Heat-stable Alkaline Phosphatase Research Articles

Development of a heat-stable alkaline phosphatase reporter system for cis-regulatory analysis and its application to 3D digital imaging of Xenopus embryonic tissues.

Xenopus is one of the essential model systems for studying vertebrate development. However, one drawback of this system is that, because of the opacity of Xenopus embryos, 3D imaging analysis is limited to surface structures, explant cultures, and post-embryonic tadpoles. To develop a technique for 3D tissue/organ imaging in whole Xenopus embryos, we identified optimal conditions for using placental alkaline phosphatase (PLAP) as a transgenic reporter and applied it to the correlative light microscopy and block-face imaging (CoMBI) method for visualization of PLAP-expressing tissues/organs. In embryos whose endogenous alkaline phosphatase activities were heat-inactivated, PLAP staining visualized various tissue-specific enhancer/promoter activities in a manner consistent with green fluorescent protein (GFP) fluorescence. Furthermore, PLAP staining appeared to be more sensitive than GFP fluorescence as a reporter, and the resulting expression patterns were not mosaic, in striking contrast to the mosaic staining pattern of β-galactosidase expressed from the lacZ gene that was introduced by the same transgenesis method. Owing to efficient penetration of alkaline phosphatase substrates, PLAP activity was detected in deep tissues, such as the developing brain, spinal cord, heart, and somites, by whole-mount staining. The stained embryos were analyzed by the CoMBI method, resulting in the digital reconstruction of 3D images of the PLAP-expressing tissues. These results demonstrate the efficacy of the PLAP reporter system for detecting enhancer/promoter activities driving deep tissue expression and its combination with the CoMBI method as a powerful approach for 3D digital imaging analysis of specific tissue/organ structures in Xenopus embryos.

USEFULNESS OF THERMO LABILE ALKALINE PHOSPHATASE AS A BIOMARKER IN DIAGNOSIS OF POST-MENOPAUSAL OSTEOPOROSIS

Background: Osteoporosis is characterized by demineralization of bones and it is a complication of women in post-menopausal life. Bone alkaline phosphatase is an isoenzyme of alkaline phosphatase which helps in the mineralization of bones and it is heat labile in nature. Its measurement facilitates the clinical diagnosis of Osteoporosis. Objectives: To assess the status of total alkaline phosphatase and heat-labile isoenzyme fraction (bone fraction) in serum of pre and post-menopausal women and to relate the findings to the clinical condition /final diagnosis to make out the correlation between the pre and postmenopausal women. Methodology:A total of 70 subjects, consisting of 35 pre-menopausal women (38.11 ± 4.3 years of age) and 35 post-menopausal women (54.40 ± 4.6 years) were constituted for the study. Their total alkaline phosphatase was measured directly and the heat stable alkaline phosphatase was measured after heating the serum at 56℃ for 10 minutes. Spectrophotometric P- Nitro-Phenyl Phosphate method was followed for the estimation. Heat labile alkaline phosphatase levels were obtained by subtracting the heat stable fraction from total Alkaline phosphatase. Result: The measured serum total alkaline phosphatase level was slightly increased in post menopause compared to premenopausal women but it was not statistically significant. The levels of heat-labile Alkaline phosphatase showed significant difference between pre and post-menopausal women by ***: p < 0.01: ***: p < 0.001 respectively. Conclusion: Heat labile alkaline phosphatase can be used as a biomarker to diagnose Osteoporosis in post-menopausal women.

Visual Vignette

Case Presentation: A 68-year-old Indian gentleman presented to our general practice clinic complaining of lower back pain with shooting pain radiating to the right posterior leg. The general practitioner ordered an X-ray of the lumbosacral spine and biochemistry. The X-ray showed sclerosis and a picture frame appearance in the L2 vertebra with degenerative changes in the rest of the spine (Fig. 1 A and Fig. 1 B). Biochemistry showed an adjusted calcium at 2.56 mmol/L (normal, 2.15 to 2.50 mmol/L), phosphate at 1.3 mmol/L (normal, 0.8 to 1.4 mmol/L), alkaline phosphatase (ALP) at 140 U/L (normal, 40 to 120 U/L), heat-stable ALP at 4% (<20% signifies bone origin), parathyroid hormone at 5.2 mmol/L (normal, 0.8 to 6.6 mmol/L), and 25-hydroxyvitamin D (total) at 20 μg/L (normal, 30 to 50 μg/L). He was referred to the endocrinologist for further management. A technetium 99m-methylene diphosphate triphasic bone scan showed intense tracer uptake in the entire L2 vertebra, including the posterior elements (spinous process) with no other focal increased tracer uptake at other sites (Fig. 2). What is the diagnosis? Fig. 2 View Large Image Figure Viewer

Diagnostic utility of heat stable alkaline phosphatase in hypertensive disorders of pregnancy.

