abnormal hybrid intestinal/tissue non-specific isoform in Down's syndrome amniotic fluids.

Quantitative and qualitative analysis of alkaline phosphatases (AP) was performed on amniotic fluid in 59 normal pregnancies and 14 Down's syndrome (DS) pregnancies at 16, 18 and 19 weeks of gestation. In DS cases, intestinal and placental isoenzyme levels were significantly reduced (P<0.001) and the AP electrophoretic pattern was seen to be modified on polyacrylamide gel electrophoresis. A unique component was detected. After extraction and purification of the abnormal isoenzyme, peptide fragments obtained after cyanogen bromide cleavage indicated a hybrid heterodimeric AP composed of intestinal and tissue non-specific subunits, as evaluated by SDS polyacrylamide gel electrophoresis.

Bile acid secretion during rat liver carcinogenesis

Retro-differentiation of liver parenchyma during neoplastic processes is characterized by the expression of tumor antigens, such as α-fetoprotein and the placental isoenzyme of glutathione-S-transferase (GST-P). To investigate whether this may also affect a typical liver function such as bile acid secretion was the aim of this work. Rat hepatocarcinogenesis was induced by diethylnitrosamine (i.p., 200 mg/Kg body weight at day 0) and promoted by two-thirds partial hepatectomy (at day 21) plus 2-acetamidofluorene administration (50 mg/Kg body weight, subcutaneously, twice a week from day 14 to day 35). In order to carry out planimetric measurements of neoplastic tissue after immunohistochemical staining, a novel monoclonal antibody (MAb 14.1.3) against GST-P with no cross-reactivity against the major liver isoform of GST (GST-H) was raised. Analysis of total biliary bile acid output using the 3α-hydroxysteroid dehydrogenase method indicated that a significant reduction (−26%) occurred during the formation of GST-P-positive foci (12 wk). This was restored to normal values during adenoma formation (16–20 wk), but decreased again during carcinoma transformation (32 wk). These changes were not parallel to that observed in bile flow, which was progressively but slightly decreased throughout the whole period under study. HPLC analysis of bile samples collected for 1 h at different time points during hepatocarcinogenesis revealed that in contrast to what happens during cholestatic disease, a continuous and progressive increase in the cholic acid-to-chenodeoxycholic acid ratio (from 4.4 ± 0.5 in control animals to 15.1 ± 1.9 in rats with hepatocellular carcinoma) occurs. A significant and transient increase at 16 wk (+120%) in the proportion of bile acids amidated with glycine as compared to those conjugated with taurine was also observed. These results indicate that the mechanisms accounting for the secretion of major bile acids are modified differently at various steps of rat liver tumor development.

Heat stable and urea resistant alkaline phosphatase in maternal neutrophils from normal and Down syndrome pregnancies

Activity levels of total and placental alkaline phosphatase (AP) were determined in maternal serum and neutrophils of normal and Down syndrome pregnancies. The placental iso-enzyme (PAP) was identified by its relative stability to urea and heat. Significant increase in the activity of all iso-enzymes across gestational stages was observed in maternal sera of 28 normal pregnancies. However, in the neutrophil extracts of the same blood samples no differences were detected between trimesters. Another set of experiments was aimed at finding diagnostic differences of PAP activity in maternal neutrophils of normal and trisomy 21 affected pregnancies. No differences of heat stable AP activity were found in maternal samples of 19 normal and 19 Down syndrome affected pregnancies. Urea resistant AP proportions were also similar when measured after 40 minutes of exposure (13 samples in each group). However, a marginally significant increase was observed in the mean value of the Down syndrome affected samples, after 60 minutes of urea treatment. In view of the above results we conclude that neutrophil AP activity is not as yet a useful marker for the screening of trisomy 21 fetuses. Copyright © 1999 John Wiley & Sons, Ltd.

