Pre- and Post-prandial Plasma Ghrelin Levels Do Not Correlate with Satiety or Failure to Achieve a Successful Outcome after Roux-en-Y Gastric

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1 Obesity Surgery, 15, Pre- and Post-prandial Plasma Ghrelin Levels Do Not Correlate with Satiety or Failure to Achieve a Successful Outcome after Roux-en-Y Gastric Bypass Nicolas V. Christou, MD, PhD; Didier Look, MD; Alexander Peter McLean, MD Section of Bariatric Surgery, Division of General Surgery, McGill University Health Center, Montreal, QC, Canada Background: We tested the hypothesis that the amount of weight lost after Roux-en-Y gastric bypass (RYGBP) correlates with plasma ghrelin levels. Methods: 36 morbidly obese patients were studied 3 years after RYGBP (6 men, 30 women) with mean initial BMI 51 kg/m 2 and 8 healthy controls (2 men, 6 women) with mean BMI 25 kg/m 2. Subjects consumed a light breakfast, and the first blood sample was drawn at 1200 hrs immediately before lunch and the second sample at 1400 hrs. Satiety was assessed using a Visual Analog Scale (VAS). Patients were stratified as success (current BMI <35) or failures (current BMI 35). Results: Plasma ghrelin levels were significantly lower in patients after RYGBP (269 ± 66 pcg/ml) compared with lean controls (616 ± 112 pcg/ml, P<0.001). Ghrelin levels pre or post meals were not different between patients who had a successful weight loss (preoperative BMI 47, current BMI 29, 72% EWL) or those who achieved a less then ideal weight loss (preoperative BMI 48, current BMI 41, 29% EWL). There was no correlation between any of the VAS scores and plasma ghrelin. There was a strong inverse correlation between pre-prandial ghrelin levels and the preoperative or current BMI. Conclusion: Failure to lose weight after RYGBP does not correlate with pre- or post-prandial ghrelin plasma levels. Ghrelin levels were inversely proportional to BMI and did not correlate with satiety. These data do not support a role for higher plasma ghrelin levels for inadequate weight loss after RYGBP. Key words: Morbid obesity, ghrelin, weight loss, satiety, gastric bypass Reprint requests to: Nicolas V. Christou, 687 Pine Ave. W., Montreal, Quebec, Canada H3A 1A1. Fax: ; Nicolas.christou@muhc.mcgill.ca Introduction The age-adjusted prevalence of obesity in the United States was 30.5% in While a precise estimate of the change in the prevalence of obesity over time is difficult because of changing definitions, nearly all clinical authorities agree that obesity is reaching epidemic proportions worldwide. 2-7 In response, pharmacological and surgical treatments for weight loss have become both more numerous and more commonly used. Weight reduction achieved by dieting, exercise, or medical therapy often elicits compensatory changes in appetite and energy expenditure that make weight loss of >5-10% unlikely to be sustained. 8 Gastric bypass is thought to undermine these compensatory mechanisms. Ghrelin is a recently discovered orexigenic hormone that is secreted primarily by the stomach and duodenum and has been implicated in both mealtime hunger and the long-term regulation of body weight. 9 In humans, plasma ghrelin levels rise shortly before and fall shortly after every meal, a pattern that is consistent with a role in the urge to begin eating. Cummings et al 10 showed that gastric bypass is associated with markedly suppressed ghrelin levels, possibly contributing to the weightreducing effect of the procedure. We are following several postoperative patients who have not achieved adequate weight loss after gastric bypass. We measured their plasma ghrelin levels and satiety scores before and after a 600-calorie meal, to deter- FD-Communications Inc. Obesity Surgery, 15,

