Alkaline phosphatases in biology and medicine.

© 1991 S. Kargcr AG, Basel 0257-2753/91/0094-0189S2.75/0

Dig Dis 1991:9:189-209

Alkaline Phosphatases in Biology and Medicine Vlaclo Sim ko SUNY Health Science Center. Brooklyn VA Medical Center. Brooklyn, N.Y., USA

followed by a second symposium in Ant werp, Belgium, in 1985 and a third at La Jol la, Calif., USA. in 1989 [20],

Structure

Mammalian AP is a dimeric zinc-contain ing glycoprotein which requires magnesium ion for the hydrolysis of a wide range of phosphomonoesters. Chemically, AP is an orthophosphoric monoester phosphohydrolase which exists in the tissue as a membranebound glycoprotein. Although optimum ac tivity occurs at pH between 9.3 and 10.3. it is active also at a physiological pH [21]. The carbohydrate moiety of AP isoen zymes contains variable proportions of ga lactose, glucose, mannose, fucose, sialic acid, galactosamine and glucosamine [22], The molecular mass of an intact enzyme varies between 70 and 280 kilodaltons, with the smallest enzyme molecules originating in the placenta, and the largest in the liver [23], AP in mammalian cells is usually located on the plasma membrane. Regarding the subcellular components [24], AP was also found in the microsomes, nucleus and Golgi apparatus. The physicochemical properties of AP have been studied on a simple monomer of

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Alkaline phosphatase (AP) is one of the most useful enzymes in the routine clinical diagnosis [1]. Most of the voluminous litera ture on AP originates after 1950. This was a landmark when separation of AP with starch gel electrophoresis rapidly expanded our un derstanding of this enzyme. Our knowledge on this important enzyme is closely related to the evolution of analytic techniques. Initial attempts to determine AP isoenzymes using heat inactivation were of some help in differentiating the bone, liver and placental APs. Chemical inhibition tech niques using phenylalanine and other amino acids further assisted in distinguishing the placental subgroup of AP. Column chroma tography proved to be of little help and also the zonal electrophoresis separated the bone and liver bands of AP rather poorly. Further important progress in research on AP has been achieved with the advent of the monoclonal antibodies and also by isoelec tric focusing on agarose-containing specific separators. Numerous excellent reviews [2-19] on AP attest to the enormous importance of this enzyme in biology and medicine. Although we still know only little about the biological functions of AP, this intriguing enzyme war ranted convocation of several international symposia. The first in Umea, Sweden, was

AP in Escherichia coli. In this species. AP is synthesized on the polysomes bound to the inner membrane of this microorganism [II], The complete amino acid sequence of this monomer has been determined to be a single unglycosylated polypeptide chain of 449 amino acid residues [25]. This sequence re sults in a molecular weight of 47,029 daltons, a native dimer having twice this molec ular mass. The crystal structure of AP from E. coli (a prototype of mammalian APs) has been re fined [26], Based on this structure and on the sequences of mammalian APs (25-30% strict homology), these authors have mod eled the core of the three-dimensional mam malian AP. Mammalian APs are larger than AP from E. coli. They contain carbohydrate and are membrane-associated through a phosphatidylinositol moiety. AP from E. coli has been considered one of the most attractive marker enzymes for studying the possibility of joining proteins at the level of their genes, to facilitate the pro duction of translational gene fusions [27], Purified preparations of liver, bone, in testinal. placental and kidney AP have been used to raise antisera for immunologic stud ies. There appears no cross-reactivity be tween liver and bone antisera, with intestinal or placental antigens and vice versa [28], Attempts to produce antibodies specific for bone or liver AP have resulted in cross-reac tive antibodies [29].

Origin

APs constitute multiple molecular forms of enzymes in which the heterogeneity is due to genetic factors and to posttranslational modifications. There is a generalized expres

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sion of one or more of these genes upon which there is superimposed a localized ex pression of genes that determine the specific isoenzyme [4], Also, various posttransla tional modifications generate additional en zyme variants which have tissue-specific dis tribution. Multiple forms of an enzyme such as AP may result, first, from isoenzymes coded by distinct genes which exist in separate loci on the chromosome. Second, isoenzymes bio synthesized from a single gene subsequently may become distinguishable as a result of posttranslational modification by proteases and by carbohydrates or lipids, attaching to the enzyme molecule [30], For AP isoenzymes derived from the same gene (e.g. the ‘tissue-unspecific' AP). the difference in biological and biochemical properties is probably related to differences in the carbohydrates constituting the mole cule. These carbohydrate moieties make the tissue-unspecific AP separable by the ana lytic techniques. Various AP isoenzymes are produced in tumor cells. The precise sites of genes which determine mutation or splicing of AP pro duced by neoplastic cells remain unclear. There are three, possibly four, structural genes which were detected by radioactive peptide mapping, reactions with substrates and inhibitors, and immunologic reactivity. These structural genes are related to the in testinal AP. placental AP and a tissue-unspe cific gene expressed in the liver, bone and kidney [31]. The existence of a fourth struc tural gene of AP. related to fetal intestinal AP, is probable [32]. Thus, the three isoenzymes, intestinal, placental and tissue-unspecific APs, are en coded at separate genetic loci [31]. Comple mentary DNA clones for these three major