Hypertensive disorders in pregnancy (HDP) complicate 3-10% of all pregnancies. Though there are several biochemical parameters which aid in predicting hypertension of pregnancy, human placental alkaline phosphatase (PLAP), synthesized in placenta during pregnancy by placental syncytiotrophoblast, assumes diagnostic relevance. The purpose of this study was to compare the total alkaline phosphatase (ALP) and heat stable placental alkaline phosphatase (PLAP) levels in the serum of normotensive and hypertensive disorders of pregnancy and to evaluate the clinical utility of ALP and PLAP as a reliable, sensitive, specific and economical biochemical marker of HDP. This was a case control study, carried out on pregnant women with hypertension, of south Indian population. Study included pregnant women, 60 patients with hypertension and 60 controls. Biochemical assays were carried out by the IFCC approved procedures based on spectrophotometric method and using fully automated random access chemistry analyser. Data was compared by using student t-test. ROC was drawn to find out optimum cut off for ALP, PLAP and PLAP/ALP ratio in HDP. Pearson's correlation was performed to ascertain the association among markers. Serum total ALP, PLAP and PLAP/ALP ratio levels were significantly higher in hypertensive pregnant women when compared to controls (p<0.05). There was significant correlation among ALP, PLAP and DBP. ROC analysis of ALP (169.5), PLAP (69) and PLAP/ALP (0.44) ratios showed optimum cut-offs in diagnosis of hypertension in pregnancy. Serum heat stable ALP isoenzyme and PLAP/ALP ratio could be useful adjuvant markers in diagnosis of HDP in association with other relevant and economically viable biochemical tests.

An Epidermal Growth Factor Motif from Del1 Protein Increases the Efficiency of In Vivo Gene Transfer with a Non-Viral Vector

Increasing the efficiency of gene transfer using non-viral vectors, which have the potential to be safe and economical, would improve upon available options for gene therapy. We previously reported that the third EGF motif of the extracellular matrix protein Del1 (E3) increases the transfection efficiency of non-viral vector methods. Here, we asked if E3 could increase the in vivo transfection efficiency of a polyplex-based approach. To test this, cDNA encoding a heat-stable alkaline phosphatase (AP) was first injected intravenously into mice along with recombinant E3. After 24h, exogenous AP activity in serum was measured. We found that the introduction of E3 resulted in 50% more AP activity as compared to the control. We next tested transfection into a tumour explant of SCCKN cells, an oral carcinoma-derived cell line. To do this, a cDNA encoding yellow fluorescent protein was locally injected into a tumour explant, followed by local injection of recombinant E3. Use of E3 increased the number of transfected cells to 2.5 times that of the control. Histochemical staining revealed that E3-induced apoptosis in a tumour explant. The data suggest that E3 might be a useful tool for cancer gene therapy using non-viral vectors.

母体末梢血中胎盤起原酵素、特にHeat-Stable Alkaline Phosphataseの産生-遊出機構、およびその胎盤機能検査への応用に関する研究

母体末梢血中胎盤起原酵素、特にHeat-Stable Alkaline Phosphataseの産生-遊出機構、およびその胎盤機能検査への応用に関する研究

ABO blood groups and alkaline phosphatase isoenzymes in the 2nd trimester of pregnancy in Nigerian women.

Heat stable and heat labile alkaline phosphatase isoenzymes were estimated in 162 healthy pregnant Nigerians between 10–26 weeks gestation. No significant association was found between the levels of these isoenzymes and maternal ABO blood groups. Eleven percent (11%) of these patients with apparently uncomplicated pregnancies had no detectable heat stable alkaline phosphatase isoenzyme even as late as 18 weeks gestational age. No association was identified between this non-secretor status and maternal ABO blood groups.

