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Technical Briefs |
1 School of Medicine and Pharmacology, University of Western Australia and Western Australian Institute for Medical Research, Perth, Western Australia
2 Biochemistry Department, University of Texas Southwestern Medical Center, Dallas, TX
aaddress correspondence to this author at: School of Medicine and Pharmacology, GPO Box X2213, Perth, Western Australia 6847, Australia; e-mail kcroft{at}cyllene.uwa.edu.au
Arachidonic acid can be metabolized by cytochrome P450 enzymes to a range of compounds that play a central role in the regulation of vascular tone, renal function, and blood pressure (1)(2). In the vasculature, smooth muscle cells produce 20-hydroxyeicosatetraenoic acid (20-HETE) as a major product of CYP450 metabolism. 20-HETE can cause vasoconstriction by inhibition of potassium channels and is thought to contribute to the vasoconstrictor action of hormones such as angiotensin II and endothelin (3)(4). Despite the physiologic importance of CYP450 metabolites of arachidonic acid, very little is known about the regulation of the concentration of 20-HETE in biological fluids or the relationship of these concentrations with physiologic state in healthy individuals. This has in part been attributable to the lack of reliable sensitive and specific assays to measure endogenous concentrations of these compounds. Gas chromatography–mass spectrometry (GCMS) has been used successfully to measure 20-HETE in biological samples. However, available methods rely on one or more thin-layer chromatography steps (5)(6), and for human urine the presence of interfering peaks can be a problem (7). An alternative procedure has recently been reported that uses a sensitive fluorescent HPLC assay (8), although this may lack the specificity of MS.
We have developed a simplified and reliable method for the analysis of urinary 20-HETE and analyzed 20-HETE concentrations in 24-h urine samples from a group of 30 healthy individuals. Our method involves the use of a single solid-phase extraction cartridge containing both reversed-phase and strong anion-exchange packing followed by HPLC separation before derivatization and GCMS analysis. We have found that preparation of the tert-butyldimethylsilyl derivative (tBDMS), as originally used by Prakash et al. (6), gives better chromatographic separation from interfering peaks present in urine.
20,20-[2H2]-20-HETE was prepared according to previously published procedures (9). Unlabeled 20-HETE was purchased from Cayman Chemicals. ß-Glucuronidase (Escherichia coli), pentafluorobenzyl bromide (PFB Br), N,N-diisopropylethylamine, and tert-butyldimethylsilyl-N-methyltrifluoroacetamide were purchased from Sigma-Aldrich. Pyridine was purchased from Fluka. Bond Elut-Certify II (200 mg, 3 mL) columns were purchased from Varian Inc. Volunteers were recruited from the general population. We monitored 24-h blood pressures (BP) by use of an ambulatory device (Spacelabs 90207). All studies with samples from humans were approved by the Human Ethics Committee of Royal Perth Hospital.
The internal standard [2H2]-20-HETE (2 ng) was added to urine (2 mL). Each sample was left at room temperature for 10 min to equilibrate before incubation with 0.2 mg of ß-glucuronidase from E. coli (in 0.075 mol/L potassium phosphate buffer, pH 6.8, containing 1 g/L bovine serum albumin) for 2 h at 37 °C. After hydrolysis, samples were diluted with 2 mL of 0.1 mol/L sodium acetate solution (pH 7) containing 50 mL/L methanol, and the pH was adjusted to 6.0 with 100 mL/L acetic acid.
Bond Elut-Certify II columns were preconditioned with 2 mL of methanol, followed by 2 mL of 0.1 mol/L sodium acetate solution (pH 7) containing 50 mL/L methanol before application of the urine samples. The columns were washed with 2 mL of methanol–water (1:1 by volume), and urinary 20-HETE and internal standard were eluted with 2 mL of hexane–ethyl acetate (75:25 by volume) containing 10 mL/L acetic acid. The organic extracts were evaporated to dryness under reduced pressure and reconstituted in 50 µL of methanol for HPLC purification on an Agilent 1100 system. Separations were carried out with a LiChrospher® RP-18 (length, 100 mm; 5 µm bead size; Agilent) column with a linear gradient mobile phase starting from acetonitrile–water–acetic acid (50:50:0.05 by volume) to acetonitrile at a flow rate of 1 mL/min for 20 min. 20-HETE and the internal standard eluted at a retention time of 6.9 min, and 1-min fractions were collected between 6.5 and 7.5 min with an automated fraction collector (Gilson).
