ISRAEL JOURNAL OF
VETERINARY MEDICINE
Vol. 63 - No. 1 2008
CLINICAL INTERPRETATION OF ENZYME ACTIVITIES AND
CONCENTRATIONS:
A REVIEW OF THE MAIN METABOLIC FACTORS AFFECTING
VARIATION
Braun JPa,b ,Médaille Cc ,Trumel Ca
(a) Département des Sciences Cliniques, (b) UMR181 Physiopathologie et
Toxicologie Expérimentales, INRA, ENVT, Ecole Nationale Vétérinaire, 23 Chemin
des Capelles-67013 ,Toulouse Cedex 3, France, (c) Laboratoire Vébiotel, 41bis Av
Aristide Briand, 94110 Arcueil, France
Corresponding author: JP Braun
jp.braun@envt.fr
+33 561 193 844
Keywords: dog, horse, cattle, enzyme, plasma, metabolism
Summary
The activity of enzymes in plasma and other body fluids can be altered by their rate
of release from organs, by the distribution of the enzyme in the extracellular
compartment, and by the rate and routes of enzyme elimination and inactivation.
These factors are influenced by individual variability, disease, drugs, exercise, etc.,
which need to be considered to ensure a more efficient diagnostic use of enzymes in
veterinary clinical practice.
INTRODUCTION
The diagnostic use of enzymes in veterinary and human clinical pathology is mostly
aimed at detecting, evaluating and monitoring organ damage based on the increase
in“ organ-specific enzymes( .”See reviews 1, 2). However, enzymes are also used to
evaluate the synthetic capacity of an organ, to diagnose the adverse effects of toxic
compounds which are enzyme inhibitors, and to monitor the inductive activity of
exogenous compounds or enzyme activation by minerals or vitamins (Table I).In all
these cases, interpretation is usually based on physiopathological data regarding
input of the enzyme into a body fluid, most often blood plasma, and sometimes urine,
digestive contents, cerebrospinal fluid, etc., with little consideration for the distribution
of enzymes within the body and the clearance of enzymes from the body fluids.
When interpreting the decrease in plasma activity of an enzyme used as a marker of
organ damage, the plasma half-life of enzymes is sometimes used to address their
clearance .
1. Criteria of interpretation of an enzyme activity or concentration
Enzyme activities in plasma and other body fluids should be interpreted by
integrating all the factors that may influence them, i.e. rate of release from the organ
or tissue, distribution within the body, and rate and route(s) of clearance from the
body (Figure 1). Unfortunately the answers to some of the following questions are
often lacking, e.g. in which organ is the enzyme located in the species of interest?
What is the concentration of the enzyme in this organ? By which route does the
enzyme reach the plasma ?Is the enzyme (or an isoenzyme) present in blood cells
and can haemolysis interfere with interpretation? In which body compartments is the
enzyme distributed? Does it remain in the plasma ,urine, or CSF or is it distributed
into the extracelllular compartment? Is it internalized by organs, for example, the
liver ?What are the mechanisms of enzyme clearance? Which organs are involved in
enzyme clearance? What is the route of elimination ? What is the kinetics of
elimination or inactivation?
2. Normal mechanisms
.1.2.2 Diffusion from intact cells
Cells have long been viewed as impermeable structures allowing only limited
exchanges between the intracellular and extracellular compartments. More
specifically, protein escape from cells has been considered to be abnormal except for
secreted proteins and enzymes such as coagulation factors or digestive enzymes. It
is now accepted that all cells‘ leak ’some of their contents including proteins, without
any sign of cell damage. Only a few precise data are available. For instance, it has
been calculated that about 0.1% to 0.01 % of the total amount of trypsinogen
synthesized leaks from canine pancreas acinar cells into the extracellular
compartment and thence into the plasma where it is measured as TLI (Tryspin-Like
Immunoreactivity). Using pharmacokinetic tools, it was shown that the normal release
of CK from muscles in the horse was in the range of 0.30 ± 4..2 U.kg.7-h
7, while
the
intracellular concentration of this enzyme ranged from 3800 to 5440 U/g of fresh
equine muscle (3). Similar studies on cows showed a rate of release of 0.69 0.74 ±
U.kg.7-h 7-and a muscle concentration of 2900 U/g (4). This amounts to a daily total
release of the amount of enzymes present in a few mg of muscle tissue, e.g. 5.8 ±
7.0mg/kg BW in the cow (5.)
