Multiple forms of human serum alkaline phosphatase: detection and quantitation

Transcription

1 Review Article Ann cu«biochem 1993; 30: Multiple forms of human serum alkaline phosphatase: detection and quantitation Christopher P Price From the Department of Clinical Biochemistry, London Hospital Medical College, Turner Street, London 1 2AD, UK Additional key phrases: alkaline phosphatase; multiple forms; techniques; serum The measurement of serum alkaline phosphatase (EC ALP) has been popular in clinical practice for over 50 years despite a limited knowledge ofthe structure, function and pathophysiology of this enzyme family. The existence of multiple forms of the enzyme has provided a great deal of opportunity for research and this has helped to refine ideas on the clinical utility of measurement of the enzymes in serum as well as to elucidate their structure and function. 1,2 STRUCTURE, FUNCTION AND MULTIPLE FORMS The alkaline phosphatases (ALP) are non-specific phosphomonoesterases that are capable of hydrolysing a wide range of phosphate monoesters; in vitro studies have shown that the specific activity for each of the isoenzymesshows some substrate dependence.? In fact, the enzymes hydrolyse orthophosphate esters and inorganic pyrophosphate and also exhibit transphosphorylation activity. The pyrophosphatase activity reduces significantly with respect to orthophosphatase activity as the ph increases. The transphosphorylation involves direct transfer of phosphate from substrate to an acceptor alcohol; a range of alcohols can act as acceptors with aminoalcohols such as diethanolamine and 2-amino 2-methyl-1 propanol being amongst those that elicit the highest levels of activity in the presence of a phosphate ester such as p-nitrophenyl phosphate. The reactions catalysed can be summarized as follows: orthophosphatase ROP + H 20 ROH + HOP pyrophosphatase PPi+Hp - 2P i phosphotransferase ROP + R6H ROH + R6p A role for alkaline phosphatase in the transport of specific substances has been implied from its presence at plasma membranes, e.g. hepatocyte canalicular surface, intestinal and kidney brush border. It has also been suggested that the enzyme has a role in hydrolysis ofintracellular metabolites and it has always been assumed, although not yet proven, that it is involved in the hydrolysis of phosphate esters. A deficiency of the enzyme in humans has been reported in the case of the liver/ bone/kidney/isoenzyme and is accompanied by defective skeletal mineralization. Certainly, its presence in many species also suggests involvement in fundamental biological processes.l-" It is thought that there are four gene loci encoding the protein moieties of the enzymes: (a) the liver/bone/kidney locus which determines the so called non-specific ALP, predominantly the isoenzyme found in plasma membrane of hepatocytes and osteoblasts; (b) the intestinal locus which determines the isoenzyme found in the intestinal brush border; (c) the placental locus which determines the isoenzyme found in the placenta from about the twelfth week ofgestation; and (d) the germ cell locus which determines an isoenzyme that is similar but not identical to placental ALP and is found in small amounts in the testis and thymus of healthy individuals. 4 The intestinal, placental and germ cell loci are closely linked and found near the end of the long arm of chromosome 2 whilst the liver/bone/ kidney/locus is found near the end of the short arm of chromosome 1. 5,6 Whilst there are only four true isoenzymes recognized at present, there are many more forms 355

2 356 Price of the enzyme; 7 furthermore the placental isoenzyme, in particular, exhibits considerable polymorphism.s The major forms of the enzyme found in sera from patients with liver and bone disease differ mainly in the amount of sialic acid present and the major enzyme found in the bile contains an additional lipid moiety to the usual liver enzyme." Another form of the enzyme found in serum of patients with obstructive liver disease again contains a lipid moiety which may be present as a membrane vesicle; it is thought to be derived from the biliary canalicular membrane and is often referred to as the biliary fraction (also particulate, high molecular mass, fast liver) The foetal and adult forms of the intestinal ALP differ in several respects but it is not clear whether they are the results of different gene 10ci. 13 Clearly, the number of variants identified will reflect the separation technique employed, as well as extraction and purification techniques. Variants may also reflect the presence of endogenous proteo- and lipolytic agents; in addition, the predominant isoforms in serum may be bound to other proteins which may be a specific antibody to the enzyme;" or another immunoglobulin. IS Several of the genes coding for the mammalian isoenzymes have been cloned and the proteins sequenced, but X-ray diffraction structure data has only been published for the E coli enzyme. There is about homology between the mammalian and E coli enzymes although the core of the three dimensional structure is common to both. Detailed analysis of the primary structures of the enzymes indicates that the liver/bone/ kidney isoenzyme comprises 507 amino acids, the intestinal 509 and the placental and the germ cell 513. The enzymes found in serum are soluble forms and thought to be dimers, being symmetrical with two active sites involving two zinc atoms and a magnesium atom;16they are thought to be released from tetrameric membrane forms by the action of a specific phospholipase. There is evidence to suggest that the soluble forms ofthe enzyme are attached to the membrane by a phosphatidylinositol glycan bridge to the C terminus of the protein. 17 The mammalian enzymes are glycosylated, this being tissue rather than species specific; the level of glycosylation may also be age related. IS In the case of the bone enzyme, differences between the neonatal and adult forms have been suggested'? whilst others have shown differences between the serum enzyme in children and adults." PATHOPHYSIOLOGY AND CLINICAL APPLICATIONS In addition to the unique tissue distribution of the multiple forms of alkaline phosphatase the following observations form the basis oftheclinical application of serum measurements of the enzyme: (a) osteoblastic activity is closely associated with bone ALP activity and is reflected in serum levels of the bone isoform; (b) obstruction to the flow of bile results in the de novo synthesis of ALP at the canalicular face of the hepatocyte and regurgitation of bile constituents into the systemic circulation including ALP; (c) placental tissue ALP levels increase during pregnancy, reflected in the appearance of this isoenzyme in the maternal serum in increasing amounts as term approaches; (d) some tumours express the placental (Regan), germ cell (Nagao) or fetal intestinal (Kasahara) isoenzymes; (e) high levels ofintestinal ALP are found in the lymph; it is thought that the general galactosyl receptors on the hepatocyte are responsible for its rapid sequestration from the circulation. There is a limited amount of information on the half life of ALP isoenzymes in the circulation. Walton et al. calculated the half life of the bone enzyme at I.7 days following a single exponential decay curve.s' In studies where pure enzyme has been administered to healthy volunteers the placental enzyme has shown a half life of 6-7 days. Tumour enzymes have been calculated to have half lives of 8-10 days.1 It is worth noting that significant increases in one form may be seen in the absence of any significant increase in the total alkaline phosphatase activity. The true value of small increases in individual forms will only be apparent when sensitive and specific techniques for the quantitation of individual forms are available. Bone disease It is now over 60 years since it was recognized that an elevated serum alkaline phosphatase was present in patients with bone disease such as rickets and Pagets disease, and that levels correlated with bone growth in children. 1There are several publications that illustrate a broad correlation between hyperphosphatasaemia and the severity of bone disease using either X_ray,22 skeletal scintigraphy/! or bone histology.22,24 However, it has been argued by some that the Ann elin Biochem 1993: 30

