Postnatal hyperglycemia alters amino acid profile in retinas (model of Phase I ROP)

**Background:** Retinopathy of prematurity (ROP) is a leading cause of childhood blindness, characterized by two phases: Phase I involves suppression of physiological retinal vascularization, and Phase II involves pathological neovascularization. Postnatal hyperglycemia is an independent risk factor for ROP, but the metabolic mechanisms underlying Phase I are poorly understood. Amino acid supplementation has been shown to reduce retinal neovascularization in mice, yet the role of amino acids in Phase I ROP remains unclear. This study aimed to characterize retinal amino acid changes in a mouse model of hyperglycemia-associated Phase I ROP (HAR) and to explore the clinical association between parenteral amino acid intake and severe ROP in preterm infants.

**Methods:** In the HAR model, neonatal C57BL/6J mice received intraperitoneal streptozotocin (STZ, 50 mg/kg/day) from postnatal day (P)1 to P9 to induce hyperglycemia (~200 mg/dL) around P8, with delayed deep retinal vascularization observed at P10. Targeted mass spectrometry-based metabolomics was performed on retinal homogenates (6 retinas pooled per sample, n=6 per group) to quantify 147 metabolites. Proteomics using selected reaction monitoring measured 21 differentially abundant proteins. Three branched-chain amino acids (BCAAs: L-leucine, L-isoleucine, L-valine) were significantly decreased in HAR retinas and were individually supplemented intraperitoneally from P7 to P9 (L-leucine 1.5 μg/g, L-isoleucine 0.6 μg/g, L-valine 2.4 μg/g). Retinal vascularization was quantified at P10 using isolectin staining and ImageJ. Clinically, data from 178 extremely preterm infants (<28 weeks gestation) enrolled in the Mega Donna Mega randomized trial were analyzed. Mean parenteral amino acid intake (g/kg) during postnatal days 2–7, 2–14, and 2–28, and total days on parenteral amino acids were compared between infants who developed Type I ROP requiring treatment (n=36) and those who did not (n=142). Multivariable logistic regression adjusted for treatment group, gestational age, birth weight, and sex.

**Key Results:** In HAR mouse retinas, 32 metabolites were significantly altered (p<0.05). BCAAs (L-leucine, L-isoleucine, L-valine) were markedly decreased, while acetyl-CoA was increased. Pathway enrichment identified glycine and serine metabolism (p<0.001), valine/leucine/isoleucine degradation (p<0.01), phenylalanine/tyrosine metabolism (p<0.01), urea cycle (p<0.01), and glutamate metabolism (p<0.01) as top pathways. Parenteral L-isoleucine supplementation significantly reduced the number of meshes and total vessel length per field in HAR mice (p<0.05), whereas L-leucine and L-valine had no significant effect. Metabolomics of L-isoleucine-treated retinas showed decreased pyruvic acid, succinic acid, and several nucleotides. Proteomics revealed increased abundance of 17 mitochondria-related proteins (including GOT2 and GLUD1) in HAR vs. control retinas. In preterm infants, those requiring ROP treatment had significantly higher mean parenteral amino acid intake during days 2–14 (1.91 vs. 1.26 g/kg, adjusted p=0.05) and days 2–28 (1.45 vs. 0.82 g/kg, adjusted p=0.046). Total days on parenteral amino acids independently predicted ROP treatment (OR 1.19, 95% CI 1.03–1.38, p=0.018), with a c-statistic of 0.79, similar to gestational age alone. The full model including days on parenteral amino acids, gestational age, birth weight, sex, and treatment group achieved a c-statistic of 0.86.

**Clinical Implications:** This study demonstrates that postnatal hyperglycemia profoundly alters retinal amino acid metabolism in neonatal mice, with decreased BCAAs and compensatory increases in mitochondrial proteins. Parenteral L-isoleucine, but not leucine or valine, worsened retinal vascularization, highlighting the specific and potentially detrimental effects of individual amino acids. In preterm infants, prolonged parenteral amino acid support was a strong independent predictor of severe ROP, suggesting that current parenteral nutrition formulations may be suboptimal for retinal health. Optimizing the composition and duration of parenteral amino acid supplementation—particularly balancing BCAA ratios—could represent a novel nutritional strategy to prevent Phase I ROP and subsequent neovascularization. Further studies are needed to determine the ideal amino acid profile and route of administration for preterm infants at risk of ROP.