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Ferrous ion oxidation in presence of xylenol orange for detection of lipid hydroperoxides in plasma
58
OXIDATIVE DAMAGE: LIPIDS
[81
[8] F e r r o u s I o n O x i d a t i o n i n P r e s e n c e o f X y l e n o l O r a n g e for Detection of Lipid Hydroperoxides in Plasma
By J A F F A R
NOUROOz-ZADEH
Introduction Hydroperoxides (ROOHs) are the initial stable products formed during peroxidation of unsaturated lipids such as fatty acids or cholesterol. A battery of assays are available for the measurement of R O O H s in liposomes, whole plasma, or plasma lipoprotein classes. These techniques are generally divided into two categories, including total and individual classes. The latter is measured by high-performance liquid chromatography linked with chemiluminescence detection (HPLC-CL) techniques. Total R O O H s have been estimated to be between 0.5 and 5.5/zM using colorimetric, chemiluminescence, or enzymatic techniques.l-6 Using HPLC-CL assays, plasma levels of phospholipid hydroperoxides (PC-OOH) have been reported to range from 10 to 500 nM. 7'8 Plasma cholesteryl ester hydroperoxide levels are reported to be as low as 3 nM 9 or as high as 920 nM. 1° In this laboratory, the ferrous oxidation in xylenol orange (FOX) version 2 assay was originally developed to measure R O O H s in liposome preparations or low density lipoprotein (LDL) suspensions, n The FOX assay is based on the oxidation of ferrous to ferric ions by R O O H s under acidic conditions [Eq. (1)]. The dye xylenol orange [o-cresolsulfonphthalein-3,3bis(methyliminodiacetic acid sodium salt)] complexes with an equal molar concentration of ferric ion to produce a color (blue-purple) with an apparent extinction coefficient at 560 nm (~:A560)of 1.5 × 104 M -1 c m -I. In the presence 1 p. j. Marshall, M. A. Warso, and W. E. M. Lands, Anal, Biochem. 145, 192 (1985). 2 G. L. Cramer, J. F. Miller, R. B. Pendleton, and W. E. M. Lands, Anal. Biochem. 193, 204 (1991). 3 A. Zamburlini, M. Maiorino, P. Barbera, A. Roveri, and F. Ursini, Anal, Biochem. 232, 107 (1995). a j. Nourooz-Zadeh, J. Tajaddini-Sarmadi, and S. P. Wolff, Anal, Biochem. 220, 403 (1994). 5 j. Nourooz-Zadeh, J. Tajaddini-Sarmadi, S. McCarthy, D. J. Betteridge, and S. P. Wolff, Diabetes 44, 1054 (1995). 6 j. Nourooz-Zadeh, A. Rahimi, J. Tajaddini-Sarmadi, H. J. Tritschler, P. Rosen, B. Halliwell, and D. J. Betteridge, Diabetologia 40, 647 (1997). 7 y . Yamamoto and B. N. Ames, Free Rad. Biol. Med. 3, 359 (1987). s T. Miyazawa, Free Rad. Biol. Med. 7, 209 (1989). 9 y . Yamamoto and E. Niki, Biochem. Biophys. Res. Comm. 165, 988 (1989). 10 A. E. Holly and T. F. Slater, Free Rad. Res. Comm. 15, 51 (1991). n Z. Y. Jaing, C. S. Woollard, and S. P. Wolff, Lipids 26, 853 (1991).
