1. Principle

Thiols are qualitatively significant components of the “plasma/serum barrier to oxidation”. In fact, thiol groups of plasma/serum compounds (e. g. proteins, P-SH) are able to oppose the propagation step of peroxidative processes by inactivating either alkoxyl (RO^*) or hydroxyl (HO^*) radicals, respectively, according to the reactions you can find here (table 1).
From a stoichiometric point of view, a couple of thiol groups can reduce a couple of alkoxyl (RO*) or hydroxyl (HO*) radicals, by the cession of two electrons (as hydrogen atoms). By means of this reaction both the radicals are inactivated. Indeed, alkoxyl radical is released as an alcohol molecule, while hydroxyl radical becomes an innocuous water molecule. In turn, thiol groups after being oxidized react among themselves thus generating a disulphide bond.
In this context, it is necessary to remember that thiol groups by oxidizing itself oppose the attack of some histolesive free radicals. However, when a disolphide bond is formed in a proteic molecule, this results in undesiderate consequences. For example, the formation of a disulphide bound between two residual of cysteine of two different proteins can results in a polymerization. If disulphide bound occurs in the same protein, this protein can modify stably its conformation. In both the cases may be that protein involved in the formation of –S–S– bonds undergoes an alteration of its functional properties.
The –SHp test is based on the ability of –SH groups to develop a colored complex photometrically calculable (maximum absorbance peak 405 nm) when reacted with 5,5-dithiobys-2-nitrobenzoyc acid (DTNB). The “titre” of thiols directly parallels with color intensity.

2. Composition of kits
A typical kit of –SHp test contains, basically, a buffer solution (R₁ reagent), a chromogenic mixture (R₂ reagent) and a calibrator (R₃ reagent) (table 3. 5). The analysis can be carried out either by means of a dedicated instrumentation (i. e. FREE system) or a normal photometer (see table 2).

3. Analytical procedure
The –SHp test can be performed on fresh serum or plasma according to the following working conditions: wavelength 405 nm, optical path 1 cm, and room temperature. The analysis can be carried only according to the fixed time mode.
To carry out –SHp test, it must be dissolved the L-cysteine powder (R₃) in 25 mL of distilled water, then prepared by dilution a solution 496 mM of thiol groups. Such solution is stable for 2-3 hr at room temperature or 2-3 days at +4°C. At room temperature, it must be performed the procedure in table 3. The antioxidant titre (or capacity), expressed as µmol -SH/L of sample, must be calculated by the formula in table 4.

4. Results interpretation
The range in healthy peoples is 450–650 µmoles -SH/L. Decreased values directly correlate to a lowered efficacy of thiols antioxidant plasma barrier.

5. Clinical studies
The –SHp test was shown useful and very reliable in the evaluation of thiol component of antioxidant plasma barrier in several clinical studies, thus integrating the results provided by OXY-Adsorbent test and d-ROMs test in the global assessment of oxidative stress.
The –SHp test was shown to found a particular indication when there are reasonable suspects of an oxidative stress status but d-ROMs values are low. Such situation can occur in subjects with solid neoplasms. In such conditions, the shift of cell metabolism toward anaerobiosis can generates, around the neoplastic mass, the production of acidic catabolites. These latter, by reducing locally the pH can favor the release of iron that catalyzes the breakdown of circulating hydroperoxides, thus generating alkoxyl radicals. Thiol group, if reduced, can react with alkoxyl radicals and this reaction results in a lowering of circulating hydroperoxides level. In this clinical situation d-ROMs test values will be apparently low, although hydroperoxides are increasingly produced. Such condition underlines the importance of a combined approach of two tests to better assess oxidative stress.