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

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Chapter | 12 Diagnostic Enzymology of Domestic Animals
expressed in the germ cell that is similar to the placental
isoenzyme (Millan and Manes, 1988). In domestic species,
there are two genes, namely the intestinal gene and the
tissue-nonspecific ALP gene that is expressed in bone,
liver, kidney, and placenta and to a lesser extent in some
other tissues. Although true ALP isoenzymes result from
specific genes, posttranslational modification (primarily
varied glycosylation) is generally organ specific and results
in additional isoforms. For example, liver ALP and bone
ALP are the products of the same gene but are glycosylated
differently, resulting in isoforms or isoenzymes that
can be differentiated by a number of techniques. True ALP
isoenzymes expressed by different genes differ enzymatically,
biochemically, and antigenically, and they respond to
inhibitors such as L-phenylalanine and levamisole in a different
manner; isoforms of ALP tend to behave enzymatically
and antigenically in a similar manner and are equally
inhibited by L-phenylalanine and levamisole, although
there are some exceptions. Although these isoforms are
not true isoenzymes, historical and clinical precedence
has resulted in these isoforms also being referred to as
isoenzymes.
Liver ALP is highly glycosylated with terminal sialic
acid residues, which results in a marked anodal migration
on electrophoresis. It is generally inhibited by greater
than 95% with levamisole but is relatively insensitive to L-
phenylalanine inhibition. LALP is only moderately sensitive
to heat inhibition at 56°C.
Bone ALP is a product of the TNS ALP gene, as is the
liver ALP, but has a slightly slower anodal electrophoretic
migration and is more sensitive to heat inhibition ( Hoffmann
and Dorner, 1975 ). The bone ALP isoform is more susceptible
to precipitation by wheat germ lectin (WGL) than
is the LALP as a result of differing glycosylation ( Hank
et al. , 1993 ; Kidney and Jackson, 1988 ; Sanecki et al. ,
1993 ). Antibodies produced against liver ALP or bone ALP
generally cross-react with the other isoforms from the same
gene. However, there are commercially available immunoassays
for human BALP that have been validated for use in
canine, feline, and equine BALP analysis ( Allen et al. , 2000 ;
Delaurier et al. , 2002 ; Jackson et al. , 1996 ).
Intestinal ALP (IALP) is a product of the intestinal ALP
gene, and it is distinctly different from BALP and LALP.
IALP is more heat stable and more readily inhibited by
L-phenylalanine, but it shows less than 10% inhibition with
levamisole at concentrations adequate to inhibit greater
than 95% of BALP and LALP (Eckersal et al. , 1986;
Hoffmann et al. , 1987 ; Nagode et al. , 1969 ). IALP is generally
thought of as an asialoglycoprotein; however, some
identifiable sialic acid is present on canine IALP ( Sanecki
et al. , 1990 ). The reduced number of sialic acid residues
is responsible for the fact that IALP has minimal anodal
migration on agarose or cellulose acetate electrophoresis.
Typically, IALP produces a broad band rather than a
sharp narrow band when compared to LALP and BALP.
Alkaline phosphatases in other organs, such as kidney
and placenta, are usually a product of the TNS gene. These
alkaline phosphatases are not fully sialated and have less
anodal migration on electrophoresis than BALP and LALP.
However, the ALP extracted from horse kidney is likely
a product of the intestinal gene as it is similar to IALP in
levamisole inhibition and antigenic recognition by antiequine
IALP antibodies ( Hoffmann et al. , 1983a ).
The so-called corticosteroid-induced isoenzyme of
ALP (CALP) has been identified in dogs treated with corticosteroids,
dogs with hyperadrenocorticism, or in some
older dogs with chronic disease or possibly chronic stress
(Sanecki et al. , 1990 ; Solter et al. , 1993 ; Wellman et al. ,
1982 ). This enzyme has not been identified in any other species;
however, it is similar to one of the two forms of ALP
expressed in the liver of rabbits (Noguchi et al. , 1987).
CALP is a highly glycosylated isoform of IALP, produced
in the liver from the intestinal gene. It has markedly greater
anodal migration than IALP on cellulose acetate electrophoresis,
but it is antigenically similar to IALP with both monoclonal
and polyclonal antibodies and responds to levamisole
and L-phenylalanine inhibition in a similar manner. It also
has the identical N-terminal amino acid sequence to IALP
but has markedly higher concentrations of N-acetylglucosamine,
mannose, galactose, and sialic acid. Hence, CALP
differs from IALP only in glycosylation. Northern blot
analysis with a 1338 bp cloned segment of the CALP gene,
which is identical to the IALP sequence, has confirmed the
liver as the origin of CALP production ( Wiedmeyer et al. ,
2002a, 2002b ).
The epididymal and seminiferous tubular epithelium
of the dog is rich in ALP activity. This ALP is a product
of the TNS gene, but it is glycosylated differently than
either BALP or LALP based on electrophoretic migration
( Kutzler et al. , 2003 ). ALP activity is also present in epididymal
fluid from horses ( Gobella et al. , 2002).
Serum alkaline phosphatase isoenzyme analyses have
been conducted in many species with numerous publications
describing techniques and diagnostic value of the analyses.
The techniques described include electrophoresis on a number
of mediums, isoelectric focusing, heat inhibition, chemical
inhibition, immunochemistry, and selective lectin binding.
Although electrophoresis is still in use, a combination of inhibition
with levamisole and selective precipitation of BALP
with wheat germ lectin is an effective means to quantitatively
evaluate BALP, LALP, and CALP in dog serum; BALP,
LALP, and IALP in rat serum; and BALP and LALP in horse,
cat, and cow serum ( Hank et al. , 1993 ; Hoffman et al. , 1988;
Hoffmann et al. , 1994; Sanecki et al. , 1993 ). Very likely, the
combination of levamisole inhibition and wheat germ lectin
precipitation will be compatible with many other species that
have not yet been tried. Immunoassays for BALP in canine,
feline, and equine serum have also been used ( Allen et al. ,
2000 ; Delaurier et al. , 2002 ; Delaurier et al. , 2004 ; Jackson
et al. , 1996 ; Price et al. , 1995 ).