ANALYTICAL

BIOCHEMISTRY

197,191-l%

(1991)

Simultaneous Separation of Malondialdehyde, Ascorbic Acid, and Adenine Nucleotide Derivatives from Biological Samples by Ion-Pairing High-Performance Liquid Chromatography’ Giuseppe

Lazzarino,2

Donato

Di Pierro,

Barbara

Tavazzi,

Loredana

Cerroni,

and Bruno

Department of Experimental Medicine and Biochemical Sciences, and *Centro di Ricerca Interuniversitario da ipossia e iperossia II University of Rome, Tor Vergata, Via 0. Raimondo, 00173 Rome, Italy

Received

March

sul danno

6, 1991

A method for a simultaneous separation of malondialdehyde (MDA), ascorbic acid and adenine nucleotide derivatives in biological samples by ion-pairing high-performance liquid chromatography is presented. The separation is obtained by an LC-18-T 15 cm X 4.6 mm 3 pm particle size column using tetrabutylammonium as the pairing ion. The starting buffer consists of 10 mM tetrabutylammonium hydroxide, 10 mM KH,PO., plus 1% methanol, pH 7.00. A step gradient is formed using a second buffer consisting of 2.8 mM tetrabutylammonium hydroxide, 100 mu KH,PO, plus 30% methanol, pH 5.5. Under these chromatographic conditions a highly resolved separation of MDA, ATP, ADP, AMP, adenosine, ascorbic acid, GTP, GDP, IMP, inosine, Hypoxanthine, Xanthine, uric acid, NAD, and NADP can be performed in about 36 min. In addition, the separation of NADH and NADPH can also be obtained; this renders the present method suitable for the detection of these reduced coenzymes in alkaline extracts from tissue samples. Data referring to PCA extracts from ischemit and reperfused isolated rat hearts and from human erythrocytes peroxidized in vitro by a challenge with 1 mM NaN, and various concentrations of H,Oz are reported. The relevance of this chromatographic method lies in the possibility to determine directly MDA concentrations avoiding the unspecific thiobarbituric acid calorimetric test, any other manipulation of the sample out of the PCA extraction, and any possible coelution of other acid soluble compounds. The simultaneous determination of MDA, ascorbic acid, and of ATP and its degradation products gives the opportunity to correlate, by a single chromatographic run, peroxida-

i This work ’ To whom

Giardina*

was partially correspondence

supported should

0003-2697191 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form

by a 40% MURS be addressed.

grant.

tive damages with the energy state of the cell which is of great importance in studies of ischemic and reperfused tissues. 0 1991 Academic Press, Inc.

Malondialdehyde (MDA)3 is a byproduct of both peroxidation of phospholipids (1) and activation of the arachidonate cycle (2). Since MDA is generally regarded as a marker of peroxidative damages induced in cell membranes by both physical and chemical oxidative stresses, its determination in biological samples is of particular interest. In effect, the increase of MDA following administration of the anthracycline anticancer drug doxorubitin (3-5) or following ischemia and reperfusion phenomena (6-8) has been reported by several authors. Notwithstanding the agreement on the existence of peroxidative damages related to increase of oxidative stresses, the data reported in the literature have been questioned because of the unspecific method used to detect MDA in tissue extracts, i.e., the TBA colorimetric assay. Recently, chromatographic methods using ion-pairing high-performance liquid chromatography (HPLC) to determine MDA have been published (9,lO). Although valid for MDA detection, apparently none of these methods allow the determination of other low molecular weight compounds present in tissue extracts. Moreover, the possibility of a coelution of some relatively low-charged metabolite with MDA has not been taken into account. On the other hand, HPLC methods

3 Abbreviations used: Ado, adenosine; ADPRib, ADP-ribose; Hyp, hypoxanthine; Ino, inosine; ip, intraperitoneal; MDA, malondialdehyde; PCA, perchloric acid; TBA, 2-thiobarbituric acid; Xan, xanthine. 191

Inc. reserved.

