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A heat-stable alkaline phosphatase, hitherto found in two families with inherited hyperphosphatasemia, was further characterized. The enzyme was similar to serum placental alkaline phosphatase from pregnant women concerning its apparent affinity constant (Km) for 4-nitrophenyl phosphate and its reactivity with H7 monoclonal anti-placental alkaline phosphatase (PLAP) antibodies, but different in the following respects: it exhibited greater heat stability, a higher pH optimum, lower sensitivity to inhibition by L-phenylalanine, and no reactivity with C2 monoclonal anti-PLAP antibodies. The low sensitivity to L-phenylalanine suggests that the enzyme might correspond to a rare phenotype of placental alkaline phosphatase found in human term placenta.

A variety of solubilised tissue extracts from cigarette smokers and non-smokers have been screened quantitatively for both placental alkaline phosphatase (PLAP) and placental-like alkaline phosphatase (PLAP-like AP) in order to identify the possible tissue origins of the circulating PLAP-like AP found in most smokers. Lung alveolar tissue, and to a lesser extent colonic tissues, contained both PLAP-like AP and PLAP. Tissues from smokers and non-smokers contained comparable proportions and amounts of PLAP and PLAP-like AP. No other tissue source of PLAP-like AP was found other than those previously reported for testicular, endometrial and thymic tissue. Selective release of PLAP-like AP from the lung in cigarette smokers seems likely to be a major source for this isoenzyme in peripheral circulation.

Four different immunological methods for the determination of the placental isoenzyme of alkaline phosphatase (Regan isoenzyme) were compared in 64 normal blood donors, 23 healthy laboratory and medical staff workers and 68 pregnant women: a.Inhibition by soluble antibodies to the placental enzyme. b.Precipitation with soluble antibodies. c.Precipitation with immobilized antibodies. d.Measurement of the activity of the placental alkaline phosphatase following binding to an immunoabsorbent that has been obtained by polymerization of anti-placental-alkaline phosphatase γ-globulin using glutaraldehyde.The immunoabsorbent method yielded the best results. The optimal conditions were evaluated for the measurement of the activity of immunoabsorbent-fixed placental (tumoral) alkaline phosphatase activity.This method was applied to 209 normal blood donors and to 239 patients with different malignant tumors: 25.5 percent of the cancer patients exhibited an elevated Regan -isoenzyme activity in the serum.

Placental alkaline phosphatase (PLAP) is normally found in enzymically measurable amounts in second and third trimester pregnancy serum. Its occurrence in sera [1,2] and tumours [3] from patients with malignant disease has led to the development of methods to specifically identify and quantitate the enzyme. Early studies utilised properties of the PLAP isozyme. such as its heat stability and its susceptibility to inhibition by particular amino acids to detect the PLAP protein specifically. Recently immunological techniques have been used. employing antibodies raised to purified PLAP; these include solid phase radioimmunoassays 14-61 and enzyme-immunoassay [7.X]. We report the development of a sensitive, specific, automated radioimmunoassay (RIA) for the measurement of PLAP in serum.

A “rocket” electroimmunoassay for human placental alkaline phosphatase is described. The method is simple and reproducible, and uses small volumes of antiserum.

Alkaline phosphatase in human milk was found to be heat stable and have a molecular size of placental alkaline phosphatase, a Mr of 160000. However, the milk alkaline phosphatase is different from placental alkaline phosphatase. The alkaline phosphatase from milk was endowed with higher surface charge and not inhibited by l -phenylalanine (2.5 mmol/l) and l -homoarginine (10 mmol/l). Both enzymes are sialyated.

A solid-phase "direct" radioimmunoassay for determination of placental alkaline phosphatase in serum was developed. The assay is highly sensitive, with a minimum detectable dose of 9 ng protein per ml. The specificity for the placental type of the enzyme, in comparison to other serum alkaline phosphatases, was found to be convincing. The choice of genetic types of either placental alkaline phosphatase or antiserm was found not to influence the radioimmunoassay. With the described technique the serum concentration of this enzyme during normal pregnancy was measured. A 25-fold increase from low levels during the first trimester up to 252 +/- 70 ng/ml (mean +/- S.D.) in gestation week 40 was observed.

