Clinical Biochemistry of Domestic Animals (Sixth Edition) - UMK ...

IV. Specific Enzymes
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and early indicator of chemical-induced nephrotoxicity with
several compounds including maleic acid, aminoglycosides,
and cyclosporine ( Clemo, 1998 ; Graur et al. , 1995 ; Nahas
et al. , 1997 ; Rivers et al. , 1996 ). Furthermore, both 24h
excretion of GGT and the GGT activity:creatinine ratio of
single urine samples have proven useful in detecting tubular
injury in dogs ( Gosset et al. , 1987 ; Rivers et al. , 1996 ).
Although there is no apparent circadian variation ( Uechi
et al. , 1994 ), it has been suggested that the within-day variation
of the GGT activity:creatinine ratio limits its usefulness
(Gosset et al. , 1987 ). As in dogs, the urine GGT activity:
creatinine ratio in horses and cattle has proven of value in
detecting nephrotoxicity ( Meyer et al. , 2005 ; Rossier et al. ,
1995 ; Ulutas and Sahal, 2005 ). The magnitude in all species
is greatest in the acute phase of injury after which the GGT
activity in urine drops rapidly during the chronic stage.
These studies support the use of urine GGT activity as a
screening test of potentially nephrotoxic drug exposure.
Evaluation of more than one enzyme and at multiple time
points is required to properly evaluate for nephrotoxicity. It
should also be noted that the urinary GGT activity:creatinine
ratio is extremely sensitive and increases can be observed
with no clinical signs of nephrotoxicity or azotemia ( van der
Harst et al. , 2005 ). For example, normal horses and horses
with pneumonia who are both treated with gentamycin show
significant increases in the urinary GGT activity:creatinine
ratio but retain normal serum creatinine concentrations
(Rossier et al. , 1995 ).
Dogs treated with glucocorticoids or dogs with hyperadrenocorticism
generally have increased serum GGT activity
( Abraham et al. , 2005 ; DeNovo and Prasse, 1983; Solter
et al. , 1994 ). However, the ratio of serum GGT-to-liver
tissue GGT activity in glucocorticoid-treated dogs is similar
to that of untreated controls, suggesting that increased
serum GGT activity is a result of increased liver tissue
GGT and increased synthesis of the enzyme ( Solter et al. ,
1994 ).
F . Alkaline Phosphatase
The alkaline phosphatases (ALP) (EC 3.1.3.1) have been
relatively well studied. They have been shown to hydrolyze
a range of monophosphates or pyrophosphates at alkaline
pH, as well as at physiological pH although at a lesser rate.
There are likely several in vivo functions of ALP. A role for
ALP in bone mineralization had been speculated since the
1920s and was first substantiated in human beings who suffer
from hypophosphatasemia, which results from several
different mutations that lead to a nonactive ALP and markedly
defective bone mineralization in children ( Mumm
et al. , 2002 ; Whyte et al. , 1996 ) . The role of ALP in bone
mineralization has been subsequently confirmed in numerous
studies using bone ALP gene knockout mice to create
hypophosphatasemia, resulting in the impaired mineralization
of bone ( Anderson et al. , 2004 ). One specific function
of ALP in bone is the hydrolysis of pyrophosphate, which
is a potent inhibitor of mineralization, thus allowing mineralization
to proceed. Other suggested functions of ALP
are the in vivo dephosphorylation of bacterial endotoxin,
which diminishes its toxic effects ( Poelstra et al. , 1997 ;
van Veen et al. , 2005 ; Xu et al. , 2002 ), and as a rate-limiting
step in intestinal fat absorption ( Narisawa et al. , 2003 ).
Although many tissues or cell types have some ALP
activity, cells from liver, bone, kidney, intestinal mucosa, and
placenta have the greatest ALP activity on a per gram of tissue
basis with intestinal mucosa having the most ( Clampitt
and Hart, 1978 ; Nagode et al. , 1969 ) . Serum ALP activity,
however, is not generally a reflection of tissue concentration.
In most domestic species, intestinal ALP (IALP) is
not found in serum, and liver, which has relatively low ALP
activity, contributes over half the serum activity.
The alkaline phosphatases are ectoenzymes attached to
the cell membrane via a hydrophobic phosphatidylinositolglycan
(PIG) anchor. Extraction of ALP from tissue requires
butanol, bile salts, or detergent in the presence of an acidic
buffer to allow the hydrolytic activity of endogenous
phosphatidylinositol-specific phospholipase D (PIPLD)
to cleave the ALP PIG anchor ( Low, 1987 ; Solter and
Hoffmann, 1995 ; Solter et al. , 1997 ). In intestine, especially
the small intestines, ALP is located on the tips of
villi of the enterocytes ( Watanabe and Fishman, 1964 ),
whereas in kidney the ALP is located on the luminal surface
of the proximal tubular epithelial cells ( Wachstein
and Bradshaw, 1965 ). Although the ALP in bone is well
recognized to be located on the osteoblasts and the matrix
vesicles derived from osteoblasts, staining liver for ALP
activity reveals that most of the ALP is found on the bile
canalicular surface of hepatocytes in normal animals
although this may vary with species. During conditions in
which hepatic ALP is increased, sinusoidal and lateral surfaces
of the hepatocytes also show substantial ALP activity
( Ogawa et al. , 1990 ; Sanecki et al. , 1987 ; Solter et al. ,
1997 ). Because many newly synthesized bile canalicular
(apical) membrane proteins traffic from the Golgi to the
sinusoidal (basolateral) membrane before their transcytosis
to the bile canalicular surface of hepatocytes, the presence
of ALP on sinusoidal membrane during disease conditions
likely represents increased synthesis of ALP rather than a
difference in enzyme trafficking ( Kipp and Arias, 2000 ;
Maurice et al. , 1994 ; Zegers and Hoekstra, 1998 ). ALP
activity is presumably present on the sinusoidal membrane
at all times, but because of the short time in residence
before transcytosis to the canalicular membrane and relatively
small amount of ALP, the insensitivity of enzymatic
staining and light microscopy make it difficult to visualize
the small fraction of ALP present.
In the human, chimpanzee, and orangutan, at least three
genes express ALP and are named for the primary organ
of their expression. These are the intestinal ALP, placental
ALP, and the tissue-nonspecific or bone/liver/kidney
ALP ( Goldstein et al. , 1982 ). In humans, a fourth gene is