192

LAZZARINO

for adenine nucleotides do not give any data concerning a simultaneous MDA separation (11,12). The present paper reports an ion-pairing HPLC technique, using tetrabutylammonium as the pairing ion, to obtain a single run separation of MDA, ascorbic acid, and adenine nucleotide derivatives in biological samples. This provides information not only on peroxidative damages but also on high-energy phosphate concentrations which are of great relevance particularly in ischemia and reperfusion studies (13-16).

ET

AL.

centration of the standard solutions of MDA was determined spectrophotometrically (9). Animals and Perfusion Conditions

Malondialdehyde-bis(diethylaceta1) and ascorbic acid were obtained from Merck (Darmstadt, Germany). Free MDA was obtained from the bis(diethylaceta1) as described by Esterbauer et al. (17). Adenine nucleotide standards were purchased from Boehringer (Mannheim, Germany). Tetrabutylammonium hydroxide was obtained from Nova Chimica (Milano, Italy) as a 55% aqueous solution, 2-thiobarbituric acid was obtained from Sigma (St. Louis, MO), and ultrapure methanol for HPLC was obtained from Merck (Darmstadt, Germany). All other reagents were of the highest purity available from commercial sources.

Male Wistar rats of 250-300 g body wt were purchased from Morini (San Polo D’ Enza, RE, Italy), fed with a standard laboratory diet and water ad l&turn in a controlled environment, and utilized after at least a week of acclimatation. Animals were anestethized by an ip injection of 200 mg/kg ketamine. The heart was quickly removed, the aorta was cannuiated, and the isolated heart was perfused according to the nonrecirculating Langendorff technique for 15 min at a constant hydrostatic pressure of 7.85 kPa (80 cm H,O) as described in detail elsewhere (6,13). A 30-min period of ischemia was induced by closing the perfusion flow above the aorta. Subsequently, the heart was reperfused for additional 30 min at the end of which the tissue was freezeclamped, immersed in liquid nitrogen, and divided into three aliquots. The first aliquot was deproteinized by ice-cold 0.6 M HClO, for the analysis of metabolites as previously described (18), the second was homogenized in 150 mM KC1 to obtain a 10% homogenate for the thiobarbituric acid reaction (19), while the third was kept for 24 h at 150°C for the dry weight determination.

HPLC Apparatus and Chromatographic Conditions

Preparation of Tissue Samples

The HPLC consisted of a Jasco 880-PU dual pump system (Tokyo, Japan) connected to a Jasco 875 ultraviolet-visible detector (Tokyo, Japan) and equipped with a Supelco LC-18-T 15 cm X 4.6 mm 3 pm particle size column (Supelco, Bellefonte, PA) provided with a guard column (2 cm X 4.6 mm) packed with the same matrix as the separative column. Chromatographic analyses were recorded using a Chromatocorder 12 integrator (System Instrument Co., Ltd., Tokyo, Japan). The mobile phase consisted of two eluants: 10 mM tetrabutylammonium hydroxide, 10 mM KH,PO,, 1.0% methanol, pH 7.0 (buffer A), and 2.8 mM tetrabutylammonium hydroxide, 100 mM KH,PO,, 30% methanol, pH 5.5 (buffer B). The buffer solutions, after pH adjustment, were filtered through a 0.22~pm Millipore filter. The gradient for the chromatographic separation reported in Fig. 1 was as follows: 12 min at 100% of buffer A, 2 min at up to 40% of buffer B, 11 min at up to 44% of buffer B, 10 min at up to 100% of buffer B, and hold for additional 5 min. The initial conditions were restored after about 5 min of washing with buffer A. The flow rate was 1.2 ml/min and the detection was performed at 266 nm, i.e., the maximum of absorption of MDA. Calibration curves for the quantitative measurements of the various compounds from biological samples were carried out by injecting standard mixtures with different known concentrations. In particular, the actual con-

The neutralized acid extract of the tissue was analyzed by HPLC (20 ~1 injected) after centrifugation for 15 min (26,890g at +4’C) in a Sorvall RC-5B refrigerated superspeed centrifuge (DuPont Instruments, Wilmington, DE) and subsequent filtration was through a 0.45-&m HV Millipore filter. For the TBA test, a 0.5-ml aliquot of the 10% homogenate in KC1 was incubated for 45 min in boiling water in the presence of 1% H,PO, and 0.6 M 2-thiobarbituric acid according to Uchyama and Mihara (19). The n-butanol extract was read at 535 and 520 nm and the difference in absorbance was used to calculate the MDA concentration in the tissue. This value was used for the subsequent comparison with that recorded by the HPLC assay.

