Free Radicals in Chemical Biology: from Chemical Behavior to Biomarker Development

1. Synthesis of Mono-trans Isomers of Cholesteryl Esters

1. Dissolve cholesteryl esters (linoleate or arachidonate cholesteryl ester) in 2-propanol (15 mM) . For a better solubilization, the samples were sonicated for 15 min under argon.
2. Transfer the solution to a quartz photochemical reactor, add 2-mercaptoethanol in 2-propanol (to reach 7 mM concentration, from a 2 M stock solution of the thiol). Flush the reaction mixture with argon for 20 min in order to eliminate the presence of oxygen in the solution.
3. Irradiate the reaction mixture by UV light using a 5.5W low-pressure mercury lamp at 22±2 °C for 4 min. Monitor by analytical Ag-TLC (silver-thin layer chromatography) to evidence the formation of the mono-trans cholesteryl esters (see Figure 1 for the chemical formulas). The TLC staining is carried out by pouring the plate in the cerium ammonium molibdate (CAM) solution, and the spots appear on heating the plate. Quench the reaction at an early stage, in order to recover the starting material, that can be reused to perform other rounds of isomerization, obtaining an increase of the overall yield.
4. Collect the reaction mixture in a round-bottom flask, washing the apparatus with a few ml of 2-propanol. Remove solvent and purify the mono-trans isomers of cholesteryl esters by Ag-TLC as described in the literature.⁵ Use hexane-diethyl ether (9:1 v/v) as the eluent for mono-trans cholesteryl linoleate isomers, whereas use hexane-diethyl ether-acetic acid (9:1:0,1 v/v) for mono-trans cholesteryl arachidonate isomers.

2. Isolation of Cholesteryl Esters Fraction from Human Serum

1. Dilute 1 ml of human serum (obtained by centrifugation of blood) with 1 ml of brine and pour the solution into a separatory funnel under a stream of argon in order to avoid artifacts (e.g. oxidation adducts). Add 10 ml chloroform-methanol (2:1 v/v) thrice. Shake the separatory funnel very slowly in order to limit the formation of the emulsion due to the presence of albumin.
2. Wash the organic layers once with brine (10 ml), then collect in an Erlenmeyer flask under an argon flow, dry over anhydrous sodium sulphate. Remove volatiles by rotary evaporator to afford a yellow oil (total plasma lipid fraction).
3. Take up the crude with 1 ml of chloroform-methanol (2:1 v/v), load onto a preparative TLC under a stream of argon and use a mixture of hexane-diethyl ether (9:1 v/v) as the eluent. Scrape off the silica portion containing the cholesteryl ester fraction and pour into a vial. Then, extract silica (3 × 5 ml) with chloroform-methanol (2:1 v/v), collect the organic layers and remove the solvent after evaporation to afford the pure fraction of cholesteryl esters (usually ~1.5 mg).
4. Keep cholesteryl esters in a dark vial covered by aluminum foil under Argon and store at -20 °C. Cholesteryl esters are sensitive to light and oxygen.

3. Characterization of Mono-trans Cholesteryl Esters by Raman Spectroscopy

1. Extract cholesteryl esters from human serum (≥0.7 mg) as described in section 2, dissolve in a small amount of carbon tetrachloride (≤10 μl) and place in a vial. CCl₄ is selected as the solvent because its Raman signal does not overlap with the region of interest of cholesteryl ester analysis.
2. Transfer the solution to the sample holder through a disposable glass pipette, then carefully remove the solvent by a slow stream of argon. Once a uniform oily film is formed on the internal wall of the sample holder, place the latter into the instrument for measurement.
3. Fourier Transformed Raman spectrum of cholesteryl esters is directly obtained on the lipid extracts without any derivatization reaction. The laser power on the sample is <100 mW to avoid sample damage. The total number of scans for each spectrum is ≥800 to minimize the background noise.
4. The 1,700-1,630 cm^-1 range of the Raman spectrum is analysed since it reflects the contribution from the C=C double bonds present in the molecule (C=C stretching mode). A curve fitting analysis is performed on this region, thus allowing the distinguishing of the superimposed contributions from cis and trans double bonds in the fatty alkyl chain and double bond in the ring B of cholesterol (see Figure 2).
5. To fit correctly the vibrational bands, some parameters should be fixed or constrained within reasonable limits. The component peak profiles are described as a linear combination of Lorentzian and Gaussian functions, whereas the half-height bandwidths are best determined by the computer optimisation routine. The number and position of the component peaks are obtained by using the fourth derivative spectra, which allow to detect also some weak component bands. Since the signal to noise ratio which deteriorates upon differentiation of the signal can preclude the use of the fourth derivative, smoothed fourth derivatives with a thirteen-point Savitsky-Golay function, are of advantage; thus, a right compromise between resolution and signal to noise ratio is obtained. Best curve fitting are obtained at the lowest possible c² values.
6. The presence of trans isomers is revealed by the component at 1671±1 cm^-1, in addition to, at least, the two components, slightly shifted towards lower frequencies, due to the fatty acid chain and cholesterol C=C double bonds. Representative Raman spectrum of plasma cholesteryl ester is shown in Figure 9. In the inset the comparison of a specific region with cholesteryl linoleate and mono-trans cholesteryl linoleic acid isomers is shown.

