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obestatin ratio in subjects with Helicobacter pylori infection
Nutrition 25 (2009) 506 –511 www.nutritionjrnl.com
Applied nutritional investigation
Circulating ghrelin/obestatin ratio in subjects with Helicobacter pylori infection Xin-Yuan Gao, M.D.a, Hong-Yu Kuang, M.D.a,*, Xiao-Min Liu, M.M.a,*, Peng Duan, M.M.b, Yi Yang, M.D.c, and Zhi-Bin Ma, M.M.d a
Department of Endocrinology, The First Clinical Hospital of Harbin Medical University, Harbin, China b Department of Endocrinology, The Third Hospital of NanChang, NanChang, China c Department of Pathology, The First Clinical Hospital of Harbin Medical University, Harbin, China d Department of Gastroenterology, The First Clinical Hospital of Harbin Medical University, Harbin, China Manuscript received August 19, 2008; accepted November 4, 2008.
Abstract
Objective: Ghrelin is a peptide hormone involved in human energy homeostasis. Obestatin is a recently discovered active peptide derived from preproghrelin. It seemed that obestatin was a physiologic opponent of ghrelin. Helicobacter pylori infection may be associated with appetite and nutrition. We compared the plasma ghrelin/obestatin ratio in H. pylori–positive and -negative groups. Methods: People undergoing an annual health checkup were included. Helicobacter pylori status was based on serologic and carbon-13 urea breath findings. Fifty adults with H. pylori infection and 50 adults matched by age and body mass index without H. pylori infection were enrolled in this study. Plasma ghrelin and obestatin levels were measured by radioimmunoassay. Results: Ghrelin concentrations and ghrelin/obestatin ratios were lower in the H. pylori–positive group than in the H. pylori–negative group. There was no significant difference in circulating obestatin between those with and without H. pylori infection. In all subjects, the ghrelin/obestatin ratio was negatively correlated with body mass index, the homeostasis model of assessment for insulin resistance, and serum levels of triacylglycerol. There was a positive correlation between circulating obestatin and ghrelin levels. Conclusion: Helicobacter pylori infection was associated with a reduction in the circulating ghrelin/obestatin ratio in Chinese adults. © 2009 Published by Elsevier Inc.
Keywords:
Ghrelin; Obestatin; Helicobacter pylori; Radioimmunoassay
Introduction Ghrelin is a 28-amino acid peptide that plays a role in food intake and energy metabolism. [1,2]. Central and peripheral ghrelin administrations lead to increased appetite and weight gain [3,4]. In 2005, a new peptide named obestatin was isolated from rat stomach, which derived from the same gene as ghrelin [5]. In contrast to ghrelin, obestatin seemed to have opposing effects to ghrelin’s actions on energy homeostasis and gastrointestinal function [5,6]. However, several recent studies could not reproduce its initially reported anorexigenic property [7,8]. The interaction between ghrelin and obestatin is still under debate. * Co-corresponding authors. Tel./fax: ⫹86-451-5364-5060. E-mail address: [email protected] (H.-Y. Kuang, X.-M. Liu). 0899-9007/09/$ – see front matter © 2009 Published by Elsevier Inc. doi:10.1016/j.nut.2008.11.002
Much of the current research is focused on the ghrelin/ obestatin ratio in obesity and related metabolic disorders. Guo et al. [9] found that obese subjects exhibited a higher plasma preprandial ghrelin/obestatin ratio than healthy normalweight controls, whereas another study confirmed that the ghrelin/obestatin ratio was decreased in obese women [10]. There is a decreased tendency of the ghrelin/obestatin ratio in obese children after weight reduction [11]. This suggests that the balance of ghrelin and obestatin might play a role in the regulation of energy homeostasis. Helicobacter pylori is a gram-negative bacteria that plays a crucial role in gastritis, peptic ulcer disease, and gastric malignant lesions [12,13]. The prevalence of H. pylori infection was high in China [14]. Furuta et al. [15] found that H. pylori infection and its eradication had an effect on nutrition. It seemed that factors other than elimi-
X.-Y. Gao et al. / Nutrition 25 (2009) 506 –511
nation of pathology could contribute to the increased appetite. Helicobacter pylori infection may affect the expression and release of gastric-originated appetite-controlling hormones. In some studies, H. pylori infection has been associated with low circulating ghrelin levels [16,17]. However, there was no significant difference of plasma ghrelin according to different H. pylori statuses in many reports [18,19]. To the best of our knowledge, no data had been published about plasma obestatin levels in asymptomatic subjects with H. pylori infection. Therefore, we hypothesized that H. pylori infection affects the expression of ghrelin and obestatin, which may play a role in nutrition and appetite. Many digestive tract diseases have an effect on circulating ghrelin and obestain levels. Plasma ghrelin levels have varied widely in diverse conditions of the upper digestive tract [20]. We previously found that chronic atrophic gastritis influenced the plasma ghrelin/obestatin ratio in elderly men [21]. In the present study, we determined the plasma ghrelin/obestatin ratio in H. pylori–positive and -negative subjects.
