Heat-stable Alkaline Phosphatase Research Articles

Inherited occurrence of a heat stable alkaline phosphatase in the absence of malignant disease

We describe a family with an inherited persistent elevation of serum alkaline phosphatase activity in the absence of malignant disease, observed for at least 15 yr. Isoenzyme studies revealed that this increased activity was due to an enzyme which showed similarities to serum placental alkaline phosphatase from pregnant women having the following properties: high heat stability; reactivity to anti-placental alkaline phosphatase antiserum; lack of inhibition by l-homoarginine; moderate inhibition by EDTA; and lack of interaction with wheat germ lectin. The enzyme was less sensitive than placental alkaline phosphatase to inhibition by l-phenyl-alanine, l-tryptophan, l-leucine, l-leucyl-glycyl-glycine and l-phenylalanyl-glycylglycine. The enzyme also differed from the placental alkaline phosphatase in its electrophoretic mobility, isoelectric heterogeneity and apparent molecular mass. We conclude that the enzyme is an inherited heat stable alkaline phosphatase variant which might correspond to a rare phenotype of placental alkaline phosphatase.

Variation in Some Enzymes in Amniotic Fluid and Maternal Serum during Pregnancy

The following 10 enzymes were assayed in 187 amniotic fluid and maternal serum samples at 15-42 weeks of gestation: alkaline phosphatase, heat-stable alkaline phosphatase (only in amniotic fluid), acid phosphatase, alanine aminotransferase, aspartate aminotransferase, alpha-amylase, gamma-glutamyltransferase, creatine kinase, lactate dehydrogenase, and lysozyme. The normal reference ranges are reported for amniotic fluid and maternal serum enzymes, together with the abnormal values accompanying neural tube defects and EPH-gestosis. The determination of gamma-glutamyltransferase, heat-stable alkaline phosphatase and creatine kinase was found to be of appreciable diagnostic significance in clinical practice.

The autorelease of alkaline phosphatase from the plasma membrane during the incubation of cultured liver cell homogenates.

When a rat hepatoma cell (R-Y121B) homogenate was incubated at 37 degrees C, 30-70% of the total alkaline phosphatase was released into the supernatant fluid from the precipitate fractions. The release reached a plateau level after 10 h of incubation at 37 degrees C. The optimum pH value for the release was 7.4. Alkaline phosphatase activity increased during the incubation of the cell homogenates, but this increase was independent of the enzyme release. Serum increased not only alkaline phosphatase activity in the cultured cells but also enzyme release in their homogenates. In addition, we examined a rat liver homogenate and the following 11 cell lines: 3 hepatoma cell lines, including the R-Y121B cell line, 4 liver cell lines, 2 human urinary bladder carcinoma cell lines, a kidney cell line, and a mouse adrenal tumor cell line. Only in the cultured liver cell line and hepatoma cell lines, 30-60% of the total enzyme was released into the soluble fraction from the precipitate fractions; the release was not observed in the other cell lines, nor in the rat liver homogenate. The release of alkaline phosphatase took place in both heat-stable and heat-labile alkaline phosphatases. Alkaline phosphatase, extracted from cell homogenates, showed two bands during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The mobilities of the two bands changed inversely with or without sodium dodecyl sulfate. In general, the alkaline phosphatase which showed slow mobility with sodium dodecyl sulfate was more readily released from the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Two gene duplication events in the evolution of the human heat-stable alkaline phosphatases

There are at least three alkaline phosphatase (AP) isoenzymes in man: a heat-stable placental enzyme (PLAP), a less heat-stable intestinal form (IAP), and the very heat-labile AP enriched in liver, bone and kidney. In addition to these enzymes, there is a heat-stable activity in the thymus and testis that is similar but not identical to the PLAP (the PLAP-like enzyme). Previous work has demonstrated a close structural relatedness among the IAP, PLAP and PLAP-like enzymes. Thus, it is possible that there are three human genes encoding heat-stable AP enzymes. To test this hypothesis, we have used a PLAP cDNA clone to screen a human genomic library cloned into the phage vector 1EMBL-3. Three sets of clones were isolated, each bearing a distinct coding region homologous to the PLAP cDNA probe. Nucleotide sequence analysis of the 5′ ends of these genes allowed comparison of their derived peptide sequences and positive identification of two of the genes. One of the genes encodes the PLAP (the PLAP-1 gene), another encodes the IAP, and a third closely resembles the PLAP-1 gene, but is distinct from it (the PLAP-2 gene). The PLAP-2 gene is highly homologous (> 95%) with the PLAP-1 except in the first exon, where sequences encoding the hydrophobic signal peptide are nearly identical with the same region of the IAP gene. These results demonstrate the existence of a small family of PLAP-related genes which is the result of at least two duplication events during the descent of man.

