Further Observationson the Incidence and - Clinical Chemistry

CLIN.CHEM.25/4, 535-54 1 (1979)

CLINICAL CHEMISTRY, Vol. 25, No. 4, 1979 535

Further Observationson the Incidence and Nature of Atypical

Creatine Kinase Activity

Sylvan M. Sax,’ John J. Moore,1 Joseph L. Giegel,2 and Muriel Welsh2

The interference of atypical creatine kinase (CK; EC2.7.3.2) with anion-exchange methods for the measure-ment of the CK-MB isoenzyme is now firmly established.False-positive results from this source are much morecommon than interferences caused by the BB isoenzyme.Atypical CK, at least in some patients, does not appear tobe a genetic variant, nor could we relate it to a specificclinical diagnosis, to hypoxia, or to administration of aparticular drug. Its behavior in an immuno-inhibition testfor CK-B subunit indicates an Immunological differencefrom the normal M subunit. Thus the change in immuno-logical properties is associated with the altered electro-phoretic properties. The variability of electrophoreticpatterns suggests that atypical CK may represent multipleforms of creatine kinase rather than a unique entity.

Addftional Keyphrases isoenzymes of creatine kinaseheart disease

Separation of CK isoenzymes on DEAE-Sephadex3 (1)shows an increase in the presumed MB fraction, sustained inthe serum of certain patients who have not suffered recentmyocardial infarction (2,3). We termed this fraction “atypicalcreatine kinase” (CK-AT) because its electrophoretic mobilityis intermediate between MM and MB (2). Its presence hasbeen confirmed by other investigators (4-7). In this report,we estimate the prevalence of factitious MB values resultingfrom interference by CK-AT and describe some additionalproperties of CK-AT.

Materials and Methods

Our original report (2) covered the period of April 9 to July29, 1975. The present study extends that period through May2, 1978. Until January 3, 1977, our procedures for measure-ment of total CK activity, separation of CK-MB activity byion-exchange column chromatography, and electrophoresisof CK isoenzymes on cellulose acetate were as originally de-scribed (2). At that time, we made two changes: we routinelybegan using Roche CPK Reagent (Roche Diagnostics, Nutley,NJ 07110) for measurement of total CK in serum and CK-MBin column eluates and the Roche CPK-CS adaptation ofMercer’s technique. The latter change resulted in a slight

1 Department of Pathology, The Western Pennsylvania Hospital,4800 Friendship Ave., Pittsburgh, PA 15224.

2 Dade Division, American Hospital Supply Corp., Box 520672,

Miami, FL 33152.Nonstandard abbreviations used: CK, creatine kinase (EC 2.7.3.2);

DEAE, diethylaminoethyl; CK-AT, atypical creatine kinase frac-tion.

Received Nov. 28, 1978; accepted Jan. 5, 1979.

upward shift in reference values. CK-MB activities exceeding2.6 U3o’c/L with normal values for total CK or accounting forover 2.6% of total activity when the latter is increased suggestrecent myocardial infarction.

Patients are classified as “suspected atypicals” if three ormore results for CK-MB by column are increased during atleast one week. These become “confirmed atypicals” when oneor more electrophoretograins demonstrate the presence of anatypical band.

Dade CK-Isoenzyme Marker (Dade Div., American Hos-pital Supply Corp., Miami, FL 33152) was used as a controlin electrophoresis and in an experiment in which pooled fresh

human serum was fractionated by column (Roche CPK-CSprocedure), and the medium salt fraction was separated byelectrophoresis after concentration. An Amicon concentrator(Amicon Corp., Lexington, MA 02173) was used to effect a25-fold concentration of the eluates. Electrophoresis of theconcentrates was performed as described, substituting 10applications of concentrate for the usual four of native serum.The final concentration factor is therefore: (1 mL serum/8 mLeluate) X 25-fold Amicon concentration X (10 applicationsof concentrate/4 applications of serum) = 7.8.

Sera containing CK-AT isoenzymes were assayed by animmuno-inhibition method for the quantitation of the MBisoenzyme without prior separation of the serum CK isoen-zymes (Cardiozyme CK-MB, Dade). In this method an anti-body (goat) specific for the human CK-M subunit is used. Theantibody inhibits both MM isoenzyme activity and the Msubunit portion of the MB isoenzyme in an ultraviolet, kineticprocedure for CK activity. Thus, the MB activity of serum isestimated from the uninhibited B subunit activity in the re-action mixture.

