Alkaline phosphatase (AP, ALP, SAP)


Alkaline phosphatase is a non-specific metalloenzyme which hydrolyzes many types of phosphate esters at an alkaline pH in the presence of zinc and magnesium ions. There are different isoenzymes (gene products) and isoforms (posttranslationally modified gene products). The main use of ALP is as a sensitive indicator of cholestasis in the dog (it will increase before bilirubin), however it is non-specific because corticosteroids (exogenous or endogenous "stress") induce increases in this enzyme. In the cat, ALP is a very specific indicator of liver disease, whereas in large animals, the enzyme is not very useful as it is insensitive, cholestatic disorders are infrequent, and reference intervals are quite broad.

Isoenzymes and isoforms of ALP

There are 2 isoenzymes (products of different genes) and several isoforms (produced from posttranslational modification of isoenyzmes) of ALP. The isoenzymes are produced from intestinal and tissue non-specific ALP genes and differ in amino acid sequence. Isoforms differ in catalytic sites and activity, immunogenecity, and electrophoretic mobility. The major isoforms that can be measured in animals are liver-ALP (L-ALP), corticosteroid-ALP (C-ALP, only in dogs), bone-ALP (B-ALP) and intestinal-ALP (I-ALP).
a) Intestinal-ALP gene/isoenzyme: This produces the C-ALP and I-ALP isoforms. C-ALP is unique to dogs and is induced by corticosteroids (endogenous or exogenous). Even though it is a product of the intestinal gene, it is actually produced in hepatocytes not intestinal epithelium.
b) Tissue non-specific-ALP gene/isoenzyme: This produces the remaining isoforms of ALP, including L-ALP, B-ALP, placental-ALP and leukocyte -ALP.

Organ specificity

The enzyme is associated with microsomal and cell membranes and is present in many tissues. ALP is anchored to cell membranes by glycophosphatidylinositol (GPI) proteins. Cleavage of these proteins by bile acids, phospholipase D and proteases releases ALP from membranes resulting in increased ALP levels in serum/plasma.

  • Liver - hepatocytes, epithelium of biliary tract. These cells are the source of the L-ALP isoform (all species) and C-ALP isoform in the dog. ALP expression is normally restricted to the canalicular membrane of hepatocytes, but can be induced (secondary to cholestasis typically) on the sinusoidal membrane (where it can be readily liberated into blood).
  • Bone - this isoform is produced by osteoblasts and increases in serum in association with osteoblastic activity (young animals, certain bone disorders).
  • Intestinal, renal, mammary, placental tissues - these are not usually important sources of increased serum ALP activity, though some placental isoform is present in serum of normal pregnant queens and mares. High levels of ALP in dogs with mammary tumors has been attributed to myoepithelial expression of ALP.
  • Leukocytes - ALP is found within myeloid cells, including neutrophils, eosinophils and monocytes. Cell lineage expression is species-dependent, i.e. monoblasts (immature monocytes) in dogs are particularly rich in ALP. It is commonly used as a marker for myeloid cells in acute leukemia. Its presence within a cell can be detected by applying an ALP-specific substrate to cytology smears (called cytochemical staining). Cytochemical staining for ALP is also used to differentiate osteoblasts from other mesenchymal cells in cytologic aspirates from tumors, i.e. high expression is expected in osteosarcomas, but not chondrosarcomas or fibrosarcomas.
The serum half life of ALP varies with species and isoform:
  • L-ALP isoform: dog - 66 hours, cat - 6 hours
  • C-ALP isoform: dog - 70 hours
  • B-ALP isoform: ?
  • Placental, renal, and intestinal isoforms: dog - less than 6 minutes, cat - less than 2 minutes. This may contribute to serum ALP values in late pregnancy, but serum ALP values are not usually above reference intervals in pregnant cats and horses.

Analysis

Routine measurement of ALP gives total serum activity (all isoforms) without specificity as to source. In healthy animals, L-ALP is the predominant isoform in blood, followed by B-ALP. The proportion of B-ALP is higher in young animals (indicates osteoblastic activity with growth). The C-ALP isoform contributes only a small amount to total serum ALP activity. This proportion increases with age in dogs.

Isoform measurement is most commonly applied to canine samples to distinguish L-ALP and C-ALP isoforms in cases with increased total serum ALP activity of uncertain cause (to identify whether total ALP is increased due to liver disease or endogenous corticosteroids). However, this is not a very reliable test for this purpose, because any chronic disease (including that affecting the liver) can result in endogenous corticosteroid release (chronic stress), which would increase C-ALP. Also, corticosteroids induce the synthesis of both C-ALP and L-ALP isoforms. Differentiation of isoforms can be accomplished by electrophoretic (affinity agarose electrophoresis, cellulose acetate electrophoresis or isoelectric focusing on agarose) or differential inhibition methods using levamisole (inhibits L-ALP and B-ALP especially), heat (inactivates L-ALP and B-ALP) and wheat germ lectin (precipitates B-ALP and C-ALP).