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dimers by an enzyme which is not proteo lytic and is probably a phosphatidase of the C or D type. Since secretion of enzymes is stimulated by factors unique to each organ, the mecha nism of release of AP may differ from organ to organ. A variety of modes has been pro posed to explain AP release, including deter gent action, proteolysis, membrane fragmen tation and lipolysis [47],

Function

Despite several decades of research which resulted in thousands of scientific reports, the physiological role of AP is unclear. Mam malian tissues high in AP usually have a vital role in transport mechanisms. AP resides in liver cells, in the brush border of the intesti nal epithelium, in osteoblasts, kidney tu bules and in the syncytium of the placenta [24, 48]. In newer reports, however, AP was found to be much more ubiquitous. The function of AP seems to depend on its location. Relative activity of AP (per mass of tissue) [2] is highest in the adrenal cortex, followed by the placental syncytiotrophoblast. microvillous part of epithelium of the ileum, stomach and colon, osteoblasts and chondroblasts in the bone, proximal and distal kidney tubules and the loop of Henle, hepatocytes close to sinusoid branches of the portal vein and bile canaiiculi. Much smaller activities of AP were found in the respiratory epithelium of the lung, in blood polymorphonuclears and monocytes, in the parotid, pancreas, testis, thyroid and endothelium of small vessels. While there is no known function for AP in plasma, within the cellular cytoplasm AP regulates phosphate metabolism, maintain


isoenzymes have been cloned and sequenced within the past 5 years, providing details of the structure of the enzyme and of differ ences between isoenzymes [33-35]. Monoclonal antibodies have been devel oped specific to each of these genetic isoen zyme forms of AP [36-39]. Monoclonal anti body techniques have been successfully used to separate placental and intestinal APs in liver métastasés from gastric cancer [40] and to characterize the AP in human colon can cer as an allelic variant encoded at the germ cell AP locus [41 ]. There is no clear understanding of the dif ference between placental AP and the 'placen tal-like' AP [42] even when monoclonal anti bodies are capable of differentiating between these two isoenzymes [43]. The latter is present in small amounts in testis and lungs. Its presence dramatically increases when derepression of peptide synthesis occurs in ma lignant tumors arising from these and other tissues. Attempts to differentiate between these two types of AP are based on reaction with monoclonal antibodies and on the sensi tivity to inhibition by L-leucine [44], Enzymes in plasma may be bound by nat urally occurring immunoglobulins (naturally occurring monoclonal antibodies) to give en zyme-immunoglobulin complexes. In addi tion to amylase, creatine kinase and lactate dehydrogenase, in autoimmune disorders AP belongs to the most frequent enzymeimmunoglobulin complexes in plasma [45]. Such enzyme binding may lead to an altered total enzyme activity, with changes in the biological behavior and electrophoretic pat terns. With regard to mechanisms releasing AP from the cells, this appears to be in most tis sue cells and body fluids an endogenous en zyme [46], AP is released as hydrophilic

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ing phosphoryl metabolites in a steady state and synthesizing and hydrolyzing phosphate esters [47]. At cell membranes AP supervises active transport of inorganic phosphate, fat, protein, carbohydrate and Na/K.

Spectrum of AP in Physiological and Pathological Conditions

Distinction should be made between AP physiologically present and the enzyme forms found in disease, especially in cholestasis. Under a physiological state, the AP in the plasma consists of multiple forms derived from many different tissues. These include primarily the liver, bone, intestine, kidney and placenta. Plasma levels of AP in normal subjects vary with age. sex and reproduction [7], Polyacrylamide gel electrophoresis indi cates approximately equal amounts of liver and bone AP in normal plasma [49]. In cer tain subjects, dependent on the blood group, the intestinal AP may represent up to 20% of total plasma level [50]. Because osteoblasts in the growing bone prominently contribute to plasma AP. the proportion of skeletal AP in childhood is 3 times the amount found in adults [51]. In a study in 857 infants born before term, high plasma AP was related to slower growth in the neonatal period and it reflected early bone mineral substrate deficiency which later resulted in metabolic bone disease [52]. In the third trimester of gestation, maternal placental AP represents a significant propor tion of the total plasma AP [53]. In adult males the AP in plasma is slightly higher than in females [54, 55]. Since various forms of AP in plasma orig inate from different tissues, there is a practi cal need to differentiate these apart. Sim

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plest distinction is by testing the heat sensi tivity when serum sample is exposed to 56 °C for 10 min. After heating, the remaining phosphatase activity (hydrolysis of the sub strate /.’-nitrophenvl phosphate) is compared with the activity of unheated serum. De crease of AP activity to 20% or less is indic ative of presence of the heat-labile bone AP. Residual activity more than 20% indicates a predominance of AP from the liver. As men tioned above, under circumstances the intes tinal and placental AP may constitute a sig nificant proportion of the total AP [10]. AP derived from the bile is more heatresistant than AP derived from liver cells [56]. Placental and intestinal AP are even more heat-resistant than AP from the liver [ 17], APs associated with neoplasms, some times called the Regan isoenzyme and the Nagao isoenzyme, are heat-resistant at the level of the placental AP [57, 58], Although heat inactivation is easy, it is not very reli able and it should now be replaced by more sophisticated separation of AP isoenzymes. More accurate separation techniques than heat resistance are high-performance liquid chromatography [59], high-perfor mance affinity chromatography [60] and the electrophoretic methods, based on separa tion of AP molecules by their size and elec tric charge. Polyacrylamide gel is the most suitable of these carriers [61, 62], but cellu lose acetate [63] and agar gel [64] can also be used as support media for electrophoresis. Much progress in our understanding of AP has been accomplished with the monoclonal antibody method [43, 65] and isolectric fo cusing [66]. APs distinguished by analytic procedures seem also to differ in their rate of catabo lism. When disappearance of infused AP preparations from blood was carefully ana-