2 Christou et al mine whether we could correlate plasma ghrelin levels and satiety scores to the degree of weight loss. Materials and Methods Study Subjects This study was approved by the McGill University Health Center institutional review board. Patients were selected from our prospectively collected outcomes database following open Roux-en-Y gastric bypass (RYGBP). We studied 16 patients with unacceptable weight loss defined as BMI >35 at 3-5 years after RYGBP. They were compared to a cohort of 20 patients who had an acceptable weight loss defined as BMI <35 at 3-5 years after RYGBP. All patients had upper gastrointestinal series to exclude a gastro-gastric fistula and estimate their pouch size, as well as to look for any other radiological evidence for inadequate weight loss. We also studied 8 healthy lean controls (2 men, 6 women) with mean BMI 25. The characteristics of the study sample are shown in Table 1. Surgical Procedure Both cohorts received the identical open Roux-en-Y isolated gastric bypass. Patients were given 2 g of sodium cephazolin I.V. and 7,500 units of unfractionated heparin S.C. with induction of anesthesia. Exposure was obtained through an upper midline incision. Blunt finger dissection was used to encircle the cardia of the stomach at the angle of His and the lesser curvature of the stomach approximately 5 cm distal to the gastroesophageal junction. At this point, a 2-cm window was made along the lesser curvature of the stomach. A previously placed 32-Fr bougie was held against the lesser curve and a 25- mm EEA (Ethicon Endo-Surgery, Cincinatti, OH, USA) was used to create a circular opening into the stomach. The PI-90 instrument (US Surgical, Norwalk, CT, USA) was passed through this opening (with the help of a 28-Fr chest tube), which was positioned in a vertical orientation against the bougie and fired. A second firing of the PI-90 created a quadruple row of staples, and the stomach was completely separated between the staple-lines. The ligament of Treitz was identified and the jejunum was transected 150 cm distal to it. A Roux limb of jejunum 150 cm long was brought up in a retrocolic, retrogastric fashion and an end-to-side gastrojejunostomy was fashioned using a 3-0 PDS (Ethicon Endo-Surgery) single continuous suture around an 18-Fr nasogastric tube. The jejunojejunostomy was completed, and the mesenteric defects were closed. The nasogastric tube was removed after a satisfactory methylene blue and airbubble leak test. The fascia was closed with a #2 Maxon double suture in a continuous fashion. The subcutaneous tissue was irrigated and carefully dried with clean sterile sponges that had not touched skin. Clips were used to close the skin and an occlusive dressing was applied. Ghrelin and Satiety Measurements Study subjects were asked to come to the Bariatric Clinic at 0800 hrs and were given a light breakfast of apple juice, coffee or tea and 1 slice of dry toast. They Table 1. The demographics and weight characteristics of the controls and patients in this study (mean ± SEM) Controls Patients Successful Unsuccessful P Weight Loss Weight Loss (BMI<35) (BMI>35) Number Age (yrs) 34 ± 4 36 ± 4 37 ± 3 NS Women:Men 6:2 18:2 14:2 NS Preoperative Weight (kg) 61.4 ± ± ± 6.1 NS Preoperative BMI (kg/m 2 ) 24 ± ± ± 2.1 NS Final Weight (kg) ± ± 9.3 P<0.001 % Initial Excess Weight Loss - 72% 29% P<0.001 Final BMI - 29 ± 2 41 ± 3 P< Obesity Surgery, 15, 2005

3 Ghrelin and Weight Loss after Gastric Bypass were kept under observation and away from food until noon (1200 hrs) when the first blood sample was collected in EDTA tubes and stored at 4 C during the collection, after which plasma was stored at -80 C. Following this, they were given a standardized balanced 600-calorie lunch and the second blood sample was collected at 1400 hrs. Plasma immunoreactive ghrelin was measured in duplicate with a radioimmunoassay involving an iodine 125 -labeled bioactive ghrelin tracer and a rabbit polyclonal antibody against full-length, octanoylated human ghrelin that recognizes the acylated and desacyl forms of the hormone (Phoenix Pharmaceuticals, Belmont, CA, USA). The lower and upper limits of detection were 80 and 2500 pg/ml. The ghrelin levels measured in the control subjects is well within the reported values using this particular type of RIA kit (a plethora of publications citing Phoenix's total human Ghrelin RIA kit in their research universally reported levels of normal plasma values ranging from