Lipoperoxides Kit Evaluated for MeasuringLipoperoxides in Biological Samples: Reference

Intervals for Human Plasma

JULIAN DIAZ,1,2 ENRIQUE SERRANO,1 FRANCISCO ACOSTA,3 and LUIS F. CARBONELL2

1Department of Biochemistry, University Hospital “Virgen de la Arrixaca,” 2Department of Physiology,University of Murcia School of Medicine, 3Department of Anesthesiology,

University Hospital “Virgen de la Arrixaca,” Murcia, Spain

Introduction

Oxygen free radicals and lipid peroxydation areassumed to play an essential and causative role

in the pathogenesis of tissue and celular damageinduced by different affections such as inflamation,infection, chemical and physical irritations, andreperfusion injury (1,2). Increased lipoperoxide pro-duction has been demostrated in a wide variety ofclinical conditions, including oxidative stress, myo-cardial infarction, stroke, rheumatic disorders, dia-betes mellitus, hepatic diseases and burns (3,4).This process leads to the destruction of membranelipids and production of lipoperoxides (LPO) andtheir products such as aldehydes. Aldehydes arealways produced when lipid hydroperoxydes breakdown in biological systems, and it is of interest toidentify and measure these compounds as an indexof the extend of lipid peroxidation and as an aid toelucidate the role of aldehydes as causative agentsin certain pathological conditions (1,5,6).

Malonaldehyde (MDA) and 4-hydroxyalkenals (4-HNA) are the end products derived from breakdownof polyunsaturated fatty acids and related esters.MDA is the many instances the most abundantindividual aldehyde resulting from lipid peroxida-tion. The 4-HNA possesses cytotoxic, hepatotoxic,mutagenic, and genotoxic properties and increasedlevels of 4-HNA were found in plasma and variousorgans under conditions of oxidative stress (3,6).

Lipoperoxides can be measured by several meth-ods (3–6). Reported reference intervals for plasmalipoperoxides levels vary widely. This may be due, atleast in part, to methological variations, technicalpitfalls, problems related to the sampling, the time

required for analysis, and limitations correspondingto each method which make it difficult to choose thebest method. Nonetheless, measuring the degree ofoxidative stress a subject is undergoing is not inwide clinical use, although no standardized methodhas been accepted as measuring the oxidative stressstatus in humans (3,5). Because of the importance oflipoperoxide measurements, we undertook the cur-rent study to evaluate a kit for measuring lipoper-oxides in biological samples and provide reliablereference intervals for plasma, including the stab-lishment of possible sex-related differences.

Materials and methods

REAGENTS AND KIT

The LPO-586 assay (Colorimetric Assay of LipidPeroxidation, LPO-586. Bioxytecht. OXIS Inter-national S.A.. Bonneuil sur Marne Cedex, France)is based on the reaction of a chromogenic reagent(N-methyl-2-phenylindole in acetonitrile), withmalonaldehyde and 4-hydroxyalkenals at 45° C.One molecule of either MDA or 4-HNE reacts withtwo molecules of N-methyl-2-phenylindole in acidmedium (methanesulfonic acid), to yield a stablechromophore with maximal absorbance at 586 nm.Calibration solutions consisting of 1,1,3,3,-tetra-ethoxypropane in Tris-HCL buffer pH 7.4 and4-hydroxynonenal as the diethyacetal in acetoni-trile (6).

The effective measurement of MDA concentra-tions in the plasma was confirmed by high perfor-mance liquid chromatography (HPLC) analysis ac-cording to Esterbauer et al. (5).

PATIENTS, SAMPLE COLLECTIONS AND SPECIMENS

PREPARATION

With the consent of our hospital’s Clinical Re-search Committee we measured plasma lipoperox-

Correspondence: Julian Diaz, M.D., C/ Jose MariaMortes Lerma, 32 Dpl, Pta 14, 46014-Valencia, Spain.

Manuscript received October 30, 1997; revised andaccepted February 9, 1998.

Clinical Biochemistry, Vol. 31, No. 4, 277–279, 1998Copyright © 1998 The Canadian Society of Clinical Chemists

Printed in the USA. All rights reserved0009-9120/98 $19.001 .00

PII S0009-9120(98)00013-7

CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998 277

ides in 200 adults subjects selected for absence ofknown organic disease and were carefully screenedfor infectious, malignant, and other serious disor-ders. To additionally check their state of health theywere subjected to a conventional biochemical screen-ing and hematological analysis (7). Subjects wereclassified in two subgroups according to sex, group A(men) and group B (women).

