Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low density lipoprotein

ANALYTICAL

BIOCHEMISTRY

202,384-389

(19%)

Ferrous Ion Oxidation in the Presence of Xylenol Orange for Detection of Lipid Hydroperoxide in Low Density Lipoprotein Zhen-Yue Jiang, James V. Hunt, and Simon P. Wolff1 Department of Clinical Pharmacology, Toxicology Laboratory, and Middlesex School of Medicine, London WClE 6JJ

Received

September

University

11, 1991

A simple and sensitive method for the direct measurement of lipid peroxides in lipoprotein and liposomes is described. The method is based on the principle of the rapid peroxide-mediated oxidation of Fe2+ to Fe’+ under acidic conditions. The latter, in the presence of xylenol orange, forms a Fea+-xylenol orange complex which can be measured spectrophotometrically at 560 nm. Calibration with standard peroxides, such as hydrogen peroxide, linoleic hydroperoxide, t-butyl hydroperoxide, and cumene hydroperoxide gives a mean apparent extinction coefficient of 4.52 X lo* Me1 cm-’ consistent with a chain length of approximately 3 for ferrous ion oxidation by hydroperoxides. Endoperoxides are less reactive or unreactive in the assay. The assay has been validated in the study of lipid peroxidation of low density lipoprotein and phosphatidyl choline liposomes. By pretreatment with enzymes known to metabolize peroxides, we have shown that the assay measures lipid hydroperoxides specifically. Other methods for measuring peroxidation, such as the assessment of conjugated diene, thiobarbituric acid reactive substances and an iodometric assay have been compared with the ferrous oxidation-xylenol orange assay. 0 1992

Academic

Press,

Inc.

Oxidative stress, which is associated with the formation of lipid peroxides, is suggested to contribute to pathological processes in ageing and many diseases, such as diabetes, atherosclerosis, and cataract (1). Increasing interest in the assessment of lipid peroxides in lipoproteins and tissue membranes in these diseases requires, however, a simple and reliable assay for their

1 To whom 384

College

correspondence

should

be addressed.

measurement. However, many of the currently employed assays either are nonspecific or can be inconvienent and problematic (2-4): for example, if they rely upon strict anoxia (2). We have modified a method (5), initially described by Gupta (6), which involves the oxidation of Fe2+ by peroxides at low pH in the presence of the ferric-complexing dye xylenol orange. We originally applied this ferrous oxidation/xylenol orange method (FOX’ assay) to the study of nanomolar H,O, formation during transition metal-catalyzed glucose oxidation (5). In this paper we have adapted the method further for the study of lipid peroxidation in human LDL and phosphatidyl choline liposomes. We have compared this method with the currently widely used iodometric assay, thiobarbituric acid assay, and conjugated diene measurement. MATERIALS

AND

METHODS

Xylenol orange [o-cresolsulfonephthalein-3’-3”-bis(methyliminodiacetic acid sodium salt)] was obtained from Aldrich. Ammonium ferrous sulfate, Hz02, cumene hydroperoxide (CuOOH), t-butyl hydroperoxide (BuOOH), di-cumyl peroxide, butylated hydroxytoluene (BHT), potassium bromide (KBr), cupric sulfate, reduced glutathione (GSH), thiobarbituric acid (TBA), linoleic acid, acetic acid (glacial), potassium iodide, phosphatidyl choline (egg yolk, sigma Type V-E), lipoxidase (EC 1.13.11.12, Type V, soybean), catalase (Type C-40), glutathione peroxidase (bovine erythrocyte Type G6137), and phospholipase A, (porcine pancreas Type

’ Abbreviations used: CuOOH, cumene hydroperoxide; BuOOH, tbutyl hydroperoxide; BHT, butylated hydroxytoluene; TBA, thiobarbituric acid; TBARS, TBA-reactive substances; FOX, ferrous oxidation-xylenol orange; FOXRS, FOX-reactive substances; LDL, low density lipoprotein; PBS, phosphate-buffered saline. cloo3-2697/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

