Elevated Circulating Level of Ghrelin in Cachexia Associated With Chronic Heart Failure. Relationships Between Ghrelin and Anabolic/Catabolic Factors

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1 Elevated Circulating Level of Ghrelin in Cachexia Associated With Chronic Heart Failure Relationships Between Ghrelin and Anabolic/Catabolic Factors Noritoshi Nagaya, MD; Masaaki Uematsu, MD; Masayasu Kojima, MD; Yukari Date, MD; Masamitsu Nakazato, MD; Hiroyuki Okumura, MD; Hiroshi Hosoda, MD; Wataru Shimizu, MD; Masakazu Yamagishi, MD; Hideo Oya, MD; Hideki Koh, MD; Chikao Yutani, MD; Kenji Kangawa, PhD Background Ghrelin is a novel growth hormone (GH)-releasing peptide, isolated from the stomach, that may also cause a positive energy balance by stimulating food intake and inducing adiposity. We sought to investigate the pathophysiology of ghrelin in the cachexia associated with chronic heart failure (CHF). Methods and Results Plasma ghrelin was measured in 74 patients with CHF and 12 control subjects, together with potentially important anabolic and catabolic factors, such as GH and tumor necrosis factor (TNF- ). Patients with CHF were divided into two groups, those with cachexia (n 28) and those without cachexia (n 46). Plasma ghrelin did not significantly differ between all CHF patients and controls ( versus fmol/ml, P NS). However, plasma ghrelin was significantly higher in CHF patients with cachexia than in those without cachexia ( versus fmol/ml, P 0.001). Circulating GH, TNF-, norepinephrine, and angiotensin II were also significantly higher in CHF patients with cachexia than in those without cachexia. Interestingly, plasma ghrelin correlated positively with GH (r 0.28, P 0.05) and TNF- (r 0.31, P 0.05) and negatively with body mass index (r 0.35, P 0.01). Conclusions Plasma ghrelin was elevated in cachectic patients with CHF, associated with increases in GH and TNF- and a decrease in body mass index. Considering ghrelin-induced positive energy effects, increased ghrelin may represent a compensatory mechanism under catabolic-anabolic imbalance in cachectic patients with CHF. (Circulation. 2001; 104: ) Key Words: heart failure hormones growth substances nutrition Cardiac cachexia, which is a catabolic state characterized by weight loss and muscle wasting, occurs in some patients with end-stage heart failure 1 3 and is a strong independent risk factor for mortality in patients with chronic heart failure (CHF). 3 Growth hormone (GH) and its mediator, insulin-like growth factor-1 (IGF-1), are anabolic hormones that are essential for skeletal and myocardial growth and metabolic homeostasis. 4,5 Elevated serum GH with normal or low IGF-1 level was observed in cachectic patients with CHF, 6,7 suggesting an important role of the GH/IGF-1 axis in cardiac cachexia. Ghrelin is a novel GH-releasing peptide, isolated from the stomach, that is identified as an endogenous ligand for growth-hormone secretagogues receptor (GHS-R). 8 Ghrelin stimulates GH secretion through an independent mechanism from that of hypothalamic GH-releasing hormone (GHRH). On the other hand, recent studies have shown that ghrelin causes a positive energy balance by stimulating food intake and decreasing fat use through GH-independent mechanisms. 9,10 Recently, we have demonstrated a considerable plasma concentration of ghrelin in healthy volunteers, although ghrelin is synthesized predominantly in the stomach. 11 These findings raise the possibility that ghrelin may play an important role as a circulating factor in the regulation of metabolic balance in patients with CHF. However, little information is available regarding the pathophysiology of ghrelin in the cachexia associated with CHF. In addition, the relationships between plasma ghrelin and potentially important anabolic and catabolic factors remain unknown. Thus, the purpose of this study was to investigate whether plasma ghrelin level is associated with cardiac cachexia with reference to the GH/IGF-1 axis and anabolic/catabolic factors in patients with CHF. Receibed June 8, 2001; revision received August 9, 2001; accepted August 14, From the Department of Internal Medicine, National Cardiovascular Center (N.N., H.O., W.S., M.Y., H.O., H.K.); Department of Internal Medicine, Osaka Seamen s Insurance Hospital (M.U.); Department of Biochemistry, National Cardiovascular Center Research Institute (M.K., H.H., K.K.); and Department of Pathology, National Cardiovascular Center (C.Y.), Osaka, Japan; and Third Department of Internal Medicine, Miyazaki Medical College (Y.D., M.N.), Miyazaki, Japan. Correspondence to Noritoshi Nagaya, MD, Department of Internal Medicine, National Cardiovascular Center, Fujishirodai, Suita, Osaka , Japan. nagayann@hsp.ncvc.go.jp 2001 American Heart Association, Inc. Circulation is available at