3 Multiple forms of human serum alkaline phosphatase 357 serum alkaline phosphatase activity is a relatively insensitive test of metabolic bone disease.> whilst others would claim that, with the aid of bone isoform measurement, both the sensitivity and the specificity is improved, the activity measurement being more sensitive thanimaging techniques and clearly less invasive than histological evaluation.p The elevation of bone alkaline phosphatase in the serum is a reflection of bone rebuilding and this is supported by the higher levels seen in children-" and conditions such as osteomalacia. There is no direct relation with bone resorption and thus levels may be normal in the early stages of osteoporosis although they may be increased as the condition deteriorates and resorption and remodelling co-exist. Some of the largest increases ofserum bone ALP are seen in patients with Pagets disease and in broad terms the increase reflects the extent of the disease. Treatment of the disease with cytotoxicagents, calcitonin or bisphosphonates leads to a decrease in the enzyme activity.27 Elevated levels of serum bone ALP are also present in patients with osteomalacia and rickets, resulting from malnutrition, malabsorption following gastrectomy, chronic liver disease, chronic renal failure, heavy metal poisoning, long-term anticonvulsant therapy or the consequence of resistance to vitamin D.28 However, there are several reports ofosteomalacia findings on histological examination with normal ALP activity, although this may be due to the poor sensitivity of early enzyme methods.p Treatment of osteomalacia with vitamin D or its analogues may initially result in an increased serum ALP activity as a result ofincreased osteoblasticactivity with a subsequent fall as the condition resolves.f The pattern of change in patients with primary hyperparathyroidism is less clear although this may be due to many studies only measuring total enzyme activity. Thus, there are several reports ofpatients with parathyroid disease having total ALP activity only up to twice the upper limit of normal, resolving on removal of the tumour. The increase in serum bone ALP in patients on haemodialysis is now well established and measurement of the enzyme activity is now an accepted test for monitoring the onset of bone disease; delineation of the bone isoform is, however, important as increases in the liver isoform can also occur due to the co-existence of liver disease." Malignancy of the bone can lead to elevations of the serum ALP as a result of a primary bone tumour or due to invasion by metastases. Patients with primary bone tumours may demonstrate variable increases in the serum ALP activity with little apparent relation to tumour burden, with osteosarcoma generally showing elevated levels whilst osteoclastoma often show no abnormality. The serum ALP is not however, usually elevated in multiple myeloma. In the case of metastases, estimation of the serum ALP isoform is regarded as less sensitive than imaging tecbniques," although it may be valuable in monitoring the progress of the disease. Liver disease Increases in serum ALP activity are commonly seen in patients with obstructive liver disease with minimal changes in patients with primarily hepatocellular damage. The presence of the biliary component (referred to earlier) is an important observation in patients with obstructive liver disease;" in broad terms the proportion of this fraction correlates with the severity of obstruction.p The largest increases in liver ALP (including the biliary fraction) are seen in patients with complete biliary obstruction, e.g. carcinoma of the bile duct or head of pancreas, advanced primary liver cancer and primary biliary cirrhosis. The pattern in patients with gallstones is, on the other hand, often transitory with a persistent elevation only occurring when stones lodge in the bile duct. A similar pattern is seen in patients with widespread secondary hepatic metastases; in the early stages of hepatic infiltration an elevation of the liver isoforrn of ALP may be the only abnormal biochemical finding. The changes in the liver alkaline phosphatase levels in other types of liver disease are more variable. In general, there are only small increases in infectious diseases of the liver, often only in the latter stages ofthe condition. Largerincreases are seen in diseases associated with fibrotic or infiltrative conditions, with the highest levels often in chronic active hepatitis and primary biliary cirrhosis. Increases may also be seen as a consequence of a reaction to drug therapy. Overall, the relative increases in liver related ALP isoforms in serum have little differential diagnostic value Increasesin the intestinal ALP isoenzyme are also often seen in patients with chronic liver disease, especially cirrhosis. Patients with chronic liver disease may also develop a secondary metabolic bone disease with elevation ofthe bone isoform in serum; however, the increasewill only be recognized with the more discriminating methods of separation. Malignancy Some tumours may give rise to the appearance of placental, germ cell or intestinal isoenzymes

4 358 Price of alkaline phosphatase in the serum, the levels overall reflecting the tumour burden. 18 Increased expression of placental ALP is particularly associated with ovarian cancer or testicular seminoma; other tumours show a much lower level of expression." However, heavy smokers may have much higher concentrations ofplacental ALP in the circulation than non-smokers and this may give rise to 'false positives'. An increased expression of fetal intestinal ALP (Kasahara isoenzyme) is especially common in hepatoma. As mentioned earlier, metastatic disease may also result in secondary elevation of the liver or bone isoforms. Miscellaneous conditions A wide range of clinical disorders may also give rise to an increase in the serum alkaline phosphatase activity without, in some instances, any clinical signs of involvement of the organ. Thus patients with rheumatoid arthritis may demonstrate increases in ALP, which are reported to be of hepatic origin,34 whilst patients with ankylosing spondylitis may show elevations of the bone enzyme; however, the literature illustrates a wide variation in experience. The most common elevation of serum ALP seen in patients with renal disease is due to the bone isoform although some patients on chronic haemodialysis may develop cirrhosis leading to an increase in the liver form. Renal tubular acidosis and other tubular disorders are also associated with an increase in the serum enzyme, although the tissue source of the enzyme is not clear. It is rare to see increases in the kidney enzyme in serum although Rosalki has reported such a finding in a patient who had taken an overdose of colchicine." A normal finding in pregnant women is the appearance of placental enzyme, rising to a peak at weeks gestation." Some women may develop cholestasis during pregnancy leading to increases in the liver and biliary enzymes. Disorders of the endocrine systems may be associated with increases in serum ALP. Thus patients with hyperthyroidism often show increases in ALP with increases in liver and bone ALP although the latter is more frequently and more markedly elevated." Diabetics may also show an increase of the serum ALP, most likely to be hepatic in origin.l" Several benign forms ofhyperphosphatasaernia have been recognized in the absence of disease. Some of these are familial: both autosomal dominant and other, more complex forms of inheritance have been reported and various isoforms may be elevated. 39,40 Non-familial forms ofhyperphosphatasaemia may, in some cases, be due to the formation of a complex with an immunoglobulin, commonly G or A. Transient increases in the serum alkaline phosphatase may be observed in infants and children with changes often more than ten times the upper limit of normal. Increases in both the liver and the bone isoenzyme have been seen although the mechanism of the increase is not known. The changes may reflect a sudden stimulus to synthesis or a reductionin the removal of the enzyme." The intestinalisoenzyme is increased in two rare clinical conditions, a-chain disease or small intestinal lymphoma and graft versus host disease of the small intestine. There is a rare inborn error of metabolism associated with a low serum alkaline phosphatase and characterized by abnormal skeletal mineralization; high levels of phosphoethanolamine are also observed in the serum and urine. The expression of the liver/bone/kidney isoenzyme is defective'! although that of intestinal and placental isoenzymes appear normal. The demonstration of a gene defect in hypophosphatasaemia has added support to the idea that alkaline phosphatase acts in skeletal mineralization.p Clinical requirements The main clinical interest centres on the elevation of serum alkaline phosphatase in patients with diseases involving the liver or bone although it should be recognized that a placental or intestinal-like enzyme may be found in certain malignant conditions. The main clinical questions are concerned with: (a) detecting the presence of liver or bone disease; (b) determining the origin ofan unexpected elevation of serum ALP and; (c) monitoring changes in the activity of the disease (particularly in the case of metabolic bone disease). Thus, the major requirement is for methods that provide accurate and precise quantitation of the bone, placental and intestinal isoenzymes together with a separation technique that will enable semi-quantitative discrimination between the bone and liver forms of the enzyme as well as detecting unusual variants. There appears to be little clinical value in having a sensitive and specific quantitative method for the liver isoform.

