Iodometric measurement of lipid hydroperoxides in human plasma

ANALYTICAL

BIOCHEMISTRY

193,204-211

(19%)

lodometric Measurement in Human Plasma

of Lipid Hydroperoxides

Gregory L. Cramer, James F. Miller, Jr., Robert B. Pendleton, and William E. M. Lands Departments

of Medicine

Received

20,199O

July

and Biological

Chemistry,

University

Many assay techniques have been used to measure lipid hydroperoxides in plasma, including absorbance of conjugated dienes and reactivity with thiobarbituric acid. Because these measurements are not specific for lipid hydroperoxides, we modified an existing iodometric method to correct for interfering phenomena and to provide a more specific measurement of the lipid hydroperoxide content of plasma. To ensure reproducible extraction of hydroperoxides from the many possible forms in plasma, the plasma was treated to hydrolyze enzymatically cholesterol ester, triglycerides, andphospholipids, and the nonesterified fatty acid peroxides were then extracted with ethyl acetate. Extracted lipids were reacted with potassium iodide in acetic acid and methylene chloride, and the resulting triiodide ion (I,) was measured spectrophotometrically. Correction for nonoxidizing chromophores was made after back-titration of the triiodide ion to iodide with sodium thiosulfate and other non-peroxide oxidants were estimated by their resistance to reduction with glutathione peroxidase. Recovery of added hydroperoxide standards provided routine validations of the procedure’s efficiency. The method indicated that insignificant amounts of hydroperoxide may be in the less polar lipids, but the total amount of lipid hydroperoxide esterAed in the plasma lipids of apparently healthy humans may be as much as 4.0 f 1.7 PM. o lee1 Academic ~r-6, IN.

Lipid peroxidation is thought to play a role in the pathogenesis of many disease states. Possible mechanisms include peroxidative damage of phospholipid bilayer membranes, modification of sulfhydryl groups on enzymes (1,2), and stimulation of prostaglandin and leukotriene synthesis by stimulation of fatty acid oxygenase (cyclooxygenase and lipoxygenase) activity (3,4). Prostaglandins and leukotrienes produced by the latter mechanism mediate inflammation (5).

of Illinois

at Chicago, Chicago, Illinois

60612

Most evidence supporting the role of lipid peroxidation in human disease has come from spectrophotometric or fluorometric measurements of the red-colored compound formed when the serum or plasma of patients is treated with thiobarbituric acid (TBA)’ (6). Degradation of lipid hydroperoxides (ROOH) during heating yields malondialdehyde which forms a red chromophore with TBA (6). Unfortunately, the development of color with thiobarbituric acid is known to be nonspecific, and it occurs with many substances, including bilirubin, DNA, sucrose, and aldehydes (6-8). Reported measurements of TBA-positive materials have estimated a content of peroxide in plasma from normal subjects equivalent to 3.0 pM MDA (6,9-11). However, this value is equivalent to 60 pM of ROOH (12). It is apparent that the assays for lipid peroxides have inherent inadequacies and that there is a need for a more specific, reproducible assay for lipid peroxides in biological samples. Conjugated dienes occur in lipid hydroperoxides and they have been reported to be elevated in the serum and tissue of patients with certain diseases (13,14). However, gas chromatography-mass spectroscopy indicates that much of the conjugated diene materials is not an oxidized acid and does not contain the hydroperoxide functional group (15,16). Iodometric methods based upon the reactivity of the hydroperoxide group have been used to measure the peroxide content of biological samples (8,17-19). These methods use the ability of iodide ions (I-) to reduce ROOH in an acidic solution to the corresponding hydroxy acid (31- + ROOH + 2H+ + 13 + ROH + H,O). The resulting triiodide complex (I;) is measured spectrophotometrically. We report further modifications of the

1 Abbreviations used: TBA, thiobarbituric dehyde; BHT, butylated hydroxytoluene; dase; NEM, N-ethylmaleimide; HPETE, oic acid; HETE, hydroxyeicosatetraenoic

acid; MDA, malondialGSPx, glutathione peroxihydroperoxyeicosatetraenacid.

204 All

Copyright 0 1991 rights of reproduction

0003-2697/91$3.00 by Academic Press, Inc. in any form reserved.

