Aromatic Hydroxylation

Abstract

With the recognition that the imbalance between oxidant and antioxidant compounds is an important causative agent of aging and of various chronic diseases (1), there has been growing interest in elucidating the pathways involved in oxidative-stress initiation. As oxidative stress is an inescapable repercussion of aerobic life, all organisms that use oxygen protect themselves against free radicals (1). Although cells are endowed with impressive equipment of antioxidant defenses (2), they are not completely efficient to resist an increased free-radical formation.

1 Introduction

With the recognition that the imbalance between oxidant and antioxidant compounds is an important causative agent of aging and of various chronic diseases (1), there has been growing interest in elucidating the pathways involved in oxidative-stress initiation. As oxidative stress is an inescapable repercussion of aerobic life, all organisms that use oxygen protect themselves against free radicals (1). Although cells are endowed with impressive equipment of antioxidant defenses (2), they are not completely efficient to resist an increased free-radical formation.

Because of the high reactivity of oxygen free radicals and the lack of specific and sensitive methodologies for their measurements, the assessment of oxidative stress in living systems in vitro and in vivo has been difficult. In particular, hydroxyl radical (OH^•) has a very short half-life, and it is merefore present in extremely low concentrations.

Aromatic hydroxylation (3) is one of the most specific methods available for its measure in biological systems and it has been largely used.

The method is based on the ability of OH^• to attack the benzene rings of aromatic molecules and produce hydroxylated compounds that may be directly and specifically measured by electrochemical detection. Formation of o-tyrosine and m-tyrosine from the OH^• attack on phenylalanine has been used to study the production of OH^• by isolated cells (4) and by human saliva (5).

Salicylate (2-hydroxybenzoate) was largely used as a marker of hydroxyl free-radical production both in vitro (6,7) and in vivo (8–10). The success of salicylate as a probe of OH^• production is mainly owing to three factors:

• 1. It has a high reaction rate constant with OH^• (5×10⁹/M/s) (11);

• 2. It may be used at concentrations sufficient to compete with other possibly present scavenger molecules,

• 3. Its hydroxylated products are rather stable and some of these are not metabolically produced (see Fig. 1 ). In fact, upon salicylate, the attack of OH^• produced by different systems such as Fenton reactions and γ-radiolysis, give rise to a set of hydroxylated by-products (2,3-dihydroxybenzoate, 2,5-dihydroxybenzoate, and catechol) (12–14)

Microsomal fractions from mammals treated with inducers of cytochrome P-450, produce 2,5-dihydroxybenzoate (2,5-dHB), but not the 2,3 isomer (2,3-dHB) (15); catechol is only a minor product (14), so that 2,3-dHB appears to be the only reliable marker of OH^• production in vivo.

Salicylates are analgesics that are used in the treatment of a variety of pain syndromes. Aspirin is commonly used for the relief of headaches, myalgia, and arthralgia. Once ingested, it is quickly hydrolyzed to salicylate by esterases in the gastrointestinal tract, in the liver, and to a smaller extent in the serum (16); salicylate reaches its peak in plasma about 1.5 h after the oral intake of aspirin (17) and is in part metabolized by conjugation with glycine (liver glycine N-acetylase) (18), by conjugation with glucuronic acid to form salicyl acyl and salicylphenolic glucuronide (19), and by hydroxylation (liver microsomalhydroxylases) to form 2,5-dihydroxybenzoic acid (15). About 60% of the salicylate remains unmodified (19) and can undergo OH^• attack ( Fig. 1 ).

The aim of this chapter is to provide a series of validated procedures for detecting OH^• production in different models, in vitro and in vivo.

We used salicylate hydroxylation both in simple biochemical systems for assessing the antioxidant capacity of selected compounds, and in cultured cells for evaluating the activity of different antioxidants and to study the mechanisms involved in cell OH^• production. Moreover, the versatility of the technique suggests its application also in humans, for evaluating the oxidative stress status both in pathology and in physiology.

2 Materials

2.1 Instruments and Column

2.1.1 High-Pressure Liquid Chromatography (HPLC) System

• 1. Reverse-phase HPLC analysis is carried out on a Supelcosil™ LC-18,5 μM (250 × 4 6 mm) analytical column, using a Perkin Elmer series 410 LC pump equipped with a Perkin Elmer SEC-4 solvent environmental control.

