- Email: [email protected]
A significant proportion of F2-isoprostanes in human urine are excreted as glucuronide conjugates
Analytical Biochemistry 403 (2010) 126–128
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
A significant proportion of F2-isoprostanes in human urine are excreted as glucuronide conjugates Zhao Yan a, Emilie Mas b, Trevor A. Mori b, Kevin D. Croft b, Anne E. Barden b,* a b
Fourth Military Medical University, Xi’an 710032, China Cardiovascular Research Centre, School of Medicine and Pharmacology, University of Western Australia, Perth, Western Australia 6847, Australia
a r t i c l e
i n f o
Article history: Received 16 March 2010 Accepted 15 April 2010 Available online 18 April 2010
a b s t r a c t The measurement of urinary F2-isoprostanes is noninvasive and widely used to assess in vivo oxidative stress in humans. Most studies measure urinary F2-isoprostanes in the free form; however, many eicosanoids are excreted as urine glucuronide conjugates. Using gas chromatography–mass spectrometry, we examined the extent of glucuronide conjugation of F2-isoprostanes in urine collected from healthy men (n = 20) and women (n = 15). Incubation of urine with exogenous glucuronidase led to F2-isoprostane concentrations that were approximately 40% higher than untreated samples (P < 0.001). We conclude that a significant proportion of F2-isoprostanes in urine are conjugated as glucuronides. Ó 2010 Elsevier Inc. All rights reserved.
Measurement of F2-isoprostanes, free radical oxidation products of arachidonic acid (AA),1 is a useful tool for assessment of in vivo lipid peroxidative damage [1]. Plasma F2-isoprostanes are mainly esterified to plasma lipids and are measured after base hydrolysis as indexes of lipid peroxidation. In contrast, F2-isoprostanes in urine are measured in the free form. Glucuronic acid conjugates are formed by enzymecatalyzed glucuronidation of compounds that contain either a hydroxyl group (ROH [alcohols], Ph-OH [phenols]) or a carboxyl group (RCOOH [acids]) [2]. A variety of compounds are metabolized by conjugation with glucuronic acid to yield water-soluble derivatives for excretion from the body. These include drug metabolites, polyphenols, and some eicosanoids. The glucuronidation process plays an important role in homeostasis. Glucuronides have been generally regarded as ‘‘detoxified” because they are most often water-soluble metabolites readily excreted in urine by the kidney. However, in some instances biologically active or toxic glucuronide conjugates exist. For example, morphine-6-glucuronide has been shown to exhibit morphine-like activity in humans [3]. The F2-isoprostanes have three hydroxyl groups that could potentially be available for glucuronidation. Given that some of these compounds are biologically active, formation of F2-isoprostane–glucuronides may be important in moderating their action. Although there is one published abstract suggesting that F2isoprostanes in urine might exist as glucuronide conjugates [4], to date there are no published studies that examine glucuronide conjugates of F2-isoprostanes in a systematic manner. Because urinary lev-
* Corresponding author. Fax: +61 8 9224 0246. E-mail address: [email protected] (A.E. Barden). 1 Abbreviations used: AA, arachidonic acid; BSTFA, bis-trimethylsilyltrifluoroacetamide; TMCS, trimethylchlorosilane; ECNI, electron capture negative ionisation; SIM, selected ion monitoring; 20-HETE, 20-hydroxyeicosatetraenoic acid. 0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2010.04.016
els of F2-isoprostanes are increasingly used as biomarkers of oxidative stress, it is important to know the extent of glucuronidation so that more accurate estimates of oxidative stress can be made. To assess the extent of glucuronide conjugation of F2-isoprostanes in human urine, we first examined the effect of urine pH on the activity of the b-glucuronidase enzymes to ensure that the hydrolysis of F2-isoprostanes was optimal. The extent of glucuronide conjugation of F2-isoprostanes was then studied in 35 human urine samples analyzed in the presence or absence of b-glucuronidase using a modification of the method of Mori and coworkers [5]. Briefly, urine samples were thawed