FERROUS

OXIDATION-XYLENOL

ORANGE

P6534) were obtained from Sigma and were of the highest purity available. Benzoyl peroxide and lauroyl peroxide were bought from Fluka. Methanol was purchased from British Drug Houses (BDH, Limited, Poole, England). Linoleic

Acid Hydroperoxide

Linoleic hydroperoxide was prepared by a method described previously (7,2). Linoleic acid (10 mM) was oxidized by exposure to lipoxidase (30,000 units/ml) in borate buffer (10 mM, pH 9.0) at 37°C for 45 min. The concentration of hydroperoxide in methanol was quantified at 233 nm using the molar extinction coefficient of 2.8 X lo4 M-’ cm-’ (8,9). Lipoprotein

Preparation

LDL was isolated from the plasma of normal volunteers in the 1.019-1.063 g/ml density range by sequential ultracentrifugation as previously described (10). In brief, blood was centrifuged in the presence of the chelating agent ethylenediaminetetraacetic acid (EDTA, 1 mg/ml) to prevent oxidation and coagulation. The pooled plasma solvent density was adjusted to 1.019 g/ ml with a high density salt solution (containing NaCl, KBr, and EDTA). After centrifugation for 18 h at lOO,OOOg, 16°C the top layer of supernatant was removed. The density of pooled infranatant was adjusted to 1.063 g/ml and was recentrifuged as before. The LDL, which floats at a relative density of 1.063, was collected and exhaustively dialyzed against phosphate-buffered saline (PBS, pH 7.4) at 4°C. In order to prevent LDL oxidation during dialysis, the buffer contained a cationexchange resin (chelex 100) to chelate transition metal. Preparation

of Liposomes

and Liposome

Incubation

Phosphatidyl choline (2 ml) was dried under nitrogen and then redissolved in 10 ml PBS (10 mM, pH 7.4) in the dark at 4°C for 1 h. The liposome suspension was then ultrasonicated on ice for 2 min to make small liposomes. Liposomes were incubated at a final concentration of 10 mg/ml in 10 mM PBS (pH 7.4) with suitable additions in a shaking water bath at 37°C. Liposomes (0.1 ml) were removed at appropriate intervals, dissolved in 0.9 ml methanol, and centrifuged at 12,000g for 3 min. Supernatant (0.1 ml) was used for the measurement of lipid peroxide or conjugated diene. Another O.lml liposome sample was used directly for the measurement of TBA-reactive substances (TBARS, for details see figure legends). Measurement of Lipid Peroxide Assay and the FOX Assay

with

the Iodometric

Linoleic hydroperoxide was determined tablished iodometric method (2). Solution

using an esA consisted

ASSAY

MEASURING

LIPID

385

HYDROPEROXIDE

of a 1:l mixture of HPLC-grade methanol and glacial acetic acid containing 1 mg/ml ethylenediaminetetraacetic acid (disodium salt) degassed with a stream of nitrogen for 20 min. Solution B consisted of HPLCgrade methanol degassed with N, for 20 min followed by addition of 20% (w/v) potassium iodide and degassed for a further 20 min. One milliliter of solution A was added to 1.5 ml of solution B in a 3-ml cuvette adapted to permit degassing with nitrogen as well as the addition of small volumes of fluid under anoxic conditions. The cuvette contents were treated with N, for 5 min and the cuvette was sealed. Absorbance was monitored at 290 nm for 5 min. The solution was discarded if absorbance increased by more than 0.005 AU. A small volume (l-10 ~1) of a N,-treated standard hydroperoxide solution was then injected. The cuvette contents were gently mixed by careful agitation. Absorbance at 290 nm was monitored for 5 min after which time no further absorbance change was observed. A standard curve generated with H,O, gave an extinction coefficient for tri-iodide at 290 nm of 4.44 X IO4 M-’ cm-‘, in good agreement with the value published previously (2). Lipid peroxides were also measured with the FOX assay, for details see Results and Discussion. Measurement of Conjugated Diene and Stable End Products of Lipid Peroxidation with the TBA Assay

