Subjects and design of the study page 1. Biochemical Analysis page 1. Image page 2. Set of neuropsychological tests page 2

Transcription

1 SUPPLEMENTAL METHODS Subjects and design of the study page 1 Biochemical Analysis page 1 Image page Set of neuropsychological tests page Statistical analysis page Subjects and design of the study The study chose severely obese women who had been submitted to Roux-en-Y gastric bypass between 0 and 01, that were selected from the bariatric surgery outpatient in the Hospital das Clínicas of the University of the Sao Paulo Medical School and women of normal weight with equal age and level of education, that were selected in same hospital. Patients were excluded with type mellitus diabetes, previous history of neurological disease, previous history of head, skull or brain injury, current or previous severe psychiatric illness, current or previous drug or alcohol abuse, chronic renal failure, liver disease, known rheumatologic disease, uncompensated hypothyroidism, use of corticoids or psychotropic medication in the last months, parents with Alzheimer s disease and dementia before the age of 0 and those with visual or hearing impairment which could undermine communication. The patients were submitted to open Roux-en-Y gastric bypass and evaluated twice (before and months after the surgical procedure) with the collection of laboratory exams, FDG-PET and neuropsychological tests. The patients were followed up post-operatively every months and received nutritional supplements following the recommendations of the American Association of Clinical Endocrinologists Guidelines (1). The patients of normal weight were assessed on only one occasion. For inclusion, each participant signed the informed consent form. The protocol of the study was approved by the ethics committee on research of the Hospital das Clínicas of the University of Sao Paulo Medical School.and done according with the Declaration of Helsinki. Biochemical Analysis The plasma glucose concentration was measured using an enzymatic colorimetric method with reference values between 0 and mg/dl. Insulinemia was measured by electrochemiluminescence with reference values between. and. uu/ml. Blood glucose and insulin were used to calculate the Homeostasis Assessment HOMA-IR, a method used to assess insulin resistance and calculated as: insulin (μu/ml) x glucose (mmol/l) /., in order to do so the results for blood glucose were multiplied by 0.01 (). An enzymatic colorimetric method was used to measure lipids, with reference values of: less than 00 mg/dl for total cholesterol and less than 0mg/dl for triglycerides. The high sensitivity C-reactive protein (hs- CRP) was determined by nephelometry using reference values greater or equal to mg/l. The interleukin (IL-) and tumor necrosis factor-alpha (TNF-a) were measured using 1

2 immunosorbent assay (High Sensitivity ELISA), with reference values of 0.-. pg/ml and pg/ml, respectively. Image Images of patients were acquired by initially submitting them to an intravenous injection with 0MBq (mci) of the fluorodeoxyglucose radiopharmaceutical marker (F- FDG). The patients then remained in the supine position for 0 minutes, in a quiet room, with low light, minimal environmental noise; and were instructed to keep their eyes closed. Sixty minutes after the injection, the metabolic images of the PET were taken using the PET/CT Biograph (CTI/ Siemens, Knoxville, TN, USA) equipment. The images were taken for minutes using the standard D protocol for the brain (matrix = x, zoom =. and pixel size = 1.0 mm). The images were reconstructed using the OSEM method ( iterations and subsets) and filtered with a mm Gaussian filter. The studies were corrected for scatter, attenuation and decay. Subsequently, the images were archived in DICOM format for quantitative analysis Processing the Images In the pre-handling, the images were converted to standard NIFTI, using the program dcmniigui ( After conversion, the images were reoriented according to the neurologic convention and the anterior commissure defined as the point of origin (x=0, y=0, z=0), in accordance with the Tailarach-Tournoux Brain Atlas () and then the images were aligned in parallel to the line that joins the anterior and posterior commissures. The images were processed using the Statistical Parametric Mapping (SPM) software, 00 version () (SPM). First, the images were spatially normalized using the PET template, based on the Montreal Neurological Institute (MNI) model. The normalized images were re-sampled using linear interpolation, producing xx mm sized voxels. Subsequently, the isotropic Gaussian filter of mm was used to smooth the images, eliminating small differences at their edges and increasing the signal/noise ratio. Set of neuropsychological tests. Six tests were selected to evaluate executive function and memory, in addition to the evaluation of the intellectual potential using the intelligence quotient: Intelligence Quotient ( IQ - Wechsler Adult Intelligence Scale - WASI) (): was used to evaluate intelligence and cognitive aspects such as: verbal understanding, processing of information and spatial and non-verbal reasoning. The Wisconsin Card Sorting Test (WCST) (). Evaluates problem solving strategies, cognitive flexibility and the capacity to solve problems using feedback. Faced with four modelcards, the subjects are instructed to classify cards by similarity (combination of color, for example) decided and only known by the examiner. After every ten correct answers the item is changed. A score is given according to the ability to perceive the similarities between the cards (number of correct and wrong answers, time and learned common characteristics) Stroop Color Test (). Evaluates the inhibitory control on the visual-verbal stimuli. It involves the reading and naming of the printed color names on cards. Both tasks consist of the names of colors printed in a different color. It evaluates the time of execution and the errors

