Justificación e importancia de la investigación

The levels o f NO in the plasma o f all subjects participating in the study was measured using a commercially available assay that measured total nitrites, from R&D, UK.

8.3.1 Basics

Direct measurement o f NO is difficult, as it is a transient and volatile gas. As most o f the NO is oxidised to both nitrite (NO2’) and nitrate (NO3 ), measurements o f these

anions can be used as indirect measurements o f NO production. The conversion o f NO into nitrate and nitrite is via the following reactions:

Eq 1. N 0 + O2 -4. ONO2 -> N O3 +

Eq 2. 2 NO + O2 —> N2O4 —> N O2 + N O3 + 2

Eq 3. NO + NO2 —> N2O3 —> 2NO2 + 2

The total NO assay involves the enzyme nitrate reductase, converting all o f the nitrate to nitrite and then via the Griess reaction, which allows the spectrophotometric measurement (OD) o f nitrite at 540 nm. The Griess reaction is based on a two step diazotization reaction in which acidified NO2 produces a

nitrosating agent, which reacts with sulfanilic acid to produce the diazonium ion. This ion is coupled to N- (1-naphthyl) ethylenediamine, to form the chromophoric azo-derivative which absorbs light at 540 nm. The intensity o f the colour change is proportional to the amount o f nitrite in the system. In order to interpret the results, a nitrate standard ‘calibration curve’ is produced in each plate. This consists o f a

|imol/l, plotted against their respective OD readings. In this way, OD readings can be compared against the OD values produced by the ‘calibration curve’ and a concentration measurement made.

8.3.2 Samples

All plasma samples were diluted two-fold in Reaction Buffer (30 mis o f 10 x Reaction Buffer concentrate diluted with de-ionised water to produce 300 mis o f 1 x Reaction Buffer). 100 pi o f serum was mixed with 100 pi o f Reaction Buffer in a 10,000 molecular weight (MILIPORE, UK) cut-off filter and spun at 400 x g, for 40 minutes at 4°C to eliminate proteins.

8.3.3 Reagents

All reagents, unless specified, were provided by the manufacturer and prepared according to their instructions. All reagents were kept at room temperature, for the duration o f the assay, except the reconstituted beta-Nicotinamide adenine dinucleotide (NADH) and Nitrate Reductase that were kept on ice.

8.3.4 Greiss reagent determination o f total nitrites

A total NO Assay kit (R&D Systems, UK) was used. The procedure was performed using a 96 well plate (supplied) with the nitrate standard arranged in pairs. The nitrate standard was supplied as a 1,000 pmol/1 concentration. To produce the dilution series with 1 0 0 pmol/1 as the high standard and 0 pmol/1 as the zero

standard, six tubes were labelled 100, 50, 25, 12.5, 6.25 and 3.12 pmol/1. 900 pi of reaction buffer was pipetted into the 100 pmol/1, tube while 500 pi o f the reaction buffer was pipetted in to each o f the remaining tubes. Using the 1,000 pmol/1 stock.

100 ni was pipetted in to the 100 pmol/1 tube. It was mixed thoroughly and after changing the pipette-tip, 500 pi was drawn up from the 100 pmol/1 tube and mixed in the 50 pmol/1 tube. The pipetting action was repeated with a fresh pipette-tip each time until the final mix in the 3.12 pmol/1 tube. Reaction buffer by itself served as the ‘zero standard’. The first two rows o f the 96 well plate were set aside for the ‘nitrite standard’. In descending concentration, starting with 100 pmol/1, 50 pi o f each concentration was pipetted in to the two wells o f each column as a pair, the last column containing 50 pi o f reaction buffer representing the ‘zero standard’. 200 pi o f reaction buffer was pipetted into the remaining wells o f the first two rows. 50 pi o f the already diluted and filtered serum was added in duplicate in to the remaining wells.

The next step required the addition o f NADH reagent to all wells. This had been reconstituted with 1 ml o f de-ionised water, mixed and allowed to sit for three minutes, on ice. Immediately before use, 900 pi was diluted with 1.8 mis o f de­ ionised water and 25 pi added to each well.

The addition o f nitrate reductase involved its reconstitution in 1 ml o f nitrate reductase storage buffer followed by two rounds o f vortexing and sitting at room temperature for 15 minutes, with a last vortexing after which it was kept on ice. Before use, the nitrate reductase was to be diluted according to the following protocol;

Step Process

1. nitrate reductase(|il) = (number o f wells + 5) x 5 |il

2. reaction buffer (pil) = (volume from 1) x 4 |il

3. volumes from (1.) and (2.) were added in a tube

and vortexed

4. final volume was placed on ice and 25 pi was

added to each well within 15 minutes o f its, dilution

The wells were mixed by tapping gently on the side o f the plate and then covered with an adhesive strip for 30 minutes (at room temperature). 50 pi each o f both Griess Reagent I (sulfanilamide in 2N Hydrochloric acid) and II (7V-( 1-naphthyl)

ethylenediamine in 2N Hydrochloric acid) were added to each well, except the blank wells, mixed by gentle tapping on the side o f the plate and then, left to incubate for 10 minutes at room temperature. Finally, after 60 minutes, the plates were read using a microplate reader (Dynex) set at 540 nm and OD values recorded.

Standard curves were performed for each experiment and for analysis the mean o f at least two separate experiments, each with duplicate standard/sample wells, were calculated and used for further analysis.