Metabolic flux analysis through de novo purine nucleotide synthesis was performed by injecting 13C2;15N-Glycine intravitreally (to 4 mM final concentration in the vitreous) to dark-adapted mice, that were then kept in the dark or exposed to 1600 lux light for 4 hr. Total nucleotide levels and labeled nucleotide levels (that incorporated the labeled carbon and nitrogen atoms from labeled glycine) were determined by LC/MS-MS, and normalized per μg of protein in each sample. (A) Retinal levels of total cGMP, IMP, AMP, GMP, ATP and GTP in dark-adapted mice (D); or bright-light-exposed mice (BL). Nucleotide levels are expressed in arbitrary units (LC-MS/MS peak integration values) normalized per μg of protein in each sample. cGMP levels decreased with light exposure, as expected [p=0,024, n = 12]. IMP levels showed a clear increase with light exposure [p=0004]; that was accompanied by an increase in AMP and GMP levels [p=0002 for AMP; p=0089 for GMP]; whereas the ATP and GTP levels were maintained. (B) Retinal levels of labeled IMP, GMP and AMP, normalized per μg prot. The labeled atoms from Gly* that are incorporated into the purine ring scaffold are shown in the diagram. The inset on the diagram shows that the levels of Gly* measured at the retinas of injected eyes were similar in dark and bright light samples. Incorporation of Gly* into IMP, AMP and GMP increased under bright-light exposure, indicative of an increase in the overall flux of de novo purine nucleotide synthesis [IMP*, p=0,020; AMP*, p=0,003; GMP*, p=0,013, with n > 7]. In four eyes of the ‘dark’ group of mice, and five eyes of the ‘bright light’ group of mice the injection failed [the injected Gly* did not reach the retina, and Gly*~0 in retinal extracts] and therefore could not be taken into account for determination of labeled nucleotides.