Alkaline Phosphatase
The Isoforms of Alkaline Phosphatase
Serum alkaline phosphatase (ALP) is the most commonly used biochemical marker of bone metabolism. While the measurement of this enzyme has long been associated with osteoblastic activity (Robinson, 1923), not all ALP activity in serum is derived solely from bone. It has recognised for over sixty-five years that serum ALP is increased in patients with hepatobiliary disease (see Moss, 1988). Although some workers postulated a failure of the liver to excrete bone derived ALP, the application of various techniques of isoenzyme-enzyme analysis identified the liver as the source of the increased ALP activity (Hül & Sammons, 1967; Hodson et aly 1967).
There are four genetic loci for human alkaline phosphatase. Those for the placental, intestinal and germ cell isoforms are found on chromosome 2, while the gene for the tissue non-specific form is on chromosome 1 (Fishman, 1990). This gene codes for the bone, liver and kidney isoforms of ALP which result from differences in post- translational glycosylation of the protein (Crofton et a/., 1982; Price, 1993; Weiss et aly
1986). The bone and liver isoforms are the most abundant in normal serum and usually account for elevated total ALP activity (Van Hoof et a l y 1990). Placental or germ cell ALP may be the primary isoform in patients with cancer, while in subjects with various diseases of the digestive tract the intestinal isoenzyme predominates (Price & Thompson, 1995).
ALP is a membrane-bound enzyme. The activity of the bone isoform rises when osteoblasts are actively laying down osteoid. Overflow of osteoblastic ALP may be due to breakdown of the plasma membrane (or its vesicles), overflow of enzyme binding sites or a combination of both (Moss, 1988). The major isoforms have relatively long half- lives of about 1-3 days (approximately 1.5 days for the bone isoenzyme) (Walton et a l y
1975). The dominating influence on serum levels of liver and bone ALP is therefore thdr rate of entry into the general circulation. Serum total ALP concentrations correlate with bone mineralisation as measured by calcium isotope studies (Charles et al. y 1985; Klein et a l y 1964), and have long been used as an indicator of bone formation. The major application total ALP in the field of metabolic bone disease is as an index of disease activity and response to treatment in Paget's disease (Russell et a l y 1981).
The lack of specificity provided by the assay of this heterogeneous group of isoenzymes manifests as a lack of sensitivity with less marked changes in bone formation such as those found in post-menopausal osteoporosis. This situation is exacerbated by the
induction of the liver isoenzyme by numerous drugs and the variable contribution of the intestinal form (Nielsen et aL, 1990a; Tohmé et a i, 1991).
The Function of Osteoblastic ALP
The hereditary disorder hypophosphatasia was first described in 1948 (see Smith, 1993). In child sufferers, low ALP activity is coupled with the presence of severe rickets, suggesting an important role for the enzyme in bone mineralisation. Furthermore, inorganic pyrophosphate, a potent inhibitor of hydroxyapatite crystal formation and thus mineralisation, is markedly elevated in this condition. This led to the suggestion that the function of bone alkaline phosphatase is to hydrolyse pyrophosphate, permitting hydroxyapatite crystal growth on newly synthesised osteoid. Other possible functions of ALP include the release of the inorganic phosphate required for hydroxyapatite formation. As the enzyme is membrane bound, a transport function of some type has been postulated. At present the precise function of osteoblastic ALP remains unclear. Measurement of Total Alkaline Phosphatase
Alkaline phosphatase is quantified by measuring the catalytic activity of the enzyme against one of its substrates. Many methods, based on differing substrates, buffer type and concentration, assay temperature and unit of measurement have been used (for review see McComb et o/., 1979). Substrates utilised include P-glycerylphosphate, and 1-, or 2- napthylphosphate. King and Armstrong developed a widely adopted method using disodium phenolphosphate with units of measurement bearing their name. All of the above methods have inconveniences such as requirements for a second reaction step, pH alteration or protein precipitation. Fifty years ago Bessey published a method that was rapid to perform and is easily applicable to the autoanalyser (Bessey et a l, 1946). This gained in popularity as the availability of the pure 4-nitrophenylphosphate (4-NP) substrate increased, and 4-NP has been universal in all recommended methods since 1972 (McLauchlan 1988). Recommendations on buffer type and concentration do vary however, delaying complete standardisation of ALP measurement.