5 Multiple forms of human serum alkaline phosphatase 359 METHODOLOGY There are a wide range of techniques available for the characterization and quantitation of the multiple forms of alkaline phosphatasein serum. The techniques depend on the physicochemical properties, the nature of the catalytic site or the protein structure of the different isoforms and any post-translational modifications. Quantitation of the catalytic activity may be undertaken using a range of substrates and buffers; however, the quantitative methods based on inhibition or inactivation almost exclusively employ the p nitrophenyl phosphate substrate with either 2-amino-2-methyl-l propanol or diethanolamine as trans phosphorylating buffers; although there may be some differences in sensitivityto substrate' for the different isoenzymes this is generally ignored, as it applies primarily to the intestinal isoenzyme. The electrophoretic techniques commonly use either a naphthyl or indoxyl phosphate substrate with a secondary visualization reagent as appropriate. 44,4~ Electrophoresis The most common approach to the identification of the alkaline phosphatase depends on an electrophoretic separation. Early methods included a paper 46 or starch gel 47 support; however, either cellulose acetate" agarose'? or polyacrylamide gel SO are now favoured. None of these techniques alone, however, are capable of producing a distinct separation of the most important isoforms, namely liver and bone, to enable accurate quantitation by densitometry. In the case of paper, agarose and cellulose acetate supports, the main liver isoform migrates as a compact band in the (lzglobulin region. The bone isoform is to be found immediately behind the liver band and is characterized by a more diffuse staining. The proximity of the bands mean that it will be difficult to recognize a small increase in one fraction in the presence of a large excess of the other and discriminatory quantitation by densitometry is not reliable. The mobility of the liver and bone isoforms in molecular sieving supports such as starch and polyacrylamide gels will depend, in part, on the concentration of the gels. However, the close proximity of the liver and bone fractions will still be seen even with these gel supports. In patients with obstructive liver disease a second liver related isoform appears in the serum (referred to earlier as the biliary fraction). This isoform migrates ahead of the main liver band when using the majority of paper, cellulose acetate and agarose supports.'" however, in the case of sieving supports such as starch and polyacrylamide, this isoform is retained at the origin. The adult intestinal isoenzyme demonstrates a slower mobility than the liver and bone isoforms in the case of all supports and yet the foetal isoenzyme has a faster mobility than the adult isoenzyme" illustrating that there are differences between the two. The migration of the placental isoenzyme is more variable with different supports but commonly appears as a discrete band overlaying the diffuse bone fraction. The placental fraction may show significant microheterogeneity in starch and polyacrylamide gel electrophoresis systems.i The electrophoretic mobility of the bone isoform may be retarded to improve its resolution from the liver fraction. Moss and Edwards" reported improved resolution of liver and bone fractions by brief preincubation of the sample with neuraminidase enabling densitometric scanning for quantitation; prolonged preincubation, however, limited the discrimination between the two bands. Rosalki and Foo~3 described the use of wheat germ lectin to retard the bone isoform mobility and, in this way, were able to achieve quantitation of both fractions even when only one component was present in small amounts. Both of these procedures depend on differences in the sialic acid residues of the liver and bone fractions. The lectin method demonstrated good precision, with between batch coefficients of variation of less than SOJa for both liver and bone components. Kuwana et al. ~4 similarly found good precision with between batch coefficients of variation of 4' 9 and 1 7% for liver (mean 38 lull) and bone (mean 112lUlL), respectively. These authors noted that the intestinal fraction coincided with the retarded bone fraction and thus, for accurate quantitation, the intestinal fraction had to be inhibited or, alternatively, removed by immunoprecipitation. In a comparison between lectin affinity and neuraminidase modification of electrophoretic mobility Muira et al. 2O found that the latter method appeared to give a higher estimate of the bone fraction; these authors also noted differences between the neonatal and adult forms of the bone enzyme. Examples of the separation that can be achieved with an agarose support using lectin or neuraminidase retardation are shown in Fig. 1.

6 360 Price lal lbl (c I of electrophoretic techniques is to identify whether an increase in the total ALP is of hepatic or osseous origin, the technical merits of IEF methods do not significantly improve the clinical benefits FIGURE 1. Agaf'tJMgel electrophoresis ofserum alkaline phosphatase isoforms showing diffenntiation betwffn mqjor liver and bone fractions. (a) Effect of tfwitment with neuraminidase; (b) and (c) effect of ntardation by wheat germ lectin. (a) Track I = patient with obstructive liver disease; track 2 = patient with PagelSdisease; track 3 = mixtun (I.' I) ofsqmp/es from tracks I and 2; track <4 = SQmplefrom track 3 after incubotion with neuraminidasefor 30 min at 37 C. (b) Aglll'OSe gel (30 gil).' track I = patient with liver disease showing prominent intestinal bond; track 2 = patient with liver disease; track 3 = patient with PagelSdisease; track <4 = mixtun ofsqmples from tracks 2 and3. (c) With agaf'tjm gel incorporating lectin (20 mgll) tracks as in (b). The electrophoresis was run in each case for 25 min at 150 Y and activity stained with 5-brom0-4 chloro-3-indolyl phosphate. IsoeIectrk 'oculln. It is possible to improve the resolution ofelectrophoretic techniques by employing isoelectric focusing (IEF). The first published methods employed a free solution approach but subsequently IEF has been widely used with polyacrylamide,55 agarose and cellulose acetate supports.56 The superior resolving power of IEF has meant that micro heterogeneity has been demonstrated within most ofthe major isoenzyme bands; in this respect polyacrylamide gel IEF will result in more bands than cellulose acetate. Thus Cocco et al. 56 demonstrated two liver bands with cellulose acetate, compared with only one for conventional electrophoresis; these authors also found three distinct bone bands. Griffiths and Black" on the other hand have identified 12 zones of ALP activity on isoelectric focusing of sera from healthy adults, with five bands predominating. Sinha" in a similar study found ten bands in the sera of healthy adults. Unfortunately, the large number of bands appears to confuse, rather than clarify, the position regarding the clinical significance of changes in enzyme patterns and despite the enhanced resolving power of IEF, the clinical significance of the individual bands associated with any tissue fraction has not been clearly established. In so far as the major value Arnnlty precipitation Wheat germ lectin binds to both N-acetylglucosamine and sialic acid residues and relative affinities for individual proteins will depend on the relative amount and accessibility ofeither or both of these sugars; this phenomenon has been investigated as a means of differentiating bone from the liver, intestinal and placental isoenzymes. Rosalki and FOO53 first reponed this approach showing that SO'l. of the bone fraction was precipitated, the liver appearing to be mildly 'activated' (by 10'7.) whilst intestinal and placental components were not affected. The authors pointed out that the biliary fraction had to be dissociated by treatment with Triton XlOO otherwise this enzyme form was partially precipitated (on average 23'1.). The authors obtained within and between batch coefficients of variation for the bone fraction of 3'2and 6 9'l., respectively (mean 67 lull) and a good correlation with a sequential heat inactivation procedure (r = O'98, slope=o 97). Certainly, the technique does provide a precise means ofquantitating the bone fraction and is relatively easy to use; however, personal experience would indicate that the quality of the lectin preparation has to be carefully controlled to ensure long term reproducible performance. Behr and Bamerr" subsequently reponed a modified lectin precipitation method in which they claimed complete precipitation of the bone isoform. These authors used cord blood as a source ofbone enzymeobtaining a 99'1. extraction of the enzyme activity. However, it was interesting to note that one batch of lectin yielded only a 72'1. extraction, and the authors suggested that incomplete extraction of bone phosphatase by the lectin may have been due to a poor batch oflectin, a point also raised by Desoize et al. 6D An alternative view was that the discrepancy was the result of the bone fraction being validated by a differential heat inactivation technique which did not give an accurate assessment of the bone enzyme. Chemical denaturation It has been shown in several reports that the bone enzyme is more susceptible to denaturation by urea compared with the liver, intestinal and A"" eli" Biocllem 1993: 3t