Little information is available about the factors of variation in enzyme release by
normal tissues .
Intra-individual variations are moderate; for instance in cows individual CVs ranged
from 6% to 29 %, which probably accounts for its moderate variability in plasma
enzyme activity (6). Even if release by cells is only one of the factors determining
plasma activity ,its variability is likely significant as shown by relatively high day-today variations in healthy animals. For instance, it was shown in healthy dogs that the
CV of intra-individual variability was 31% and 36 % for plasma ASAT and ALAT
respectively, as compared to 10% for plasma glucose concentrations .)1(
Inter-individual variability is high: CK release from muscle in 6 normal resting
horses ranged from 1.6 to 3.7 U.kg.7-h .)6( 7-One possible reason for this difference
may be the inter-individual variation of enzyme concentration in organs which is high
in horses and other species (8-12). This may be one of the causes of the large
reference intervals for plasma enzyme activities, for instance 60 to 280 U/L for ASAT
in
sheep
according
to
Kaneko (13 .)
Enzyme release from tissues can be increased by factors such as physical activity
or decreased by inactivity. In dogs, the kinetics of CK entry into the thoracic duct
following experimental muscle damage to the hind leg is accelerated by movement of
the leg (14). Physical effort( without any clinical sign of damage) leads to an
increased enzyme release by muscles. In trotting horses, this increase was scarcely
measurable for distances less than 30 km but became more intense after 60 km,
although it caused only a twofold increase of plasma CK in this case (3). Similar
effects were observed in untrained Beagle dogs after a 1hr run at 9 km/h (15, 16 .)
Nutritional factors can influence enzyme concentration in organs and their release.
This has been especially studied in the fattening of birds and ruminants .)47-71( Drug
treatment may also increase the intracellular concentration of enzymes and their
release from tissues. This has long been known for canine liver enzyme induction by
phenobarbital and glucocorticoids in dogs (22, 23), which induce the synthesis of
GGT and ALP, thus increasing the plasma activity of these enzymes ,even after
topical application of glucocorticoids (24, 25.)
Induction of enzyme synthesis can also be caused by cancer. As a consequence,
the increased concentration of enzymes leads to their increased leakage and thus of
their plasma activity. This is the case of liver GGT in rats and mice for which the
activity is high in foetuses and newborns and low in adults (26). When liver cancer is
induced or grafted ,the hepatocytes synthesize more GGT which leads to an increase
in plasma GGT activity. In contrast, the amount of enzyme synthesized by an organ
can decrease when most of its cells have been destroyed, in the case of liver fibrosis
or cirrhosis or in pancreatic insufficiency of the dog. In these cases, leakage of
enzymes from the cells is reduced causing a decrease in their activities or
concentrations. For instance ,in dogs and cats, a decrease in TLI concentration is the
gold standard for pancreatic insufficiency (27, 28), and an increase in coagulation
times is a diagnostic criterion for liver insufficiency.
.1.2.1 Absorption from the digestive tract
This is a minor cause of increased plasma enzyme activity which is only observed
in newborns, especially ruminants. During the first hours of life, the intestinal mucosa
is still permeable to macromolecules, thus allowing absorption of colostrum proteins,
including enzymes. If the concentration of a given enzyme in the colostrum is high,
the resulting plasma increase is very high. In cattle, sheep, goats ,and buffaloes the
increase of plasma GGT in newborns can be used as an efficient and inexpensive
test of colostrum intake (29-32), as the concentration of plasma immunoglobulins is
highly correlated to plasma GGT activity.
.1.1 Routes of abnormal enzyme release into body fluids
.1.1.2 Cell damage
Cell damage can range from total irreversible cell necrosis to moderate reversible
alteration of membrane impermeability. In any case, the flux of enzymes released
from the intracellular compartment is increased, but the extracellular compartment is
not the same for all organs or tissues ,as shown in the three following examples.
