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

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Chapter | 12 Diagnostic Enzymology of Domestic Animals
this pathway was provided by evidence of disruptive changes
to tight junctions in cholestasis and by experimental observations
of infused horseradish peroxidase moving through
tight junctions (Boyer, 1993; Lowe et al. , 1988 ). However,
increases of serum ALP and GGT activity have been shown
to occur in the absence of increased biliary pressure and any
evidence of alterations in tight junctions, and it is unlikely
that this paracellular pathway is a significant contributor
to the appearance of cholephilic enzymes in serum in most
cases ( Debroe et al. , 1985 ; Putzki, 1989; Toyota et al. ,
1983). An alternative pathway of movement of cholephilic
enzymes to blood has been suggested as a retrograde vesicular
transport system following the observation that retrograde
infusion of ferritin and polymeric and secretory forms
of IgA undergo reversed transcytosis from the biliary or apical
surface of the hepatocytes to the basolateral or sinusoidal
surface during cholestasis (Carpino et al. , 1981; Jones et al. ,
1984 ). However, neither of these pathways allows explanation
of the appearance of ALP and GGT activity in blood in
the absence of cholestasis. Studies using a choledochocaval
shunt model show that within 12 h of shunting of bile or taurocholic
acid into blood, there is a marked induction of ALP
synthesis, appearance of ALP on the basolateral membranes,
and a parallel increase in serum ALP activity ( Ogawa et al. ,
1990 ). This occurs in the absence of increased biliary pressure
and any evidence of alterations in tight junctions
(Toyota et al. , 1983). These observations along with others
led to a third and more likely mechanism of the appearance
of cholephilic enzymes, especially ALP, in blood. The basolateral
appearance of enzymes typically considered to be on
the apical membrane or bile canalicular surface is not unexpected
as following synthesis all apical membrane proteins
are believed to first be transported to the basolateral surfaces
before vesicular transport to their final site on the bile
canalicular membrane ( Bartles et al. , 1987 ; Maurice et al. ,
1994 ; Schell et al. , 1992 ). Therefore, these so-called biliary
enzymes or proteins have a brief period of residence on the
sinusoidal surface of the hepatocytes with the enzyme on the
external surface in the space of Disse so that they can potentially
be released into blood if and when a suitable release
mechanism exists. In addition, the quantity of enzyme available
on the basolateral membrane and accessible for release
into blood is increased at any time there is increased synthesis
of the enzyme as described previously with the choledochocaval
shunt model, as occurs with cholestasis and
as may occur during hormonal or drug-driven induction of
enzyme synthesis ( Ogawa et al. , 1990 ; Putzki et al. , 1989 ;
Solter and Hoffmann, 1999 ; Solter et al. , 1997 ). Although
positioned on the basolateral membrane facing the space of
Disse for a brief period allows the possibility of release into
blood, this does not occur without appropriate conditions to
cleave the hydrophobic anchor. Alkaline phosphatase and
5 nucleotidase are anchored to the membrane via a hydrophobic
phosphatidylinositol glycan anchor, whereas GGT is
anchored via a transmembrane peptide therefore requiring
different mechanisms of release. These release mechanisms
will be discussed specifically in the sections dealing with
each of these enzymes.
E . Blood Clearance Rates of Enzymes
The amount of enzyme activity in blood is very dependent
on the rate of clearance of the enzyme from the blood
following its release from cells. The half-lives of various
enzymes range from minutes to hours to days, and the
mechanisms or factors that determine the half-life of the
various enzymes vary.
The actual mechanisms of removal of enzymes from
blood are not well established but likely are varied.
Some small-molecular-weight enzymes such as amylase
and lipase are, in part, filtered through the glomerulus.
Enzymes that are glycoproteins are likely endocytosed by
the galactose receptors on hepatocytes either directly via
exposed galactose molecules or after loss of terminal sialic
acid molecules resulting in exposed galactose residues, or
are endocytosed by mannose receptors on Kupffer cells.
Other enzymes may be degraded by proteases or are labile
and activity is lost while the protein continues to circulate.
The rate of clearance of enzymes from blood can be
affected by disease and may complicate the correct interpretation
of diagnostic test results. For example, pancreatic
amylase activity, which is normally cleared by the kidneys,
will increase in patients with renal failure because of the
decreased glomerular filtration rate. A false-positive test
result for pancreatitis could result.
F . Enzyme Induction
Changes to serum enzyme activity may in some cases
reflect changes in enzyme production by the cells, rather
than cell injury. Although there is certainly evidence of
varying concentrations of cytoplasmic enzymes in cells,
these generally do not result in dramatic changes in the
serum activity of these enzymes. Marked increases in
serum enzymes as a result of induction are most often
associated with enzymes that are membrane bound where
they can readily be released from the membrane into the
lymphatics or blood or secreted by the cell. This induction
can be as a result of hormonal changes, pathophysiological
events such as cholestasis, or can be drug induced.
IV . SPECIFIC ENZYMES
A . Alanine Aminotransferase
Alanine aminotransferase (EC 2.6.1.2) (ALT), formerly known
as glutamic pyruvate transaminase, catalyzes the reversible
transamination of L-alanine and 2-oxoglutarate to pyruvate
and L-glutamate. ALT, along with other transaminases,