Data Control, Riviera Beach, FL 33404) to be satisfactory for this work. Our. Model 6000A pump (Waters Associates,. Milford, MA 01757) was unsatisfactory.
LiChrosorbC-18 10 m column (Cole Scientific, Calabasas,CA versed-phase
91302). Passivation, to place the surface of the stainless steel column in the oxidized state, was performed with each new column used by pumping HNO3 (6 molfL) through the column only, for 30 mm. This strong acid was pumped from the column with the mobile phase, which was methanol (55 mL/L) in acetic and sodium acetate (70 mmol of acetate per liter, pH 5.8), with sodium heptanesulfonate (3.5 mmol/L) and disodiurn ethylenediaminetetraacetate (0.1 mrnol/L). Water used in this solvent was do-ionized by repeated passage through a four-cartridge (organic removal, two mixed-bed ion-exchangers, and 0.2-zm pore exclusion) filter system (Barnstead Nanopure; Barnstead/Sybron, Boston, MA 02132). The water supplied to this unit was from a commercial de-ionizer. All mobile phase was de-gassed by bubbling helium through it for 5 mm before use. We used the Model LC-4 electrochemical detector with glassy carbon electrode (Bioanalytical Systems, West Lafayette, IN 47906). We found the Altex 1IOA pump and the Constatmetric II pump (Laboratory Data Control, Riviera Beach, FL 33404) to be satisfactory for this work. Our Model 6000A pump (Waters Associates, Milford, MA 01757) was unsatisfactory in that it increased our baseline noise by 10- to 20-fold because of electrical interference, although Moyer et al. (3) appear to have found the Model 6000A pump satisfactory for this type of work. Both the phosphate buffer of Moyer et al. (3) and the citrate-phosphate buffer of Shoup and Kissinger (4) as used in their mobile phases contributed chemical noise in the detector signal, which was reduced by 40 and 80%, respectively, by use of the acetate buffer we describe. The 0.1 mob/L citratephosphate buffer of Shoup and Kissinger (4) required maximum off-set current of 999 nA at a potential of 500 mV. At a sensitivity of 50 nA/V the background noise was as much as half-scale on the recorder chart. To minimize noise in the system, particularly at high sensitivity, it is important to minimize the polarity of the mobile phase without sacrificing the desired resolution of compounds. Also, the pH of the mobile phase should be close to the auto-oxidation point of these compounds, for maximum sensitivity. Mobile phase de-gassing with helium markedly decreases problems of bubble formation. Thorough grounding of the pump, recorder, and detector through their respective power-cord grounds is required. Also, the electrostatic shield around the glassy carbon detector should be grounded to the house plumbing by a separate wire. One should not overlook the possibility that the
glassy carbon electrode may have an excessively high internal resistance. If polishing does not solve this, a substitute electrode may be required. Work in progress in our laboratory and the work of Mon (5) indicate that the preliminary sample clean-up procedure before chromatography for catecholamines can be greatly simplified over those now described in the litera-
CII (U/Li
N
*0
. I’ ‘I
ture.
I
:
References 1. Kissinger, P. T., Refshauge, C., Dreiling, R., and Adams, R. N., An electrochemical detector for liquid chromatography with picogram sensitivity. Anal. Lett. 6, 465 (1973). 2. Riggins, termination
R. M., and Kissinger, P. T., Deof catecholamines in urine by
reverse-phase liquid chromatography with electrochemical detection. Anal. Chem. 49, 2109 (1977).