4. Derivatization to Fatty Acid Methyl Esters (FAME) and Analysis by Gas Chromatography

1. Dissolve cholesteryl esters (~1.5 mg) with 0.5 ml of a 1 M solution of sodium hydroxide in benzene-methanol (2:3 v/v) and place in a dark vial covered by aluminum foil under argon.
2. Leave the mixture stirring at room temperature and monitor by TLC using a mixture of hexane-diethyl ether (9:1 v/v) as the eluent. Reaction time is usually 30 min but a careful reaction monitoring is required. In fact, once the corresponding methyl esters are formed, the reaction must be quenched in order to avoid the formation of degradation and side products. TLC showed the quantitative formation of methyl esters.
3. Quench the reaction mixture by adding 1 ml of brine and extract methyl esters with n-hexane (3 × 2 ml). Collect the organic layers into a vial and remove the solvent by rotary evaporation to afford the methyl ester fraction, which is then used for GC analysis without further purification.
4. Dissolve the methyl esters in the minimum quantity of n-hexane (usually 1 mg in 50 μl) and inject in the GC equipped with a 60 m × 0.25 mm × 0.25 μm (50%-cyanopropyl)-methylpolysiloxane column. Use a flame ionization detector (FID) with the following oven program: temperature start at 165 °C, hold for 3 min, followed by increase of 1 °C/min up to 195 °C, hold for 40 min, followed by a second increase of 10 °C/min up to 240 °C, and hold for 10 min. A constant pressure mode (29 psi) is chosen. Helium or hydrogen can be used as the carrier gas. Methyl esters are identified by comparison with the retention times of authentic samples. Figure 10 shows a representative GC analysis of FAME obtained from plasma cholesteryl esters.

5. Synthesis of (5’R)- and (5’S)-5′,8-cyclo-2′-deoxyadenosine

1. Dissolve 33 mg of 8-bromo-2′-deoxyadenosine (8-Br-dAdo) in acetonitrile (100 ml) in order to reach 1 mM concentration. Regulate gas flow (Ar or N2) in the photoreactor and fill the apparatus with the solution of 8-Br-dAdo.
2. Prepare a septum with the appropriate size for the photoreactor’s input and pass a needle connected to the inert gas line through the center of it. Close the system with the septum. Flush the inert gas for 30 min.
3. Irradiate by UV light using a 125W medium-pressure mercury lamp at 22±2 °C for 15 min, collect the solution into a 250 ml round-bottomed flask. Wash the photoreactor with 10 ml acetonitrile and collect washings in the same flask.
4. Quench the crude reaction mixture with 1 M NH₄OH solution and evaporate the solvent in the rotary evaporator.
5. Run high performance liquid chromatographic analysis (HPLC-UV) with analytical column (see instrumentation section) under known conditions , and compare the profile with reference compounds.
6. Run high performance liquid chromatographic analysis (HPLC-UV) with an analytical column (see instrumentation section) by using the following solvents and gradients: 2 mM ammonium formate as solvent A and acetonitrile as solvent B. Set the flow rate to 1 ml/min and the gradient from 0% solvent B to 0.3% solvent B to be reached in 2.2 min. Then 0.8% solvent B in in 4.0 min, then 1% solvent B in 4.8 min. Remain at 1% solvent B for 9 min and then go to 8% solvent B in 6 min. After go to 10 % solvent B in 4 min and to 30% solvent B in 5 min and remain at 30% solvent b for other 5 min. Collect the chromatographic peaks corresponding to the two diastereomeric products in two different vials.
7. Measure the absorbance of the two collected fractions in the UV spectrophotometer and calculate the exact concentration based on the Lamber-Beer law (A = εlC, where A is the absorbance, ε the extinction coefficient and l the length of the cell). Use the extinction coefficient ε of 2′-deoxyadenosine (dAdo) (15,400 M^-1 cm^-1 at 260 nm).