We also used an immunoprecipitation method involvingprecipitating anti-CK-MM and anti-CK-BB antibodies(sheep) (12). Unlike the rapid immuno-inhibition procedure,the immunoprecipitation method requires overnight incu-bation of serum and antibody mixtures followed by determi-nation of the residual CK activity in the supernatant fluidafter centrifugation of the mixtures.

In an experiment designed to see the extent to whichCK-AT could be separated from CK-MB in the columnmethod, three specimens were selected: serum from a con-firmed CK-AT subject (patient V.P.), serum from a myocar-dial infarct patient near the peak MB value (patient F.A.), andthe Dade CK Marker. Each was run six times by the RocheCPK.CS column method, modified by increasing the NaC1concentration of the “low salt buffer” from 0.10 mol/L to 0.15mol/L in 0.01 mol/L increments. Low and medium salt eluateswere analyzed for CK activity. Aliquots of those eluates ofpatient V.P. were concentrated 10-fold in an Amicon con-centrator and analyzed by electrophoresis and by the inhibi-tion method.

Table 1. Incidence of Atypical CK at the Western Table 2. Final Discharge Diagnoses of 62 PatientsPennsylvania Hospital (April 9, 1975-May 2, with Confirmed Atypical CK

1978)%oftotal

assays21 000a 100

13800 64

% ofpositiveassays

Gastrointestinal disease4 700a 22 100 Diabetes mellitus

Acute myocardial infarction381 1.8 8.1 Anxiety or depression

519

Includes arteriosclerotic heart disease, previous myocardlal Infarction,

ischemic heart disease, angina pectouis. and congestive heart failure.2.4 11.0 b Includes pulmonary embolism, chronic pulmonary disease, and pneumo-

nia.

4.2 19.1

536 CLINICAL CHEMISTRY, Vol. 25, No. 4, 1979

Total assaysAssays on specimens from

intensive-, coronary-, orprogressive-care units

Assays positive for CK-MB by

column (true MB or atypical)Assays positive for CK-MB by

column and confirmed to be

atypicalAssays positive for CK-MB by

column and suspected to be

atypicalAssays positive for CK-MB by 900

column on combined,

confirmed, and suspected

atypicalsA Extrapolated from data obtained from five separate months throughout the

total time period.

Results and Discussion

Review of Data on Patients

Some idea of the incidence of CK-AT may be gainedbyinspection of Table 1. The 21 500 assays were performed on6100 patients. Of those patients, 62(1.0%) wereconfirmedand80 (1.3%) were suspected atypicals. These figures are conser-vative, because we have been systematically searching for suchresults only since January 1978. The high rate of false-positiveCK-MB results by the column chromatographic method canbe partially explained by the need to repeat CK-MB assaysto evaluate and confirm patients with CK-AT. Jeanmet et al.(4) observed an 8.3% incidence of false positives in a smallerseries. In only one out of our series of patients who showed avery prolonged elevation of CK-MB by column was the frac-tion demonstrated to be true MB by electrophoresis; theclinical diagnosis in that case was polymyositis. It is apparentthat the frequent appearance of CK-AT calls for awareness,both on the part of the clinical chemist who is using ion-ex-change methods to separate CK isoenzymes and on the partof the clinician interpreting such test results. As will be shownlater, similar awareness by those who use the immuno-inhi-bition method is also required.

We examined themedical charts of the 62 confirmed casesshowing CK-AT, seeking some common denominator. Themost frequent diagnostic categories cited upon final dischargeareshown inTable 2. It is not surprising that heart disease oflong standing was present in many patients, because 64% ofthe specimens analyzed came from the intensive care, coro-nary care, and progressive care units. However, the presenceof CK-AT was confirmed in many patients not suffering fromchronic heart disease. Thus the presence of CK-AT does notappear to be associated with a particular clinical diagnosis,a finding that is in agreement with those of others (4, 5, 7).

Patients’ records were checked for medications being re-ceived at the time of sampling. No drug was common to allCK-AT patients. Digoxin, the most commonly prescribeddrug, was being administered to 63% of this series of patients.A sizable proportion (23%) had received no drug within 24 h

Diagnostic

category

Chronic heart disease a

Other vascular diseasePulmonarydiseaseb

HypertensionCardiac arrhythmia

No. Perc.ntag.