Bone Specific Alkaline Phosphatase
In normal individuals, the bone and liver isoforms of ALP are present in roughly equal amounts (Van Hoof et a/., 1990). Individuals with blood group types B and O exhibit elevations in intestinal ALP after fatty meals, while placental ALP increases in pregnant women. Specific measurement of the bone isoform therefore adds sensitivity to the use of ALP in metabolic bone disease. The major forms of ALP are products of the same genetic locus, differing only in the number of sialic acid and N-acetyl glucosamine residues attached to the carbohydrate side chains of the molecule (Hitz et a/., 1980; Kerkhoff et aly 1968). The differentiation of the bone and liver isoforms, required in
about half of all routine requests for ALP analysis and the majority of those concerned with metabolic bone disease, is therefore a difficult process (Moss, 1988).
Methods for the Differentiation of the Isoenzymes of ALP
Heat/Chemical Inhibition
Historically, ALP isoenzyme measurement has depended on minor differences in response to chemical inhibitors and temperature which result from tissue-specific post- translational modifications of the isoforms.. Heat inactivation methods are based on the differing half lives of the bone and liver isoforms at 56°C. (Moss & Whitby, 1975). This allows the proportion of each isoenzyme contributing to the total activity is indirectly calculated according to the residual activity after incubation. High concentrations of both major isoforms, or increased placental or intestinal ALP make quantitation of the relative contributions difficult. Heat inactivation methods have been more commonly adopted than alternatives utilising chemicals such as urea, phenylalanine, or neuraminidase (Gonchoroff et a l, 1991). However all these methods exhibit poor resolution and offer only indirect quantitation. The requirement for sample pre-treatment and multiple analysis has made these techniques technically cumbersome, time consuming and generally unpopular.
Gel Electrophoresis
Isoenzyme separation by electrophoresis on agarose (Van Hoff et aly 1988), cellulose acetate (Rosalki & Foo, 1984), and (most commonly), polyacrylamide gels (Ramasamy, 1991) may be undertaken. Subsequent quantitation is then facilitated by densitometic scanning of the electrophoresis gel. While sample pre-treatment improves the generally poor resolution afforded by these methods, electrophoretic techniques are generally time
consuming and lack sensitivity. ^
Lectin Precipitation
A method based on the precipitation of bone alkaline phosphatase (B-ALP) by a lectin derived from wheat germ was first described in 1984 (Rosalki & Foo, 1984). While this method was employed clinically (Behr & Bamert, 1986; Mazda & Gyure, 1988; Sorenson, 1988), between batch variability in the reactivity of lectin and the lack of a suitable control material obstructed standardisation. The introduction of a commercial version of the assay (‘Iso-ALP’, Boehringer Maimheim GmbH, Mannheim, Germany), lessened such problems (Rosalki et aly 1993). Although the method is both rapid and technically simple falsely elevated B-ALP levels have been noted in patients with liver disease. This may be due to a second form of the liver isoenzyme, bound to phospholipid and membrane fragments, which also interferes with electrophoretic
methods (Price, 1993). While such species may be excluded by biochemical tests of liver disease, the non-specificity of the method is illustrated. While some have suggested lectin precipitation lacks the quantitative accuracy of the best electrophoretic methods (Day et aly 1992) others have found it to provide both the sensitivity and specificity required for the precise measurement of B-ALP (Price, 1993).
