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
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
b) Tissue non-specific-ALP gene/isoenzyme:
This produces the remaining isoforms of ALP, including L-ALP, B-ALP,
placental-ALP and leukocyte -ALP.
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.
The serum half life of ALP varies with species and isoform:
- 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
- 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.
- 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.
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
- 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.
- 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.
- 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
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).
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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