Causes of increased AP

  1. Drug effects
    • Glucocorticoids: In dogs, increased total ALP is due mainly to synthesis of the C-ALP isoform. Marked increases are possible (50-100 fold). Total ALP may remain high for three to six weeks, depending on the drug preparation administered (ie, short-acting vs. depot forms). Interestingly, it takes approximately 10 days for C-ALP to be induced by corticosteroids; therefore the initial increases in total ALP with corticosteroid administration is due to increases in the L-ALP, and not the C-ALP, isoform.
    • Anticonvulsants: phenobarbital, primidone, phenytoin - mild to marked increases in total activity occur, due mainly to raised L-ALP isoform. This is probably secondary to cholestasis because studies in dogs with phenobarbitone show that liver synthesis of ALP is not induced.

  2. Age effects
    ALP activity in young, growing animals of all species may be 2 - 10 times higher than in adults, due to increased B-ALP isoform. Values decrease within 3 months of age and are within adult ranges by 15 months of age. Note that some Siberian Huskies have benign (transient) familial hyperphosphatasemia. This is characterized by high ALP values (> 1100 U/L at 11 weeks of age and > 700 U/L at 16 weeks of age). It is not associated with any clinical effects and is due primarily to the bone isoform. High ALP values have also been reported in older Scottish Terriers, but this may be secondary to underlying disease, rather than a true-breed related phenomenon.

  3. Disease effects
    • Hepatobiliary disease: Increases in ALP (primarily the L-ALP isoform) is used as an indicator of cholestasis (intra- or extrahepatic) in animals. In cats, ALP is a specific but insensitive marker of hepatobiliary disease. Increases in ALP do occur in hepatobiliary disease, but the increase is less reliable and of lower magnitude compared to the situation in dogs (feline hepatic tissue contains much less ALP and serum half life is only six hours). Therefore, any increases in ALP in the cat are considered clinically relevant. The wide range of ALP activities and insensitivity of this test to cholestasis in large animals limits utility of ALP in these species.
      a) Extrahepatic cholestasis (bile duct obstruction): This causes very dramatic increases in ALP. Increases in ALP may occur before development of icterus, especially in the dog. Pancreatitis (acute or chronic) may result in increased ALP levels from swelling and/or fibrosis around the bile duct (especially in cats which have a common bile and pancreatic duct).
      b) Intrahepatic cholestasis: Localized or generalized cholestasis from hepatocyte swelling will induce ALP. Lesions that are primarily centrilobular generally cause only mild increases in ALP while lesions affecting the periphery (periportal areas) of the lobule usually result in more dramatic elevations as a result of impaired bile flow. Causes of intrahepatic cholestasis include neoplasia (primary or metastatic), hepatic lipidosis (marked increases are possible with idiopathic hepatic lipidosis in cats - lipidosis is the cause of the most dramatic increases in ALP in this species, often without concurrent elevations in GGT, which is a useful diagnostic feature), acute hepatocellular injury (intrahepatic cholestasis occurs due to hepatocellular swelling; elevated ALT levels are expected concurrently), bile sludging (occurs with anorexia, especially in cats) and periportal fibrosis and inflammation (marked increases are possible).
      c) Functional cholestasis: This is defined as decreased bile flow due to downregulation or inhibition of transporters responsible for excreting bile salts or conjugated bilirubin into bile. It occurs without any physical obstruction or impairment to bile flow. It is frequently mediated by inflammatory cytokines and has been reported in dogs with E coli infections. It likely occurs in other species as well. Cytokine-mediated cholestasis is usually characterized by high total bilirubin (due to direct and indirect bilirubin) with mild increases in hepatocellular leakage enzymes (ALT, SDH, GLDH). ALP levels may be normal in this condition.
      d) Neoplasia: In primary liver cancer (hepatocellular/biliary), marked increases in ALP are possible (due to L-ALP or C-ALP in dogs). Metastatic neoplasia often increases ALP due to localized cholestasis. In many cases of hepatic neoplasia, ALP may be the only enzyme that is increased on a panel.
      e) Acute hepatocellular injury: Mild to moderate elevations in ALP are attributed to intrahepatic cholestasis associated with hepatocellular swelling rather than hepatocellular injury per se. Concommitantly high values of ALT, SDH and GLDH would be expected.
    • Hyperadrenocorticism - Levels vary from moderate to marked (up to 100- fold) and are frequently due to induction of the C-ALP isoform (although L-ALP increases are also seen) in dogs. Up to 83-100% of dogs with Cushings disease have high C-ALP levels, but chronic endogenous stress (due to any underlying disease) may increase C-ALP and total serum ALP (up to 2-3 x normal). Therefore, C-ALP levels are a sensitive, but not specific, test for hyperadrenocorticism in dogs.
    • Increased osteoblastic activity - osteoblastic activity in response to hormones (PTH, thyroxine) or neoplasia (osteosarcoma) may increase total serum ALP due to the B-ALP isoform.
      1) Primary and secondary hyperparathyroidism (2-3x increase).
      2) Osteosarcoma
      3) Fracture healing in dogs.
      4) Hyperthyroidism in cats: Affected animals may have mild increases in total serum ALP, which is mostly due to high B-ALP with a lesser increase in the L-ALP isoform.
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