Fractions containing the HETEs were dried under reduced pressure, and the residue was treated with 40 µL of 100 g/L PFB Br in acetonitrile and 20 µL of 100 g/L N,N-diisopropylethylamine in acetonitrile for 30 min at room temperature. After drying under nitrogen, the residues were treated with 20 µL of N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide and 10 µL of pyridine for 20 min at 45 °C. Samples were dried under nitrogen and dissolved in 30 µL of isooctane for analysis on an Agilent 5973 GCMS. Samples (1 µL) were injected on a HP-1MS column [15 m x 0.25 mm (i.d); 0.25-µm film thickness; Agilent] with a temperature program of 160 °C initially held for 0.50 min and increased to 300 °C at a rate of 15 °C/min. Helium was used as the carrier gas, and injections were made in the splitless mode. The mass spectrophotometer was operated in the negative chemical ionization mode with methane reagent gas at a source pressure of 2 x 10-4 Torr. For selected-ion monitoring analyses, the m/z 433 (endogenous 20-HETE-PFB-tBDMS) and m/z 435 ([2H2]-20-HETE-PFB-tBDMS) ions were monitored. Urinary 20-HETEs were identified by comparison against the retention time of an authentic 20-HETE standard, and concentrations were determined by peak-area ratios of the analyte to internal standard [2H2]-20-HETE.
This highly sensitive and specific assay for 20-HETE in human urine uses a dideuterated autologous 20-HETE as internal standard for GCMS analysis. As reported previously (6), the tBDMS-PFB derivative has good chromatographic properties, with major negative ion fragments at m/z 433 (M-PBF) for 20-HETE and m/z 435 for 2H2-20-HETE. Fig. 1
shows a typical selected-ion chromatogram obtained from a human urine sample. Although most urine samples show a range of peaks in the region of 20-HETE, using this purification system and the tBDMS derivative we were able to clearly detect 20-HETE in each of >30 individual urine samples analyzed. However, when we used the trimethylsilyl derivative, we were not able to clearly determine 20-HETE because of contaminating peaks. The limit of detection for this assay was <0.2 pg injected on the column (signal-to-noise ratio = 28), and the assay has good intraassay reproducibility (CV = 5%; n = 10) for a urine sample containing 264 pmol/L 20-HETE. The interassay variation for the same urine sample over 10 assays was 10%.
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We examined urinary excretion of 20-HETE in 30 healthy individuals. For the 24-h urine samples (Table 1
; results expressed as either pmol/L or pmol/24 h). All samples were treated with glucuronidase because it has previously been shown that most 20-HETE in urine exists as the glucuronide (6). There is little information available on 20-HETE excretion in normal human urine. Prakash et al. (6) studied four healthy individuals with a mean 20-HETE concentration of 
400 ng/L (1250 pmol/L). Although our mean value was somewhat lower than this, we did have some individuals falling within that range (Table 1
). Sacerdoti et al. (7) studied eight healthy individuals with 20-HETE expressed as the excretion rate (1.6 ng/min). It is difficult to compare these data with our own because these individuals were receiving an infusion of aminohippurate in 50 g/L dextrose in water at 1.5 mL/min for 5 h to determine renal plasma flow.
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Information on the physiologic effects of 20-HETE in humans is limited. Sacerdoti et al. (7) showed that the rate of 20-HETE excretion was increased in individuals with hepatic cirrhosis. Laffer et al. (5) recently showed a positive correlation between diastolic BP and 20-HETE excretion rate in a group of 13 salt-sensitive hypertensive individuals. In some rat models of hypertension, high BP has been associated with increased 20-HETE production (2), but this is yet to be confirmed in human studies.
In conclusion, the sample preparation procedure using solid-phase cartridge extraction and HPLC purification would lend itself to automation and enable the convenient analysis of 20-HETE excretion in large human studies. Such studies could examine in more detail the potential role of this vasoactive CYP450 metabolite in vascular function and human hypertension.
Acknowledgments
This study was funded in part by grants from the National Health and Medical Research Council of Australia (NHMRC; Grant 139067) and NIH Grant GM31278 (to J.R.F.).
References
The following articles in journals at HighWire Press have cited this article:
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  P. Minuz, H. Jiang, C. Fava, L. Turolo, S. Tacconelli, M. Ricci, P. Patrignani, A. Morganti, A. Lechi, and J. C. McGiff Altered Release of Cytochrome P450 Metabolites of Arachidonic Acid in Renovascular Disease Hypertension, May 1, 2008; 51(5): 1379 - 1385. [Abstract] [Full Text] [PDF]
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N. C. Ward, I-J. Tsai, A. Barden, F. M. van Bockxmeer, I. B. Puddey, J. M. Hodgson, and K. D. Croft A Single Nucleotide Polymorphism in the CYP4F2 but not CYP4A11 Gene Is Associated With Increased 20-HETE Excretion and Blood Pressure Hypertension, May 1, 2008; 51(5): 1393 - 1398. [Abstract] [Full Text] [PDF]
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 N. C. Ward, J. Rivera, J. Hodgson, I. B. Puddey, L. J. Beilin, J. R. Falck, and K. D. Croft Urinary 20-Hydroxyeicosatetraenoic Acid Is Associated With Endothelial Dysfunction in Humans Circulation, July 27, 2004; 110(4): 438 - 443. [Abstract] [Full Text] [PDF]
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