Heat stable and urea resistant alkaline phosphatase in maternal neutrophils from normal and Down syndrome pregnancies

Activity levels of total and placental alkaline phosphatase (AP) were determined in maternal serum and neutrophils of normal and Down syndrome pregnancies. The placental iso-enzyme (PAP) was identified by its relative stability to urea and heat. Significant increase in the activity of all iso-enzymes across gestational stages was observed in maternal sera of 28 normal pregnancies. However, in the neutrophil extracts of the same blood samples no differences were detected between trimesters. Another set of experiments was aimed at finding diagnostic differences of PAP activity in maternal neutrophils of normal and trisomy 21 affected pregnancies. No differences of heat stable AP activity were found in maternal samples of 19 normal and 19 Down syndrome affected pregnancies. Urea resistant AP proportions were also similar when measured after 40 minutes of exposure (13 samples in each group). However, a marginally significant increase was observed in the mean value of the Down syndrome affected samples, after 60 minutes of urea treatment. In view of the above results we conclude that neutrophil AP activity is not as yet a useful marker for the screening of trisomy 21 fetuses.

Serum beta-hexosaminidase isoenzymes are precursor forms.

The lysosomal enzyme beta-hexosaminidase (Hex, EC 3.2.1.52) was purified from human serum and placental tissue. On gel chromatography, Hex isoenzymes (Hex A, B and P) from serum showed a molecular weight of about 150 kDa, i.e. higher than the 120 kDa found for the placental (tissue) isoenzymes (Hex A and B). Sodium dodecyl sulphate polyacrylamide gel electrophoresis of non-reduced serum Hex A revealed two different subunits, denoted alpha and beta, corresponding to apparent molecular weights of 70 and 67 kDa, respectively. Serum Hex B and Hex P were composed of beta-subunits only. In contrast, the apparent molecular weights of the alpha- and beta-subunits originating from tissue were both 55 kDa. Reduction of tissue Hex A and Hex B resulted in cleavage of the beta-subunit into several fragments, whereas the beta-subunit in serum forms was resistant to this treatment. These results are consistent with the serum forms never having entered the lysosome prior to their release to the extracellular environment and serum forms can therefore be classified as precursor molecules. No mature (tissue) forms were found in serum. Isoelectric focusing before and after neuraminidase treatment of the serum isoenzymes showed that they all contained sialic acid and that Hex B and Hex P had an identical focusing pattern after desialylation. Serum beta-hexosaminidase is frequently elevated in liver disease, alcoholism and pregnancy and the results of the present study indicate that this is due to an increased synthesis and secretion of hypersialylated beta-subunits, rather than to release of intralysosomally stored enzyme.

Multicomponent analysis for alkaline phosphatase isoenzyme determination by multiple linear regression

Alkaline phosphatase (EC 3.1.3.1) isoenzymes in serum may be determined by multicomponent analysis of the enzyme activities in the presence of multiple inhibitors. To determine inhibition coefficients of the isoenzymes, we used multiple linear regression analysis to compare alkaline phosphatase activities in the presence of known inhibitors with electrophoretically determined isoenzyme activities in plasma and serum samples. All possible combinations of exactly determined and overdetermined linear systems of inhibitors were ranked according to their prediction error to select an optimum set. The best multicomponent system for prediction included the use of levamisole, phenylalanine, and heat inhibition at 56 degrees C and 65 degrees C to determine bone, hepatic, intestinal, and placental isoenzymes. Consideration of the hepatic isoenzyme as liver and macromolecular fractions resulted in significantly worse predictions. Error analysis involving repeat determinations and a simplex optimization of the inhibition coefficients indicated that the inaccuracy of the comparison electrophoretic method may have been a major factor affecting poor isoenzyme prediction in some samples.

Rat Plasma Clearance Rate and Organ Distribution of β-Hexosaminidase Isoenzymes from Human Serum

beta-Hexosaminidase (EC 3.2.1.30) is markedly increased in human serum in liver disease, chronic alcoholism, and pregnancy. Knowledge of the clearance rate of plasma/serum beta-hexosaminidase is necessary to evaluate this increase. We studied the plasma clearance of beta-hexosaminidase isoenzymes (purified from human serum and placenta) after their infusion into rat circulation. A recently developed enzyme immunoassay method was used to measure the human beta-hexosaminidase isoenzymes; this method relies on both immunoreactivity and enzyme activity, so intact human isoenzymes were measured. In comparison with the placental isoenzymes (t1/2 less than 2 min), the serum forms showed a considerably slower clearance (t1/2 = 2-4 h). However, desialylation of the serum forms resulted in their rapid clearance (t1/2 less than 2 min). The organ localization of the enzyme eliminated from the circulation was investigated 24 h after infusion. Placental and native serum isoenzymes accumulated mainly in the liver and the spleen. Desialylated serum forms were almost exclusively localized to the liver.