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lvzed, intestinal and bone AP were cleared most rapidly while the liver AP required sev eral days to clear [67], Human placental AP was slowest to disappear, having a half-life of approximately 7 days [68]. Hepatectomy in dogs did not affect the rate of clearance of intestinal AP [67] from plasma. There seems to be no specific function for AP circulating in plasma. Its levels reflect abnormal tissue metabolism of the enzyme. A substantial increase in plasma AP by infu sion of the enzyme had no consequences on the calcium or phosphate metabolism [68], Microvascular endothelium is strongly APpositive. Using specific inhibitors of AP. the isoenzyme in the endothelium was found to be the tissue-unspecific liver/bone/kidney AP [69], Intestinal AP. which is a glycoprotein of a molecular mass of 138 kilodaltons. was the first of the AP isoenzymes to be intensively studied. Intestinal AP is almost never ele vated in diseases of the gut and only rarely in some patients with liver cirrhosis [70], The enterocyte is a polarized cell with an apical brush border membrane containing most of the cellular AP. This apical compo nent of the enterocyte is exposed to high con centrations of bile salts and phospholipases from the lumen [47]. The intracellular enterocytic AP is soluble and with 62 kilodaltons mass smaller than the membranous intestinal AP [47]. The two intestinal APs, soluble and membranous, differ not only in carbohydrate composition but also in peptide structure. Two separate mRNAs have been detected for intestinal AP by Northern blot analysis, using a human placental AP cDNA as a probe [71]. A full-length rat intestinal AP clone was isolated and sequenced with a primary se quence of 519 amino acids and with an addi

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tional signal peptide of 20 amino acids [72]. Of the amino acids from residues 1-474, 80% were identical when compared with the human intestinal AP, but there was only 31% identity in the COOH terminus of 45 amino acids. None of the intestinal APs in plasma has been found on membranes or particles. Analysis by isoelectric focusing of the intesti nal AP secreted into plasma demonstrated that secretion of particulate AP, after extra cellular release from the membrane, can ac count for the intestinal isoenzyme found in plasma [73]. On the other hand, particles containing intestinal AP have been found in the intestinal lumen, partly as a response to cholecystokinin. Elucidation of the biology and function of this secretory particle in the intestinal lumen may bring us more informa tion on the nature of intestinal AP [47], The effect of starvation was studied in rats [74]. A 3-day starvation caused a signif icant fall in plasma AP which was restored to normal after feeding. In fed rats the serum during the day showed a pattern compatible with the presence of a circadian rhythm which may be related to feeding habits. Elevation of plasma AP in patients with idiopathic sprue was attributed to osteoma lacia from calcium malabsorption, rather than to disease of the intestinal mucosa [75], However, it is known that intestinal AP may be elevated in extensive mucosal damage to the stomach or intestine [48], Intriguing are the data on behavior of intestinal AP during digestion and absorp tion of fat. An increase in serum intestinal AP has been reported in rats ingesting fat [76]. Oleic and stearic acid were found to induce this rise but not glycerol [77], The magnitude of rise of intestinal AP was re lated to the length of the fatty acid [78]. A



finding [79] that the AP in the thoracic lymph duct increases in man after a fatty meal but not after protein or carbohydrate was in agreement with other reports. On the basis of these experiments, it was hypothe sized [78] that AP may play a role in the absorption of fat. Intestinal AP in the rat is known to have much of its activity released into plasma after feeding, especially after ingestion of fat [80]. In this species, fat administration sig nificantly increased intestinal AP in the in testinal lumen and in intestinal lymph [81], but it did not change biological characteris tics of the four various intestinal AP frac tions (brush border, cytosol, luminal and lymphatic). This suggests that fat adminis tration induces quantitative changes in in testinal AP but it does not modify the physi ological route of AP transport into the intes tinal lymph. Relation between digestion and absorp tion of dietary fat and AP is further strength ened by an observation that intravenous ad ministration of both secretin and cholecystokinin leads to a marked increase in AP in the intestinal content [82], This rise also occurs when there is a complete obstruction of the biliary and pancreatic duct [82], Administra tion of secretin and cholecystokinin did not increase AP in the bile from a patient with a T tube in the common bile duct [83]. Both these hormones are released by intraduodenal fat. There is a possibility that the rise in intestinal AP after ingestion of fat is me diated by secretin and cholecystokinin. Mammalian feces contains high amounts of AP which was first reported to be antibi otic- and intestinal-microflora-independent [84]. It was concluded that fecal AP repre sents material shed from the surface of intes tinal epithelium. However, a more recent