6. Synthesis of (5’R)- and (5’S)-5′,8-cyclo-2′-deoxyguanosine

1. Dissolve 35 mg of 8-bromo-2′-deoxyguanosine (8-Br-dGuo) and 30 mg of sodium iodide (NaI) in distilled water (100 ml) in order to reach 1mM and 2mM concentration, respectively. Regulate inert gas (Ar or N₂) flow connected to the photoreactor and fill the photoreactor with the reaction solution.
2. Degass the reaction mixture as described in procedure 5 (step b).
3. Irradiate by UV light using a 125W medium-pressure mercury lamp at 22±2 °C for 30 min, collect the solution into a 250 ml round-bottomed flask. Wash the photoreactor with 10 ml of water and collect washings in the same flask.
4. Quench the crude reaction mixture with 1 M NH₄OH solution and evaporate the solvent under vacuum.
5. Perform analysis as described in Procedure 5 (steps e to g). Use the extinction coefficient ε of 2′-deoxyguanosine (dGuo) (11,700 M^-1 cm^-1 at 260 nm).

7. Synthesis of Isotopic Labeled Purine (5’R)- and (5’S)-5′,8-cyclonucleosides

1. Prepare 2 ml of 1mM aqueous solution (distilled water) of the ¹⁵N isotopic labeled purine nucleosides ([15N⁵]dAdo or [15N⁵]dGuo) in a glass vial (4 ml) with a septum cap.
2. Connect the vial with the septum cap and connect to the gas line (N₂O for saturation of the solution) through a needle in the septum that reaches the bottom of the vial. Another short needle in the septum cap works as the gas exit.
3. Flush the solution with N₂O for 30 min. The gas flow is regulated to be very low.
4. The exit needle is first taken out and after 1′ the long one follows, in order to have a small pressure inside the vial.
5. Put the solution in the apparatus of gamma radiolysis for 8 hr (calculation on the basis of a dose rate ca. 4.5 Gy/min).
6. After reaction quench the crude with 1 M NH₄OH solution.
7. Run high performance liquid chromatographic analysis (HPLC-UV) under known conditions,⁶ and compare the chromatogram with the standard references.

8. Gamma Radiolysis of DNA Aqueous Solutions

1. Prepare 1 ml of 0.5 mg/ml solution (distilled water) of calf thymus DNA and put in a glass vial (2 ml) with a septum cap.
2. Degass the reaction as described in Procedure 7 (steps b-d).
3. Put the solution in the apparatus of gamma radiolysis for 30 min (calculation on the basis of dose/rate ca. 4.5 Gy/min).

9. Enzymatic Digestion of DNA

1. To an Eppendorf tube containing 80 μg of DNA, add 8 U of nuclease P1, 0.01 U of phosphodiesterase II, 20 nmol of EHNA in 20 μl of 300 mM sodium acetate (pH 5.6) and 10 mM zinc chloride. Incubate the mixture at 37°C for 48 hr.
2. Next add a mixture of 8 U of alkaline phosphatase, 0.02 U of phosphodiesterase I, in 40 μl of 0.5 M Tris-HCl buffer (pH 8.9) to mixture digestion. The sample is incubated at 37°C for 2 hr.
3. The mixture is neutralized by addition of 10% formic acid.
4. Transfer the sample to an Amicon Ultra-0.5 centrifugal filter device (cutoff 3 kDa), add 200 μl of tridistilled H₂O. Place the Ultra-filter into the centrifuge rotor at 14,000 x g for 15 min. Then separate the Ultra filter device from the microcentrifuge tube. Lyophilize the enzyme-free solution in the microcentrifuge tube.

10. DNA Sample Desalinization Prior to Analysis

1. Run high performance liquid chromatographic analysis (HPLC-UV) with analytical column (see instrumentation section) following known conditions.⁶
2. Dissolve the lyophilized digested DNA in 20 μl of H₂O and inject in HPLC-UV.
3. Collect the sample in a vial after the salt elution (first minutes) and right before the elution time of the first nucleoside (5’R-cdGuo), until the end of the chromatographic program.
4. The sample is lyophilized and resolubilized in 20 μl water.

11. LC-MS/MS Quantitative Analysis

1. Prepare the HPLC-MS/MS (triple quadrupole) before starting the analysis by loading the analytical method and the method for the MS detector (ESI).⁶
2. Add 1 μl of the isotopic labelled compounds mixture in the sample prepared in Procedure 7.
3. Inject 20 μl of the spiked digested DNA solution in distilled water.
4. The obtained chromatographic data are elaborated on the basis of previous calibration with reference compounds. A representative HPLC run containing adenosine and guanosine 2′-deoxyribonucleosides and their oxidative and cyclo derivatives is shown in Figure 11.