3. Moyer, T. P., Jian, N. S., Tyce, G. M., and Sheps, S. G., Analysis for urinary catecholamines by liquid chromatography with amperometric detection: Methodology and clinical interpretation of results. Clin. Chem. 25, 256 (1979). 4. Shoup, R. E., and Kissinger, P. T., Determination of urinary normetanephrine, metanephrine, and 3-methoxytyramine by liquid chromatography, with amperometric detection. Clin. Chem. 23, 1268 (1977). 5. Mori, K., Automated measurement of cat.echolamines in urine by high-speed liquid chromatography with fluorometric reaction detection. md. Health 16,41(1978). E Dale Behner
Richard Clinical Laboratory Loma Linda Univ. Med. Loma Linda, CA 92350
W. Hubbard Center
Immunoglobulin-Bound Creatine
KinaseBB (“Macro CK”) inThree Patientswith DifferentDiseases To the Editor: While we were investigating three cases above-normal total creatine kinase (CK, EC 2.7.3.2) activity, Urdal and Landaas (1) demonstrated the existence of “macro CK.” Independently of them, we have been able to attribute the abnormal CK activities in three patients to macro CK, based on isoenzyme CK-BB. Case 1, a 58-year-old woman with coronary heart disease (myocardial infarction in August 1978): In the serum of this patient, CK activities are still above normal (March 1979). Exclusion chromatography of 200 tL of serum on a column of Sephadex G-200 sf(15 X 0.9 cm, 50 mmol/L Tris buffer, pH 7.3) shows two peaks with CK activity. The first one (more than 90% of the total activity) is macro CK (Me> 300 000); the second peak is normal CK (Mr 80
of persistently
30
Fig. 1. Pattern from exclusion chroma-
tography 000) (Figure 1: #{149}-u-.). Electrophoresis of the serum also revealed the atypical macro CK (Figure 2, lane 2), migrating between CK-MM and CK-MB (Sigma Chemical GmbH, M#{252}nchen). Case 2, a 77-year-old woman with acute myocardial infarction and an apoplectic stroke. Figure 1 (#{149} - - -#{149}) shows the elution pattern of the CK activity after exclusion chromatography. Two distinct peaks are detectable. The ratio of the peak areas is about 3:2 (macro CK:normal CK). The isoenzyme pattern after electrophoresis is shown in Figure 2, lane 4. In addition to CK-MM and CK-MB, we found the atypical macro CK. Case 3, a 45-year-old woman with colitis ulcerosa. Exclusion chromatography of this serum again shows macro CK (Figure 1: A - - A .. A) and only a small peak of normal CK. The isoenzyme pattern after electrophoresis is shown in figure 2, lane 6. We classified the CK isoenzyme [in the complex] by immunological methods [precipitation test (2)] as CK-BB. By precipitating the CK activity with anti-immunoglobulin G serum and by staining the precipitin lines against anti-IgG in the Ouchterlony technique for CK activity, we determined that immunoglobulins G form the macromolecular part of the CK-BB-IgG complexes in case 1. In contrast to Urdal and Landaas (1), we were able to split off the CK-BB from its macromolecular IgGs by prolonged treatment with DEAE-Sephadex A-50 (3) and directly determine the CK-BB
-
BB
-
MB
#{149}
MM 12
3
Fig. 2. Results of creatine
L5
6
kinase isoen-
zyme electrophoresis
CLINICAL CHEMISTRY. Vol. 25, No. 8, 1979
1513
after electrophoresis. The results
are
shown in Figure 2: lane 1, isoenzyme marker; lane 3, CK-BB from case 1; and lane 5, CK-BB from case 2. The stoichiometric composition of the complexes and the specificity and nature of the CK-protein binding are now under
Table 1. Parametersof the Log-LinearRegressionLine y 1.699
0.032
1.076
2.000
1.059
250
2.301 2.398
0.025 0.017 0.013 0.01
300
2.477
0.008
References
1. Urdal, P., and Landaas, S., Macro creatine kinase BB in serum, and some date on its prevalence. Clin. Chem. 25, 461 (1979). 2. Wurzburg, U., Hennrich, N., Orth, H.-D., et al., Quantitative determination of creatine kinase isoenzyme catalytic concentrations in serum using immunological methods. J. Clin. Chem. Clin. Biochem. 15, 131 (1977). 3. Klein, B., Foreman, J. A., Jeunelot, C. L., and Sheehan, J. E., Separation of serum creatine kinase isoenzymes by ion-exchange chromatography. Clin. Chem. 23, 504 (1977).
D-7400
Tubingen,
2.176
West
Temperature.