pg/ml). Immediately before the blood collection, subjects were asked to complete a satiety questionnaire using a validated Visual Analog Scale (VAS) 11 as shown in Table 2. Subjects were asked to place a vertical mark along a horizontal line 100 mm long for each question asked. The response was converted to mm and analyzed as described below. Statistical Analysis Table 2. The Visual Analog Scale (VAS) used to determine satiety On each line below (this varies from 0 to 10), place a vertical mark that corresponds to how you feel about each question asked. For example, if you feel no hunger mark closer to 0; if you are extremely hungry mark closer to 10. I am hungry 0 10 I am thirsty 0 10 I feel nauseous 0 10 Amount of food I can eat 0 10 I feel full 0 10 Differences of continuous variables such as starting weight, weight loss, BMI, age, etc. between successful weight loss and failure were examined for statistical significance by Student s t-test. Ghrelin levels and VAS differences between controls, successful weight loss and failed weight loss were analyzed by analysis of variance with Bonferroni correction for multiple t-tests. Linear regression analysis was used to assess the correlation between preoperative weight and ghrelin levels, preoperative BMI and ghrelin, as well as between ghrelin and VAS measurements. The statitistical software used was SYSTAT version 10. Data are reported as mean ± standard error of the mean (SEM). Results The demographics of the study subjects are shown in Table 1. There were no differences in age, men to women ratios, and preoperative weights or BMI of the patients. There were no gastro-gastric fistulas or pouch dilatation to account for the differences in weight loss. The final weights, BMI and % initial excess weight loss was significantly different between the two patient cohorts. The pre-meal satiety scores and ghrelin levels are shown in Table 3. The only significant difference noted was in the higher amount of food that the controls thought they could eat. Ghrelin levels in the controls were 616 ± 100 pcg/ml, significantly higher from that of the patients (Figure 1). Both patient groups had significantly reduced plasma ghrelin compared with non-operated controls. There was no difference in the plasma ghrelin levels between patients who had a successful weight loss after RYGBP, 312 ± 35, compared with those who failed to achieve the expected weight loss, 238 ± 25 (NS). The 1400 hrs post-prandial satiety scores are shown in Table 4. All three groups showed improved satiety scores post-meal. A significant reduction was detected in the plasma ghrelin level of the lean controls after a meal compared with pre-prandial ghrelin as shown in Figure 2. No differences were detected in pre- and post-prandial plasma ghrelin levels in the two patient groups. Multiple relationships were examined using correlation analysis, and no correlation was found between any of the satiety scores at any time-point with their comparable plasma ghrelin levels. A very strong and highly significant inverse correlation was found between preoperative BMI and plasma ghrelin levels at the pre-prandial time-points but not the at 1400 hrs post-prandial time-point. Obesity Surgery, 15,

4 Christou et al Table 3. Visual Analog Scale data and plasma ghrelin levels immediately before lunch (mean ± SEM) Controls Patients (C) Successful (S) Unsuccessful (U) P Weight Loss Weight Loss (BMI<35) (BMI>35) Number I am hungry 49 ± ± 9 33 ± 6 NS I am thirsty 49 ± ± 9 68 ± 5 NS I feel nauseous 12 ± 5 12 ± 6 11 ± 6 NS Amount of food I can eat 52 ± ± 6 31 ± 4 P<0.05 C vs S, U I feel full 26 ± 8 16 ± 7 43 ± 8 NS Ghrelin pcg/ml 616 ± ± ± 25 P<0.05 C vs S, U Discussion The mechanism by which RYGBP causes substantial, permanent weight loss is not known. A hypothesis put forth by Cummings et al 10 is that the exclusion of food from the main part of the stomach results in a reduced production and secretion into the blood of the orexigenic peptide ghrelin. Ghrelin is secreted primarily by the stomach and duodenum and has been implicated in both mealtime hunger and the long-term regulation of body weight. 