Materials and methods Subjects and research design The study was approved by Harbin Medical University ethics committee. All samples were obtained with written informed consent of the patients before their inclusion, in accordance with the Helsinki Declaration. Asymptomatic adults who underwent health checkups at our hospital between June 2007 and June 2008 were observed. All subjects underwent upper gastrointestinal endoscopy after ⬎6 h of fasting. None presented a macroscopic lesion of the esophageal, gastric, or duodenal mucosa by endoscopic examination. A complete medical history and physical examination were carried out. The exclusion criteria were an age ⬍18 or ⬎70 y, a body mass index (BMI) ⬎25 or ⬍18.5 kg/m2, use of medications effective against H. pylori, use of analgesics or non-steroidal anti-inflammatory drugs during the preceding 3 mo, alcohol abuse, and pregnancy. None had diabetes mellitus, systemic infection, thyroid and liver diseases, heart failure, renal impairment, psychiatric disease, or any other conditions known to affect endocrine and gastrointestinal functions.
507
⫺80°C until assayed. Plasma glucose levels were measured by an automated glucose oxidase method. Serum levels of total cholesterol, triacylglycerol, and high-density lipoprotein cholesterol were measured by enzymatic methods using an autoanalyzer. Serum anti–H. pylori and anti–Cag immunoglobulin G (IgG) was measured using a commercial ELISA kit (EIAGEN HP IgG, Clone Systems, Italy). Results showing an anti-Hp IgG ⬎15 arbitrary units/mL and an anti-CagA ⬎0.3 optical density were considered seropositive [22]. Carbon-13 urea breath tests were analyzed using a mass spectrometer, with 2.5 over baseline being a positive result. Helicobacter pylori infection was considered present when the urea breath test and the serologic tests were positive. Helicobacter pylori infection was considered negative if all test results were negative. Fasting plasma obestatin levels were measured with a commercial radioimmunoassay (RIA) kit (Phoenix Pharmaceuticals). The same protocol was used in another clinical study that has been published [23]. Levels of plasma ghrelin were determined using commercially RIA kits (Phoenix Pharmaceuticals), as described previously [24]. This RIA system can measure the acylated and des-acyl forms of ghrelin. RIA has been demonstrated by high-performance liquid chromatography to measure the intact peptide in the type of blood sample being analyzed [25]. Plasma insulin levels were measured using an enzyme-linked immunoassay kit. Insulin resistance was calculated by the homeostasis model of assessment for insulin resistance (HOMA-IR) approach, calculated as fasting insulin (microunits per milliliter) ⫻ fasting blood glucose (millimoles per liter)/22.5 [26]. Statistics Data are expressed as mean ⫾ standard deviation. All variables were normally distributed as tested by the Kolmogorov-Smirnov test. Homogeneity of variances was calculated by Levene’s test. Unpaired Student’s t test was used to assess significant differences in two groups where appropriate. The relation between the plasma ghrelin/obestatin ratio and BMI, lipid profile, or HOMA-IR was examined by bivariate correlations. P ⬍ 0.05 was considered statistically significant. Analyses were performed using SPSS 13.0 for Windows (SPSS Inc., Chicago, IL, USA).
