Box plots of urinary alanine and histidine in relation to NEC sub-types versus controls. Urine samples collected during days 4 to 9, restricted to one sample per infant (31 samples). Analysis of urinary alanine, pyridoxine and histidine among NEC-I, NEC-II, death and control subjects using multiple samples per individual (60 samples) is shown in Supplementary Information, Table S2 of Additional file 2. (A) Urinary alanine. Using the Kruskal–Wallis test, urinary alanine was significantly higher in NEC-I vs controls (P = 0.044) and NEC-I than NEC-II (P = 0.023), but did not differ between NEC-II vs controls or all NEC vs controls. (B) Urinary histidine. Using the Kruskal–Wallis test, urinary histidine was significantly lower in NEC-II vs controls (P = 0.023). Histidine also tended to be lower in NEC-II vs NEC-I (P = 0.059), but did not differ between NEC-I vs controls or all NEC vs controls. (C) Ratio of urinary alanine to histidine. Using the Kruskal–Wallis test, the ratio of urinary alanine to histidine was significantly higher in NEC cases overall vs controls (P = 0.023), but did not differ between the NEC sub-types. The optimal cut-point in the alanine:histidine ratio to differentiate NEC cases from controls was identified as a ratio greater than 4 (predictive value 78%, P = 0.007). NEC: necrotizing enterocolitis.