and 1 ng of the internal standards d4-15-F2t-isoprostane and d4-8-F2t-isoprostane was added to 0.2 ml of urine. The pH of the urine was adjusted to 6.8, and the samples were flushed with nitrogen and incubated for 2 h at 37 °C in the presence or absence of b-glucuronidase from Escherichia coli (Sigma, product no. 016K8614, 0.2 mg in 0.075 mol/L phosphate buffer [pH 6.8] containing 1 g/L bovine serum albumin). After incubation, 2 ml of 0.1 mol/L sodium acetate (pH 4.6) was added to each sample and, where necessary, the pH was adjusted to 4.6 with 0.1 mol/L HCl. Samples were applied to Bond Elut-Certify II columns (Varian) preconditioned with 2 ml of methanol, followed by 2 ml of 0.1 mol/L sodium acetate buffer (pH 7.0) containing 50 ml/L methanol. The columns were washed with 2 ml of methanol/water (1:1 by volume), followed by 2 ml of ethyl acetate/hexane (1:3), and the F2-isoprostanes were eluted with 2 ml of ethyl acetate/methanol (9:1). The samples were evaporated to dryness under vacuum and treated with 40 ll of pentafluorobenzylbromide in acetonitrile (100 g/L) and 20 ll of N,N-diisopropylethylamine in acetonitrile (100 g/L) for 30 min at room temperature. Samples were dried under nitrogen and treated with 20 ll of bis-trimethylsilyltrifluoroacetamide/ trimethylchlorosilane (BSTFA/TMCS, 99:1) and 10 ll of anhydrous
127
Notes & Tips / Anal. Biochem. 403 (2010) 126–128
pyridine at 45 °C for 20 min. The sample was dried and reconstituted in isooctane (30 ll) and was analyzed on an Agilent 6890 gas chromatograph coupled to an Agilent 5975 mass selective detector using a J&W Scientific DB-5MS column (25 m 0.2 mm 0.33 lm) with helium as the carrier gas. The mass spectrometer was operated in electron capture negative ionization (ECNI) mode using methane as the ionizing gas. F2-isoprostanes were identified by retention time of authentic 15-F2t-isoprostane standard using selected ion monitoring (SIM) m/z 569 and 573 for the 15-F2t-isoprostane and d4-8-F2t-isoprostane and d4-15-F2t -isoprostane internal standards, respectively. The effect of pH on glucuronidase hydrolysis of F2-isoprostanes in urine was determined by adjusting the pH of seven aliquots of pooled urine with HCl (0.1 mol/L) to pH 4.1, 4.5, 5.0, 5.8, 6.2, 7.1, and 7.7. The aliquots were then incubated with b-glucuronidase and assayed as described above. Glucuronidase hydrolysis yielded F2-isoprostanes in urine of 9.52 ± 1.69 nmol/24 h at pH 4.1, 10.55 ± 0.98 nmol/24 h at pH 4.5, 12.45 ± 1.26 nmol/24 h at pH 5.0, 13.1 ± 1.97 nmol/24 h at pH 5.8, 13.74 ± 1.13 nmol/24 h at pH 6.2, 13.21 ± 0.42 nmol/24 h at pH 7.1, and 12.24 ± 1.83 nmol/24 h at pH 7.7. The pH of the urine incubated without glucuronidase was 6.3, and the F2-isoprostane level of this urine was 10.22 ± 1.26 nmol/24 h. The results showed that glucuronidase release of F2-isoprostanes is dependent on urine pH. A pH less than 5.0 resulted in a substantial reduction of hydrolysis of F2-isoprostanes by the glucuronidase enzyme compared with a pH between 6.0 and 7.0. This is consistent with the pH optimum of 6.8 (published by Sigma) for this glucuronidase enzyme. To confirm that the increase in urine F2-isoprostanes was specific to the glucuronidase enzyme, an experiment was carried out with inactivated glucuronidase. In this experiment, the glucuronidase enzyme was heat inactivated for 1 h at 90 °C prior to incubation with the urine. F2-isoprostane levels in urine incubated with heat-inactivated glucuronidase were similar to those in urine incubated without enzyme (3.75 ± 0.20 and 3.60 ± 0.20 nmol/24 h, respectively) and lower than when incubation took place with active glucuronidase (6.33 ± 0.35 nmol/24 h). This confirms that the hydrolysis of F2-isoprostanes was due to the glucuronidase enzyme. The effect of glucuronidase on urinary F2-isoprostanes was then studied in 20 men and 15 postmenopausal women who were recruited from the general population by advertisement. The volunteers were nonsmokers and were not taking any medication or vitamin supplements at the time of study. After telephone screening to assess suitability, they attended the research unit where height, weight, and blood pressure were measured and a fasted blood sample was collected for measurement of serum lipids. All volunteers collected a 24-h urine sample for measurement of F2isoprostanes. The urine volume was recorded, and aliquots were frozen at 80 °C until assay. The study was approved by the University of Western Australia human ethics committee, and all subjects gave written informed consent to participate. The characteristics of the men and women in the study are listed in Table 1. Analysis of urinary F2-isoprostanes in the 35 vol-