Is maternal serum heat stable alkaline phosphatase an index of fetal maturity?

A cross sectional estimation of serum heat stable alkaline phosphatase (HSAP) levels in 145 women with uncomplicated pregnancies between 24–41 weeks gestation showed increasing levels with advancing gestation and a steep rise at about 34 weeks gestation from a mean level of 17.3±6.5 μg/ml to a mean level of 36.1±1.5 μg/ml (t = 7.4 p < .001) at about 37 weeks gestation. Ninety-three point seven percent (93.7%) and 24.4% of women between 31–35 weeks gestation and 27–34 weeks gestation respectively had levels equal to or exceeding 20 μg/ml. It is postulated that serum HSAP represent stages of fetal maturity rather than gestational age per se and that there is a possible relationship between serum HSAP and amniotic fluid lecithin viewed against a background of a similar rise of amniotic fluid lecithin levels at about 33 weeks gestation and the common role of steroid receptor sites in the formation of both HSAP and Lecithin.

Heat-stable alkaline phosphatase as a useful marker in cancer prevention

Heat-stable alkaline phosphatase as a useful marker in cancer prevention

Valuable tumor marker, heat-stable alkaline phosphatase assayed by fluorometric analysis to detect the risk of lung cancer and other cancers by cigarette smoking

Valuable tumor marker, heat-stable alkaline phosphatase assayed by fluorometric analysis to detect the risk of lung cancer and other cancers by cigarette smoking

Alkaline phosphatase and protein kinase(s) activities in free cytoplasmic mRNPs from human term placenta.

Free mRNPs isolated from human term placental tissue were examined for protein kinase and phosphoprotein-phosphatase activities. Free mRNPs incubated with [gamma-32P]ATP in a protein kinase standard buffer show self-phosphorylation in the absence of exogenous substrates. Treatment of phosphorylated products with alkali showed a significant phosphorylation of tyrosine residues within the mRNP-proteins. An alkaline-phosphatase activity was found to be tightly associated with the mRNPs. Both heat stable and heat labile alkaline phosphatase activities were found in the mRNPs. Heat labile alkaline phosphatase is the major isoenzyme form of the mRNPs. The existence of both protein kinase(s) and alkaline phosphatase activities in placental free cytoplasmic mRNPs might suggest that a balance between phosphorylation, specifically on tyrosine residues, and dephosphorylation states of some of the mRNP-proteins is relevant for their physiological functions, and may therefore play a role in the regulation of mRNPs' metabolism and, consequently, in mRNA translation.

Sensitive fluorometry of heat-stable alkaline phosphatase (Regan enzyme) activity in serum from smokers and nonsmokers.

We developed a simple, sensitive enzymatic assay involving the fluorogenic substrate naphthol AS-MX phosphate [(3-hydroxy-2-naphthoic acid 2,4-dimethylanilide) phosphate] to measure heat-stable alkaline phosphatase (EC 3.1.3.1), the Regan isoenzyme, in human serum. The day-to-day CV was 5.7% for a serum activity of 0.080 arbitrary units/L. Measurable amounts of enzyme were detected in most normal individuals. The mean for 51 nonsmokers was 0.068 (SD 0.037) arb. units/L; for 25 smokers it was 0.440 (SD 0.360) arb. units/L. Activity of this isoenzyme in smokers was as much as 10-fold the upper normal limit for nonsmokers. Activation of this tumor marker by smoking has not received attention hitherto. We conclude that a truly normal range can only be established among nonsmokers. The isoenzymes in smokers, nonsmokers, and pregnant women were similar in their heat stability, immunologic cross reactivity, and inhibition by L-phenylalanine.