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report [85] described that the intestinal wall of conventional rats, when compared with germ-free rats, contains only 50% of the intestinal mucosal AP. When germ-free rats were fed sterilized microorganisms, this de crease in mucosal AP did not occur. Viabil ity of the intestinal microorganisms was thus essential to cause decrease in the intestinal mucosal AP. AP has been shown to be re leased from mammalian tissue by phosphati dylinositol phospholipase enzymes from bacterial sources such as Bacillus cereus [46, 47, 86], The relation between the intestinal AP and the AP found in the adult human kidney was explored [87], The kidney was found to contain not only the tissue-unspecific AP but also another AP isoenzyme which by mono clonal antibodies, specific for intestinal AP, was different from the adult intestinal AP and the tissue-unspecific AP, but identical with the fetal (méconial) AP. The existence of different allelozvmes of intestinal AP was suggested in a biochemical case study of a 47-year-old woman with hy perphosphatemia who after an agarose gel electrophoresis showed the presence of three isoforms of intestinal AP [88]. Like the intestinal AP, placental AP is also of considerable interest because it origi nates from a structural gene and because of its difference from other APs. Placental AP rises in pregnancy and bears a specific rela tion to certain malignancies. Monoclonal an tibodies with specificity to human placental AP demonstrated AP radioimmunolocalization in animal Hep2 tumors [89], With advancing gestation there is a pro gressive rise in serum AP [90]. During the second half of pregnancy, up to 50% of total serum AP consists of the heat-stable fraction [91]. Because of these findings, it was pro


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AP [97]. AP derived from a mammalian periosteal cell culture system reflected the sensitivity of these cells to oxygen tension [98] . AP production was optimal at lower oxygen tensions. In Paget’s disease, plasma AP reaches some of the highest values observed in clini cal medicine. Its elevation generally reflects the activity and extent of bone involvement [99] . In rheumatoid arthritis and ankylosing spondylitis, the bone isoenzyme of AP was a much more sensitive indicator of disease ac tivity than the total AP. and it correlated with the number of affected extravertebral joints and with pain [100]. With regard to bone malignancy, AP has only limited use as a marker. In skeletal neo plasms, serum AP may be lower than when pathological fractures occur, or when skele tal metastases have an osteoblastic character [ 101].

Leukocyte AP has been known for several decades and it has been in focus of interest after the first cytochemical demonstration of leukocyte AP in 1946 [102]. Subsequent ly it was found that leukocyte AP is virtu ally restricted to neutrophil polymorphonuclears. especially to specific granules in ma ture neutrophils [103], and that leukocyte AP activity in neutrophils decreases with cell age [104]. There is no relationship of leuko cyte AP to serum AP [ 105], Leukocyte AP is higher in newborns [106]. Elevation of leu kocyte AP in ‘stress' conditions can be repro duced by the administration of adrenocorticotropin or corticosteroids [107], Values of leukocyte AP were reported to be low in chronic myelogenous leukemia, in paroxysmal nocturnal hemoglobinuria and in hypophosphatasia [108], Conditions where leukocyte AP is elevated include poly cythemia vera, agnogenic myeloid metapla


posed that placental AP may have diagnostic value in disorders of placental function. Spe cifically. it was tested if serial assay of pla cental AP may predict the progression of ges tation. the fetal weight and fetal prognosis. Careful evaluation of numerous reports on this subject [92] unfortunately yielded a neg ative answer to all these questions. Placen tal-type AP was found to be elevated in the peritoneal fluid obtained from women with endometriosis [93], Placental AP and placental-like AP are present in trace amounts also in other tis sues. especially in the lung. It is of interest that this placental-like AP may be released into the circulation during smoking [94]. A normal lung may express eutopically trace amounts of this isoenzyme and this expres sion may be enhanced by smoking. The relation of placental AP to malig nancy will be discussed in more details in the segment on the role of AP in neoplasia. With regard to AP in the pancreas, this was reported by histochemical staining to be present in pancreatic islet cells and in the endothelium of blood vessels in the pancreas, but not in the exocrine pancreatic cells [95], It remains controversial if the pancreatic AP is organ-specific. Regarding the diagnostic value, pancreatic AP proved unreliable as a histochemical marker for different categories of pancreatic cells, due to its variability in the same type of cells and because of exten sive species differences [95]. AP which has its source in the bone is a consequence of osteoblastic and chondroblastic processes. Its elevation occurs in peri ods of bone growth, in bone fractures, vita min D deficiency and osteomalacia [96]. Os teoporosis was also observed to lead to an increase in AP while estrogen therapy in postmenopausal women resulted in a drop in

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tients with osteoarthritis, rheumatoid arthri tis and ankylosing spondylitis [115]. Since benoxaprofen affects the monocytes, it was hypothesized that the reduction of AP under its influence was via the osteoblasts, second ary to osteoclasts which are derived from monocyte precursors. Also, there are reports that in rare cases patients with primary sclerosing cholangitis, a typical cholestatic disorder, may have a normal serum AP. A recent article on 2 such cases, combined with an extensive review of other reports [116], documented that up to 3% of patients with sclerosing cholangitis may have a normal serum AP. This creates a compelling need to use a cholangiogram for diagnosis, whenever there is a clinical suspi cion for primary sclerosing cholangitis. Finally, clinicians occasionally encounter patients where an abnormally elevated AP cannot be explained even after a very thor ough investigation [117], Severe familial hy perphosphatemia with an elevated serum AP has been described [118]. This biochemical abnormality was found to be inherited as an autosomal dominant trait.