unteers showed that F2-isoprostane levels were higher after the urine was incubated with glucuronidase (Fig. 1) (P < 0.001). Correction of urinary F2-isoprostanes for creatinine excretion gave similar results (0.43 ± 0.02 vs. 0.61 ± 0.03 nmol/mmol creatinine after glucuronidase treatment) (P < 0.001). There was a significant effect of gender on urinary F2-isoprostane excretion. Men excreted more F2-isoprostanes than women regardless of whether the urine was treated with glucuronidase (P < 0.006) (Fig. 1). This study shows that a substantial amount of F2-isoprostanes are excreted in human urine as glucuronide conjugates. The welldescribed gender differences in urinary F2-isoprostanes were still observed when urine was incubated with glucuronidase. The effect of pH on hydrolysis F2-isoprostanes by glucuronidase was highlighted and is important because the pH of urine can be altered when samples are frozen. Other AA metabolites, such as 20-hydroxyeicosatetraenoic acid (20-HETE), are also excreted as glucuronides in urine [6]. 20-HETE, a cytochrome P450 metabolite of AA, is increased in human urine by 13- to 28-fold after treatment with glucuronidase [7]. In contrast, the increase in F2-isoprostanes with glucuronidase treatment was approximately 40%. The differences between glucuronidation of F2-isoprostanes and 20-HETE may relate to the structure and relative availability of the hydroxyl and carboxyl moieties for glucuronidation. Several of the F2-isoprostanes are biologically active [8]. For example, 8-iso-prostaglandin F2a is a potent vasoconstrictor in the kidney and has bronchoconstrictor actions [8]. Therefore, it is possible that formation of glucuronide conjugates of F2-isoprostanes may be a homeostatic mechanism to limit any damaging effects. Numerous endogenous metabolites are substrates for glucuronidases, including steroids and polyphenols. This raises the possibility that competition with these substrates for glucuronidation could affect the level of free isoprostanes measured in urine. For example, in our own studies examining the effects of alcohol or tocopherol on F2-isoprostanes, we have observed that the effects of the intervention on F2-isoprostanes in plasma and urine are not always concordant [9,10]. In those studies, plasma F2-isoprostanes were measured after hydrolysis, whereas urine F2-isoprostanes were measured in the free form, raising the possibility that the intervention could have affected the extent of conjugation of F2-isoprostanes in urine. Further studies are required to establish whether background diet or drug treatment can affect glucuronidation of F2-isoprostanes in urine.
(nmol/24 h)
*
10.0
- glucuronidase + glucuronidase
*
7.5
5.0 Table 1 Characteristics of volunteers
Age (years) Body mass index (kg/m2) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Cholesterol (mmol/L) Triglycerides (mmol/L)
2.5 Men (n = 20)
Women (n = 15)
55 ± 1.5 27.5 ± 0.7 125 ± 2 76 ± 1 5.1 ± 0.2 1.0 ± 0.1
58 ± 1.4 24.4 ± 0.8* 109 ± 2* 68 ± 2* 5.6 ± 0.2 0.9 ± 0.1
Note. Values are means ± standard errors. An asterisk () signifies differences between genders (P < 0.01).
0.0
All volunteers
Men
Women
Fig. 1. Effect of incubation with glucuronidase on urinary F2-isoprostane excretion in men and women. A dagger ( ) indicates paired t test (P < 0.001) comparing F2isoprostane excretion with and without glucuronidase incubation, and an asterisk () indicates P < 0.006 comparing F2-isoprostane excretion between men and women using a general linear model.
128
Notes & Tips / Anal. Biochem. 403 (2010) 126–128
Acknowledgments This work was supported by a National Health and Medical Research Council of Australia Project Grant (458514) and a National Heart Foundation of Australia Project Grant (GP05 P2186).