(po,)m’
COVTSCtOd
mmHg
Germany
1.040
1.030 1.023 1.018
Patient’s
(P02)t’
body
#{176}C
37.7
39.7
37.6
66.2
62.0
42.4
32.0
89.7
94.2
92.7
36.7
96.9
112.3
127.1
39.4
98.2
155.7
148.6
35.8
99.1
295.8
299.3
37.7
99.6
blood po2. Clin. Chem.
measured
(1979). 4. Burnett,
R. W., Erroneous
To the Editor: Recent developments in the field of acid-base studies suggest the need for measuring blood gas parameters instead of using derived quantities. The pK’ of the Henderson-Hasselbalch equation changes in an acutely ill patient, and the calculation of either Pco2 or total CO2 from an alignment nomogram is not valid (1). Corrections are made on the measured values at 37 #{176}C to estimate those values at the patient’s body temperature (2). Simple temperature coefficients are used to correct blood pH and p co2. Temperature correction of P02, however, requires a calculated value of oxygen saturation in addition to po2 and the patient’s body temperature (3, 4). Because oxygen saturation is calculated from pH and P02, the correction of p02 is further complicated. The equation referred to by Porter (3) can be written as (po2)t
=
(P0z)rn
(2) (P02)t = (p02)m . yt where y = lOt. Taking into consideration the sigmoidal curve of the P02 and percent oxygen saturation, and the exponential term in the f value, a log-linear relation is obtained between (P02)m and y as
y
k1+k2log(po2)m
=
(P02)m
[1.212
-
0.0786 log(p02)]t
(4)
The coefficient of correlation for the temperature-corrected values of P02 from equations 1 and 4 is r = 0.999. This is based on data from 36 patients whose po2 ranged from 38 to 295 mmHg, body temperature from 32.2 to 39.4 #{176}C, and percent oxygen saturation from 66 to 99%. Table 2 lists illustrative results.
(1)
10
References
where (P 02)1= temperature-corrected
value of po2 (P02)m
=
measured value of po2 at 37
patient’s body temperature -37 f = 0.032 - 0.0268e103’301 x = percent oxygen saturation =
It is desirable
to simplify
the daily
1. Natelson, S., and Nobel, D., Effect of the variation of pK’ of the Henderson-Hasselhalch equation on values obtained for total CO2 calculated from pco2 and pH values. Clin. Chem. 23,767 (1977). 2. Siggaard-Anderson, 0., Blood gases. In Fundamentals of Clinical Chemistry, N. Tietz, Ed., W. B. Saunders PA, 1976, p 854.
3. Porter, saturation
1514 CLINICAL CHEMISTRY, Vol. 25, No. 8, 1979
W. H., Influence on temperature
25, 499
temperature
J. AppI. Physiol.
J. W., Blood gas calculator. 21, 1108(1966).
Shirish
Shastri
Michael Bright Edward Wagman Bergen Paramus,
Pines
County
Hospital
NJ 07652
(3)
where k1 and k2 are constants. By Severinghaus’ curve (3, 5) for f values ranging from 0.032 to 0.008 and (P02)m values ranging from 50 to 300 mmHg, k1 and k2 are determined by the least-square regression (Table 1). The equation for (po2)t is then obtained as (Po2)t
%
corrections for blood pH and gas measurements. Clin. Chem. 24, 1850 (1978). 5. Severinghaus,
Correctionof BloodPo2 for the Patient’sBodyTemperature
Oxygen saturation,
temPetuT0,
mmHg
calculation of the corrected value of po2 and to provide a more direct approach that requires only the P02 and the patient’s body temperature. Equation 1 can be rewritten as
IV
k1 = 1.212 k2 = -0.0786 r = 0.993
Table 2. Temperature-CorrectedValuesof Po2 Measured
Wolfgang Stein Jurgen Bohner Abteilung
Constants
iOg(po2)m
50 100 150 200
Klinik,
y(=1O’)
(po2)m
investigation.
Medizinische
k1 + k2
=
log(p02)rn
Co., Philadelphia,
of hemoglobin correction of
is SodiumAzide Preservativea Carcinogen? To the Editor: Although sodium aside is a valuable and useful preservative and possesses significant bactericidal and herbicidal properties, its use in the clinical laboratories has created some problems. Numerous laboratory tests have been affected by sodium azide (1-5). In addition, sodium azide as a hazardous explosive in the laboratory has been documented (6). The purpose of this letter is to add yet another potential health risk of using this preservative in the laboratory. My speculation is based on the observations of McCann et al. (7), who tested a total of 300 chemicals, including sodium azide, for mutagenicity by using the Salmonella/microsome test system (8) for screening the potential carcinogens. They demonstrated that sodium azide was indeed a very potent mutagen. Because there is a high correlation between carcinogenicity and mutagenicity-90% (156/174) of carcinogens are mutagenic in the Salmonella/microsome test system (8)-I suggest that sodium azide should be