Blood was collected from overnight fasted subjectsby venipuncture into 5 mL evacuated tubes contain-ing EDTA/K3 solution as anticoagulant (BectonDickinson Vacutainer Systems). After centrifuga-tion (2500 3 g) for 10 min in a centrifuge cooled to4° C, the supernatant plasma was removed care-fully, to avoid contamination with platelets, within30 min after sample collection. During the assayperiod, plasma samples were stored at 280° C untilanalyzed (usually within 30 days) with no freeze-thaw cycles, in trace-element-free tubes to maintainthe stability of the plasma samples and preclude anyin vitro lipid peroxidation.

ANALYTICAL PERFORMANCE

Detection limit

The detection limit was determined as describedby Gatautis and Pearson (8). A sample containingMDA 1 4-HNA at a concentration three- to fivefoldthat of reagent blank was measured 10 times, andthe detection limit was calculated as 2 SD/mean.

Linearity

The levels of standard calibration solutions (1.25,2.50, 5.0, 10.0, 15.0, and 20.0 mmol/L) were deter-mined in triplicate. Linear regressions and the cor-relation coefficient were then calculated.

Precision

To determine between-run and without-run preci-sions, we froze aliquots of plasma from a controlsubject at 280° C, thawing these only before analy-sis. Within-run precision was calculated from 10assays done on the same day. Between-run preci-sions was calculated from 20 assays done over 30days.

Accuracy

Accuracy was measured by evaluating the analyt-ical recovery of standard additions. Known quanti-ties of the 20 mmol/L standard solution were addedto plasma from healthy subject before adding thereagents. When the sample is mixed with reagents(N-methyl-2-phenylindole in acetonitrile and meth-anesulfonic acid), the most of the proteins precipi-tates and extracts MDA and 4-HNA simultaneously(6). After homogenizing the sample and centrifuga-tion, MDA 1 4-HNA were measured as describedabove.

Statistical analysis

The statistical study (mean, standard deviation,fractiles, coefficient of variation, regression, un-paired Student’s t-test and Kolmogorov-Smirnovtest) was determined with the SPSS program (Mi-crosoftt, EEUU). Correlations were examined byapplying linear regression according to Pearson.

Results and discussion

Lipid peroxidation is a biochemical process sharedby several different phenomena, either physiologicalor pathological (2). The detection of an oxidation pro-cess of polyunsaturated fatty acids in vivo and assay ofsecondary molecules released (whether or not they areresponsible for tissue lesions) require the developmentof quantitative methods that satisfy the fundamentalanalytical criteria of whitin and between-run preci-sions, sensitivity and accuracy.

The linearity displayed by measuring standard so-lutions was excellent in the assay range that corre-sponded to plasma concentrations. The correlationcoefficient of the regression line (r 5 0.002, p , 0.001)in the standard range from 0 to 20 mmol/L was excel-lent, indicanting that this method could be used inmost biological samples. Detection limit was 0.10mmol/L. This excellent sensitivity is sufficient for themethod to applied to human plasma. Within-run andbetween-run precisions expresed in Table 1. Analyticalrecovery was 90–100% when the final concentration ofMDA 1 4-HNA was ,10 mmol/L and one of theadventages of using this kit for assaying biologicalsamples then monitoring patients, is primeraly, thesatisfactory analytical recovery.

The LPO concentrations showed an excellent (r 50.925) and a highly significant (p , 0.001) corre-lation with MDA levels. The regression equationbetween LPO and MDA levels was: [LPO] 5 1.11 z[MDA] 1 0.08. As thiobarbituric acid reactive sub-stances other than MDA, can be present in biologicalsamples. We have verified whether the values ob-tained by the thiobarbituric acid assay resulted fromthe presence of MDA in the plasma. The simulta-neous analysis of 25 different samples using theLPO-586 assay and a direct HPLC method for MDA,showed a close correlation between the values mea-

TABLE 1Between-Run and Within-Run Precision for

Lipoperoxides Assay

Low Pool High Pool

Within-run precision (n 5 10)Mean (mmol/L) 1.23 1.78Standard deviation (mmol/L) 0.05 0.07Coefficient of variation (%) 4.07 3.93

Between-run precision (n 5 20)Mean (mmol/L) 1.27 1.81Standard deviation (m/L) 0.06 0.08Coefficient of variation (%) 4.72 4.42

DIAZ ET AL.