Specimen collection and biochemical analysis
Results
After overnight fasting for 12 h, blood samples were drawn in all 100 subjects at 0800 h. Blood samples for measurement of obestatin were drawn into chilled polypropylene tubes containing ethylene-diaminetetra-acetic acid/ 2Na (1 mg/mL) and aprotinin (Phoenix Pharmaceuticals, Belmont, CA, USA; 100 L containing 0.6 U of trypsin inhibitor per milliliter of blood). After immediate centrifugation at 4°C for 15 min, plasma samples were stored at
Of 257 adults observed, 42 subjects had indeterminant results for H. pylori status and were excluded. Fifty H. pylori–positive and 50 H. pylori–negative adults matched for age (⫾5 y) and BMI (⫾1 kg/m2) were enrolled for the ghrelin and obestatin study. As presented in Table 1, age, gender, BMI, HOMA-IR, and lipid profile were similar in the H. pylori–negative and -positive groups. Plasma ghrelin levels (Fig. 1A) were significantly lower in the H. pylori–
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Table 1 Characteristics of the two different groups* Helicobacter pylori status
Age (y) Weight (kg) BMI (kg/m2) Men/women Fasting blood glucose (mmol/L) Fasting insulin (mU/L) HOMA-IR Total cholesterol (mmol/L) HDL cholesterol (mmol/L) LDL cholesterol (mmol/L) Triacylglycerol (mmol/L)
Negative
Positive
43.3 ⫾ 2.9 62.5 ⫾ 6.8 21.5 ⫾ 1.2 25/25 5.0 ⫾ 0.1 7.5 ⫾ 2.2 1.7 ⫾ 0.3 4.4 ⫾ 0.3 1.3 ⫾ 0.2 2.8 ⫾ 0.2 1.1 ⫾ 0.2
45.9 ⫾ 2.4 60.7 ⫾ 7.6 21.2 ⫾ 0.7 26/24 5.1 ⫾ 0.1 7.3 ⫾ 2.9 1.6 ⫾ 0.2 4.3 ⫾ 0.4 1.2 ⫾ 0.2 2.9 ⫾ 0.1 1.2 ⫾ 0.2
BMI, body mass index; HDL, high-density lipoprotein; HOMA-IR, homeostasis model of assessment for insulin resistance; LDL, low-density lipoprotein * Values are means ⫾ SDs.
positive group than in the H. pylori–negative group (P ⬍ 0.01). There was no significant difference in circulating obestatin levels (Fig. 1B) between those with and without H. pylori infection (P ⬎ 0.05). The plasma ghrelin/obestatin ratio (Fig. 1C) was decreased in subjects with H. pylori infection (P ⬍ 0.01). The ghrelin/obestatin ratio was negatively correlated with BMI and HOMA-IR in H. pylori– negative (r ⫽ ⫺0.644, P ⬍ 0.001, and r ⫽ ⫺0.505, P ⬍ 0.001, respectively) and H. pylori–positive (r ⫽ ⫺0.693, P ⬍ 0.001, and r ⫽ ⫺0.367, P ⫽ 0.009, respectively) subjects. There was no significant correlation between the ghrelin/obestatin ratio and serum lipids in H. pylori–negative and H. pylori–positive subjects. The ghrelin/obestatin ratio was negatively correlated with BMI (r ⫽ ⫺0.296, P ⫽ 0.003), HOMA-IR (r ⫽ ⫺0.224, P ⫽ 0.025), and serum levels of triacylglycerol (r ⫽ ⫺0.202, P ⫽ 0.044) in all subjects (Fig. 2A–C). There was a positive correlation between circulating obestatin and ghrelin in the H. pylori– negative (r ⫽ 0.652, P ⬍ 0.001) and H. pylori–positive (r ⫽ 0.494, P ⬍ 0.001) groups. Obestatin concentrations were correlated with ghrelin concentrations (r ⫽ 0.430, P ⬍ 0.001) in all subjects (Fig. 3).
ies have found that obestatin plays a role in the regulation of energy homeostasis [32,33]. The intricate balance of ghrelin and obestatin in humans is interesting. In our study, H. pylori–positive subjects had lower ghrelin blood levels and a lower ghrelin/obestatin ratio. Helicobacter pylori are a persistent colonizer of the human stomach and affect various cell types in gastric mucosa. The stomach is a major source of circulating ghrelin, and plasma ghrelin levels were significantly lower in patients after gastrectomy [34,35]. Ghrelin is primarily produced by X/A-like cells in the gastric mucosa [36]. There are some data in the literature about the relation between H. pylori and ghrelin. Many researchers have found that total plasma ghrelin decreases in patients with gastric atrophy secondary to H. pylori infection [16,37]. This might be explained by the loss of ghrelin-producing cells caused by inflammation and/or gastric atrophy associated with the infection. Furthermore, plasma ghrelin levels have been found to be lower in healthy subjects with H. pylori infection [17,29]. In agreement with these reports, we found that H. pylori infection was associated with a reduction in circulating ghrelin in healthy Chinese adults. It remains unclear what factors related to H. pylori infection influence plasma ghrelin levels
Discussion Helicobacter pylori infection may be responsible for dyspeptic symptoms and reduced appetite. Some studies have shown that elimination of H. pylori increases nutrition and weight not only in patients but also in healthy subjects [27,28]. Its role requires further research. Helicobacter pylori infection seemed to alter serum levels of some appetiteaffecting gastric hormones [29]. Ghrelin is an orexigenic gastrointestinal hormone. Obestatin is generated from proteolytic cleavage of preproghrelin. Although there have been some controversies concerning obestatin [30,31], many stud-
Fig. 1. Plasma ghrelin levels (A), obestatin levels (B), and ghrelin/obestain ratio (C) in the Helicobacter pylori–positive and -negative groups. *P ⬍ 0.01 compared with subjects without H. pylori infection.
