I am a student researching the effect of anthocyanins on liver function in 4-month-old male Wistar rats (Rattus norvegicus) that are induced by obesity with a high-fat diet.

What I expect from a high-fat diet (HFD) treatment are:

1. There was an increase in bilirubin levels in rats given a high-fat diet (obese group)

2. Normal bilirubin levels in control rats group with basal diet.

However, after high-fat diet treatment, i had difficulty analyzing the bilirubin level data where the bilirubin levels were:

1. There was no significant difference in bilirubin levels before (baseline) and after (obese group) HFD treatment for five weeks

2. Bilirubin levels (before and after HFD treatment) in all groups including the control group with basal diet exceeded normal limits (0.2-0.55 mg/dL) based on Mohan et al., (2016) and Prihapsara et al., (2017) (Figure below).

I continued the research by giving anthocyanins mixed with a high-fat diet for four weeks, on the blood sample test results, all bilirubin levels in all groups jumped to within normal limits.

I'm very confused as to what might be causing this oddity. I have read various journal publications but found no similar cases. Have any of you experienced this or can think of a possible cause? Thank you.