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Cardiovascular Research 2006 71(2):322-330; doi:10.1016/j.cardiores.2006.03.005
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Copyright © 2006, European Society of Cardiology

Hydroxylation of D-phenylalanine as a novel approach to detect hydroxyl radicals: Application to cardiac pathophysiology

Roberto Biondia,b,c, Giuseppe Ambrosioa,c,*, Tibaud Liebgottc, Arturo J. Cardounelc, Marco Bettinia, Isabella Trittoa and Jay L. Zweierc

aDepartment of Clinical and Experimental Medicine, University of Perugia School of Medicine, Perugia, Italy
bAzienda Ospedaliera S. Maria, Terni, Italy
cThe Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine and Center for Biomedical EPR Spectroscopy and Imaging, The Ohio State University, Columbus, OH, USA

* Corresponding author. Cardiology, Ospedale Silvestrini, S. Andrea delle Fratte, 06156 Perugia, Italy. Fax: +39 755271244. Email address: giuseppe.ambrosio{at}ospedale.perugia.it

Received 19 October 2005; revised 17 February 2006; accepted 6 March 2006


   Abstract
Top
 Abstract
1. Methods
 2. Results
 3. Discussion
 References
 
Objective Research in the pathophysiology of ischemia/reperfusion or redox signaling is hindered by lack of simple methodology to measure short-lived oxygen radicals. In the presence of hydroxyl radical (•OH), D-phenylalanine (D-Phe) yields para-, meta- and ortho-tyrosine. We have previously demonstrated that D-Phe can accurately detect •OH formation in chemical, enzymatic and cellular systems by simple HPLC methodology [Anal Biochem 290:138;2001]. In the present study, we tested whether D-Phe hydroxylation can be used to detect •OH formation in intact organs.

Methods Rat hearts were perfused with buffer containing 5 mM D-Phe and subjected to 30 min of total global ischemia at 37 °C followed by 45 min of reperfusion. Quantitative analysis of the three hydroxytyrosine isomers was achieved by HPLC-based electrochemical detection of cardiac venous effluent, with the analytical cells operating in the oxidative mode. The detection limit of this assay was <10 fmol.

Results Under baseline conditions, hydroxytyrosine release from the heart was very low ({cong}0.8 nmol/min/g). However, a prominent tyrosine burst occurred immediately upon post-ischemic reflow. In cardiac effluent collected 40 s into reperfusion, the hydroxytyrosine concentration was more than 40 times greater than at baseline; hydroxytyrosine concentration then progressively declined, to return to pre-ischemic values by 5 min of reperfusion. In parallel experiments, formation of hydroxytyrosines was markedly reduced in hearts reperfused in the presence of the •OH scavenger mannitol. Inclusion of 5 mm D-Phe in the perfusion medium altered neither basal cardiac function nor coronary vascular tone, but it enhanced recovery of myocardial function during post-ischemic reperfusion, consistent with direct reaction with •OH.

Conclusion Our results demonstrate that D-Phe is a sensitive method for detection of •OH generation in the heart. Since D-Phe is not a substrate for endogenous enzymes, it can be exploited as a reliable method to measure •OH formation under a variety of pathophysiological conditions.

KEYWORDS Ischemia; Reperfusion; Oxygen radicals; Redox signaling

Oxygen-derived free radicals (OFRs) have long been implicated in the pathophysiology of tissue injury upon ischemia/reperfusion of the heart and other organs (i.e., reperfusion injury [1–3]), as well as in other conditions, including atherosclerosis, hypertension, diabetes and aging [4–8]. More recently, it has been proposed that in small amounts OFRs may also mediate physiological processes through redox signaling pathways [9–12]. Thus, it would be important to investigate OFR generation under various in vivo conditions. However, our understanding of this phenomenon is hindered by lack of suitable methodology. Typically, radicals are detected by electron-paramagnetic resonance (EPR) spectroscopy either of tissue specimens quickly frozen in liquid nitrogen [13–15], or of an organ's venous effluent collected after intra-arterial administration of "spin-traps", i.e., chemicals which react to form more stable radical adducts [14,16–20]. However, both methodologies have significant limitations with respect to their in vivo use. Biopsies are destructive and cannot be performed often enough to provide detailed time course of radical production. As for spin-traps, their in vivo use is limited by the large amounts required, their cost, limited stability of the radical adduct formed, and possible toxic effects [21,22]. Furthermore, both approaches require relatively expensive EPR spectrometers, which further limit widespread application.

