Alkaline phosphatase placenta

Alkaline phosphatase assays based on 3-glycerophosphate now appears to be obsolete, and methods buffered by either glycine or barbital are also obsolete as these buffers inhibit ALP or are poor buffers. Serum alkaline phosphatase is known to be composed of several isoenzymes which presumably arise from bone, liver, intestine, and placenta. The placental alkaline phosphatase is known to be much more resistant to heat denaturation than the other isoenzymes, and this resistance provides a simple test for it (5). The other enzymes can be separated through the differential inhibition by phenylalanine, by electrophoresis and by specific antibodies. However, the clinical usefulness of the results obtained is in doubt (23). 

Alkaline phosphatase is an enzyme represented by various isoforms in many tissues such as liver, bone, intestine, placenta, some tumors and in leukocytes. Addition of 1 mM levamisole to the chromogen/substrate will inhibit endogenous alkaline phosphatase activity, with the exception of the intestinal isoform. If necessary, this can be blocked with a weak acid wash, such as 0.03 0.5 N HC1 or 1 M citric acid. 

Phosphates of pharmaceutical interest are often monoesters (Sect. 9.3), and the enzymes that are able to hydrolyze them include alkaline and acid phosphatases. Alkaline phosphatase (alkaline phosphomonoesterase, EC 3.1.3.1) is a nonspecific esterase of phosphoric monoesters with an optimal pH for catalysis of ca. 8 [140], In the presence of a phosphate acceptor such as 2-aminoethanol, the enzyme also catalyzes a transphosphorylation reaction involving transfer of the phosphoryl group to the alcohol. Alkaline phosphatase is bound extracellularly to membranes and is widely distributed, in particular in the pancreas, liver, bile, placenta, and osteoplasts. Its specific functions in mammals remain poorly understood, but it seems to play an important role in modulation by osteoplasts of bone mineralization. 

Serum alkaline phosphatase elevations have been reported following administration of salt-poor albumin (B5). Placenta is very rich in a heat-stable alkaline phosphatase, and albumin prepared from placental blood has a high activity of this enzyme. In one cirrhotic patient who received 1-6 units per day of albumin obtained from pooled human blood and/or human placenta, the alkaline phosphatase before infusion was 5 Bodansky units and by the thirteenth day of administration had reached a value of 160 units. The physician administering the albumin at first thought the patient was having a severe toxic liver reaction and stopped the therapy. The alkaline phosphatase then started to go down and within 10 days returned to normal levels. Analysis of the albumin indicated that it contained 470 units of alkaline phosphatase activity and was probably responsible for the observed elevations in the serum enzyme activity. Albumin prepared from venous blood did not cause an alkaline phosphatase elevation, but placenta-albumin caused elevations with a half-life of about 8 days (Ml). 

Alkaline phosphatase Human placenta AOT/isooctane Kinetics [96]... 

The last treatment is the most gentle and should be used for labile antigens 2 For alkaline phosphatase, incubate the section in 20% acetic acid for 5 min, and then wash it in tap water. This treatment may destroy the antigen. An alternative for tissues other than the intestine is to make the substrate solution (Section 3.2.2.) in 1 mM levamisole (increase to 2 mM for tissues rich in alkaline phosphatase, e.g, kidney or placenta) see Note 10)... 

The regeneration of the activity of the enzyme by cobalt differs from the behaviour of an alkaline phosphatase isolated from human placenta.228 This enzyme is reported to be re-activated by replacement of the native zinc ion by either zinc, magnesium, or mercury. No other metal is active. 

Figure 2. Placenta showing endogenous alkaline phosphatase activity (a) before, and (b) after blocking with levamisole.

Unquenched endogenous alkaline phosphatase activity may be seen in leucocytes, kidney, liver, bone, ovary bladder, salivary glands, placenta and gastro-intestinal tissue. Add levamisole to the alkaline phosphatase chromogen reagent or use another enzyme label such as horseradish peroxidase. Intestinal alkaline phosphatase is not quenched by the addition of levamisole. Pretreat the tissue with 0.03 N HCI. 115-121... 

Indicates endogenous alkaline phosphatase activity in the tissue sections. It is present in liver, kidney, Gl tract, bone, bladder, ovary, salivary gland, placenta, leukemic, necrotic or degenerated cells. 

ALP Alkaline phosphatase, found in liver, intestine, bone, placenta, others. ALP is not elevated in fiver cell injury unless cholestasis results. 

Alkaline phosphatase is an enzyme of the cellular membranes. Its isoforms can be found in liver, digestive tract, placenta, and some tumor tissues. Bone isoform, BALP, is a membrane enzyme of the osteoblasts. Bone, liver, and intestinal isoforms are the posttranslational modifications of the same isoenzyme exprimed by the same gene, and the difference among them lies in various ways of reaction with a saccharide component, sialic acid (M9). 

However, other isoenzymes of alkaline phosphatase are found in parts of the body such as bone, kidney, intestine and placenta, hence an isolated raised alkaline phosphatase may not be associated with liver dysfunction. In late pregnancy, alkaline phosphatase can increase to three times ULN, which may persist for several months after delivery, particularly if the mother is breastfeeding, owing to bone effects. In... 