Conjugated diene was detected at 233 nm by uv spectophotometry and concentrations were calculated using an extinction coefficient of 2.8 X lo4 M-’ cm-’ (8,9). A 0.2-ml methanol-extracted LDL or liposome sample was added to 0.8 ml pure methanol in a cuvette and the absorbance read at 233 nm. To correct for the absorbance of phosphatidyl choline at 233 nm, commercial phosphatidyl choline was diluted to 10 mg/ml with methanol, dried under nitrogen to remove chloroform, and redissolved in 0.9 ml methanol and 0.1 ml PBS. @-Aldehydes, which are one of the end products of lipid peroxidation, were measured using the TBA assay. LDL (0.1 ml) or liposome sample (0.1 ml) was mixed with 1 ml of 0.67% TBA and 0.5 ml 20% trichoroacetic acid and incubated at 100°C for 20 min. After cooling, the reaction mixture was centrifuged at 4000 rpm for 5 min and the absorbance of the supernatant read at 532 nm. The concentration of TBARS was calculated using an extinction coefficient of 1.56 X lo5 M-’ cm-’ (11). Pretreatment

with Peroxide-Metabolizing

Enzymes

LDL or liposome, preincubated with Cu2+ (10 PM), was incubated with different enzymes, known to metabolize peroxides, in 10 mM PBS (pH 7.4) at 37°C in a shaking water bath (for details see figure legends). After incubation for 30 min, a methanol-extracted sample was used for the measurement of lipid peroxides and conjugated dienes as described above. The incubation sam-

386

JIANG,

HUNT,

WAVELENGTH

A B

INCUBATION

TWE

(minutes)

FIG.

1. Spectral characteristics of FOX and time course. Spectrum: 100 pM xylenol orange and 250 WM Fe*+, 25 mM H,SO,, 4 mM BHT in 90% methanol before (A) and after the addition of 50 pM H,O, (B) or 25 pM linoleic acid hydroperoxide (C) and incubation for 45 min at room temperature. Time course: The development of color at room temperature using FOX reagent (166 pM xylenol orange, 250 pM Fe*+, 4 mM BHT, 25 mM H,SO, in 90% methanol and 10% distilled water) in the presence of 30 pM linoleic hydroperoxide (A) or 30 pM W, (B).

AND

WOLFF

ml 250 mM H,SO,, 880 mg BHT (in order to inhibit further peroxidation within the assay itself), 76 mg xylenol orange, and 98 mg ammonium iron(I1) sulfate hexahydrate. Methanol-extracted liposomes (0.1 ml) or LDL samples (0.1 ml, oxidized with Cu2+) were mixed with 0.9 ml FOX reagent and incubated for 30 min at room temperature prior to measurement at 560 nm (Fig. 1). Figure 1 shows that the reaction between H,O, or linoleic hydroperoxide and FOX reagent was complete in 15 min. The color was stable overnight at room temperature (data not shown). H,O, and linoleic hydroperoxide were used as standard peroxides for measurement of the apparent extinction coefficient in the modified FOX assay and for comparison with the iodometric assay. The results (Fig. 2) show that H,O, and linoleic hydroperoxide give apparent extinction coefficients for these peroxides at 560 nm of 4.56 X lo4 and 4.70 X lo4 M-’ cm-‘, respectively. Given that the true extinction coefficient of the Fe3+xylenol orange COmpleX is 1.5 X IO4 M-l Cm-’ (5) it iS clear that limited chain oxidation of Fe2+ must occur, circa approximately 3 mol of Fe3+ formed per mole hydroperoxide. Comparison of the FOX method with the iodometric assay (2) shows that the methods have similar sensitivities toward H,O, and linoleic hydroperoxide (Fig. 2). Reactivity of other peroxides in the FOX assay was tested using standard commercial BuOOH, CuOOH, dicumyl peroxide, benzoyl peroxide, and lauroyl peroxide. Table 1 lists the reactivity of these peroxides within the FOX reagent. Acylhydroperoxides and alkylhydroperoxides have similar reactivity with FOX reagent as H,O,. The endoperoxides exhibit lower or no reactivity in the assay.