Immunoassay
Improvements in the ability to differentiate B-ALP from other isoforms have resulted from the use of monoclonal antibodies. While the first of such antibodies were two to five times more specific for the bone isoform (Bailyes et aly 1987; Lawson et aly 1985; Seabrook et aly 1988), Hill & Wolfert (1989) isolated a murine antibody produced by challenge with human osteosarcoma cells which have little cross-reactivity to the liver isoform. This led to the development of a commercially available immunoradiometric assay (IRMA), (‘Ostase’, BM Browne Ltd, Reading UK), which has no significant cross-reactivity to the intestinal isoform and a seven fold preference for B-ALP over liver ALP (Panigrahi et a l y 1994). This represents a cross-reactivity of about 15% (Gamero & Delmas, 1993; Price et a l y 1995). While this assay appears to have no advantage over lectin precipitation methods in terms of assay precision (Price et a l y 1995), the variability of wheat germ lectin remains a possible problem with respect to long-term patient follow- up. Furthermore as the IRMA measures enzyme mass rather than activity it may be used on samples which have undergone long term storage at -20°C or multiple freeze thaw cycles (Panigrahi et a l y 1994). Recentiy a further commercial assay for the measurement B-ALP has become available (‘Alkphase-B’, Metra Biosystems Ltd, Oxford, UK). This enzyme immunoassay (EIA) is based on the selective capture of B-ALP with a monoclonal antibody and a subsequent colorimetric reaction with 4-nitrophenol phosphate (Gomez et a l y 1994; Gomez et a l y 1995). A greater specificity for the bone isoform has been claimed by the manufacturers of this assay and others (Hata et a l y
1996), although it is possible that in the cross-reactivity experiments undertaken, sample pre-treatment affected the immunological integrity of the liver isoform. As the EIA quantitative step is based on enzyme activity rather than mass, sample stability is likely to be less than that found with the IRMA method.
Clinical Use of Total and Bone-ALP
Bone-ALP undergoes a circadian variation, with peak values at 14:30 and 23:30 which are 30% higher than the 06:30 trough. TTie seasonal variations exhibited by total ALP in normal subjects (low in summer, high in winter), are likely to be due to changes in B- ALP and correlate negatively with concentrations of 25-hydroxyvitamin D (Devgun et a l y
1991; Tohmé et a l y 1 9 9 1 ) . As well as correlating with histomorphometric assessments of bone formation, bone alkaline phosphatase (B-ALP) is found to be a better predictor of
mineralisation in normal women than total ALP (Brixen et a l, 1989). Until one year of age, B-ALP is the predominant ALP isoform in human serum (Van Hoof et a l, 1990). Levels peak during childhood and puberty, subsequently falling to adult levels (Stepan et a l, 1985). In mature adults B-ALP levels rise with age in both sexes (Duda et a l, 1988; Gamero & Delmas, 1993; Kuwana et a l, 1988). As B-ALP is not excreted by the kidney, this rise is not an artefact of the age related decrease in glomerular filtration rate but consistent with the general increase in bone turnover known to be associated with ageing. Elevations in B-ALP activity have been reported in a number of disease states. These include Paget’s disease (Gamero & Delmas, 1993; Deftos et a l, 1990), renal osteodystrophy, (Tibi et a l, 1991), primary hyperparathyroidism, (Gamero & Delmas, 1993; Gomez et a l, 1995; Silverberg et a l, 1991) and in patients with bone métastasés (Cooper et a l, 1992; Cooper & Jones, 1993). While elevated levels of ALP are also common in these conditions, the magnitude of the increase is less marked.
Osteocalcin
Osteocalcin (OC) is, together with osteonectin, one of the two the most abundant non- collagenous bone proteins and one of the ten most plentiful proteins in the entire human body (Hauschka et a l, 1989). Apart from bone, this small (5.8kD, 49 amino acid) protein is also present in dentine, but not non-osseous tissues (Nishimoto & Price, 1979). The presence of three y-glutamic acid (Gla) residues at amino acids 17, 21 and 24, led to the older, lesser used name of Bone-Gla protein (BGP) (Price et a l, 1976). The Gla residues, derived from the post-translational carboxylation of glutamic acid, strongly bind calcium, so conferring the ability to bind the hydroxyapatite of mineralised bone. Such binding aids stabilisation of the tertiary protein structure (Hauschka & Carr, 1982).