Hepatocytes are in direct contact with plasma of the sinusoid capillaries, the
fenestrae of which allow complete exchange of macromolecules with the pericellular
space of Disse. Thus in the case of liver damage the total amount of enzyme
released from cells immediately enters the plasma compartment. Muscle cells are
irrigated by capillaries which have very small pores precluding direct transfer of
macromolecules into the plasma. When muscle cell damage occurs, enzymes such
as CK (Mr ~85000) are first released into the pericellular compartment from which
they are collected by lymphatics into the plasma .Thus there is a delay between cell
damage and plasma enzyme activity increase, and this delay varies with physical
activity which causes increased lymph drainage. Moreover, enzymes can be
degraded or inactivated during their transfer, so their bioavailability is less than .7
The estimated CK in cows, dogs, and horses was ,3. ,.1 and 75 % respectively (4 .)
The kidney tubular cells lie on a basal membrane with their apical membrane facing
the tubule lumen. In the case of tubular cell damage ,there is no increase in plasma
enzyme activity, as enzymes are released immediately and completely into the urine
(33), except in the case of very intense kidney damage. This allows early and
sensitive detection of even moderate kidney damage. Moreover ,as urine enzymes
are cleared with each urination, the amount of enzymes present in urine at any one
time precisely reflects the damage which has occurred very recently. This means that
the progress of kidney damage can be monitored. This has applications in
experimental toxicology (34, 35) (Figure 2.)
.1.1.1 Reflux from excretion route
In practice the only case is reflux from bile in the case of cholestasis. When the bile
flux is slowed down or blocked, the pressure in the bile ducts and ductules is
increased. This causes paracellular reflux of the bile contents from the
interhepatocyte ductules into the sinusoid capillaries. This is the route by which
enzymes such as GGT and ALP, which are present in high concentrations in the
membrane of the biliary pole of the hepatocytes, reach the plasma in the case of
cholestasis. Moreover ,their transfer to the plasma is increased by the detergent
effect of bile acids which solubilise the enzymes from the membranes and induce
their synthesis by hepatocytes in the case of cholestasis.
.3 Distribution of enzymes in the extracellular compartment
When enzymes reach a body fluid, it is assumed that they stay in this compartment,
especially in the blood in which exchanges with blood cells do not seem to occur .
The volume of distribution of enzymes after injection of purified enzyme preparations
was reported to be approximately the same as the plasma volume, i.e. about 5% of
the body weight. This has been verified for creatine kinase in the dog (36.)
In vivo leakage of enzymes from blood cells to plasma seems to be of little practical
relevance, even when their intracellular concentration is high (e.g. LDH). In vitro, this
leakage can be quantitatively significant, especially when some degree of haemolysis
occurs during coagulation for serum preparations in species with fragile RBCs, such
as dogs. In healthy dogs ,serum CK activity is about twice higher than in heparinated
plasma (37); however, this is not of relevance in muscle damage assessment
because the increases in this case are much more than 2-fold (38.)
.4 Elimination/inactivation/degradation
of
enzymes
in
the
vascular
compartment
.4.2 Inactivation or degradation in plasma?
During their distribution in blood, enzymes are degraded or inactivated to various
extents depending on the enzyme. Sometimes these modifications are limited to
moderate intravascular proteolysis by circulating proteases or proteases bound to the
vascular endothelium. In this case ,there is no alteration of catalytic activity. In
humans, the creatine kinase M subunit is modified by action of carboxypeptidase N,
which hydrolyzes the C-terminal lysine residue of the molecule, thus producing 3
isoforms of the MM isoenzyme : MM1( native, unmodified), MM2 (minus lysine in one
subunit), MM3 (minus lysine in the 2 subunits) and 2 isoforms of the MB isoenzyme.
This was used in the diagnosis of myocardial damage (39) before the development of
new markers such as troponins.
Most studies dealing with the fate of intracellular enzymes in plasma were based
on measurement of their catalytic activity and not of their true mass concentration.
An enzyme molecule can have lost all or part of its activity by losing a cofactor, and
not be truly degraded. Thus, most information available today does not allow
differentiation of enzyme clearance due to inactivation from that due to degradation.
Using porcine malate dehydrogenase labelled with74. I, it was shown that the
clearances of activity and radioactivity were the same in rats, but clearance of the
mitochondrial isoenzyme was slower than the clearance of the cytosolic isoenzyme.
However, these observations were made with heterologous enzyme preparations
and may not be representative of what happens with homologous ones. It was also
shown that several dehydrogenases compete for degradation (40-43.)