METHODS IN ENZYMOLOGY,VOL. 300
Copyright © 1999by AcademicPress All rightsof reproductionin any form reserved. 0076-6879/99 $30.00
[8]
LIPID HYDROPEROXIDES IN PLASMA
59
of ROOHs, the yield of ferric ion-xylenol orange complex, however, is higher than 1:1 because of limited chain oxidation of ferrous ion by ROOHs. Undesirable chain oxidation [Eqs. (2)-(4)] is prevented by inclusion of the lipid-soluble chain-breaking antioxidant butylated hydroxytoluene (BHT), which repairs the alkyl radicals produced by the reaction of alkoxyl radicals with unsaturated lipids [Eq. (5)]. The apparent ~a560for a number of R O O H s is 4.5 x 104 M -1 cm -1. R O O H + Fe 2÷ --->Fe 3÷ + RO- + OHR O . + RH---> R O H + RR- + 02 --->R O O . R O O - + R H --->R O O H + R. R. + BHT---> R H + BH-
(1) (e) (3) (4) (5)
The FOX2 assay in conjugation with triphenylphosphine (TPP) has been implemented for the measurement of plasma ROOHs. 4-6 TPP reduces R O O H s to their corresponding alcohols while itself being converted to triphenylphosphine oxide. This maneuver was also necessary to generate a proper control, since plasma contains interfering components, mainly ferric ions, that are detected by xylenol orange. There are other advantages of the FOX2 assay over existing techniques: (a) the kinetics of the reaction are independent of the chemical structure of ROOHs; (b) no extraction step is normally needed for analysis of liposomes and lipoprotein suspensions because of the use of 90% methanol/25 mM HzSO4, which denatures proteins sufficiently for access of the ferrous ions to available ROOHs.
Preparation of FOX2 Reagent The FOX2 reagent comprises two stock solutions, A and B. Solution A is prepared by dissolving ammonium ferrous sulfate (98.03 mg) in 100 ml of 250 mM H2SO4. Subsequently, xylenol orange (76.06 rag) is added to the ammonium ferrous sulfate solution and the mixture is kept under stirring for about 10 min at room temperature. Solution B is prepared by dissolving 969.76 mg BHT in 900 ml methanol (HPLC-grade). A working FOX2 reagent is prepared by mixing one volume of solution A with nine volumes of solution B. The final FOX2 reagent is composed of xylenol orange (I00 ixM), BHT (4.4 mM), sulfuric acid (25 mM), and ammonium ferrous sulfate (250/xM). This solution is stable for I month at 4°C in the dark. The molar extinction coefficient for each batch of the FOX2 reagent is determined by calibration against varying concentrations of freshly prepared hydrogen peroxide (H202). Concentration of the stock H202 solution is determined
60
OXIDATIVE DAMAGE: LIPIDS
[8]
0.5 -
E
0.4
tO
0.3 ¢-
0.2 O
< o.1 o 0
1
2
3
4
5
6
7
8
9
10
11
H202 [I.tM]
FIG 1. Calibrationcurve for H202 usingthe FOX2 assay.
using the molar g,~240 43.6 M -1 Cm -1. Figure 1 shows the calibration curve for H202 in the concentration range 0-10/~M.
FOX2 Assay Ninety microliter aliquots of plasma or lipoprotein suspensions (100 ~g/ml protein) are transferred into 8 (1.5 ml) microcentrifuge reaction vials (L.I.P. Ltd, West Yorkshire, England). TPP (10 mM in methanol; 10/.d) is added to 4 vials to remove ROOHs. Methanol alone (10 ~l) is added to the remaining 4 vials. The samples are vortexed and subsequently incubated at room temperature for 30 rain. FOX2 reagent (900 ~l) is added and the samples are vortexed. After incubation at room temperature for 30 min, the samples are centrifuged at 15,000g at 20°C for 10 rain, the supernatant is carefully decanted into a cuvette, and absorbance is read at 560 nm. Absorbance values in plasma, without TPP treatment, range between 0.050 and 0.070. The background is less if there is a very small degree of plasma hemolysis. If there is more hemolysis, the background is greater. The ROOH content in the plasma samples is determined as a function of the mean absorbance difference of samples with and without elimination of ROOHs by TPP. Absorbance differences between pre- and posttreatment with TPP range from 0.002 to 0.05. The standard deviation is taken as the larger of the standard deviations of the measurements obtained with or without TPP treatment. Figure 2 shows typical data for native plasma and plasma contaminated with a defined amount of synthetic ROOHs.