IODOMETRIC

ASSAY

Beuge and Aust iodometric method (8) which correct for interfering substances and allow a more specific and sensitive measurement of hydroperoxides in human plasma.

MATERIALS

All solvents (ethyl ether, petroleum ether, chloroform, methylene chloride, ethyl acetate) were distilled using an all-glass refluxing distillation apparatus with a 3-in. head to separate solvent from nonvolatile impurities that were iodometrically reactive. Acetic acid, butylated hydroxytoluene (BHT), citric acid, potassium iocadmium chloride (CdCl,), sodium dide (KI), thiosulfate (Na,S,O,), anhydrous sodium sulfate (Na,SO,), potassium periodate (KIO,), glutathione (GSH), glutathione peroxidase (GSPx), phospholipase A, from Naja mocambique mocambique (1580 U/mg protein), cholesterol esterase from Pseudomonas sp. (400 U/mg protein), N-ethylmaleimide (NEM), phosphatidyl choline from egg yolk, and triphenylphosphine were obtained from suppliers (Sigma Chemical Co., St. Louis, MO; Fisher Scientific, Fairlawn, NJ). Sodium taurocholate (>95% pure) (Calbiochem, San Diego, CA) and arachidonic acid (Nu Check Prep, Inc., Elysian, MN) were purchased. [1-14C]Arachidonic acid (51 Ci/mol) was purchased from Amersham, Inc. (Arlington Heights, IL). Silica gel G thin-layer chromatography (TLC) plates (250 pm) were purchased from Analtech Inc. (Newark, DE). [1-‘4C]15-Hydroperoxyeicosatetraenoic acid (15-HPETE), 260 cpm/nmol, was prepared from arachidonic acid by the method of Graff (20) and stored at -20°C in 1:l petroleum ether-ethyl acetate. [1-14C]15-Hydroxyeicosatetraenoic acid (15-HETE) was prepared by reducing the [l-14C]15-HPETE with 1.0 ml of triphenylphosphine in diethyl ether (1.0 mgl ml) at 25°C for 60 min.

METHODS

Preparation Samples

and Analysis of Pooled Human Plasma

Plasma was prepared by centrifuging at 4°C at 700g for 30 min whole blood that was mixed with sodium citrate. To have sufficient material for repeated tests to calibrate the methods developed in this work, combined pools of plasma were formed from titrated whole blood obtained by venipuncture of fasting, apparently healthy volunteers aged 20 to 60 years. The pooled plasma samples were divided into 2- to 4-ml aliquots that were frozen at -40°C for later analysis. Prior to analysis, the plasma samples were thawed at 25°C and placed on ice and divided further into l-O-ml aliquots.

OF HYDROPEROXIDES

205

Lipids were extracted from the plasma with either chloroform:methanol(2:1) or ethyl acetate (21,22). The ethyl acetate extraction was performed as follows: 1 ml of plasma at 0°C was acidified to pH 3.5 with 90 ~1of 2 M citric acid. A 2-ml volume of distilled ethyl acetate containing 0.01% BHT and 1.0 ml of 100% ethanol were added to the acidified plasma at room temperature with vortex mixing. After centrifugation at 500g for 10 min at 4”C, the top organic layer was removed with a Pasteur pipet. A 2-ml-volume of ethyl acetate and 1.0 ml of deionized water were added to the supernatant to achieve separation of phases. The upper organic layer was then separated and washed with 2.0 ml of 1 mM HCl. Following this, about 1 g of anhydrous sodium sulfate was added to the organic layer to remove the remaining water. The washed organic layer was divided into aliquots corresponding to 0.3 to 0.6 ml of original plasma and evaporated to near dryness under nitrogen in a Pyrex screw-cap tube after adding 200 ~1 of ethanol to facilitate removal of water. The resulting residue was resuspended in 50 ~1of ethanol and then reacted sequentially with the reagents for the iodometric assay: 350 ~1 of acetic acid:methylene chloride (3:2 (v/v), made with 0.001% BHT) and 15 ~1 of aqueous KI (2.4 g KI plus 2.0 ml water). Prior to use, the acetic acid:methylene chloride and water were deoxygenated by bubbling with nitrogen gas for 20 min at 0°C. To avoid photocatalyzed oxidations, the reagents were added in a dimly lit room, mixed, and then incubated for 3 min in a dark closed drawer. Then 1.0 ml of 0.35% CdCl, was added, the tube was centrifuged at 200g for 5 min at room temperature, and the aqueous phase was removed for measurement of the absorbance at 353 nm (Shimadzu 160 uv spectrophotometer; Kyoto, Japan). The absorbance was determined again after treating the contents of the spectrophotometer cuvette with 10 ~1 of 0.2 M Na,S,O, to reduce the 13 to II. This back-titration of I; allowed measurement of the absorbance at 353 nm due to other compounds with overlapping absorbances and calculation of how much absorbance was actually due to I;. To determine the recovery of ROOH during analysis of samples, we added 5 to 20 nmol of the standard organic hydroperoxide, 15-HPETE (see Materials above), to some of the l.O-ml aliquots of plasma prior to extraction. Standard curves were prepared by reacting KIO, (1 mol KIO, yields 3 mol I;) and 15-HPETE (1 mol 15HPETE yields 1 mol 13) using the reaction conditions described above. Solvent blanks were prepared by substituting 1.0 ml of 100 mM potassium phosphate buffer (pH 7.2) for the 1.0 ml of plasma. A small amount of I; was produced upon assay of the solvent blanks and reagent blanks, presumably due to autooxidation of I- to I;. This capacity of the solvent blanks to form I; was comparable to that of standard reagent blanks with no added KIO, or 15HPETE.