Biosvnthesis of Osteocalcin
Osteoblastic production of OC is stimulated by 1,25-dihydroxyvitamin D (l,25(OH)2D) (Price & Baukol, 1980; Zerwekh et a l, 1985), although production does occur in the absence of this hormone (Kaplan et a l, 1985). The OC gene, like that of alkaline phosphatase, is located on chromosome 1 (Puchacz et a l, 1989). Osteocalcin is initially produced as a lOkD pre-pro-peptide (Pan et a l, 1985), which is cleaved and carboxylated (the latter process being vitamin K dependent and stimulated by 1,25(0H)2D), before excretion into the extracellular space (Bianco et a l, 1985; Hauschka et a l, 1989; Skjodt et a l, 1985). While the precise effect of parathyroid hormone on OC synthesis is unclear, it may serve to modulate the action of l,25(OH)2D (Hauschka et a l, 1989; Nielsen et a l, 1991). Calcitonin, the other major hormone thought be involved in calcium homeostasis, appears to have no apparent effect on OC production (Beresford et a l, 1984). Hormones such as oestrogen, thyroid hormone and
cyclic AMP directly affect gene transcription and mRNA levels enhancing OC protein synthesis (Hauschka et al.^ 1989).
While a proportion of native osteocalcin is rapidly degraded by hepatic and renal metalloenzymes (Farrugia & Melick, 1986; Price etaLy 1976), 60-90% is incorporated into bone through binding to hydroxyapatite (Poser et a/., 1980). Circulating intact OC is solely derived from de novo synthesis and is not released during bone resorption (Price, 1983; Taylor et al., 1990), although some workers consider that resorptive activity may produce fragments of osteocalcin (Taylor et a/., 1990). The in vivo catabolism of circulating intact OC produces peptide fragments, which may react with antisera used to detect intact OC (Gundberg & Weinstein, 1986; Poser et a/., 1980; Rosenquist et a/., 1995; Taylor et al., 1990). As these peptide fragments are cleared by the kidney, any impairment of renal function may lead to over-estimation of OC (Delmas et aly 1983a). Function of Osteocalcin
Although several possible roles for OC have been postulated, no precise function has been discovered (Power & Fottrell, 1991a). As levels are highest in the bone matrix following completion of the mineralisation process a role in bone maturation has been suggested (Lian et a/., 1982). It is also possible OC acts as a 'messenger' for 1,25(0H)2D, facilitating bone resorption by promoting osteoclastic activity and/or osteoclast precursor differentiation (Mundy & Poser, 1983; Povolny etaLy 1987; Skjodt
et at. y 1985). Although OC is thought to inhibit leukocyte elastase (elastin plays an important role in inter chain cross-linking) (Hauschka, 1985), and the activity of growth factors (Povolny et a/., 1987; Tsutsumi et a/., 1987), the possible significance of such action is unknown.
Measurement of Osteocalcin
A fraction of de novo osteocalcin is not incorporated into the bone matrix but released into the general circulation (Hauschka et a/., 1989). Measurement of circulating OC should therefore give a reliable estimate of the level of OC production and therefore osteoblastic activity. Since development of the first assay for this protein (Price & Nishimoto 1980), many immunoassays to measure serum OC have been designed (for review see Power & Fottrell, 1991a). These assays have utilised a wide range of monoclonal and polyclonal antisera (Power etaLy 1989; Power & Fottrell, 1991a; Tracy
e t al.y 1990), in both radio-isotope and enzyme-immunoassay formats (Egsmore etaLy
1989; Hosoda et a/., 1992; Monaghan et a/., 1993). Such differences in format, as well as diversity of antibody source, epitope recognition site and matrix of calibration material have contributed to a lack of standardisation for OC assays. This effect is so marked that results from different assays are difficult to compare, even when values are normalised against those from healthy subjects (Masters et a/., 1994).