[8]
LIPID H Y D R O P E R O X I D E S IN PLASMA
61
0.18 -
"30.15 E ¢O CO
8 (o)
0.12 -
0.09 -
0.06 -
< 0.03 -
0.00 Plasma
+(5-HPETE)
+(PC-OOH)
FIG 2. Detection of authentic ROOHs in plasma. PC-OOH: phospholipid hydroperoxides; 5-HPETE: 5-hydroperoxyeicosatetraenoic acid. Application The F O X 2 assay has been successfully used to examine circulating R O O H levels in apparently healthy volunteers as well as in a variety of clinical conditions associated with oxidative stress. The m e a n R O O H content in fresh plasma f r o m healthy volunteers from three separate studies was 3.02 ___ 1.85/xM (n = 23), 3.76 + 2.48/zM (n = 23), and 4.1 + 2.2/zM (n = 41). 4-6 Values of 9.04 + 4.3/zM and 9.4 + 3.3/zM were found in fresh plasma f r o m type II diabetic patients from two separate studies (n = 22 and 81, respectively). 5'6 For 67 patients with chronic renal failure, a m e a n level of 5.95 + 3.04/xM of R O O H s was recorded. The m e a n level of plasma R O O H s in hypercholesterolemic patients (n = 52) was 4.4 _ 2.1 /xM. The coefficient of variation for individual plasma using the F O X 2 assay is <10%. 4-6 The assay is not influenced by diurnal, fasting, or nonfasting conditions. 5 It has also been shown that more than 65% of total R O O H s in native plasma is confined to the L D L fraction. 12 Precautions Precision of the F O X 2 assay for the m e a s u r e m e n t of R O O H s in whole plasma or isolated lipoprotein fraction relies on accurate assessment of the amount of ferric ions formed during oxidation of ferrous ions by R O O H s . Thus, a prerequisite for the implementation of the assay is to eliminate the following confounding factors. 12j. Nourooz-Zadeh, J. Tajaddini-Sarmadi, K. L. E. Ling, and S. W. Wolff, Biochem. J. 313, 781 (1996).
62
OXIDATIVE DAMAOE: LIPIDS
[8]
Spontaneous Oxidation. Ammonium ferrous sulfate should be dissolved in 250 mM H2SO4 immediately after weighing to prevent spontaneous oxidation of ferrous ions to ferric ions during preparation of the FOX2 reagent. Reagent Decay with Time. A freshly prepared FOX2 reagent should be yellow with an absorbance at 560 n m < 0.005, against methanol as blank. Higher values suggest dirty glassware or the presence of substantial amounts of ferric ion in the reagent derived from oxidation ferrous ions to ferric ions. A high background absorbance (<0.05 at 560 nm) does not interfere with the assay when it is used for in vitro studies (lipoprotein suspensions or liposomes). However, FOX2-reagent with high background absorbance values is not suitable for the measurement of plasma ROOHs. Metal-Chelating Agents. EDTA and D E T A P A C are often used as anticoagulants during blood collection or as metal-chelating agents during dialysis of isolated lipoprotein fractions. These agents interfere with the accurate determination of R O O H s when present in the assay. Therefore, blood samples should be collected in heparinized tubes and chelating agents should be removed prior to the FOX assay. Ferric Ions. Circulatory iron is generally bound to the protein transferrin. Iron may, however, be released in plasma as a result of hemolysis of red blood during sample collection. This would give rise to a high background absorbance as consequence of liberation of ferric ion during the assay. Again, plasma samples with absorbance values >0120, pretreatment with TPP, will not be suitable for the assay. Antioxidants. Ascorbic acid (vitamin C) undergoes oxidation in the presence of transition metals (e.g., iron and copper ions) generating H202 and hydroxyl radicals as by-products. Vitamin C does not interfere with the FOX assay under physiological concentrations (12-100/zM). However, contamination of plasma with vitamin C at concentrations higher than 200/zM would give rise to a high background signal. Vitamin E or uric acid does not interfere with the assay. Acknowledgment W e t h a n k the British Heart Foundation for financial support.