Alkaline phosphatase (ALP) Liver kidney, bone, placenta, intestine, biliary epithelia 30-300 lU/L (higher in children due to increased bone growth) Raised levels may indicate biliary inflammation/ obstruction, malignant infiltration, cirrhosis, bone destruction, Paget s disease... 

Alkaline phosphatase catalyzes the biochemical splitting of phosphoric acid ester. AP (W.M. Roberts, 1933) is found in the liver, bone, kidney, intestine, lung and placenta. A Regan isoenzyme can be detected as an ectopic variation of placental AP in tumour patients (10-30% of cases). The AP of the liver is located in the cytoplasm and in the membranes, primarily at the biliary pole. Placental AP is also present in the liver. The AP of bile duct epithelia is not elevated in healthy individuals. The serum activity of AP is predominantly due to the isoenzymes of the liver and osteoblasts only 14% are of renal origin. Half-life is 3-7 days. 

Alkaline phosphatase may arise from liver, bone, or placenta. The alkaline phosphatase in the sera of normal adults comes primarily from the liver or biliary tract. Elevated levels of alkaline phosphatase are seen in primary or secondary liver... 

Alkaline phosphatase Bone, intestinal mucosa, hepatobiliary system, placenta, kidney... 

Alkaline phosphatase Human placenta 125,000 3 Unknown function role of Zn " " unknown but needed for activity. 

From the biochemical point of view, there is merit in measuring the moiety of the serum alkaline phosphatase activity that is inhibited by L-phenylalanine. This moiety is henceforth referred to as LPSAP, L-phenylalanine-sensitive alkaline phosphatase. The conditions to be chosen should provide the maximum expression of LPSAP and the extent of inhibition of intestine and placenta should be as great as possible at a concentration of L-phenylalanine that does not at the same time inhibit... 

As reported earlier (F16, G8), placenta and intestine are two sources of alkaline phosphatase that are equally sensitive to L-phenylalanine. As can be expected, gastric and duodenal contents contain LPSAP as well. Preparations made so far from the following tissues contain much smaller amounts of LPSAP liver, bone, kidney, lung, and spleen, usually of the order of 0-15% (Table 4). 

Fia. 7. pH optima for intestinal and placental alkaline phosphatases (R) values in the absence of phenylalanine (D) in the presence of 0.005Af n-phenylalanine, and (L) in the presence of 0.005 Af L-phenylalanine. The substrate concentration was 0.018 Af phenyl phosphate. The triangles represent the intestine, and the circles represent human placenta. 

Fig. 8. Relationship between pX versus pH for intestine (rat) and placental (human) alkaline phosphatases. Filled circles represent intestine and open triangles human placenta. The plot for intestinal enzyme is made according to Ghosh and Fishman (G5). Ammonium sulfate fractionation was omitted during the purification of the placental enzyme.

Certain kinetic features of L-phenylalanine inhibition will now be described for the purified human intestinal and placental preparations of alkaline phosphatase. In experiments on the effect of pH, Ghosh and Fishman (G5) observed that the degree of stereospecific L-phenylalanine inhibition of alkaline phosphatase from rat or human intestine and from human placenta is highly pH-dependent. Rat or human intestinal alkaline phosphatase exhibited maximum inhibition at pH 9.2 with phenyl phosphate as substrate, whereas the human placental alkaline phosphatase had a peak at pH 9.6 (Fig. 10). 

Boyer s studies (B39) appear to indicate a lower degree of organ enzyme specificity than that observed by Nisselbaum et al., in that antihuman intestine alkaline phosphatase sera cross-reacted with kidney and placenta. Also, the antihuman bone preparation precipitated alkaline phosphatase from spleen, liver, kidney, and intestine. Boyer considers liver, bone, spleen, and kidney 3-phosphatases to be closely related proteins, followed by intestine and placenta, the enzyme from these latter tissues representing a second and third class of alkaline phosphatase proteins. Intestine and placenta partially cross-react with one another and with a minor kidney component. Three genetic loci are considered by Boyer, therefore, to control the synthesis of alkaline phosphatase. The most recent study in this area is reported by Birkett et al. (B19). 

The resolution of the isozymes of human alkaline phosphatase in normal individuals by starch-gel electrophoresis was systematically studied in 1961 by Boyer, who observed a characteristic alkaline phosphatase pattern similar in pregnancy sera and in placenta (B38). With regard to placenta, recent work (H5, R15, R16) has indicated genetic variation of placental alkaline phosphatase in human placenta when the starch-gel electrophoresis is carried out at two different pH s (8.6 and 6.0). Other tissues could not be differentiated by their starch-gel patterns by Boyer (B38, B39). 

D-isomer. It has been possible to separate and identify the different variants of alkaline phosphatases in human placenta by Sephadex-gel filtration, sucrose density-gradient centrifugation, and ultracentrifugation, and in these studies starch-gel electrophoresis has proved to be unique in characterization of different isoenzyme fractions. Figure 17... 