Osteocalcin contains two arginine-arginine sequences at amino acids 19-20 and 43-44. These are possible sites of lesion for protease enzymes and result in a number of potential OC fragments; 1-19; 20-43; 44-49; 1-43; and 20-49 (Hosoda et aly 1992; Prigodich et aly 1985) (see Figure 1.5). Such fragments are produced by both in vivo and in vitro
degradation of intact osteocalcin (Blumsohn et al.y 1995a). The predominant degradation product appears to be the N-terminal midregion fragment (OC^"^^), which represents approximately 30% of the total immunoreactive osteocalcin in both normal individuals and osteoporotic patients (Gamero et a l y 1994b). Assays which measure as well as the intact molecule (Gamero et a l y 1992), exhibit less apparent instability on storage than assays measuring intact OC alone (Blumsohn et a l y 1995a; Gamero et a l y 1994b). This indicates that while intact OC is highly susceptible to in vitro proteolytic degradation, the major OC^"^^ fragment is more stable. Furthermore the N-terminal, mid and mid-C-terminal fragments are detected in much smaller amounts than the N-terminal- mid fragments (Gamero et a l y 1994b). Such findings are likely to be due to the easier accessibility of the 43-44 peptide bond to proteolytic cleavage, an effect possibly due to the conformation of the a-helix structure in the presence of calcium ions (Hauschka & Carr, 1982). Short C-terminal peptides, which may be theoretically detected by some assays are thought to undergo quick in vivo degradation in serum (Gamero et a l y 1994b). Low molecular weight fragments of OC detected in HPLC studies with RIA quantitation (Taylor et a l y 1990; Gundberg & Weinstein, 1986) have previously been speculated to be due to the release of OC fragments from bone matrix during resorption. A further study using monoclonal antibodies also detected such low molecular weight fragments (Tracy et a l y 1990). The identification of such fragments and their subsequent quantitation (Gamero et al . y 1994b), showed the levels of the different immunoreactive forms to be unchanged in osteoporosis compared with normal subjects.
49
44 43
2 4
KEY: Human and bovine common sequence
G C N
Human specific sequence
g- carboxyglutamic acid (GLA) residues Carboxy terminal
Amino terminal
Potential proteolytic sites N-terminal-mid fragment (1-43) Mid-C-terminal fragment (20-49) N-terminal fragment (1-19) Mid fragment (20-43)
C-terminal fragment (43 49)
Figure 1.5: Diagrammatic representation of the osteocalcin molecule and its potential fragments.
This is strongly suggestive that such fragments are not products of bone resorption. Certainly the major 1-43 fragment is not a product of bone resorption, as serum levels of this peptide are not reduced by administration of anti-resorptive agents (Gamero et al.^
1994b).
Antisera whose epitope contains one of the three Gla residues detect OC more effectively in the presence of calcium (Egsmore et aly 1989; Tanaka et a/., 1986; Taylor et al.,
1988). Any undercarboxylaton of the glutamate residues during post-translational processing of OC would reduce their calcium-binding ability and thus the detection of OC by calcium dependent antisera. As carboxylation of OC is a vitamin K-dependent process (Hauschka et al., 1989), and many osteoporotic patients have been found to be vitamin K deficient (Hart et a l, 1985), OC values measured by such assays may be artifactually low. A further group of antisera whose recognition sites are conformational may have calcium-dependent binding even if the epitope is not in a calcium-binding region (Tracy et a l, 1990). The calcium-binding action of anticoagulants means that plasma is not suitable in any calcium dependent assay. In other assays, lithium heparinised plasma gives the best correlation with serum (Power & Fottrell, 1991b). Serum OC is sensitive to sample haemolysis, as erythrocyte hydrolases in the red cell lysate produce low molecular weight fragments not recognised by assay antibodies. It is postulated a similar effect may contribute to the lower OC values found in plasma (Power & Fottrell, 1991b; Tracy et a l, 1990).
Polyclonal antibody based assays indicate a greater loss of OC on storage than their monoclonal counterparts, yet produce results two to three times lower than assays measuring both intact and OC^"^^ (Gamero et at., 1994b). Such evidence supports the contention that conventional polyclonal based assays preferentially measure intact OC with poorer recognition of the large N-terminal midfragment. Indeed Price and Nishimoto (1980) in their original polyclonal RIA found the C-terminal region of OC necessary for antibody recognition. That polyclonal RIAs produce lower values than assays for intact OC alone is probably due to the immunochemical heterogeneity of circulating OC, for which the structural basis is unclear (Deftos et a i, 1992). In order to