:

0.6

2

ples were directly used to measure TBARS using the methods described above and in the figure legends. Unless otherwise indicated the data are presented as means f SD of triplicate determinations and are representative of at least two experiments.

P 4

0.2

0.0

RESULTS

Composition

AND

0

DISCUSSION

and Sensitivity

of the FOX Assay

As described previously (5,6), peroxides oxidize Fe2+ to Fe3+ in acidic solution and the latter ions, in the presence of xylenol orange, form a Fe3+-xylenol orange complex, which absorbs at 560 nm (Fig. 1). In the FOX assay developed here for the study of lipid peroxidation, 1000 ml of reagent contains 900 ml pure methanol, 100

3

6

9

[Peroxides] FIG. 2.

12

15

3

(PM)

Standard curves of hydrogen peroxide (H202) and linoleic hydroperoxide (LOOH) in the O-16 pM range in assay. Peroxides were measured with the FOX assay and the iodometric assay (for details see Materials and Methods). Linoleic acid (dissolved in methanol and kept at -20°C) did not generate any color either in the FOX assay or in the iodometric assay at the same concentration as linoleic hydroperoxide (data not show).

FERROUS

OXIDATION-XYLENOL TABLE

Reactivity

of Various

ORANGE

ASSAY

Lipid

1

Peroxides

to the FOX Percentage (relative

Hydrogen peroxide Linoleic hydroperoxide t-Butyl hydroperoxide Cumene hydroperoxide Di-cumyl peroxide Benzoyl peroxide Lauroyl peroxide

(lmlole/mg

Assay

100% 103% 96% 98% 12% 9% 21%

01234567

Lipid peroxidation is catalyzed by trace amounts of transition metals, such as Fe2+ and Cu2+ (12,13). Liposomes (10 mg/ml in 10 mM PBS, pH 7.4) were incubated alone or with 10 PM Cu2+ at 37°C in a shaking water bath. Figure 3 shows that the formation of lipid peroxides (measured by FOX assay), conjugated diene, and TBARS is time dependent. However, the concentration of lipid peroxide was slightly higher than that of conjugated diene, whereas the values estimated for TBARS given as malondialdehyde equivalents were much lower.

Conjugated diene CPM) 250,

lO@f) L0c”++( (ILM)

40 ,

I

0 cu++(lopM)

200 0

c0nt.r01

0 Control

20

100

30

T1

150 50

10

i/f/ ~:&4 0

Incubation

FIG. 3.

i

/

./’ .r . ,b---0.0 A

0

0 5

10

time

15

20

Conjugated (nmole/mg

LDL

diene protein)

TBARS (nmole/mg

LDL

01234567

Incubation

protein)

01234567

time

(days)

FIG. 4.

The accumulation of lipid peroxides (FOXRS), conjugated diene, and TBARS in LDL. LDL (2 mg/ml) was incubated alone (control) or with 10 pM Cu2+ in 100 mM potassium phosphate buffer (pH 7.4) at 37°C for 7 days. Before incubation, potassium phosphate buffer was treated with chelating resin and filtered (0.2 pm pore size) to limit contamination with transition metals and microbes. Samples were taken before incubation and after incubation for 6, 24, 48, and 168 h. The above sample (0.1 ml) was directly used for the measurement of TBARS (for details see Materials and Methods). Also, O.l-ml incubated samples were mixed with 0.05 ml of catalase (500 units/ml) at 25°C for 15 min. Then, 0.85 ml methanol was added and mixed for 15 min to extract lipid peroxides. After centrifugation at 12,000g for 3 min, 0.1 ml supernatant was used for the measurement of lipid peroxides with the FOX reagent. For detection of conjugated diene, O.l-ml incubated samples were directly mixed with 0.9 ml methanol for 15 min. After centrifugation, 0.2 ml supernatant was used for the detection of conjugated diene.

