Growth Hormone & IGF Research 20 (2010) 179184

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Growth Hormone & IGF Research

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Body composition, endocrine and metabolic proles in adults with Prader-Willi syndrome
Rasmus Sode-Carlsen a,*, Stense Farholt a, Kai Fr. Rabben b, Jens Bollerslev c, Thomas Schreiner c, Anne Grethe Jurik d, Jens Sandahl Christiansen e, Charlotte Hybye f
a

Centre for Rare Diseases, Department of Paediatrics, Aarhus University Hospital Skejby, DK-8200 Aarhus N, Denmark Frambu, NO-1400 Siggerud, Norway Department of Endocrinology, Rikshospitalet, NO-Oslo, Norway d Department of Radiology, Aarhus University Hospital, DK-8000 Aarhus C, Denmark e Department of Endocrinology M, Aarhus University Hospital, DK-8000 Aarhus C, Denmark f Department of Endocrinology, Metabolism and Diabetology, Karolinska University Hospital, S-171 76, Stockholm, Sweden
b c

a r t i c l e

i n f o

a b s t r a c t
Objective: Prader-Willi syndrome (PWS) is a complex genetic disease associated with hypothalamicpituitary dysfunction and severe obesity. The aim of the present study was to describe the relationships between body composition, metabolic and hormonal proles in PWS adults. Method: Forty six adults with genetically veried PWS, 25 women and 21 men, median age 28 years were studied. Body composition was evaluated by standard anthropometric procedures and with computed tomography (CT) of the abdomen and at the mid-femur level. CT of abdomen was compared to 22 healthy, unmatched adults. Circulating lipids were measured and oral glucose tolerance test (OGTT) and hormonal screening including GH secretory capacity (GHRH/arginine test) was carried out. Results: Median body mass index (BMI) was 27.2 kg/m2, with women being more obese than men. Sixteen patients had dyslipidaemia, 10 impaired glucose tolerance and seven had diabetes. Fifty percent were hypogonadal and six fullled BMI related criteria for growth hormone deciency (GHD). Visceral to subcutaneous abdominal fat ratio was reduced in PWS. Visceral abdominal fat fraction correlated with both subcutaneous fat, BMI and peak GH-response. Thigh muscle volume was about half of the thigh fat volume. Benecial effects of sex-steroid replacement on body composition were not observed. Conclusions: Body fat was primarily located subcutaneously and metabolic consequences of obesity limited. The abnormal body composition similar to that in non-PWS GHD adults increases the interest of GH treatment in the prevention of obesity in adults with PWS. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 5 May 2009 Revised 18 December 2009 Accepted 18 December 2009 Available online 3 March 2010 Keywords: PWS Adults Body composition Metabolism

1. Introduction Prader-Willi syndrome (PWS) is a complex genetic disorder caused by lack of paternally expressed genes in region q1113 on chromosome 15 [1,2]. PWS is characterized by short stature, muscular hypotonia, mild to moderate intellectual disability, hypogonadism, hyperphagia and risk of severe obesity from early childhood [14]. Body composition is abnormal with increased body fat predominantly located subcutaneously [2,59], whereas lean body mass is decreased [10,11]. Thus, at any given level of BMI, PWS patients would be expected to have a higher percent body fat compared to non-PWS, albeit with a lower ratio between visceral to subcutaneous fat [5]. The cause of the abnormal body composition is not completely known, but it has been ascribed to

* Corresponding author. Tel.: +45 89496888; fax: +45 89496023. E-mail address: Rasmus.Sode-Carlsen@ki.au.dk (R. Sode-Carlsen). 1096-6374/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.ghir.2009.12.004

the impairment in the activity of the GH-IGF system and to the partial hypogonadism [3,4,7]. To some degree the relatively reduced amount of visceral fat protects the PWS individuals from complications to the obesity [6]. However, type 2 diabetes, dyslipidaemia and cardiovascular diseases have been frequently reported in PWS adults, and it has been estimated that yearly mortality is as high as 3% [12]. No specic therapy exists for the treatment of obesity in PWS, and prevention of obesity is obtained principally through environmental control. A closer description of body composition and factors involved in its regulation as well as factors regulated by body composition in adults with PWS are important before interventions such as growth hormone (GH) therapy in such patients should be used as routine. The aim of this study was, in a large cohort of genetically diagnosed adults with PWS, to relate body composition as measured by computer tomography (CT) to simultaneously measured anthropometrics as well as metabolic and endocrine indices.