• 2. Perkin Elmer LC-95 UV/Vis Spectrophotometer detector: Set at 300 nm, 0.005 AUFS recorder range.

• 3. BAS LC-4B amperometric detector (Bio-Analytical Systems, West Lafayette, IN): Equipped with a BAS detector cell The detector potential is set at +0.76 V versus an Ag/AgCl reference electrode.

• 4. ESA (Bedford, MA) Coulochem II detector: Equipped with a conditioning cell (Model 5021) followed by a 2011 analytical cell.

Fig. 1

Diagram showing the main pathways involved in salicylate metabolism In in vivo studies, this compound may be administered in different forms: aspirin, sodium, or choline salicylate, all releasing salicylate after hydrolysis.

2.2 Reagents

All reagents should be of the highest chromatographic quality to avoid interferences and to ensure reproducibility, and all solutions are made using Milli-Q (Millipore, Bedford, MA) double distilled water (resistance > 18mΩ/cm²).

• 1. Salicylic acid and dihydroxybenzoate isomers (dHBs) (Aldrich, Steinheim, Germany).

• 2. HPLC-grade solvents (Carlo Erba, Milano, Italy).

• 3. All other chemicals are purchased from Sigma (St. Louis, MO)

• 4. The mobile phase (10 mL/min flow) consists in 95% (v/v) 30 mM sodium cit-rate/27 7 mM acetate buffer and 5% (v/v) methanol, pH 4.75. The solution is filtered through a 0.22 μM pore size Milhpore filter and continuously sparged with He gas during elution. The injection loop volume is 100 μL.

3 Methods

3.1 In Vitro Evaluation of the Scavenging Capacity of Antioxidants

• 1. Salicylate stock solutions (100 mM), freshly made in methanol are kept on ice until use. Single antioxidants or mixtures to be analyzed are incubated in 10 mM phosphate buffer, pH 7.00, containing 2.0 mM salicylate in polypropylene tubes at 37°C (see Note 1 )

• 2. A flux of OH^• is commonly generated by Fenton’s reagent consisting of 100 μM FeCl₃, 104 μM EDTA, 100 μM ascorbate and 1.0 mM H₂O₂ Iron-EDTA complex is obtained by pre-mixing stock solutions, reaction is started by ascorbate addition (7)

• 3. After 15 min incubation, the tubes were transferred to an ice-bath and 200 μM deferoxamine and 2.0 mM dimethyl sulfoxide (DMSO) were added Samples are then extracted into diethyl ether (see Subheading 3.4.1.). Under these conditions the attack of OH^• on salicylate produces 2,3 and 2,5-dHB to a similar extent (see Note 1 ). In the absence of enzymatic systems metabolizing salicylate, the sum of the two dHBs may be used to obtain a measure of the OH^• that has escaped from the antioxidant scavenging. Following this protocol, the anti-hydroxyl radical capacity of single compounds or antioxidant mixtures may be measured. The results may be expressed as the percentage of OH^• trapped, in comparison with the “blank” or standardized by a well-known OH^• scavenger and expressed as mM equivalents of it, thiourea, having a reaction-rate constant with OH^• similar to that of salicylate (11) is often used for this purpose.

3.2 Evaluation of OH^• Production by Stimulated Cells

To investigate the capacity of human neutrophils to make OH^• during the “respiratory burst,” Halhwell et al. (19) used phenylalanine hydroxylation and similar experiments have been conducted with salicylate (20).

We studied the production of OH^• by platelets stimulated to aggregate with arachidonic acid. Blood samples were taken from healthy volunteers who had not ingested salicylate-containing drugs; blood was collected in BD Vacutainer containing 0.13M Na-citrate (ratio 9:1). Platelet-rich plasma was separated by centrifugation at 100g and platelets were washed three times in phosphate-buffered saline (PBS) as described more in detail elsewhere (21). Platelets were suspended to a final count of 5×10⁸/mL in PBS containing 1.5 mg/mL fibrinogen and pre-incubated at 37°C for 5 min with 2.0 mM salicylate. Arachidonic acid (10–50 μM) was used as aggregating agent, the reaction allowed for 10 min, then stopped by adding cold 1.0 vol. of 20% trichloroacetic acid. The supernatant was extracted into diethyl ether (see Subheading 3.4.1.). Also in this system, the rate of dHB hydroxylation is similar for the 2,3- and the 2,5 isomer, thus their sum may be used.