A search for trace expression of placental-like alkaline phosphatase in non-malignant human tissues: demonstration of its occurrence in lung, cervix, testis and thymus

A search for placental or placental-like human alkaline phosphatase (ALP) was made in human tissues. The tissue extracts were assayed for ALP before and after heating at 65°C for l h. Trace amounts of heat-stable ALP activity (> 0.01 IU/g) were found in lung, testis, cervix and thymus. The heat-stable ALP in these four tissues gave in Ouchterlony double diffusion plates lines of apparent identity with placental ALP when a rabbit anti-human placental antiserum was used. Inhibition studies with L-phenyl-alanine (Phe), L-homoarginine (Har), L-phenylalanylglycylglycine (Pgg), L-leucine (Leu) and levamisole (Leva), were carried out on the heat-stable ALP and on the total ALP. The heat-stable ALPs from cervix and lung gave [ I] 50 values with each inhibitor comparable to those of placental ALP. The heat-stable ALPs from testis and thymus gave [ I] 50 values for Leu and Pgg which were significantly different from the placental isoenzyme. Electrophoresis of heat-stable lung ALP from different individuals showed polymorphic differences similar to those seen with placental ALPs. Such differences were not seen with heat-stable testis ALP. We conclude that human non-malignant testis, cervix, lung and thymus tissues contain small amounts of placental or placental-like ALPs. The heat-stable ALPs in cervix and lung appear to have the same characteristics as placental ALP and are probably encoded by the same gene locus. The heat stable ALPs in testis and thymus, though immunologically very similar to placental ALP differ from it in inhibition profile and electrophoretically. The significance of the results in relation to the ‘ectopic’ expression of placental and placental-like ALPs in malignancy is discussed.

Stimulation of human chorionic gonadotropin secretion by glucocorticoids

The effects of glucocorticoids on hormone secretion by human placenta in organ culture were studied. The addition of cortisol resulted in a fourfold increase in human chorionic gonadotropin (hCG) secretion over that in untreated cultures after 144 hours' incubation (P < 0.05), and a twofold increase in hCG was observed in the presence of cortisone (P < 0.01). Dexamethasone stimulated hCG secretion in a dose-response manner (r = 0.9542; P < 0.01). Progesterone, which suppresses hCG under these conditions, decreased the cortisol-enhanced secretion of hCG (r = −0.9794; P < 0.01). No change in the secretion of human chorionic somatomammotropin was observed, but glucocorticoids increased heat-stable alkaline phosphatase activity (P < 0.001). The physiologic significance of glucocorticoid effects on placental hormone synthesis is ducussed.

Differential determination of the heat-stable alkaline phosphatase activity in serum.

A procedure for the determination of the heat-stable alkaline phosphatase activity in serum was developed by using antibody-conjugated paper disks and inhibition by sodium thiocyanate. The placental alkaline phosphatase adsorbed on a paper disk conjugated with antibody was completely inhibited by 3M sodium thiocyanate, but the activity of intestinal alkaline phosphatase adsorbed on the paper disk was unaffected by 3M sodium thiocyanate. The results obtained by the proposed method showed a good correlation with those obtained by the heat-inactivation method (γ=0.991). These results strongly suggest that the proposed method can be utilized as a routine clinical test for the determination of serum placental-type alkaline phosphatase, i. e., placental, Regan, Nagao and Kasahara isoenzymes, from patients with cancer.

Heat-stable alkaline phosphatase in uterine cancer, with special reference to its histochemical heat-stability and the L-phenylalanine inhibition test.

We report a histochemical study of alkaline phosphatase (ALP) in normal cells of the female reproductive system, in pre-cancerous and cancerous lesions of the uterine cervix and in endometrial cancer to ascertain the incidence of ALP and its isoenzyme type. For this purpose, serial sections were subjected to heat stability and L-phenylalanine (LP) inhibition tests. The Regan-like isoenzyme, a heat-stable and LP-sensitive ALP, which has been thought to derive only from cancer or the placenta, was found in uterine cervical reserve cells and endometrial luminal surface lining cells. In contrast, ALP activity in endometrial glandular cells was found to be heat and LP sensitive. Of 183 cases of cervical neoplasia, 60 (33%) manifested non-specific ALP activity. One dysplasia and two invasive cancer cases manifested the Regan-like isoenzyme. The other 36 classifiable lesions had small-intestine ALP-like activity (marked heat and LP sensitivity) or a liver ALP-like isoenzyme (marked heat and slight LP sensitivity). Of 42 cases of endometrial cancer, all cases manifested non-specific ALP activity. Seven endometrial cancers exhibited the Regan-like isoenzyme. The other 19 cases manifested either small intestine or liver ALP-like isoenzyme. Our findings indicate that in the course of uterine carinogenesis, the ALP isoenzyme of reserve cell and endometrial glandular cells undergo a change and that enzyme deviation occurs.

