Table 1. Changes in Concentrations of Serum ... - Clinical Chemistry

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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

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