Reversible inactivation of enzymes by loss of a cofactor has no consequence on
the in vitro measurement of enzyme activity, because most analytical techniques of
enzyme activity measurement are optimized by the addition of cofactors, such as
pyridoxal phosphate for aminotransferases, and magnesium for alkaline phosphatase
(44-46). Similarly, partial in vivo oxidation of thiol radicals of the catalytic site inhibit
creatine kinase. This is reversed in vitro by addition of reducing agents such as Nacetylcysteine (47).
4.2. Renal elimination
Low molecular weight enzymes circulating in the plasma are cleared by glomerular
filtration.
This is the case of alpha-amylase, lipase and trypsinogen. As a result: 1. Their
clearance is very rapid, with plasma half-lives in the range of 1 to 4 hours; 2.Chronic
renal failure, which causes a decrease of glomerular filtration rate, produces
increases in their plasma activity of up to 4 times the upper limit of the reference
interval. In some species, filtered enzymes are reabsorbed in the tubule and
inactivated or degraded after reabsorption, whereas in other species they are
eliminated in urine where their output can be monitored, e.g. alpha-amylase in
humans but not in dogs (48, 49).
4.3. Internalization and intracellular catabolism
Some enzymes circulating in the plasma are cleared by internalization in tissue
macrophages, especially the Kupffer cells of the liver. Isolated hepatocytes can also
degrade enzymes in vitro (50). The quantitative importance of this mechanism is not
known. It is not specific to enzymes but mainly acts on asialoglycoproteins containing
sugar residues terminated by galactose, mannose or N-acetyl glucosamine (51, 52)
(Figure 3). In cultured rat liver cells, lactate dehydrogenase M4 isoenzyme is
internalized by a receptor recognizing mannose containing glycoproteins, for which it
competes with creatine kinase and malate dehydrogenase but not
with
aminotransferases (53, 54). After endocytosis, most enzymes are degraded in the
lysosomes, whereas a small proportion of some enzymes can be excreted into the
bile e.g., superoxide dismutase or lyzozyme in the rat.
The macrophage capture mechanism is modulated by diverse disease states. In
mice infected by Riley virus the clearance of LDH is reduced (55). In sheep,
inactivation of the macrophage system by gadolinium retarded the clearance of
creatine kinase thereby increasing its plasma half-life, whereas activation by
lipopolysaccharide administration had reverse effects (56).
It is likely that similar effects are also observed in spontaneous diseases of man
and animals, especially in the case of infectious disease or of immunodepression, but
this has not been documented to our knowledge.
Although precise knowledge of the fates of intracellular enzymes in the extracellular
compartment is still lacking, a consideration of all factors likely to modify plasma
enzyme activity should improve clinical interpretation.
Table I: Examples of possible diagnostic uses of enzyme levels in animal
(P-, E-, F-, B- : activity concentration in plasma, RBC, faeces, blood)
___________________________________________________________________
_______
• Detection of organ damage
P-ALT (liver, dog, cat), P-GLDH (liver, ruminants, equids), U-NAG (kidney, dog)
Reflux of secreted enzyme
P-ALP, P-GGT from bile into plasma
• Drug induction
P-ALP, P-GGT (glucocorticoids, phenobarbital, dog)
• Organ secreting activity
P-TLI (exocrine pancreas, dog, cat), F-Elastase (exocrine pancreas dog), BProthrombin time (liver, all species)
• Inhibition
E-ALAD (lead, all species, esp. cattle), P-Cholinesterases (organophosphate
derivatives, carbamates, all species, including wildlife)
• Activation
E-GSHPx (selenium), E-SOD (copper), E-Tranketolase (thiamin)
Figure 1: Main routes of release, distribution and clearance of intracellular enzymes
used as markers of cell damage.
Figure 2: Variations of total amount of GGT eliminated into the urine of rats
before (■) and after (□) IP injection of 0.5 mmol/kg NaF. Data from (34).
Figure 3: Effect of the number of galactosyl residues added to the SOD
molecule on its uptake by rat liver. Data from (52)
Figure 4. Effects of in vivo Kupffer cell activation by lipopolysaccharide (LPS)
and inhibition by gadolinium on the plasma activity (P-CK, ■) and clearance
(Cl-CK, □) of creatine kinase in sheep. Data from (56).
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