Measurement of the Effect of a Plant-Based Diet on Reactive Oxygen Metabolite Production Using the d-ROMs Test is Invalid

We read with interest the article by Colombo et al (Int J Biol Markers 2005; 20 (3): 169-76), which suggests that a plant-based diet can improve the serum fatty acid profile and decrease the production of reactive oxygen metabolites (ROMs) (1). In this study the correlation between the serum fatty acid profile and ROMs was investigated by means of the d-ROMs test (Diacron, Grosseto, Italy). This test measures ROMs by their reaction with an alkylamine chromogen. However, the basic principle of this method is invalid because alkylamine is also a substrate for the enzyme ceruloplasmin (ferroxidase), which is abundantly present in serum, and the type of buffer used and its pH are more appropriate for ferroxidase activity. This is borne out by the fact that a significant positive correlation between the assay results and ferroxidase activity has been found (r=0.911, p<0.001, n=100) (Tab. I and Fig. 6 in (2)). Also, this assay is inhibited by sodium azide (Fig. 3 in (2)); no response is observed during copper-induced lipoprotein autoxidation (Fig. 8 in (2)); and there are no linear appropriate responses for H2O2, t-butyl hydroperoxide or cumene hydroperoxide solutions (2). From these data it is clear that most of the measured absorbance pertains to the activity of ferroxidase, a known antioxidant enzyme, rather than to free oxygen radicals (2). Additionally, a clear calibration mistake has been made, in that the reported mean serum value for the method is about 300 CARR U/L in healthy subjects (3). This value is equal to 7054 μmol H2O2/L, a concentration that is about 350 times higher than actual values. Many studies have found that levels of H202 above about 50 μM are cytotoxic to a wide range of animal, plant and bacterial cells (4). The presumption that the d-ROMs test measures lipid hydroperoxides in serum has been shown to be incorrect by the lack of response during copper-induced lipoprotein autoxidation as found by Erel (Fig. 8 in (2)). A further invalidating fact is that antioxidants can reduce the colored compound in the test and thereby produce false negative results. In sum, the conclusion that a plant-based diet results in significant lowering of ROM production needs to be confirmed using a scientifically valid method – which the d-ROMs test is not. Measurement of antioxidants present would also be useful.