OXIDATIVE DAMAGE: LIPIDS
[81
[8] F e r r o u s I o n O x i d a t i o n i n P r e s e n c e o f X y l e n o l O r a n g e for Detection of Lipid Hydroperoxides in Plasma
By J A F F A R
NOUROOz-ZADEH
Introduction Hydroperoxides (ROOHs) are the initial stable products formed during peroxidation of unsaturated lipids such as fatty acids or cholesterol. A battery of assays are available for the measurement of R O O H s in liposomes, whole plasma, or plasma lipoprotein classes. These techniques are generally divided into two categories, including total and individual classes. The latter is measured by high-performance liquid chromatography linked with chemiluminescence detection (HPLC-CL) techniques. Total R O O H s have been estimated to be between 0.5 and 5.5/zM using colorimetric, chemiluminescence, or enzymatic techniques.l-6 Using HPLC-CL assays, plasma levels of phospholipid hydroperoxides (PC-OOH) have been reported to range from 10 to 500 nM. 7'8 Plasma cholesteryl ester hydroperoxide levels are reported to be as low as 3 nM 9 or as high as 920 nM. 1° In this laboratory, the ferrous oxidation in xylenol orange (FOX) version 2 assay was originally developed to measure R O O H s in liposome preparations or low density lipoprotein (LDL) suspensions, n The FOX assay is based on the oxidation of ferrous to ferric ions by R O O H s under acidic conditions [Eq. (1)]. The dye xylenol orange [o-cresolsulfonphthalein-3,3bis(methyliminodiacetic acid sodium salt)] complexes with an equal molar concentration of ferric ion to produce a color (blue-purple) with an apparent extinction coefficient at 560 nm (~:A560)of 1.5 × 104 M -1 c m -I. In the presence 1 p. j. Marshall, M. A. Warso, and W. E. M. Lands, Anal, Biochem. 145, 192 (1985). 2 G. L. Cramer, J. F. Miller, R. B. Pendleton, and W. E. M. Lands, Anal. Biochem. 193, 204 (1991). 3 A. Zamburlini, M. Maiorino, P. Barbera, A. Roveri, and F. Ursini, Anal, Biochem. 232, 107 (1995). a j. Nourooz-Zadeh, J. Tajaddini-Sarmadi, and S. P. Wolff, Anal, Biochem. 220, 403 (1994). 5 j. Nourooz-Zadeh, J. Tajaddini-Sarmadi, S. McCarthy, D. J. Betteridge, and S. P. Wolff, Diabetes 44, 1054 (1995). 6 j. Nourooz-Zadeh, A. Rahimi, J. Tajaddini-Sarmadi, H. J. Tritschler, P. Rosen, B. Halliwell, and D. J. Betteridge, Diabetologia 40, 647 (1997). 7 y . Yamamoto and B. N. Ames, Free Rad. Biol. Med. 3, 359 (1987). s T. Miyazawa, Free Rad. Biol. Med. 7, 209 (1989). 9 y . Yamamoto and E. Niki, Biochem. Biophys. Res. Comm. 165, 988 (1989). 10 A. E. Holly and T. F. Slater, Free Rad. Res. Comm. 15, 51 (1991). n Z. Y. Jaing, C. S. Woollard, and S. P. Wolff, Lipids 26, 853 (1991).