206

CRAMER

Enzymatic Hydrolysis Hydroperoxides

of Esters

and Reduction

AL.

of

Enzymatic hydrolysis of total plasma lipids was performed on l.O-ml aliquots of plasma using a mixture of 10 units of phospholipase A, (Sigma No. P7778) and 40 units of a nonspecific Pseudomonas esterase (Sigma No. C1403) in the presence of 1.5% sodium taurocholate. TLC and GC analyses demonstrated that the Pseudomonas esterase was capable of hydrolyzing both triglycerides and cholesterol esters (see below). Samples were incubated at 25°C for 20 min. Plasma contains sufficient Ca2+ to support the hydrolytic enzymes. Reduction of liberated fatty acid hydroperoxides in control samples was achieved by treating with 5 units of glutathione peroxidase in the presence of 1 mM GSH and then incubating for an additional 10 min with 2 mM NEM to complex all unreacted GSH. TLC analysis of the extracts confirmed that treatment with glutathione peroxidase effectively reduced all added [i4C]15-HPETE to [14C]15-HETE (Fig. 4). For samples in which enzymatic reduction of hydroperoxides was not desired, the sequence for adding reagents was altered, and the GSH was first inactivated by incubation separately with NEM for 30 min at 25°C before adding it to the system. In this way, all samples contained identical reagents, thereby creating a uniform matrix for the iodometric assay. Fatty acid hydroperoxides were then extracted with ethyl acetate from aliquots of the enzyme-treated plasma as described above and assayed with the iodometric reagents. The hydroperoxide content was calculated as the difference in iodometric activity between samples with and without active GSH during the glutathione peroxidase treatment. TLC Analysis

ET

of Plasma Lipids

TLC of plasma lipids was performed to confirm enzymatic hydrolysis. Cholesterol ester, triglyceride and nonesterified fatty acid were resolved with a hexane:diethyl ether:acetic acid (80:20:1 (v/v/v)) solvent system without curtains at 25”C, whereas lysophospholipids, phospholipids, and fatty acids were resolved in chloroform:methanol:water (65:35:5 (v/v/v)) with filter paper curtains at 25°C and with 30-40 min solvent equilibration. After the TLC plates were airdried, the lipids were detected by staining with iodine vapor. Using this technique, cholesterol esters, glycerides, and phospholipids were demonstrated to be extensively hydrolyzed (data not shown) after incubation with phospholipase A, and Pseudomonas esterase. The extent of hydrolysis was quantitated by gas chromatography. For some samples, the regions of the TLC plate corresponding to cholesterol ester, triglyceride, nonesterified fatty acid, mono- and diglyceride, phospholipid, and lysophospholipid were scraped and the fatty acids were methylated in boron trifluoride in methanol with

