Fluorescent electrophoretic artifacts in the CK-BB region have been reported in electropherograms of serum from patients with prostatic cancer (1) or decreased.
Table 1. Changes in Concentrations of Serum Lipids and Apo A-I in Three Men K.Y., age 37
N.M., age 25 FIrst
Total cholesterol, mg/L
Second
1380
Triglyceride, mg/L
710
HDL-cholesterol,
270
mg/L
Apo A-I, units/L a
Two
a
1970 840
2060 970
520 860
400 800
550
FIrst
second b
2090 770 410 870
2000 960 770 1580
b
1980 470 870 1400
months after first sampling. #{176} 18 months after first sampling. The second determination on N.M.
determinations of total and HOL cholesterol gave values of 1970, 510; 2170, 500; and 2130, 530 rng/L. was done on 2/26/81. Fujther
concentration
by
the single radial
im-
munodiffusion method (3). The changes in apo A-I almost paralleled those of HDL-C, indicating that the observed changes were not artefactual (Table 1). All of these subjects were closely questioned as to all possible factors capable of affecting the HDL-C content, but no reason for the variation was found. Hence, we strongly suggest that investigators should be alert for some new factor or factors that regulate HDL-C. We also propose that a concentration of
HDL-C can be considered “inherent” to each individual subject if the value determined one or more months later is close
N.T., age 33
Second
First
to the result
of the initial
deter-
on 4/30/81,
5/12/81,
and 6/4/81
study (2) a metabolite pyridoxal-5-phosphate,
of pynidoxine, was suggested as a possible cause of the fluorescent band, but no confirming data were presented.
Fig. 1 . Cellulose acetate electrophoresis electrophoretic migration of patterns of (from top to bottom) serum certain fluorescent substances on celwith the fluorescent artifact, and normal lulose acetate can lead to possible misserum pools with added salicyiate, pyriinterpretation of the serum creatine kidoxine, pyridoxal-5-phosphate, pyridoxai, or 4-pyridoxic acid nase (EC 2.7.3.2) electrophoretic patNative fluorescence is shown with no CK substrate; terns. anode at left We tested specimens from 23 prostatic cancer patients and five patients with decreased renal function for the presence of a fluorescent artifact in the CK-BB region. Ten microliters of serum was applied to a cellulose acetate plate (Titan Ill Iso-Flur, cat. no. 3906; Helena Laboratories, Beaumont, TX 77704)
The
mination.
that
References
buffer (pH 8.8; ionic strength 0.029) for 30 mm. After electrophoresis in the same buffer for 10 mm at 300 mA, the
1. Nicoll,
A., Miller,
N. E., and
Lewsi,
B.,
High-density lipoprotein metabolism. Adv. Lipid Res. 17, 54-106 (1980). 2. Noma, A., Nezu-Nakayama, K., Kits, M., and Okabe, H., Simultaneous determination ofserum cholesterol in high- and low-density lipoproteins with use of heparin, Ca2, and an anion-exchange resin. Clin. Chem. 24, 1504-1508 (1978). 3. Cheung, M. C., and Albers, J. J., The measurement of apolipoprotein A-I and A-Il levels in men an women by immunoassay. J. Clin. Invest. 60, 43-50 (1977).
Atsushi Murai Tadao Miyahara Masakuni Kameyama Dept. of Geriatrics Kyoto Univ. Hosp. Sakyoku, Kyoto 606 Japan
FluorescentAcidIc Substances with ElectrophoretlcMigration Similar to CK-BB To the Editor:
Fluorescent electrophoretic artifacts the CK-BB region have been reported in electropherograms of serum from patients with prostatic cancer (1) or decreased renal function (2, 3). In one in
had been presoaked in Tris/barbital
electropherogram
was dried
med under an ultraviolet prostatic
cancer
patients
and exam-
lamp. The 23 showed
no
electrophoretic fluorescent artifacts in their sera in the CK-BB region, but two of the five patients with decreased renal function had a fluorescent artifact detectable in the pattern for their sera, which migrated anodic to the CK-BB position.
