Abstract
Background
Macrocreatine kinase (MCK) type 1 is a high molecular weight form of CK that is non-pathological. Identification of MCK is beneficial in preventing unnecessary investigations that may follow a persistently elevated CK of unknown origin. Currently, gel filtration chromatography can be used as a confirmatory technique, but it is laborious, time-consuming and expensive. The aim of this work, carried out as part of a larger investigation into the prevalence of MCK, was to determine whether trypsin can be used as an alternative to confirm the presence of MCK on isoenzyme electrophoresis.
Methods
Five samples found to have bands running in the MCK region on isoenzyme electrophoresis were treated with trypsin. Electrophoresis was carried out using the Helena Biosciences Sas-1 Plus system. These samples were also analysed by the confirmatory technique of gel filtration chromatography.
Results
Of the five samples treated with trypsin, three were found to be MCK-positive and two MCK-negative. These results correlated with those obtained by the reference method of gel filtration chromatography.
Conclusions
There appears to be a potential use of trypsin in confirming the presence of MCK following isoenzyme electrophoresis. If these findings were verified, this would provide a less labour-intensive, less time-consuming and more cost-effective confirmatory technique. Further study is required, which needs to be expanded to include a larger number of patients, before this method can be adopted routinely.
Introduction
Two types of macrocreatine kinase (MCK) have been described: MCK type 1 (MCK1) is a CK-immunoglobulin complex, whereas MCK type 2 (MCK2) represents a high molecular weight form of CK thought to be of mitochondrial origin. MCK1 is not known to be pathologically significant, whereas MCK2 is thought to be associated with diseases of high cell turnover. 1 The presence of MCK causes an increase in measured CK activity, postulated to be due to decreases in enzyme inactivation, clearance or excretion. 2 Detection of MCK1 in samples with unexplained persistently elevated CK activity could be beneficial to prevent diagnostic confusion and unnecessary clinical investigation. 1
Methods that can be used to detect the presence of MCK include polyethylene glycol (PEG) precipitation, isoenzyme electrophoresis and gel filtration chromatography (GFC). 3 Detection may also be possible by using the technique of ultrafiltration, but this method is not in routine use as the equipment is not widely available and the current evidence base focuses on the detection of macroprolactin. 2–4
In PEG precipitation, PEG causes protein precipitation by decreasing the availability of solvent to the molecules such that the higher the PEG concentration, the more protein will be precipitated. 1,5,6 At optimum PEG concentrations, the technique is relatively specific for immunoglobulin precipitation. However, as the PEG concentration increases, proteins of a smaller size will also be precipitated. 1,5,6 As a result of the low specificity, the main application of this technique is a screening test. 5,6 Conversely, isoenzyme electrophoresis and GFC are more widely used as confirmatory techniques. 1,3 Electrophoresis, on agarose gel or cellulose acetate, can separate CK isoenzymes, including MCK, on the basis of differing mass to charge ratio. Densitometry can then be used for quantification of the bands. 7,8 GFC separates CK from MCK by size, with the larger MCK eluting earlier than normal dimeric CK as MCK is not retarded by the pores present within the column packing. The relative activities of the fractions can then be measured by routine analysis. 1
The reference method for the detection of macroenzymes and macrohormones is considered to be GFC. 7,9 However, this technique is costly, time-consuming and laborious, meaning that its routine use is not practicable. 1,3,10 Isoenzyme electrophoresis could be used as an alternative, but during the wider part of this study (a method comparison to compare PEG precipitation and isoenzyme electrophoresis for the detection of MCK), atypical bands were found to migrate in this region, causing false-positive results. This phenomenon does not appear to have been reported in the literature, but necessitates the use of a method to confirm the presence of MCK1. In alkaline phosphatase (ALP) isoenzyme electrophoresis, samples that demonstrate unusual bands can be treated with trypsin to determine whether the ALP is complexed with an immunoglobulin. Trypsin dissociates ALP from the immunoglobulin, thus allowing the appropriate characterization of the enzyme. 11 This study investigated whether this principle could be translated to CK isoenzyme electrophoresis to confirm the presence of MCK1.
Methods
As part of a wider study investigating MCK, surplus samples with a raised CK were collected and stored frozen at −20°C. A raised CK was taken to be any sample with a CK above the upper limit of normal of the laboratory reference intervals in use at the time of the study: men 185 IU/L and women 145 IU/L. There were no further criteria for inclusion into the sample population.
A total of 443 samples were collected and analysed by both PEG precipitation and isoenzyme electrophoresis. Samples that exhibited a low percentage recovery following PEG precipitation, or unusual bands on electrophoresis, were sent for confirmation by GFC at Southend University Hospital. The reference method of GFC was carried out as defined by Fahie-Wilson et al. 1
Isoenzyme electrophoresis was carried out using the Helena Biosciences SAS-1 Plus instrument (Tyne and Wear, UK) and the fractions were quantitated by densitometry. Of the 443 samples analysed by electrophoresis, five, with bands found running in the MCK1 region, were treated with trypsin for this study. One 250 μL aliquot of each sample was treated with 1 mg of crystalline trypsin (from bovine pancreas, 391362V; VWR International Ltd, Leicestershire, UK) whereas a second aliquot was left untreated. Both aliquots were incubated in a water bath at 37°C for two hours. Following incubation, the samples were prepared for electrophoresis. One microlitre of CK isoenzyme activator (supplied in the Helena Biosciences kit) was added to 100 μL of the patient's serum and left to incubate at room temperature for 10 min. Following incubation, 35 μL of this mixture was used for electrophoresis. The two aliquots for each patient were run in adjacent lanes. Electrophoresis was performed according to the manufacturer's instructions, but with a voltage increase to 120 V to improve the separation of the isoenzyme bands. This change was identified during in-house optimization for the identification of MCK as part of the wider study.