6-Phosphofructo-1-kinase of rat placenta

6-Phosphofructo-1-kinase (PFK) of rat placenta was purified to homogeneity with a recovery of 56% of the enzyme activity in the original extract. The purified enzyme is a tetramer and the Mr value of the subunit is 85,000 +/- 1500 as shown by gel filtration and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Considering the properties of the native rat placental PFK isoenzyme, it is clear that this tissue is a complex mixture of homotetramer and heterotetramer. Purified placenta PFK displayed little cooperativity at pH 7.0 with respect to fructose 6-phosphate and was markedly inhibited with high concentrations of ATP. The affinity of the enzyme for fructose 6-phosphate was increased by fructose 2,6-biphosphate. The purified enzyme was highly inhibited by citrate, whereas it was only slightly inhibited by phosphoenol pyruvate. ADP, AMP and fructose 2,6-bisphosphate showed little stimulation towards placental PFK. The present study suggests that the placental PFK is a relatively active enzymic form and it is also probably characterized with a high rate of glycolysis possibly because this tissue requires a high energy production for the development and maintenance of the fetus as the placenta tends to be a semipermeable membrane through which substances are exchanged between mother and fetus.

Characterization and expression of uterine and placental alkaline phosphatases in the mouse

Alkaline phosphatase (ALP) is rapidly induced in the uterine subepithelial stroma after a natural or artificial decidual stimulus. During gestation ALP-specific activity peaked at Day 7 to 8 (Day 1 is day of detection of the copulation plug) followed by a rapid decline to control levels by Day 9. This elevation in enzyme activity was preceded by an 8-fold induction of a 2.6 kilobase (kb) mRNA. This mRNA was not preferentially localized to implantation sites. ALP activity was detected in the placenta at Day 9 and reached maximum specific activity at Day 19. The placental ALP was also encoded by a 2.6 kb mRNA. Uterine and placental ALPs were inhibited to the same extent by levamisole, L-tryptophan and homoarginine. The calculated Ki values for these inhibitors were not statistically different between the uterine and placental forms. Km values towards the substrate p-nitrophenylphosphate, however, were statistically different between the uterine and placental forms. Both uterine and placental ALPs were stimulated 3-4-fold by addition of 2 mM-Mg2+. Electrophoretic mobilities on SDS polyacrylamide gel, where the enzyme migrated as a single band, were the same. The uterine form, however, could be distinguished from the placental isoenzyme by separation on non-denaturing polyacrylamide gels; the uterine form had a single zone of activity which migrated with an intermediate mobility between the two zones of activity detected for the placental enzyme. These differences in mobility could be ascribed to the sialic acid content of the enzyme because treatment with neuraminidase resulted in the uterine and placental forms migrating with comparable but slower mobilities in non-denaturing gels.(ABSTRACT TRUNCATED AT 250 WORDS)

An alkaline phosphatase reacting with both monoclonal antibodies to intestinal and placental isoenzymes

An alkaline phosphatase reacting with both monoclonal antibodies to intestinal and placental isoenzymes

Modulation of enzymatic activities during spontaneous and induced differentiation in a human pancreatic adenocarcinoma cell line CAPAN-1

Different enzymatic activities were studied in the human pancreatic cancer cell line CAPAN-1 in order to analyze their relation to differentiation. Alkaline phosphatase (Alk Ph), acid phosphatase, aminopeptidase, dipeptidyl peptidase IV, acid and neutral alpha-glucosidases, and acid beta-galactosidase were present. Especially alkaline phosphatase, which we have found to be of the placental type isoenzyme, is being highly expressed. Spontaneous cell differentiation at confluence as well as differentiating agents: sodium butyrate and DMSO, modulated the levels of three enzymes: Alk. Ph., aminopeptidase, and acid alpha-glucosidase. The exposure of the cells to the differentiating agents amplified the modulations occurring during the spontaneous differentiation. Aminopeptidase and acid alpha-glucosidase were found to be induced by differentiation. Alk Ph specific activity was significantly increased by the spontaneous and the butyrate-induced differentiations; whereas DMSO exerted an opposite effect, probably related to its biphasic action on cell proliferation.