METHODS IN ENZYMOLOGY,VOL. 300
Copyright © 1999by AcademicPress All rightsof reproductionin any form reserved. 0076-6879/99 $30.00
[8]
LIPID HYDROPEROXIDES IN PLASMA
59
of ROOHs, the yield of ferric ion-xylenol orange complex, however, is higher than 1:1 because of limited chain oxidation of ferrous ion by ROOHs. Undesirable chain oxidation [Eqs. (2)-(4)] is prevented by inclusion of the lipid-soluble chain-breaking antioxidant butylated hydroxytoluene (BHT), which repairs the alkyl radicals produced by the reaction of alkoxyl radicals with unsaturated lipids [Eq. (5)]. The apparent ~a560for a number of R O O H s is 4.5 x 104 M -1 cm -1. R O O H + Fe 2÷ --->Fe 3÷ + RO- + OHR O . + RH---> R O H + RR- + 02 --->R O O . R O O - + R H --->R O O H + R. R. + BHT---> R H + BH-
(1) (e) (3) (4) (5)
The FOX2 assay in conjugation with triphenylphosphine (TPP) has been implemented for the measurement of plasma ROOHs. 4-6 TPP reduces R O O H s to their corresponding alcohols while itself being converted to triphenylphosphine oxide. This maneuver was also necessary to generate a proper control, since plasma contains interfering components, mainly ferric ions, that are detected by xylenol orange. There are other advantages of the FOX2 assay over existing techniques: (a) the kinetics of the reaction are independent of the chemical structure of ROOHs; (b) no extraction step is normally needed for analysis of liposomes and lipoprotein suspensions because of the use of 90% methanol/25 mM HzSO4, which denatures proteins sufficiently for access of the ferrous ions to available ROOHs.
Preparation of FOX2 Reagent The FOX2 reagent comprises two stock solutions, A and B. Solution A is prepared by dissolving ammonium ferrous sulfate (98.03 mg) in 100 ml of 250 mM H2SO4. Subsequently, xylenol orange (76.06 rag) is added to the ammonium ferrous sulfate solution and the mixture is kept under stirring for about 10 min at room temperature. Solution B is prepared by dissolving 969.76 mg BHT in 900 ml methanol (HPLC-grade). A working FOX2 reagent is prepared by mixing one volume of solution A with nine volumes of solution B. The final FOX2 reagent is composed of xylenol orange (I00 ixM), BHT (4.4 mM), sulfuric acid (25 mM), and ammonium ferrous sulfate (250/xM). This solution is stable for I month at 4°C in the dark. The molar extinction coefficient for each batch of the FOX2 reagent is determined by calibration against varying concentrations of freshly prepared hydrogen peroxide (H202). Concentration of the stock H202 solution is determined
60
OXIDATIVE DAMAGE: LIPIDS
[8]
0.5 -
E
0.4
tO
0.3 ¢-
0.2 O
< o.1 o 0
1
2
3
4
5
6
7
8
9
10
11
H202 [I.tM]
FIG 1. Calibrationcurve for H202 usingthe FOX2 assay.
using the molar g,~240 43.6 M -1 Cm -1. Figure 1 shows the calibration curve for H202 in the concentration range 0-10/~M.
FOX2 Assay Ninety microliter aliquots of plasma or lipoprotein suspensions (100 ~g/ml protein) are transferred into 8 (1.5 ml) microcentrifuge reaction vials (L.I.P. Ltd, West Yorkshire, England). TPP (10 mM in methanol; 10/.d) is added to 4 vials to remove ROOHs. Methanol alone (10 ~l) is added to the remaining 4 vials. The samples are vortexed and subsequently incubated at room temperature for 30 rain. FOX2 reagent (900 ~l) is added and the samples are vortexed. After incubation at room temperature for 30 min, the samples are centrifuged at 15,000g at 20°C for 10 rain, the supernatant is carefully decanted into a cuvette, and absorbance is read at 560 nm. Absorbance values in plasma, without TPP treatment, range between 0.050 and 0.070. The background is less if there is a very small degree of plasma hemolysis. If there is more hemolysis, the background is greater. The ROOH content in the plasma samples is determined as a function of the mean absorbance difference of samples with and without elimination of ROOHs by TPP. Absorbance differences between pre- and posttreatment with TPP range from 0.002 to 0.05. The standard deviation is taken as the larger of the standard deviations of the measurements obtained with or without TPP treatment. Figure 2 shows typical data for native plasma and plasma contaminated with a defined amount of synthetic ROOHs.
