Table 1. PartitIonCoefficients(C)a and ... - Clinical Chemistry

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L serum samples, 5-(p-methyl)-phe- nyl-5-phenylhydantoin, 5-ethyl-p-tolyl barbituric acid, and a .... We thank the John P. Kelly Mater Re- search Foundation for a ...
a study on citrate metabolism in recurrent stone formers we wished to mea-

Table 1. PartitIonCoefficients(C)a and PrecisionStudieson Antiepileptic Drugs in Various Blood-Collection Tubes in

so

0.000 0,54

126.

gin.

.7/1.

10

4.07

CV

$9/I

.5/1.

.

00

25.44

1, 0.5 .36

PPM

25.46

1$

07 PPM CC “IA Ge

07 C’fl. POlO Clx

V?

3 Si.

In

CV

00

S ml.

,,b

50

17.31

16

2.07

7.47

0.53

21.43

20

0.77

2.42

1.00

16

0.30

3.23

1.00

C0

CV

20

N.11

C,

0542.04

‘I

CO

1

CC

5.45

1.0$

150.31

.7/1. 16

26.16

I.64

0.67

Sq/i.

5 7.40

0.55

20 0.73 2.13 0.54 ‘.5, LI 0.24 2.60 1.01 10.14 13 0.26 2.7 0.55 6.61 16 0.20 4.22 1.03 7.11 170.12 1.73 0.55 14.61 II 0.3 2.46 0.04 15.45200.20 1.34 1.04 1S.01 17 2.3 4 .30 1.04 67.17 20 1.50 2.23 1.03 00.7I 18 1.54 4.13 0.52 ‘I.”

I.’.

10.21 15 0.22 1.14 6.40 0.35 0.55 7.07 20 0.14 L.51 0.53 15.53 1, 0.15 I1.4$ 14.06 0.00 34.2$ 16 1.10 0.87 #{149}4.77 08 0.44 0.23 44.0$ 19 4.’, 703

0Yieldsdrugsratiosof tested

tubes

to glass

PA 16823),

a1m x 2

mm column. Before extraction of phenobarbital (PB in Table 1), carbamazepine (CZ), phenytoin (DPH), primidone (PD), or ethosuximide (ETX), we added,to 500L serum samples, 5-(p-methyl)-phe-

nyl-5-phenylhydantoin, 5-ethyl-p-tolyl barbituric acid, and a,a-dimethyl-methylsuccinirnide

as internal

stan-

dards. The acidified serum was then extracted with methylene chloride. For extraction of valproic acid (VPA), a solution of cyclohexanecarboxylic acid (internal standard) in methylene chloride was added to 500 L of acidified serum For the PIA we used “Abbott TDx” kits. Fluorescence polarization was measured with the TDx Analyzer (Abbott Diagnostics, North Chicago, IL 60064). Table 1 shows CC and FPIA results for the anticonvulsant drugs in serum

stored in the B-D and Venojet serum separators and without

this treatment.

Becausethe m is expensive,I tested fewer tubesthan in the GC method. In general, the partition coefficients (ratios for tested tubes to glass tubes) were near

to 1.0.

Means and their CVs for the serumseparator tubes and the glass tubes were within the expectedexperimental limits by both methods. The recognized better precision of spx (Table 1) is observed. Paired student’st-test analysis of the means (Table 1) indicated significant differences (p 0.05)-but inspection of the individual data pairs

J

5

C,

5 16

1.56

7; 16

15

13.6015 2.04

CV

1.57

7$.’,

0.24 2.31 1.00 6.00 14 0.46 7.64 0.05 7.45 10 0.20 1.04 14.36 16 1.07 0.52

10.32

3.71 1.31

1$

2.01

.05

1.00 1.01

-

14.l 140.133.64

1.54

0.55

$1.10ii

3.17

5.14 1.05

0.67

.1S

5.,.

4.54 1.01

--...

-

57.40

tubes. bNumber of

nants. We useda Perkin-Elmer 3920B chromatograph equipped with a flame ionization detector. Chromatography was on GP 2% SP 2100/1% SP 2510 on Supelcoport, 100/120 mesh (Supelco Inc., Bellefonte

5.45

/1.

00

$0

sure citrate in plasma. Most methods

10 Si,

7-?

