Alkaline Phosphatase Activity as a Clinical Chemistry Diagnostic Aid

J. Leland Raymond, DVM; Heather L. Tarpley, DVM; Kenneth S. Latimer, DVM, PhD; Perry J. Bain, DVM, PhD

Class of 2004 (Raymond) and the Department of Pathology (Tarpley, Latimer), College of Veterinary Medicine, University of Georgia, Athens, GA 30602-7388

Introduction

Alkaline phosphatase (E.C. 3.1.3.1, ALP) is an enzyme that encompasses a family of phosphatases that carry out their enzymatic activity in an alkaline environment (Fig. 1).^1,5 Although their exact physiologic roles are not recognized, they are typically membrane-bound enzymes that are often associated with brush borders. Increases in membrane permeability does not cause their release into plasma.^1,5 Two ALP genes, designated intestinal and tissue nonspecific genes, have been recognized.¹ The various isoenzymes of ALP found in different body tissues are produced from these two genes.¹ These isoenzymes vary in their glycosylation.¹ The isoenzymes of ALP that are of clinical use in veterinary medicine include the hepatic, corticosteroid-induced, bone, intestinal, and placental forms.¹ The corticosteroid-induced isoenzyme of ALP is unique to the dog. Although ALP is present in many tissues and organs throughout the body, only those listed above produce enough ALP to increase plasma or serum ALP activity.⁵

Figure 1. Molecular model of alkaline phosphatase.

Clinical Importance of Measuring Alkaline Phosphatase Activity

A serum ALP measurement’s most useful clinical attribute is its sensitivity in distinguishing hepatobiliary disease.² However, due to its numerous isoenzymes, its presence in non-hepatic tissues, and its sensitivity to drug induction, it has a low specificity for hepatobiliary disease.² Only increases in serum ALP measurements are helpful clinically. There are no significant causes of decreased ALP.⁶ Also, there is no correlation between the magnitude of increase in ALP and the prognosis or seriousness of the disease.⁶ Although mainly used for evaluation of liver disease, there are some other uses for ALP measurement. The measurement of corticosteroid-induced ALP can be used as a screening test for hyperadrenocorticism in dogs.⁵ ALP has been shown to be a prognostic indicator for canine osteosarcoma.³ Measurement of ALP concentration in urine has been used as an indicator of early toxic tubular injury.⁷ The reference ranges for ALP in large animals species are quite wide, making its measurement less useful in these animals.¹ In cats, ALP is present in low levels and has a short half life, both of which lessen its usefulness as a diagnostic aid.¹

Differentiation of ALP Isoenzymes

Individual ALP subtypes can be quantified by electrophoresis. Increases in liver and corticosteroid-induced ALP in dogs can be differentiated by heat inactivation or levamisole treatment in vitro. The corticosteroid-induced ALP is resistant to levamisole or heat inactivation, while the liver and bone isoenzymes are inhibited.¹ Wheat germ lectin can be used to differentiate bone ALP from hepatic ALP by causing precipitation of the bone ALP.¹

Hepatic Isoenzyme

The hepatic isoenzyme of alkaline phosphatase (L-ALP) is considered to be an induced (cholestatic) enzyme^1,5 that is relatively specific for cholestasis in dogs and cats.¹ Cholestasis may be defined as decreased secretion of bile from extrahepatic or intrahepatic causes.¹ Increase serum ALP activity in animals commonly is the result of the L-ALP isoenzyme because its production by biliary epithelium and hepatocytes is induced during obstructive cholestasis.^1,5 When considering various types of hepatic disease (Table 1),⁵ focal or diffuse intrahepatic or extrahepatic cholestasis causes the greatest increases in serum ALP activity.² When bile concentrations in the liver are increased, L-ALP production is triggered and L-ALP gathers on the sinusoidal (as opposed to canalicular) side of the hepatocyte (Fig. 1). This gathering of more of the enzyme on the sinusoidal (blood sinus) side is the ultimate reason for the measured increase in serum ALP activity.⁵ Clinical studies have described increases in hepatic ALP activity following the administration of anticonvulsants such as phenobarbital.^2,5 Induced ALP activity is not apparent until several days after drug administration and serum ALP activity may persist for 2-4 weeks after the drug is discontinued.⁵

Table 1. Conditions or disorders resulting in increased L-ALP.⁵

Corticosteroid-Induced Isoenzyme

The corticosteroid-induced isoenzyme of ALP (C-ALP) is unique to the dog.^2,5 This induced isoenzyme was found to be located in the area of the hepatocyte membrane that constitutes the bile canaliculi.⁴ Serum C-ALP activity increases approximately one week after steroid treatment has begun.⁵ Two main theories exist to explain the formation of C-ALP. The first theory suggests that an up-regulation of a certain gene in canine hepatocytes results in production of the C-ALP isoenzyme. The second theory speculates that corticosteroids chemically modify intestinal ALP that is transported to the hepatocytes by portal blood.⁵