Of various oxidant species, hydroxyl radical (•OH) plays a prominent role in biological phenomena [4,5,10–12,23]. •OH undergoes addition reactions with aromatic compounds leading to specific hydroxylated products, which are chemically stable and can be detected by HPLC methodology. Accordingly, high-performance liquid chromatography (HPLC) assay of hydroxylated derivatives of salicylate or L-phenylalanine (L-Phe) has been proposed as an alternative to EPR-based spin-trapping to measure OFRs in vivo [24–29]. However, salicylate may directly inhibit the activity of cycloxygenase and phospholipase [30], two enzymes involved in the pathophysiology of tissue injury and inflammation. Furthermore, concentrations of salicylate required for in vivo measurements may cause cardiotoxicity [31]. Finally, salicylate can be hydroxylated in vivo by cytochrome P-450 enzyme systems [32]. The amino acid L-phenylalanine (L-Phe) is also an efficient substrate for •OH-mediated hydroxylation, with formation of 2-hydroxy-o-tyrosine (o-tyr), 3-hydroxy-m-tyrosine (m-tyr) and 4-hydroxy-phenylalanine (p-tyr), which can be separated and detected by HPLC. As it is devoid of pharmacological effects and it is well tolerated in vivo even at millimolar concentrations [33], HPLC measurement of transcardiac hydroxytyrosines formation after infusion of L-Phe has been successfully used to detect •OH in reperfused hearts [34,35]. However, in vivo L-Phe can give rise to hydroxytyrosines also by enzymatic reaction catalyzed by endogenous L-phenylalanine hydroxylase [36]: this •OH-independent formation of hydroxytyrosines obviously affects the specificity of the measurement. Furthermore, several endogenous pathways can metabolize L-Phe and p-tyr, thus altering their concentration in blood.

The D-form of phenylalanine (D-Phe) shares with its enantiomer L-Phe identical chemical reactivity, but it does not serve as a substrate for L-Phe hydroxylase or other endogenous enzymes and may therefore be a better probe to detect •OH in vivo. We have recently evaluated its reactivity pattern toward •OH in chemical, enzymatic and cellular systems. We found that whereas L-Phe and D-Phe share similar propensity to reaction with •OH and, comparable sensitivity for •OH detection, D-Phe provides much higher specificity due to the lack of enzymatic conversion [37].

In the present study, we expand our previous observations to an intact organ. Here, the capability of D-Phe as a probe for •OH is investigated in a well-characterized model of cardiac OFR generation [13–18,28,29,38–42].


   1. Methods
Top
 Abstract
 1. Methods
2. Results
 3. Discussion
 References
 
1.1 Animals
Male Sprague–Dawley rats (250–300 g) were employed, unless specified differently. Animals were purchased from Charles River (Wilmington, MA and Milan, Italy), and allowed at least 3 days of in-house acclimatization before experimental use, with ad libitum access to standard laboratory food and water. The experimental protocols had been approved by the Institutions' Committees on Animal Welfare. Experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (US National Institutes of Health, NIH Publication 85-23, revised 1996).

1.2 Isolated heart preparation
Under pentobarbital anesthesia and following heparinization, hearts were quickly excised, the aorta cannulated and retrograde perfusion started at 37 °C under constant pressure of 70 mm Hg with non-recirculating filtered Krebs–Henseleit buffer of the following composition (mM): NaCl 118.0, KCl 6.1, CaCl2 3.0, ethylenediamine-tetraacetic acid disodium salt 0.5, MgSO4 1.2, NaHCO3 25.0, NaH2PO4 1.0, glucose 11.1, pH 7.4. The perfusate was equilibrated with 95/5% O2/CO2 gas mixture. Coronary flow was measured using a Transonic HT 107 (Ithaca, NY, USA) flow meter with a 2N in-line flow probe. The hearts were let beat spontaneously. Cardiac contractile function was assessed by means of a saline-filled latex balloon inserted into the left ventricle, with diastolic pressure set at 5–10 mm Hg and connected to a pressure transducer. Heart rate, left ventricular systolic pressure, left ventricular end-diastolic pressure and the first derivative of LV pressure (dp/dt) were continuously recorded by MacLab system (AD Instruments, Colorado Springs, CO, USA). Perfusate draining from coronary sinus was collected at various time points to measure concentration of hydroxytyrosines in the cardiac venous effluent (see below). Hearts were then subjected to one of several different protocols, as outlined below.