48 77%

18 29%

17 27%16 26%15 24%14 23%9 15%6 10%

6 10%

of venipuncture. This would appear to rule out binding of adrug or drug metabolite to one of the threecommon isoen-zymes ofCK, resulting in a change in effective charge, as anexplanation for anomalous behavior on electrophoresis orion-exchange columns.

The 62 patients in the confirmed atypical group were 19men and 43 women. The men ranged in age from 48 to 87(mean age, 65.7 years). The women ranged in age from 33 to94, with a mean age of 67.3 years.

In our earlier publication (2), we noted the persistence ofCK-AT, measured as apparent CK-MB by column chroma-tography in 14 patients. In two patients undergoing coro-nary-bypass surgery, CK-AT was seen to decrease consider-ably after such surgery, but in neither case did the apparentCK-MB return to normal.

We have examined our expanded series of patients in searchof unusual trends in serial determinations. Illustrative casesare summarized in Table 3.

Patient V.P. showed no CK-MB, only CK-MM and CK-ATon electrophoresis. Her CK-AT value is quite constant overa 1.5-year period; the longest time span during which thepresence of CK-AT was followed in any patient in our earlierstudy was nine weeks.

Patient A.L., with electrophoretic findings similar to thosefor patient V.P., showed CK-AT during a period spanning 13months and three hospital admissions. The considerablyhigher apparent CK-MB seen during the most recent admis-sion was not associated with any notable change in the clinicalstate.

Patient C.B., again with similar electrophoretic findingsduring a 1976 admission, was admitted moribund in 1978.Despite the dramatic deterioration in her physical state, re-sults for total and CK-MB on admission were normal; sub-sequent CK-MB results were not suggestive of increases ineither true CK-MB or C K-AT. This case is illustrative becauseit shows that (a) column-chromatographic increases in ap-parent CK-MB, but resulting from CK-AT, although quiteconstant over a period of days or weeks, may revert to normalover a longer time span; (b) such a reversion to normal limitscannot be associated with an improvement in clinical status;and (c) such a reversion to normal makes it unlikely thatCK-AT represents a genetic variant.

Patient J.T. illustrates a reverse change, from normalCK-MB tothe abrupt emergenceofa high result on February21, 1978. The slight increases in total CK and CK-MB seenin 1977 were accompanied by parallel changes in other cardiacenzymes, and, although not confirmedelectrophoretically,

Table 3. Data on Selected Patients with Electrophoretically Confirmed CK-AT

Total CKDate U/I Apparent MB % MB Clinical diagnosis and comments

V.P.,a 73-year-old woman8/6/76 50 12 24 Hemachromatosis diagnosed in 1967;

8/6/76 41 11 27 cardiomyopathy secondary to hema-8/8/76 38 10 26 chromatosis. Admitted on 3/12/788/10/76 36 11 31 with chest pain, anxiety, and depres-3/13/78 37 12 32 sion. Serial WH (total and sum of fractions3/13/78 35 10 29 I and II) and AST activities normal3/14/78 28 10 36 throughout 1978 admission.

A.L., a 79-year-old man2/24/77 56 5.7 10 Admitted 3/15/78 with possible myocardial infarct.

2/25/77 43 4.8 11 Long history of hypertensive heart

2/26/77 42 3.0 7 disease and old cerebrovascular

5/1/77 50 4.4 9 accident

5/2/77 34 4.4 13

5/3/77 31 3.4 11

5/4/77 27 2.8 10

3/15/78 46 11 243/15/78 50 12 253/17/78 82 11 14

C.B., a 52-year-old woman5/15/76 23 14 60 Diabetes mellitus diagnosed in 1961;

5/16/76 21 13 61 exogenous obesity and atrial fibrilla-

5/17/76 23 13 58 tion noted on 1976 admission.

5/18/76 18 11 62 Admitted in cardiac arrest5/19/76 20 12 60 and suffering cerebral hypoxia on5/20/76 21 13 61 1/28/78; died 2/2/78.1/28/78 66 2.6 4.0

1/29/78 274 1.8 0.71/30/78 225 1.4 0.6

1/31/78 168 1.4 0.8

J. T., a 64-year-old man12/16/76 35 0 0 Cardiomyopathy, congestive heart

7/19/77 100 2.7 2.7 failure, alcoholism, and cardiac7/19/77 72 2.9 4.0 arrhythmia.