Both endogenous and exogenous glucocorticoids (including topical and ophthalmic medications) can lead to noticeable increases in ALP activity in dogs.¹ Following corticosteroid treatment, the initial rise in ALP activity is due to the hepatic isoenzyme of ALP (L-ALP). After several weeks of corticosteroid therapy, C-ALP progressively becomes the major isoenzyme in plama or serum.¹ In dogs with hyperadrenocorticism, C-ALP may be the major isoenzyme present when increased serum ALP activity is first detected.¹ As stated earlier, levamisole can be used to differentiate between the different isoenzymes. The steroid and intestinal isoenzymes of ALP are resistant to levamisole inhibition, while all the other isoenzymes (hepatic, bone, placental) are susceptible to levamisole inhibition. Therefore, if the ALP assay is repeated in the presence of levamisole and the serum ALP activity remains relatively constant, then the most prominent isoenzyme is C-ALP.¹ Use of drugs such as anticonvulsants may also cause an increase in the corticosteroid induced ALP.¹

Bone Isoenzyme

The bone isoenzyme of ALP (B-ALP) causes increased enzymatic activity in the serum or plasma of young dogs (< 6 to 8 months old). ALP activity often is increased as much as 3 times above the upper end of the adult reference interval.^1,6 Lytic or proliferative lesions of bone result in increased osteoblastic activity. The increased activity of B-ALP adds to the total activity of ALP in serum.^1,5 Furthermore, active resorption of bone also can result in increased B-ALP activity.¹ As stated above, wheat germ lectin can be used to precipitate B-ALP, allowing its differentiation from L-ALP.¹ Benign familial hyperphosphatasemia is another cause of increased B-ALP activity in the serum or plasma in some lines of Siberian Huskies.⁵

Intestinal and Placental Isoenzymes

The intestinal isoenzyme has a very short half-life and does not significantly add to the serum ALP level in dogs and cats.¹ Rats have a high intestinal ALP activity, especially after feeding. Thus, determination of serum ALP activity in rats as a marker of cholestasis is only valid in fasted animals. Cecal ALP activity in horses contributes significantly to plasma ALP activity.¹ Because of the broad reference range for ALP activity in large animals, gamma-glutamyl transpeptidase is a better marker of cholestasis in these species.

A placental isoenzyme of ALP also has been described. This isoenzyme may cause increased serum or plasma ALP activity during late term pregnancy.¹

Conclusion

ALP is most helpful in detecting hepatobiliary disease in small animals. If an increase in serum ALP activity is seen, it is important to consider young age, drug therapy, and hyperadrenocorticism as causes of increased enzyme activity before proceeding with a hepatic biopsy.⁶ Once these possibilities have been excluded, hepatobiliary disease is a more likely diagnosis. With the correct diagnostic approach, serum ALP activity can be a useful diagnostic aid (Fig. 2).

References

1. Bain PJ: Liver. In: Latimer KS, Mahaffey EA, Prasse KW: Duncan and Prasse's Veterinary Laboratory Medicine: Clinical Pathology, 4th ed. Ames, Iowa State Press, 2003, pp. 193-214.

2.Ettinger SJ, Feldman EL: Textbook of Veterinary Internal Medicine, Diseases of the Dog and Cat, 5th ed, vol 2. Philadelphia, W.B. Saunders Co., 2000, pp. 1282-1283;1465.

3. Kirpensteijn J, Kik M, Rutteman GR, Teske, E. 2002. Prognostic significance of a new histologic grading system for canine osteosarcoma. Vet Pathol 39:240-246.

4. Sanecki RK, Hoffmann WE, Gelberg HB, Dorner JL. 1987. Subcellular location of corticosteroid-induced alkaline phosphatase in canine hepatocytes. Vet Pathol 24:296-301.

5. Stockham SL, Scott MA. Fundamentals of Veterinary Clinical Pathology. Ames, Iowa State Press, 2002, pp. 438, 446-450.

6.Willard MD, Tvedten H, Turnwald GH. Small Animal Clinical Diagnosis by Laboratory Methods, 3rd ed. Philadelphia, W. B. Saunders Company, 1999, pp. 199-201.

7. Wisløff H, Flåøyen A, Ottesen N, Hovig T. 2003. Narthecium ossifragum (L.) huds causes kidney damage in goats: Morphologic and functional effects. Vet Pathol 40:317-327.

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