The placental alkaline phosphatase variant D and natural selection. Relationship to complications of pregnancy and to "the Regan isoenzyme".

The placental alkaline phosphatase variant D is controlled by a gene which in most populations is rare (0.001–0.007). A significant increase of abortions in previous pregnancies was found in a series of deliveries from Sweden where the placenta (fetus) showed the D-variant. Among deliveries after various complications during pregnancy the D-variant was found in an increased frequency compared to a series of unselected, consecutive deliveries. In 1973 Inglis et al. claimed that the D-variant occurs in a frequency of 40–50% among cancer patients displaying ectopic (derepressed) heat-stable alkaline phosphatase (“the Regan isozyme”). A reexamination of their data suggests that the D-variant is not associated with cancer and that the identity between “the Regan isozyme” and placental alkaline phosphatase is still an open question.

Biochemical diagnosis of placental infarction/damage: acutely rising alkaline phosphatase

There are currently no simple tests in clinical use to detect acute placental damage. A case is described to demonstrate that a routinely used measurement such as alkaline phosphatase (ALP) can be employed to detect acute damage to the placenta. Seventeen serial blood samples, three pre-delivery, were collected from a 22-year-old primigravida who delivered a stillborn baby. Retrospectively, blood samples were analysed for total and heat-stable ALP as well as human chorionic gonadotropin (hCG) and alpha-fetoprotein (AFP) as a measure of placental function when an unusual pattern of change in ALP was noticed. Histological examination of the placenta revealed new and old placental infarcts. Total and heat-stable ALPs as well as AFP peaked by more than eight-, 19- and two-fold, respectively over 16 h. Plasma hCG fell sharply even before delivery of placenta by five-fold over 16 h before further falling slowly to baseline. The fall in hCG is also consistent with the placental damage being acute and critical. As far as we are aware this is the first description of changes in circulating proteins reflecting placental damage.

Development of a monoclonal antibody-based immunoassay for specific quantification of bovine milk alkaline phosphatase

The detection of alkaline phosphatase (ALP) activity is used as a legal test to determine whether milk has been adequately pasteurized or recontaminated with raw milk. However, a wide variety of microorganisms produce both heat labile and heat stable ALPs which cannot be differentiated from the milk ALP by current enzymatic methods. Monoclonal antibodies specific of the bovine milk ALP were obtained in mice from a raw bovine milk ALP preparation. Coated in microtitre plates, these antibodies specifically capture the bovine milk ALP from dairy products. After washing, the enzymatic activity of the captured ALP is revealed by adding p-nitrophenyl-phosphate as a substrate. This simple immunoassay does not react with ALPs of intestinal or bacterial origin and, once optimized, was found to be the first immunoassay suitable to detect raw milk in boiled milk down to a 0.02% dilution. Moreover, in contrast with competitive indirect ELISA formats, the capture immunoassay does not require purified ALP.

Reference interval of serum heat-stable alkaline phosphatase activity in pregnant women in Zaria

The study was designed to establish a reference interval for serum heat-stable ALP activity in pregnancy, which is not available in this environment. One hundred (100) apparently healthy pregnant women in their third (3rd) trimester who were attending the antenatal clinic (ANC) of Ahmadu Bello University Teaching Hospital (ABUTH), Zaria were recruited for the study. The serum heat-stable ALP activity was measured using the 4-nitrophenyl phosphate (4-NPP) method introduced in 1946 by Bessey, Lowry and Brock. The reference interval for the serum heat-stable ALP activity was found to be 24-161 IU/L. This was similar to some earlier reports elsewhere. The finding will help in the evaluation/diagnosis of patients with placental insufficiency.