2 Nagaya et al Elevated Plasma Ghrelin in Cardiac Cachexia 2035 Methods Study Subjects We studied 74 patients with CHF (48 men and 26 women; mean age, 60; range, 18 to 87 years). The diagnosis of CHF was based on a history of dyspnea and symptomatic exercise intolerance with signs of pulmonary congestion. The cause of CHF was idiopathic dilated cardiomyopathy in 36, ischemic cardiomyopathy in 18, and valvular heart disease in 20 patients. Nine patients were classified as New York Heart Association (NYHA) functional class II, 48 patients as class III, and 17 patients as class IV. Left ventricular ejection fraction (LVEF) was 40% (mean, 28 1%), which was assessed by cine-ventriculography, radionuclide ventriculography, or echocardiography. All patients were clinically stable at the time of evaluation and had no evidence of active infection, gastric ulcer, or other primary cachectic states, such as cancer, thyroid disease, and severe liver disease. Patients with renal failure (serum creatinine 1.5 mg/dl) were excluded. The study included 12 control subjects (8 men and 4 women; mean age, 57; range, 33 to 78 years). The age, sex, and body mass index of the control subjects were similar to those of the 74 patients with CHF. All subjects provided informed consent. Patients with cardiac cachexia were defined as those with documented nonedematous and nonintentional weight loss of 7.5% of the previous normal nonedematous weight over a period of at least 6 months. 3 The weight loss was assessed by taking a history or body weight measurement. Consequently, 28 of 74 patients with CHF were defined as having cardiac cachexia. The weight loss amounted to kg or % loss of previous body weight during 20 4 months. Fat mass was measured by dual x-ray absorptiometry (DPX-L, Lunar Radiation) to investigate the relationship between plasma ghrelin level and fat mass in 17 patients with a similar amount of weight loss ( kg or % loss of previous body weight) during 18 2 months. The significant weight loss was not observed in noncachectic patients ( kg or % loss of body weight) during 25 4 months. Blood Sampling and Assay for Plasma Ghrelin Blood samples were taken from the antecubital vein in the morning between 7 AM and 8 AM after an overnight fast, because plasma ghrelin level has been shown to be altered by food intake. 9 The blood was immediately transferred into a chilled glass tube containing disodium EDTA (1 mg/ml) and aprotinin (500 U/mL) and centrifuged immediately at 4 C. Plasma samples were frozen and stored at 80 C and then were extracted before radioimmunoassay (RIA). Briefly, Sep-Pak C18 cartridges (Waters) were preconditioned with 5 ml each of chloroform, methanol, 60% acetonitrile containing 0.1% trifluoroacetic acid (TFA), and saline. Plasma (1000 L) was diluted with 1000 L saline and then loaded into a Sep-Pak C18 cartridge. After the column was washed with 5 ml each of saline and 5% acetonitrile containing 0.1% TFA, the absorbed materials were eluted with 3 ml 60% acetonitrile containing 0.1% TFA. The eluate was then lyophilized. RIA for plasma ghrelin was performed as described previously. 