1.3 Experimental protocols
1.3.1 Effect of D-Phe on cardiac function
Possible effects of D-Phe on cardiac function were preliminarily investigated in hearts not subjected to ischemia/reperfusion, which were perfused for 45 min with either standard perfusate (n=6), or perfusate to which 5 mM D-Phe had been added (n=6).

1.3.2 Ischemia/reperfusion experiments
After 15 min of equilibration with standard perfusate, in eight hearts perfusion was switched to Krebs–Henseleit buffer containing 5 mM D-Phe; after 10 min, normothermic (37 °C) total global ischemia was induced by completely arresting aortic inflow. At the end of 30-min ischemic period, flow was restored and the hearts reperfused for 45 min with perfusate containing 5 mM D-Phe. Previous experiments had shown that this protocol of ischemia/reperfusion results in major alterations of cardiac function and it is associated with a remarkable production of oxygen radicals as directly measured by EPR methodology [13,40].

Cardiac effluent was collected at baseline just prior to ischemia, then every 20 s during the initial 2 min of reflow, and after 5, 10 and 15 min of reperfusion. The effluent (1 ml aliquots) was dried under vacuum, resuspended with 0.1 ml of HPLC water, centrifuged and the supernatant utilized for measurement of hydroxytyrosines by HPLC (see below). In preliminary experiments, we established that this concentration procedure gives a percent recovery of 95±2, 93±3 and 96±4 for p-tyr, m-tyr and o-tyr, respectively.

1.3.3 Hydroxylation of D-Phe in vitro by Fenton reaction
Hydroxylation of D-Phe by •OH formed via reaction of H2O2 with the ferric iron-nitrilotriacetic acid (NTA) complex was conducted either in 10 mM sodium phosphate, pH=7.4, or in K–H buffer equilibrated with 5% CO2, under various experimental conditions (see Results). The reaction was initiated by adding H2O2 (0.4 mM) to a solution containing 5.0 mM D-Phe and Fe+3/NTA (0.1/0.2 mM). After 1 h of incubation, the reaction was stopped with 1 mM deferoxamine mesylate. The yield of hydroxylation products (p-, m- and o-tyr) was evaluated by HPLC separation (see below).

1.3.4 Effect of mannitol scavenging
Additional hearts were subjected to the ischemia/reperfusion protocol and randomly assigned to either a control group (n=8), or a group, which, in addition to 5 mM D-Phe, also received the •OH scavenger D-mannitol (100 mM) during reperfusion (n=8). Sampling of coronary effluent was as under section 1.3.2.

1.3.5 Validation experiments
A series of experiments were performed to ascertain whether under our conditions hydroxytyrosines (i.e., the product of D-Phe reaction with •OH) remained stable, or whether they were metabolized or chemically modified secondary to possible interactions with either the cardiac effluent or the cardiac perfusion system. Accordingly, the following tests were carried out: (a) in additional hearts, a known amount of an equimolecular mixture of hydroxytyrosines (p-, m- and o-tyr; 10 µM each) was added exogenously to the cardiac effluent as it was being collected, both at baseline and at various time points into post-ischemic reperfusion (see Results); (b) in another group, an equimolar mixture of p-, m- and o-tyr (10 µM each) were directly added to perfusate entering the hearts (see Results); (c) finally, in additional hearts subjected to ischemia and reperfusion, a mixture of hydroxytyrosines was infused into the coronary circulation upon reflow. In all cases, the effluent samples were processed and assayed as per standard procedures.

Another series of experiments was undertaken to ascertain whether D-Phe retained its efficiency as a detector probe when •OH were formed within the cardiac vasculature. Accordingly, hearts were perfused at a constant flow of 15 ml/min with K–H medium equilibrated with 95% O2–5% CO2, at 37 °C. After equilibration, D-Phe (5 mM) was added to the perfusate; Fenton reaction was performed adding Fe3+/NTA 10/20 mM to the perfusate, via a syringe pump connected to the perfusion line through a side cannula, at a flow of 0.15 ml/min, and was started by infusing H2O2 (final concentration 0.4 mM) through another side port. The cardiac effluent was collected, the reaction stopped with 1 mM deferoxamine mesylate and hydroxytyrosine content assayed by HPLC. Parallel experiments were performed employing identical set up and incubation conditions, infusing D-Phe and reagents through the perfusion apparatus but in the absence of hearts.