AP in Disorders of the Hepatobiliary

System

Pathophysiological mechanisms affecting the hepatic and biliary AP. due to their spe cific clinical significance, are described in this separate segment. Liver AP is elevated in extrahepatic and intrahepatic cholestasis, in parenchymal liver disease and in primary or secondary malignancy of the liver. The mechanisms responsible for the rise in AP originating from the liver are not quite clear [119]. Our understanding of the mode of escape of hepatic AP in liver disorders


sia, leukemoid reaction, pregnancy and oral contraceptives. Leukocyte AP is not of a diagnostic value when considered outside of the clinical context. Although the pathophysiological mecha nisms leading in decompensated diabetes mellitus to a rise in leukocyte AP are unclear, leukocyte AP correlated with the levels of glycosylated hemoglobin [109]. Also, the to tal AP in plasma was observed elevated in unstable diabetes [110]. Uncontrolled dia betes may lead to hepatomegaly. Serum AP elevation was associated with increased size of the liver. Liver biopsies in diabetics with elevated AP demonstrated marked accumu lation of glycogen in the hepatocytes [110]. An interesting clinical application of AP was reported in the field of parasitology [111]. Several enzymes of the parasitic worm Schis tosoma mansoni, including its tissue AP. be have as antigens useful in immunodiagnosis. Of the coproparasitologically proven S. manion/'-infected patients. 93% had antibodies, in an immunoadsorption technique, against AP from the worm extract. This antibody response against S. mansoni AP was progres sively reduced after treatment. This review on AP in health and disease should be concluded with a discussion of clinical situations when serum AP has an atypical behavior. Factors which abnormally decrease plas ma AP may occasionally confuse the clinical diagnosis, in conditions where one would expect an elevation of AP [112]. Decreases in serum AP were found in hypophosphatasia. hypothyroidism, pernicious anemia and in blood samples anticoagulated with oxalacetate [113, 114], Benoxaprofen, a nonsteroidal anti-in flammatory agent used in the UK, was found occasionally to normalize serum AP in pa

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which vary in the etiology and pathogenesis, like extrahepatic bile stasis, primary biliary cirrhosis and alcohol liver injury, has been promoted by newer techniques of separation of AP isoenzymes. There is an agreement that plasma AP increases to a lesser level in parenchymal liver damage, for example in duced by alcohol, than in intrahepatic and extrahepatic cholestasis [120]. Using chromatography on diethylaminoethyl cellulose, biliary AP was measured in 182 patients with various hepatobiliary disorders [121], In patients with nonbiliary disease and in 'healthy’ carriers of hepatitis B virus, biliary AP activities were very low. In cirrhosis and chronic hepatitis, the activ ity of this enzyme was somewhat higher but still very low when compared with obstruc tive jaundice and primary and secondary liver neoplasms. There was no correlation between biliary AP and serum bilirubin in obstructive jaundice. Diagnostic sensitivity for detecting hepatocellular carcinoma sig nificantly increased when biliary AP was added to serum a-fetoprotein [121], In liver cirrhosis an elevated serum AP may be derived from the hepatobiliary and also the intestinal AP. When cirrhosis is ac companied by portal hypertension, there is an increase in the thoracic duct lymph flow. This lymph contains higher concentration of the intestinal AP [122], Portal hypertension and liver disease may explain the concomi tant rise of liver and intestinal AP in liver cirrhosis. Important clinical implications regarding changes in serum AP are related to bile duct obstruction limited to one lobe of the liver [123], In 8 such patients, after an initial rise of serum AP. unrelieved bile duct obstruc tion was associated with a gradual decrease in serum AP. This return to normal coin

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cided with atrophy of the segment of liver subjected to bile stasis. An animal model confirmed this association between reduc tion of hepatocyte mass and a decrease in AP activity [123]. Patients suspected of long term partial bile duct obstruction should be subjected to thorough clinical investigation, including cholangiography - even when se rum AP is normal [116, 123], Increase in plasma AP in cholestasis is not a simple consequence of an enzyme leak from the damaged hepatocyte or of bile stasis. Cli nicians know that AP may be elevated in dis proportion to the aminotransferases or to the bilirubin. Infiltrative liver disorders may stimulate de novo synthesis, regurgitation and plasma elevation of AP even before the obstruction of the biliary system becomes so prominent that it also induces an increase in bilirubin [16]. While bilirubin excretion can be effectively compensated by the nonobstructed liver, de novo synthesis and regurgi tation of AP in the obstructed part of the liver result in a rise in plasma AP [68]. Of the numerous theories proposed to ex plain abnormal level of plasma AP in liver disorders, increased de novo synthesis and regurgitation from the liver seem to be plau sible and acceptable [9], while the retention theory could not be confirmed. Making it even more complex, it has been known for quite some time [ 124] that the AP in bile differs from AP derived from the liver cells. The biliary AP was thought to consist from a protein-phospholipid complex [125] which was more heat-stable than the liver AP [56], Improvement in analytic procedures re sulted in a substantial elucidation of the pro cess of regurgitation of AP from the liver [47. 126]. The hepatocyte has an apical (or lumi nal) membrane facing the bile canaliculus, a