Stevenson (S47) and Port and Van Venrooy (P14) were able to demonstrate alkaline phosphatase activity following agar-gel electrophoresis. This technique was further developed by Haije and DeJong (HI), who obtained characteristic separate bands for liver and bone alkaline phosphatases. Dymling (D24) applied the technique to pregnancy serum and placenta. 

Sites of alkaline phosphatase activity are frequently in endothelial cells of blood capillaries, mucous glandular cells (F3), microvilli of intestine (C6, CIO, D2, H21, P8, W7), bile canaliculi (D21, F27, W2), and placenta (W3), as well as in the brush border of the lumenal surface of epithelial cells of the proximal convoluted renal tubules (M22, Wl). The location of L-phenylalanine-sensitive alkaline phosphatase in human intestine and placenta is illustrated in Fig. 30. Electron micrographs (Fig. 31) show the details of the alkaline phosphatase, and illustrate the... 

Fig. 30. Enzyme staining reactions for L-phenylalanine-sensitive alkaline phosphatase in human placenta and intestine [conditions were those of Watanabe and Fishman (W7)l (a) human intestine in the presence of D-phenylalanine, X400 (b) high power view of human intestine showing brush border (arrow), terminal web, and apical concentration of alkaline phosphatase, xl200 (c) human intestine in the presence of L-phenylalanine, X400 (d) human placenta in the presence of D-phenylalanine, X400 (e) human placenta in the presence of L-phenylalanine, X400. Note that the enzyme location is on the peripheral absorptive surfaces of the intestine and placenta.

French workers (C4, CIS) discovered in 1934 that pregnancy serum exhibits elevations in alkaline phosphatase. The origin of the enzyme has been attributed variously to maternal osteoblastic activity, to fetal osteoblastic activity, and to placenta. Following Boyer s electrophoretic studies (B38), which showed that serum exhibits a strong enzyme band in the placental region, and Posen s demonstration of heat-stable placenta-type alkaline phosphatase in serum (C13), the reasonable view now is that the placenta supplies alkaline phosphatase to the circulation. 

Harris and his group (R15, R16) in London recently initiated genetic studies on human placental alkaline phosphatase, and demonstrated phenotypic differences in this enzyme. In this study, butanol extracts of alkaline phosphatase from each of 338 placentas were prepared, and the enzyme preparations were subjected to starch-gel electrophoresis at two different pH s (8.6 and 6.0). The electrophoretic patterns obtained were classified in six different groups representing six distinct phenotypes. The... 

In the pregnant subject, the placenta and intestine become the two major sources of serum alkaline phosphatase. Like intestine, placenta is a rich source of alkaline phosphatase and it enriches the outermost cell membranes of the microvilli of the syncytiotrophoblast, which is bathed by the maternal circulation. The properties of the placental enzyme differ sufficiently from those of the intestinal isozyme to permit their differentiation. ... 

The alkaline phosphatase of both human intestine and placenta are L-phenyl-alanine-sensitive and undergo uncompetitive inhibition to the same extent (nearly 80%) by 0.005 M L-phenylalanine. However, we have been able to find several distinguishing biochemical characteristics of the two enzymes (1) the anodic mobility of intestinal alkaline phosphatase remains unchanged after neuraminidase treatment, whereas the placental enzyme is sialidase-seusitive and hence the electrophoretic mobility on starch gel is considerably reduced by such treatment, (2) the Michaelis constant of placental alkaline phosphatase at a definite pH is appreciably higher than that of the intestinal enzyme (at pH 9.3 the Km values of placenta and intestine are 316 and 160 ixM, respectively), and (3) the pH optima (with 0.018 Af phenyl phosphate as substrate) of the two enzymes are different the values for intestinal and placental enzymes with 0.006 Af n-phenylalanine are 9.9 and 10.6, respectively, and the respective values in the presence of 0.005 Af L-phenylalanine are 10.2 and 11.1. Finally, contrary to the behavior of intestinal alkaline phosphatase, placental enzyme is completely heat stable (P19). 

From physiological and ultrastructural considerations, it would appear that the alkaline phosphatase of the bile canaliculi (the only liver structures that are histochemically positive) would have the same function as that assigned to the brush border of intestine and placenta. It may serve in membranes participating in the active transport of metabolites and ions into and out of the bile duct epithelium. The bile normally contains relatively low alkaline phosphatase activity, which can be considered enzyme produced by the bile canaliculi only. 

In general, it would seem that osteoblasts could not compare as a source of alkaline phosphatase with intestine or placenta. In pregnancy the level of alkaline phosphatase is rarely increased above 10 Bodansky or Shinowara units, and yet the placenta s microvilli, which are extremely rich in alkaline phosphatase, are directly immersed in the ample and efficient maternal blood supply (H3). In the nonpregnant individual, therefore, before hyperphosphatasemia can be attributed to bone it would appear necessary to evaluate the intestinal contribution that is evident as heat-sensitive non-LPSAP protein. 

Hitherto unreported are examples of patients such as Case D (Fig. 39) whose cancer of the lung and its metastases exhibited a placenta-like alkaline phosphatase that enriched the serum. This case may well be classified with those neoplasms that have apparently undergone derepression and produce proteins normally unrelated to the tissue of origin. The study of this interesting tumor is continuing (F15). 

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