The inhibition of normal human placental alkaline phosphatase, its rare "D-variant" phenotype, and the "Nagao isoenzyme" found in an ovarian cancer fluid by peptides containing L-leucine were studied. The Nagao isoenzyme and the D-variant phenotype were distinct from the normal placental enzyme, but similar to each other in their inhibition by L-leucine and dipeptides containing L-leucine in their amino termini. On the other hand, L-leucinamide, L-Leu-L-Leu-L-Leu and L-Leu-Gly-Gly inhibitions did not readily discriminate between D-variant and normal phenotypes, but did so between D-variant and Nagao isoenzymes. The distinctions among these enzymes in their responses to peptide inhibitors may reflect differences in their primary structures.

An immunoprecipitation method for the determination of human intestinal alkaline phosphatase (I-AP) in feces is described using anti-human I-AP IgG. Antibodies to human I-AP are monospecific after absorption with human placental alkaline phosphatase and show no cross-reactions with human liver alkaline phosphatase or alkaline phosphatase from E. coli. Normal controls demontrate a logarithmic normal distribution of I-AP activity in feces. The standard deviation of the intra- as well as of the inter-assay variation is 11.7%. This method represents a simple quantitative non-invasive in vivo assay for brush border damage under experimental and/or clinical conditions.

Crystalline human alkaline phosphatase from placenta and intestine was isolated by butanol extraction, acetone precipitation, a heat step (for the placental enzyme), ammonium sulfate precipitation, anion exchange chromatography, gel filtration and crystallisation with ammonium sulfate. Rabbit antibodies showed a partial cross-reaction between both enzymes in double diffusion, quantitative precipitation experiments and in serial precipitin curves. There was no reaction with human alkaline phosphatases from liver, kidney and bone. Three phenotypes of placental alkaline phosphatase with different electrophoretical mobility exhibited identical immunological reactions with their respective antisera. Monospecific antisera were obtained by absorption with crystalline intestinal or placental alkaline phosphatase. These monospecific antisera against the placental or the intestinal alkaline phosphatase can be used for an immunological determination of these two alkaline phosphatases without contamination by other alkaline phosphatases in human serum.

Human placental alkaline phosphatase incorporates radioactive phosphate specifically and covalently at acid pH. By titration of solutions of the purified enzyme with radioactive orthophosphate, the enzyme was shown to incorporate up to 2 phosphate groups per molecule. No evidence was found to suggest that the two sites had different affinities for phosphate. Similar titrations can be used to determine the molarity of solutions of non-placental alkaline phosphatases of unknown purity, if these also are assumed to possess 2 binding sites per molecule.

The polymorphism of placental alkaline phosphatase (ALP) is usually studied by butanol extraction followed by starch gel electrophoretic separation at two different pH's, 8.6 and 6.0. Acrylamide gel electrophoresis (7.5%) in the presence of Triton X-100 is shown to be preferable in speed, reliability and reproducibility of results. All the placental ALP phenotypes detected had the same molecular mass by gradient acrylamide electrophoresis. The enzyme butanol extract is not sufficiently pure to enable the different placental ALP's to be distinguished by isoelectric focusing on polyacrylamide gels.

Specific assays for human intestinal and liver alkaline phosphatases were developed by use of isozyme specific monoclonal antibodies bound to paper discs. The assays are fast, specific and convenient to use as demonstrated by determinations of alkaline phosphatase isozymes in sera and tissues. In sera from forty healthy individuals the activity of the tissue unspecific alkaline phosphatase was determined to 32 +/- 12 IU/l (mean +/- SD). The activity of the intestinal alkaline phosphatase was found to be ten-fold lower, 3.5 +/- 6.3 IU/l (mean +/- SD), and of the placental alkaline phosphatase another ten-fold lower, 0.3 +/- 0.2 IU/l (mean +/- SD), than that of the tissue unspecific alkaline phosphatase. Several normal tissues contained all three isozymes, the intestinal mucosa, for example, which besides intestinal alkaline phosphatase also expresses trace amounts of placental and liver-bone-kidney alkaline phosphatase. Seminomas, known to express eutopically placental-like alkaline phosphatase were demonstrated to contain increased levels of both intestinal and liver-bone-kidney alkaline phosphatases as compared to the normal testis.

