Supplementary Information
Biochemical analysis (preparation and analysis)
Blood samples were drawn into different tubes (fluoride tubes for glucose; chilled dry tubes for insulin, cpeptide, FGF-19 and bile acids; chilled EDTA tubes for total GLP-1, PYY, total ghrelin, FFA, CCK, leptin,
triglycerides (TG), thyroid stimulating hormones (TSH), free thyroxine (T 4) , free triiodothyronine (T3), highsensitivity C-reactive protein (hsCRP), high-density lipoprotein (HDL) and low-density lipoprotein (LDL).
Samples in dry tubes were left to coagulate for 30 min before centrifugation, whereas the remaining samples
were immediately cooled on ice and centrifuged at 4°C. All blood and urine samples were frozen at -80°C until
analyzed as described in appendix.
Samples were analyzed for concentrations of glucose [enzymatic assay (ABX Pentra Glucose HK CP)], insulin
[solid-phase enzyme-labeled chemiluminescentimmunometric assay (Immulite 1000 Insulin Kit, LKIN1)], cpeptide [solid-phase enzyme-labeled chemiluminescentimmunometric assay (Immulite 1000 C-peptide Kit
(LKPEP1)], total ghrelin [enzyme-linked immunosorbent assay (Millipore)], TG, HDL/LDL cholesterol, FFA,
hs-CRP [ABX Pentra 400 (Horiba ABX, Montpellier, France)], TSH, free T4 and free T3 [solid-phase enzymelabeled chemiluminescentimmunometric assay (Siemens immunilite 1000; Siemens Medical Solutions)], total
GLP-1 [radioimmunoassay (RIA) (antiserum no. 89390)], PYY3-36 [commercially available RIA kit (Linco
Research , USA)], leptin [human gut hormone Milliplex assay (Linco Research, USA)],CCK [RIA (antiserum
no 92128)](1), total bile acids [enzyme cycling based TBA assay kit, (cat. no. DZ042A-K, Diazyme, San Diego,
CA, USA], and FGF19 [human FGF-19 ELISA assay (cat. no. RD191107200R,Biovendor)]. Urine samples
were frozen at -80°C and assessed for concentrations of nitrogen and adrenaline and noradrenaline [solid-phase
extraction by ion exchange followed by high performance liquid chromatography with electrochemical detection
(HPLC-EC)].
Statistical analysis
Descriptive data (gender, age, body weight, FFM, FM and BMI) collected at the screening visit and changes
over time were evaluated using post hoc t-tests for the appropriate contrasts defined in linear mixed models
including the group-time interaction as a fixed effect and participants as random. Data were summarized as mean
± standard deviation (SD).
Primary and secondary endpoints, EE and appetite, respectively, and all biochemical measures, were analyzed by
means of linear mixed models. Baseline values (week 7) were included in the model to account for potential
differences present already before the intervention. Analysis of EE additionally included a number of covariates
as fixed effects to adjust for differences in body composition and spontaneous physical activity. Likewise, leptin
was analyzed in a model adjusted for FM. Results were reported as estimated means with corresponding 95%
confidence intervals (CI).
The postprandial outcomes were analyzed by a repeated measurement model including the group-time
interaction as a fixed effect and participants as random effects. In addition, a spatial Gaussian serial correlation
structure was included to describe serial correlation between repeated measurements on the same subject,
ensuring that measurements close in time were more correlated than measurements taken far apart. Time was
measured as weeks elapsed since baseline and within a study week (on the test day) as minutes elapsed after
ingesting a test meal. Postprandial measures were reported as model-based AUC estimates with 95% CI. Area
under the curve (AUC) was calculated in R version 2.15.0 as total area (tAUC) using the trapezoidal rule. All
models used were validated using residual plots and normal probability (QQ) plots. Likelihood ratio tests were
used to evaluate the group effect.
If a difference between RYGB and CON was found in the repeated measurement model, each time point was
compared by model based post hoc analysis. To explore if post-surgical effects could be explained by alterations
in hormonal responses we considered exploratory analyses where additional explanatory variables were included
in the models. Hence GLP-1, PYY3-36 and bile acids were included to explain potentially positive effects of
RYGB on EE, whereas EE and leptin were included if negative effects of RYGB were found. Likewise, we
investigated if appetite-regulating hormones GLP-1, PYY3-36, CCK, leptin, ghrelin explained effects seen in
appetite.
Only participants who attended the baseline visit were included in the analysis. P-values <0.05 were considered
significant. Figures were based on raw data, unless otherwise stated, whereas conclusions were based on the
fitted model.