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Formamidopyrimidines, 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), are among major lesions in DNA generated by hydroxyl radical attack, UV radiation, or photosensitization in vitro and in vivo. FapyAde and FapyGua exist in living cells at detectable background levels and are formed by exposure of cells to DNA-damaging agents. Numerous prokaryotic and eukaryotic DNA glycosylases exist for the repair of formamidopyrimidines by base excision repair pathways in cells, indicating their biological significance. Moreover, they are premutagenic lesions, albeit to different extents, revealing a possible role in disease processes. Methodologies using gas chromatography/mass spectrometry (GC/MS) with capillary columns have been developed to accurately measure FapyAde and FapyGua in DNA in vitro and in vivo. Stable isotope-labeled analogues of these compounds have been synthesized and are commercially available to be used as internal standards for accurate quantification. GC/MS with isotope dilution provides excellent sensitivity and selectivity for positive identification and accurate quantification, and has widely been applied in the past to the measurement of formamidopyrimidines under numerous experimental conditions. This paper reports on the details of this GC/MS methodology.

Familial Mediterranean fever (FMF) is an autosomal recessively inherited disorder characterized by recurrent, inflammatory self-limited episodes of fever and other symptoms. This disease is caused by more than 25 mutations in the gene MEFV. During fever attacks, there is a substantial influx of polymorphonuclear leukocytes into the affected tissues. Attack-free periods are accompanied by the up-regulation of neutrophil and monocyte phagocytic activity and oxidative burst. These facts led us to hypothesize that oxidative damage by free radicals to DNA may accumulate in FMF patients. To test this hypothesis, we investigated oxidative DNA damage in polymorphonuclear leukocytes of FMF patients during the attack-free period in comparison with FMF-free control individuals. DNA was isolated from polymorphonuclear leukocytes of 17 FMF patients and 10 control individuals. DNA samples were analyzed by liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry to measure the levels of various typical oxidatively induced products of DNA. We show, for the first time, that FMF patients accumulate statistically significant levels of these lesions in their DNA when compared to FMF-free control individuals. This work suggests that the persistent oxidative stress with excess production of free radicals in FMF patients may lead to accumulation of oxidative DNA damage. Defective DNA repair may also contribute to this phenomenon, perhaps due to mutations in the MEFV gene. The accumulation of mutagenic and cytotoxic DNA lesions may contribute to increased mutations and apoptosis in FMF patients, thus to worsening of the disease and well-being of the patients. Future research should deal with prevention of oxidative DNA damage and apoptosis in FMF patients, and also the elucidation of a possible role of DNA repair in this disease.

Background: About a quarter of familial Mediterranean fever (FMF) patients are partially or totally resistant to colchicine. A previous observation reported that acute attacks may be shortened by administration of interferon alpha (IFN)., Objective: We designed a double-blind, placebo-controlled trial to test our initial observations of a beneficial response with IFN in FMF attacks., Methods: We treated 34 acute abdominal attacks with IFN 5 million IU or placebo sc in the early phase of the attack. Leucocytes, thrombocytes, the erythrocyte sedimentation rate, fibrinogen, C-reactive protein (CRP), serum amyloid A protein (SAA), haptoglobin, transferrin, IL-1beta and TNF-alpha were measured at hours 0, 6, 12, 24 and 48., Results: The median time to recovery in those treated with IFN and placebo was not significantly different, while the leucocytosis and high levels of fibrinogen were significantly more prolonged in placebo-treated patients. CRP and SAA were extremely elevated and peaked at 24h, remaining less marked in the IFN-treated patients but the difference was not statistically significant. Observations regarding the other parameters were unremarkable., Conclusions: Although there were some clues indicating a depressed inflammatory response with IFN, we could not demonstrate a definitive effect of this agent in this double-blind trial. The drug may suppress the acute inflammation of FMF only if administered at the earliest phase. CRP and SAA may be more sensitive indicators of an attack than ESR or fibrinogen.