3.3 Evaluation of OH^• Production in Humans

The investigation of the rate of in vivo oxidative stress in humans certainly represents the most engaging application of salicylate hydroxylation.

• 1. We evaluated the in vivo OH^• production in two groups of subjects, who were presumably exposed to a high rate of oxidative stress, diabetic patients (8) and heavy smokers (manuscript in preparation) Diabetics were 20 subjects suffering from insulin-dependent diabetes mellitus in good metabolic control. Smokers (24 healthy subjects, who smoked more than 250 mg tar/d) were studied before and after receiving a daily antioxidant supplementation consisting of ascorbic acid, tocoferol and b-carotene (1000, 600, and 25 mg, respectively) for 28 d (see Note 2 ).

• 2. All subjects suffering from renal and hepatic failure, asthma, bleeding disorders, and peptic diseases were excluded. Each fasting subject took 1.0 g aspirin (Flectadol 1000®, Maggioni, Italy) by mouth and had a breakfast (150 mL whole milk and 50 g carbohydrates) to avoid gastric distress; 3 h after the aspirin intake a blood sample was withdrawn into heparinized BD-Vacutainer and immediately centrifuged at 2000g for 10 min at +4°C.

• 3. Portions of plasma were extracted for benzoates as described below (see Subheading 3.4.1.).

3.4 Assay Conditions

• 1. Extraction Procedures: To 500 μL of sample to be extracted, 25 μL 1.0 N HCl and 50 nM final concentration 3,4-dHB as internal standard are added with mixing. Samples pre-treated with trichloroacetic acid (platelets) are only added with the internal standard. The resulting solution is twice extracted into 50 mL of HPLC-grade diethyl ether by thoroughly mixing for 2 min, and subsequently centrifuged for 1 min at 1000g to separate the phases. The organic phases are joined, and evaporated to dryness under nitrogen

• 2. Owing to the instability of benzoates in biological fluids (13), samples should be extracted as soon as possible. Evaporated samples may be stored at −80°C until the analysis.

• 3. Just before the injection into the HPLC system, evaporated samples are reconstituted to 10 mL with mobile phase added with 20 mM DMSO and 200 μM deferoxamine. These two compounds do not interfere with the chromatographic separation and detection, and by avoiding autooxidation of salicylate and dHBs, ensure accurate and reproducible results.

3.5 Choice of Working Conditions

• 1. Electrochemical detectors provide significant benefits over other means. There are two approaches to electrochemical detection for HPLC amperometry and coulometry. Amperometric detectors, such as BAS LC-4B are designed so that the eluent flows by the electrode surface The fraction of the electroactive compound that reacts is about 10%. On the contrary, coulometric detectors are made so that the eluent flows trough a porous electrode and all the electroactive compounds react. Hence the sensitivity of coulometric detectors is 10 (and more) times higher than that of amperometric detectors. This may be observed in Fig. 2 , where the hydrodynamic voltammograms relative to the dHB isomers are made with amperometric and coulometric detector. Hydrodynamic voltammetry represents not only the fingerprint of a selected molecule, but it is also used to determine the potential to be applied to the analysis Coulometric detection provides important advantage respect to amperometry. A sensitivity at least 20 times higher than amperometry, with a significant rise of the signal-to-noise ratio is obtained. According to the results of Fig. 2 , BAS LC-4B detector was set at 0.76 V in oxidation mode (the potential giving the highest signal-to-noise ratio). To minimize the noise, mobile phase may be prepared the d before the analysis and recycled by placing the outlet tube from the detector into the solvent reservoir. This procedure gives an increase in signal-to-noise ratio without affecting HPLC separation (methanol evaporation is negligible). As ESA Coulochem II oxidizes 100% of analytes the recycle of the mobile phase may persist during working procedures and does not produce interferences with the analysis. Moreover the high voltage necessary to produce detectable peaks in amperometry may shorten the lifetime of electrodes.

• 2. ESA Coulochem II settings were the following. Conditioning cell and the first electrode of the analytical cell −0.1 V; the second electrode was the analytical one and was set at +0.3 V

• 3. The retention times expressed in min, relative to dHBs and salicylate, are 6.8±0 6 for 2,3-dHB, 7.7±0.5 for 2,5-dHB, 9.8±0.7 for 3,4-dHB, and 23.5±1.0 for salicylate. If salicylate values are considered unessential for the experimental protocol, the chromatographic assay is complete within 12 min.