278 CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998

sured by the two tests and demonstrating that thetwo tests are actually measuring the same phenom-enon (5,6)

For each group, there was a representative sam-ple as reference population, according to the Inter-national Federation of Clinical Chemistry (IFCC)guidelines (9,10). From the initial 100 individuals ineach group, we discarded the results of those who, inthe diagnosis, had some disease and more than onebiochemical or hematological measurement altered.For both groups, the distribution of LPO values wassuch that, in each group, plasma lipoperoxides(MDA 1 4-HNA) values followed a gaussian fre-quency distribution, as verified by the Kolmogorov-Smirnov test. Aberrant values were excluded ac-cording to the IFCC guidelines (9). Between groups,the composite distributions were not significantlydifferent from the distribution for either sex sepa-rately. Accordingly, we calculated both parametric(mean 6 2SD) and nonparametric (0.05–0.95 frac-tiles) reference intervals (Table 2). When all menwere compared with all women, the men had signif-icantly higher values for plasma (p , 0.01).

In conclusion, this simple, reproducible, rapid andsensitive assay is adapted to screening patients whomay be subjected to oxidative stress. It can beadapted by clinical laboratories for the routine mon-itoring lipid peroxidation in human disorders.

Acknowledgements

Supported by grants from Fondo de InvestigacionesSanitarias, Madrid, Spain (FIS 96/1631 and FIS 97/5249)and Fundacion para el Desarrollo del Trasplante Hepaticoand Novartis Farmaceutica S.A., Madrid, Spain.

References

1. Duthie GG. Lipid peroxidation. Eur J Clin Nutr 1993;47: 759–64.

2. Halliwell B. Free radicals, reactive oxygen species andhuman disease: a critical evaluation with specialreference to atherosclerosis. Br J Exp Pathol 1989; 70:737–57.

3. Janero DR. Malonaldehyde and thiobarbituric acidreactivity as diagnostic indices of lipid peroxidationand peroxidative tissue injury. Free Radic Biol Med1990; 9: 515–40.

4. Gutteridge JMC, Halliwell B. The measurement andmechanism of lipid peroxidation in biological systems.Trends Biochem Sci 1990; 15: 129–35.

5. Esterbauer H, Lang J, Zadravec S, Slater TF. Detec-tion of malonaldehyde by high-performance liquidchromatography. Met Enzymol 1984; 105: 319–25.

6. Esterbauer H, Cheeseman KH. Determination of al-dehydic lipid peroxidation products: malonaldehydeand 4-hydroxynonenal. Meth Enzymol 1990; 186: 407–21.

7. Diaz J, Tornel PL, Martinez P. Reference intervals forblood ammonia in healthy subjects determined bymicrodiffusion. Clin Chem 1995; 7: 1048.

8. Gatautis V, Pearson KH. Separation of plasma caro-tenoids and quantitation of beta carotene usingHPLC. Clin Chim Acta 1987; 166: 195–206.

9. IFCC. The theory of reference values. Part 5. Statis-tical treatment of collected reference values. Determi-nation of reference limits. J Clin Chem Clin Biochem1983; 21: 749–60.

10. Solberg HE. Establishment and use of reference val-ues. In: Burtis CA, Ashwood ER, Eds. Tietz textbook ofclinical chemistry. 2nd ed. Pp. 454–84. Philadelphia:WB Saunders, 1994.

TABLE 2Reference Intervals for Human Plasma Lipoperoxides

Concentraction (mmol/L)

Groups Mean

Reference Intervals

(Mean 6 2SD)(0.05–0.95Percentile)

Group A (men;n 5 87)a

1.48 0.98–1.98 1.10–1.95

Group B (women;n 5 83)

1.33 0.91–1.75 1.05–1.76

aSignificantly greater than women (p , 0.01).

HUMAN PLASMA LIPOPEROXIDES

CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998 279