7 Multiple forms of human serum alkaline phosphatase 361 placental forms; the inactivation is time, temperature and urea concentration dependent and is irreversible, being described by first order reaction kinetics. Bahr and Wilkinson'" and Birkett et al. 62 were two of the first groups to report on inactivation by urea as a means of differentiating the bone and liver forms, and many more reports have subsequently appeared employing a range of conditions. The wide variety of reaction conditions, particularly with respect to incubation times and urea concentrations has contributed to there being poor agreement between results, particularly with respect to discrimination where one or both of the isoforms is within normal limits. However, the situation has been improved by the use of continuous reaction monitoring techniques, probably because of the closer control of reaction temperature and incubation times and the improved photometric precision, although some authors have expressed concern about the influence of variations in inactivation half lives between different samples. 66,67 Brown and Lewis 66 attempted to overcome the variation in inactivationhalf lifeby usingcontinuous reaction monitoring through to complete denaturation. The individual isoform levels were then calculated from the constituent exponential components. They employed this approach analysing the decay curve for the longer half-life component (the liver isoenzyme) in the first case and then by subtraction of the shorter half-life component. Forsman and O'Brien'" pointed out that a simple solution was to plot the natural logarithm ofactivity against time until the most labile fraction had completely disappeared with a least squares regression of the remaining decay curve being subtracted from the total to give the proportion of each. Analysis of radionucleide decay would suggest that the strategy adopted by Forsman and O'Brien 67 is valid if the decay constants differ by a factor of four, and in fact the ratio is about four. However, as pointed out earlier the rates of decay are time, temperature and urea concentration dependent and these variables must be strictly controlled to ensure consistent performance. Several of the early methods showed poor discrimination and poor precision when the total activity was normal or only slightly elevated and in several cases could not discriminate a small increase, or normal level of one fraction, in the presence of an excess of the other, particularly in the case of an elevated liver fraction. Bergstrom and Lefvert 68 have also pointed out that, in some procedures, the combination of the analytical errors in each of the activity measurements may produce large errors in the final result, a point also made forcibly by Tilyer. 69 Gerhardt et al. 7o also showed good agreement between estimates of liver and bone fractions and the clinical diagnosis. However, these authors did not explore the clinical sensitivity of the test, limiting their study to patients with established liver and bone disease. Thus, in several papers it has not been possible to assess the precision of the assays and ascertain whether the procedure would have any value in detecting mild changes in the bone or liver fraction. Forsman and O'Brien'" obtained a good correlation of results with a high-performance liquid chromatography (HPLC) technique and further claimed their method could be used to determine liver and bone isoform when the total activity was normal. However, these authors did not report precision or recovery data for small amounts of the liver and bone fraction. More recently, Shephard and Peake?' have proposed the use of guanidine hydrochloride as an alternative to urea, on the basis that it is a more potent inactivator and requires a shorter incubation period. At a concentration of 0'3 M and preincubating for 170s with a 60 s read time, of the bone isoform was inactivated whilst only 53% of the liver fraction was lost. More pronounced differences were seen between the placental and intestinal isoenzymes with the former being activated; this necessitated a four parameter algorithm to quantitate the fractions accurately. Shephard et al. subsequently showed'? that the combination of guanidine hydrochloride inactivation, heat inactivation at 60 C (see later) and L-phenylalanine inhibition enabled all four isoforms to be determined within an accuracy of ± 3% with coefficients of variation of less than 5% for 50:50 mixtures, albeit at elevated levels. A clinical study showed agreement with the clinical diagnosis in of the patients, with 10% of the results leading to a new diagnosis, the remainder not being resolved. Fitzpatrick and Pardue have recently studied the kinetic behaviour of inhibition reactions in detail and reported their experience for two, three and four component models using both p-nitrophenyl phosphate" and methylumbelliferyl phosphate'" as substrates. They found that the choice of conditions for buffers, inhibitors and substrate in order to discriminate between liver and bone forms were different, thus again stressing the importance of close attention to optimized reaction conditions to ensure continuing reproducible performance.

8 362 Price Heat inactivation Variations in the thermostability of the various isoforms have also been recognized for many years and have formed the basis of a wide range ofmethods. In broad terms the placental enzyme is stable, it being possible to inactivate all of the other tissue forms completely by heating at 65 C for 10 min." At the other extreme the bone isoform is very labile, being almost completely destroyed by heating for 10min at 56 C. The liver and intestinal isoenzymes demonstrate intermediate levels ofstability. There is some data to suggest that liver and biliary fractions in serum exhibit different thermal stability although this is not taken into account in the heat inactivation techniques. Some early methods for differentiating the liver and bone forms employed a single incubation mixture for a fixed interval (usually 10 min) at 56 C. The methods required careful control of the temperature, the use of reproducibly thin walled glass tubes which were placed in an ice bath at the end of the incubation period and a constant sample volume to ensure reproducible heat transfer. Naik et al. 25 used this approach to quantitate the bone fraction in patients with chronic renal failure on haemodialysis and achieved reasonable levels of precision. Stepan et al." found agreement with a similar technique and electrophoretic separations of the isoforms. An alternative approach was used by Petitclerc?? who employed continuous monitoring of heat inactivation to quantitate placental, intestinal and liver isoenzymes using a continuous flow autoanalyser. The bone fraction could not be determined by this method; however, the author claimed that the method could be used to quantitate an isoenzyme contributing as little as of a mixture. The rate of inactivation of alkaline phosphatase at 56 C was described by Whitby and Moss'" as having two phases: the first exponential decay having an average half-life of 112s and the second having a half-life of 456 s. The more rapid decay reflected denaturation ofthe bone isoform, the slower being due to the liver and intestinal forms, with a wide degree ofvariation on these estimates. After taking into account method variability, Whitby and Moss found the true variability of the half-lives to be ± 16 3 and ± 86'6 s (SD) for bone and liver isoforms, respectively. These observations invalidate the use of a single inactivation period and use ofa simple simultaneous equation calculation of the type described by Gerhardt et al. 64 and Statland et al. 65 Moss and Whitby79 subsequently described a simplified approach to following the decay curve, monitoring the progress of the denaturation at a time corresponding to the decay of the more stable liver fraction. It was found to be important to choose a monitoring period such that all bone activity had disappeared; however, excessive delay led to poorer precision as the residual activity was small. Moss and Whitby chose to measure the residual activity after 15and 25 min incubation at 56 C. This method has been shown to provide good discrimination between the liver and bone enzyme although accurate quantitation of the bone enzyme to monitor disease is not feasible. Samuelson and Moss8D reported ethanol precipitation as a means of differentiating between bone and liver enzymes and showed that it gave similar information to heat inactivation. The precipitation technique was less critically dependent on temperature or time but was less precise probably due to errors associated with separation and resolution of the precipitate. At this stage, therefore, it is not possible to endorse inactivation techniques for the estimation of small increases in liver and bone isoforms although delineation of the cause of the major increase is good. Furthermore, the imprecision of fully automated procedures is unlikely to meet the requirements for monitoring elevated levelsof one of the isoforms (e.g., bone enzyme in patients on haemodialysis with metabolic bone disease) particularly when there are only small increases (e.g., %). Chemical inhibition There are many reports of the inhibition of intestinal and placental alkaline phosphatase by L phenylalanine at a concentration of 5-10 mm,81 the inhibition being approximately 80%, whilst there is little effect on the liver or bone enzymes. The inhibition is non-competitive and there is evidence to suggest that it is due to prevention of dephosphorylation of the enzyme. In contrast to the inactivation procedures, the inhibition is not time or temperature dependent and has proved to be a robust technique. The use of L phenylalanine has been combined with heat, urea and guanidine inactivation as mentioned earlier in a variety of three (or four) parameter protocols to evaluate liver, bone intestinal (and placental) isoenzymes.65,66,72 It has been shown that other amino acids, such as histidine, lysine and glutamic acid, may also influence alkaline phosphatase activities to differing

9 Multiple forms of human serum alkaline phosphatase 363,.., /00 (0 ) 100.;;; n o e; ~ o,.., :E ~ :~ '0..- u Cl> 0 C' 40 ~ 0 40 c :;:::: Cl> C u... ~ '0 20'0 0'0 1'0 2'0 3'0 At 56 C (min) Urea in cuvette (mrnol/l) 100 (c) 100 (d) ~ s;,.., ~ 60,..,.,...-., :;:::: :;:::: u u a 40 :;:: 0 :;:::: c H 'c O+--r-""""T-.--""T"""--.---,--r--r , O r---r--.,----r--r-""""T , 0 0 2'0 4'0 6'0 8' B O 10 0 L-phenylaline in cuvette (rnrnol/l) L-p- bromotetramisole in cuvette (mmol/ll FIGURE 2. Illustration ofthe influence ofdenaturation and inhibition effectson liver (.. ) bone (.) and intestinal (e) alkaline phosphatases: (a) heating at 56 Cprior to measurement; (b) in the presence ofurea; (c) in the presence ofis-phenylalanine; and (d) in the presence ofl-p-bromotetramisole. degrees. L-homoarginine, at a concentration of 10mM, is a particularly powerful inhibitor of the bone and liver enzymes whilst the intestinal and placental enzymes are less affected. 82 Van Belle 83 showed that the drug levamisole is a strong competitive inhibitor of the liver, bone and kidney alkaline phosphatases whilst having little effect on the intestinal or placental form. The same author also showed that another similar compound L-p-bromotetramisole can also be used to inhibit the liver/bone/kidney isoenzyme.p' The inhibition by levamisole or L-p-bromotetramisole (althoughnotcomplete) has proved to be a useful property in attempting to identify abnormal phosphatase activity bands after electrophoresis whilst little use has been made of the less discriminating influence of L-homoarginine. An illustration of the sensitivity of the major isoforms to heat, urea, L-phenylalanine and L p-bromotetramisole is shown in Fig. 2. Chromatography There has recently been increasing interest in the use of high performance liquid chromatographic techniques for separation and quantitation of the serum ALP isoforms. Schoenau et al. 85 described the separation of ALP isoforms using a strong anion exchange column (Mono Q HR 5/5) with stepwise elution using lithium chloride. These authors found two heat sensitive fractions in a bone tissue extract and in serum from a growing child, although only one of the fractions was found in a sample from a patient with rickets. Similarly, two bands were found in a liver extract and serum from a patient with cholestasis, together with the high molecular weight biliary Ann Ctin Biochem 1993: 30