TABLE

Absorbance

1

Values at 353 nm for the Iodometric Standard and Plasma Extracts Absorbance Added KI

KIO, (neq 0.0

(353 nm)

Before N+Wh

After N+%O,

0.000 0.000 0.149 0.000 0.243 0.000 0.408 0.000

0.000 0.000 0.001 0.000 0.001 0.000 0.000 0.000

0.047 0.007 0.116 0.008 0.228 0.021 0.325 0.027

0.006 0.007 0.008 0.008 0.020 0.021 0.029 0.027

1~)~ + -

4.7

+ -

9.4

+ -

18.8

+ -

Plasma 0.1

Assay of

volume

(ml) * + -

0.2

+ -

0.5

+ -

1.0

+ -

a I; was generated by reaction of KIO, with KI. Three 13 are formed from each nanomole of original KIO,. * Aliquots of the chloroform extract were equivalent cated amounts of normal human plasma.

nanomoles

of

to the indi-

added heneicosanoic acid as an internal standard. The methyl esters were extracted with hexane, mixed with methyl heptadecanoate, and analyzed by gas chromatography with a 30-m Supelcowax 10 capillary column (Supelco, Bellefonte, PA). Cholesterol esters were 92% hytriglycerides 98% hydrolyzed, and drolyzed, phospholipids 95% hydrolyzed to lysophospholipid, and no mono- or diglycerides were detected (data not shown). [ 14C] 15-HPETE and [“Cl 15-HETE added to plasma were resolved by TLC in diethyl ether:petroleum ether:acetic acid (50:50:0.5 (v/v/v)) and were detected by analyzing radioactivity on TLC plates with a System 400 imaging scanner (Bioscan, Washington, DC).

RESULTS

Calibration of I; Absorbance I; was generated in 1.0 ml of 0.01 N HCl by addition of 15 ~1 of the aqueous KI to varying amounts of standard KIO, (1 mol KIO, yields 3 mol I;). The absorbance at 353 nm (Table 1) was linear from 0 to 20 nmol of I;: A,, = 0.023 (neq I;) + 0.007 (r = 0.999, n = 10). The solution was back-titrated with Na,S,O, to confirm that no absorbance other than that due to I; was present.

IODOMETRIC

ASSAY

OF

207

HYDROPEROXIDES

4 325

350

375

Wavelength

400

425

450

(nanometers)

325

350

375

Wavelength

400

425

450

(nanometers)

FIG. 1. Absorbance spectra for the iodometric assay. A: KIO, in the indicated number of nanoequivalents produced the spectra of 13 shown. Note that a small amount of I; is generated even when no KIO, is added to baseline autooxidation of I- to I;. Following treatment with Na,S,O,, all samples exhibited spectra like the lowest curve. B: 15HPETE in amounts from 0.0 to 10.2 neq generated the absorbance spectra of 1; shown by the upper curve. The lowest curve represents the absorbance spectra after adding Na,S,O,.

Standard

Curves

Using the overall assay conditions described under Methods, a standard curve prepared with KIO, in Pyrex screw-cap tubes resulted in A,,, = 0.023 (neq 13) + 0.048 (r = 0.988, n = 9). Spectral recordings from 325 to 450 nm for these samples are shown in Fig. lA, confirming that the peak absorbance of 13 occurs at 353 nm. All of the absorbance at 353 nm was removed by treating these standards with Na,S,O, (bottom curve in Fig. IA), indicating that only 13 was responsible for the absorbance at 353 nm in these standards. Standard curves were also prepared with 0 to 20 nmol of 15HPETE in alcohol. When pure 15-HPETE was reacted with the above standard assay reagents, the result was a linear increase in absorbance: A 353 = O.O23(neq HPETE)

+ 0.048

(r = 0.988, n = 8).

