Fig 2: Amnionless is detected in serum and increases with age while inversely correlating with vitamin B12 levels in rats. (A) Amnionless (AMN) protein was detected by western blot using a specific AMN antibody on three independent human sera diluted 1/10, and compared with 1, 10, and 100 ng of human recombinant AMN. Black arrows indicate the size of full‐length AMN at 48 kDa. (B‐C) levels of AMN protein were measured in the serum (B) and urine (C) of adult, middle‐aged, and aged rats (n = 9–10 per group). (D) Comparative protein and mRNA levels of AMN in ileum and kidney from adult rats. (E) Association between circulating levels of AMN and vitamin B12 in the same animals as in (B). Dashed line: linear regression. Data are represented as mean +/− SEM. *P < 0.05 **P < 0.01 ***P < 0.001 with a one‐way ANOVA test.

Fig 3: Renal vitamin B12 reabsorption is impaired with age. (A) Kidney protein level of amnionless (AMN), cubilin (CBN), and megalin (LRP2) in adult and aged rats quantified by western blot (n = 6 per group). (B) Graphs showing the protein quantification of the six replicates. (C,D) vitamin B12 (C) and creatinine (D) levels in urine of adult and aged rats over 24 h (n = 14 per group). Data are represented as mean +/− SEM. For all graphs *P < 0.05 **P < 0.01 ***P < 0.001 with an unpaired t‐test.

Fig 4: Serum amnionless levels increase with age and positively correlate with MMA levels in humans. (A) Levels of amnionless (AMN) were measured in the serum of young adult controls (n = 30) and aged participants of the Singapore longitudinal aging study (n = 238). P‐value was calculated with a Wilcoxon rank sum test with continuity correction. (B) Scatter plot of AMN levels and methyl‐malonic acid (MMA) levels in serum. Spearman rank correlation (ρ) and its P‐value are reported. Dashed line: linear regression. (C) Levels of AMN in individuals with normal MMA levels or presenting a vitamin B12 deficiency (high MMA, i.e. above the 0.28 μM threshold for deficiency). P‐value was calculated with a Wilcoxon rank sum test with continuity correction. Detailed statistics are provided in Table S1 and graphs presented in linear scale are provided in Fig. S4.