10 364 Price fraction commonly seen in patients with cholestasis. However, Bielby and Chin 86 were unable to reproduce these results. A weak anion exchange column (Synchropak AX 3(0) with in-line detection ofenzyme activity and elution with an increasing sodium acetate gradient was employed by Parviainen et al. 87 These authors again found two fractions derived from bone and two from liver in addition to the biliary fraction, and, furthermore, noted that one ofthe bone fractions was preferentially elevated in osteomalacia, osteoporosis and bone metastases and particularly in Pagets disease. In addition, they were able to resolve the various enzyme fractions in normal subjects suggesting that this method could be used to study the significance of minor changes in individual fractions. Severini et al. 88 also employing ion exchange chromatography with a DEAE column eluting with an increasing gradient of KCI, were able to identify two liver, one bone and one intestinal fraction in normal serum; the biliary fraction was also separated in cases of patients with liver disease. The authors claimed a detection limit of 2 lull, the lowest found in any published paper to date; between day coefficients of variation less than 8070 were reported. The affinity of the bone isoform for wheat germ lectin has also been employed in a low performance affinity chromatography technique by Gonchoroff et al. 89 with the lectin coupled to Sepharose 4B and elution with N-acetylglucosamine; however, the method could not completely resolve the liver and bone isoforms. Anderson et al. 90 subsequently developed a high performance affinity chromatography technique with the lectin coupled to silica particles, but were also unable to completely resolve the liver and bone fractions with calculations of true activity being achieved with the aid of correction factors. An exponential gradient ofn-acetylglucosamine produced one single peak each for liver and bone enzymes but a shallow stepwise gradient resolved the liver and bone enzymes into three fractions of each, although there was one predominant peak in each case. The authors noted a positive bias for liver enzyme in the presence of a large amount of bone enzyme, but no bias when the proportions were reversed. The correlation of bone enzyme with urea inactivation studies was also found to be poor at low levels of activity which may reflect differences in the bone peaks. It is unfortunate that the ability to resolve small increases in the bone enzyme is not possible despite claims of a low detection limit, as this would have improved the clinical utility of the method. The high molecular weight form of the liver enzyme has been clearly recognized in electrophoretic studies and may interfere in certain lectin precipitation procedures. However, it is readily resolved from the other fractions in most of the chromatographic techniques. Indeed, ion exchange chromatography?' and gel filtration chromatography92 have both been used specifically to identify and quantitate this isoform of the enzyme. Immunoassay The first documented production of an antibody against alkaline phosphatase was in 1954 and was raised against the dog intestinal enzyme." Although the antibody was shown to be specific for the intestinal isoenzyme, subsequent work by the same author" demonstrated that an antibody raised against the bone isoform cross reacted with the liver enzyme. Subsequently, several groups have demonstrated that liver, bone and kidney enzymes are antigenically very similar whilst intestinal and placental enzymes are quite distinct. The intestinal and placental isoenzymes show some structural similarities and some early polyclonal antisera demonstrated significant cross reactivity." Whilst there have been some reports of polyclonal antisera that are claimed to be specific for either the intestinal or placental isoenzymes, the achievement of specificity has only been successfully accomplished with the selection of monoclonal antibodies. Indeed, the allelic forms of placental ALP have been distinguished with monoclonal antibodies, having been indistinguishable with polyclonal antisera. 96 Several reports of assays for the placental isoenzyme using monoclonal antibodies have now been published.p'?" In an attempt to differentiate between fetal and adult intestinal ALP, Bailyes et al. 98 used meconiumas a source ofantigen, producing seven monoclonal antibodies, two of which were specific for the intestinal isoenzyme the remainder cross reacting with the placental isoenzyme; the antibodies specific for the intestinal enzyme reacted with both adult and fetal forms although differences in binding were noted. These authors used one of the antibodies to develop a capture assay for intestinal ALP. Vockley et al. were also unable to fully differentiate between adult and fetal isoenzymes using monoclonal antibodies" although again differences in relative binding affinities were noted. Several groups have attempted to differentiate between liver and bone forms using polyclonal antibodies without success, which is perhaps not

11 Multiple forms of human serum alkaline phosphatase 365 surprising bearing in mind their close structural similarities. 100 However. Sussman'?' prepared an antiserum using liver ALP as antigen which was claimed not to cross react with the bone enzyme; in a study of samples from patients with various clinical disorders this author claimed that the elevation of ALP in Pagets disease was hepatic in origin. a finding at odds with the vast literature on this subject. Singh and Tsang HJ2 also reported the preparationof a polyclonal antiserumspecific for the bone enzyme using a cultured cell line as antigen; however. there have been no subsequent reports on the use of this reagent. There have been several reports of the production of monoclonal antibodies to the bone and liver enzyme. Andrews et a/. I03 raised monoclonal antibodies to the liver enzyme but demonstrated cross reactivity to the bone enzyme. Meyer et al. 104 also demonstrated antibodies with some specificity for the bone enzyme, and Lawson et al. 105 found an antibody with a threefold preference for the liver enzyme with incubation at room temperature which increased to fivefold after a 20 h incubation at 30 DC. Bailyes et al. employed both purified bone and liver enzyme as antigens and generated 27 antibody producing cell lines; 106 several of the antibodies showed at least twofold preference for the bone enzyme but no antibodies with a preference for the liver enzyme were found. Epitope mapping produced a total of six major epitope groups. None of the monoclonal antibodies bound the high molecular weight biliary enzyme found in sera from patients with obstructive liver disease; however, binding did occur after treatment with detergent. In a further evaluation of these monoclonal antibodies Seabrook et al. 107 used two antibodies, one showing a twofold preference for the bone enzyme and the other showing no preference, to establish an assay based on knowledge of the proportions of the bone and liver enzyme in a serum standard. Deng and Parsons.P' interestingly have raised a monoclonal antibody to the high molecular weight form of the enzyme found in serum that does not react with liver. bone. intestinal or placental ALP or the ALP found in bile. A more recent paper by Hill and Wolfert l O9 has reported the use of bone alkaline phosphatase from a human osteosarcoma cell line (SAOS-2) as immunogen, producing monoclonal antibodies that react preferentially with the bone enzyme, one antibody showing less than 3070 cross reactivity with the liver form. In a total of clones screened from 19 fusions, 354 were found to be positive for the bone enzyme and five showed differences in response between bone and liver enzyme using an antibody screening format. When one of these antibodies was used in an RIA assay format 6 ng of bone enzyme from a crude bone extract was required to displace 50% ofthe radiolabelled antigen whilst 200 ng of liver enzyme was required to achieve a 50% displacement; the remaining four antibodies had cross reactivities of between 4 and 8'5%. The most recent claim for a monoclonal antibody that is specific for the bone isoform has been made by Masuhara et a/yo These authors produced a monoclonal antibody that demonstrated excellent specificity toward bone ALP from a human osteosarcoma cell line, using purified rat bone ALP as an immunogen. However, the antibody did not react with bone ALP in serum from patients with Pagets disease. REFERENCE VALVES The intraindividual variability of the total as well as liver and bone isoforms of alkaline phosphatase is low in adults especially when compared with the inter individual variability although it is higher in children; the intra individual variability in adults is quoted as 6'5%.1 There is considered to be no diurnal rhythm in phosphatase activity and neither mild exercise nor prolonged bed rest has any marked effect. I The serum total alkaline phosphatase does show small increases after a fatty meal predominantly due to an increase in the intestinal isoenzyme. However, there are no major influences by other dietary components (with the obvious exception of vitamin D) although deficiencies of protein intake. magnesium and zinc may be associated with low levels of activity in serum. I The age relationship for serum total ALP reference ranges are well known, the major determining factor being the association between bone growth, osteoblastic activity and the appearance of bone ALP in the circulation. The bone isoform demonstrates a maximum level of activity in the first 6 months of life falling to a short plateau period between 2 and 9 years rising to a variable degree around the time of puberty before falling to adult levels about the age of years.iil-114 There is some evidence to suggest that levels are higher in young males althoughthe peak may be reached earlier in girls. Although several authors have recognized more than one form of the bone enzyme, there is no evidenceto suggest that delineation of the different forms has any clinical significance. The plateau of Ann C/in Biochem 1993: 30

