Inherited disorders affecting mitochondrial function are associated with glutathione deficiency and hypocitrullinemia

Abstract

Disorders affecting mitochondria, including those that directly affect the respiratory chain function or result from abnormalities in branched amino acid metabolism (organic acidemias), have been shown to be associated with impaired redox balance. Almost all of the evidence underlying this conclusion has been obtained from studies on patient biopsies or animal models. Since the glutathione (iGSH) system provides the main protection against oxidative damage, we hypothesized that untreated oxidative stress in individuals with mitochondrial dysfunction would result in chronic iGSH deficiency. We confirm this hypothesis here in studies using high-dimensional flow cytometry (Hi-D FACS) and biochemical analysis of freshly obtained blood samples from patients with mitochondrial disorders or organic acidemias. T lymphocyte subsets, monocytes and neutrophils from organic acidemia and mitochondrial patients who were not on antioxidant supplements showed low iGSH levels, whereas similar subjects on antioxidant supplements showed normal iGSH. Measures of iROS levels in blood were insufficient to reveal the chronic oxidative stress in untreated patients. Patients with organic acidemias showed elevated plasma protein carbonyls, while plasma samples from all patients tested showed hypocitrullinemia. These findings indicate that measurements of iGSH in leukocytes may be a particularly useful biomarker to detect redox imbalance in mitochondrial disorders and organic acidemias, thus providing a relatively non-invasive means to monitor disease status and response to therapies. Furthermore, studies here suggest that antioxidant therapy may be useful for relieving the chronic oxidative stress that otherwise occurs in patients with mitochondrial dysfunction.

Fig. 1.

iGSH levels are lower in patients with mitochondrial disorders. iGSH levels were measured by the MCB assay on whole blood and analyzed by Hi-D FACS within 3 h of staining (see Materials and Methods). iGSH values are normalized to iGSH levels of a standard PBMC preparation stained and analyzed at the same time as patient samples. Top panel, iGSH levels in CD4 T cells; bottom panel, iGSH levels in monocytes. Statistical significance was determined by Wilcoxon/Kruskal Wallis non-parametric test. Each point represents a single sample. Adult controls (solid circles, n = 21); subjects not on antioxidant supplements (solid circles, n = 10); and subjects on antioxidant supplements (open circles, n = 11).

Fig. 2.

iGSH levels are lower in patients with organic acidemias. iGSH levels were measured by the MCB assay on whole blood and analyzed by Hi-D FACS within 3 h of staining (see Materials and Methods). iGSH levels are normalized to iGSH levels of a standard PBMC preparation stained and analyzed at the same time as patient samples. Top panel, iGSH levels in CD4 T cells; bottom panel, iGSH levels in monocytes. Statistical significance was determined by Wilcoxon/Kruskal Wallis non-parametric test. Each point represents a sample. Two subjects had 2 repeat measurements, and 1 subject had 3 repeat measurements. Adult controls (n = 21); inpatient subjects during an acute episode (n = 7); and outpatient subjects while clinically stable (n = 6).

Fig. 3.

Plasma protein carbonyl levels are increased in subjects with organic acidemias. Plasma carbonyl levels were measured in 100 μL of platelet-free plasma as described in Materials and Methods. Statistical significance was determined by Wilcoxon/Kruskal Wallis non-parametric test for ranked sums using JMP software. Each point represents a single subject. Solid circles represent subjects not on antioxidants and open circles represent subjects on antioxidants. Adult controls (n = 10): subjects with mitochondrial disorders (n = 12); and subjects with organic acidemias (n = 8).

Fig. 4.

Mitochondrial disorders and organic acidemias are associated with hypocitrullinemia. Citrulline levels were measured in platelet-free plasma as described in Materials and Methods. Statistical significance was determined by Wilcoxon/Kruskal Wallis non-parametric test for ranked sums using JMP software. Each point represents a single subject. Solid circles represent subjects not on antioxidants and open circles represent subjects on antioxidants. Adults controls (n = 5): subjects with mitochondrial disorders n = 8; subjects with organic acidemias (n = 6).