Page 129 - Human Bile Acid Metabolism: a Postprandial Perspective

identical liquid meals (Ensure Plus; 1.5 kcal/ml, 54E% carbohydrates, 29E% fat, 17E% protein; Abbott Nutrition, Columbus, Ohio, USA) per day. The total daily amounts of calories was set at 25 kcal/kg bodyweight, except for one healthy control who had very high baseline daily food intake and was provided with 35 kcal/kg bodyweight. The type 2 diabetes patients were instructed to pause metformin use from two days prior to the measurements until study end (in total 5 days).
At the evening of day 2 subjects were admitted to the clinical research unit. Subjects slept undisturbed in darkness (0 lux) during their habitual sleep times. On day 3 at ZT 0, the light was turned on at ~150 lux at eye level. Participants were provided with the three identical liquid meals at ZT 0:30, ZT 6:00 and ZT 11:30. Blood samples for BA measurements were obtained from a cannula in a peripheral arm vein prior to every meal and with 60-min intervals in the postprandial period. Blood samples were centrifuged for 10 minutes at 3000 rpm at 4°C and plasma was stored at -20°C.
Endpoints
Primary endpoints were the effect of mealtime on postprandial BA excursions
of total BAs, CA, CDCA, DCA, UDCA, taurine conjugated, glycine conjugated
and unconjugated BAs in lean subjects and in obese patients with type 2 diabetes. 7 Secondary endpoints were the differences between lean control subjects and obese
patients with type 2 diabetes in fasting total BAs, CA, CDCA, DCA, UDCA,
taurine conjugated, glycine conjugated and unconjugated BAs.
Measurement of bile acids
Taurine- and glycine-conjugated internal standards [2,2,4,4-2H4]taurocholic acid (tauro-CA), [2,2,4,4-2H4] taurochenodeoxycholic acid (tauro-CDCA), [2,2,4,4- 2H4]glycocholic acid (glyco-CA) and [2,2,4,4-2H4] glycochenodeoxycholic acid (glyco-CDCA) were synthesized as described by Mills et al (29). Plasma (50μL) was diluted with 50μL internal standard solution (29) and 500μL acetonitrile was added while mixing. After centrifugation, the sample was dried under N2 and reconstituted in 100μL methanol:H2O (1:3). Subsequently, 10μL of this solution was injected onto a UPLC column (Waters Acquity BEH C18; length 10cm, i.d. 2.1mm, particle size 1.7μm). The BAs were separated using a gradient from 98% 5mM ammoniumformate (pH8.1):methanol (3:1 v:v) to 98% acetonitrile:H2O (9:1 v:v) in 5 minutes at a flow rate of 350μl/min.
Diurnal rythm of bile acids
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