References [1] L.J. Roberts, J.D. Morrow, Measurement of F2 isoprostanes as an index of oxidative stress in vivo, Free Radic. Biol. Med. 28 (2000) 505–513. [2] C.T. Bedford, Glucuronic acid conjugates, J. Chromatogr. B 717 (1998) 313–326. [3] R. Osborne, S. Joel, D. Trew, M. Slevin, Analgesic activity of morphine-6glucuronide, Lancet 1 (1988) 828. [4] D. Callewaert, R. McGowen, C. Sloan, K. Godshalk, T. Solomon, S. Basu, J. Morrow, S. Gupta, Isoprostane glucuronides in human urine and the evaluation of oxidative stress (abstract), Free Radic. Biol. Med. 39 (2005) S110.
[5] T.A. Mori, K.D. Croft, I.B. Puddey, L.J. Beilin, An improved method for the measurement of urinary and plasma F2-isoprostanes using gas chromatography–mass spectrometry, Anal. Biochem. 268 (1999) 117–125. [6] J. Rivera, N. Ward, J. Hodgson, I.B. Puddey, J.R. Falck, K.D. Croft, Measurement of 20-hydroxyeicosatetraenoic acid in human urine by gas chromatography– mass spectrometry, Clin. Chem. 50 (2004) 224–226. [7] C. Prackash, J.Y. Zhang, J.R. Falck, K. Chauhan, I.A. Blair, 20Hydroxyeicosatetraenoic acid is excreted as a glucuronide conjugate in human urine, Biochem. Biophys. Res. Commun. 185 (1992) 728–733. [8] J.D. Morrow, The isoprostanes—unique products of arachidonate peroxidation: their role as mediators of oxidant stress, Curr. Pharm. Des. 12 (2006) 895–902. [9] J.H.Y. Wu, N.C. Ward, A.P. Indrawan, C.A. Almeida, J.M. Hodgson, J.M. Proudfoot, I.B. Puddey, K.D. Croft, Effects of a-tocopherol and mixed tocopherol supplementation on markers of oxidative stress and inflammation in type 2 diabetes, Clin. Chem. 53 (2007) 511–519. [10] A. Barden, R.R. Zilkens, K. Croft, T. Mori, V. Burke, L.J. Beilin, I.B. Puddey, A reduction in alcohol consumption is associated with reduced plasma F2isoprostanes and urinary 20-HETE excretion in men, Free Radic. Biol. Med. 42 (2007) 1730–1735.
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
A significant proportion of F2-isoprostanes in human urine are excreted as glucuronide conjugates Zhao Yan a, Emilie Mas b, Trevor A. Mori b, Kevin D. Croft b, Anne E. Barden b,* a b
Fourth Military Medical University, Xi’an 710032, China Cardiovascular Research Centre, School of Medicine and Pharmacology, University of Western Australia, Perth, Western Australia 6847, Australia
a r t i c l e
i n f o
Article history: Received 16 March 2010 Accepted 15 April 2010 Available online 18 April 2010
a b s t r a c t The measurement of urinary F2-isoprostanes is noninvasive and widely used to assess in vivo oxidative stress in humans. Most studies measure urinary F2-isoprostanes in the free form; however, many eicosanoids are excreted as urine glucuronide conjugates. Using gas chromatography–mass spectrometry, we examined the extent of glucuronide conjugation of F2-isoprostanes in urine collected from healthy men (n = 20) and women (n = 15). Incubation of urine with exogenous glucuronidase led to F2-isoprostane concentrations that were approximately 40% higher than untreated samples (P < 0.001). We conclude that a significant proportion of F2-isoprostanes in urine are conjugated as glucuronides. Ó 2010 Elsevier Inc. All rights reserved.