basal membrane facing the sinusoid and a lateral membrane connecting these other two areas. A simplified concept is that under physiological conditions the liver AP circu lating in plasma is derived from the sinusoi dal membrane of the hepatocytes. In choles tasis, the AP in plasma is derived largely from the canalicular membrane. In hepatic parenchymal disorders, the apical brush bor der membrane of hepatocytes is the major source of AP. In the bile, AP appears to be in a particulate form while that in blood is largely soluble [126], The mechanisms involved in regurgita tion of AP from the liver are quite complex [127], Studies on kinetics of transfer of hépa tocyte proteins have helped to reveal some of the mechanisms of transfer of AP in hepato biliary disease. In cholestasis, de novo synthesis of AP contributes to increased amounts of AP which regurgitate from the canalicular mem brane by transcellular processes or via a paracellular route. Transcellular regurgitation would result in partial endocytosis of the canalicular membrane, followed by exocytosis of the sinusoidal membrane of hepato cytes [128], In case of paracellular route of regurgitation of AP. the enzyme from the canalicular membrane would leak through the tight junctions [129]. During cholestasis the distribution of AP is altered. The apical AP becomes equally spread over the sinusoi dal membrane of the hepatocyte [ 130], The AP in cholestasis, whether intra- or extrahepatic, is a high-molecular complex, related to the hepatic isoenzyme of AP [12, 126]. During cholestasis, particulate (mem brane-associated) AP appears in serum of about 50% of patients. When the abnormal AP found in extrahepatic cholestasis was pu rified and compared with other APs, it was

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found that this cholestatic AP was similar, but not identical, with normal AP from liver or bile [126], In advanced cholestasis, sufficient distor tion of the canalicular tight junctions occurs, so that large molecules of bile lipoproteins can enter the circulation. Association of this cholestatic AP with a lipoprotein carrier was proposed [ 126] and later confirmed by elec trophoretic separation, with the finding of a typical 'biliary band" [131, 132]. To explain the presence of this cholestatic lipoprotein in plasma, evidence for a passage through the tight junctions [133] and transcellular regur gitation [ 134] have been reported. This lipoprotein, termed lipoprotein X, consists of a phospholipid, nonesterified cholesterol and a small amount (2-3%) of bile acids. Bile acids are represented pri marily by lithocholate, which may be hepatotoxic. Various investigators assigned dif ferent names to this high-molecular complex of AP in cholestasis. Attempts to use termi nology related to the electrophoretic mobil ity of cholestatic AP were criticized, because mobility depends on the gel used for electro phoresis [12]. Association of AP with lipo protein into high-molecular complexes was considered to be related to the cholestatic process. Lipoprotein X is found in plasma as a result of bile regurgitation [ 135]. It was also noted that complexes of the lipoprotein X with AP occur in cholestasis, but not in hepatic malignancy [131]. At tempts to distinguish cholestatic AP from AP in hepatic malignancy, by studying the rela tive contribution of the sinusoidal or canalic ular membranes of the hepatocyte to the ori gin of AP, could not clarify this difference. In neoplastic disease of the liver, a highmolecular AP appears in the ot|-l globulin area on serum protein electrophoresis [136].


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The diagnostic sensitivity of this type of AP is high for primary and secondary neoplasm of the liver [ 137], Unfortunately, the specific ity of this AP is much less acceptable because this fraction also occurs in cholestasis. Processes leading to the damage of liver cells and to an increase in AP have been studied in animals with bile duct ligation and also in human disease [9]. Unfortunate ly, comparing animal and human cholestasis is problematic because of important species differences in the phospholipids of the hepatocytic membrane and also in the spectrum of a normal and cholestatic bile. In a normal liver, parts of the hepatocytic membrane are continuously solubilized and repaired by regeneration [138]. This process is altered in cholestasis. When bile salts rise, there occurs an impressive increase in de novo synthesis of AP [139. 140]. Increased synthesis of AP seems to be related to en hanced translation of mRNA in the hepatocytes. rather than to an increase in transcrip tion [141]. Newly synthesized AP reaches the canalicular membrane of the hepatocyte and is then released into the bile by the solu bilizing effect of bile salts [142], AP level in plasma rises mostly because of regurgitation of AP from the bile [126]. In an acute bile duct obstruction in a rat. the rise of AP in plasma is too high to be accounted for only by the mechanical block to bile secretion [143]. Twelve hours after a com plete bile duct ligation in a rat. de novo syn thesis of AP is 4 times normal [139, 140, 144]. Plasma AP reaches a peak 24 h after ligation and, although AP in the liver contin ues to rise until 72 h after ligation, plasma AP decreases in the mean lime to a steady state [145]. In rat livers with chronic bile duct ob struction and in experimental cirrhosis, en