Alkaline phosphatase activity from human osteosarcoma cell line SaOS-2: an isoenzyme standard for quantifying skeletal alkaline phosphatase activity in serum.

Earlier we described a kinetic assay for quantifying skeletal alkaline phosphatase (ALP) isoenzyme activity in serum. The precision of the assay depends on including ALP standards for the skeletal, hepatic, intestinal, and placental isoenzymes. We wondered whether human osteosarcoma cells could provide an efficient alternative to human bone or Pagetic serum as a source of the skeletal ALP standard. ALP activities prepared from five human osteosarcoma cell lines were compared with a bone-derived ALP standard with respect to heat stability and sensitivity to chemical effectors. Two of the cell lines (SaOS-2 and TE-85) contained ALP activities that resembled the bone-derived standard. We selected SaOS-2 cells for additional evaluation (as a potential source of isoenzyme standard), because they contained 40-50 times more ALP activity than did the TE-85 cells. To include the SaOS-2 cell-derived ALP activity in the quantitative isoenzyme assay, we diluted the enzyme in a solution containing heat-inactivated (i.e., ALP-negative) human serum. Surprisingly, this dilution caused a 60-125% increase in maximum enzyme activity. In the quantitative assay of ALP isoenzyme in serum, the SaOS-2 derived ALP was indistinguishable from the serum skeletal ALP standard, with respect to the above criteria and assay variations. Evidently ALP from SaOS-2 cells is suited as a standard for measuring skeletal ALP activity in this assay.

Levamisole inhibition of alkaline phosphatase and 5'-nucleotidase of bovine milk fat globule membranes

1. 1. The effect of levamisole (LMS) on alkaline phosphatase (EC 3.1.3.1) and 5'-nucleotidase (EC 3.1.3.5) activities of bovine milk fat globule membranes (MFGM) was examined. 2. 2. LMS inhibited MFGM alkaline phosphatase activity in a concentration-dependent manner with 50% inhibition produced by 49 ± 23 μ M LMS. 3. 3. 5'-Nucleotidase was resistant to LMS inhibition with 30.9% inhibition produced by 10 mM LMS, the highest concentration tested. 4. 4. LMS was an uncompetitive inhibitor of MFGM alkaline phosphatase with a K i of 45 ± 6μM. 5. 5. The extent of LMS inhibition of alkaline phosphatase was dependent on the substrate utilized in the assay. 6. 6. The effect of LMS on bovine MFGM alkaline phosphatase was similar to LMS effects on other mammalian alkaline phosphatases of liver/kidney/bone/placental isoenzyme origin.

Alkaline phosphatase activities in human amniotic fluid, chromatographic separation of the foetal intestinal component

Hydrophilic gel permeation chromatography of 14-36 wk human amniotic fluid on Fractogel columns divides the total alkaline phosphatase (AP) activity in a higher and a lower mol wt zones. Differential inhibition testing, isoelectric focusing, cellulose acetate, agarose and polyacrylamide gel electrophoreses before and after neuraminidase treatment show the higher mol wt zone to be homogeneous and to be made of the higher mol wt foetal intestinal isoenzyme form whereas the lower mol wt zone represents an unresolved mixture of hepatic, placental and lower mol wt foetal intestinal isoenzymes. In the early stages of pregnancy, the activity associated with the higher mol wt zone outweighs by far that of the lower mol wt zone; however from the 24 th week one notes a steady increase in the relative magnitude of this second zone until at the end of the gestation period both zones assume near equal importance albeit within a lower total AP activity. Satisfactory quantitation of the higher mol wt foetal intestinal isoenzyme form in one ml amniotic fluid can be attained after a 3-h chromatography run using p-nitrophenylphosphate as substrate.

