PhysiologyCreatine kinase (CK) is a "leakage" enzyme present in high concentrations in the cytoplasm of myocytes (skeletal and cardiac primarily) and brain and is the most widely used enzyme for evaluation of muscular disease. In muscles, CK functions by making ATP available for contraction by phosphorylating ADP from phosphocreatinine. Phosphocreatine is the major storage form of high energy phosphate in muscle. Creatine kinase is a dimeric molecule composed of two types of monomers, the M and B subunits. Various combinations of these monomers make up different CK isoenzymes which are found in specific locations in brains and cells (see below). The routine activity assay measures total CK activity and not the individual isoenzymes. Isoenzymes may be quantitated by electrophoretic or immunologic methods. This is most commonly used in humans with ischemic cardiac events (heart attack) but has been supplanted by measurement of cardiac troponin in many hospitals. Due to the difficulties in identifying CK isoenzymes in animals and the perceived scarcity of cardiac injury, we do not quantitate the different isoenzymes. In addition, we use troponin as a marker of cardiac injury in animals. There are four principle isoenzymes of CK:
- CK-1: BB isoenzyme, found mostly in the brain. Injury to brain tissue may increase CK-1 activity in CSF, but rarely results in raised total serum CK activity.
- CK-2: MB isoenzyme, highest concentration in cardiac muscle, with lesser amounts in skeletal muscle.
- CK-3: MM isoenzyme, found in skeletal muscle, with lesser amounts in cardiac muscle. This isoenzyme accounts for the majority of CK in plasma of animals.
- CK-Mt: Found in mitochondrial membranes, and comprises 15% of total cardiac CK activity. There are ubiquitous and sarcomeric forms of CK-Mt.
MethodologyThe routinely used enzymatic assay takes advantage of the normal ability of CK to catalyze the conversion of phosphocreatine to creatine, releasing ATP in the process.
Reaction typeKinetic with UV measurement
ProcedureNicotinamide adenine dinucleotide phosphate (NADPH) and ATP are produced in equal quantities at the same rate. The increase in NADPH formation is directly proportional to the CK activity.
Creatine phosphate + ADP CK > creatine + ATP
Glucose + ATP Hexokinase > Glucose-6-P + ADP
Glucose-6-P + NADP+ G6P-DH > Gluconate-6-P + NADPH + H+
Units of measurement
Creatine kinase activity is measured in U/L (conventional units) and µkat/L (SI units), which is defined as the amount of enzyme that catalyzes the conversion of 1 µmol of substrate per minute under specified conditions. The conversion formula is shown below:
U/L x 0.0167 = µkat/L
Sample typeSerum, plasma, cerebrospinal fluid.
AnticoagulantHeparin (lithium, ammonium) or EDTA
StabilityAccording to the reagent manufacturer, stability of CK in human serum and plasma samples is as follows: 2 days at room temperature, 7 days refrigerated, or 4 weeks frozen at around -20°C.
- Lipemia: Severe lipemia (>1000 lipemia index) may increase concentrations.
- Hemolysis: May increase with hemolysis (>100 hemolysis index). This is due to the reasons indicated below.
- Icterus: Severe icterus may increase activity (>60 icteric index)
Increased concentration/activityIncreases in serum or plasma CK is most commonly used as a marker of skeletal muscle injury (by sheer mass, it is unlikely to see increases in total CK activity in serum or plasma after cardiac injury, however increases in CK-MB or CK-2 may be seen if electrophoretic separation is done).
- Artifact: Hemolysis will increase CK activity as constituents within red blood cells (ADP or glucose-6-phosphate) or in their membranes (adenylate kinase) contribute to the enzymatic reaction for CK, falsely increasing activity. Inadvertant penetration of muscle during venipuncture can cause 3- to 4-fold increases in CK activity in the sample and is a common cause of mildly increased CK values in healthy (or sick) animals. We have seen that CK activity can be as high as 3-4000 U/L from a muscle stick (which we consider a mild increase).
- Physiologic: CK activity in young puppies is higher than in adults, although this is apparently without explanation. Moderate increases (2-3x) are possible in exercising horses, with the post-exercise increase being less in well-conditioned animals. Intramuscular injections will increase CK activity to various degrees depending on the irritant or toxic effects of the drug or if the drug impedes local blood flow.
- Muscle disease: Detection of increased activity in serum is useful as an indicator of muscle injury. The assay is quite sensitive in this regard, but increased activity is not specific as to cause (e.g., trauma, inflammation, degeneration). High CK activity are observed in inherited muscular dystrophies, exercise-induced rhabdomyolysis, polymyositis, vitamin E-selenium deficiency, snake bite poisoning, etc. In one study, increased CK activity was seen in 60% of sick cats with various diseases, with marked increases (>30x upper reference limit) only being seen in 7% of cats. Some of these increases could potentially be due to inadvertent muscle sticks (Aroch et al 2010). Note that animals which are recumbent ("downer" cows or post-surgical patients) will have high CK activity (up to 10 x normal) from muscle injury or ischemia. Similarly, horses and cattle after shipping have moderate increases in CK activity.
- Miscellaneous: Critically ill anorectic cats have been reported to have high CK activity (median CK in one study was 2,500 U/L). The exact mechanism is uncertain, but it is thought to be due to increased muscle catabolism or membrane permeability (blood was withdrawn through an indwelling catheter in these cats, arguing against a muscle puncture during collection) (). In another study, anorectic cats with a nasopharyngeal tube had a higher CK activity than non-anorectic cats. The high CK values were associated with other evidence of muscle injury in these cats, including high AST and lactate dehydrogenase (Fascetti et al 1997). It is possible that the muscle was secondary to the primary disease process versus anorexia per se.
- Neurologic disease: Creatine kinase activity has been measured in the cerebrospinal fluid of animals as a marker of neurologic injury. Studies in horses have shown that high CK activity is seen in various neurologic conditions, including equine protozoal neuritis (Sarcocystis neurona) but that high values are neither sensitive or specific for the latter disease. Furthermore, contamination with epidural fat is a major confounding variable that falsely increases CK activity (Jackson et al 1996).