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Fig. 2. Correlation between ghrelin/obestatin ratio and BMI (A), HOMA-IR (B), and triacylglycerol (C) in all subjects. BMI, body mass index; HOMA-IR, homeostasis model of assessment for insulin resistance.
in healthy subjects. There is a probable direct effect of H. pylori on the survival of the ghrelin-producing cells, but Cindoruk et al. [38] found that H. pylori infection had no effect on plasma ghrelin levels in patients without atrophic gastritis. The discrepancy of these results may be due to the different ages and ethnicities of subjects across different studies. Obestatin has proved to be primarily produced by the same neuroendocrine cells in the oxyntic glands as ghrelin [39]. However, we did not find any effect on plasma obesta-
tin levels of H. pylori infection. The mechanisms of this result are not yet known. One possibility is that obestatin is released from many tissues, contributing to a certain amount of circulating obestatin. Including the gastrointestinal mucosa, obestatin immunoreactivity is expressed in several other tissues, such as the perinatal pancreas [40], myenteric plexus, Leydig cells [41], and the salivary gland [42]. Although obestatin and ghrelin are derived from the same prohormone, their main source seemed to be different. Plasma obestatin levels tended to be lower in patients after
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In summary, ghrelin/obestatin ratios were decreased in H. pylori–infected subjects. The balance of ghrelin and obestatin was changed in the presence of H. pylori infection.
References
Fig. 3. Correlation between plasma obestatin and ghrelin levels in all subjects.
gastrectomy, but did not reach statistical significance [34]. The significant quantities of obestain outside the stomach may have been sufficient to compensate for the altered plasma obestatin levels in the H. pylori–positive group. Furthermore, ghrelin and obestatin may be regulated differently in the presence of H. pylori infection. In this study, we confirmed that the ghrelin/obestatin ratio was lower in the H. pylori–positive group than in the H. pylori–negative group. The alteration in circulating blood levels may play a role in the pathophysiology of H. pylori infection. Moreover, the ghrelin/obestatin ratio was negatively correlated with BMI, HOMA-IR, and triacylglycerol. A negative correlation of the ghrelin/obestatin ratio with BMI suggests that it may have a role in body weight control. It seemed that insulin may affect the regulation of ghrelin and obestatin secretion. Insulin excess has been proved to decrease circulating ghrelin concentrations in rodents [43]. Ren et al. [44] found that obestatin inhibits glucose-stimulated insulin secretion from isolated rat islets and in vivo. The balance of ghrelin and obestatin may have a role in glucose and lipid metabolism. The present study showed that plasma ghrelin decreased in the H. pylori–positive group compared with controls. Helicobacter pylori infection had no effect on plasma obestatin concentration. This implies that obestatin is not a non-functional connective peptide followed by the release of ghrelin. In agreement with previous reports [34,45], plasma obestatin levels were positively correlated with ghrelin in all subjects. This supports the notion that expression of the ghrelin gene may regulate the two peptides.
