Clinical Interretation of Enzyme Activities and Concentrations

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ISRAEL JOURNAL OF VETERINARY MEDICINE
CLINICAL INTERPRETATION OF ENZYME ACTIVITIES AND CONCENTRATIONS: A REVIEW OF THE MAIN METABOLIC FACTORS AFFECTING VARIATION
Braun JP , Medaille C , Trumel C
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(a) Departement des Sciences Cliniques, (b) UMR181 Physiopathologie et Toxicologic Experimentales, INRA, ENVT, Ecole Nationale Veterinaire, 23 Chemin des Capelles, 31076-Toulouse Cedex 3, France, (c) Laboratoire Vebiotel, 4Ibis Av Aristide Briand, 94110 Arcueil, France Corresponding author: JP Braun jp.braun@envt.fr 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 "organspecific 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 2.1.1. 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 (TryspinLike Immunoreactivity). Using pharmacokinetic tools, it was shown that the normal release of CK from muscles in the horse was in the range of 2.54 ± 0.69 U.kg '.h" 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 ±0.12 U.kg'.h" 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 ± 1.0 mg/kg BW in the cow (5).
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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 VOLUME 63 (1) 2008
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(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-to-day variations in healthy animals. For instance, it was shown in healthy dogs that the CV of intraindividual variability was 31% and 36 % for plasma ASAT and ALAT respectively, as compared to 10% for plasma glucose concentrations (7). Inter-individual variability is high: CK release from muscle in 6 normal resting horses ranged from 1.6 to 3.7 U.kg '.h" (3). 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).
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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. 2.2. Routes of abnormal enzyme release into body fluids 2.2.1. 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.
Enzyme release from tissues can be increased by factors such as physical activity or decreased by inactivity. In dogs, the kinetics of C K 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 lhr 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 (17-21). 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. 2.1.2. Absorption from the digestive tract This is a minor cause of increased plasma enzyme activity which is only observed in newborns, especially ruminants. VOLUME 63 (1) 2008
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 1. The estimated CK in cows, dogs, and horses was 57,65, 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). 2.2.2. 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. 13
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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 compartment of enzymes in the extracellular
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 4.2. Renal elimination preparations was reported to be approximately the same as the Low molecular weight enzymes circulating in the plasma are plasma volume, i.e. about 5% of the body weight. This has been cleared by glomerular filtration. verified for creatine kinase in the dog (36). This is the case of alpha-amylase, lipase and trypsinogen. As a In vivo leakage of enzymes from blood cells to plasma seems result: 1. Their clearance is very rapid, with plasma half-lives in to be of little practical relevance, even when their intracellular concentration is high (e.g. LDH). In vitro, this leakage can the range of 1 to 4 hours; 2.Chronic renal failure, which causes a be quantitatively significant, especially when some degree of decrease of glomerularfiltrationrate, produces increases in their haemolysis occurs during coagulation for serum preparations plasma activity of up to 4 times the upper limit of the reference in species with fragile RBCs, such as dogs. In healthy dogs, interval. In some species,filteredenzymes are reabsorbed in the serum C K activity is about twice higher than in heparinated tubule and inactivated or degraded after reabsorption, whereas plasma (37); however, this is not of relevance in muscle damage in other species they are eliminated in urine where their output assessment because the increases in this case are much more can be monitored, e.g. alpha-amylase in humans but not in dogs (48,49). than 2-fold (38). 4. Elimination/inactivation/degradation of enzymes in the vascular compartment 4.1. Inactivation or degradation in plasma ? 4.3. Internalization and intracellular catabolism
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 N-acetylcysteine (47).