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2. Method 2.1. Study protocol The current study presents baseline data from a complete sample of 46 consecutively enrolled adults with PWS participating in a Nordic multi-centre investigator sponsored trial of growth hormone treatment. Enrolment was from December 2004 to January 2007. Eligibility criteria were genetically diagnosis of PWS and that the subjects had reached nal adult height. Twenty-one men and 25 women, median age 28 years (range 1641 years) were included. All had previously been genetically diagnosed as having PWS by standard genetic testing (methylation positive). In 32 cases further genetic analyses were available (27 deletion, three UPD and two translocation). Seven patients were treated with steroid inhalation and b2-agonist due to asthma. One woman aged 41 years suffered from sick sinus node syndrome. Thirteen were treated with neuroleptics. Data from 22 healthy subjects (11 women and 11 men) in whom a CT scan of the abdomen had been performed as part of unrelated metabolic studies and who were of similar age (median 30 years, range 1945), and had a similar BMI (median, 29 kg/m2, range 2137 kg/m2), was included for comparison. The study was approved by local Ethical Committees at the University of Oslo, the Karolinska Institute and the University of Aarhus, and the study of the healthy subjects by the Ethical Committee at the University of Aarhus. Signed informed consent to study participation was obtained from all participants and their legal guardians prior to sample collection and examinations. Baseline examinations included measurements of anthropometry and body composition as well as endocrinological and metabolic parameters as described below. 2.2. Anthropometric methods Physical examination included measurements of height, weight, waist and hip. Waist and hip were measured in standing position. Waist circumference was measured halfway between the costal edge and iliac crest and cut-off points for obesity was set to 88 cm in women and 102 cm in men [13]. Hip was measured at the greatest circumference and waist-to-hip (WH) ratio above 0.85 in women and 1.0 in men dened obesity [14]. Body mass index (BMI) was calculated as weight divided by the square height, kg/m2. BMI from 18.5 to 25 kg/m2 was dened as normal, between 25 and 30 kg/m2 as overweight and above 30 kg/m2 as obese according to WHO criteria. 2.3. Body composition studies Examinations of fat and muscle volume were performed with computed tomography (CT) as previously described [15]. Comparable 16 slice scanners were used; in Denmark Philips MX 8000 and in Sweden and Norway GE LightSpeed pro 16. CT measurements were all evaluated at a Philips Extended Brilliance Workspace; Version v 7.1. The basic thin slices, either two or four dependent on the slice thickness, were combined to slices with a thickness of 625 mm to be used for measurements. In the calculation of volumes measurements were standardized to a width of 12 mm. For the calculation of total volume, fat volume and muscle volume, Hounseld units in the range of 200 to 3000, 200 to 20, and 0100, respectively, were used. CT scans of the abdomen were made at the L2L3 level. Total abdominal volume, total abdominal fat volume and visceral abdominal fat (VF) were measured and subcutaneous abdominal fat (SF) was calculated by subtracting VF from total abdominal