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zymatic histochemical methods showed most increase of AP in the portal tracts [ 146]. AP was localized in the walls of prolif erating blood vessels. Rise in total serum AP was similar in these rats whether they had chronic bile duct obstruction or experimen tal cirrhosis. The metabolism of AP is closely related to bile acids. Although the nature of the inducer of AP in the hepatocytes remains unknown, the observation that bile acids can induce AP in cultured rat hepatocytes [147] suggests that the accumulation of bile salts in the liver may be a causative factor in the ele vation of AP. Clinical observations of AP elevation in localized bile duct obstruction are consistent with this hypothesis. Local ized bile stasis and bile salt accumulation will lead to a rise in AP, while bilirubin secretion continues essentially unimpaired. If the bile acid pool is depleted, an acute extrahepatic obstruction results in a smaller elevation of hepatic AP [7]. The molecule of AP possesses a hydrophobic part which binds this enzyme to cell membranes. Inter action of bile acids with cell membrane in tegrity may explain the interference of bile acids with AP in liver disorders. Concentration of bile acids in the hepato cellular microenvironment affects the aggre gation of AP to proteins of different molecu lar weights [148]. A high concentration of bile salts competes for the membrane phos pholipids which are normally used for mi celle complexes in the bile [ 138], When bile salt concentration is high, AP is present pre dominantly in a low-molecular form [148]. Membrane-damaging properties of bile acids in acute cholestasis or in liver disease accompanied by cholestasis are related to the detergent effect of bile salts [138]. Also im portant are the number and orientation of



hydroxyl groups in the bile acid molecule and the presence of conjugation with taurine or glycine [149], A trihydroxy bile acid (cholate) is less damaging than the dihydroxy- or even the monohydroxy bile acid lithocholate. Urso deoxycholic acid, presently used for gallstone dissolution, has favorable effects on cholesta sis in primary biliary cirrhosis [150]. Urso deoxycholic acid was reported to elevate only the AP in the liver, not AP in plasma [151]. Cholic and chenodeoxycholic acids elevate, besides the liver AP. also the AP in plasma. To determine the effect of various bile acid loads [152], the endogenous bile acid pool in rats was substituted with equimolar amounts of a single bile acid. Hepatic synthesis and serum activity of AP were influenced by the location of the hydroxyl group in the core of the bile acid molecule, by the bile acid deter gent effect and hydrophobicity. Hepatic AP activity was highest when taurocholic acid, the most detergent of the bile acids tested, replaced the endogenous pool. Taurohyocholic and tauroursodeoxycholic acid did not induce a rise in the hepatic AP. It appears that bile acids with lower secre tory pressure have a higher potential to dis rupt the tight junctions between hepatocytes and to induce a leak of AP and other mole cules into the sinusoidal space [6].

Metabolism of AP in Malignancy

Abnormal AP detected in various neo plasms appears to be a variant of placental AP, produced by the same gene [153], prob ably as an ectopic polypeptide produced by the tumor. This AP is occasionally named the Regan isoenzyme, after a patient who died of bronchogenic cancer [154]. In Japan,

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a similar AP was named Nagao isoenzyme, after another patient with carcinomatous pleuritis [58], Nagao AP differs somewhat from the Regan isoenzyme, with respect to the enzyme kinetics [58]. As a tumor marker, the diagnostic use of placental AP is not so widespread as carcinoembryonic antigen or a-fetoprotein be cause the frequency of elevated serum pla cental AP in malignancy is lower. Studies on purification and partial characterization of placental AP in the lung indicate new diag nostic potential for this isoenzyme [155]. When human gastric tumor cell lines were biochemically and biologically characterized according to the monophenotypic expression of placental AP-like enzymes, the cells de rived from adeno- and squamous carcinoma showed properties of the Regan isoenzyme while cells derived from gastric choriocarci noma had properties identical with the Na gao isoenzyme [156], In a rat model of gas tric cancerogenesis. marked enhancement of tissue-unspecific AP production was demon strated in the stroma of the adenocarcinoma [157], AP isoenzyme associated with malig nancy is sharply reduced after elective sur gery or chemotherapy of the tumor, and it reappears with the recurrence of malignancy [14], Regan isoenzyme was also detected in familial polyposis of the colon and in ulcer ative colitis [158]. In 8 patients with colorectal carcinoma, the progression of the tumor correlated with serum placental AP and other tumor mark ers (carcinoembryonic antigen. CA 19-9 and Ca-50) but not with the tissue-unspecific AP and with intestinal AP [159]. AP in hepatocellular carcinoma resem bles placental AP but it differs from the Regan isoenzyme [160],


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in 87 patients with ovarian cancer, the diagnostic sensitivity of a tumor marker CA125 was substantially increased when serum placental AP activity and concentration, measured with a combination of enzymelinked immunosorbent assay and mono clonal antibodies, were added to the screen ing [161]. Although it is recognized that specific AP isoenzymes can be ectopically produced by a variety of human neoplasms, and that cul tured human cancer cells retain the capacity to produce these enzymes [162], no conclu sions were made why only some tumors pro duce this AP. Part of this ignorance is related to our lack of understanding of normal met abolic processes of the AP. The demonstration of small amounts of placental AP in normal testes, lung and uter ine cervix suggests that the expression of this type of AP in tumors derived from these tis sues is an amplification of a gene locus which is normally expressed at a low level [163]. The elusive nature of this 'carcinoplacentaf AP is obvious from an experience that it is not a reliable marker even for an early detec tion of ovarian cancer [164], Unlike the placental isoenzyme, the bone isoenzyme of AP is an indicator of the activ ity of the osteoblasts, and in malignancy it reflects the involvement of the bone with primary disease or metastases but not the biological process of prostatic [ 165] or breast [ 166] malignancy.