4.46

.?I

14

testedtubes.

for anticonvulsant by CC and rii.

drugs monitoring

References 1. Kessler KM, Leech RC, Spann JF. Blood collection techniques dine protein binding.

heparin, and quiniClin Pharmacol Ther

25, 204-210 (1979). 2. Cothain RH, Shand D. Spuriously

low concentrations resulting from blood collection methods. Ibid., 18, 535-538 (1975). 3. Frematad D, Bergerud K. Plasma protein binding ofdrugsas influenced by blood plasma propanolol

methods. Acta Pharmacol Toxicol 39, 570-572 (1976). Letter. 4. Borg#{226} 0, Piafsky KM, Nilsen 00. Plasma protein binding of basic drugs. I. Seleccollection

tive displacement from a1-acid glycoprotein by tris(2-butoxyethyl) phosphate. Clin Pharmacol Ther 22, 539-544 (1977). 5. Shah VP, Knapp G, SkellyJP, Cabana BE. Interference with measurements of certhin drugs in plasma by a plastizer in Vacutamer Tubes. Clin Chem 28, 2327-2328 (1982). Letter. 6. Shang-Qiang J, Evenson ME. Effects of contaminAnts in blood collection devices on measurements of therapeutic drugs. Clin Chem 29, 456-461 (1983). 7. Arranz MI. Rapid determination of anticonvulsant drugs by isothermal gas liquid chromatography. J Chrornstogr 222, 486490 (1981).

8. Arranz ME. Rapid gas chromatographic of valproic acid in serum. Ibid., 225,459-462 (1981). determination

Maria Isabel Arranz

Pefla

Biochem. Service Ramon y Cajal Special Center Carretera de Coirnenar Km 9.100 Madrid 34, Spain

An AutomatedMethodfor Measuring PlasmaCitrateWithout

demonstratedno clinically significant

Protein Precipitation

differences. VPA and PD means showed some larger differences. In conclusion: these separator tubes with their well-known advantagescan be validly used in collecting samples

To the Editor: Urinary citrate is an important inhibitor of both the formation and growth of renal calculi (1,2). As part of

(>10%)

1578 CLINICALCHEMISTRY,Vol.31, No. 9, 1985

for plasma citrate involve either protein removal by precipitation or ultrafiltration (3-7). One group found protein removal to be unnecessary (8); however, the method was manual and required a relatively large plasma sample (0.5 mL). After protein removal, the plasma filtrate is then monitored for citrate by manual methods. The more specific methods for measuring citrate involve the useof citrate (pro-3S)-lyase (EC 4.1.3.6) coupled with malate dehydrogenase (MDH; EC 1.1.1.37) and lactate dehydrogenase (LDH; EC 1.1.1.27) (3-8). We describe here an automated enzymic method for measuring plasma citrate that does not

involve removal of plasma proteins,an adaptation of our recently published method for urinary citrate (9). Plasma from heparinized blood is stored at -20 #{176}C until analysis. Prepare the following: triethanolamine (TEA) buffer (0.5 mol/L; pH 8.2); zinc chloride, 30 mmol/L in water MDH and LDH (Sigma Chemical Co., St. Louis, MO) 10 kU/L, use as supplied; citric acid stock standard, 1 g citric acid H20 in 100 mL ofH2S04 (0.1 mol/L); citric acid working standard, citric acid stock standard diluted with water to 100, 200, and 400 p.mol/L; NADH, 10 mg in 10 mL of TEA buffer. The reagent mixture is 5.2 mL of TEA buffer, 1.5 mL of NADH, 0.3 mL of zinc chloride, 15 pL of MDH and 15 L of LDH. The start reagent: 7 mg of citrate lyase (product no. C 0897; Sigma Chemical Co.) in 1.0 mL of TEA buffer. Prepare the citrate lyase, NADH, working standards, and reaction mixture immediately before use; store all

other solutionsat 4#{176}C. Program the Cobas Bio as follows: Units, mol/L; Calculation factor, 0; Standard 1, 100; Standard 2, 200; Standard 3, 400; Limit, 800; Temperature, 37 ‘C; Type of analysis, 6; Wavelength, 340 urn; Sample volume, 40 LL;

Diluent volume, 40 L; Reagent volume, 200 L; Incubation time, 600 s; Start reagent volume, 30 pL; Time of first

reading,

5 s; Time

interval,

30 s;