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[33] Zamrazilová H, Hainer V, Sedlácková D, Papezová H, Kunesová M, Bellisle F, et al. Plasma obestatin levels in normal weight, obese and anorectic women. Physiol Res 2008;57(suppl 1):S49 –55. [34] Huda MS, Durham BH, Wong SP, Deepak D, Kerrigan D, McCulloch P, et al. Plasma obestatin levels are lower in obese and post-gastrectomy subjects, but do not change in response to a meal. Int J Obesity (Lond) 2008;32:129 –35. [35] Ariyasu H, Takaya K, Tagami T, Ogawa Y, Hosoda K, Akamizu T, et al. Stomach is a major source of circulating ghrelin, and feeding state determines plasma ghrelin-like immunoreactivity levels in humans. J Clin Endocrinol Metab 2001;86:4753– 8. [36] Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 1999;402:656 – 60. [37] Isomoto H, Ueno H, Saenko VA, Mondal MS, Nishi Y, Kawano N, et al. Impact of Helicobacter pylori infection on gastric and plasma ghrelin dynamics in humans. Am J Gastroenterol 2005;100:1711–20. [38] Cindoruk M, Yetkin I, Deger SM, Karakan T, Kan E, Unal S. Influence of H pylori on plasma ghrelin in patients without atrophic gastritis. World J Gastroenterol 2007;13:1595– 8. [39] Zhao CM, Furnes MW, Stenström B, Kulseng B, Chen D. Characterization of obestatin- and ghrelin-producing cells in the gastrointestinal tract and pancreas of rats: an immunohistochemical and electronmicroscopic study. Cell Tissue Res 2008;331:575– 87. [40] Chanoine JP, Wong AC, Barrios V. Obestatin, acylated and total ghrelin concentrations in the perinatal rat pancreas. Horm Res 2006; 66:81– 8. [41] Dun SL, Brailoiu GC, Brailoiu E, Yang J, Chang JK, Dun NJ. Distribution and biological activity of obestatin in the rat. Endocr J 2006;191:481–9. [42] Ozbay Y, Aydin S, Dagli AF, Akbulut M, Dagli N, Kilic N, et al. Obestatin is present in saliva: alterations in obestatin and ghrelin levels of saliva and serum in ischemic heart disease. BMB Rep 2008;31:55– 61. [43] McCowen KC, Maykel JA, Bistrian BR, Ling PR. Circulating ghrelin concentrations are lowered by intravenous glucose or hyperinsulinemic euglycemic conditions in rodents. Endocrinology 2002;175:R7–11. [44] Ren AJ, Guo ZF, Wang YK, Wang LG, Wang WZ, Lin L, et al. Inhibitory effect of obestatin on glucose-induced insulin secretion in rats. Biochem Biophys Res Commun 2008;369:969 –72. [45] Park WH, Oh YJ, Kim GY, Kim SE, Paik KH, Han SJ, et al. Obestatin is not elevated or correlated with insulin in children with Prader-Willi syndrome. J Clin Endocrinol Metab 2007;92:229 –34.
Applied nutritional investigation
Circulating ghrelin/obestatin ratio in subjects with Helicobacter pylori infection Xin-Yuan Gao, M.D.a, Hong-Yu Kuang, M.D.a,*, Xiao-Min Liu, M.M.a,*, Peng Duan, M.M.b, Yi Yang, M.D.c, and Zhi-Bin Ma, M.M.d a
Department of Endocrinology, The First Clinical Hospital of Harbin Medical University, Harbin, China b Department of Endocrinology, The Third Hospital of NanChang, NanChang, China c Department of Pathology, The First Clinical Hospital of Harbin Medical University, Harbin, China d Department of Gastroenterology, The First Clinical Hospital of Harbin Medical University, Harbin, China Manuscript received August 19, 2008; accepted November 4, 2008.
Abstract
Objective: Ghrelin is a peptide hormone involved in human energy homeostasis. Obestatin is a recently discovered active peptide derived from preproghrelin. It seemed that obestatin was a physiologic opponent of ghrelin. Helicobacter pylori infection may be associated with appetite and nutrition. We compared the plasma ghrelin/obestatin ratio in H. pylori–positive and -negative groups. Methods: People undergoing an annual health checkup were included. Helicobacter pylori status was based on serologic and carbon-13 urea breath findings. Fifty adults with H. pylori infection and 50 adults matched by age and body mass index without H. pylori infection were enrolled in this study. Plasma ghrelin and obestatin levels were measured by radioimmunoassay. Results: Ghrelin concentrations and ghrelin/obestatin ratios were lower in the H. pylori–positive group than in the H. pylori–negative group. There was no significant difference in circulating obestatin between those with and without H. pylori infection. In all subjects, the ghrelin/obestatin ratio was negatively correlated with body mass index, the homeostasis model of assessment for insulin resistance, and serum levels of triacylglycerol. There was a positive correlation between circulating obestatin and ghrelin levels. Conclusion: Helicobacter pylori infection was associated with a reduction in the circulating ghrelin/obestatin ratio in Chinese adults. © 2009 Published by Elsevier Inc.