Measurement of F2-isoprostanes, free radical oxidation products of arachidonic acid (AA),1 is a useful tool for assessment of in vivo lipid peroxidative damage [1]. Plasma F2-isoprostanes are mainly esterified to plasma lipids and are measured after base hydrolysis as indexes of lipid peroxidation. In contrast, F2-isoprostanes in urine are measured in the free form. Glucuronic acid conjugates are formed by enzymecatalyzed glucuronidation of compounds that contain either a hydroxyl group (ROH [alcohols], Ph-OH [phenols]) or a carboxyl group (RCOOH [acids]) [2]. A variety of compounds are metabolized by conjugation with glucuronic acid to yield water-soluble derivatives for excretion from the body. These include drug metabolites, polyphenols, and some eicosanoids. The glucuronidation process plays an important role in homeostasis. Glucuronides have been generally regarded as ‘‘detoxified” because they are most often water-soluble metabolites readily excreted in urine by the kidney. However, in some instances biologically active or toxic glucuronide conjugates exist. For example, morphine-6-glucuronide has been shown to exhibit morphine-like activity in humans [3]. The F2-isoprostanes have three hydroxyl groups that could potentially be available for glucuronidation. Given that some of these compounds are biologically active, formation of F2-isoprostane–glucuronides may be important in moderating their action. Although there is one published abstract suggesting that F2isoprostanes in urine might exist as glucuronide conjugates [4], to date there are no published studies that examine glucuronide conjugates of F2-isoprostanes in a systematic manner. Because urinary lev-
* Corresponding author. Fax: +61 8 9224 0246. E-mail address: [email protected] (A.E. Barden). 1 Abbreviations used: AA, arachidonic acid; BSTFA, bis-trimethylsilyltrifluoroacetamide; TMCS, trimethylchlorosilane; ECNI, electron capture negative ionisation; SIM, selected ion monitoring; 20-HETE, 20-hydroxyeicosatetraenoic acid. 0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2010.04.016
els of F2-isoprostanes are increasingly used as biomarkers of oxidative stress, it is important to know the extent of glucuronidation so that more accurate estimates of oxidative stress can be made. To assess the extent of glucuronide conjugation of F2-isoprostanes in human urine, we first examined the effect of urine pH on the activity of the b-glucuronidase enzymes to ensure that the hydrolysis of F2-isoprostanes was optimal. The extent of glucuronide conjugation of F2-isoprostanes was then studied in 35 human urine samples analyzed in the presence or absence of b-glucuronidase using a modification of the method of Mori and coworkers [5]. Briefly, urine samples were thawed and 1 ng of the internal standards d4-15-F2t-isoprostane and d4-8-F2t-isoprostane was added to 0.2 ml of urine. The pH of the urine was adjusted to 6.8, and the samples were flushed with nitrogen and incubated for 2 h at 37 °C in the presence or absence of b-glucuronidase from Escherichia coli (Sigma, product no. 016K8614, 0.2 mg in 0.075 mol/L phosphate buffer [pH 6.8] containing 1 g/L bovine serum albumin). After incubation, 2 ml of 0.1 mol/L sodium acetate (pH 4.6) was added to each sample and, where necessary, the pH was adjusted to 4.6 with 0.1 mol/L HCl. Samples were applied to Bond Elut-Certify II columns (Varian) preconditioned with 2 ml of methanol, followed by 2 ml of 0.1 mol/L sodium acetate buffer (pH 7.0) containing 50 ml/L methanol. The columns were washed with 2 ml of methanol/water (1:1 by volume), followed by 2 ml of ethyl acetate/hexane (1:3), and the F2-isoprostanes were eluted with 2 ml of ethyl acetate/methanol (9:1). The samples were evaporated to dryness under vacuum and treated with 40 ll of pentafluorobenzylbromide in acetonitrile (100 g/L) and 20 ll of N,N-diisopropylethylamine in acetonitrile (100 g/L) for 30 min at room temperature. Samples were dried under nitrogen and treated with 20 ll of bis-trimethylsilyltrifluoroacetamide/ trimethylchlorosilane (BSTFA/TMCS, 99:1) and 10 ll of anhydrous
127
Notes & Tips / Anal. Biochem. 403 (2010) 126–128
pyridine at 45 °C for 20 min. The sample was dried and reconstituted in isooctane (30 ll) and was analyzed on an Agilent 6890 gas chromatograph coupled to an Agilent 5975 mass selective detector using a J&W Scientific DB-5MS column (25 m 0.2 mm 0.33 lm) with helium as the carrier gas. The mass spectrometer was operated in electron capture negative ionization (ECNI) mode using methane as the ionizing gas. F2-isoprostanes were identified by retention time of authentic 15-F2t-isoprostane standard using selected ion monitoring (SIM) m/z 569 and 573 for the 15-F2t-isoprostane and d4-8-F2t-isoprostane and d4-15-F2t -isoprostane internal standards, respectively. The effect of pH on glucuronidase hydrolysis of F2-isoprostanes in urine was determined by adjusting the pH of seven aliquots of pooled urine with HCl (0.1 mol/L) to pH 4.1, 4.5, 5.0, 5.8, 6.2, 7.1, and 7.7. The aliquots were then