A simple in vivo approach to investigate invasive trophoblast cells

Intrauterine trophoblast cell invasion is an essential part of hemochorial placentation. Aberrant trophoblast cell invasion has been associated with pathologies including preeclampsia and fetal growth restriction. In this study, we describe an in vivo method to assess trophoblast cell invasion using a transgenic rat model, constitutively expressing heat stable human placental alkaline phosphatase (Rosa 26 promoter driven human placental alkaline phosphatase, R26-hAP). Wild-type female Fischer 344 inbred rats were mated with hemizygous R26-hAP transgenic male Fischer 344 rats and sacrificed during the second half of pregnancy. Heat stable alkaline phosphatase (AP) activity associated with the invasive transgenic trophoblast cells was monitored in the wild-type uterine mesometrial compartment and used as an index of trophoblast cell invasion. The expression pattern of cytokeratins by invasive trophoblast cells mimicked the uterine mesometrial distribution of AP activity. Trophoblast cell invasion exhibited a gestation-dependent profile with peak invasion between days 18-20 of pregnancy. In summary, we have devised a simple in vivo method for assessing intrauterine trophoblast cell invasion. This technique should facilitate the discovery of endogenous regulatory mechanisms controlling trophoblast cell invasion and should represent an effective method of testing the impact of various environmental stressors on an essential part of hemochorial placentation.

Relationship between serum 5′-nucleotide phosphodiesterase activity and thrombophilia: enzyme activity in fetus and in patients with acute coronary syndrome

Inhibition of ADP-induced platelet aggregation by a placental substance was first reported by Myatt et al. in 1977 [1]. This substance was assumed to play an important role in maintaining fetal–placental circulation under extremely low oxygen pressure. However, the principle behind this activity has not been fully characterized as yet. Hutton et al. [2] reported in 1980 that this activity was derived from placental heat stable alkaline phosphatase. Sakura et al. [3] proposed that placental 5Vnucleotide phosphodiesterase (PDEase) is involved in the inhibition of the platelet aggregation mentioned above. 5V-Nucleotide phosphodiesterase is an ectoenzyme, and hydrolyzes phosphodiester bonds of DNA and RNA releasing nucleoside 5V-monophosphate. This enzyme also hydrolyzes pyrophosphate bond of ATP, ADP and NAD [4,5]. Enzyme activity in serum is elevated in hepatic disease [6,7]. The physiological function of the enzyme in umbilical cord serum remains to be clarified. We have shown that the enzyme purified from human umbilical cord serum hydrolyzed ADP and suppressed ADP-induced platelet aggregation depending on the enzyme concentration in platelet-rich plasma [8]. These data suggest that serum PDEase is a potent inhibitor of platelet aggregation. In neonates and infants, thromboembolic events have been shown to be rare, possibly due to low vitamin K-dependent coagulation factors and poor ATP storage in neonate platelet [9–12]. Serum PDEase

Phage displayed antibodies to heat stable alkaline phosphatase: framework region as a determinant of specificity.

Human antibodies against specific targets of tumor cells are the most desirable molecules for possible immunotherapy. They could be developed by using the combinatorial antibody library displayed on a phage. We selected four human antibody fragments (scFv) binding to the oncoplacental antigen Heat Stable Alkaline Phosphatase (HSAP, the placental isozyme of alkaline phosphatase) from a synthetic human antibody library. Characterization of these scFvs showed they bound HSAP with moderate affinity but did not have isozyme specificity, as determined by binding to cell lines exhibiting differential expression of isozymes of alkaline phosphatase. The V(H) sequences of two of these scFvs were similar and although both bound to HSAP only one was cross-reactive with albumin. The sequences revealed a difference in the framework region (FR1) of these antibodies, indicating a role for this region in the determination of specificity. This is also significant considering that the heavy chains generated the diversity of the synthetic library used in this study, and only a single light chain showing binding to BSA was used for the entire library.

Direct antigen capture by soluble scFv antibodies. A method for detection, characterization, and determination of affinity.

For ease of detection, soluble forms of phage-displayed scFv antibodies are usually expressed with a tag, e.g., c-myc or His (Histidine). The binding is then assayed by a monoclonal antibody to the tag. In this article, we describe the use of biotinylated antigen for detecting soluble scFv antibodies without utilizing the peptide tag detection system. The scFv antibodies were against the oncoplacental antigen heat-stable alkaline phosphatase (HSAP). The method essentially consisted of either reverse Western or antigen capture enzyme-linked immunosorbent assay (ELISA). In the reverse Western, periplasmic extract was electrophoresed, and binding to biotinylated antigen was detected by the detection system based on streptavidin-horseradish peroxidase. The antigen capture ELISA utilized the binding of periplasmic extract to a polystyrene plate. We have also demonstrated the use of antigen capture ELISA for studying specificity and affinity of the selected clones. Although these techniques have been developed for antibodies to HSAP, they have general utility for phage expression systems without a peptide tag.