7/28/77 51 1.3 2.61/25/78 72 2.0 2.7

2/21/78 66 5.8 8.8F.C., a 75-year-old woman

10/17/77 44 6.4 14 Congestive heart failure.10/19/77 31 3.0 1012/2/77 22 3.0 1312/2/77 16 3.5 2212/3/77 13 2.4 18

12/4/77 12 2.0 173/31/78 24 4.4 184/1/78 18 4.3 244/2/78 18 4.3 24

J.H., a 53-year-old man1/13/76 192 19 10 Classical myocardial infarction in 1975. Coronary by-1/14/76 1519 105 6.9 pass operation on 1/13/76. Admitted1/15/76 1093 18 1.6 in April 1976 after suffering first1/16/76 703 5.7 0.8 anginal-type attacks since surgery.1/21/76 29 1.2 4.0

4/21/76 86 9.5 114/23/76 70 8.0 11

continued


+ +

I

‘I,I

MarkerNormal Patient

Control

+

I

1’’C’ - +

Marker $Patient #77230

Normal PatientControl

+

Marker

Patient F508Normal Patient

ControlPatient #17374

Patient W508

Patient #14958


Table 3. Continued

Total CK% MB Clinical dIagnosis and commentsDate U/L Apparent MB

4/25/76 90 14.3 164/26/76 98 10.4 114/28/76 68 7.0 104/29/76 105 11.5 11

probablyrepresenta transientreleaseofCK-MB. Electro-phoresis ofthe last specimen indicates thepresence ofCK-ATand theabsenceCK-MB.

PatientF.C.showstwo normalvaluessandwichedbetween

a,


Patient Sl14

Marker

b1

Patient 10231

abnormal ones. Impermanence of CK-AT elevation has beenobserved in 13 of the confirmed atypical patients.

Patient J.H. is unique, because thespecimen ofApril 25,1976 shows both CK-MB and CK-AT on electrophoresis.

Marker

Patient 3607

Normal Patient

Control

Patient #3630

Marker


#{247} fi

d. Patient M0661


Patient T432

Marker

Fig. 1. Some examples of different electrophoretic patternsa. Patient sample S 114 shows two CK-AT bands in region between MM and MB b. Patient sample 10231 shows a narrow CK-AT band with little or no CK-MM C

Patient sample 77230 shows a CK-AT which is somewhat fused with but distinguishable from an MM band d. Patient sample T 432 shows two CK-AT bands, oneslightly anodalto CK-MM and onebetweenMB and BBe. Patient3607 shows both CK-MM and a distinct CK-AT between MM and MB f. Patient 3630 has noCK-MMand what appears to be a dual CK-AT band between MM and P g. Samples W 508 and F 508, from the same patient but collected on different days, show a CK-ATband migratingcloser to CK-MB than CK-MM.Some CK-MMactivity can be seen. Patient sample 17374 has a broad CK-AT band Just slightly anodal to the usualMM position. Patient M 0661 has a narrowCK-AT betweenMM and MB and an unidentified band cathodai to MM. Patient 14958 has both an MM and a cK-AT bandanodal to MM

(I appl.)

(4 appl.)

(I appl.)

MarkerUnfractionatedNormal SerumUnfractionatedMarkerMB Eluate

Normal SerumMB Eluate

MarkerMB EluateMarkerUnfractionated

Table 4. Analysis of Seven Confirmed CK-ATSera by DEAE-Sephadex Column,

Electrophoresis, and an immuno-InhibitionSystem in Which the CK-M Subunit is inhibited by

Anti-M antibody

Total MB byCK column

MB byimmuno-inhibition

Seen onelecirophoreels


(10 appl.)

(2 appl.)

(lappl.)

Fig. 2. Electrophoresis of concentrated MB eluates of normalhuman serum and Dade Marker vs. the unfractionated sam-ples“AppI.”Is application(s)

Serial determinations of aspartate aminotransferase (EC2.8.1.1), total and lactate dehydrogenase (EC 1.1.1.27), andits isoenzymes Iand IIconfirm the presence ofa mild myo-cardial infarction.Perhaps the most significant result of this retrospective

studyisan awarenessthatCK-AT valuescan increaseorde-cline. Although we have not confirmed an increase and sub-sequent decrease within a period of a few days, mimicking atypical post-infarct CK-MB curve, such an occurrence isconceivable.