fat volume. The measurements of visceral fat were made by manually outlining the intra abdominal wall and excluding retrorenal fat. Visceral to subcutaneous fat ratio (VS) was calculated as VF divided by SF, subcutaneous fat fraction (SFF) was calculated as SF divided by total abdominal volume, and visceral fat fraction (VFF) as VF divided by total abdominal volume. Measurements of thigh muscle and fat volumes were made midway between the right greater trochanter and the lateral epicondyl as previously described [16]. The procedures was performed and controlled by two examiners independently. 2.4. Endocrinological and metabolic investigations The patients visited the clinics for two consecutive days for endocrine and metabolic investigations. On day 1 serum TSH, free T4, LH, FSH, testosterone, estradiol, total-IGF-I, HbA1c, triglycerides and total, low (LDL) and high (HDL) density lipoprotein cholesterol were measured in the fasting state. Analyses were performed locally according to standard procedures, except from IGF-I which was analyzed centrally by a TR-IMFA inhouse assay [17]. Classication of gonadal status as eugonadal or hypogonadal was based upon the judgment of the local physician. Total cholesterol >5.0 mmol/L, LDL > 3.0 mmol/L, HDL < 1.2 mmol/L for women and <1.0 mmol/L for men and triglycerides >1.7 mmol/L was considered as dyslipidaemic values [13]. After a 10-min rest, systolic and diastolic blood pressure were measured three times with local equipment, and the lowest value was used. Blood pressure above 140/90 mm Hg was dened as hypertension according to WHO criteria [18]. A standard 75 g oral glucose tolerance test (OGTT) was performed in the morning in the fasting state. Impaired glucose tolerance and diabetes was diagnosed based upon the 120 min plasma glucose values (between 7.8 and 11.0 and above 11.1 mmol/L, respectively, in accordance with the WHO criteria [19]). Insulin resistance was calculated by the homeostasis model assessment index (HOMA-IR) as insulin (mU/L) plasma glucose (mmol/L)/ 22.5 using single fasting samples [20]. The threshold for insulin resistance was set at 2.77, as suggested in the Bruneck study [21]. Insulin was measured by ouroimmunoassays (autodela insulin Wallac Oy, Turku, Finland) in Sweden and Denmark, and by radio immunoassay (DPC, Los Angeles, CA, USA) in Norway. Cross reactivity with proinsulin was low. The second day a GH stimulation test was performed with bolus infusion of GHRH (1 lg/kg) and infusion of arginine (0.5 g/kg, maximally 35 g) during 30 min. GH was measured every 10 min, and GH peak levels were recorded [22]. The cut-off points for growth hormone deciency (GHD) was 11.5 lg/L for normal body weight, 8.0 lg/L for overweight and 4.2 lg/L for obese [23]. GH was determined by ouroimmunoassays (Dela hGH Wallac Oy, Turku, Finland) in Sweden and Denmark, and by radio immunoassays (Orion Diagnostica, Espoo, Finland) in Norway. 2.5. Statistics Data are presented as median (10th and 90th percentile) unless otherwise stated, since only a few of the data followed the Gaussian distribution, as determined by standard plots. Relationships were analysed with either Spearman correlations, Wilcoxon matched pairs signed rank test or Wilcoxon signed rank test. Statistical signicance was set at P < 0.05. Statistical analysis was performed with Stata/IC 10.1 (StataCopr LP, College Station, Texas).

R. Sode-Carlsen et al. / Growth Hormone & IGF Research 20 (2010) 179184 Table 1 Anthropometric characteristics for 46 adults with PWS, median (10th90th percentile). Women Number Age, range (years) Height (m) BMI (kg/m2) Waist circumference (cm) WH ratio 25 27 (1641) 1.55 (1.421.71) 28.3 (21.243.4) 93 (72119) 0.81 (0.730.95) Men 21 30 (1839) 1.69 (1.541.80) 24.9 (19.334.4) 92 (77112) 0.90 (0.820.99) P-value 0.44 <0.001 0.05 0.88 0.01

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3.3. Endocrine and metabolic assessment (Table 3) Two women received treatment for primary hypothyroidism and were euthyroid on replacement. Nine men were treated with testosterone and seven women received estrogen replacement. The number of PWS women with spontaneously menstrual cycle was not registered. No abnormalities were observed in thyroid levels. IGF-I values were in general low with a median SDS value of 0.4, lowest value seen was 50 lg/L (2.9 SDS) and highest value seen was 464 lg/L (4.9 SDS). Two patients had IGF-I levels below 2.0 SDS, while four had levels above 2.0 SDS. The peak GH response to the GHRH-arginine test was below the cut-off points for GHD in two patients with BMI <25 kg/m2, two with BMI between 25 and 30 kg/m2 and in two with BMI >30 kg/m2. Median peak GH was 17.7 lg/L. The test was not performed in ve patients. There was no signicant correlation between peak GH and SDS IGF-I. Two patients were treated for hypertension with ACE-inhibitors, one of them in combination with hydrochlorothiazide, in addition only one patient had elevated blood pressure (>140/ 90 mm Hg). Median total, LDL- and HDL-cholesterol as well as triglycerides were all within the normal range. However, in 10 patients total cholesterol was above 5 mmol/L, and in 14 LDL-cholesterol was above 3.0 mmol/L, with the highest value being 4.7 mmol/L. In four men and four women HDL-cholesterol levels were under the cutoff limit and three patients had triglycerides above 1.7 mmol/L. No gender differences were seen. Twenty-nine patients had normal OGTT, whereas 10 (ve women and ve men) had an impaired glucose tolerance. Seven had diabetes (all women) of whom three were already diagnosed with diabetes (two with diabetes type 2 and one with diabetes type 1). Only four patients had a HOMA index >2.77, two of whom with known diabetes. The three known diabetic patients had HbA1c above normal, the reminder all had normal HbA1c values. No other gender differences were discovered in the above endocrine or metabolic parameters. 3.4. Correlations (Table 4) Both SF and VF correlated signicantly to BMI (r = 0.9313, P < 0.001 and r = 0.7295, P < 0.001, respectively) (Fig. 1) and to waist circumference (r = 0.8099, P < 0.001 and r = 0.8135, P < 0.001, respectively). The correlation between SF and VF was positive (r = 0.6793, P < 0.001). The correlations between SFF and BMI and between VFF and BMI were positive, whereas the correlations between SFF and peak GH and between VFF and peak GH were negative. Thigh fat and muscle volume showed positive correlations to BMI and negative correlations to peak GH.