11 Briefly, a polyclonal antibody was raised against the C-terminal fragment [13-28] of rat ghrelin in a rabbit. A maleimide activated mariculture keyhole limpet hemocyanin (mcklh)-[cys 0]-ghrelin [13-28] conjugate was used for immunization. The RIA incubation mixture consisted of 100 L of standard ghrelin or unknown sample, normal rabbit serum, and 200 L of antiserum at a dilution of 1: After 12-hour incubation at 4 C, 100 L of 125 I-labeled ligand ( cpm) was added to the mixture. After a 36-hour incubation at 4 C, 100 L of goat anti-rabbit IgG antiserum was added. Free and bound tracers were separated by centrifugation at 3000 rpm for 30 minutes after incubation for 24 hours at 4 C. Pellet radioactivity was quantified using a counter. The minimum detectable dose of ghrelin was 6 fmol/tube. The antiserum exhibited 100% cross-reactivity with rat or human ghrelin [13-28]. No significant cross-reactivity with other peptides was observed. Intraobserver variability of ghrelin measurement was 6%, and its interobserver variability was 9%. Day-to-day variation was 9%. Other Biochemical Measurements Serum GH and IGF-1 were measured by immunoradiometric assay (Ab Bead HGH Eiken, Eiken Chemical Co, sensitivity 0.1 ng/ml; Somatomedin CII Bayer, Bayer Medical Ltd, sensitivity 0.3 ng/ ml). Serum tumor necrosis factor (TNF- ) and interleukin-6 were measured by enzyme immunoassay (Quantikine HS, R and D Systems Inc, sensitivity 0.18 pg/ml; TFB kit, TFB Co, sensitivity 0.3 pg/ml). Plasma atrial natriuretic peptide and brain natriuretic peptide were measured directly with specific assay kits (Shiono RIA ANP assay kit, sensitivity 2 pg/ml; Shiono RIA BNP assay kit, sensitivity 1 pg/ml, Shionogi Co). Plasma norepinephrine and epinephrine were measured by high-performance liquid chromatography (HLC8030, Tosoh Co, sensitivity 6 pg/ml). Plasma renin and aldosterone were measured with RIA kits (RENIN RIABEAD, sensitivity 0.1 ng/ml; ALDOSTERONE RIAKIT II, sensitivity 2 ng/dl, DAINABOT Co). Plasma angiotensin II was determined by a RIA kit (angiotensin II kit, SRL Inc, sensitivity 3 pg/ml). EDTA, but not an ACE inhibitor, was added into the sample tube according to the protocol from the angiotensin II kit. 12 Serum dehydroepiandrosterone surfate (DHEAS) was measured by a RIA kit (DPC DHEA-S kit, DPC Co, sensitivity 20 ng/ml). Serum cortisol and insulin were measured by enzyme immunoassay (AIA-PACK CORT, sensitivity 0.2 g/dl; AIA-PACK IRI, sensitivity 2 U/mL, Tosoh Co). Statistical Analysis Data are expressed as mean SEM. Comparisons of parameters between the two groups were made by Fisher s exact test or unpaired Student s t test. Comparisons of parameters among three or four groups were made by one-way ANOVA followed by the Scheffe s multiple comparison test. Correlation coefficients between plasma ghrelin and clinical parameters were calculated by linear regression analysis. Multiple regression analysis was applied to determine independent relations of clinical parameters with plasma ghrelin. P 0.05 was considered statistically significant. Results Patient Characteristics According to Cachexia Body mass index was significantly lower in cachectic patients than in noncachectic patients and in control subjects (Table 1). CHF patients with cachexia had a significantly longer disease period than those without cachexia. Peak oxygen consumption assessed by cardiopulmonary exercise testing, an index of exercise capacity, was significantly lower in cachectic patients than in noncachectic patients. The cause of CHF, NYHA functional class, LVEF, and medication use did not significantly differ between cachectic and noncachectic patients. Serum albumin and total cholesterol tended to be lower in cachectic patients than in noncachectic patients (P 0.083; P 0.057, Table 2). Plasma Ghrelin and Other Hormones in Cardiac Cachexia Plasma ghrelin level did not significantly differ between all CHF patients and control subjects ( versus fmol/ml, P NS). However, plasma ghrelin level was significantly higher in CHF patients with cachexia than in those without cachexia and in control subjects (Figure 1). Serum GH level was also significantly higher in cachectic patients than in those without cachexia and in control subjects, although serum IGF-1 level did not significantly differ among the 3 groups. Plasma ghrelin level tended to increase with the severity of NYHA functional class (Figure 2), but the differences did not reach statistical significance. Circulating levels of catabolic factors, such as TNF-, norepinephrine,