Isoelectric focusing on cellulose acetate membrane: a separation procedure for alkaline phosphatase isoenzymes

An isoelectric focusing technique for the separation of alkaline phosphatase isoenzymes on cellulose acetate membrane is described. Optimal conditions for isoelectric focusing were established by changing ampholine concentration and focusing conditions. Bone, liver, intestinal, and placental isoenzymes can be resolved into vacious sub-bands in a pH range of 4.1 to 5.2. These sub-bands were correlated with the findings of electrophoretic isoenzyme separation. The whole procedure proves very simple to perform and comparatively time saving (4 h). This procedure may help clarify the problems of ALP isoenzyme differention when electrophoretic patterns are unresolved.

Identification of a human osteosarcoma-associated glycoprotein with monoclonal antibodies: relationship with alkaline phosphatase.

The molecular nature of an osteosarcoma-associated antigen was investigated with the three monoclonal antibodies Ost6 (immunoglobulin (IgG1), Ost7 (IgG1), and Ost15 (IgG2a), which selectively react with frozen sections of osteosarcoma and chondrosarcoma tissues. When tested with a panel of 41 human cell lines in the mixed hemadsorption assay, the antibodies reacted similarly with three of six osteosarcomas, one choriocarcinoma, one teratoma, and one osteoblast-like culture, but failed to react with 32 lines of normal and other tumor cell types. Immunoprecipitation plus sodium dodecyl sulfate (SDS)--polyacrylamide gel electrophoresis and sequential immunoprecipitation studies revealed that in [35S]methionine- or [14C]glucosamine-labelled osteosarcoma cells the three antibodies detected a single glycoprotein, with an apparent molecular mass of 86 kilodaltons (kDa), which was not affected by reducing conditions. Tunicamycin treatment and pulse-chase experiments showed glycosylation of this molecule to be N-linked; it arose from a 54-kDa polypeptide precursor. Alkaline phosphatase activity was detected in the material rich in 86-kDa molecules that was immunoprecipitated from serologically reactive cell lines with each antibody. These antibodies also cross-reacted with two isoenzymes of alkaline phosphatase (strongly with the liver and bone, and moderately with the placental isoenzyme), but not with the intestinal form.

Cloning and sequencing of human intestinal alkaline phosphatase cDNA.

Partial protein sequence data obtained on intestinal alkaline phosphatase indicated a high degree of homology with the reported sequence of the placental isoenzyme. Accordingly, placental alkaline phosphatase cDNA was cloned and used as a probe to clone intestinal alkaline phosphatase cDNA. The latter is somewhat larger (3.1 kilobases) than the cDNA for the placental isozyme (2.8 kilobases). Although the 3' untranslated regions are quite different, there is almost 90% homology in the translated regions of the two isozymes. There are, however, significant differences at their amino and carboxyl termini and a substitution of an alanine in intestinal alkaline phosphatase for a glycine in the active site of the placental isozyme.

Quantitative method for determining serum alkaline phosphatase isoenzyme activity I. Guanidine hydrochloride: new reagent for selectively inhibiting major serum isoenzymes of alkaline phosphatase.

The potential use of the protein denaturant guanidine hydrochloride to inhibit selectively the enzyme activity of serum alkaline phosphatase isoenzymes from liver, bone, intestine, and placenta was investigated. Inhibition of each isoenzyme was shown to be dependent on time and concentration of inhibitor. In the presence of 0.3 mol/l (28.7 g/l) guanidine hydrochloride for 170 seconds 14%, 47%, and 90% of the total alkaline phosphatase activity remained in samples of bone, liver, and intestinal origins, respectively. In contrast, the activity of the placental isoenzyme increased by 24%. The degree of inhibition was shown to be independent of total alkaline phosphatase activity. Investigations were performed at 37 degrees C using the Cobas Bio centrifugal analyser. We conclude that this reagent has several practical advantages over urea as a selective inhibitor of alkaline phosphatase isoenzymes, including a faster and more reproducible inhibition at a much lower reagent concentration.