incubated with b-glucuronidase and assayed as described above. Glucuronidase hydrolysis yielded F2-isoprostanes in urine of 9.52 ± 1.69 nmol/24 h at pH 4.1, 10.55 ± 0.98 nmol/24 h at pH 4.5, 12.45 ± 1.26 nmol/24 h at pH 5.0, 13.1 ± 1.97 nmol/24 h at pH 5.8, 13.74 ± 1.13 nmol/24 h at pH 6.2, 13.21 ± 0.42 nmol/24 h at pH 7.1, and 12.24 ± 1.83 nmol/24 h at pH 7.7. The pH of the urine incubated without glucuronidase was 6.3, and the F2-isoprostane level of this urine was 10.22 ± 1.26 nmol/24 h. The results showed that glucuronidase release of F2-isoprostanes is dependent on urine pH. A pH less than 5.0 resulted in a substantial reduction of hydrolysis of F2-isoprostanes by the glucuronidase enzyme compared with a pH between 6.0 and 7.0. This is consistent with the pH optimum of 6.8 (published by Sigma) for this glucuronidase enzyme. To confirm that the increase in urine F2-isoprostanes was specific to the glucuronidase enzyme, an experiment was carried out with inactivated glucuronidase. In this experiment, the glucuronidase enzyme was heat inactivated for 1 h at 90 °C prior to incubation with the urine. F2-isoprostane levels in urine incubated with heat-inactivated glucuronidase were similar to those in urine incubated without enzyme (3.75 ± 0.20 and 3.60 ± 0.20 nmol/24 h, respectively) and lower than when incubation took place with active glucuronidase (6.33 ± 0.35 nmol/24 h). This confirms that the hydrolysis of F2-isoprostanes was due to the glucuronidase enzyme. The effect of glucuronidase on urinary F2-isoprostanes was then studied in 20 men and 15 postmenopausal women who were recruited from the general population by advertisement. The volunteers were nonsmokers and were not taking any medication or vitamin supplements at the time of study. After telephone screening to assess suitability, they attended the research unit where height, weight, and blood pressure were measured and a fasted blood sample was collected for measurement of serum lipids. All volunteers collected a 24-h urine sample for measurement of F2isoprostanes. The urine volume was recorded, and aliquots were frozen at 80 °C until assay. The study was approved by the University of Western Australia human ethics committee, and all subjects gave written informed consent to participate. The characteristics of the men and women in the study are listed in Table 1. Analysis of urinary F2-isoprostanes in the 35 vol-
unteers showed that F2-isoprostane levels were higher after the urine was incubated with glucuronidase (Fig. 1) (P < 0.001). Correction of urinary F2-isoprostanes for creatinine excretion gave similar results (0.43 ± 0.02 vs. 0.61 ± 0.03 nmol/mmol creatinine after glucuronidase treatment) (P < 0.001). There was a significant effect of gender on urinary F2-isoprostane excretion. Men excreted more F2-isoprostanes than women regardless of whether the urine was treated with glucuronidase (P < 0.006) (Fig. 1). This study shows that a substantial amount of F2-isoprostanes are excreted in human urine as glucuronide conjugates. The welldescribed gender differences in urinary F2-isoprostanes were still observed when urine was incubated with glucuronidase. The effect of pH on hydrolysis F2-isoprostanes by glucuronidase was highlighted and is important because the pH of urine can be altered when samples are frozen. Other AA metabolites, such as 20-hydroxyeicosatetraenoic acid (20-HETE), are also excreted as glucuronides in urine [6]. 20-HETE, a cytochrome P450 metabolite of AA, is increased in human urine by 13- to 28-fold after treatment with glucuronidase [7]. In contrast, the increase in F2-isoprostanes with glucuronidase treatment was approximately 40%. The differences between glucuronidation of F2-isoprostanes and 20-HETE may relate to the structure and relative availability of the hydroxyl and carboxyl moieties for glucuronidation. Several of the F2-isoprostanes are biologically active [8]. For example, 8-iso-prostaglandin F2a is a potent vasoconstrictor in the kidney and has bronchoconstrictor actions [8]. Therefore, it is possible that formation of glucuronide conjugates of F2-isoprostanes may be a homeostatic mechanism to limit any damaging effects. Numerous endogenous metabolites are substrates for glucuronidases, including steroids and polyphenols. This raises the possibility that competition with these substrates for glucuronidation could affect the level of free isoprostanes measured in urine. For example, in our own studies examining the effects of alcohol or tocopherol on F2-isoprostanes, we have observed that the effects of the intervention on F2-isoprostanes in plasma and urine are not always concordant [9,10]. In those studies, plasma F2-isoprostanes were measured after hydrolysis, whereas urine F2-isoprostanes were measured in the free form, raising the possibility that the intervention could have affected the extent of conjugation of F2-isoprostanes in urine. Further studies are required to establish whether background diet or drug treatment can affect glucuronidation of F2-isoprostanes in urine.