Clinical records were checked on six patients from thesuspected atypical series. In only one was evidence of a pos-sibly concurrent myocardial infarction provided by electro-cardiogram or by other enzyme results. These records revealno obvious differences between the confirmed and suspectedatypical groups.

Studies on the Nature of CK-AT

The appearance of cellulose acetate electrophoretogramsshowingCK-AT isquitevariablefrom onepatienttoanother.Illustrationsofdifferenttypesare illustrated in Figure 1. Al-though CK-AT usually appears between MM and MB, wehave observed atypical bands between MB and BB in the caseof three of the 62 electrophoretically confirmed patients. Twoof the three showed a second atypical band in the usual regionbetween MM and MB. Nothing in the clinical records of thesepatients, all three of whom had chronic heart disease, wasrevealing. Lim (8) reported two “sub-bands” observed in CKelectrophoretograms of human sera, one type occurring be-tween MM and MB, the other between MB and BB. We agreewith his observation that the former type occurs much morefrequently than the latter, but our patients do not fit his ob-servation that the latter type is seen only in patients withneurological disorders and the former only in those sufferingsevere angina pectoris.

In fact, we observe thatCK-AT bands lying between MMand MB comprise a variety of sub-types. Most are foundsomewhat closer to MM than to MB, but are clearly resolv-able. However, some, which appear as slight broadenings ofMM, are difficult to detect. Some CK-AT bands are relativelysharp, others much more diffuse. Electrophoretograms ofdifferent subtypes retain their characteristic appearance inrepeated runs. Ljungdahl and Gerhardt (7) also observedseveral subtypes of CK-AT lying between MM and MB.

U/L (at 30 #{176}C)

35 10 20 CK-MM and CK-AT

33 15 7 CK-MM and CK-AT19 7 10 CK-MMand CK-AT68 20 28 CK-MM and CK-AT42 15 24 CK-MM and CK-AT33 16 17 CK-MM and CK-AT

46 11 26 CK-MMandCK-AT

Since early 1977, we have sometimes encountered runs inwhich all or nearly all patients’ sera, but not the Dade CK-Isoenzyme Marker, show a small band in the usual CK-ATregion. We are not certain whether this variability is due totechnique, reagents, or membranes. This has necessitatedincluding two fresh normal sera in each run for comparison;only those patterns showing relatively heavy staining in theappropriate region are termed positive for CK-AT. Onetherefore asks, is the 0 to 2.6 U of MB activity per liter ofnormal sera found by the ion-exchange method actuallyCK-MB, or does CK-AT make up all or part of that fraction?With a radioimmunoassay that involved anti-B-subunit,Roberts et al. found a range for MB of 0-2.2 U/L in sera from100 healthy controls (9). This did not prove the presence ofCK-MB, becausebehavior of CK-AT toward anti-B-subunitwas not known.

We attempted to establish the presence of CK-MB orCK-AT in normal serum more definitively by fractionatingnormal serum by column chromatography, concentrating thedesired fraction, and electrophoresing the concentrate (seeMaterials and Methods). The results (Figure 2) are incon-clusive, because the pattern of the normal serum concentrateshows two bowed, somewhat diffuse bands, neither of whichresembles the CK-AT or CK-MB seen in patterns of neatserum. On the other hand, the MB fraction of the Dade CPKMarker survived the same treatment without apparentchange. At least three interpretations cometo mind: (a) thatmost of the apparent CK-MB present in normal serum results,not from true MB, but from several fractions of differentelectrophoretic mobility; (b) that human CK-MB, but not thesimilar CK-MB of the marker, is altered by the experimentalconditions; or (c) that the very high concentration of albuminin concentrates of the medium eluate causes distortion of thepattern.

We next examined the behavior of CK-AT-containing serain the Cardiozyme CK-MB immuno-inhibition test system.Results are shown in Table 4. All were falsely positive for MBby the column method and all but one by the immuno-inhi-bition method. Results of testing sera containing CK-AT withthe precipitating antibodies are seen in Table 5. The anti-CK-MM quantitatively precipated CK-MM and CK-MBisoenzymes; anti-CK-BB precipitated CK-MB and CK-BBisoenzymes. The control test represents the total CK activityof the sample. Although in our hands the precision of themethod was not good, residual activity in the anti-CK-MMsupernate, not found in normal serum samples, was a constantfinding for these samples which gave CK-AT activity betweenthe MM and MB positions on the electrophoretogram.

