1. Principle

The d-ROMs test is a spectrophotometric test that allows assessing, in a biological sample, the concentration of hydroperoxides (ROOH). Such compounds are generated into the cells by the oxidative attack of ROS on a number of organic substrates (e. g. carbohydrates, lipids, amino acids, proteins, nucleotides etc.).
The initials “ROMs” underlines that the analites measured by this test, i. e. hydroperoxides, belong to the reactive oxygen metabolites (ROMs).
By means of d-ROMs test the hydroperoxides of a biological sample, e. g. the blood serum, after reacted with a chromogenic substrate develop a colored derivative (pink to red). Such colored complex is detectable and then quantifiable by a spectrophotometric methodic. Hydroperoxides concentration, which directly correlates with detected color intensity, is expressed as arbitrary units which are of easy use in clinical practice. Such unities are indicated with the initials “CARR U”, i. e. Carratelli Units, by the name of the Italian research chemist Mauro Carratelli (Pienza, Italy), the “inventor” of the d-ROMs test.
In the d-ROMs test, therefore, hydroperoxides of a biological sample, e. g. blood serum, are posed in the same conditions of the Fenton’s reaction to generate in vitro alkoxyl and peroxyl radicals. Practically, a small amount of serum is diluted in a acidic buffered solution (pH 4.8). In these conditions, iron ions before bonded to the
serum proteins become available to catalyze in vitro the breakdown of blood hydroperoxides to alkoxyl and peroxyl radicals (figure 1).
A compound (chromogen) that has the ability to change its color when is oxidized by hydroperoxyl and alkoxyl radicals is then added to this solution. The chromogenic substrate used in the d-ROMs test is N,N,-diethylparaphenylen-diamine, that possess the feature of being oxidized by hydroperoxyl and alkoxyl radicals, thus transforming itself in a pink to red colored cation. Such cation is a radical but it is sufficiently stable so that it is possible to assess its quantity by means of a photometer (work conditions: wavelength 505 or 546 nm, optical path 1 cm, temperature 37 °C, kinetic or endpoint mode). The concentration of colored complex will be directly related to the hydroperoxides levels of the tested biological sample.
Because the chemical heterogeneity of alkoxyl and peroxyl radicals generated by hydroperoxide breakdown, the results of d-ROMs test are expressed as arbitrary units, the CARRATELLI UNITS (CARR U), where 1 CARR U correspond to 0.08 mg/100 mL H₂O₂.

2. Composition of kits
Several kits of d-ROMs test are available depending either on biological sample tested (e. g. whole blood, plasma or serum blood, inflammatory fluids, cell extracts etc.) or on instrumentation required to carry out the analysis. In fact, in this subject it must be remembered that d-ROMs can be performed either by means a common multiple analyzer or by means of a dedicated instrumentation (FRAS and FREE systems). In any case, a typical kit of d-ROMs test contains, basically, a cromogenic mixture (R₁ reagent) and a buffer (R₂ reagent) (table 1). To calibrate the analytical instrumentation a lyophilized control serum with assigned value is available.

3. Analytical procedure
As mentioned above, the working conditions are: wavelength 505 or 546 nm, optical path 1 cm, and temperature 37 °C. Moreover, the analysis can be carried out either in kinetic or endpoint mode, depending on instrumentation used to carry out the test.

4. Results interpretation
The availability of a method precise and reliable has allowed to define the blood reference levels of d-ROMs test in healthy people. Indeed, in a population of 5,000 healthy subjects was shown that blood hydroperoxide levels as measured by d-ROMs test have an unimodal distribution that picks between 250 and 300 CARR U (i. e. between 20 and 24 mg/dL H₂O₂). Therefore, these values were taken as reference value of the test (Cornelli U and Coll., The International Union Angiology’s Bulletin, 1999).
Obviously, higher value indicate a border-line condition (301-320 CARR U) or an oxidative stress status (>320 CARR U) (table 2). Moreover it was shown that d-ROMs test results are not influenced by age or gender. However, newborns, independently of the gender, have significantly lower levels of hydroperoxides than adults, while pregnancy is associated with medially higher value of d-ROMs test.

5. Clinical studies
The d-ROMs test was shown to be very useful in the monitoring of health state in smokers and alcohol-consumers. Very interesting results were obtained in sport medicine, with a number of trials performed on cycloergometer, on cyclists and on walkers.
The d-ROMs test was demonstrated to be also an important parameter to evaluate the metabolism in obese patients. These subjects, in fact, possess significantly higher hydroperoxides level than individuals with normal body mass index. A preliminary report demonstrates, moreover, that the level of plasma lipids is relatively independent respect to the plasma hydroperoxide levels but it can lower with specific therapy and, particularly, in diabetics, by following a regular diet.
According to the scientific literature (over 70 papers, see references), actually, d-ROMs test can take application in any field of medicine either in the prevention or therapy of oxidative stress-related diseases. In this subject, very eloquent are the results obtained from trials performed on cardiovascular diseases, in rheumatoid arthritis, in Alzheimer’s disease, in Down syndrome, in dislipidaemia, in AIDS, in oncology, and in women that intake the pill. The results of published studies have allowed defining not only the indications and the purposes of the d-ROMs test but also the primary interest areas of this test. Among the more recent areas of application it must be remembered: nephrology, geriatrics, neuropsychiatry, cardiology and angelology, bronchopneumology, rheumatology, gynecology, infectivology and oncology.In conclusion, d-ROMs test was demonstrate to possess a wide range of advantages. Therefore, d-ROMs test is a very useful tool in preventing and monitoring of any situation related to the oxidative stress.

6. The d-ROMs test in clinical practice

The d-ROMs test should be performed periodically on all healthy subjects, because do not exist any subject that are not exposed to the risk to produce exaggerate amounts of reactive species. In these cases, the aim of the test is to prevent oxidative stress and its consequences (ageing and oxidative stress related diseases). Special categories of subjects that are candidates to d-ROMs test are: sport-lovers and elite athletes, cigarette smokers, alcohol drinkers, any subject that intake some drug (such as oral contraceptives or chemiotherapeuticals) and any subject at risk for or suffering by oxidative stress-related diseases (e. g. blood hypertension, stroke, infarction, peripheral vasculopathies, obesity, diabetes, rheumatoid arthritis, Parkinson’s diseases, Alzheimer’s disease, Chron’s disease and others). In these cases, the aim of the test is to prevent and/or monitor oxidative stress, especially when patients undergo specific and/or antioxidant therapies.