The concentration of the standard 15-HPETE determined by comparison with a standard curve of the primary standard, KIO,, confirmed that expected from the absorbance of conjugated dienes at 233 nm. Spectral recordings of the assay mixtures after reaction of 15HPETE with KI are shown in Fig. 1B. Again, all of the absorbance at 353 nm was removed by Na,S,O,, indicating that no substance absorbing at 353 nm other than I; was present. Correction

for Interfering

Materials

in Plasma

The iodometric assay procedure described under Methods was performed with varied amounts of standard KIO, and with varied amounts of an extract of plasma (Table 1). Although all of the standard KIO, samples were completely decolorized by reduction of I; with thiosulfate, a significant fraction of the absorbance of the extract from plasma was not decolorized by adding thiosulfate. The A,,, of plasma samples after treat-

ment with thiosulfate was similar to that of plasma samples that had not contained KI (Table l), indicating that thiosulfate reduced only the I; present. The Choice of Extraction

Solvent

The original procedure employed chloroform:methano1 (21), but we noted that increased amounts of the material extracted from plasma (dried in the assay tubes and then dispersed in ethanol) gave a nonlinear and proportionally lower response for larger aliquots, suggesting the presence of an inhibitory factor (Fig. 2A). The chloroform extract contained extracted plasma phospholipids which may interfere with the iodometric assay. To test this hypothesis, we performed the iodometric assay in the presence of increasing amounts of phosphatidylcholine keeping the amount of KIO, constant. Increasing the amount of phosphatidylcholine resulted in a decreasing spectrophotometric response (Table 2) which was not overcome by altering the concentrations of KI or CdCl, (results not shown). Recovery of the Ii-generating activity of [14C]15-HPETE that had been added to plasma and then extracted with chloroform:methanol was only 47 + 9%, whereas recovery of the radioactivity averaged 81+ 3% (mean + SE, n = lo), indicating a selective loss of detectable I,-generating activity. Extraction with ethyl acetate from 0.1 to 1.0 ml plasma of the lipids removed a predominant percentage of the free fatty acids but a lesser part of the phospholipids, and these extracts produced more linear results (Fig. 2B) than were noted for the chloroform extracts (Fig. 2A). Increasing the amount of material from ethyl acetate extracts by two- to fourfold resulted in a linear increase in recovered iodometric activity (Fig. 2B) in contrast to the results obtained with the chloroform: methanol extracts (Fig. 2A). In addition, the recovery of IT-generating activity of HPETE that was added di-

208

CRAMER

10--

ET

AL.

” A

PLASMA VOLUME (ml)

PLASMA VOLUME (ml)

FIG. 2. Assay response with extracts of plasma. A: Iodometric assay of material extracted by chloroform:methanol from increasing volumes of different plasma samples from healthy volunteers and from hospitalized patients. Each point represents the average of duplicate determinations. Linear least-squares fits of the samples gave r values that averaged 0.84 + 0.21 (n = 4, mean + SD). B: Iodometric assay of material extracted by ethyl acetate corresponding to increasing volumes of different plasma samples. Each point represents the average of duplicate determinations. Linear least-squares fits gave an average r value of 0.998 + 0.004 (n = 7, mean f SD).

rectly to the ethyl acetate extract was 100% (result not shown), indicating that no interfering material was present and that no loss occurred during handling and drying of the extract under nitrogen, When [14C]15HPETE was added to plasma before extraction with ethyl acetate, the recovery of Ii-generating activity averaged 66 f 7% (mean + SE, n = 10) and the recovery of radioactivity was 74 + 5%. Thus, although not all of the added material was extracted, the similar recovery indicated that little Ii-generating activity was selectively lost in handling with ethyl acetate. Iodometric

Analysis

of Plasma

after Enzyme

Treatment

Aliquots of four different pools of plasma (A-D) were treated in eight different ways (conditions l-8) with reagents and enzymes as depicted in Table 3. The resulting lipids were then extracted with ethyl acetate and assayed with the iodometric reagents. Values for conditions 1 and 2 indicate that most of the Ii-generating activity extracted from plasma (prior to treatment with

TABLE Phosphatidvlcholine Phosphatidyl choline hd 0.0

Inhibition

2 of Iodometric Absorbance

8.4

neq 13

0.5

0.220 0.183

1.0 5.0

Response

(353 nm)

8.4 neq

I;

8.7

neq 1;

0.224

0.226

0.144

0.192 0.160

0.081

0.064

0.162 0.110

Note. Increasing the amount of phosphatidylcholine results in a decrease in the absorbance of the I; generated by a constant amount of KIO,. Absorbance values represent the mean of two determinations. Three separate experiments are shown.