Keywords:
Ghrelin; Obestatin; Helicobacter pylori; Radioimmunoassay
Introduction Ghrelin is a 28-amino acid peptide that plays a role in food intake and energy metabolism. [1,2]. Central and peripheral ghrelin administrations lead to increased appetite and weight gain [3,4]. In 2005, a new peptide named obestatin was isolated from rat stomach, which derived from the same gene as ghrelin [5]. In contrast to ghrelin, obestatin seemed to have opposing effects to ghrelin’s actions on energy homeostasis and gastrointestinal function [5,6]. However, several recent studies could not reproduce its initially reported anorexigenic property [7,8]. The interaction between ghrelin and obestatin is still under debate. * Co-corresponding authors. Tel./fax: ⫹86-451-5364-5060. E-mail address: [email protected] (H.-Y. Kuang, X.-M. Liu). 0899-9007/09/$ – see front matter © 2009 Published by Elsevier Inc. doi:10.1016/j.nut.2008.11.002
Much of the current research is focused on the ghrelin/ obestatin ratio in obesity and related metabolic disorders. Guo et al. [9] found that obese subjects exhibited a higher plasma preprandial ghrelin/obestatin ratio than healthy normalweight controls, whereas another study confirmed that the ghrelin/obestatin ratio was decreased in obese women [10]. There is a decreased tendency of the ghrelin/obestatin ratio in obese children after weight reduction [11]. This suggests that the balance of ghrelin and obestatin might play a role in the regulation of energy homeostasis. Helicobacter pylori is a gram-negative bacteria that plays a crucial role in gastritis, peptic ulcer disease, and gastric malignant lesions [12,13]. The prevalence of H. pylori infection was high in China [14]. Furuta et al. [15] found that H. pylori infection and its eradication had an effect on nutrition. It seemed that factors other than elimi-
X.-Y. Gao et al. / Nutrition 25 (2009) 506 –511
nation of pathology could contribute to the increased appetite. Helicobacter pylori infection may affect the expression and release of gastric-originated appetite-controlling hormones. In some studies, H. pylori infection has been associated with low circulating ghrelin levels [16,17]. However, there was no significant difference of plasma ghrelin according to different H. pylori statuses in many reports [18,19]. To the best of our knowledge, no data had been published about plasma obestatin levels in asymptomatic subjects with H. pylori infection. Therefore, we hypothesized that H. pylori infection affects the expression of ghrelin and obestatin, which may play a role in nutrition and appetite. Many digestive tract diseases have an effect on circulating ghrelin and obestain levels. Plasma ghrelin levels have varied widely in diverse conditions of the upper digestive tract [20]. We previously found that chronic atrophic gastritis influenced the plasma ghrelin/obestatin ratio in elderly men [21]. In the present study, we determined the plasma ghrelin/obestatin ratio in H. pylori–positive and -negative subjects.
Materials and methods Subjects and research design The study was approved by Harbin Medical University ethics committee. All samples were obtained with written informed consent of the patients before their inclusion, in accordance with the Helsinki Declaration. Asymptomatic adults who underwent health checkups at our hospital between June 2007 and June 2008 were observed. All subjects underwent upper gastrointestinal endoscopy after ⬎6 h of fasting. None presented a macroscopic lesion of the esophageal, gastric, or duodenal mucosa by endoscopic examination. A complete medical history and physical examination were carried out. The exclusion criteria were an age ⬍18 or ⬎70 y, a body mass index (BMI) ⬎25 or ⬍18.5 kg/m2, use of medications effective against H. pylori, use of analgesics or non-steroidal anti-inflammatory drugs during the preceding 3 mo, alcohol abuse, and pregnancy. None had diabetes mellitus, systemic infection, thyroid and liver diseases, heart failure, renal impairment, psychiatric disease, or any other conditions known to affect endocrine and gastrointestinal functions.