Results
Figure 1 shows the results of trypsin treatment on the samples run by isoenzyme electrophoresis. Of the five samples that were treated with trypsin, two were classified as MCK1-negative (samples 2 and 3), as the atypical bands were still present after treatment, and three as MCK1-positive (samples 1, 4 and 5), because the bands running in the MCK1 region were removed by trypsin treatment.
Gel produced for samples treated with trypsin. Samples 1–5 were run in pairs in adjacent lanes; the first lane contained the untreated sample and the second lane contained the trypsin-treated sample. Note, lane 8 is empty because the control (purchased from Helena Biosciences) was not treated with trypsin. CK, creatine kinase; MCK, macrocreatine kinase
Four of the five samples were correctly classified as MCK1-positive or -negative when compared with the reference method results of GFC. Figure 2 shows the GFC traces obtained for samples 1, 3, 4 and 5. GFC results were not available for sample 2 as there was insufficient serum remaining to send for confirmation.
Gel filtration chromatography traces for (a) sample 1 (macrocreatine kinase [MCK]-positive); (b) sample 3 (MCK-negative); (c) sample 4 (MCK-positive); and (d) sample 5 (MCK-positive). CK, creatine kinase
Recorded details for the MCK electrophoresis-positive subjects
MCK, macrocreatine kinase; DM, diabetes mellitus; IHD, ischaemic heart disease; F, female; M, male; P, positive; N, negative
Results of quantitation of MCK1 by GFC and isoenzyme electrophoresis (quantitation by densitometry)
MCK1, macrocreatine kinase type 1; GFC, gel filtration chromatography; EP, electrophoresis
*Insufficient sample remained to send this sample for confirmation by GFC
Discussion
MCK1 is reported as a non-pathological finding and has been found, most commonly, to be the CK-BB isoenzyme linked to IgG or IgA in either a 1:1 or 2:1 molar ratio. 8,12 Currently, GFC is considered the reference method for the detection of MCK. 7,9 However, this study has demonstrated the potential use of trypsin digestion to confirm the presence of MCK1 following isoenzyme electrophoresis. It has shown that MCK1 bands are susceptible to digestion by trypsin as the bands in this region are removed following incubation with the protease, whereas they are not in MCK1-negative subjects. As MCK1 has been mostly identified as the CK-BB isoenzyme complexed with an immunoglobulin, it would be expected that the uncomplexed CK would appear as a separate band in the CK-BB region. However, in 2005, Ercan and Grossman 13 investigated the susceptibility of CK isoenzymes to trypsin digestion and found that the CK-BB isoenzyme was much more susceptible to proteolysis than the CK-MM fraction. This would explain the absence of the MCK1 band following treatment with trypsin and also the lack of a second band in the CK-BB region of the gel. During their study, Ercan and Grossman 13 also found that CK-MM was highly resistant to trypsin digestion, although digestion will occur with higher concentrations of trypsin and with longer incubation periods. 13 Therefore, the decrease in intensity of some of the CK-MM bands in the present study could also be attributed to this mechanism, as the samples in this study were incubated for longer and with higher concentrations of trypsin than were used by Ercan and Grossman. 13
In order to fully understand the mechanisms involved in trypsin digestion of MCK1 bands, further study is required. First, the effect of the trypsin treatment on the isoenzyme control would have to be investigated to determine whether the same decreases in CK-BB and CK-MM are observed as seen in the patients used in this study. Second, it would also be necessary to run the sample by GFC pre- and post-trypsin digestion to confirm the residual activity of the CK fractions.
The present study has shown the potential value of isoenzyme electrophoresis to confirm the presence of MCK1 by the removal of the band in the MCK1 region post-trypsin digestion. In addition, all samples that were also analysed by GFC were correctly classified as MCK1-positive or -negative using this method. Samples 1, 4 and 5 all demonstrated the removal of the MCK1 band following trypsin treatment, whereas samples 2 and 3 were visibly negative. However, the bands causing the false-positive results in samples 2 and 3 remain unidentified. Furthermore, the quantification of monomeric CK and MCK concentrations by isoenzyme electrophoresis have been found in this study to be comparable to the reference technique of GFC. Therefore, an estimate of the pathological rise in CK, if present, can be provided to aid patient management. This would be beneficial as one patient in this study (sample 4) had a concurrent pathological increase in CK alongside the presence of MCK1. Insufficient numbers of MCK1-positive patients were identified by this study to allow significant conclusions to be drawn regarding its pathological significance.
Identification of MCK1 would be beneficial to help avoid unnecessary further clinical investigation in patients with a persistently raised level of CK and to allow correct therapeutic decisions to be made, for example on the use of statins. 1,7 From this study, it appears that the use of trypsin may provide a less laborious and more cost-effective method than the current confirmatory technique of GFC. However, a greater number of samples need to be examined, the effect of trypsin treatment on the control sample must be studied, and further investigation carried out into the mechanism of trypsin digestion of MCK before more significant conclusions can be made and this method can be used in routine practice.
DECLARATIONS