phospholipase or esterase) was not diminished by glutathione peroxidase treatment (6.8 vs 6.2, 13.5 vs 14.4, 16.1 vs 16.8, and 5.4 vs 5.4). However, values for conditions 3 and 4 indicate that the oxidative activity of 15 HPETE that was added to plasma pools A and B (6 and 10 nmol, respectively) was reduced by the peroxidase, indicating that the glutathione peroxidase was capable of reducing any nonesterified lipid hydroperoxide that may have been present. Addition of 6 nmol of HPETE to l.O-ml aliquots from pool A led to detected amounts of 3.8 nmol I; (condition 3 minus condition 1) and 4.0 nmolI3 (condition 7 minus condition 5), equivalent to about 65% recovery of added peroxide. Similarly, the 10 nmol added to pool B showed recoveries of 12.0 and 8.7 nmol. The combined overall recovery of the 32 nmol added to the four tubes was 28.5 nmol(89%). The individual results are provided to illustrate the treatments and the calculations involved. The greater values in the iodometric assay for conditions 5 and 6 compared to conditions 1 and 2 indicate that treatment with hydrolytic enzymes (phospholipase A, and esterase) increased the amount of extracted oxidant. However, the values for condition 6 indicate that all of the increased oxidant activity could not be reduced by peroxidase to levels seen with conditions 1 and 2. Nevertheless, values for conditions 7 and 8 indicate that the peroxidase was able to reduce any lipid hydroperoxide that may have been present in hydrolyzed plasma. Figure 3 shows the iodometric spectra resulting from analysis of hydrolyzed plasma with added 15-HPETE (pool A) without (condition 7) and with (condition 8) treatment with glutathione peroxidase. The difference in absorbance at 353 nm (AA) between curve 1 (condition 7) and curve 2 (condition 8) was used to calculate the amount of hydroperoxide present (AA divided by 0.023 A/nmol I; equals the nmoles of hydroperoxide present in the plasma sample).

IODOMETRIC

ASSAY

OF

TABLE

Iodimetry Incubation Condition used

period

209

HYDROPEROXIDES 3

of Extracts

of Plasma Plasma pool (nmol I;)

(25°C)

1st (20 min)

2nd (10 min)

A

B

C

D

Nonhydrolyzed 1 2 3 4

0 0 HP HP

0 GSPx 0 GSPx

N/G GSH N/G GSH

GSPx NEM GSPx NEM

6.8 6.2 10.6 7.4

13.5 14.4 25.5 12.7

16.1 16.8 nd nd

5.4 5.4 nd nd

Enzyme 5 6 7 8

0 0 HP HP

0 GSPx 0 GSPx

N/G GSH N/G GSH

GSPx NEM GSPx NEM

13.7 10.7 17.7 11.0

26.2 23.6 34.9 25.1

23.1 18.6 nd nd

a.7 3.1 nd nd

hydrolyzed

Note. All incubations contained 1.0 ml of plasma and 15 mg taurocholate with other reagents added in the sequence listed for each condition. Conditions 1 to 4 were not treated with hydrolytic enzymes, whereas 5 to 8 contained esterase and phospholipase in the first incubation period. The four different pooled plasma samples are indicated as A, B, C, and D. Where indicated HP, 6 nm HPETE was added to samples from pool A and 10 nm HPETE was added to samples from pool B. Abbreviations are: GSPx, glutathione peroxidase; GSH, glutathione; NEM, N-ethylmaleimide; N/G, glutathione precomplexed with NEM; 0 indicates an absence of the indicated reagent; nd indicates value not determined. Each value is the average of two separate measurements.

The content of lipid hydroperoxides in the pooled plasma samples was estimated as follows. The nonesterified (or neutral) hydroperoxide was calculated as the difference in iodometric activity with and without reduction by glutathione peroxidase (condition 1 minus condition 2, Table 3). The values for this difference ranged from 0.6 to -0.9, giving a negligible average of -0.3 f 0.2 (Table 4). Similarly, the total extracted hydroperoxides from hydrolyzed samples were estimated from the difference in iodometric activity between con-

0.30

ditions 5 and 6 (Table 3). This calculation gave an estimate (uncorrected for recovery) for the total plasma hydroperoxide of 2.6 to 5.6 (average 4.0 f 1.7) nmol/ml plasma (Table 4). DISCUSSION

Interfering Materials Two potential sources of interference were recognized for the iodometric analysis of hydroperoxides in plasma, and they had to be circumvented by chemical and enzymatic means. First, it was clear that extracts of plasma contained substances that absorb at 353 nm which could cause overestimation of the amount of hydroperoxide. This phenomenon has been noted by other

0.20 TABLE

Estimated

0.10

Lipid

Plasma

0.00 325

350

375

Wavelength

400

425

FIG. 3.