Placental-like alkaline phosphatase (PLAP) was measured by its catalytic activity (CA), using an amplified enzyme-linked immunoassay, and by its immunologie activity (IA), using an enzyme-linked immunosorbent assay. In both assays the same monoclonal anti-PLAP antibody was used as the primary reagent. This antibody reacts with the main epitope on PLAP that is common to PLAP of ovarian and testicular origin, as well as of PLAP that is induced by smoking. Determinations of CA and IA of PLAP were carried out in serum samples from 101 patients with epithelial ovarian cancer (49 patients with progressive or recurrent disease, 52 patients with no evidence of disease), 20 patients with testicular cancer (8 non-semi-noma testis, 12 seminoma testis) and 61 healthy controls. Smoking status was taken into consideration. The main findings from this study are: 1. In prolonged follow-up of patients who were treated for ovarian cancer, progressive or recurrent disease was never accompanied by rising values of CA or IA of PLAP. Therefore in this study, PLAP was not a good monitor for this disease. 2. In all instances where expression of PLAP was reflected by raised serum levels of these antigens, the measurement of the catalytic activity proved to be a more sensitive parameter than that of the immunologie activity. The value of PLAP measurements in the follow-up of patients with testicular cancer remains to be elucidated.

The microheterogeneity of Kasahara isozyme was investigated by affinity electrophoresis with Con A as the affinity ligand in combination with polyacrylamide gradient gel electrophoresis. On two-dimensional Con A-containing agarose gel electrophoresis, the Kasahara isozyme was separated into three molecular species. Kasahara isozyme electrophoresed as two distinct bands with enzyme activity on polyacrylamide gradient gel, but liver, intestinal or placental alkaline phosphatase showed only one distinct spot or band on both electrophoreses. One of the three molecular species of Kasahara isozyme separated by Con A-containing agarose gel electrophoresis was extracted from the gel and applied to the polyacrylamide gradient gel electrophoresis again, resulting in the same electrophoretic pattern as that of the original Kasahara isozyme. These findings indicated that the Kasahara isozyme consists of at least four molecular species. The same analysis was conducted with alkaline phosphatase of the HuH-6 cl-5 cell line, which has been reported to release an alkaline phosphatase closely resembling the Kasahara isozyme, and the results were compared with those obtained with the Kasahara isozyme.

1. I. Partially inhibitory antibodies against highly purified human placental alkaline phosphatase were prepared in rabbits. The specificity and sensitivity of immunochemical reactions were employed to develop a specific, reliable assay for the placental isoenzyme in pregnancy sera. 2. II. Monospecific antiserum to the placental isoenzyme was polymerized using ethylchloroformate. Incubation of pregnancy serum with the polymerized antiserum for 30 min resulted in over 90% binding of the placental isoenzyme to the surface of the antiserum, which can then be removed by centrifugation or filtration.

A method of radioimmunoassay for human placental alkaline phosphatase was first elaborated using the highly purified enzyme labeled with 125I and the antiserum. For the purpose of precipitating its antigen-antibody complex, goat anti-rabbit γ-globulin was employed. The lowest measurable amount by this method was 1 ng, and the specificity was also satisfactorily high, because this antibody reacted only with the alkaline phosphatase of placental origin and A1-P4 circulating in human serum, which had been previously named by the authors1 according to the migration site on agar gel plate by electrophoresis. The two enzymes; placental alkaline phosphatase and A1-P4 in the serum, have similar enzymic properties.

Isoenzymes of alkaline phosphatase from human placentas, pregnancy sera, non-pregnancy sera, cord blood, intestines, and bones were studied. Placental alkaline phosphatase and the heat-stable fraction in the pregnancy serum were found to be comparable regarding heat stability (the critical temperature of inactivation was found to be 65° for 30 min), optimum pH, sensitivity to l -phenylalanine and neuraminadase. All these aspects were similar in the two isoenzymes. Also the starch gel electrophoretic pattern of the two enzymes showed the same bands.