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. 2017 Feb 7:7:41984.
doi: 10.1038/srep41984.

Metabolomic profiling of reactive persulfides and polysulfides in the aqueous and vitreous humors

Hiroshi Kunikata  1   2 , Tomoaki Ida  3 , Kota Sato  1 , Naoko Aizawa  1 , Tomohiro Sawa  4 , Hiroshi Tawarayama  2 , Namie Murayama  1 , Shigemoto Fujii  3 , Takaaki Akaike  3 , Toru Nakazawa  1   2   5   6
Affiliations

Metabolomic profiling of reactive persulfides and polysulfides in the aqueous and vitreous humors

Hiroshi Kunikata et al. Sci Rep. .

Abstract

We investigate the metabolomic profile of reactive persulfides and polysulfides in the aqueous and vitreous humors. Eighteen eyes of 18 consecutive patients with diabetes mellitus (DM) and diabetic retinopathy underwent microincision vitrectomy combined with cataract surgery. Samples of the aqueous and vitreous humors were collected and underwent mass spectrometry-based metabolomic profiling of reactive persulfides and polysulfides (polysulfidomics). The effect of reactive polysulfide species on the viability of immortalized retinal cells (the RGC-5 cell line) under oxidative stress (induced with H2O2) was also evaluated with an Alamar Blue assay. The experiments showed that cysteine persulfides (CysSSH), oxidized glutathione trisulfide (GSSSG) and cystine were elevated in the aqueous humor, and CysSSH, Cys, and cystine were elevated in the vitreous. Furthermore, GSSSG, cystine, and CysSSH levels were correlated in the aqueous and vitreous humors. A comparison, in DM and control subjects, of plasma levels of reactive persulfides and polysulfides showed that they did not differ. In vitro findings revealed that reactive polysulfide species increased cell viability under oxidative stress. Thus, various reactive persulfides and polysulfides appear to be present in the eye, and some reactive sulfide species, which have a protective effect against oxidative stress, are upregulated in the aqueous and vitreous humors of DM eyes.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Metabolomic profile of the plasma of diabetes mellitus (DM) patients with diabetic retinopathy and normal controls.
GSH, GSSH, Cys, CysSSH, GSSG, GSSSH and cystine were all detected in the plasma of both DM patients (n = 7) and controls (n = 7). There were no significant differences in the concentrations of these reactive sulfide species in the DM patients and controls.
Figure 2
Figure 2. Metabolomic profile of the aqueous humor in diabetes mellitus (DM) patients with diabetic retinopathy and normal controls.
GSH, GSSH, Cys, CysSSH, GSSG, GSSSG and cystine were all detected in the aqueous humor of both DM patients (n = 12) and controls (n = 20). There were no significant differences in the concentrations of GSH, GSSH, Cys, or GSSG in the DM patients and controls. CysSSH, GSSSG and cystine had higher concentrations in the aqueous humor of the DM patients than the controls (P < 0.05, P < 0.001, and P < 0.05, respectively).
Figure 3
Figure 3. Metabolomic profile of the vitreous humor in diabetes mellitus (DM) patients with diabetic retinopathy and normal controls.
GSH, GSSH, Cys, CysSSH, GSSG, GSSSG and cystine were all detected in the vitreous of both DM patients (n = 18) and controls (n = 11). There were no significant differences in the concentrations of GSH, GSSH, GSSG or GSSSG in DM patients and controls. Cys, CysSSH and cystine had higher concentrations in the vitreous humor of DM patients than controls (P < 0.05, P < 0.001, and P < 0.001, respectively).
Figure 4
Figure 4. Relationship between metabolomic profiles of the aqueous and vitreous humor in diabetes mellitus (DM) patients with diabetic retinopathy and normal controls.
In the controls (n = 9), the aqueous and vitreous concentrations of GSSSG, cystine, Cys, and CysSSH were not correlated (upper). In the DM patients (n = 11), the aqueous and vitreous concentrations of GSSSG, cystine and CysSSH were correlated (r = 0.63, P < 0.05; r = 0.73, P = 0.01; r = 0.72, P = 0.01), although the aqueous and vitreous concentrations of Cys were not (lower).
Figure 5
Figure 5. Cell viability after oxidative stress exposure with or without reactive sulfide species.
Exposing glutathione-depleted RGC-5 cells to high levels of hydrogen peroxide (H2O2; 100 μM) resulted in a remarkable reduction in cell viability (H2O2(−): 100.0 ± 3.8%; H2O2(+): 68.1 ± 1.5%; n = 4; **P < 0.001). However, this H2O2-mediated negative effect on cell survival was significantly attenuated by GS(S)nSG (80.4 ± 2.5%; n = 4; *P < 0.01), although not by GSSG (68.4 ± 5.6%; n = 4), at a final concentration of 25 μg/ml.
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