Importance of Blood-Collectiontubes in Plasma Lidocaine Determinations

CLIN. CHEM. 25/4, 617-619 (1979)

Importance of Blood-CollectionTubes in Plasma Lidocaine

Determinations

W. W. Stargel, Charles R. Roe,2 P. A. Routledge,3 and 0. G. Shand3 Downloaded from https://academic.oup.com/clinchem/article/25/4/617/5671205 by guest on 24 September 2021

In 25 clinical samples serum lidocaine concentrations patients and transferred to Vacutainer Tubes from which the fell from a mean of 6.5 ± 2.1 mg/L (mean ± SD) to 4.9 ± red stopper had been removed. One aliquot was allowed to 1.8 mgIL(p < 0.001)whenthe blood sample was allowed clot,while another aliquot was placed in a red-stoppered tube, to make contact with the stopper of the Vacutainer#{174}col- allowed to contact the stopper, and clot. The two serum lection tube. In vitro experiments showed that this effect samples were assayed for lidocaine with one lot of EMIT5-cad of the stopper occurred only with whole blood and was (Syva Corp., Palo Alto, CA) reagents. dependenton sample concentration. The plasma binding In Vitro Experiments of lidocaine decreasedfrom a normalvalueof 56% ± 2.2 (mean ± SD)to 28% ± 2.2 (p < 0.001)whenexposedto One hundred milliliters of whole blood from a normal vol- the Vacutainer stopper. We conclude that a chemical unteer receiving no medication was collected by direct veni- leached from such stoppers displaces lidocaine from its puncture in a plastic syringe and immediately transferred to plasma- binding sites and that the drugis then redistributed a glass tube containing 500 USP units of heparin. Lidocaine into the erythrocytes, producing spuriously low lidocaine hydrochloride was added to give a final concentration of 3 g concentrations in plasma or serum. Such artifacts are of lidocaine per milliliter of blood. Two-milliliter aliquots were important in therapeutic drug monitoring and can lead to then transferred to five glass-stoppered tubes, five green- erroneous clinical decisions. stoppered Vacutainer Tubes, fivered-stoppered Vacutainer Tubes, and five red-stoppered Monojecte tubes (Sherwood Medical Industries, St. Louis, MO). The remaining blood was Additional Keyphrases drug assay . blood-collection tubes then centrifuged, and 1-mL aliquots of the plasma layer were - variation, source of added to various tubes, as described for whole blood. All tubes Therapeutic monitoring of drug concentrations in plasma were then inverted 10 times and plasma obtained from the has now become an accepted part of clinical practice, and blood samples. All plasma samples were assayed for lidocaine many new assay procedures are being developed. Although with one lot of EMIT reagent. itisknown that spurious values can resultfrom the use of The effect of increased concentrations of lidocaine(up to certaintubes forblood collection(1), thisinformationhas not 12 izg/mL) added to blood collected in Vacutainer Tubes or been very widely disseminated. Itwas firstshown that lower in an all-glass system was also investigated in quadrupli- plasma propranololconcentrationsresultedwhen Vacutainer cate. Tubes (Becton-Dickinson,Rutherford,NJ 07070) ratherthan Plasma Binding an all-glasssystem were used forblood collection.Since then, the same artifacthas been noted with the other basicdrugs: Plasma was obtained afterblood collectionintoVacutainer alprenolol,imipramine, chlorimipramine, and nortriptyline Tubes or glass-stopperedtubes,and lidocainewas added to (2), as wellas meperidine (3). Diminished plasma binding of a finalconcentrationof 2 zg/mL. Five 1-mL samples of plasma quinidine from blood collectedinVacutainer Tubes has also from each collectionsystem were dialyzed against I mL of been noted, but the resultingeffecton plasma concentration Sorensen’s phosphate buffer (pH 7.38)at 37 #{176}Cfor 6 h by was small (4,5). means of Teflon dialysiscellsand Spectrapor membranes We recently noted erraticconcentrations of the antiar- (Spectrum Medical Industries,Los Angeles,CA). Lidocaine rhythmic drug lidocainein plasma, and have investigated concentrationsinboth plasma and bufferwere measured and whether this involvesbinding displacement and redistribution calculated from appropriatestandard curves,and the per cent because of the use of the blood-collectiontubes. plasma binding was calculated. Materials and Methods Results Patients’ Samples In the 25 clinical samples, exposure to the rubber stopper lowered lidocaine concentration in serum from 6.5 ± 2.1 Blood sampleswerecollectedinto plastic syringes from tg/mL (mean ± SD) to 4.9 ± 1.8zg/mL (p <0.001 by a paired t-test). Departments ofPharmacy’ and Pediatrics,2and the Divisionof Clinical Pharmacology,3 Duke University Medical Center, Durham, In the in vitro experiment, there was a significant reduction NC 27710.Correspondence to W. W. S., Box 3028, Divn. Pediat. in the lidocaine concentration of plasma obtained from blood Metabolism,at the Center. collectedin the commercial tubes (Table 1).This was not seen Received Dec. 22, 1978; accepted Jan. 29, 1979. when plasma was added to the tubes,which shows that er-