MATERIALS

AND

METHODS

Chemicals

Peroxidation of Human Erythrocytes Blood samples were obtained from healthy volunteers in our laboratory. Thoroughly washed red cells were suspended in 38 mM citric acid + 77 mM trisodium citrate dihydrate + 123 mM glucose, pH 7.4. After the addition of 1 mM NaN, and various concentration of H,O, (0.5,1, 2, 5, and 10 mM), the erythrocytes were incubated at 37°C for 1.5 h. The neutralized HClO, extracts of both suspending medium and packed erythrocytes were used to quantitate the MDA produced during the challenge

CHROMATOGRAPHIC

SEPARATION

OF

MALONDIALDEHYDE

AND

ADENINE

NUCLEOTIDES

193

uncrowded area of the chromatogram allowing the assignment of the peak without any uncertainty. Using the same gradient composition it is possible to obtain a highly resolved separation also of NADH and NADPH (Fig. 1); the present method is also useful for the quantitation of these reduced coenzymes in the alkaline extracts from tissue samples (12). Calibration curves with standard mixture revealed a linearity between the area of each compound and its respective amount injected. Referring to MDA, the minimum concentration detectable is 167 mM which corresponds to 3.34 pmol injected using a 20-~1 loop. Another advantage, with respect to other methods used for adenine nucleotides separation (11,12), lies in the possibility of performing the simultaneous separation of ascorbic acid and uric acid. HPLC Analysis of PCA Extract of Ischemic and Reperfused Rat Heart and of Peroxidized Human Erythrocytes

30

MINUTES

FIG. 1.

Ion-pairing HPLC on a 3-pm (15 cm X 4.6 mm) Supelcosil LC-18-T column of 20 ~1 of a standard mixture of MDA, ascorbic acid, and adenine nucleotide derivatives using tetrabutylammonium as the pairing ion. Concentration of the standards in the mixture ranged from 26 to 150 pM. Absorbance was fixed at 266 nm. Separation conditions are described under Materials and Methods.

with the oxidant by either HPLC or thiobarbiruric reaction (20). RESULTS

AND

acid

DISCUSSION

HPLC Separation of Standard Mixture Figure 1 reports the separation of a mixture containing 17 different compounds including MDA, ascorbic acid, uric acid, NADH, and NADPH performed in about 36 min by using tetrabutylammonium as the ion-pairing reagent. Although a previously published method (9) states that tetrabutylammonium does not permit an acceptable retention time for MDA, we found that MDA elutes, under the present chromatographic conditions, with a it’ = 6.41. This could be explained on the basis of three main differences in our starting buffer, i.e., a much lower concentration of tetrabutylammonium (10 versus 50 mM), a much higher concentration of inorganic phosphate (10 versus 1 mM), and a change and decrease in the organic solvent (1% methanol versus 14% acetonitrile). Therefore, it is not the tetrabutylammonium ion itself that prevents the MDA retention but rather an improper composition of the eluant. It is worth noting that MDA elution occurs in a relatively