1.4 HPLC analysis of tyrosines
Analyte separation was conducted on a TOSOHAAS (Montgomeriville, PA) reverse-phase OSD 80-TM C-18 analytical column (46 x 25 cm, 5 µm particle size) using isocratic elution consisting of 30 mM citric acid, 30 mM lithium acetate and 4% methanol, pH 3.1, at flow 1.0 ml/min. Analyte detection was performed on an eight-channel ESA model 5600 CoulArray instrument (Chelmsford, MA); electrode oxidizing potentials were set at channel/potential=1:120 mV, 2:240 mV, 3:350 mV, 4:600 mV, 5:700 mV, 6:750 mV, 7:830 mV, 8:900 mV. Concentrations of the compounds were evaluated by analytical channel 6, which was set at 750 mV, equipped with Coul Array Win software (ESA). Using this technique, the detection limit was <10 fmol for hydroxytyrosines. Reproducibility was assessed in repeated (n=12) runs of aliquots of authentic p-, m- and o-tyr standards, and found to be <2% for each hydroxytyrosine.

1.4.1 Reagents
Methanol (HPLC grade) and HPLC water were obtained from JT Baker (Phillipsburg, NJ). D-Phenylalanine, L-tyrosine, DL-o-tyrosine, DL-m-tyrosine, citric acid, lithium acetate, sodium heparin, sodium pentobarbital, ferric chloride, NTA, H2O2 (30% w/w) and deferoxamine mesylate were from Sigma Chemical Company (St. Louis, MO). All other chemicals were of reagent grade and obtained from standard sources.

1.4.2 Statistical analysis
Data are presented as mean±S.D. Means for each of the measured variables were compared by one-way ANOVA and differences were considered significant at the level P<0.05.


   2. Results
Top
 Abstract
 1. Methods
 2. Results
3. Discussion
 References
 
2.1 Effect of D-Phe on hydroxytyrosines generation in post-ischemic hearts
In preliminary experiments, we ascertained that infusion of 5 mM D-Phe for 45 min has no effect on cardiac function (Table 1).


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  		Table 1 Effect of D-Phe on cardiac function and coronary flow

 
Fig. 1 depicts the resolution of p-, m- and o-tyr in typical HPLC-ECD chromatograms. Separation of authentic hydroxytyrosine standards is shown in panel A, demonstrating that the procedure of concentrating effluent followed by EC detection afforded excellent resolution of peaks. Analysis of samples obtained from cardiac effluent immediately after reflow following 30 min of ischemia (Fig. 1, panel C) revealed three peaks, exhibiting retention times identical to those of authentic standards; the three peaks showed marked differences in amplitude, the peak corresponding to m-tyr being particularly prominent (see also below). In contrast to the findings obtained during very early reperfusion, hydroxytyrosine concentrations were low or negligible in coronary effluent collected either before ischemia (panel B) or after 5 min of reperfusion (panel D).


Figure 1
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  		Fig. 1 Representative HPLC chromatograms of p-, m- and o-tyr (electrochemical detection). (A) Authentic hydroxytyrosine standards (1 µM each) in K–H buffer; (B) cardiac effluent at baseline; (C) cardiac effluent collected at 40-s reperfusion following 30-min ischemia; (D) effluent collected after 5 min of post-ischemic reperfusion.

 
Detailed time course of myocardial production of hydroxytyrosines at baseline (pre-ischemia) and during post-ischemic reperfusion is shown in Fig. 2 for all hearts. Concentration of hydroxytyrosines was very low in the effluent of pre-ischemic hearts, as values averaged 0.21±0.02 nmol/min/g, 0.47±0.12 nmol/min/g and 0.14±0.06 nmol/min/g for p-, m- and o-tyr, respectively. However, immediately after reperfusion, a prominent increase in hydroxytyrosines release was observed in the cardiac effluent, which peaked at 40-s reflow (Fig. 2). Cardiac release of hydroxytyrosines then declined, returning to baseline values by 5 min of reperfusion. It should be noted that, on a qualitative basis, there were no differences among the three hydroxytyrosines isomers, as they were formed and released according to a time course identical for all three species (Fig. 2). However, differences were evident concerning the absolute amount of each isomer produced during reperfusion, with m-tyr representing the most abundant species (Figs. 1 and 2Go). This issue was further addressed in specific experiments (see below).