CLINICAL CHEMISTRY, Vol. 25, No.4, 1979 617 Table 1. Effect of Various Blood-Collection Tubes on Measured Lidocaine Concentration (g/mL) in Plasma Control (all glass) Green Vacutalner Tube Rod Vacutalner Tube Red Monoject Mean a SD b Mean a SD b Mean a SD b Mean a so b Whole blood 3.12 0.13 2.42’ 0.08 2.22c 0.15 2.28c 0.08 Plasma 3.16 0.06 3.02 0.19 3.08 0.13 3.18 0.28

Mean of five separatealiquots of the sameblood sample or plasma derivedfrom the blood sample; lidocaine was added to freshly withdrawn blood tomake a concentrationofabout3 sg of lidocalne per milliliter of whole blood. b SD = standard deviation.

C p < 0.001 by analysis of variance.

ythrocytes were involved in producing the spuriously low glycoprotein,a probable major binding sitefor these basic Downloaded from https://academic.oup.com/clinchem/article/25/4/617/5671205 by guest on 24 September 2021 concentrations. ligands (2). In our studies we have found a similar phenome- The effect of increasing concentrations of lidocaine from non with lidocaine, with reduced binding in plasma confirmed. 2 to 12 zg/mL in whole blood from Vacutainer Tubes or from It is therefore likelythat TBEP isthe compound that dis- an all-glass system on the subsequent lidocaine concentrations placeslidocainefrom its binding sitesinplasma, although the in plasma isshown inFigure 1.With eithercollectionsystem, relevant binding protein for lidocaine has not yet been iden- increasingthe initialblood concentration produced a lesser tified (8). It is interesting that the magnitude of the effect of increaseinsubsequent plasma concentration,which implies this phenomenon on lidocaine concentrations in plasma is very a decrease in the blood/plasma concentration ratio.Fur- similar to the mean percentage effect on plasma propranolol thermore, the effect of using Vacutainer Tubes diminished concentration (28%) in plasma, calculated from the data of progressively as whole blood concentration was increased, Cotham and Shand (1), despite the fact that propranolol is until there was no effect at 12 zg/mL. 90-95% bound at therapeutic concentrations whereas lido- Finally, when Vacutainer Tubes were used in blood col- caine is only about 60% bound at a plasma concentration of lection, lidocaine binding in plasma fell significantly from a 3 tg/mL (8). Presumably, the relative affinity of TBEP for normal value of 56% ± 2.2 (mean ± SD) to 28% ± 2.2 (p < the binding sites in plasma is much greater than the affinity 0.001 by an unpaired t-test). of lidocaine. Tucker and coworkers also showed that the binding of ii- Discussion docaine varied from approximately 70% at 1 ig/mL of plasma Cotham and Shand (1) postulated that there was in some to around 40% at 12 ig/mL (8). Theoretically, therefore, the rubber stoppers a chemical that could leach intoblood and effect of Vacutainer Tubes on the lidocaine concentration in reduce the plasma binding of propranolol.The increasedfree plasma measured from a whole blood sample should fall with drug could then partitionwith the drug bound to erythrocytes, increasing lidocaine concentration, and we found this to be which would lead to spuriously low plasma concentrations. the case (Figure 1). However, the percentage change of lido- The plasticizertris(2-butoxyethyl)phosphate (TBEP) has caine concentration in plasma was still marked throughout been reported to be a contaminant in plasma collectedin the “therapeutic” range (1.5 to 5.0 ig/mL of plasma). The Vacutainer Tubes (6)and identifiedas an inhibitorof drug figure also shows that the concentration ratioof blood-to- binding to protein (7).Borga and coworkers subsequently plasma increases with increasing lidocaine concentration in have showed that TBEP-inhibited protein binding of aipre- whole blood. nololand imipramine isdue to a selectiveeffecton a,-acid The enzyme immunoassay (EMIT) of lidocaine inserum or plasma has been shown to be rapid, specific, and sensitive and therefore particularly suitable for therapeutic monitoring (9). IC We feel it is important to emphasize that these advantages may be nullified and patients exposed to risk if clinical deci- 0 sions are made on the basis of spuriously low concentrations of lidocaine in plasma or serum because of the use of certain types of commercially available collection tubes. 8 Plasma We are grateful to Jan Admiraal and Peggy Rogers for their ex- Lidocoine cellent technical assistance and to Syva Corporation for the re- 6 Concentration agents. (ug/ml) References 4 - - - . au 9Ia ystea 1. Cotham, R. H.,and Shand, D. C.,Spuriouslylowplasma propra- - acutaIne, laDe, nolol concentrations resulting from blood collection methods. GUn. 2 Phormacol. Ther. 18,535 (1975). 2. Borga, 0., Piafsky, K. M., and Nilsen, 0. C., Plasma protein binding of basic drugs. 1. Selective displacement from a,-acid glycoprotein by tris(2-butoxyethyl) phosphate. Clin. Pharmacol. Ther. 2 4 6 8 10 12 22,539(1977). Whole Blood Lidocaine Concentration 3. Wilkinson, G. R., and Schenker, S., Pharmacokinetics of meperi- Fig. 1. Effects of Increasing lidocaine concentrations in whole dine in man. Gun. Pharmacol. Ther. 19, 486 (1976). Letter. blood collected In Vacutainer Tubes or In an all-glass system on 4. Fremstad, D., Reduced binding of quinidine in plasma from Va- the subsequent lidocalne concentration in plasma cutainers. GUn. Pharmacol. Ther. 20, 120 (1976). Letter. Each dot represents the mean and range offour samples; line of Identity. 5. Shand, D. G., Reduced binding of quinidine in plasma from Va- cutainers. Clin. Pharmacol. Ther. 20, 120 (1976). Letter.