3 2036 Circulation October 23, 2001 TABLE 1. Patient Characteristics According to Cachexia in Patients With CHF Control (n 12) CHF Without Cachexia (n 46) CHF With Cachexia (n 28) Age, y Sex, male/female 8/4 31/15 17/11 Body mass index, kg/m * Cause of CHF, n NA Dilated cardiomyopathy Ischemic cardiomyopathy 13 5 Valvular heart disease 12 8 CHF period, mo NA NYHA functional class, n NA II 7 2 III IV 7 10 LVEF, % NA Peak V O2, ml/kg per min (n 12) * (n 30) * (n 19) Medication use, n None Digoxin ACE inhibitors Ang II blockers 5 4 -Blockers 12 9 Diuretics Furosemide dose, mg Peak V O2 indicates peak oxygen consumption; Ang II, angiotensin II. Data are mean SEM. *P vs controls; P 0.001, P 0.05 vs CHF without cachexia. and angiotensin II, were significantly higher in CHF patients with cachexia than in those without cachexia and in control subjects (Table 2). Plasma DHEAS was significantly lower in CHF patients with cachexia than in those without cachexia and in control subjects. Correlations Between Plasma Ghrelin and Clinical and Hormonal Parameters Plasma ghrelin level correlated negatively with body mass index in the patients with CHF (r 0.35, P 0.01, n 74, Figure 3). Plasma ghrelin did not significantly correlate with LVEF (r 0.03, P NS). In addition, there was no significant difference in plasma ghrelin between patients with LVEF above and below 25% ( versus fmol/ml, P NS). Plasma ghrelin correlated modestly with circulating GH (r 0.28, P 0.05) and IGF-1 (r 0.29, P 0.05). Plasma ghrelin correlated significantly with plasma TNF- (r 0.31, P 0.05) and epinephrine (r 0.29, P 0.05). However, it did not significantly correlate with any other hormones, such as norepinephrine, renin, angiotensin II, and aldosterone. In multivariate analysis, plasma ghrelin was related to the presence of cachexia but was independent of age, sex, body mass index, NYHA functional class, and LVEF (Table 3). For cachectic CHF patients with a similar amount of weight loss whose percent body fat was %, there was no significant correlation between plasma ghrelin level and percent body fat (r 0.23, P NS). Changes in Plasma Ghrelin in Cardiac Cachexia Plasma ghrelin level increased significantly ( to fmol/ml, P 0.05) in 10 patients who developed cachexia ( % loss of previous body weight) during a mean follow-up period of 12 1 months. In contrast, plasma Figure 1. Plasma levels of ghrelin (left), GH (middle), and IGF-1 (right) in control subjects, noncachectic patients with CHF (NC- CHF), and cachectic patients with CHF (C-CHF). *P 0.05 vs control subjects; P 0.05 vs NC-CHF. Figure 2. Plasma ghrelin level in patients with CHF according to New York Heart Association (NYHA) functional class.

4 Nagaya et al Elevated Plasma Ghrelin in Cardiac Cachexia 2037 TABLE 2. Hormone Analysis According to Cachexia in Patients With CHF Control (n 12) CHF Without Cachexia (n 46) CHF With Cachexia (n 28) Total protein, g/dl Albumin, g/dl Total cholesterol, mg/dl Triglycerides, mg/dl Fasting glucose, mg/dl ANP, pg/ml * * BNP, pg/ml * * TNF-, pg/ml * IL-6, pg/ml * * NE, pg/ml * EPI, pg/ml Renin, ng/ml per hr * * Angiotensin II, pg/ml Aldosterone, ng/dl * Cortisol, g/dl DHEAS, ng/ml * Insulin, U/mL ANP indicates atrial natriuretic peptide; BNP, brain natriuretic peptide; IL-6, interleukin-6; NE, norepinephrine; EPI, epinephrine; and DHEAS, dehydroepiandrosterone sulfate. Data are mean SEM. *P 0.05, P 0.01 vs controls; P 0.01, P 0.05 vs CHF without cachexia. ghrelin was unchanged ( to fmol/ml, P NS) in 24 patients who did not develop cachexia ( % gain of previous body weight) during a mean follow-up period of 11 1 months. Discussion In the present study, we demonstrated that plasma ghrelin was elevated in cachectic patients with CHF and that plasma ghrelin correlated positively with serum GH and TNF- and negatively with body mass index. Earlier studies have shown that cardiac cachexia is associated with hormonal changes and cytokine activation. 2,6,13 15 In fact, some catabolic factors, including TNF- and norepinephrine, were significantly increased in cachectic patients with CHF in the present study. A recent study has shown that angiotensin II induces skeletal muscle wasting by enhancing protein degradation via its inhibitory effect on the IGF-1 system. 