9 In humans, plasma ghrelin levels rise shortly before and fall shortly after every meal, a pattern that is consistent with a role in the urge to begin eating. Cummings et al 10 showed that gastric bypass is associated with markedly suppressed ghrelin levels, possibly contributing to the weight-reducing effect of the procedure. More recently, they measured plasma ghrelin levels among human subjects initiating meals voluntarily without cues related to time or food. Ghrelin levels decreased shortly after the first meal in all subjects. A subsequent pre-prandial increase occurred over a wide range of inter-meal intervals ( min). Hunger scores and ghrelin levels showed similar temporal profiles and similar relative differences in magnitude between lunch and dinner. The pre-prandial increase in ghrelin levels and the overlap between these levels and hunger scores are consistent with a role for ghrelin in meal initiation. 12 Lin et al 13 observed that a divided RYGBP that creates a small proximal gastric pouch resulted in sig- m / g c p n i l e r h G Ghrelin pcg/ml Pre-Lunch Ghrelin Levels P<0.001 vs F and S P<0.001vs. F and C NS vs. F C F S m / g c p n i l e r h G Ghrelin pcg/ml Post-Meal 1400 hrs Ghreliin P=0.067 P<0.067 vs. F and vs F vs. S and vs S C F S Figure 1. Ghrelin levels immediately before meals and 2 hours after a meal between normal-weight control subjects (C) and morbidly obese subjects that had successful (BMI <35, S) and unsuccessful (BMI >35, F) weight loss Obesity Surgery, 15, 2005

5 Ghrelin and Weight Loss after Gastric Bypass Table 4. Visual Analog Scale data and plasma ghrelin levels at the 1400 hrs post-prandial sample (mean ± SEM) Controls Patients (C) Successful (S) Unsuccessful (U) P Weight Loss Weight Loss (BMI<35) (BMI>35) Number I am hungry 20 ± 9 12 ± 7 13 ± 6 NS I am thirsty 46 ± ± 9 32 ± 8 NS I feel nauseous 6 ± 2 7 ± 6 12 ± 5 NS Amount of food I can eat 17 ± 7 17 ± 6 12 ± 6 NS I feel full 68 ± 8 78 ± 7 66 ± 36 NS Ghrelin pcg/ml 430 ± ± ± 57 NS nificant early declines in circulating ghrelin levels that were not observed with other gastric procedures that did not divert food from the main stomach. They suggested that this may explain, in part, the loss of hunger sensation and rapid weight loss observed following gastric bypass surgery. 13 Adami et al 14 found no evidence upholding a relationship between serum ghrelin concentration and food intake after BPD. A sharp drop was observed in body weight and BMI values 12 months after BPD, whereas serum ghrelin levels increased at 12 months, when food intake had returned to preoperative levels. Geloneze et al 15 found that fasting plasma ghrelin concentrations were 56% lower in patients after gastric bypass compared with lean controls. They suggested that reduced production of ghrelin after gastric bypass could be partly responsible for the lack of hyperphagia and thus for the weight loss. This hypothesis was not corroborated by the study of Stoeckl et al 16 who found that plasma ghrelin levels failed to increase during substantial weight loss after gastric bypass, but did increase in response to lesser weight loss after adjustable gastric banding. Morinigo et al 17 measured ghrelin response to a standardized test meal in eight morbidly obese patients before and 6 weeks after RYGBP. Ghrelin response was compared with that of an age-matched group of six normal-weight individuals. They found that fasting serum ghrelin levels were lower in obese subjects compared with controls (P<0.05). Meal ingestion significantly suppressed ghrelin concentration in controls (P<0.05) and obese subjects (P<0.05), albeit to a lesser degree in the latter group (P<0.05). Despite a 10% excess weight loss, fasting Ghrelin mcg/ml Ghrelin pcg/ml P<0.05 Pre-Meal Post-Meal Lean Controls Success Failure Figure 2. The pre-/post-prandial ghrelin levels between lean controls, patients with acceptable weight loss after RYGBP and those with an unacceptable weight loss. Obesity Surgery, 15,