618 CLINICAL CHEMISTRY, Vol. 25, No. 4, 1979 6. Misson, A. W., and Dickson, S. J., Contamination of blood samples Binding of anilide-type local anesthetics in human plasma. 1. Rela- by plasticizer in evacuated tubes. Gun. Chem. 22, 765 (1976). tionships between binding physiochemical properties and anesthetic activity. Anesthesiology 33, 287 (1976). 7. Piafsky, K. M., and Borga, 0., Inhibitor of drug-protein binding 9. Pape, B. E., Whiting, R., Parker, K. M., and Mitra, R., Enzyme in “Vacutainers.” Lancet ii, 963 (1976). immunoassay and gas-liquid chromatography compared for deter- 8. Tucker, G. T., Boyes, R. N., Bridenbaugh, P. 0., and Moore, DC., mination of lidocaine in serum. Gun. Chem. 24, 2020 (1978).

CLIN. CHEM. 25/4, 619-621 (1979) Downloaded from https://academic.oup.com/clinchem/article/25/4/617/5671205 by guest on 24 September 2021

Improved Automated Kinetic Determination of Uric Acid in Serum by Use of Uricase/Catalase/Aldehyde Dehydrogenase

Knut Bartl, Max Brandhuber, and Joachim Ziegenhorn

The enzymatic determination of serum uric acid by use of H202/catalase equals the amount of uric acid present in the of uricase, catalase, and aldehyde dehydrogenase ac- sample. If, e.g., ethanol-converting enzymes such as alcohol cording to Haeckel [J. Clin. Chem. Clln. Biochem. 14, 101 dehydrogenases are present in the sample, acetaldehyde is (1976)] showed interferences from ethanol-converting continuously formed from ethanol, thus giving falsely high enzymes, which are present in some patients’ sera. We values for uric acid: have identified these enzymes as alcohol dehydrogenase ADH isoenzymes. Among other substances, a mixture of py- Ethanol + NADP4 acetaldehyde (EC 1.1.1.1) razole and oxalate can be used to eliminate these interferences. This inhibitor system gives good results when + NADPH + H used in the automated kinetic uric acid determination, as aldehyde is shown by a comparison with the manual assay for uric Acetaldehyde + NADP4 acetate dehydrogenase acid according to Kageyama [Gun. Chim. Acta 31, 421 + NADPH + H4 (1971)]. During our evaluation of the method, a few serum samples Additional Keyphrases: centrifugal analyzer . variation, from patients suffering from acute liver or renal disease caused

source of ‘ uremia liver disease kidney disease interferences, which led to an overestimate of uric acid. A similar effect has already been reported by Haeckel (1), who, A new kinetic method for the automated determination of to increasethe accuracy ofthe assay system, proposed use of uric acid by use of uricase (EC 1.7.3.3), catalase (EC 1.11.1.6), NADP instead of NAD4 as coenzyme and determination of and aldehyde dehydrogenase (EC 1.2.1.5) according to a sample blank. However, using NADP4 diminished but did Haeckel (1) has been developed in our laboratory (2). The not eliminate the interference; furthermore, determination reaction sequence is as follows: of a sample blank is inconvenient in an automated assay. To uricase improve the specificity and practicability of the assay system, Uric acid + 2 H20 + 02 allantoin+ CO2 + H202 we investigated the molecular basis of the interference described above. The results allowed us to modify the kinetic catalase Ethanol + H202 acetaldehyde + 2 H20 assay so that it yields precise results in each case without determination of sample blanks (3). aldehyde Acetaldehyde + NADP4 acetate Materials and Methods dehydrogenase Apparatus + NADPH + H4 We used an ENI GEMSAEC centrifugal analyzer, including The amount of acetaldehyde formed from ethanol by means DEC-PDP 8 and DEC tape (Electro-Nucleonics, Fairfield, NJ 07006), an Abbott ABA-100 analyzer (Abbott Scientific Boehringer Mannheim GmbH, Biochemica Werk Tutzing, For- Products Division, S. Pasadena, CA 91030), and a LKB 8600 schungszentrum, D-8132 Tutzing, Germany. reaction rate analyzer (LKB-Produkter, Bromma, Sweden) Received Nov. 28, 1978; accepted Jan. 23, 1979. for the automated experiments. Manual assays were done with

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