Phosphate-Hydrolyzing Enzymes

The substrate specificity of AP is of con siderable interest. The traditional view has been that AP hydrolyzes organic phosphate esters in an alkaline environment. Regarding

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the hydrolysis of inorganic pyrophosphate (PPi), the role of AP deserves further com ments [167], Nucleotide triphosphates are the major source of PPi. PPi phosphorolysis accom panies the synthesis of nucleotides, nucleic acids, lipids, steroids, proteins and structural polysaccharides. Catalysis of PPi hydrolysis by enzymes can occur at an acid, neutral or alkaline pH. At neutral and alkaline pH, the PPi phosphohydrolytic activities are attributable main ly to AP. Basically, PPi phosphohydrolysis can involve an orthophosphoric monoester phosphohydrolase, an inorganic pyrophos phatase and a glucose-6-phosphatase [167]. Clinical evidence for a potential role of AP in hydrolysis of PPi was indicated by observing an inborn error of metabolism, hypophosphatasia [168]. In this disorder, plasma AP is low and there is an abnormal urinary excretion of PPi. AP enzymes which hydrolyze PPi have been reported in plasma, liver, intestine, kid ney, bone, placenta, brain, bile and endome trium. in a variety of species including the man, dog, sheep, calf. pig. rat, mouse and hamster [167], Enzymatic activity hydrolyz ing PPi was also reported in E. coli [ 167], Tissues differ with respect to hydrolytic sub strate specificity of various types of AP. Hy drolysis of organic phosphomonoesters was higher than hydrolysis of PPi in the liver, placenta and bone, while in the intestine PPi phosphohydrolysis was more prominent [169], APs with organic phosphate- and PPiphosphohydrolytic activity are in the mam malian tissue firmly attached to membranes containing lipoproteins, especially microsomes [170]. Inorganic pyrophosphatases have their specificity restricted to PPi phos-



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AP in the Urine

In the 1960s, much attention was devoted to the diagnostic potential of enzymes ex creted in the urine. AP was one of several enzymes studied [176, 177], Urinary AP was found elevated in up to 57% of patients with neoplasms of the urinary tract, in 68% of patients with nonurinary malignancy, in 68% of chronic glomerulone phritis, after a myocardial infarction, in some

cases with kidney transplant rejection and almost invariably in patients with chronic pyelonephritis. In extrarenal malignancy and in liver cirrhosis, the rise in urinary AP was independent of the level of serum AP. In liver cirrhosis with ascites, the clear ance of AP was found to be increased [178]. It was not determined if this increase in uri nary AP in cirrhosis was the consequence of altered AP production or of the renal dys function associated with liver disease. Initial enthusiasm for AP as a urinary diagnostic aid proved excessive. It was nec essary to dialyze the urine to eliminate the high concentration of phosphate, which acts as a competitive inhibitor [179], Even after dialysis, storage of urine specimens causes an increase in the AP [ 180]. The main prob lem, however, was the high rate of false neg atives and a lack of specificity [178],

Other Enzymes Used in Indications Similar to AP

Three enzymes are occasionally used in clinical indications similar to the use of AP. Of these, only the 5'-nucleotidase is a phos phatase which catalyzes the hydrolysis of nu cleotides, releasing PPi from the pentose ring. y-Glutamyl transferase catalyzes the transfer of the y-glutamyl group between peptides. Leucine aminopeptidase activates the hydrolysis of the amino acid terminus from peptide molecules. Leucine aminopeptidase is used less fre quently than 5'-nucleotidase or y-glutamyl transpeptidase. The last two enzymes help to distinguish between the AP elevation from the liver and the bone. Compared to the AP, these enzymes are not elevated in bone dis orders [181, 182],


phohydrolysis. These were reported in the liv er. bone, brain, kidney and heart [167], Glucose-6-phosphatase is present in the kidney, small intestine, pancreas and other tissues in a wide variety of species. PPi phosphohydrolytic activity in crude homogenates of tissues characterizes all three of these enzymes. Although the molecular mass of AP is similar to that of the inorganic pyrophospha tases. APs are zinc metalloproteins while inorganic pyrophosphatases are not [171], There is a difference in the amino acid se quence around the active site of the molecule of these three enzymes. Thus, these three enzymes, possessing PPi phosphohydrolvtic activities at a neutral and alkaline pH. de spite their overlapping effect are distinct proteins [171]. Further studies trying to separate the phosphohydrolytic activities of organic phosphomonoesters and PPi found that these two were not separable by extraction of tissue homogenates with organic solvents, deter gents. using differential centrifugation, gel fil tration, ion-exchange chromatography, elec trophoresis [167, 172. 173], immunoelectrophoresis or immunodiffusion [ 174], Also, the response to heat at 50-60 °C appeared simi lar in both these hydrolytic activities [175].


Conclusion

Although AP is one of the most thor oughly studied enzymes by basic researchers and clinicians, its metabolism has not yet been fully elucidated. Over many decades the diagnostic value of routine serum AP has been firmly established and it became one of the most useful laboratory tests to assess cholestasis and bone disease. Molecular biol ogy and genetic techniques are rapidly in creasing our understanding of basic mecha nisms involved in the metabolism of AP. There is a good potential for an accurate identification of various subtypes of AP as sociated with specific clinical disease enti ties. If this happens, the isoenzymes of AP may evolve as diagnostic tests to differen tiate between various liver disorders and as a marker of malignancy.

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