We also examined electropherograms of protein-free tungstic acid filtrates of a patient’s serum containing the artifact. The fluorescent artifact was still detectable in the filtrate. We next added 10 iL of 2.5 mol/L NaOH or 6 mol/L HCI to 50 tL of an artifact-containing patient’s serum before electrophoresis. Acidification totally inhibited the anodic
migration
of the
artifact;
substrate
used to develop bands with CK sample is a cornmercial CK control shown for reference (left to right: BB, MB, and MM isoen-
activity; the bottom
zyrnes)
alkalin-
ization caused complete loss of fluorescence. We tested pyridoxine, 4-pyridoxic acid, pynidoxal, pyridoxal-5-phosphate, and salicylic acid added to aliquots of normal serum and compared the resulting fluorescent electropherograms with the pattern observed for one of the artifact-containing specimens (Figure
1). We also compared
Fig. 2. Cellulose acetate electrophoresis patterns of same samples as in Fig. 1, in the same order, but with creatine kinase
these patterns (Figure 2) with those of three creatine kinase isoenzymes in control material from Helena Laboratories, using CK isoenzyme reagent (Helena Laborato-
ries, cat. no. 5130). Salicylate and pyridoxal-5-phosphate migrated in essentially the same position as the patient’s fluorescent artifact. However, the concentration
of
pyridoxal-5-phosphate
in
the patient’s serum (see Figures 1 and 2) was 0.2 g/L (measured by BioScience Laboratories, Van Nuys, CA 91405), far below the usual value of 3.6 to 18.0 .tg/L, while the same patient’s salicylate concentration was 560 mgIL, considerably above the therapeutic range of 20 to 290 mg/L.
CLINICAL CHEMISTRY,
Vol. 28, No. 7, 1982
1717
In an attempt
to identify
the sub-
stance causing the fluorescent artifact, after electrophoresis of 50 L of the
patient’s
artifact-containing
serum, we
cut out the cellulose acetate area contaming the artifact and treated this material with 10 tL of6 mol/L HC1 and 1.5 mL of absolute methanol. After centrifugation (1550 X g) to remove the
cellulose
acetate,
we recorded
the ul-
traviolet spectrum (140 to 360 nm) of the supernate and compared it with the ultraviolet spectra of pyridoxal-5phosphate and salicylate. The spectrum of the methanol-extracted material
coincided
with
that
of salicylate
methanol.
We also used
directly
artifactual
band,
confirming
the pres-
fluorescent was observed
electroin serum
ence of salicylate. The
identical
Ritzmann, S., Salicylate mimicking an abnormal CK-isoenzyme. Clin. 1622-1623 (1981). Letter.
phoretic artifact from another patient with decreased renal function; yet another patient with a high plasma salicylate concentration (1.48 g/L) had the fluorescent electro-
phoretic artifact but no evidence of renal
impairment. Follow-up testing
of the
Chem.
27,
Timothy T. Dick Kenneth E. Buck’
Trinder’s
over the fluorescent
Univ. of Oklahoma Health Sci. Center Dept. of Pathol. BMSB 455 P.O. Box 26901 Oklahoma City, OK 73190 1
Address correspondence
to this author.
2), and
may
also
electrophoretic
interfere
with
CK
interpretations.
Note: While this manuscript was in preparation, a Letter by McCoy et al. (5) reported a rapidly migrating fluorescent electrophoretic artifact in serum from a
patient
with salicylate
concluded
toxicity.
They
was probably
that the artifact
due to salicylate.
Heparin Interference in Creatine Kinase Assays To the Editor:
for measuring
work
was supported
by the Fraternal
Order of Eagles, Aerie 427. References 1. Jaggarao,
N., and Moss, D., A fluorescent
artifact resembling sera of patients with
BE-creatine prostatic
kinase in disease. Clin.