• 4. Salicylate is not electrochemically active in the conditions described and it needs higher voltage to be detected (1.0 V). Owing to its high concentrations both in vitro (20 mM) and in plasma samples (about 400 mM) it may easily be detected using an ultraviolet (UV) detector set at 300 nm. Figure 3 reports a typical chromatographic run

Fig. 2

Hydrodynamic voltammometry of all di-hydroxybenzoate isomers (A) Amperometric detector, 50 pmol injected. Full-scale current was set at 10 nA (B) Coulometric detector, 2.5 pmol injected. Full-scale current was set at 100 nA The dotted lines show the potential used for analytical runs (o), 2.3-dHB, (•), 2.5-dHB, (X), 3.4-dHB.

3.6 Experimental Results

The results of the analytical procedures are reported in Subheadings 3.5.1.–3.5.4. Here are reported the main results obtained by employing salicylate hydroxylation in proposed expenmental protocols for measuring in vitro and in vivo OH^• production (see Methods).

Fig. 3.

HPLC-separation of dHB isomers (EC Coulometric detector) and salicylate (UV detector) in human plasma sample of a healthy subject after a single dose of 1.0 g Aspirin

3.6.1 OH^• Production by Stimulated Platelets

A dose-dependent production of hydroxyl radicals by platelets stimulated to aggregate with increasing amounts of arachidonic acid is shown in Fig. 4 . Salicylic acid at this concentration does not affect cyclooxygenase pathway (21), consequently it does not interfere with platelet aggregation.

The amount of OH^• produced by platelets under these conditions probably originates by Fenton-like reactions triggered by the disruption of cell membranes with subsequent release of metal ions. The production of OH^• by platelets would be necessary for aggregation itself (22,23), and could contribute to the understanding of the relationship between platelets and endothelial damage in atherosclerosis (see Note 3 ).

Fig. 4.

OH^• production by platelets stimulated to aggregate with increasing amounts of arachidonic acid. Values represent the sum of 2,3 and 2,5-dHB

3.6.2 Assessment of Oxidative Stress in Humans

The results obtained by our in vivo studies are reported in Table 1 . For the reasons previously discussed, when the experimental protocols are applied in vivo, only 2,3-dHB can be used as marker of OH^• production; the 2,5 isomer is produced by enzymatic pathways.

Diabetic patients (in the left part of the table), even in good metabolic control, are exposed to an hyperproduction of OH^• and at that time, the increase in salicylate hydroxylation is the sole sign that they are exposed to oxidative stress; this observed oxidative-stress status could help to explain why these patients will undergo late complications. Salicylate hydroxylation reveals, therefore, that a certain amount of OH^• is eluding antioxidant defenses before that the phenomenon becomes evident, and produces angiopathy, neuropathy, or cataract.

In contrast heavy smokers (the right hand of the table) did not show any significant difference in comparison with controls, although they showed a trend toward higher values. After 28 d of antioxidant supplementation, however, they showed significantly reduced values of plasma 2,3-dHB. Probably their rate of OH^• production did not change, but the increased antioxidant defenses may have trapped a higher amount of radicals Controls did not show any significant change (49±6 nmol/l) after antioxidant supplementation.

4 Notes

• 1. The protection of salicylate and dHBs against auto-oxidation during the extraction and analysis times is extremely important in that unexpected oxidation may occur during these phases giving false results. An antioxidant cocktail containing

  Table 1 Values of Hydroxyl Radical Production in Humans Exposed to Two Different Sources of Oxidative Stress ^a

  deferoxamine and DMSO, as previously described, is sufficient in our assay condition to minimize salicylate oxidation. All solution should be made in metal ion-freed distilled water.

• 2. Plasma-salicylate concentration in humans who ingested 1.0 g aspirin ranges from 200–500 μM Other salicylate salts (see Fig. 1 ) can be administered in higher doses (Na-salicylate or choline salicylate, Arthropan®); they are able to reach higher plasma concentrations, thus trapping a larger amount of OH^•.

• 3. Salicylate salts, moreover, unlike aspirin, do not inhibit platelet function (21). Because platelets are able to generate OH^• ( Fig. 4 ) through the cyclooxygenase pathway, their inactivation could give an underestimate of the real amount of OH^• produced, especially in patients suffering from degenerative diseases that often are coupled with platelet hyperfunction. The administration of sodium or choline salicylate could alleviate this concern.