Methods

Table 2. RegressIon Analysis of ComparIson of

Methods Test

Slope

ALP, U/L

3.521

ALT, U/L Amylase,U/L AST, U/L

0.833 1.992 1.006 0.947

CK, U/L

GGT, U/L Sodium,mmol/L Potassium,mmol/L Bilirubin, zmol/L Creatinine,/Lmol/L Glucose, mmoVL Total protein,g/L Urea, mmoVL

1.202

0.993 0.974 0.995 0.842 1.060

0.968 1.011

Intercept 5.4

3.4 -9.5

2.2 -2.7 4.2 -0.7

-0.1 0.9

S

r

7.04 2.24 10.51 1.90 9.32 3.76 1.14

0.997 0.997

0.11

0.990 0.997

18.4 -0.3 -1.4

3.14 7.11 0.22 2.01

-0.2

0.23

0.995 0.998 0.996 0.998 0.976 0.999 0.997 0.969

0.999

50 patients samples for each method, in duplicate. y-glutamyltransferase (530 UIL) and creatinine (1200 moI/ L) did not meet the limits stated by the manufacturer. Sample carryover was insignificant. The 20-day imprecision evaluation and comparison of methods were performed according to the guidelines of the NCCLS (EP5-P and EP9-P). Total imprecision (Table 1) was within the allowable limit of error as calculated according to Tonks’ criteria. Results obtained with the Easy analyzer correlated well with those from the SMAC II, Astra 8 (amylase), and RA-1000 (CK) methods. For most of the tests the slope of the regression line was near unity (Table 2). The difference in slope for alkaline phosphatase (3.521), and amylase (1.992) presumably is attributable to the different substrates used. Creatinine showed a difference in both slope (0.842) and intercept (18.4 unoI/L). However, the difference between the Easy and comparison methods can be corrected by use of factors for slope and intercept. The low sample volume required and a dead volume of only 20 zL economize on patients’ serum. Sample throughput is limited and ranges, according to the pattern of requests, from 30 to 100 results per hour. In general, the analyzer is easy to use and its analytical performance is acceptable.

the erythrocytes were partly depleted of AlT by incubation at 37 #{176}C for 6 h before analysis. For measurement of cellular ATP, the EDTA-treated blood samples were diluted 1000fold with a pH 7.5 buffer containing, per liter, 0.1 mol of Tris acetate, 10 mmol of MgSO4, 2 mmol of EDTA, and 1 g of Triton X-100. ATP was determined in the hemolysates within 10 mm. Parallel AlP determinations by the accepted perchloric acid (PCA) extraction procedure (1, 4) were also performed on the same sample population. Al? was measured in both extracts by use of the firefly luciferin-luciferase system, with photon counting and internal standardization technique (4, 5), as follows. Mix 0.1 mL of reconstituted ATP (constant light signal kit; Boehringer, Mannheim, cat. no. 567 736) with 1.0 mL of the Thton-containing buffer and pipet into this AlT reagent 50 tL of the Triton or PCA extract. Measure the light output, then add 20 &L of an aqueous 1.2 imol/L ATP standard solution to the samples and redetermine the luminescence. Refer the measured AlP values to the hematocrit of the blood samples. Our results showed that AlP in the Triton X-100 hemolysate is stable for at least 30 mm at room temperature. Intraassay precision of the Triton method was acceptable (CV 3.0% for 1.55 mmol ofATP per liter oferythrocytes, n = 15). Accuracy of the Triton technique was investigated by comparison of the two methods. Statistical analysis by an unpaired t probe revealed no significant differences between the Triton and the PCA extraction procedure. The equation of the computed regression line (where y is the new method) was y = 1.066x 0.071 mmol of AlT per liter of erythrocytes, with a coefficient of correlation of r = 0.942 (n = 48). The mean AlP value for freshly drawn blood was 1.60 (SD 0.24) mmol per liter ofcells by the Triton method, whereas that for the PCA technique was 1.65 (SD 0.28) mmoIJL. These values are consistent with those reported (6, 7). The presence of the minute amount of hemoglobin in the AlP reagent in the case of the detergent extracts (i.e., hemoglobin originating from approximately 2 x i0 cells per sampie) did not interfere with the AlP measurements. I conclude that the Triton extraction procedure for the determination of erythrocyte AlP is not only simple and rapid but is also precise and accurate, with applicability for automation. -

References 1. Trautachold I, Lamprecht W, Schweitzer G. Adenosme 5’-triphosphate. UV-method with hexokinase and glucose-6-phosphate dehydrogenase. In: Bergmeyer HU, Bergmeyer J, Grassi M, eds. Methods of enzymatic analysis.Vol. 7. Weinheim: VCH Verlags-

Rapid BIoluminescent Measurement of Human Erythrocyte ATP Content, Tam#{225}s Koszegi (Dept. Clin. Chem., Univ. Med. School of Pecs, Pecs, Ius#{225}gu. 13, H-7624,

Hungary)

Determination of intracellular ATP can be of great importance, not only in studies of the biochemistry of the erythrocyte, but also in certain pathological conditions. Unfortunately, current methods usually require a rather timeconsuming protein-denaturing step for the extraction of cellular ATP (1, 2). Here I suggest, based on our earlier experiences with Triton X-100 nomonic detergent extraction (3, 4), a simple and rapid procedure, not involving protein denaturation, for measurement of erythrocyte ATP. Freshly drawn EDTA-anticoagulated venous blood was collected from randomly selected outpatients. In 10 cases, 2578 CLINICAL CHEMISTRY, Vol.34, No. 12, 1988

gesellschaft, 1985:350. 2. Godwin IR, Agar NS, Roberts J. Measurement of erythrocyte ATP, DPG, glucose, and lactate with a Cobas-Bio centrifugal analyzer [Letter]. Clin Chem 1983;29:1855. 3. Koezegi T, Kellermayer M, Berenyi E, Jobst K, Hazlewood CF. The bulk of ATP is associated to proteins in the living cell: a release kinetics study. Physiol Chem Phys Med NMR 1987;19:143-6. 4. Koszegi T, Kellermayer M, Kovecs F, Jobst K. Bioluminescent monitoring of ATP release from nonionic detergent treated human red blood cells. J Clin Chem Clin Biochem 1988; in press. 5. Turner GK. Measurement of light from chemical or biochemical reactions. In: Van Dyke K, ed. Bioluminescence and chemiluminescence: instruments and applications. Vol I. Boca Raton, FL: CRC Press, 1985:56. 6. Beutler E, Baluda MC. Simplified determination of blood adenosine triphosphate using the firefly system. Blood 1964;23:688-97. 7. F#{233}o CJ, Leblond F. The discocyte-echinocyte transformation: comparison of normal and ATP-enriched human erythrocytes. Blood 1974;44:639-47.