Highly efficient gene transfer into preterm CD34+ hematopoietic progenitor cells

Objective: Retrovirus-mediated gene transfer has been shown to transduce CD34+ cells from term gestation umbilical cord blood with relatively high efficiency. The purpose of this study was to compare the efficiencies of retrovirus-mediated gene transfer into early (23-28 weeks’ gestation) and term (37-41 weeks’ gestation) umbilical cord blood CD34+ hematopoietic progenitor cells. Study Design: CD34+ cells were purified from cyropreserved early (23-28 weeks’ gestation) and term (37-40 weeks’ gestation) umbilical cord blood specimens with fluorescence-activated cell sorting. The CD34+ cells were then transduced in virus-containing medium (gibbon ape leukemia virus pseudotype vector LAPSN [PG13]) in wells coated with the recombinant human fibronectin fragment CH-296 and in the presence of multiple hematopoietic growth factors (interleukin 6, stem cell factor, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and megakaryocyte growth and development factor) and protamine sulfate. The LAPSN (PG13) virus–containing medium was changed every 12 hours for 48 hours, after which time colony-forming cells were assayed in soft agar. The gibbon ape leukemia virus pseudotype vector LAPSN (PG13) contains the human placental alkaline phosphatase and neomycin phosphotransferase (neo ) genes. The efficiency of gene transfer was assessed by histochemical staining of colony-forming cells in agar for expression of heat-stable alkaline phosphatase. Results: Gene transfers, as assessed by alkaline phosphatase staining of colony-forming cells (granulocyte-macrophage colony-forming units and erythroid burst-forming units), were similar for CD34+ hematopoietic progenitor cells from early (58.4% ± 11.8%) and term (63.2% ± 12.5%) gestation fetal umbilical cord blood. Conclusion: CD34+ hematopoietic progenitor cells from midgestation fetal blood can be transduced with high efficiency using techniques optimized for postnatal samples with a gibbon ape leukemia virus pseudotype vector. The early fetus may be a preferable target for gene therapy because of the higher number of circulating CD34+ and CD38– cells relative to term cord blood, their greater proliferative capacity, and the rapid expansion of the fetal hematopoietic system that occurs from the second trimester to delivery. Because in vitro studies of gene transfer into hematopoietic progenitor cells and long-term culture-initiation cells have not been predictive of the efficiency of gene transfer into marrow-repopulating cells in vivo, studies that examine clinically applicable approaches to in utero gene therapy in appropriate animal models are still needed. (Am J Obstet Gynecol 2000;183:732-7.)

ENHANCEMENT OF LIPOSOME-MEDIATED GENE TRANSFER TO HUMAN AIRWAY EPITHELIAL CELLS BY REPLICATION-DEFICIENT ADENOVIRUS

We hypothesized that replication-deficient adenovirus (Ad), when complexed with plasmid DNA (pl) and cationic liposomes (L), would enhance liposome-mediated gene transfer in cultured human airway epithelial cells. Pl/L/Ad complexes were formed using charge-charge interactions. A gel electrophoresis retardation assay showed plasmid DNA to be associated with the virus in a high-molecular weight, low-mobility complex, the diameter of which was 300 to 350 nm. Compared to pl/L alone, pl/L/Ad enhanced luciferase expression on average by 1 log-fold in human airway epithelial cells which express either mutant or wild-type cystic fibrosis transmembrane conductance regulator (CFTR). Transgene expression was sustained at high levels for up to 7 days following transfection with pl/L/Ad. Using a heat-stable alkaline phosphatase reporter gene, we showed that a larger fraction of cells was transfected by pl/L/Ad compared to pl/L. Finally, cells were exposed to Ad for 0 to 24 hours prior to pl/L or exposed to pl/L prior to Ad. We found that enhancement was significantly greater using pl/L/Ad compared to the simultaneous addition of Ad with the pl/L complexes. In addition, when pl/L was added 4 to 24 hours prior to Ad, some enhancement was found, suggesting that plasmid DNA remained in a compartment in the cell for several hours and became available for transcription with the addition of Ad. When Ad was added prior to pl/L, enhancement was found suggesting that the effect of the virus on cell membranes may persist for up to 24 hours. We conclude that the tripartite pl/L/Ad complex significantly enhances liposome-mediated transgene expression for a prolonged period of time in human bronchial epithelial cells.