Figure 2
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  		Fig. 2 Time course of myocardial release of p-, m- and o-tyr in the cardiac effluent at baseline and at various time points during reperfusion after 30-min ischemia. Data are mean±S.E.; n=8; *P<0.05 vs. pre-ischemic value.

 
2.2 Analysis of tyrosine isomers obtained in vitro by Fenton •OH generating system
The effect of exposing D-Phe in vitro to a known source of •OH was investigated under various experimental conditions. Under any condition, we observed appearance in the chromatogram of three distinct peaks, the retention time of which was the same in the various conditions, and identical to that of authentic p-, m- and o-tyr standards (not shown). Interestingly, the relative proportion of each hydroxytyrosine isomer was affected by the medium used to carry out the reaction. In phosphate buffer, oxidation of D-Phe (Fig. 3, open bars) yielded equimolar amount of the three hydroxytyrosine isomers, consistent with our own earlier observations [37]; this occurred regardless of changes in oxygen tension (Fig. 3, open bars) or temperature (not shown). In contrast, greater formation of m-tyr (75–80%) than either p-tyr (15–17%) and o-tyr (9–10%) was consistently observed when D-Phe oxidation was carried out in bicarbonate/CO2 buffer (Fig. 3, closed bars), irrespective of oxygen tension (Fig. 3, closed bars) or temperature (not shown). Importantly, this difference in relative yield of hydroxytyrosines was entirely due to a shift among isomers, not to differences in the absolute amount of hydroxytyrosine formed, which was unaffected.


Figure 3
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  		Fig. 3 Formation of p-, m- and o-tyr by hydroxylation of D-Phe in H2O2-driven •OH generating system at 37 °C either in 10 mM phosphate buffer, pH 7.4, equilibrated with ambient air or 100% O2 (open bars), or in K–H buffer pH 7.4, equilibrated with 95% N2–5% CO2 or 95% O2–5% CO2 (closed bars). Data are mean±S.E. of six experiments.

 
2.3 Effect of •OH scavenging on hydroxytyrosine formation in post-ischemic hearts
Having established that D-Phe infusion during ischemia/reperfusion is associated with cardiac release of hydroxylated derivatives identical to those formed when D-Phe is exposed in vitro to a known source of •OH: in subsequent experiments, we evaluated the effect of scavenging •OH on cardiac release of hydroxytyrosines in post-ischemic hearts (Fig. 4). Addition of the •OH scavenger D-mannitol (100 mM) to perfusate entering the heart resulted in marked reduction in the release of all three tyr isomers, compared with control ischemic/reperfused hearts receiving only standard perfusate. Decreased hydroxytyrosines concentrations were consistently observed throughout reperfusion, as values in mannitol-treated hearts at each time point were always about half or less of corresponding concentrations in controls (Fig. 4). The effect of mannitol scavenging was identical for all three hydroxytyrosines (data not shown). Fig. 4 reports the sum of all three species, for the sake of simplicity (i.e., total hydroxytyrosines release).


Figure 4
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  		Fig. 4 Effect of reperfusion with 100 mM D-mannitol on tyrosines release in cardiac effluent at 20, 40, 60 and 80 s of reperfusion after 30 min of total global ischemia at 37 °C. Data are mean±S.E.; n=8. *P<0.05 vs. control experiments.

 
2.4 Effect of D-Phe perfusion on post-ischemic functional recovery
In reacting with •OH, D-Phe scavenges this highly reactive radical. Thus, one should predict that infusion of D-Phe during post-ischemic reperfusion may also result in cardiac protection, secondary to •OH scavenging [34]. The effect of 5 mM D-Phe on post-ischemic functional recovery after 45-min reperfusion is summarized in Table 2. Recovery of cardiac function was significantly improved in the group perfused with 5 mM D-Phe, compared to the control group of hearts perfused with KH buffer alone (Table 2).


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  		Table 2 Influence of 5 mM D-Phe on functional recovery at 45 min of reperfusion in hearts subjected to 30 min of ischemia

 
2.5 Effect of cardiac perfusion on hydroxytyrosines yield
Additional experiments were performed to further assess the reliability of D-Phe as a •OH probe. We could already rule out concerns based upon possible enzymatic modification, since the substrates for endogenous enzymes are the L-derivatives of Phe, whereas we employed the D-isomer. Yet, it remained to investigate whether our experimental conditions might affect the yield of hydroxytyrosines formation, due to non-enzymatic reactions or physical interaction with the system.