12 366 Price the bone enzyme in adult life may, on careful study, show a trough around the age of 40 years with minor increases seen in later life.!" There is no good evidence to suggest any significant differences between levels in men and women although there are reports of significant increases in post-menopausal women, thought to reflect increased bone IOSS.1I6 In addition to an appearance of the placental enzyme in pregnancy, an increase of the bone enzyme in pregnancy has also been noted presumably reflecting increased bone turnover."? The changes in the level of bone isoform in serum throughout life are illustrated in Fig. 3. In the case of the liver isoform there is little or no enzyme present in the plasma in the first few months of life lu rising to a level at about 6 months that remains fairly constant thereafter. Similarly, there is little change in the intestinal enzyme. The intestinal isoenzyme activity in serum is related to diet and blood group status with lower levels seen with groups A and AD.liS A summary of data on reference ranges is given in Table I, illustrating the reference ranges for different techniques and giving the ranges for total activity to allow some means of comparison. The distribution of the activity of each of the isoforms in serum is skewed as is that of the total activity measurements Yeors FIGURE 3. Illustration of the trends in the reference rangesforserum bone alkalinephosphataseactivity with age. The data is expressed as a proportion ofthe upper reference limit for bone alkaline phosphatase activity over the period 3D-50 years to take account of methodological variations and is gleaned from several references. including J. 54. II J- II 5. CALIBRATION OF ASSAYS AND QUALITY ASSURANCE Purified preparations of all of the major tissue forms of alkaline phosphatase have been reported for use either as immunogens or to determine 70 TABLE I. Quoted reference ranges for main alkaline phosphatase isoenzymes in serum for adults: (a) quoted as mean ±2SD; (b) quoted as range of results; (c) '1 range Total Bone Liver Intestinal Author Reference Gender (lull) (lull) (lull) (lull) Method Rosalki and Foo (a) 53 M Lectin F electrophoresis M Lectin F precipitation Behr and Barnet (a) 59 M Lectin F precipitation Brown and Lewis (a) 66 M+F Urea Statland et 01. (a) 65 M+F Urea Shephard et 01. (b) 72 M Guanidine F Parviainen et 01. (a) 87 M+F HPLC Anderson et 01. (a) 90 M+F Urea Schiell (a) 115 M Heat Kuwana et 01. (c) 54 M Lectin F Bailyes et 01. (b) 98 M+F '5-14 Immuno Hirano et 01. (a) 119 M+F Immuno HPLC = High-performance liquid chromatography. Ann Clin Biochem t993: 30

13 Multiple forms ofhuman serum alkaline phosphatase 367 inactivation/inhibition characteristics or mobility; they have also been used for the preparation of calibration material. Specific activities reported have ranged from five to 1357 when recalculated as activity in micromols per minute at 30 C per milligram of protein. l,9s The majority of purification protocols have included the use of a detergent or solvent to solubilizethe enzyme and, in the case of the major enzyme fractions, this results in a preparation with electrophoretic mobilities identical to those found in serum. Although it might be expected that purificationof enzyme from tissue or serum might alter mobility or inhibition characteristics, there is little data to support this expectation. Decay constants have been produced using either tissue homogenates or sera containing predominantly the fraction of interest, validated by an alternative technique (e.g., electrophoresis or HPLC). In the case of electrophoretic techniques more bands are seen in tissue extracts than sera as a generalobservation, presumably reflecting variations in purification protocols. However, there is some evidence to suggest that enzyme prepared from tissue may not be suitable for lectin based assays, or immunoassays, particularly if a surfactant has been used in the processing of the tissue. Thus whilst tissue extracts diluted in heat inactivated serum may provide a reasonable serum based calibration and control material for inactivation/inhibition techniques, they may not be suitable for electrophoretic,lectinaffinity and immunoassay methods. In the future, enzymes obtained from specific cell lines will no doubt prove to be a valuable source of enzyme for these purposes. 120 There are no recognized external quality assurance programmes available for monitoring the detection of multiple forms of ALP. Furthermore, there are no quality control materials available that are widely applicable to the various enzyme forms and the range of techniques available. Thus, the clinical biochemist must depend on control serum provided for use with specific methods (e.g., immunoassay for placental ALP), aliquots of pooled serum stored at - 70 C or tissue extracts that are diluted in heat inactivated serum for qualitycontrol purposes. The long-termstability may be enhanced by freeze drying of such hybrid materials although this may lead to variable recoveries and possible matrix effects. SAMPLE REQUIREMENTS The majority of studies involving the detection of different forms of ALP have been undertaken l 120 <, 2 ~ 100 ~ o 80 ~ >...g Ul c s: : m;: Ul ~ o s: ~ ~ <r 20 ~ ,----, ~ ~ ( o l (b) (c) FIGURE 4. Data to illustrate the stability ofbone (.) and liver (e) alkaline phosphatase in serum: (a) fresh sample; (b) after 72 h at 4 C; and (c) after five cycles offreezing and thawing. Data were generated using a lectin precipitation method and with eight samples shown by electrophoresisto contain predominantly bone or liver isoforms. on serum samples, although in the case of total activity measurement an heparinized plasma sample is satisfactory. A plasma sample is probably also adequate for many of the physical separation techniques and methods based on heat or chemical denaturation. However, serum is the preferred sample for affinity and immunological methods due to the problems associatedwith matrix effects. The majority of data on the stability of ALP in serum refers to the assessment of total activity and indicates that lessthan 100/0 of activity is lost over 3 days at 4 C or 3 months at - 20 C. It is preferable that a sample is stored at - 20 C prior to analysisalthough all of the major forms of ALP show reasonable stability at 4 C. The stable placental isoenzyme will retain all ofits catalytic and immunological activity at 4 C for at least a week whilstthe more labile bone isoform will retain activity for at least 3 days at 4 C (Fig. 4). Repeated freezing and thawing of the sample should be avoided and, in particular, may lead to a distortion ofthe electrophoretic pattern ofalp fraction in patients with liver disease reflecting modification of the lipid containing biliary fraction. CONCLUSION The existence of a major text devoted to a single enzyme family, containing many hundreds of