6 Christou et al serum ghrelin levels were paradoxically further decreased in obese subjects 6 weeks after gastric bypass and food intake did not elicit a significant ghrelin suppression, indicating that the adaptive response of ghrelin to body weight loss was already impaired 6 weeks after RYGBP. Holdstock et al 18 measured serum ghrelin in 12 men and 54 women with mean age 39 years, mean weight 127 kg and mean BMI 45 kg/m 2 before and 12 months after RYGBP. Ghrelin increased from 55 fmol/ml to 85 fmol/ml (+58%) at 12 months after surgery. The changes were all related to the reduction in BMI. Schindler et al 19 studied 23 morbidly obese subjects who underwent laparoscopic adjustable gastric banding. Six months after surgery, body weight decreased significantly by 15.7 ± 1.4 kg which was accompanied by an increased cognitive restraint of eating, and by a decreased disinhibition of eating and susceptibility to hunger. Plasma ghrelin increased by 27% from 100 ± 13 to 127 ± 13 fmol/ml. The change in plasma ghrelin correlated with changes in body weight and fat mass, but not with changes in fat-free mass and in the three dimensions of eating behavior. They suggest that changes in eating behavior, which promote reduction of food intake and not fasting ghrelin, determine weight loss achieved by adjustable gastric banding. Nijhuis et al 20 measured ghrelin levels before and 2 years after gastric restrictive surgery (vertical banded gastroplasty or adjustable gastric banding), and found that BMI decreased from 47.5 ± 6.2 kg/m 2 to 33.2 ± 5.8 kg/m 2 whereas plasma ghrelin increased from 742 ± 246 pg/ml preoperatively to 904 ± 127 pg/ml 2 years postoperatively. Korner et al 21 measured plasma ghrelin in the fasted state and in response to a liquid test meal in lean, weight-stable post-rygbp (BMI 33 kg/m 2 at 35 ± 5 months) and obese non-operated subjects matched to the surgical group for BMI and age. Fasting plasma ghrelin levels were nearly identical between RYGBP (425 ± 54 pg/ml) and the matched controls (424 ± 28 pg/ml), and highest in lean controls (564 ± 103 pg/ml). The response to the test meal was comparable between lean and RYGBP groups with 27% and 20% maximal suppression, respectively, whereas the magnitude of suppression was significantly diminished in the matched controls (17%) compared with the lean group. The absence of a compensatory increase in ghrelin concentrations that usually occurs with diet-induced weight loss, may contribute to weight loss and to the ability of an individual to maintain weight loss after RYGBP. Frühbeck et al 22 studied 24 matched obese male patients in whom similar weight loss had been achieved by either adjustable gastric banding (n=8), RYGBP (n=8) or dieting (n=8) before and 6 months after treatment, as well as 6 patients undergoing total gastrectomy. Comparable weight loss after 6 months exerted significantly different effects on plasma ghrelin concentrations, depending on the procedure applied (adjustable gastric banding: ± 32.8 pg/ml; gastric bypass: ± 13.5; dieting: ± 18.7; P<0.001). Without significant differences in body weight and BMI, patients who had undergone the gastric bypass exhibited a statistically significant decrease in fasting ghrelin concentrations, while the other two procedures (adjustable gastric banding and dieting) showed a weight loss-induced increase in ghrelin levels. Despite significant differences in BMI between gastric bypass and total gastrectomy patients after 6 months (31.9 ± 2.2 vs 22.0 ± 0.7 kg/m 2, respectively), both groups showed similar ghrelin concentrations. They suggest that the reduction in circulating ghrelin concentrations in RYGBP patients after 6 months of surgery are not determined by an active weight loss but rather depend on the surgicallyinduced bypass of the ghrelin-producing cell population of the fundus. Our study shows similar findings to these previously reported studies in that RYGBP resulted in significant reduction in pre- and post-prandial plasma ghrelin levels up to 3-5 years after RYGBP. This reduction in ghrelin levels does not correlate with satiety scores and is strongly but inversely correlated with preoperative BMI. We could not detect differences in ghrelin levels between patients who demonstrated a successful weight loss (70% initial excess weight loss) and those who we considered failed to achieve sufficient weight loss (29% initial excess weight loss). We also could not detect a difference in satiety scores, e.g. increased hunger or food craving in those with failed weight loss compared with those with successful weight loss. It is clear from our data and those of others that RYGBP that excludes food from the main part of the stomach results in marked reduction in ghrelin production despite permanent reduction of the initial BMI Obesity Surgery, 15, 2005