Table 3 shows that, when a known amount of a mixture of authentic hydroxytyrosines (p-, m- and o-tyr) was exogenously added to cardiac effluent as soon as it was being collected, either before ischemia or at different time points into reperfusion, recovery of each isomer was not affected. This finding rules out chemical modification of hydroxytyrosines upon reaction with other substances released in the cardiac effluent.


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  		Table 3 Effect of incubation with heart perfusate on relative tyr isomers

 
In another set of experiments, we evaluated whether the total yield and/or the relative distribution among the three tyr isomers might be affected via interactions between hydroxytyrosines and the heart itself, or any components of the perfusion system. Table 4 shows that hydroxytyrosines infused into normal hearts were recovered unaffected in the coronary effluent; similar findings were observed when hydroxytyrosines were infused into post-ischemic hearts (Table 4).


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  		Table 4 Recovery of hydroxytyrosines in the cardiac effluent after intracoronary injection

 
Finally, additional experiments aimed at ascertaining whether D-Phe retained its efficiency as a detector probe for •OH when radicals were formed within the cardiac vasculature. Formation of hydroxytyrosines from D-Phe exposed to a source of •OH that was being infused intracoronary was equivalent to that obtained when D-Phe was exposed to identical oxidant conditions in the perfusion apparatus but in the absence of the heart (Table 5).


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  		Table 5 Effect of perfusing rat hearts on D-Phe oxidation by Fenton chemistry

 
Collectively, these various findings rule against any metabolism/interaction occurring between hydroxytyrosines formed upon •OH reaction and the heart and/or the perfusion apparatus.


   3. Discussion
Top
 Abstract
 1. Methods
 2. Results
 3. Discussion
References
 
Oxygen radicals are important mediators of cell injury and of various physiological processes [1–12]. Among oxygen radicals, the hydroxyl radical •OH is by far the most reactive and hence the most relevant to pathophysiology. To better understand the biological role of •OH, specific assays are required to accurately detect •OH formation in vivo. However, due to its reactivity (half-life{cong}10–9 s), direct measurement of •OH in cells and tissues is not possible. Currently, •OH detection is based on electron-paramagnetic resonance (EPR) analysis of the product of its reaction with "spin-traps", to form more stable adducts. EPR spin-trapping has enabled measurement of •OH in chemical systems, cultured cells and isolated organs. In vivo studies have also been successfully performed [19,20,43]; however, concerns over possible toxic effects of the traps as well as instability of the adducts limit the applications of this technique [21,22,44]. EPR spin-trapping also requires relatively expensive instrumentation and these reagents can be prohibitively expensive in the amounts required for in vivo studies of large animals. Therefore, there is a need for assays that enable convenient and reliable measurement of •OH in vivo.

The capability of •OH to react with aromatic compounds, yielding characteristic hydroxylated products that can be simply assayed by HPLC, provides another means to assess •OH production. Hydroxylation of salicylate, or of L-Phe, has been employed to measure •OH in a variety of systems [24–29,33–35]. However, both probes have major limitations. Salicylate may exert unwanted pharmacological effects; more importantly, both salicylate and L-Phe can be hydroxylated in vivo by endogenous enzyme systems, leading to formation of hydroxylated products unrelated to •OH production [32,36]. In addition, L-Phe and its hydroxy-derivative p-tyrosine are naturally occurring amino acids; therefore, on the one hand, they are already present in vivo; on the other hand, they can undergo metabolism by a variety of endogenous pathways. As a result, the effective amount of L-Phe present, as well as that of the hydroxy-derivatives formed, cannot be precisely known, thus affecting estimation of the actual amount of •OH formed during the conditions under investigation. To circumvent these problems, we have recently documented that D-Phe (the enantiomer of L-Phe) may represent a more accurate probe for •OH detection [37]. In fact, while D-Phe and L-Phe have the same propensity for reaction with •OH and identical sensitivities for •OH detection, D-Phe is not a substrate for endogenous enzymes and, therefore, it can achieve much more specific detection of •OH in biological systems [37].