Absorbance spectra after iodometric assay of plasma ethyl acetate extracts. Curve 1 is a spectrum of the aqueous phase of the iodometric assay mixture from enzyme hydrolyzed plasma containing 6 @M 15-HPETE. Curve 2 is a spectrum of a similar sample that was treated with GSH peroxidase. The difference in absorbance between curves 1 and 2 at 353 nm (AA) is used to calculate the amount of hydroperoxide present. Curve 3 represents the spectrum resulting after reduction of I; in the above samples with Na,S,O,. Vertical line designates 353 nm.

Mean

in Pooled

Less polar ROOH (PM)

A B C D

(nanometers)

4

Hydroperoxides

0.6 -0.9 -0.7 0.0 + SE

-0.3

+ 0.2

Human

Plasma

Total ROOH (M-f) 3.0 2.6 4.7 5.6 4.0 f 1.7

Note. Nonesterified fatty acid hydroperoxide (less polar ROOH) was calculated as the difference in iodometric activity of plasma before and after reduction with glutathione peroxidase. Total ROOH is the difference in oxidant activity of enzyme hydrolyzed plasma (see Table 3) before and after reduction with glutathione peroxidase.

210

CRAMER

investigators using iodometry to determine the content of peroxides in rat liver tissue (17). Certain retinoid compounds were found to absorb light at the same wavelength as 13 (17). By extracting 1; from the organic phase into an aqueous phase with cadmium acetate, Buege and Aust partially eliminated interference by these lipoidal chromophores (8). We found some interfering colored material was still present in the aqueous layer of the iodometric assay mixture, and it was greater in the plasma of jaundiced patients (data not shown). Back-titration of I; to I- with Na,S,O, allowed us to determine the exact degree of this calorimetric interference and to calculate more accurately the amount of 13 generated. If the Na,S,O, back-titration correction is not used, the amount of lipid oxidant material will tend to be overestimated. The second source of interference was due to phbspholipid and perhaps other similar amphipathic molecules from plasma which could cause an underestimation of the amount of hydroperoxide. Chloroform: methanol is widely used as a solvent for extracting total lipids (21,22), and it was our first choice in early studies of plasma hydroperoxides. Unfortunately, the phospholipids present in chloroform:methanol extracts of plasma appeared to decrease the amount of triiodide (I,) formed in response to peroxide thereby causing serious underestimates of hydroperoxide content. We theorized that the inhibitory effect of phospholipid may be due to interactions between the cadmium chloride:triiodide complex and the positively charged portion of the phospholipid head which alter the distribution of triiodide between the aqueous and organic phases of the assay mixture. In addition, this polar solvent may promote loss of hydroperoxide which does not occur in nonprotic solvents. (However, see comments on specificity below.) For this reason, we used ethyl acetate to extract only the less polar lipids for iodometry. Specificity. Many substances which are not hydroperoxides may also oxidize I- to I;. Thus, although the lipid oxidants extracted from the hydrolyzed samples include most of the lipid hydroperoxides of plasma, other oxidants may also be present. Reduction of the nonesterified hydroperoxides by the selective action of glutathione peroxidase (Fig. 4) enabled us to observe directly the amount of nonhydroperoxide oxidant. Most of the total I; measured in the assay of extracts from plasma was due to this type of peroxidase-resistant substance (Table 3 and Fig. 3). The less polar hydroperoxides extracted from plasma by ethyl acetate could include those in the nonesterified fatty acids, triglycerides, and cholesterol esters. The detection of a negligible average value of -0.3 f 0.2 indicates that very little hydroperoxide may be in those lipid fractions. In contrast, the significant amount of hydroperoxide extracted after treatment with hydrolytic enzymes suggests that hydroperoxides may be attached to the circulating phospholipids of apparently healthy hu-

ET

AL.