In the LDL oxidation experiment, 2 mg/ml of LDL were incubated with 10 PM Cu2+ in 100 mM potassium phosphate buffer (pH 7.4) at 37°C. The values of lipid peroxides (FOX-reactive substances, FOXRS) generated upon oxidation of LDL were compared with those of the conjugated diene and TBARS, measured at different time points. Figure 4 shows a slight increase in the concentrations of lipid peroxides, conjugated diene, and TBARS in an LDL sample after incubation for 7 days in the absence of added catalyst. There was, by contrast, a large accumulation of lipid peroxides, conjugated diene, and TBARS in LDL incubated in the presence of Cu2+ over the first 24 h. The levels of lipid peroxides and TBARS then dropped dramatically, as reported by Jurgens and colleagues (14). However, the level of conjugated diene, as judged by absorbance at 233 nm, continually increased in Cu2+-incubated LDL over 7 days of oxidation. The time-related changes in the level of lipid peroxides can be explained by the activity of Cu*+, which catalyzes lipid peroxidation, as well as peroxide decomposition (15). After 24 h of incubation, lipid peroxidation of LDL reaches the highest level and then decomposition of lipid peroxides becomes the major process of LDL oxidation with the formation of carbonyls, including malondialdehyde and other aldehydes (14,16-20). Malondialdehyde and other aldehydes, which are major TBA reactive substances, form poly-