In the present study, we show that •OH generation in post-ischemic hearts can be investigated by intracoronary D-Phe infusion and subsequent HPLC analysis of cardiac venous effluent. We chose this model since cardiac post-ischemic reperfusion is a condition previously documented by EPR methodology to be associated with prominent oxygen radical generation, the characteristics of which have been largely investigated [13–18,28,29,38–42]. Our results demonstrate that reperfusion following 30 min of ischemia is associated with a burst of hydroxytyrosines release occurring early upon reflow, followed by rapid decline and complete cessation within a few minutes of reperfusion, consistent with the findings of previous studies in this experimental model [13–18,28,29,39,40]. Several lines of evidence concur to demonstrate that hydroxylated species recovered in the effluent from post-ischemic hearts are indeed hydroxytyrosines produced upon interaction of D-Phe with •OH formed during reperfusion. (1) The HPLC characteristics of the products appearing in cardiac effluent are identical to those of authentic hydroxytyrosine standards, and (2) to hydroxytyrosines formed in vitro upon reaction of D-Phe with a known source of •OH. (3) The time course of hydroxytyrosine formation and release in the cardiac effluent closely matches data from previous studies in which •OH production in hearts subjected to ischemia and reperfusion was documented by EPR techniques [13–18,39,40]. (4) Assay of D-Phe hydroxylation products in hearts treated with mannitol demonstrated the expected reduction in hydroxytyrosine release secondary to reduced •OH concentration by mannitol scavenging. (5) Finally, although D-Phe infusion did not influence function of normal hearts, it did improve functional recovery when administered to post-ischemic hearts, consistent with direct chemical interaction with (and, hence, inactivation of) •OH [34]. Indeed, D-Phe seems to be substantially more efficient than HPLC assays in which salicylate was used as a probe for •OH, as dihydroxybenzoic acids (the product of •OH reaction with salicylate) in cardiac effluent during post-ischemic reperfusion were detected in the f molar to picomolar range [28,29], much lower than our study.

All three hydroxytyrosines species were found in the effluent from ischemic hearts during early reperfusion period, but with large differences in relative distribution of each isomer (m-tyr being the predominant species formed); these differences in the release were observed throughout tyr production. This finding apparently contrasts with our previous report that the three hydroxytyrosines were produced in equimolar amounts when D-Phe was exposed to •OH formed by either chemical (Fenton or hypoxanthine–xanthine oxidase reaction) or biological systems (activated human neutrophils [37]). However, preferential formation of m-tyr was shown to be a characteristic of the specific experimental conditions and it does not affect the reliability of the assay, since: (a) we present data demonstrating that the unbalanced yield of the three hydroxytyrosine isomers found in perfused hearts is due solely to the presence of bicarbonate/CO2 in the perfusate (Fig. 3); of note, this occurs in the absence of changes in total hydroxytyrosines production; (b) the time course of hydroxytyrosines formation during reperfusion was identical for all three species; (c) •OH scavenging by mannitol influenced each hydroxytyrosine in a similar fashion; (d) extensive validation experiments demonstrate that neither the yield nor the recovery of any isomer is affected under our experimental conditions and, therefore, there appear to be no preferential recovery of one species over the others. Finally, preliminary experiments (not shown) indicated that all three hydroxytyrosines are formed when testing D-Phe added to human plasma in presence of defined amounts of H2O2.

Because of its short half-life, •OH undergoes rapid inactivation/reaction within cells. Thus, although D-Phe and D-tyrs may to some extent diffuse across cell membrane, our assay mostly detects •OH formed in the vascular lumen or its vicinity. This limitation, however, is shared by all methodologies that rely on infusion of a water-soluble chemical to detect oxygen radicals, either by HPLC (L-Phe, salicylate) or EPR (DMPO spin-trapping).

In conclusion, hydroxytyrosine formation after administration of D-Phe can reliably document cardiac •OH formation. Lack of toxicity, absence of non-specific reaction and ease of detection may make HPLC analysis of D-Phe hydroxylation a widely applicable methodology to investigate •OH formation in vivo.


   Acknowledgements
 
This work was supported by National Institutes of Health Grants HL63744, HL65608 and HL38324, and grants #RBNE01HLAK_005 and #2003064224_008 from MIUR, Italy.


   Notes
 
Time for primary review 25 days


   References
Top
 Abstract
 1. Methods
 2. Results
 3. Discussion
 References
 

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