BMI, body mass index; WH, waist/hip.

3. Results 3.1. Anthropometry (Table 1) Median height in women was lower than in men (1.55 m vs 1.69 m) while median BMI was higher in women than in men (28.3 vs 24.9 kg/m2). In the whole group median BMI was 27.2 kg/m2 (19.940.9 kg/m2); 17 had normal BMI (six women and 11 men), 12 were overweight (seven women and ve men) and 17 were obese (12 women and ve men). Median waist circumference did not differ in women (0.93 m) and men (0.92 m); but 15 women and two men were above the gender related cutoff points for waist circumference. Median WH ratio in women was lower (0.81) than in men (0.90). Ten women but only two men were above the gender related cut-off points for WH ratio. 3.2. Body composition (Table 2) The PWS adults accepted the CT examinations well and no major problems were encountered. However, in one very obese woman examination was not possible and in two the examination was not performed. Thus, results from the CT scans are available in 43 patients only. Median SF was 371 ml and 162 ml (P = 0.02); median VF 88 ml and 124 ml (P = 0.11); median VS 0.24 and 0.65 (P = 0.001) in the PWS group vs control group, respectively. Among the PWS adults, two had VF in excess of 300 ml. None of these had overt diabetes, HOMA index was below 2.77 and lipids were normal. Median SFF fraction was higher than VFF (0.54 vs 0.12, P < 0.001). No signicant differences in the results of the abdominal examinations between men and women were found. Median thigh fat volume was 179 ml and thigh muscle volume 104 ml. Thigh fat volume was increased in women as compared to men (219 vs 124 ml, P = 0.01), and the muscle/fat ratio of the thigh was signicantly different between patients with normal weight and both overweight (0.91 vs 0.46, P = 0.002) and obese (0.91 vs 0.45, P < 0.001), whereas no signicant difference was found between the overweight and the obese group.

Table 2 Comparison of body composition characteristics of 43 adults with PWS and 22 healthy adults as measured with CT of abdomen and thigh, median (10th90th percentile). PWS N SF (ml) VF (ml) SFF (%) VFF (%) VS Thigh fat (ml) Thigh muscle (ml) 43 43 43 43 43 43 43 Median (10th90th percentile) 371 (144793) 88 (34170) 0.54 (0.300.70) 0.12 (0.050.18) 0.24 (0.120.44) 179 (86267) 104 (76149) Healthy adults N 22 22 Median (10th90th percentile) 162 (37659) 124 (51193) P-value* 0.02 0.11

22

0.65 (0.220.80)

0.001

SF, subcutaneous abdominal fat; VF, visceral abdominal fat; SFF, subcutaneous abdominal fat fraction. VFF, visceral abdominal fat fraction; VS, visceral-subcutaneous abdominal fat ratio. * Wilcoxon signed rank test.

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Table 3 Endocrine and metabolic characteristics of 46 adults with PWS, median (10th90th percentile). N Cholesterol (mmol/L) LDL-cholesterol (mmol/L) HDL-cholesterol, men (mmol/L) HDL-cholesterol, women (mmol/L) Triglycerides (mmol/L) HbA1c Total IGF-I (SDS) TSH (mIU/L) Free T4 (pmol/L) Fasting glucose (mmol/L) 2 h Glucose (mmol/L) Fasting insulin (pmol/L) HOMA-IR Peak GH response (lg/L) Systolic BP (mm HG) Diastolic BP (mm HG) 45 43 21 24 45 44 46 45 44 46 46 43 43 41 46 46 Median (10th90th percentile) 4.50 (3.765.66) 2.90 (2.004.02) 1.20 (0.822.08) 1.40 (0.901.45) 0.75 (0.411.70) 0.054 (0.0480.060) 0.40 (1.561.81) 1.57 (0.803.46) 16.6 (12.424.5) 4.9 (3.96.7) 6.8 (4.914.2) 32 (1576) 1.02 (0.382.98) 16.7 (5.9544.55) 118 (100140) 67 (5290) Normal range <5.0 <3.0 >1.0 >1.2 <1.7 0.0510.062 0.34.50 1133 3.07.0 <7.8 569 <2.77 <140 <90

HOMA-IR, homeostasis model assessment index; BP, blood pressure.