Since one of the aims of the present method is to obtain clear and reproducible information on both peroxidative damage (as evaluated by the MDA content) and energy metabolism of ischemic and reperfused tissues (as evaluated by adenine nucleotide derivative concentrations) we chose the model of isolated rat heart subjected to ischemia and reperfusion to verify the validity of the method on biological samples. In Fig. 2 a typical elution pattern of PCA extract from a control (A) and a reperfused rat heart (B) is shown; the presence of a given amount of ADPRib in the samples, also detectable in the standard chromatogram (Fig. l), was attributed to a partial degradation of NAD, whose actual concentration was therefore obtained by adding to its area that corresponding to the ADPRib peak. Table 1 reports data referring to ATP, ADP, AMP, IMP, Ino, Ado, Hyp, Xan, uric acid, NAD, NADP, GDP, GTP, MDA, and ascorbic acid of both control and reperfused rat hearts. The assignment of the peaks to the corresponding compounds was performed by comparing the retention times of the samples with respect to those obtained with a standard mixture. In addition, as the separation and identification of MDA is the main critical issue of our method, we carried out runs of: (i) tissue extracts of reperfused hearts; (ii) MDA standard with a concentration similar to that of the samples; and (iii) a mixture composed by the sample plus an internal standard of MDA with a similar concentration. A typical cochromatogram is reported in Fig. 3. It is evident that MDA standard and MDA in the sample show the same chromatographic behavior and, moreover, the total MDA content in the run with the internal standard (sample + MDA standard) is exactly the sum of the two other separate runs. On the basis of these data, some observations can be

194

LAZZARINO

ET

AL.

9 z

1 ;,

FIG.

2. Determination (A) and ischemic-reperfused

I

1

0

15

of MDA, ascorbic (B) rat heart.

I

,

3 ’

MINUTES

acid, and adenine Chromatographic

0

15

MINUTES

nucleotide compounds and perfusion conditions

made: (i) The separation of MDA can be obtained only at low methanol concentrations in buffer A (l%), therefore rendering useless other similar methods used for adenine nucleotide separation (11,12). In fact, by using the method of Stocchi et al., we found that MDA elutes as a broad peak, partly coeluting with Xan and Ino (data not shown). Hence, since in postischemic rat hearts the concentration of MDA is very similar to the concentration of these low charged compounds (see Table 1) a possible overestimation of Xan or Ino should be taken into account. (ii) Data referring to MDA concentration in both control and reperfused rat hearts, as determined by the TBA calorimetric test, gave 0.199 (SD 0.014) and 0.287 (SD 0.032) pmol/g dry wt, respectively. Ischemic and reperfused hearts showed 2.2-fold less MDA determined by HPLC than by TBA reaction, and no detectable amount of MDA in unperoxidized myocardial tis-

30

by ion-pairing are described

HPLC in 20 pl of PCA extract of control in detail under Materials and Methods.

sue (control hearts) was found by the present HPLC method. This confirms previous results (10) and supports the hypothesis that higher values obtained by the TBA assay may be due either to artificially produced MDA during the acid and heat exposure (21) or to the presence of some interferants in the tissue homogenates able to develop a positive TBA reaction (22-26). However, the possibility of an incomplete extraction of MDA by the PCA deproteinization of the tissue, which would then result in lower MDA values determined by HPLC than by TBA, should always be taken into account. (iii) Values referring to ATP and to products of its catabolism (as well as the pyridine coenzymes NAD and NADP), of both control and reperfused rat heart, are in line with previously reported data obtained under similar experimental conditions (6,13), thus supporting the validity of our HPLC method for their separation simul-

CHROMATOGRAPHIC

SEPARATION

OF

MALONDIALDEHYDE TABLE

AND

ADENINE

195

NUCLEOTIDES

1

Values of MDA, Ascorbic Acid, and Adenine, Nucleotide Derivatives Detected by HPLC in PCA Extracts of Control and Ischemic-Reperfused Isolated Rat Hearts Ascorbic Control Hearts Ischemic and reperfused hearts

HYP

Xan

acid

In0

Uric acid

MDA

Ado

NAD

IMP

AMP

GDP

NADP

ADP

GTP

ATP

ND

ND

ND

ND

ND

0.149 (0.027)

0.115 (0.026)

0.382 (0.037) 0.077 (0.044)

0.549 (0.163)

0.300 (0.045)

0.129 (0.031)

0.080 (0.021) 0.155 (0.049)

8.61 (0.08) 6.20 (0.51)