Similar results were observed by Ljungdahl and Gerhardt

P02,mmH

Electro-

MMMM and BB


Table 5. Residual Activity of Sera ContainingCK-AT and of Normal Sera after incubation with

Precipitating AntibodiesControl Anti-CK.BB Anti-CK.MM

t.st Mipernate supernate

Activity, U/L (at 30 #{176}C)

Atypical sera20.3 9.6 8.5

12.8 7.5 6.413.9 6.4 6.4

24.5 12.2 7.5

21.5 15.3 2.1

Normal sera18.0 21.7 0

7.8 10.6 0

14.0 16.7 0

(7), using immuno-inhibition of CK-M subunits in agarose gel.In their system, CK-MM was completely inhibited, CK-MBpartly inhibited, and CK-AT not inhibited. Those authorsconcluded that CK-AT is not aberrant CK-MM, but is prob-ably an abnormally migrating CK-BB. We have made severalobservations that are difficult to reconcile with their inter-pretation. We have observed a large number of electropho-retograms of sera with a very high proportion of CK-AT. Inmany of these, the MM band is very faint or even undetect-able. In a patient with increasing CK-AT, we had the oppor-tunity to perform serial assays by column and electrophoresis.As the CK-AT band increased in intensity, the CK-MM banddecreased, and could no longer be seen in the last pattern run.Because a prominent MM band is practically always seen,even in normal sera of low total CK activity, one might askwhat happens to CK-MM when CK-AT forms? Is CK-MMa precursor, which is subjected either to post-translationalmodification or to binding with an unknown substance, tocause an increasingly negative charge? Might such structuralmodification or binding also change the ability to bind toanti-M? Caution is suggested in attempting to define the CKisoenzyme composition of serum on the basis of behavior inthe presence of an antibody against a single subunit (see ref.10).

We previously have shown that CK-AT in one serumspecimen was eluted from an uncharged Sephadex column atthe same time as CK-MM or CK-MB, which argues againstmacromolecular binding (2). However, several observationssuggest that binding of one of the usual isoenzymes to anothersubstance, small or large, may account for the formation ofCK-AT. Partial dissociation of such a complex during elec-trophoresis might account for the multiplicity of patterns il-lustrated in Figure 1. Moreover, staining of the CK-AT bandis far more intense than that of MM, MB, or BB, againsuggesting removal of a binding substance that inhibits ac-tivity in the total CK assay system. We sought evidence oflipid binding by incubating serum containing C K-AT with anonionic detergent (Triton X-100; Rohm and Haas Co.,Philadelphia, PA 19105) as described by Epstein et al. (11).Subsequent electrophoresis showed no change of the CK-ATband. Other attempts to alter the electrophoretic mobility ofthe CK isoenzyme were unsuccessful. Jacobs et al. (13) sug-gested that a change in mobility might be caused by formationof a mixed disulfide on one of the polypeptide chains of thedimeric CK molecule. We were unable to affect the mobilityof the MM isoenzyme by adding L-cystine to normal sera.Neuraminidase (EC 3.2.1.18) added to a serum containing

Table 6. Test in Eight Patients of Correlationbetween Arterial p (Patient Breathing Room

Air) and CK-ATCK-MB

By immuno-- Total CK By column Inhibition

Activity, U/L (at 30 #{176}C)

58 83 3.1 -13

38 38 3.3 -648 44 1.3 -12 MM

35 305 3.0 -6 MM50 54 1.2 -7 MM43 38 1.6 -16 MM29 23 0.3 not run MM48 9 0.3 not run MM

CK-AT did not change the mobility of the atypical isoenzyme.The mobility of neither CK-AT nor of the simian isoenzymesof the Dade Marker was affected by batch treatment for 30mm to 2 h with an equal volume of a Con ASepharosee (10mg/mL suspension; Pharmacia Fine Chemicals, Inc., Pisca-taway, NJ 08854), a concanavalin-A adsorbant for polysac-charides and glycoproteins.

Lim (8) postulated that the sub-band on agarose gel elec-trophoresis, lying between MB and BB, is a conformationaltransformation of BB arising as an adaptation to ischemicconditions in the brain; he also suggested that the morecommon sub-band lying between MM and MB is a similarconformationally altered MB arising from ischemic conditionsin myocardium. A considerable number of our patients withCK-AT had low arterial P02. We therefore examined patientswith moderate to severe hypoxia for the presence of CK-AT;the results are summarized in Table 6. The first two of thesepatients showed borderline increases in apparent CK-MB byDEAE-Sephadex column chromatography. All subsequentpatients were normal by this procedure. Electrophoresis andthe immuno-inhibition assay system provided no evidence forthe presence of CK-AT in these eight patients with hypox-emia. These data therefore provide no support for Lim’spostulated mechanism.