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.

OPTIMIZATION OF CATIONIC LIPOSOME-MEDIATED GENE TRANSFER TO HUMAN BRONCHIAL EPITHELIAL CELLS EXPRESSING WILD-TYPE OR ABNORMAL CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR)

We determined optimum conditions for delivering DNA to transformed human bronchial epithelial cells expressing wild-type (BEAS) or abnormal (2CF) cystic fibrosis transmembrane conductance regulator (CFTR) using cationic liposomes (Lipofectin, [N-(N,N-dimethylaminoethane) carbamyl] cholesterol [DC-Chol] dioleoylphosphatidylethanolamine [DOPE], or LipofectAMINE) and reporter genes which measured overall transgene expression (luciferase) or the fraction of cells transfected (heat stable alkaline phosphatase). All liposomes showed dose-related toxicity. Optimal liposome and lipid: DNA ratios were different for BEAS than for 2CF cells. For all 3 liposome preparations, small particle size and net cationic charge related to transfection efficiency. Both LipofectAMINE and DC Chol/DOPE transfected a maximum of 3% of BEAS cells, but luciferase expression could be increased without increasing the fraction of cells transfected. LipofectAMINE transfected a maximum of 6% of 2CF cells, and luciferase expression could be increased with no further increase in fraction of transfected cells. DC-Chol/DOPE transfected over 12% of 2CF cells with relatively small increases in luciferase expression. We conclude that an optimal cationic liposome and lipid: DNA ratio for transfecting bronchial epithelial cells depends on: (1) small particle size and net cationic charge, (2) whether the cells have the cystic fibrosis defect, and (3) whether the desired outcome is transfection of the maximum fraction of the cells or maximum total expression of the transgene.

Second trimester calcium-phosphorus-magnesium homeostasis in women with threatened preterm delivery.

The effect of threatened preterm delivery on calcium, phosphorus, magnesium homeostasis in the second trimester of pregnancy was investigated. Serum concentrations of total and ionized calcium, inorganic phosphorus, magnesium, total protein, albumin, total estrogens and human placental lactogen were determined in women with threatened preterm delivery at 23-28 weeks of gestation (the studied group) and in women with uncomplicated pregnancy of the same duration (the control group). Additionally activities of total alkaline phosphatase and heat-stable alkaline phosphatase fraction were measured. Patients of the studied group compared to the control group showed decreased concentration of total calcium (2.15 +/- 0.073 vs. 2.25 +/- 0.11 mmol/l, P < 0.001), inorganic phosphorus (1.21 +/- 0.26 vs. 1.34 +/- 0.22 mmol/l, P < 0.01) and magnesium (0.63 +/- 0.053 vs. 0.71 +/- 0.12 mmol/l, P < 0.001), total protein (64.0 +/- 5.4 vs. 68.6 + 1.0 g/l, P < 0.001), albumin (546.3 +/- 55.1 vs. 579.6 +/- 49.3 micromol/l, P < 0.003) and placental lactogen (3664 +/- 1806 vs. 4651 +/- 1858 ng/ml, P < 0.02); they also demonstrated decreased activity of total alkaline phosphatase (42.17 +/- 16.99 vs. 50.66 +/- 6.56 IU/l, P < 0.001) and its heat stable fraction (22.65 +/- 7.89 vs. 31.89 +/- 9.09 IU/l, P < 0.001). Patients of the studied group showed normal values of ionized calcium and total estrogens. Premature uterine contractility in women in the second trimester is accompanied by lowered serum concentrations of total calcium, inorganic phosphorus, magnesium, total protein and albumin. There is also decreased activity of total alkaline phosphatase, its heat-stable fraction and placental lactogen which may have diagnostic value. Premature uterine contractility in women in the second trimester may be related to the disturbances of calcium-phosphorus-magnesium homeostasis and calcium supplementation should be considered.