U-J E ‘0 0

200

4

8

12

16

Centimeters FIG. 4. Reduction of [“C]lS-HPETE to [“C]15-HETE in plasma by glutathione peroxidase. Plasma plus 5 nmol [“C]lB-HPETE were incubated with and without added glutathione peroxidase. Radioisotope scanning was done on the ethyl acetate extract of the plasma sample after TLC in petroleum ether:diethyl ether:acetic acid (50:50:0.5). 1, [“C]15-HPETE standard; 2, [“C]l5-HETE standard; 3, [“C]lB-HPETE incubated in normal plasma at 25’C for 30 min; 4, [‘*C]lS-HPETE incubated in normal plasma in the presence of GSH peroxidase. The shift in R, from 0.60 to 0.52 indicates conversion of [‘%]lS-HPETE to [“C]15-HETE.

mans. The increase in hydroperoxide oxidants in extracts after treatment with the hydrolytic enzymes, phospholipase A,, and Pseudomonas esterase may be due either to hydrolytic release of some oxidants from esterified forms or to some oxidant in the enzyme preparation. Irrespective of the actual source, treatment with glutathione peroxidase allows for correction of the nonhydroperoxide substances which oxidize iodide. Recovery of added hydroperoxide standards (Table 3) provided an estimate of the recovery of hydroperoxides that were exposed to potentially degradative conditions during treatment with the hydrolytic enzymes. Further experience with esterified and nonesterified hydroperoxides (23) has confirmed the usefulness of this iodometric assay in conjunction with other quantitative measurements of hydroperoxide abundance and with treatment by hydrolytic enzymes. A satisfactory use of chloroform:methanol for extraction of plasma-free samples (23) lends support to the hypothesis that interfer-

IODOMETRIC

ASSAY

ence by plasma phospholipids may be a major concern in assaying plasma with iodometric procedures. Sensitiuity. The molar absorbance of the triiodide ion obtained in our assays (2.3 X lo* M-’ cm-‘) was similar to the reported values of 2.8 X lo* and 1.73 X lo* M-’ cm-’ (8,23). The value of 2.8 X lo* M-’ cm-’ was obtained after performing the reaction in methanol rather than chloroform (24), and the value of 1.73 X lo* M-' cm-’ was obtained using cumene hydroperoxide (an aromatic hydroperoxide) as standard rather than 15 HPETE (8). Possibly, altering the organic phase or using different organic hydroperoxides may alter the observed extinction coefficient. The absorbance observed in our modified iodometric assay (0.023 absorbance unit per nanoequivalent 1, or nanomole hydroperoxide) theoretically permits detection of as little as 0.2 nmol of standard hydroperoxide (0.005 A). If a l-ml sample of plasma contained 0.6 nmol/ml of nonesterified lipid hydroperoxide (24) that was extracted with an 80% efficiency, the extracted lipid could be expected to exhibit an absorbance of about 0.012 above the thiosulfate-treated control reading. Unfortunately, the absorbance due to colored impurities and non-hydroperoxide oxidants in the plasma combines to give an overall absorbance that can be greater than 0.30. Although the thiosulfate treatment has reliably permitted subtraction of the calorimetric interference, the peroxidase-resistant oxidant material contributed the major part of the remaining absorbance. This creates an unfavorable condition of attempting to determine accurately a small number by subtracting one large number from another, producing a difference value that has a high percentage error. Thus although our improved iodometric method is sensitive and accurate with standard materials, the large amount of peroxidase-resistant oxidant in plasma detected no significant nonpolar hydroperoxides, and it permitted only a rough estimate of the content of total esterified plus nonesterified hydroperoxides in normal human plasma. Without correcting for an estimated recovery of 70%, the estimated value was approximately 4.0 -t 1.7 nmol hydroperoxide/ml. This value is lo-fold greater than an apparent value for nonesterified fatty acid hydroperoxide (25) and it is 15- to 20-fold lower than that predicted by the conventional TBA analysis. research was supported in part by funds from Hoffmann-LaRoche, Inc., and a Pfizer Biomedical (W.E.M.L.).

211

HYDROPEROXIDES

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