Sensitive fluorometric detection of heat-stable alkaline phosphatase from bloodstains for the forensic diagnosis of pregnancy

Sensitive and simple procedures are established for the forensic pregnancy test from bloodstains. This paper describes new techniques based on heat stability of placental alkaline phosphatase. In this study the total and heat-stable alkaline phosphatase activities were determined using 4-methylumbelliferyl-phosphate as substrate. The ratio of heatstable alkaline phosphatase to total alkaline phosphatase was calculated. A more simple screening test which was visible by exposure to UV light is described. These may be helpful in the diagnosis of pregnancy from only a slight bloodstain.

Maternal serum alkaline phosphatase and fetal maturity.

Total as well as heat stable alkaline phosphatase activity was determined in sera of 70 normal pregnant mothers. Rise in activity was observed with advancement of gestation and was due to increased concentration of heat stable fraction. Statistically significant differences in levels between various gestational age groups suggest the assay of this enzyme for assessment of fetal maturity. Total alkaline phosphatase activity of 15 K.A. units/dl or more, with more than 50% of it being heat stable fraction, indicated a mature fetus (37 weeks or more gestation).

Assessment of placental functions using placenta-originating enzymes in maternal serum (author's transl)

The purpose of the present investigation was to know whether the measurements of enzyme activities were of use in assessing the placental functions and of diagnostic value. A total of 2, 660 samples from the sera of 506 pregnant women and 42 samples from placental tissues were assayed for cystine aminopeptidose (CAP), leucine aminopeptidase (LAP) and heat stable alkaline phosphatase (HSAP). And the following results were obtained.(1) As regards enzyme concentrations in the placental tissue, CAP and LAP in a toxemic pregnancy showed lower values than those in a normal pregnancy. Nevertheless, in the case of a toxemic pregnancy, CAP and LAP in the maternal serum exhibited higher values than those in the placental tissue, and there were no correlations between the enzyme activities in the placental tissue and those in the maternal serum. As for HSAP, its values were low in both the placental tissue and the maternal serum. In the case of SFD, HSAP had a tendency to have a low value in both the maternal serum and the placental tissue.(2) In a normal pregnancy, there was a correlation between the concentration of the given enzyme in the maternal serum and the total quantity of the enzyme in the placental tissue (γ= 0.54 in CAP, γ =0.57 in LAP, r=0.60 in HSAP). On the other hand, in an abnormal pregnancy, there was a rise in the correlation cofficient for HSAP (γ =0.74) whereas the correlation cofficient for LAP was 0.44.(3) The activities of CAP, LAP and HSAP in serum increased as the gestational week advanced. Curves representing the variations in concentrations of these enzymes during a normal pregnancy consisted of two parts, i. e., the initial part where concentrations increased at a fixed rate and the late part where they increased at a continually decreasing rate.(4) The enzyme activity index (actual value-to-predictional value ratio) was derived. The index clearly showed the difference between the actual and the predicted values. Furthermore, a conversion table was made to simplify the calculation of the predictional value.(5) According to the activity index, one or more of these enzymes exhibited abnormal values in 76.46% of SFD (small for dates baby), 92.6% of toxemia of pregnancy and 50% of mild toxemia of pregnancy.