3 made by the individual being examined (reading the written color instead of the color in which the word is written). Iowa Gambling Task (). Evaluates the ability to make decisions. It simulates a game of cards involving the losing and winning fictional money, with a pre-determined reward and punishment scheme. The capacity of the examinee to choose which choices yield more between four sets of cards is evaluated. Rey Auditory Verbal Learning Test (RAVLT) (). This test measures recent memory, learning, susceptibility to interference, retention after other activities and memory recall. A list of nouns (list A) read aloud for the subject, with a one second interval between the words, five times consecutively, each time followed by a test of immediate recall. After the fifth attempt, a list of interference, also of words (list B), was presented, next follows a memory test. Afterwards, the subject is asked to record the words of list A. Finally, the memory recall was evaluated, presenting a list of 0 words which contains all of the items from list A and B and another 0 words, which were phonetically similar to those on list A and B. Rey Complex Figure Test (). Evaluates the capacity to plan actions. The examinee is asked to copy a standard figure, paying attention to the proportions and the details. People without impairment start copying the central rectangle and then the internal or external elements of the figure. A score is given according to the time and the richness of detail to which the figure is copied. Trail Making Test () Evaluates the capacity of self-monitoring. The test is divided into types A and B. In the first, one must join a sequence of numbers in increasing order without taking the pencil from the paper. In type B, 1 numbers and 1 letters must be joined alternatively (1-A, -B, etc.). The test is stopped after either errors or minutes. Statistical analysis The quantitative variables with normal distribution were compared between two groups by using the unpaired t-test. For the non-normal distributed variables, these comparisons were performed by the Wilcoxon rank-sum test. Statistical analyses were conducted in paired samples using paired t-test or Wilcoxon signed-rank test when adequate. Normally-distributed quantitative variables were presented as mean ± standard deviation and non-normally distributed quantitative variables as median (interquartile range (IQR)). Normality was assessed by the Shapiro-Wilks test and visual inspection of histograms. All statistical analyses were performed using SAS. (). All p-values are two-sided and a p-value < 0.0 was considered statistically significant. SPM was used for the analysis of the PET images, which generated statistical maps showing the areas of significant difference in the cerebral metabolism of the obese patients before and after surgery. On SPM, each voxel is considered as an independent variable and the Student t test is applied to each voxel, which is transformed to the Z statistic, as the cerebral volume in each voxel has its own statistic a statistical map can be created. For the statistical maps the level of significance was defined as p < 0.001, uncorrected for multiple comparisons, for the identification of the differences between the groups. The statistical maps obtained were designed according to the coordinates of the stereotaxic brain atlas by Talairach and Tourneaux. The quantitative analysis was performed by means of the comparison between the established conditions (Obese pre-surgery x normal weight group; Obese post-surgery x normal weight group). For the statistical analysis the paired Student t test was used for each voxel. These analyses produced statistical parametric maps of the t statistic, which was then converted into a unit of normal distribution (Z scores). The statistical level of significance adopted was: Threshold: p<0.001 (Z>.0). The maps obtained followed pre-established hypotheses aimed to determine areas with altered glycolytic metabolism of the obese group compared to the group of patients of normal weight group recruited for this study.

4 References cited in the Supplemental Methods Mechanick JI, Kushner RF, Sugerman HJ, Gonzalez-Campoy JM, Collazo-Clavell ML, Guven S, Spitz AF, Apovian CM, Livingston EH, Brolin R, Sarwer DB, Anderson WA, Dixon J 00 American Association of Clinical Endocrinologists, The Obesity Society, and American Society for Metabolic & Bariatric Surgery Medical Guidelines for Clinical Practice for the perioperative nutritional, metabolic, and nonsurgical support of the bariatric surgery patient. In: Surg Obes Relat Dis. United States; S-. Muniyappa R, Lee S, Chen H, Quon MJ 00 Current approaches for assessing insulin sensitivity and resistance in vivo: advantages, limitations, and appropriate usage. Am J Physiol Endocrinol Metab :E-. Talairach J, Tournoux P Co-Planar Stereotaxic Atlas of the Human Brain: -D Proportional System: An Approach to Cerebral Imaging. 1st Edition ed. Stuttgart, Germany: Thieme. Friston KJ Statistical Parametric Mapping and Other Analysis of Functional Imaging Data. In: Brain Mapping: The Methods. USA: Academic Press; -. Spreen O, Strauss E A compendium of neuropsychological tests - admintration, norms and commentary. ed. USA: Oxford University Press. Walker GA, Shostak J 0 Common Statistical Methods for Clinical Research with SAS Examples Third Edition ed. North Carolina, USA: SAS Institute