l+-.+l TBARS

1

387

HYDROPEROXIDE

~~~~~~~~

Comparison of Lipid Peroxides with Conjugated Diene and TBARS in Oxidized Phosphatidyl Choline Liposome and LDL

peroxide CPM)

peroxide LDL protein)

LIPID

reactivity to H,O,)

Note. 100% refers to an apparent “extinction coefficient” of 4.56 X lo4 M-l cm-’ for H202 in the FOX reagent. Note that the extinction coefficient of the Fe3+-xylenol orange complex is 1.5 X lo4 M-’ cm-’ and thus there is a chain length of 3 mol Fe2+ oxidized per mole H,O, under these conditions.

Lipid

MEASURING

0

5

10

15

20

(hours)

The accumulation of lipid peroxides (FOXRS), conjugated diene, and TBARS in liposomes. Liposomes (10 mg/ml) were incubated alone (control) or with 10 pM Cu*+ in PBS (10 mM, pH 7.4) at 37°C for 20 h. Samples were taken at zero time and at varying time intervals up to 20 h. The above sample (0.1 ml) was directly used for the measurement of TBARS (for details see Materials and Methods). Also, 0.1 ml incubated sample was dissolved in 0.9 ml pure methanol and after centrifngation at 12,000g for 3 min, 0.1 ml supernatant was mixed with FOX reagent for the measurement of lipid peroxides and 0.2 ml supernatant was used for detection of conjugated diene.

388

JIANG,

HUNT,

malondialdehyde and aldehyde polymers which appear less reactive to TBA (21). They are also able to form conjugated Schiff bases, which absorb in the ultraviolet region (19), by covalent attachment to the amino group of protein (22,23). The reaction of carbonyls with lipoprotein, after a long incubation at 37°C may contribute to the absorbance increase at 233 nm and the decrease in the level of TBARS (14). Other oxidation products in oxidized LDL may also contribute to the absorbance increase at 233 nm.

AND

WOLFF

a Lipid b==wmg

peroxide

Conjugated

LmJ Protein)

of Peroxide

Content

in LDL

Confounding

Factors

in the Measurement

(umole/mg

-

TBABS LDL protein)

2ol-----

150

12

100

6

60

4

0 !ul A

and Liposome

The substances reactive with FOX reagent in LDL and liposome were confirmed as lipid peroxides by pretreatment with enzymes involved in peroxide metabolism. It is well known that glutathione peroxidase can metabolize peroxides, such as phospholipid hydroperoxide, fatty acid hydroperoxide, cholesterol hydroperoxide, and H,O,, in the presence of GSH and phospholipase A, (24-26). The latter hydrolizes esterified peroxide (24-26). In the experiment shown in Fig. 5, LDL and liposome were preincubated with 10 I.LM CC for 6 and 20 h, respectively. To the samples of oxidized LDL (Fig. 5a) and liposome (Fig. 5b) were added (A) control buffer, (B) catalase, and (C!) GSH, glutathione peroxidase, and phospholipase A,. After incubation for 30 min at 37”C, the yields of FOXRS, conjugated diene, and TBARS were compared. Catalase reduced the color yield of FOX by lo-15%, which indicates the formation of H,O, during oxidation of LDL and liposomes, while glutathione peroxidase, in the presence of GSH and phospholipase A,, produced a 90-95% decrease in the color yielded with the FOX assay. By contrast, levels of conjugated diene and TBARS were not influenced by this enzyme pretreatment.

diene LDL protein)

18 1

0

Confirmation

(nmole/mg 250 ,

B

C

A

B

C

-

I

.111

A

B

C

A

B

C

b Lipid

peroxide Wo

400

250

15

200

12

160

0

100

6

50

3

350 300 260 200 150 100 60 0

0

0

A

B

C

A

B

C

FIG. 5. The comparison of FOXRS with conjugated diene and TBARS after pretreatment with enzymes of lipid peroxides in LDL and liposomes. LDL (2 mg/ml, a) and liposomes (10 mg/ml, b) were preincubated with 10 pM Cu2+ in 10 mM PBS, (pH 7.4) for 6 and 20 h, respectively. LDL and liposome samples (diluted twofold in PBS) were exposed in PBS for 30 min at 37°C to (B) catalase (lOOunits/ml) and (C) GSH (500 PM), glutathione peroxidase (10 units/ml), and phospholipase A, (10 units/ml). (A) The control. At the end of the incubation period the samples were diluted 1:9 with methanol and centrifuged at 12,000g for 3 min. Supernatant (0.2 ml) was then analyzed for peroxides and conjugated diene as described under Materials and Methods. Enzyme-treated sample (0.1 ml) was directly used for the detection of TBARS.

of Peroxides

In its application to different biological systems the FOX assay may be subject to interference by redox materials, present in biological extracts, which may oxidize generating H%O,. For example, in a linoleic hydroperoxide (4 PM) recovery experiment it was observed that ascorbic acid, 100 PM in the assay, slowly contributed to color development (20% increase in color yield). At the same concentration, uric acid increased color yield about 6%. For membrane or lipoprotein peroxide determination such interference need not be considered since washing and extracting or precipitation procedures would remove these low-molecular-weight agents. In other circumstances, however, separation of low-molecular-weight components by HPLC followed by postcolumn derivatization with FOX could be a useful strategy to adopt. We also observed the effects of other materials, such as glucose (25 mM), fructose (25 mM), galactose (25 mM), glycerol (25 mM), cholesterol (100 PM),

triglyceride (100 yM), linoleic acid (100 PM), reduced glutathione (100 PM), vitamin E (500 FM), lens crystallins (100 pg/ml), and bovine serum albumin (100 pg/ml), on color development in the FOX assay. None of these contributed to the color yield in either the presence or the absence of 4 pM linoleic hydroperoxide or 4 pM H,Oz in this FOX system. CONCLUSIONS Simple and sensitive methods for the determination of peroxides will assist evaluation of the “oxidative stress” hypothesis of pathophysiology. The method described here provides a new alternative and may be useful in this respect. The FOX assay is a sensitive, convenient, and simple assay for the direct measurement of lipid peroxide. By comparison with conjugated diene and TBA assays, which measure products at different

FERROUS

OXIDATION-XYLENOL

ORANGE

ASSAY

MEASURING

stages of lipid peroxidation, the FOX assay has the advantage of direct detection of lipid peroxides. We have employed the FOX assay in a study of LDL oxidation in vitro (27,28), lipid peroxidation in the cataractous lens (29), and erythrocyte membranes in diabetes mellitus (Z.-Y. Jiang and S. P. Wolff, unpublished data).

11. Slater,

ACKNOWLEDGMENTS

16. Esterbauer,

We are grateful to Research into Ageing, the Sir Jules Thorn Trust, and the Medical Research Council for financial support. We thank Wim Koppenol for helpful discussions. Z. Y. Jiang wishes to thank Wendy Slemen for her comment on the manuscript.

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