No. readings, 20; Blanking mode, 1; Printout mode, 1. The plasma citrate concentration is calculated by the analyzer and printed out in molJL. The standard curve is linear to 800 anol of citrate per liter and satisfies the following equation: absorbancechangeat 340 urn x 965 = citrate, in j.mo1/L. In the assay, a 10-mm preincubation of all reagents and enzymes, except citrate lyase, was necessary to allow side reactions due to plasma and reagent enzymes and substrates to come to completion.In general, after several minutes

of preincubation the absor-

per minute for the was essentially the same as for

bance change (A) sample

the blank (-0.002 to -0.006 A/mm). Citrate lyase, used in amounts ranging from 0.21 to 0.49 mg per assay, gave similar results for citrate. Citrate lyase concentrations sO.12 mg per assay did not result in quantitative conversions of citrate to oxaloacet.ate, and the results obtained were lower. For the assay we decidedto use 0.21 mg of citrate lyase for each citrate analysis. The smallest measurable concentration of citrate was 20 moI/L. The within-day CV for a plasma containing 69 pmol of citrate per liter was 3.5% (n = 10). The same plasma supplemented with 200 pmol of citrate per liter gave a CV of 1.0% and an analytical recovery of 106.0% (SD 1.6%, n = 10). These plasmas, analyzed 10 times during two weeks, gave CVs of 6.5 and 4.4%, respectively, for the low and high concentrations. Recovery was 102.2% (SD 4.4%) and the citrate concentrations did not change during this two-week period. The preliminary normal reference interval, from data on 20 healthy volunteers, is 71 to 139 (mean 101) zmo1/ L This correlates well with two other methods in which the same specific enzyme assay is used (5, range 70-140, mean 100 rnoI/L, n = 31; 7, range 80170, mean 120 pmol/L, n = 20). We thank the John P. Kelly Mater Research Foundation for a grant supporting this project. References 1. Ligabue A, Fini M, Robertson WG. Influence of urine on “in vitro” crystallization

rate of calcium oxalate; determination of inhibitory activity by a [“C]oxalate technique. Clin Chim Acts 98, 36-46 (1979). 2. Schwille P0, Scholtz D, Engelhardt W, Sigel A. Citrate in daily and fasting urine:

Stabilizationof the Reaction MixtureUsed In Urinary Citrate

Results of controls, patients with recurrent idiopathic calcium nephrolithiasis and primary hyperparathyroidism. Invest Urol 16, 457-462 (1979). 3. Moellering H, Gruber W. Determination of citrate with citrate lyase. Anal Biochem 17, 369-375 (1966). 4. Welshman SG, McCambridge H. The estimation of citrate in serum and urine using a citrate lyase technique. Clin Chem Acts 46, 342-246 (1973). 5. Tomisek .AJ, Winkler EM, Natelson S. Fluorometry of citrate in serum with use of citrate (pm-3S)lyase. Clin Chem 21, 730734 (1975). 6. Toftegaard-Nielsen T. A method for enzymic determinationofcitratein serum and urine. Scand J Clin Lab Invest 36, 513-519 (1976).

We recently published a Letter on rapid estimation of urinary citrate with a centrifugal analyzer (1). We have experienced instability with certain batches of citrate (pm-3S)-lyase (EC 4.1.3.6) used in this assay, resulting in a nonlinear standard curve. As a result we have attempted to stabilize the citrate lyase used in the reaction mixture of this assay. By using a strong triethanolamine buffer (0.5 mol/ L compared to 0.1 mol/L) and dissolving 100 mg of bovine serum albumin (product no. A-7638; Sigma Chemical Co., St. Louis, MO) in 10 mL of buffer before adding the enzymes, we found that the reaction mixture was stable for 1 hat 37 #{176}C. At present, we prepare this mixture within 1 h of use, at room temperature (22 #{176}C). With the new reaction mixture, the standard curve was linear and stoichiometric from 0.5 to 4.8 mmol citrate per liter. We thank the John P. Kelly Research Foundationfor a grant supporting this proj-

7. Tompkins D, Toffaletti J. Enzyxnic determination of citrate in serum andurine with

use of the Worthington “Ultrafree” device. Clin Chem 28, 192-195 (1982). 8. ZenderH, Torrents C, Schneider U. Analyse

du citrate plasmatique par voie enzy-

matique sans deproteinisation. Clin Chim Acta 24, 335-340 (1969). 9. Holt C, Cowley DM, Chalmers AH. Rapid estimation of urinary citrate by use of a centrifugal analyzer. Clin Chem 31, 779780 (1985). Letter. B.

C. McWhinney D. M. Cowley A. IL Chalmers

Dept. of Pathol. Mater Misericordiae Public Hospitals South Brisbane, Queensland Australia 4101

Estimations To the Editor:

Reference

L HoltC, Cowley DM, Chalmers All. Rapid estimation of urinary citrate by use of a centrifugal analyzer. Clin Chem 31, 779780 (1985). Letter. A. H. Chalmers D. M. Cowley Dept. of Pathol. Mater Misericordiae Public Hospitals South Brisbane, Queensland Australia 4101

CUNICAL CHEMISTRY, Vol. 31, No. 9, 1985 1579