Finally, we explored the possibility of reducing the inci-dence of false positives in the column method by increasingthe concentration of NaCl in the low salt buffer sufficientlyto remove both CK-MM and CK-AT without eluting CK-MB(see Materials and Methods), but we couldnot effectivelyseparate CK-AT without inordinate lossof CK-MB. However,Clejan (14) reports successwith a similar approach.

A reviewer of this paper noted the presence of atypical ac-tivity in control specimens sent by the Center for DiseaseControl in a recent CK survey. Behavior on ion-exchangecolumns (Mercer method) and with use of the Cardiozymeimmuno-inhibition method was similar to that described inour study.

We wish to thank Grace Bell, supervisor of the Pulmonary FunctionLaboratory of The Western Pennsylvania Hospital, for her coopera-tion in obtaining specimens from patients with low arterial p02 ten-sion.

References1. Mercer, D. W., Separation of tissue and serum creatine kinaseisoenzymes by ion-exchange column chromatography. Clin. Chem.20, 36-40 (1974).2. Sax, S. M., Moore, J. J., Giegel, J. L., and Welsh, M., Atypical in-crease in serum creatine kinase activity in hospital patients. Clin.Chem. 22, 87-91 (1976).


3. Mercer, D. W., and Varat, M. A., Detection of cardiac-specificcreatine kinase isoenzyme in sera with normal or slightly increasedtotal creatine kinase activity. Clin. Chem. 21, 1088-1092 (1975).

4. Jeanmet, A., Cramer, J., Goulle, J. P., et al., L’iso-enzyme MB deIa cr#{233}atinephosphokinase dam l’infarctus du myocard. Nouv. PresseMed. 7, 177-181 (1978).

5. Fiolet, J. W. T., Willebrande, A. F., Lie, K. I., and Ter Welle, H.F., Determination of creatine kinase isoenzyme MB (CK-MB):Comparison of methods and clinical evaluation. Clin. Chim. Acta 80,23-35 (1977).6. Leroux, M., Jacobs, H. K., Nabkin, S. W., and Desjardins, P. R.,Measurement of creatine kinase Z in human sera using a DEAE-cel-lulose mini-column method. Clin. Chim. Acta 80, 253-264 (1977).7. Ljungdahl, L., and Gerhardt, W., Creatine kinase isoenzyme var-iants in human serum. Clin. Chem. 24,832-834 (1978).8. Lim, F., Significance and applications of CPK isoenzyme sub-bands in the clinical laboratory. Clin. Chem. 21,975 (1975). AbstractNo. 181.9. Roberts, R., Parker, C. W., and Sobel, B. E., Detection of acute

myocardial infarction by radioimmunoassay for creatine kinase MB.Lancet ii,319-322 (1977).10. Sieg, J. F., Gauchel, F. D., and Herrschaft, H., Hohe Aktivitat desKreatin-kinase-BB-isoenzyms als seltene Anomalie? Dtsch. Med.Wochenschr. 102, 1647-1648 (1977).

11. Epstein, E., Baginski, E. S., and Zak, B., Detergent alteredalka-line phosphatase patterns of liver disease. Ann. Clin. Lab. Sd. 8, 34(1978).

12. Prellwitz, W., Neumeier, D., Knedel, M., et al., Isoenzyme derKreatin kinase bei extra kardialen Erkrankungen und nach diag-nostischen und therapeutisehen Eingriffen. Dtsch. Med. Wochenschr.101,983-988 (1976).

13. Jacobs, H. K., Philipp, K. H., Sundmark, V., and Weselake, R.J., Isolation and identification of a new potent inhibitor of creatinekinase from human serum. Clin. Chim. Acta 85, 299-309 (1978).

14. Clejan, S., Modification of the anion exchange chromatographicmethod for separation of creatine kinase isoenzymes. Its use formeasurement of an abnormal creatine kinase. Mikrochim. Acta 2,275-284 (1978).

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Further Observationson the Incidence and - Clinical Chemistry