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⫺80°C until assayed. Plasma glucose levels were measured by an automated glucose oxidase method. Serum levels of total cholesterol, triacylglycerol, and high-density lipoprotein cholesterol were measured by enzymatic methods using an autoanalyzer. Serum anti–H. pylori and anti–Cag immunoglobulin G (IgG) was measured using a commercial ELISA kit (EIAGEN HP IgG, Clone Systems, Italy). Results showing an anti-Hp IgG ⬎15 arbitrary units/mL and an anti-CagA ⬎0.3 optical density were considered seropositive [22]. Carbon-13 urea breath tests were analyzed using a mass spectrometer, with 2.5 over baseline being a positive result. Helicobacter pylori infection was considered present when the urea breath test and the serologic tests were positive. Helicobacter pylori infection was considered negative if all test results were negative. Fasting plasma obestatin levels were measured with a commercial radioimmunoassay (RIA) kit (Phoenix Pharmaceuticals). The same protocol was used in another clinical study that has been published [23]. Levels of plasma ghrelin were determined using commercially RIA kits (Phoenix Pharmaceuticals), as described previously [24]. This RIA system can measure the acylated and des-acyl forms of ghrelin. RIA has been demonstrated by high-performance liquid chromatography to measure the intact peptide in the type of blood sample being analyzed [25]. Plasma insulin levels were measured using an enzyme-linked immunoassay kit. Insulin resistance was calculated by the homeostasis model of assessment for insulin resistance (HOMA-IR) approach, calculated as fasting insulin (microunits per milliliter) ⫻ fasting blood glucose (millimoles per liter)/22.5 [26]. Statistics Data are expressed as mean ⫾ standard deviation. All variables were normally distributed as tested by the Kolmogorov-Smirnov test. Homogeneity of variances was calculated by Levene’s test. Unpaired Student’s t test was used to assess significant differences in two groups where appropriate. The relation between the plasma ghrelin/obestatin ratio and BMI, lipid profile, or HOMA-IR was examined by bivariate correlations. P ⬍ 0.05 was considered statistically significant. Analyses were performed using SPSS 13.0 for Windows (SPSS Inc., Chicago, IL, USA).
Specimen collection and biochemical analysis
Results
After overnight fasting for 12 h, blood samples were drawn in all 100 subjects at 0800 h. Blood samples for measurement of obestatin were drawn into chilled polypropylene tubes containing ethylene-diaminetetra-acetic acid/ 2Na (1 mg/mL) and aprotinin (Phoenix Pharmaceuticals, Belmont, CA, USA; 100 L containing 0.6 U of trypsin inhibitor per milliliter of blood). After immediate centrifugation at 4°C for 15 min, plasma samples were stored at
Of 257 adults observed, 42 subjects had indeterminant results for H. pylori status and were excluded. Fifty H. pylori–positive and 50 H. pylori–negative adults matched for age (⫾5 y) and BMI (⫾1 kg/m2) were enrolled for the ghrelin and obestatin study. As presented in Table 1, age, gender, BMI, HOMA-IR, and lipid profile were similar in the H. pylori–negative and -positive groups. Plasma ghrelin levels (Fig. 1A) were significantly lower in the H. pylori–
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Table 1 Characteristics of the two different groups* Helicobacter pylori status
Age (y) Weight (kg) BMI (kg/m2) Men/women Fasting blood glucose (mmol/L) Fasting insulin (mU/L) HOMA-IR Total cholesterol (mmol/L) HDL cholesterol (mmol/L) LDL cholesterol (mmol/L) Triacylglycerol (mmol/L)
Negative
Positive
43.3 ⫾ 2.9 62.5 ⫾ 6.8 21.5 ⫾ 1.2 25/25 5.0 ⫾ 0.1 7.5 ⫾ 2.2 1.7 ⫾ 0.3 4.4 ⫾ 0.3 1.3 ⫾ 0.2 2.8 ⫾ 0.2 1.1 ⫾ 0.2
45.9 ⫾ 2.4 60.7 ⫾ 7.6 21.2 ⫾ 0.7 26/24 5.1 ⫾ 0.1 7.3 ⫾ 2.9 1.6 ⫾ 0.2 4.3 ⫾ 0.4 1.2 ⫾ 0.2 2.9 ⫾ 0.1 1.2 ⫾ 0.2
BMI, body mass index; HDL, high-density lipoprotein; HOMA-IR, homeostasis model of assessment for insulin resistance; LDL, low-density lipoprotein * Values are means ⫾ SDs.