5 SUPPLEMENTAL TABLE Supplemental Table Remaining neuropsychological performance of the studied subjects* Obese (N=) Normal weight (NW) (N=) Obese after RYGB (N=) P value Obese vs. NW P value Obese vs. obese after RYGB** WCST Categories 0. B 0. C Mean±SD.±1..±1.1.±1. Median (q-q).0 (.0-.0).0 (1.0-.0).0 (1.-.) PE B 0.0 C Mean±SD 1.±. 1.±. 1.±. Median (q-q).0 (.0-.0).0 (.0-.0). (.0-.) PR 0. B 0. C Mean±SD.±..±1. 1.±. Median (q-q) 1.0 (.0-0.0).0 (.0-.0) 1.0 (.0-.0) FMS 0. B 0. C Mean±SD 0.±0. 0.±0. 0.±0. Median (q-q) 0.0 ( ) 1.0 ( ) 0.0 ( ) Stroop 1-Time 0. B 0. C Mean±SD.±..±..±.1 Median (q-q).0 (1.0-.0).0 (1.0-.0).0 (1.0-.0) -Time 0. B 0.1 C Mean±SD.1±.0 1.±. 0.±. Median (q-q) 0.0 (.0-.0).0 ( ).0 (.0-.) -Time 0.1 B 0. C Mean±SD.±1. 1.1±..±. Median (q-q).0 (.0-0.0).0 (.0-.0).0 (.0-.) -Error 0. B 0. D Mean±SD.1±.1.0±1. 1.±.1 Median (q-q) 1.0 (0.0-.0) 1.0 (1.0-.0) 1.0 (0.0-.) IGT Step B 0. C Mean±SD -1.±. -.±. -1.±. Median (q-q) -.0 ( ) 0.0 (-.0-.0) 0.0 (-.0-.0) Step 0. B 0.0 C Mean±SD -1.±. -.±. 1.±.1 Median (q-q) -.0 (-.0-.0) -.0 (-.0-.0).0 ( ) Step 0. B 0.0 C Mean±SD -0.±. -.1±. 1.±. Median (q-q) 0.0 (-.0-.0) -.0 (-.0-.0) 0.0 (-.0-.0) Step 0. A 0. D Mean±SD 0.±. 1.1±. -1.0±.0 Median (q-q) 0.0 (-.0-.0).0 (-.0-.0) 0.0 (-.0-.0) Step 0. A 0.1 C Mean±SD 0.±. -1.1±. -0.±. Median (q-q) 0.0 (-.0-.0) 0.0 (-.0-.0) 1.0 (-.0-.0) Net score 0. A 0. D Mean±SD -.±. -.± ±.1 Median (q-q).0 (-.0-.0) 0.0 ( ).0 (-.0-.0) Continuation

6 RAVLT Total 0. A 0.0 C Mean±SD.±.1.±..±. Median (q-q).0 (.0-.0).0 (.0-.0).0 (.0-1.) Recognition 0.0 B 0. C Mean±SD.±..±..±1. Median (q-q) 1.0 (.0-1.0).0 (.0-1.0) 1.0 (.0-1.) After interference 0. A 0.1 C Mean±SD.±.0.±..±.0 Median (q-q).0 (.0-.0).0 (.0-.0).0 (.0-.) After B 0.0 C Mean±SD.±..1±..±. Median (q-q).0 (.0-.0).0 (.0-1.0). (.0-.) True recognition 0. B 0.1 C Mean±SD.±1..±1..±1. Median (q-q).0 (.0-.0).0 (.0-.0).0 (.0-.0) Rey Planning 1 0. B 0. C Mean±SD.±.0.±. 1.±1. Median (q-q).0 (1.0-.0).0 (1.0-.0) 1.0 (0.-.0) Planning 0. B 0. C Mean±SD.1±.1.±..1±.0 Median (q-q).0 (0.0-.0).0 (1.0-.0).0 (0.0-.0) Praxis B 0.1 C Mean±SD.±.1.±..±.0 Median (q-q).0 (.0-.0).0 (.-.0). (.-1.) Praxis 0. B 0. C Mean±SD.±..±..±.b Median (q-q). (.-.).0 (.0-.). (.-.) WCST: Wisconsin Card Sorting Test PE: Perseverative Erros PR: Perseverative Responses FMS: Failure to Maintain Set Stroop: Stroop Color Test IGT: Iowa Gambling Task RAVLT: Rey Auditory Verbal Learning Test Rey: Rey Complex Figure Test A : p-value obtained from the unpaired t-test. B : p-value in unpaired samples obtained from the Wilcoxon rank-sum test C : p-value in paired samples obtained from the paired t-test. D : p-value in paired samples obtained from the Wilcoxon signed-rank test * Variables expressed as mean ± standard deviation (SD) and as median and interquartile range (q-q) **Analysis performed with subjects in each group