16 In the present study, plasma angiotensin II was higher in cachectic patients than in noncachectic patients. In contrast, some anabolic hormones Figure 3. Correlation between plasma ghrelin level and body mass index in patients with CHF. Log transformation was used to normalize the distribution of plasma ghrelin levels. were decreased (DHEAS) or unchanged (IGF-1, insulin). These results indicate a catabolic/anabolic imbalance in cachectic patients with CHF. On the other hand, serum GH was elevated in cachectic patients with CHF, consistent with the results reported by Anker et al. 6 Interestingly, plasma ghrelin was significantly higher in CHF patients with cachexia than in those without cachexia and in control subjects, and the increase in plasma ghrelin was associated with the increase in serum GH. These results suggest that ghrelin as well as GHRH may be involved in the regulation of GH in patients with CHF. Because the GH-releasing effects of ghrelin are mediated by GHS-R, mainly present at the pituitary, it is likely that increased ghrelin circulates in the blood stream to act on the anterior pituitary. Considering that activation of the GH/IGF-1 axis induces anabolic effects, such as maintenance of skeletal muscle mass and myocardial growth, 4,5 elevated circulating ghrelin may have a role in attenuating catabolic-anabolic imbalance via its potent GHreleasing effects. Recently, peripheral administration of ghrelin has been reported to induce a positive energy balance and weight gain TABLE 3. Multivariate Analysis of Variables Associated With Plasma Ghrelin Level in Patients With CHF Variables Standardized Coefficient P Age Male sex Body mass index NYHA functional class LVEF Presence of cachexia

5 2038 Circulation October 23, 2001 by decreasing fat use and increasing carbohydrate use through a GH-independent mechanism. 9 In addition, both intracerebroventricular and peripheral administration of ghrelin has been shown to elicit a potent, long-lasting stimulation of food intake via activation of neuropeptide Y neurones in the hypothalamic arcuate nucleus. 10,17,18 In the present study, plasma ghrelin increased in association with a decrease in body mass index and an increase in plasma TNF- level in patients with CHF. In addition, plasma ghrelin increased when patients developed cardiac cachexia. These results raise the possibility that increased plasma ghrelin may represent a compensatory mechanism under conditions of anabolic/catabolic imbalance in cachectic patients with CHF. Multivariate analysis demonstrated that plasma ghrelin was related to the presence of cachexia but was independent of age, sex, body mass index, NYHA functional class, and LVEF. Plasma ghrelin was not significantly correlated with fat mass in CHF patients with a similar amount of weight loss, although plasma ghrelin has been shown to be negatively correlated with fat mass in humans. 19 These results suggest that not only the absolute value of fat mass but also the progressive weight loss (cachexia) may influence plasma ghrelin in patients with CHF. Interestingly, the majority of ghrelin is produced by X/A-like cells in the stomach, 8,11,20 although a small amount of ghrelin is produced by the arcuate nucleus of the hypothalamus, where it seems to play a role in GH release. 21 Thus, it is interesting to speculate that the stomach has a role as an endocrine organ in the regulation of energy balance. Investigating the relationship between ghrelin production and other cachexia attributable to chronic obstructive pulmonary disease, liver cirrhosis, and cancer may additionally constitute interesting studies. More recently, we have shown that a specific receptor for ghrelin exists not only in the hypothalamus and pituitary but also in blood vessels and the heart and that intravenous injection of ghrelin causes beneficial hemodynamic effects via reducing cardiac afterload and increasing cardiac output without an increase in heart rate. 22 Thus, it is possible that increased plasma ghrelin may have beneficial hemodynamic effects in patients with CHF. Because cardiac cachexia is a strong independent risk factor for mortality in patients with CHF, 3 it would be interesting to investigate whether additional