Though the pathophysiology of CBSD is not well understood, it has been proposed that Hcy excess or cysteine deficiency, rather than methionine accumulation, is more likely to be involved in the pathogenesis of the disease 1. On the other hand, oxidative stress has been suggested to play an important role in the pathophysiology of homocystinuria 5,25–27. This was also rooted to researches that reported an association between Hcy and oxidative stress 28,29.
NAD+ and its reduced form NADH are key components in redox reactions 30. The intracellular NAD+ concentration is directly proportional to the redox potential. A decreased amount of mitochondrial signalling molecules, particularly NAD+, has been postulated to be responsible for compromised mitochondrial function and repair mechanisms in chronic disease states 9,10. In our study, the NAD+ level and NAD+/NADH ratio were found to be decreased in CBSD patients. In contrast, healthy controls showed normal NAD+ and NAD+/NADH levels. These findings suggest the presence of oxidative stress in CBSD patients. Another study similarly showed that Hcy induces oxidative stress by upregulating protease-activated receptor-4 (PAR-4) and enhancing iNOS and NAD(P)H oxidase expression 31.
FGF-21 and GDF-15 have been identified as reliable tools for the diagnosis of mitochondrial diseases 20. The fact that FGF-21 and GDF-15 levels were statistically significantly different in patients with CBSD compared to healthy controls suggests that these patients suffered oxidative damage and mitochondrial dysfunction similar to mitochondrial diseases. The levels of these two biomarkers have been documented to be increased in various inherited metabolic diseases 18,19,32.
Dysfunctional mitochondria may release DAMPs, which can trigger inflammasomes and a systemic inflammatory response 33,34. Levels of mitochondrial DAMPs were found to be higher in CBSD patients than in healthy subjects. Also, levels of FGF-21, GDF-15, NAD+, NADH and NAD+/NADH, which are markers of oxidative stress and mitochondrial dysfunction, differed between CBSD patients and healthy subjects, suggesting the presence of oxidative stress and mitochondrial dysfunction in CBSD patients.
Previous studies have reported that oxidative damage can occur in hepatocytes of CBSD patients and in animal experiments with induced hyperhomocystinemia 29,35. Our study suggests that patients with CBSD experience mitochondrial dysfunction besides endoplasmic reticulum damage due to oxidative stress.
Accumulation of the pro-oxidant Hcy 29 and decreased hepatic antioxidant capacity due to reduced activity of antioxidant enzymes, including glutathione peroxidase, are possible causes of increased mitochondrial and oxidative damage in homocystinuria 36. Vanzin et al discovered a significant negative correlation between antioxidant content and Hcy levels 37. They also observed a positive correlation between malondialdehyde levels and Hcy levels, implying a possible mechanistic role for Hcy in the oxidative damage observed in homocystinuria. The positive correlation between total homocysteine level and GDF-15, as well NAD+/NADH levels and negative correlation with total NAD++NADH and NADH levels of patients with CBSD in our study, were consistent with these previous findings.
Biomarkers of CBSD patients shows an increase in oxidative damage and a reduction in antioxidant defences, likely due to the generation of reactive species induced by Hcy 27. The alterations in NAD+, FGF-21, GDF-15 levels, and NAD+/NADH ratio in patients with homocystinuria suggest that oxidative damage coexists with mitochondrial dysfunction in CBSD patients. Vanzin et al. demonstrated that patients with homocystinuria experience protein and lipid oxidative damage, which can be ameliorated through therapy 27,38. In this respect, assessment of oxidative damage and addition of anti-oxidant therapy together with mitochondrial support may have additional benefits in reducing long-term morbidity in CBSD patients. Reproducibility is important, especially in rare diseases, and more studies on this subject are needed to support these results.