7 Ghrelin and Weight Loss after Gastric Bypass Conclusions In summary, our findings suggest that weight loss after gastric bypass is controlled by multiple mechanisms and that plasma ghrelin levels do not explain the failure of some morbidly obese patients to achieve a successful weight loss. References 1. Flegal KM, Carroll MD, Ogden CL. Prevalence and trends in obesity among US adults, JAMA 2002; 288: Mokdad AH, Bowman BA, Ford ES. The continuing epidemics of obesity and diabetes in the United States. JAMA 2001; 286: Centers for Disease Control and Prevention. Obesity epidemic increases dramatically in the United States. Available at URL: Accessed 6/11/ Kuczmarski RJ, Flegal KM, Campbell SM. Increasing prevalence of overweight among US adults. The National Health and Nutrition Examination Surveys, 1960 to JAMA 1994; 272: Katzmarzyk PT. The Canadian obesity epidemic, CMAJ 2002; 166: Friedrich MJ. Epidemic of obesity expands its spread to developing countries. JAMA 2002; 287: Pi-Sunyer X. A clinical view of the obesity problem. Science 2003; 299 (5608): Bray GA, Tartaglia LA. Medicinal strategies in the treatment of obesity. Nature 2000; 404 (6778): Kojima M, Hosoda H, Date Y et al. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 1999; 402 (6762): Cummings DE, Weigle DS, Frayo RS et al. Plasma ghrelin levels after diet-induced weight loss or gastric bypass surgery. N Engl J Med 2002; 346: Flint A, Raben A, Blundell JE et al. Reproducibility, power and validity of visual analogue scales in assessment of appetite sensations in single test meal studies. Int J Obes 2000; 24: Cummings DE, Frayo RS, Marmonier C et al. Plasma ghrelin levels and hunger scores among humans initiating meals voluntarily in the absence of time- and food-related cues. Am J Physiol Endocrinol Metab 2004; 287: E Lin E, Gletsu N, Fugate K et al. The effects of gastric surgery on systemic ghrelin levels in the morbidly obese. Arch Surg 2004; 139: Adami GF, Cordera R, Andraghetti G et al. Changes in serum ghrelin concentration following biliopancreatic diversion for obesity. Obes Res 2004; 12: Geloneze B, Tambascia MA, Pilla VF et al. Ghrelin: a gut-brain hormone: effect of gastric bypass surgery. Obes Surg 2003; 13: Stoeckli R, Chanda R, Laanger I et al Changes of body weight and plasma ghrelin levels after gastric banding and gastric bypass. Obes Res 2004; 12: Morinigo R, Casamitjana R, Moize V et al. Shortterm effects of gastric bypass surgery on circulating ghrelin levels. Obes Res 2004; 12: Holdstock C, Engstrom BE, Ohrvall M et al. Effect of bariatric surgery on adipose tissue regulatory peptides and growth hormone secretion. Asia Pac J Clin Nutr 2004; 13 (Suppl): S Schindler K, Prager G, Ballaban T et al. Impact of laparoscopic adjustable gastric banding on plasma ghrelin, eating behaviour and body weight. Eur J Clin Invest 2004; 34: Nijhuis J, van Dielen FM, Buurman WA et al. Ghrelin, leptin and insulin levels after restrictive surgery: a 2-year follow-up study. Obes Surg 2004; 14: Korner J, Bessler M, Cirilo L et al. Effects of Rouxen-Y gastric bypass surgery on fasting and postprandial concentrations of plasma ghrelin, PYY and insulin. J Clin Endocrinol Metab First published October 13, 2004 as doi: /jc Frühbeck G, Rotellar F, Hernandez-Lizoain JL et al. Fasting plasma ghrelin concentrations 6 months after gastric bypass are not determined by weight loss or changes in insulinemia. Obes Surg 2004; 14: (Received May 10, 2005; accepted June 3, 2005) Obesity Surgery, 15,