[8]
LIPID H Y D R O P E R O X I D E S IN PLASMA
61
0.18 -
"30.15 E ¢O CO
8 (o)
0.12 -
0.09 -
0.06 -
< 0.03 -
0.00 Plasma
+(5-HPETE)
+(PC-OOH)
FIG 2. Detection of authentic ROOHs in plasma. PC-OOH: phospholipid hydroperoxides; 5-HPETE: 5-hydroperoxyeicosatetraenoic acid. Application The F O X 2 assay has been successfully used to examine circulating R O O H levels in apparently healthy volunteers as well as in a variety of clinical conditions associated with oxidative stress. The m e a n R O O H content in fresh plasma f r o m healthy volunteers from three separate studies was 3.02 ___ 1.85/xM (n = 23), 3.76 + 2.48/zM (n = 23), and 4.1 + 2.2/zM (n = 41). 4-6 Values of 9.04 + 4.3/zM and 9.4 + 3.3/zM were found in fresh plasma f r o m type II diabetic patients from two separate studies (n = 22 and 81, respectively). 5'6 For 67 patients with chronic renal failure, a m e a n level of 5.95 + 3.04/xM of R O O H s was recorded. The m e a n level of plasma R O O H s in hypercholesterolemic patients (n = 52) was 4.4 _ 2.1 /xM. The coefficient of variation for individual plasma using the F O X 2 assay is <10%. 4-6 The assay is not influenced by diurnal, fasting, or nonfasting conditions. 5 It has also been shown that more than 65% of total R O O H s in native plasma is confined to the L D L fraction. 12 Precautions Precision of the F O X 2 assay for the m e a s u r e m e n t of R O O H s in whole plasma or isolated lipoprotein fraction relies on accurate assessment of the amount of ferric ions formed during oxidation of ferrous ions by R O O H s . Thus, a prerequisite for the implementation of the assay is to eliminate the following confounding factors. 12j. Nourooz-Zadeh, J. Tajaddini-Sarmadi, K. L. E. Ling, and S. W. Wolff, Biochem. J. 313, 781 (1996).
62
OXIDATIVE DAMAOE: LIPIDS
[8]
Spontaneous Oxidation. Ammonium ferrous sulfate should be dissolved in 250 mM H2SO4 immediately after weighing to prevent spontaneous oxidation of ferrous ions to ferric ions during preparation of the FOX2 reagent. Reagent Decay with Time. A freshly prepared FOX2 reagent should be yellow with an absorbance at 560 n m < 0.005, against methanol as blank. Higher values suggest dirty glassware or the presence of substantial amounts of ferric ion in the reagent derived from oxidation ferrous ions to ferric ions. A high background absorbance (<0.05 at 560 nm) does not interfere with the assay when it is used for in vitro studies (lipoprotein suspensions or liposomes). However, FOX2-reagent with high background absorbance values is not suitable for the measurement of plasma ROOHs. Metal-Chelating Agents. EDTA and D E T A P A C are often used as anticoagulants during blood collection or as metal-chelating agents during dialysis of isolated lipoprotein fractions. These agents interfere with the accurate determination of R O O H s when present in the assay. Therefore, blood samples should be collected in heparinized tubes and chelating agents should be removed prior to the FOX assay. Ferric Ions. Circulatory iron is generally bound to the protein transferrin. Iron may, however, be released in plasma as a result of hemolysis of red blood during sample collection. This would give rise to a high background absorbance as consequence of liberation of ferric ion during the assay. Again, plasma samples with absorbance values >0120, pretreatment with TPP, will not be suitable for the assay. Antioxidants. Ascorbic acid (vitamin C) undergoes oxidation in the presence of transition metals (e.g., iron and copper ions) generating H202 and hydroxyl radicals as by-products. Vitamin C does not interfere with the FOX assay under physiological concentrations (12-100/zM). However, contamination of plasma with vitamin C at concentrations higher than 200/zM would give rise to a high background signal. Vitamin E or uric acid does not interfere with the assay. Acknowledgment W e t h a n k the British Heart Foundation for financial support.