positive group than in the H. pylori–negative group (P ⬍ 0.01). There was no significant difference in circulating obestatin levels (Fig. 1B) between those with and without H. pylori infection (P ⬎ 0.05). The plasma ghrelin/obestatin ratio (Fig. 1C) was decreased in subjects with H. pylori infection (P ⬍ 0.01). The ghrelin/obestatin ratio was negatively correlated with BMI and HOMA-IR in H. pylori– negative (r ⫽ ⫺0.644, P ⬍ 0.001, and r ⫽ ⫺0.505, P ⬍ 0.001, respectively) and H. pylori–positive (r ⫽ ⫺0.693, P ⬍ 0.001, and r ⫽ ⫺0.367, P ⫽ 0.009, respectively) subjects. There was no significant correlation between the ghrelin/obestatin ratio and serum lipids in H. pylori–negative and H. pylori–positive subjects. The ghrelin/obestatin ratio was negatively correlated with BMI (r ⫽ ⫺0.296, P ⫽ 0.003), HOMA-IR (r ⫽ ⫺0.224, P ⫽ 0.025), and serum levels of triacylglycerol (r ⫽ ⫺0.202, P ⫽ 0.044) in all subjects (Fig. 2A–C). There was a positive correlation between circulating obestatin and ghrelin in the H. pylori– negative (r ⫽ 0.652, P ⬍ 0.001) and H. pylori–positive (r ⫽ 0.494, P ⬍ 0.001) groups. Obestatin concentrations were correlated with ghrelin concentrations (r ⫽ 0.430, P ⬍ 0.001) in all subjects (Fig. 3).
ies have found that obestatin plays a role in the regulation of energy homeostasis [32,33]. The intricate balance of ghrelin and obestatin in humans is interesting. In our study, H. pylori–positive subjects had lower ghrelin blood levels and a lower ghrelin/obestatin ratio. Helicobacter pylori are a persistent colonizer of the human stomach and affect various cell types in gastric mucosa. The stomach is a major source of circulating ghrelin, and plasma ghrelin levels were significantly lower in patients after gastrectomy [34,35]. Ghrelin is primarily produced by X/A-like cells in the gastric mucosa [36]. There are some data in the literature about the relation between H. pylori and ghrelin. Many researchers have found that total plasma ghrelin decreases in patients with gastric atrophy secondary to H. pylori infection [16,37]. This might be explained by the loss of ghrelin-producing cells caused by inflammation and/or gastric atrophy associated with the infection. Furthermore, plasma ghrelin levels have been found to be lower in healthy subjects with H. pylori infection [17,29]. In agreement with these reports, we found that H. pylori infection was associated with a reduction in circulating ghrelin in healthy Chinese adults. It remains unclear what factors related to H. pylori infection influence plasma ghrelin levels
Discussion Helicobacter pylori infection may be responsible for dyspeptic symptoms and reduced appetite. Some studies have shown that elimination of H. pylori increases nutrition and weight not only in patients but also in healthy subjects [27,28]. Its role requires further research. Helicobacter pylori infection seemed to alter serum levels of some appetiteaffecting gastric hormones [29]. Ghrelin is an orexigenic gastrointestinal hormone. Obestatin is generated from proteolytic cleavage of preproghrelin. Although there have been some controversies concerning obestatin [30,31], many stud-
Fig. 1. Plasma ghrelin levels (A), obestatin levels (B), and ghrelin/obestain ratio (C) in the Helicobacter pylori–positive and -negative groups. *P ⬍ 0.01 compared with subjects without H. pylori infection.
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Fig. 2. Correlation between ghrelin/obestatin ratio and BMI (A), HOMA-IR (B), and triacylglycerol (C) in all subjects. BMI, body mass index; HOMA-IR, homeostasis model of assessment for insulin resistance.
in healthy subjects. There is a probable direct effect of H. pylori on the survival of the ghrelin-producing cells, but Cindoruk et al. [38] found that H. pylori infection had no effect on plasma ghrelin levels in patients without atrophic gastritis. The discrepancy of these results may be due to the different ages and ethnicities of subjects across different studies. Obestatin has proved to be primarily produced by the same neuroendocrine cells in the oxyntic glands as ghrelin [39]. However, we did not find any effect on plasma obesta-
tin levels of H. pylori infection. The mechanisms of this result are not yet known. One possibility is that obestatin is released from many tissues, contributing to a certain amount of circulating obestatin. Including the gastrointestinal mucosa, obestatin immunoreactivity is expressed in several other tissues, such as the perinatal pancreas [40], myenteric plexus, Leydig cells [41], and the salivary gland [42]. Although obestatin and ghrelin are derived from the same prohormone, their main source seemed to be different. Plasma obestatin levels tended to be lower in patients after
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In summary, ghrelin/obestatin ratios were decreased in H. pylori–infected subjects. The balance of ghrelin and obestatin was changed in the presence of H. pylori infection.
References
Fig. 3. Correlation between plasma obestatin and ghrelin levels in all subjects.
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