0.024 (0.004) 0.395 (0.073)

1.50 (0.01) 2.24 (0.38)

0.227 (0.092) 0.272 (0.088)

0.465 (0.171) 0.289 (0.041)

5.39 (0.41) 7.33 (0.83)

1.54 (0.27) 0.884 (0.217)

25.93 (0.67) 13.65 (1.67)

Note. ND, not detectable; For other control hearts and six ischemic-reperfused

abbreviations hearts.

see text. Values are expressed Perfusion and chromatographic

taneously with MDA. (iv) Data concerning ascorbic acid in postischemic hearts indicate that this antioxidant molecule is strongly affected by oxidative stresses. The reliability of the present method is supported by data obtained on human erythrocytes peroxidized in uitro by increasing concentrations of hydrogen peroxide (Table 2). A typical chromatogram of a supernatant of erythrocytes incubated with 1 InM NaN, and 5 InM H,O,

as pmoles/g dry wt and represent the means conditions are fully explained under Materials

(SD) of four and Meth-

is reported in Fig. 4. This separation method permits the detection of the presence of significant amounts of Hyp and Ino as well as the peak corresponding to MDA. A dose-response relationship was observed between H,O, concentration and MDA released in the suspending medium (Table 2). It is worth noting that data referring to MDA in the supernatants, obtained by the TBA test, were similar to those recorded by HPLC only when 5 InM H,O, was present in the erythrocyte suspensions (2.97 nmol/ml supernatant by HPLC and 3.94 nmol/ml supernatant by TBA test). On the contrary, using lower concentrations of the oxidant (from 0.5 to 2 mM H,O,) no detectable amount of MDA could be revealed by the TBA test; with 10 mM H,O,, a 3.6-fold higher value was observed in comparison with the HPLC method. Analysis carried out by HPLC on neutralized acid extract of

TABLE

2

Values of MDA Detected Either by HPLC or by TBA Test in the SuspendingMedium of Erythrocytes Incubated with 1 IIIM NaN, and various Concentrations of H,O, MDA by HPLC

I

I

I

0

5

10

I

HA%

0.5 mM

KO,

1mM

HA

2mM

f&O,

5mM

I

20 15 MINUTES

FIG. 3. Cochromatogram of PCA extract from reperfused heart with a standard containing approximately the same concentration of MDA as the sample. Separation conditions are fully explained under Materials and Methods. (1) MDA in 20 ~1 of myocardial extract, (2) MDA in 20 pl of a standard solution with a concentration similar to that of the tissue sample, (3) MDA in 20 ~1 of myocardial extract supplemented with the standard solution of MDA reported in (2).

WA

Note. Values sent the means able. For other and incubation Methods.

10

mM

0.248 (0.018) 0.680 (0.074) 1.020 (0.096) 2.970 (0.151) 3.260 (0.212)

MDA by TBA test ND ND ND 3.940 (0.410) 11.880 (2.190)

are expressed as nmoles/ml supernatant and repre(SD) of three different experiments. ND, not detectabbreviations see text. Preparation of erythrocytes conditions are fully explained under Materials and

196

LAZZARINO

ET

AL.

metabolism, both of which are of great relevance emia and reperfusion studies.

in isch-

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T-z-75

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MINUTES FIG. 4.

Chromatogram of 20 al of a suspending medium of human erythrocytes peroxidized in vitro by 1 mM NaN, + 5 mM H,Oz obtained by ion-pairing HPLC. Incubation and chromatographic conditions are reported in detail under Materials and Methods.

erythrocytes did not show any trace of MDA, while the development in the same samples of an orange color at the end of the TBA reaction prevented the quantitation of MDA by this calorimetric technique. This comparison confirms the poor reliability of the TBA test for quantitating peroxidative damage in terms of MDA production. In conclusion, the present ion-pairing HPLC method for the simultaneous determination of MDA, ascorbic acid, and adenine nucleotide derivatives from biological samples offers, for the first time to the best of our knowledge, the opportunity to obtain clear, reliable, and reproducible information on peroxidative injuries and energy

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