Figure 2: Effect of Biotin Supplementation on Restless Legs Syndrome Score (A) and Biotin Status (B). International Restless Legs Study Group [16] scores are depicted as box-whisker plots; the upper and lower limits of the box depict the 75th and 25th quartiles, the midline depicts the medians, and the whiskers depict the ranges. IRLS scores decreased with biotin supplementation (P = 0.001, n = 16) but also decreased with placebo (P = 0.01, n = 15); the decrease was not significantly different between biotin and placebo. Lymphocytes (Figure 2B) were isolated from Study 2 participants before and after supplementation with biotin or placebo for 8 weeks. Isolated lymphocytes were incubated with and without 10 nmol/L of biotin before the activity of propionyl-CoA carboxylase (PCC) was determined. The boxwhisker plots (as described in 2A) depict the change in PCC activity after biotin incubation; in addition, outlier values greater than the upper quartile + 1.5* IQD (inter-quartile distance) or less than the lower quartile - 1.5* IQD are depicted as individual points. The activation coefficient decreased (P = 0.01) with biotin supplementation (n = 8) but not with placebo (n=7).