Chem. 25, 477-480 (1979). 2. Coolen,
1718
R. B., Herbstman,
R., and
Her-
either serum is the preferred creatine
(Beckman Instruments, CA 92634). Depending
kinase
or heparispecimen
(CK) ac-
Inc., Fullerton, on the concen-
tration of heparin, the CK activity of heparinized plasma can be higher than that of serum or as low as zero. We studied the magnitude of the interference and elucidated the mechanism by which this interference is
caused. Materials pies
were
and methods. collected
Blood
sam-
simultaneously
without anticoagulant and in 7-mL heparinized tubes (Becton Dickinson
and Co., Rutherford,
Serum
172
164
Plasma Sample 3
48
164
118
122
0
122
113 0
108 108
56 0
62 62
Sample
2
Serum Plasma
Plasma a Heparin
concentration.
of
20 USP unIts/mi
the aca. CK values for plasma (obtained from a completely filled 7-mL heparinized tube), analyzed in the Rotochem, were either zero or much lower than those of the corresponding sera. Examination of the absorbance values revealed zero-order kinetics for serum, while
with
plasma
proceeding
the
reaction
in the opposite
was
direction
(Figure 1). Furthermore, the Post Mixing Absorbance (PMA) and the absorbance difference between 160 s and 10 5 (LAG-PMA) of plasma (PMA, 0.660 A; LAG-PMA, 0.296 A) were much
higher than those of the corresponding serum A).
(PMA,
0.259 A; LAG-PMA,
The peculiar
phenomenon
0.041
was ex-
plained visually when serum and heparinized plasma were added to
Beckman
substrate in test tubes. The mixture was clear, the
serum-substrate plasma-substrate
mixture
turbid.
This
NJ 07070): these
tubes contain approximately 143 USP units of heparin. We performed CK assays at 37 #{176}C in the Rotochem hA centrifugal analyzer (Travenol Instrument Division, Jessup,
MD 20794), using the Beckman
sub-
strate (2) and on the Du Pont aca (Du Pont, Wilmington, DE 19898). Assay conditions for the Rotochem were: sample volume, 20 zL; water, 80 tL;
substrate, This
174 172
Plasma
blood.
tivity (1 ), but we encountered a serious interference from heparin when using the “CK-UV, Liquid STAT Substrate”
the CK-BB region in patients with decreased renal function. We have identifled the substance causing the artifact to be salicylate in two patients with decreased renal function. Pyridoxal-5phosphate and 4-pyridoxic acid also migrate in the CK-BB region (see Figure
178 0
Sample 1 Serum
Sample 5 Serum
470 mg/L. We did not electrophoretic
studies support earlier reports (2, 3) of a fluorescent electrophoretic artifact in
aca
Serum Plasma
Reportedly, nized plasma
retic artifact in the CK-BB region in 28 of 28 prostatic cancer patients. Our
CK U/L Rotochem
Sample 4
latter patient revealed a faintly visible fluorescent electrophoretic artifact and a much lower salicylate concentration of detect the fluorescent artifact in the CK-BB region in our series of 23 prostatic cancer patients, although Jaggarao and Moss (1 ) reported a fluorescent electropho-
Table 1 . CK Activitiesof Serum and Heparinized Plasma Compared a
M., Natural fluorescence in serum of patients with chronic renal failure not to be confused with creatine kinase-BB isoenzyme. Clin. Chem. 24, 492-494 (1978). 4. Richterich, R., Clinical Chemistry. Theory and Practice, Academic Press, New York, NY, 1969, p 289. 5. McCoy, M. T., Aquanno, J., and
in
reagent (4 ) to test for salicylate on the patient’s cellulose acetate plate after the fluorescent band had been separated electrophoretically. A faint purple color
formed
mann, P. , Spurious brain creatine kinase in serum from patients with renal disease. Clin. Chem. 24, 1636-1638 (1978). 3. Aleyassine, H., Tonks, D. B., and Kaye,
500 zL.
Absorbance
was
measured about 10 s after initiating the run (Post Mixing Absorbance), at the end ofthe lag-phase (150 s), and at 20-s intervals thereafter. Table 1 shows representative results. On the aca , identical CK activities were obtained for both serum and plasma. Serum CK values measured in the Rotochem agreed very well with those from
CLINICAL CHEMISTRY, Vol. 28, No. 7, 1982
Fig. 1. Absorbance changes. at 340 nm,
in creatine kinase assays on serum and heparinized plasma (obtained simultaneously from the same subject) with use of Beckman CK substrate and the Rotochem hA PMA, Post Mixing Absorbance; L-P, absorbance at the end of lag-phase (160 5). Heparin, 20 use
units/mL of blood