supplementation of ghrelin attenuates the development of cachexia in the most severe forms of CHF. Conclusions Plasma ghrelin level was elevated in cachectic patients with CHF, associated with increases in serum GH and TNF- and a decrease in body mass index. Considering ghrelin-induced positive energy effects, increased ghrelin in plasma may represent a compensatory mechanism under catabolicanabolic imbalance in cachectic patients with CHF. Acknowledgments This work was supported by the Research Grant for Cardiovascular Disease (12C-2) from the Ministry of Health, Labor and Welfare, the Uehara Memorial Foundation, and the Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research of Japan. References 1. Pittman JG, Cohen P. The pathogenesis of cardiac cachexia. N Engl J Med. 1964;271: Anker SD, Coats AJS. Cardiac cachexia: a syndrome with impaired survival and immune and neuroendocrine activation. Chest. 1999;115: Anker SD, Ponikowski P, Varney S, et al. Wasting as independent risk factor for mortality in chronic heart failure. Lancet. 1997;349: Amato G, Carella C, Fazio S, et al. Body composition, bone metabolism, heart structure and function in growth hormone deficient adults before and after growth hormone replacement therapy at low doses. J Clin Endocrinol Metab. 1993;77: Fuller J, Mynett JR, Sugden PH. Stimulation of cardiac protein synthesis by insulin-like growth factors. Biochem J. 1992;282: Anker SD, Chua TP, Ponikowski P, et al. Hormonal changes and catabolic/anabolic imbalance in chronic heart failure and their importance in cardiac cachexia. Circulation. 1997;96: Niebauer J, Pflaum CD, Clark AL, et al. Deficient insulin-like growth factor I in chronic heart failure predicts altered body composition, anabolic deficiency, cytokine and neurohormonal activation. J Am Coll Cardiol. 1998;32: Kojima M, Hosoda H, Date Y, et al. Ghrelin is a growth-hormonereleasing acylated peptide from stomach. Nature. 1999;402: Tschop M, Smiley DL, Heiman ML. Ghrelin induces adiposity in rodents. Nature. 2000;407: Wren AM, Small CJ, Ward HL, et al. The novel hypothalamic peptide ghrelin stimulates food intake and growth hormone secretion. Endocrinology. 2000;141: Hosoda H, Kojima M, Matsuo H, et al. Ghrelin and des-acyl ghrelin: two major forms of rat ghrelin peptide in gastrointestinal tissue. Biochem Biophys Res Commun. 2000;279: Tsutamoto T, Wada A, Maeda K, et al. Angiotensin II type 1 receptor antagonist decreases plasma levels of tumor necrosis factor, interleukin-6 and soluble adhesion molecules in patients with chronic heart failure. J Am Coll Cardiol. 2000;35: Levine B, Kalman J, Mayer L, et al. Elevated circulating levels of tumor necrosis factor in severe chronic heart failure. N Engl J Med. 1990;323: McMurray J, Abdullah I, Dargie HJ, et al. Increased concentrations of tumor necrosis factor in cachectic patients with severe chronic heart failure. Br Heart J. 1991;66: Dutka DP, Elborn JS, Delamere F, et al. Tumour necrosis factor in severe congestive cardiac failure. Br Heart J. 1993;70: Brink M, Price SR, Chrast J, et al. Angiotensin II induces skeletal muscle wasting through enhanced protein degradation and down-regulates autocrine insulin-like growth factor I. Endocrinology. 2001;142: Nakazato M, Murakami N, Date Y, et al. A role for ghrelin in the central regulation of feeding. Nature. 2001;409: Shintani M, Ogawa Y, Ebihara K, et al. Ghrelin, an endogenous growth hormone secretagogue, is a novel orexigenic peptide that antagonizes leptin action through the activation of hypothalamic neuropeptide Y/Y1 receptor pathway. Diabetes. 2001;50: Tschop M, Weyer C, Tataranni A, et al. Circulaing ghrelin levels are decreased in human obesity. Diabetes. 2001;50: Date Y, Kojima M, Hosoda H, et al. Ghrelin, a novel growth hormonesreleasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans. Endocrinology. 2000; 141: Date Y, Murakami N, Kojima M, et al. Central effects of a novel acylated peptide, ghrelin, on growth hormone release in rats. Biochem Biophys Res Commun. 2000;275: Nagaya N, Kojima M, Uematsu M, et al. Hemodynamic and hormonal effects of human ghrelin in healthy volunteers. Am J Physiol Regul Integr Comp Physiol. 2001;280:R1483 R1487.