Some enzymes circulating in the plasma are cleared by internalization in tissue macrophages, especially the Kupffer During their distribution in blood, enzymes are degraded cells of the liver. Isolated hepatocytes can also degrade enzymes or inactivated to various extents depending on the enzyme. in vitro (50). The quantitative importance of this mechanism Sometimes these modifications are limited to moderate is not known. It is not specific to enzymes but mainly acts on intravascular proteolysis by circulating proteases or proteases asialoglycoproteins containing sugar residues terminated by bound to the vascular endothelium. In this case, there is no galactose, mannose orN-acetyl glucosamine (51, 52) (Figure 3). alteration of catalytic activity. In .humans, the creatine kinase In cultured rat liver cells, lactate dehydrogenase M4 isoenzyme M subunit is modified by action of carboxypeptidase N, which is internalized by a receptor recognizing mannose containing hydrolyzes the C-terminal lysine residue of the molecule, thus glycoproteins, for which it competes with creatine kinase and producing 3 isoforms of the M M isoenzyme : MM1 (native, malate dehydrogenase but not with aminotransferases (53, 54). unmodified), MM2 (minus lysine in one subunit), MM3 (minus After endocytosis, most enzymes are degraded in the lysosomes, lysine in the 2 subunits) and 2 isoforms of the MB isoenzyme. whereas a small proportion of some enzymes can be excreted This was used in the diagnosis of myocardial damage (39) before into the bile e.g., superoxide dismutase or lyzozyme in the rat. the development of new markers such as troponins. The macrophage capture mechanism is modulated by diverse Most studies dealing with the fate of intracellular enzymes in disease states. In mice infected by Riley virus the clearance of plasma were based on measurement of their catalytic activity LDH is reduced (55). In sheep, inactivation of the macrophage and not of their true mass concentration. An enzyme molecule system by gadolinium retarded the clearance of creatine kinase can have lost all or part of its activity by losing a cofactor, thereby increasing its plasma half-life, whereas activation by and not be truly degraded. Thus, most information available lipopolysaccharide administration had reverse effects (56). today does not allow differentiation of enzyme clearance due to inactivation from that due to degradation. Using porcine It is likely that similar effects are also observed in spontaneous malate dehydrogenase labelled with I, it was shown that diseases of man and animals, especially in the case of infectious the clearances of activity and radioactivity were the same in disease or of immunodepression, but this has not been rats, but clearance of the mitochondrial isoenzyme was slower documented to our knowledge. than the clearance of the cytosolic isoenzyme. However, these Although precise knowledge of the fates of intracellular observations were made with heterologous enzyme preparations and may not be representative of what happens with homologous enzymes in the extracellular compartment is still lacking, a ones. It was also shown that several dehydrogenases compete consideration of all factors likely to modify plasma enzyme activity should improve clinical interpretation. for degradation (40-43).
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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), B-Prothrombin 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 Figure 2: Variations of total amount of GGT eliminated of intracellular enzymes used as markers of cell damage. into the urine of rats before (•) and after (•) IP injection of 0.5 mmol/kg NaF. Data from (34). Urine
CNI
10 9" 87•
hO
6"
C•
D
e> 4z 3> 2" 1o-
2
4
6
i
8
ii
10 12 14 16 18 Time (h)
20
24
99
Inactivation Degradation
Elimination
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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).
n
LPS
gadolinium
control P-CK 0 Cl-CK 0 500 0,5 U/L mL/min/kg
0
50 Hepatic uptake (% dose)
100
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COVER PAGE Short-toed Eagle Ciraetus gallicus
The species spread throughout the Mediterranean basin, Russia, the Middle East and into parts of Asia (Pakistan, India and some Indonesian islands). In Israel it can be found in summer and during migration in spring and autumn. Adults are 63-68 cm long with an 185-195 cm wingspan and weigh 1.1-1.6 kg. The Short-toed Eagle is an accomplished flyer and spends more time on the wing than do most members of its genus. He is hunting from the air at heights of up to 500 meters. Its prey is mostly reptiles, mainly snakes, but also some lizards. Occasionally small mammals to the size of a rabbit;rarely birds and large insects. This eagle is generally very silent. On occasions it emits a variety of musical whistling notes. When breeding it lays only one egg, and it can live up to 17 years.
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