(nmol/24 h)
*
10.0
- glucuronidase + glucuronidase
*
7.5
5.0 Table 1 Characteristics of volunteers
Age (years) Body mass index (kg/m2) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Cholesterol (mmol/L) Triglycerides (mmol/L)
2.5 Men (n = 20)
Women (n = 15)
55 ± 1.5 27.5 ± 0.7 125 ± 2 76 ± 1 5.1 ± 0.2 1.0 ± 0.1
58 ± 1.4 24.4 ± 0.8* 109 ± 2* 68 ± 2* 5.6 ± 0.2 0.9 ± 0.1
Note. Values are means ± standard errors. An asterisk () signifies differences between genders (P < 0.01).
0.0
All volunteers
Men
Women
Fig. 1. Effect of incubation with glucuronidase on urinary F2-isoprostane excretion in men and women. A dagger ( ) indicates paired t test (P < 0.001) comparing F2isoprostane excretion with and without glucuronidase incubation, and an asterisk () indicates P < 0.006 comparing F2-isoprostane excretion between men and women using a general linear model.
128
Notes & Tips / Anal. Biochem. 403 (2010) 126–128
Acknowledgments This work was supported by a National Health and Medical Research Council of Australia Project Grant (458514) and a National Heart Foundation of Australia Project Grant (GP05 P2186).
References [1] L.J. Roberts, J.D. Morrow, Measurement of F2 isoprostanes as an index of oxidative stress in vivo, Free Radic. Biol. Med. 28 (2000) 505–513. [2] C.T. Bedford, Glucuronic acid conjugates, J. Chromatogr. B 717 (1998) 313–326. [3] R. Osborne, S. Joel, D. Trew, M. Slevin, Analgesic activity of morphine-6glucuronide, Lancet 1 (1988) 828. [4] D. Callewaert, R. McGowen, C. Sloan, K. Godshalk, T. Solomon, S. Basu, J. Morrow, S. Gupta, Isoprostane glucuronides in human urine and the evaluation of oxidative stress (abstract), Free Radic. Biol. Med. 39 (2005) S110.
[5] T.A. Mori, K.D. Croft, I.B. Puddey, L.J. Beilin, An improved method for the measurement of urinary and plasma F2-isoprostanes using gas chromatography–mass spectrometry, Anal. Biochem. 268 (1999) 117–125. [6] J. Rivera, N. Ward, J. Hodgson, I.B. Puddey, J.R. Falck, K.D. Croft, Measurement of 20-hydroxyeicosatetraenoic acid in human urine by gas chromatography– mass spectrometry, Clin. Chem. 50 (2004) 224–226. [7] C. Prackash, J.Y. Zhang, J.R. Falck, K. Chauhan, I.A. Blair, 20Hydroxyeicosatetraenoic acid is excreted as a glucuronide conjugate in human urine, Biochem. Biophys. Res. Commun. 185 (1992) 728–733. [8] J.D. Morrow, The isoprostanes—unique products of arachidonate peroxidation: their role as mediators of oxidant stress, Curr. Pharm. Des. 12 (2006) 895–902. [9] J.H.Y. Wu, N.C. Ward, A.P. Indrawan, C.A. Almeida, J.M. Hodgson, J.M. Proudfoot, I.B. Puddey, K.D. Croft, Effects of a-tocopherol and mixed tocopherol supplementation on markers of oxidative stress and inflammation in type 2 diabetes, Clin. Chem. 53 (2007) 511–519. [10] A. Barden, R.R. Zilkens, K. Croft, T. Mori, V. Burke, L.J. Beilin, I.B. Puddey, A reduction in alcohol consumption is associated with reduced plasma F2isoprostanes and urinary 20-HETE excretion in men, Free Radic. Biol. Med. 42 (2007) 1730–1735.