14 368 Price references bears witness to the amount of research activity in this area,' and the vast literature on the characterization and quantitation of different forms of the enzyme in serum may be confusing to the reader. However, there are now assays for individual enzyme forms that are beginning to show distinct clinical benefits. Incertain cases it willbe necessary to account for an unexplained elevation of the serum alkaline phosphatase and this can be achieved with the aid of an electrophoretic separation. A choice of a nonsieving support, such as cellulose or agarose, furthermore, enables the recognition of the high molecular weight fraction (the biliary fraction) and any enzyme immunoglobulin complexes. Immunoassay is undoubtedly the one singletechnique that will ultimately provide the sensitivityand specificity required for quantitative measurements in clinical practice. This is now readily achievable for the intestinal and placental enzymes and may soon be possible on a routine basis for the bone isoform although a full validation of such an assay has yet to be published. In the past, most clinicianshave mainly been concerned with determining the origin of an unexpected elevation of the total alkaline phosphatase in serum. However, it is now becoming increasingly obvious that minor changes in the bone isoform may provide a sensitiveindicator of increased bone remodelling. Thus, in the context of bone disease, the specifications for the assay of the future are that an assay is specific for the bone fraction and capable of detecting changes of less than or equal to 5 lull. At present, the only techniques that have the potential for meeting this specification are lectin precipitation with quantitation of supernatant activity, lectin or neuraminidase modified electrophoretic techniques with densitometry, HPLC, and immunoassay; of these the lectin precipitation, lectin or neuraminidase mediated electrophoresis and immunoassay techniques are the most practical for use in the routine laboratory. However, there are several published reservations about the lectin precipitation assay, particularly with regard to lotto-lot variability in the lectin reagent and interference from biliary ALP. Only a complete reevaluation of the clinical utility of a sensitive specific assay will ascertain which of these techniques can truly meet the new goals in relation to assessment of bone disease. REFERENCES 1 McComb RB, Bowers GN, Posen S. Alkaline Phosphatase, New York: Plenum Press. 1979: Moss DW. Multiple forms of acid and alkaline phosphatases: genetics, expression and tissue-specific modification. Clin Chim Acta 1986; 161: Wolf M, Dinwoodie A, Morgan HG. Comparison of alkaline phosphatase isoenzyme activity using five standard methods. Clin Chim Acta 1969; 24: Harris H. The human alkaline phosphatases: what we know and what we don't know. CUnChim Acta 1989; 186: Griffin CA, Smith M, Henthorn PS, Harris H, Weiss MI, Raducha M, Emanuel BS. Human placental and intestinal alkaline phosphatase gene maps to 2q34-q37. Am J Hum Genet 1987; 41: Smith M, Weiss MI, Griffin CA, Murray JC, Buetow KH, Emanuel BS, et al. Regional assignment of the gene for human liver/bone/kidney alkaline phosphatase to chromosome Ip36.1-p34. Genomics 1988; 2: Crofton PM. Biochemistry of alkaline phosphatase isoenzymes. CRC Crit Rev Clin Lab Sci 1982; 16: Robson EB, Harris H. Further genetics of placental alkaline phosphatase. Ann Hum Genet 1%7; 30: Price CP, Hill PG, Sammons HG. The nature of the alkaline phosphatases of bile. J Clin PathoI1972; 25: Price CP, Sammons HG. The nature of the serum alkaline phosphatases in liver diseases. J CUnPathol 1974; 27: De Brae ME, Borgers M, Wieme RJ. The separation and characterisation of liver plasma membrane fragments circulating in the blood of patients with cholestasis. CUn Chim Acta 1975; 59: Crofton PM, Smith AF. High molecular-mass alkaline phosphatase in serum and bile: physical properties and relationship with other high-molecularmass enzymes. CUn Chem 1981; 27: Mueller HD, Leung H, Stinson RA. Different genes code for alkaline phosphatases from human fetal and adult intestine. Biochem Biophys Res Comm 1985; 126: Rosalki SB. Monoclonal antibodies to enzymes. Clin Chim Acta 1989; 183: Hattori Y, Yamamoto K, Urabe C, Furuya M, Eguchi H, Hattori H, et al. Frequency of alkaline phosphatase immunoglobulin complex among disease and healthy population. Clin Chim Acta 1985; 147: Howard AD, Berger J, Gerber L, Familletti P, Udenfriends S. Characterization of the phosphatidylinositol-glycan membrane anchor of human placental alkaline phosphatase. Proc Nat A cad Sci 1987; 84: Kim EE, Wyckoff HW. Structure of alkaline phosphatases. CUn Chim Acta 1989; 186: Stigbrand T, Fishman WHo Human Alkaline Phosphatases. New York: Alan R Liss, 1984: Miura M, Matsuzaki H, Bailyes EM, Koyama I, Sakagishi Y, Sekine T, et al. Differences between human liver and bone-type alkaline phosphatases. CUn Chim Acta 1989; 180:

15 Multiple forms of human serum alkaline phosphatase Onica D, Sundblad L, Waldenlind L. Affinity electrophoresis of human serum alkaline phosphatase isoenzymes in agarose gel containing lectin. Clin Chim Acta 1986; 155: Walton RJ, Preston CJ, Russell RGG, Kanis JA. An estimate of the turnover rate of bone-derived plasma alkaline phosphatase in Paget's disease. Clin Chim Acta 1975; 63: Barak Y. Osteoblasts and osteoclasts in bone marrow aspirated from children with rickets. Acta Paed Scand 1970; 59: Khairi MR, Wellman HN, Robb JA, Johnston CC. Pagets disease of bone (osteitis deformans): symptomatic lesions and bone scan. Ann Int Med 1973; 79: Posen S, Cornish C, Kleerekoper K. Alkaline phosphatase and metabolic bone disease. In: Avioli LV, Krane SM (eds). Metabolic Bone Disease. New York: Academic Press, 1977: Naik RB, Gosling P, Price CP. Comparative study of alkaline phosphatase isoenzymes, bone histology and skeletal radiography in dialysis bone disease. BMJ 1977; 1: Stepan 11, Tesarova A, Havranek T, JodI J, Formankova J, Packovsky V. Age and sexdependency of the biochemical indices of bone remodelling. Clin Chim Acta 1985; 151: Joplin GF, Stevenson JC. Paget's disease of bone. In: Stevenson JC (ed.), New Techniques in Metabolic Bone Disease. London: Wright, 1990: Boyle IT. Current problems with rickets and osteomalacia. In: Stevenson JC (ed.). New Techniques in Metabolic Bone Disease. London: Wright, 1990: Ewen LM. Separation of alkaline phosphatase isoenzymes and evaluation of the clinical usefulness of this determination. Am J Clin Pathol 1974; 61: Mayne PD, Thakrar S, Rosalki SB, Foo AY, Parbhoo S. Identification of bone and liver metastases from breast cancer by measurement of plasma alkaline phosphatase isoenzyme activity. J Clin Pathol 1987; 40: Crofton PM, Elton RA, Smith AF. High molecular weight alkaline phosphatase: a clinical study. Clin Chim Acta 1979; 98: Price CP, Sammons HG. An interpretation of the serum alkaline phosphatase isoenzyme patterns in patients with obstructive liver disease. J Clin Pathol 1976; 29: De Broe ME, Pollet DE. Multicenter evaluation of human placental alkaline phosphatase as a possible tumor-associated antigen in serum. Clin Chem 1988; 34: Thompson PW, Houghton BJ, Clifford C, Jones DD, Whitaker KB, Moss DW. The source and significance of raised serum enzymes in rheumatoid arthritis. Q J Med 1990; 76: Rosalki SB, Foo AY, Arntsen KW. Alkaline phosphatase of possible renal origin identified in plasma after colchicine overdose. Clin Chem 1989; 35: (letter) 36 Adeniyi FA, Olatunbosum DS. Origins and significance of the increased plasma alkaline phosphatase during normal pregnancy and preeclampsia. Br J Obstet Gynaeco/1984; 91: Tibi L, Patrick AW, Leslie P, Toft AD, Smith AF. Alkaline phosphatase isoenzymes in plasma in hyperthyroidism. Clin Chem 1989; 35: Tibi L, Collier A, Patrick AW, Clarke BF, Smith AF. Plasma alkaline phosphatase isoenzymes in diabetes mellitus. Clin Chim Acta 1988; 177: Siraganian PA, Mulvihill 11, Mulivor RA, Miller RW. Benign familial hyperphosphatasaemia. JAm Med Assoc 1989; 261: Panteghini M. Benign inherited hyperphosphatasaemia of intestinal origin: report oftwo cases and a brief review of the literature. Clin Chem 1991; 37: Stein P, Rosalki SB, Foo AY, Hjelm M. Transient hyperphosphatasaemia of infancy and early childhood: clinical and biochemical features of 21 cases and literature review. Clin Chem 1987; 33: Mueller HD, Stinson RA, Mohyuddin F, Milne JK. Isoenzymes of alkaline phosphatase in infantile hypophosphatasia. J Lab Clin Med 1983; 102: Weiss MJ, Cole DE, Ray K, Whyte MP, Lafferty MA, Mulivor R, Harris H. First identification of a gene defect for hypophosphatasaemia: evidence that alkaline phosphatase acts in skeletal mineralization. Connect Tissue Res 1990; 21: Burlina A, Galzigna L. Parallel electrophoretic fractionation of alkaline phosphatase and serum protein on cellulose acetate strips: clinical evaluation. Clin Chem 1976; 22: Anido G, Soto A, McBeth CH, Romero P. Alkaline phosphatase isoenzymes in diagnosis. Quad Sclavo Diagn Clin Lab 1972; 8: Baker RWR, Pellegrino C. Separation and detection of serum enzymes by paper electrophoresis. Scand J Clin Lab Invest 1954; 6: Jennings RC, Brocklehurst D, Hirst M. A comparative study of alkaline phosphatase enzymes using starch gel electrophoresis and Sephadex gel filtration with special reference to high molecular weight enzymes. Clin Chim Acta 1970; 30: Siede WH, Seiffert VB. Quantitative alkaline phosphatase isoenzyme determination by electrophoresis on cellulose acetate membranes. Clin Chem 1977; 23: Hagerstrand I, Skude G. Improved electrophoretic resolution of human serum alkaline phosphatase isoenzymes in agarose gel by Triton X-1OO. Scand J Clin Lab Invest 1976; 36: Walker AW, Pollard AC. Observations on serum alkaline phosphatase electrophoretic patterns on polyacrylamide gel, with particular reference to the effects ofbutanol extraction. Clin Chim Acta 1971; 34: Mulivor RA, Hannig VL, Harris H. Developmental change in human intestinal alkaline phosphatase. Proc Nat Acad Sci 1978; 75: Moss DW, Edwards RK. Improved electrophoretic resolution of bone and liver alkaline phosphatases resulting from partial digestion with neuraminidase. Clin Chim Acta 1984; 143:

16 370 Price 53 Rosalki SB, Foo AY. Two new methods for separating and quantifying bone and liver alkaline phosphatase isoenzymes in plasma. Clin Chem 1984; 30: Kuwana T, Sugita 0, Yakata M. Reference limits of bone and liver alkaline phosphatase isoenzymes in the serum of healthy subjects according to age and sex as determined by wheat germ lectin affinity electrophoresis. Clin Chim Acta 1988; 173: Blum-Skolnick 1, Pace F, Munst G, Minder W. Isoelectric focusing of serum alkaline phosphatase isoenzymes, Clin Chim Acta 1983; t:z9: Cocco C, Marini M, Rizzotti P. Isoelectric focusing on cellulose acetate membrane: a separation procedure for alkaline phosphatase isoenzymes. Clin Biochem 1987; 20: Griffiths 1, Black 1. Separation and identification of alkaline phosphatase isoenzymes and isoforms in serum of healthy persons by isoelectric focusing. Clin Chem 1987; 33: Sinha PK, Bianchi-Bosisio A, Mayer-Sabell EK, Righetti PG. Resolution of alkaline phosphatase isoenzymes in serum by isoelectric focusing in immobilized ph gradients. Clin Chem 1986; 32: Behr W, Barnert 1. Quantification of bone alkaline phosphatase in serum by precipitation with wheat germ lectin: a simplified method and its clinical plausibility. Clin Chem 1986; 32: Desoize B, Cravero L, lardillier lc. Alkaline phosphatase isoenzyme reactivity with wheat germ agglutinin. Clin Chem 1986; 32: (letter) 61 Bahr M, Wilkinson lh. Urea as a selective inhibitor of human tissue alkaline phosphatases. Clin Chim Acta 1967; 17: Birkitt DJ, Conyers RAJ, Neale Fe, Posen S, Brudenell-Woods 1. Action of urea on human alkaline phosphatases: with a description of some automated techniques for the study of enzyme kinetics. Arch Biochem Biophys 1967; 121: Winkelman 1, Nadler S, Demetriou 1, Pileggi VJ. The clinical usefulness of alkaline phosphatase isoenzyme determinations. Am J Clin Path 1972; 57: Gerhardt W, Lykkegaard Nielsen M, Vagn Nielsen 0, Olsen ls, Statland BE. Routine measurements of liver and bone alkaline phosphatase in serum. Differential inhibition by L-phenylalanine and carbamide (urea) on the LKB 8600 reaction rate analyzer. Clin Chim Acta 1974; 53: Statiand BE, Nishi HH, Young DS. Serum alkaline phosphatase: total activity and isoenzyme determination made by use of a centrifugal fast analyzer. Clin Chem 1972; 18: Brown PB, Lewis KO. Reaction rate retardation as a method for serum alkaline phosphatase isoenzyme measurement. Ann Clin Biochem 1980; 17: Forsman RW, O'Brien IF. Quantifying bone and liver alkaline phosphatase by the resolution of twocomponent inactivation data obtained with a centrifugal analyzer. Clin Chem 1991; 37: Bergstrom K, Lefvert K. Practical aspects of the determination of serum alkaline phosphatase isoenzymes using L-phenylalanine and urea. Clin Chim Acta 1975; 64: Tillyer CR. Error estimation in the quantification of alkaline phosphatase isoenzymes by selective inhibition methods. Clin Chem 1988; 34: Gerhardt W, Lykkegaard Neilsen M, Vagn Nielsen0, Olsen ls, Staiand BE. Clinical evaluation of routine measurement of liver and bone alkaline phosphatases in humanserum. Differentialinhibitionby L-phenylalanine and carbamide (urea) on the LKB 8600 reaction rate analyzer. ClinChim Acta 1974;53: Shephard MDS, Peake Ml. Quantitative method for determining serum alkaline phosphatase isoenzyme activity I. Guanidine hydrochloride: new reagent for selectively inhibiting major serum isoenzymes of alkaline phosphatase. J Clin Pathol 1986; 39: Shephard MDS, Peake Ml, Walmsley RN. Quantitative method for determining serum alkaline phosphatase isoenzyme activity II. Development and clinical application of method for measuring four serum alkaline phosphatase isoenzymes. J Clin Pathol 1986; 39: Fitzpatrick CP, Pardue HL. Simultaneous determination of liver and bone-type alkaline phosphatase by curve-fitting of inhibitor kinetic data. I. Development and evaluation of an absorbance based model. Clin Chem 1992; 38: Fitzpatrick CP, Pardue HL. Simultaneous determination of liver and bone-type alkaline phosphatase by curve-fitting of inhibition kinetic data. II. Development and evaluation of a fluorescence-based model. Ciin Chem 1992; 38: Posen S, Neale FC, Clubb ls. Heat inactivation in the study of human alkaline phosphatase. Ann lnt Med 1965; 62: Stepan J, Volek V, Kolar J. A modified inactivationinhibition method for determining the serum activity of alkaline phosphatase isoenzymes. Clin Chim Acta 1976; 69: Petitclerc C. Quantitative fractionation of alkaline phosphatase isoenzymes according to their thermostability. Clin Chem 1976; 22: Whitby LG, Moss DW. Analysis of heat inactivation curves of alkaline phosphatase isoenzymes in serum. Clin Chim Acta 1975; 59: Moss DW, Whitby LG. 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17 Multiple forms of human serum alkaline phosphatase Schoenau E, Herzog KH, Boehies H-J. Liquid chromatographic determination of isoenzymes of alkaline phosphatase in serum and tissue homogenates. Clin Chem 1986; 32: Bielby JP, Chin C. Alkaline phosphatase isoenzymes incompletely resolved by "fast protein liquid chromatography". Clin Chem 1987; 33: 2327 (letter) 87 Parviainen MT, Galloway JH, Towers JH, Kanis JA. Alkaline phosphatase isoenzymes in serum determined by high-performance anion-exchange liquid chromatography with detection by enzyme reaction. Clin Chem 1988; 34: Severini G, Aliberti LM, Di Giovannan-Drea R. Diagnostic aspects of alkaline phosphatase: separation of isoenzymes in normal and pathological human serum by high-performance liquid chromatography. J Chromatog Biomed App/199l; 563: Gonchoroff DG, Branum EL, O'Brien JF. Alkaline phosphatase isoenzymes of liver and bone origin are incompletely resolved by wheat-germlectin affinity chromatography. 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