Study of fetal reserve in intrauterine fetal growth retardation (author's transl)

caused by small fetus resulting from small maternal body of reasons unknown mostly have satisfactory courses after birth. On the other hand, fetal malnutrition often causes fetal distress and the post-natal prognosis is sometimes unfavorable.Therefore, in the case of IUGR, it is necessary to judge correctly whether the fetus is in a state of well being or latent fetal distress.In this study, fetal reserve is examined by means of fetal function testing constituted of fetal response to tone stimulation and by fetal placental function tests including the quantitative determination of urinary estriol, serum Cystine aminopeptidase (CAP), Leucine aminopeptidase (LAP), Heat-stable alkaline phosphatase (HSAP) activities in subjects whose newborns are light for dates after birth.1) Two hundred and seventy subjects light for dates after birth could be classified as follows: Fetal malnutrition, 181cases (67.04%); fetal hypoplasia, 15 cases (5.56%); uncertain, 68 cases (25.19%); maternal predisposition, 6 cases (2.22%).2) Fetal response to tone stimulation, maternal urinary estriol, maternal serum CAP, LAP, and HSAP levels were all lower in the subjects than in controls where the course of pregnancy was normal.3) In the IUGR group, many subjects with fetal malnutrition showed lower values in fetal response to tone stimulation, maternal urinary estriol maternal serum CAP, LAP, and HSAP activities in comparison with others.4) Fetal response to tone stimulation may not adequately indicate fetal reserve, since the result is sometimes negative even in a normal course of pregnancy during pre-term (less than 37 weeks). However, when the result is positive, this test is very reliable for demonstrating satisfactory fetal reserve.5) The results of the examinations including fetal response to tone stimulation, maternal urinary estriol, and maternal serum CAP, LAP, and HSAP activities showed lower valves in subjects with toxemia of pregnancy, especially in severe cases, and this tendency was more remarkable in the subjects whose newborns were light for dates after birth.If IUGR of high-risk pregnancy is clinically suspected, the appropriate consideration of the high frequency of unfavourable prognosis or clinical course, the fetal respons to tone stimulation, maternal urinary estriol, maternal serum CAP, LAP, and HSAP activities test are necessary in view point of evaluating the fetal reserve capacity.

Preparation of highly purified cytotrophoblast from human placenta with subsequent modulation to form syncytiotrophoblast in monolayer cultures.

The preparation of highly purified monolayer cultures of human cytotrophoblast cells essentially free of stromal and syncytial cells is described. Such cells subsequently form multinucleate syncytial cells in vitro. This is accompanied by the synthesis of heat-stable alkaline phosphatase and beta-HCG. A significant proportion of the multinucleate cells that form in monolayer cultures of early placentae arise as a result of an amitosis. It is suggested that this in vitro behavior may reflect an in vivo process.

HEAT-STABLE ALKALINE PHOSPHATASE IN THE NORMAL FEMALE GENITAL ORGAN

The Regan isoenzyme, a heat-stable alkaline phosphatase, which has been thought to be derived only from cancer or the placenta, has been reported in non-cancer, non-pregnant patients. We performed histochemical studies to determine whether the heat-stable alkaline phosphatase exists in normal cells of the female reproductive organ. For this purpose, the heat-stability test and the L-phenylalanine inhibition test were used. Heat-stable and L-phenylalanine-sensitive alkaline phosphatase was found in normal cells of Muellerian duct origin, e.g., uterine cervical reserve cells, cervical columnar cells and epithelial cells of the endometrial surface. Our findings suggest that heat-stable alkaline phosphatase, such as the Regan enzyme, may be produced by normal cells of Muellerian duct origin.

The Ontogony of the Human Placental Glucocorticoid Receptor and Inducibility of Heat-Stable Alkaline Phosphatase*

The human placenta was found to contain a cytosol receptor for glucocorticoids. The concentration of this receptor in term placenta was 27-fold higher than that found in cytosol from first trimester placenta. The levels of cytosol glucocorticoid receptor in three trophoblastic cell lines (JAr, BeWo, and JEG) were also determined and all were found to be low. The ability of prednisolone, a potent glucocorticoid, to stimulate heat-stable alkaline phosphatase activity found in these cells was tested. Although control experiments demonstrated that the conditions were adequate to stimulate HeLa cell alkaline phosphatase, none of the trophoblastic lines responded to prednisolone administration. This result may be explained by the observation that the JAr cells lacked any detectable glucocorticoid receptor and the receptor levels in cytosol prepared from JEG and BeWo cells were 12% and 2%, respectively, of those measured in HeLa cytosol. Our studies also suggest that the increase in serum levels of heat-stable alkaline phosphatase observed during pregnancy may reflect increasing placental sensitivity to glucocorticoids as a result of increased receptor levels.