Table 4 Spearman correlations r and (P-value) of body composition characteristics in 43 adults with PWS. BMI SFF VFF Femur fat Femur muscle 0.7616 0.2800 0.8480 0.4014 (<0.001) (0.041) (<0.001) (0.008) Peak GH 0.4218 0.4368 0.4117 0.4226 (0.008) (0.005) (0.008) (0.007)

indices. There was a tendency towards a higher thigh muscle/fat ratio in eugonadal compared to hypogonadal men (0.96 vs 0.66, P = 0.14). No differences between the men with or without testosterone treatment were found, except for a tendency towards a higher thigh muscle/fat ratio in those receiving testosterone (1.23 vs 0.88, P = 0.14). Seven women received oestrogen replacement with no apparent differences between those with and without treatment. 4. Discussion The data from this study showed that in both PWS men and women the amount of visceral fat was low in relation to the amount of subcutaneous fat and as a consequence the metabolic prole was only affected to a modest degree. Hormonal levels were in general normal but about 50% were hypogonadal and six patients fullled the established criteria for GHD in non-PWS individuals. The abdominal fat volume correlated to BMI and waist circumference, but not to metabolic parameters except the HOMA-index. PWS women had a higher BMI, similar waist circumference, subcutaneous and visceral abdominal fat but augmented thigh fat volume as compared to the men. Testosterone and oestrogen replacement did not seem to improve body composition signicantly.

SFF, subcutaneous abdominal fat fraction. VFF, visceral abdominal fat fraction.

No other signicant correlations with measured metabolic parameters were found. 3.5. Differences in body composition in hypogonadal vs eugonadal men and women with PWS Nine of the men in the cohort were treated with testosterone, seven men had normal testosterone levels without replacement and ve men had secondary hypogonadism. No signicant differences were found between hypogonadal and eugonadal men regarding the body composition measurements or metabolic

(a) 1000
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400

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20 25 30 35 40 45

600

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Fig. 1. Legend to Fig. 1a + b: subcutaneous abdominal fat 1a (SF) and visceral abdominal fat 1b (VF) (ml) in relationship to BMI (kg/m2) in 43 adults with PWS.

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In contrast to previous studies [7,11,24,25] approximately one third of the present cohort could be classied as non-obese according to their BMI. Examinations with CT of the abdomen showed, that abdominal fat was to a higher degree localised subcutaneously in the PWS group as compared to the comparator group. Furthermore, the relation VF/SF was lower in the PWS adults without gender differences. The CT results of the thigh showed similar volumes for the PWS adults with normal weight and obese PWS patients but the muscle/fat ratio was different, and for both clearly below the mean of 2.8 and 2.0, as shown for healthy lean and obese individuals [26]. Although, BMI does not correctly reect the nutritional state in PWS, because of the increased body fat and reduced muscle mass in addition to the short stature [5,11], the highly signicant correlation between BMI and abdominal (both SF and VF) and thigh fat was seen in the PWS adults. As expected body fat parameters showed a positive correlation with BMI and waist circumference, but not with metabolic parameters, reecting the less pronounced metabolic consequences of the relatively low VF, as also reported previously [6]. Anyhow, due to the positive correlation between BMI and VF as well as between VF and waist circumference and between BMI and waist circumference, the latter can in clinical practice be used as a marker for VF in PWS patients. This may be of convenience, particularly during clinical follow-up. The GH response to GHRH-arginine stimulation test was below the BMI related cut-off limits for GHD in six patients, and thus the number of patients with GHD in our cohort was lower, than what has been shown in previous studies [7,24,25,27]. However, BMI-related cut-off limits were only considered by Grugni et al. [24]. The chromosomal defect in PWS is suspected to cause a hypothalamic dysfunction resulting in impairments in gonadotropins and GH. The GHRH-arginine stimulation test is of limited value in patients with hypothalamic damage for example after irradiation to this area [28]. It can therefore be argued that this particular stimulation test might be a concern in PWS. However, GHRH-arginine stimulation has previously been used successfully in the investigation of GH secretion in PWS [27,29], and the results in the current study can probably not be ascribed to the test as such but rather to the lower BMI in our cohort. As expected there was a strong negative association between the amount of body fat and the GH peak upon stimulation. Despite the fact that the GH peak following GHRHarginine stimulation was normal in the majority of patients, there are still ndings pointing towards a dysfunction of the GH-IGF system; the inborn short stature [2,3] and low free IGF-I [30]. Also the reduced amount of muscle relative to the increased body fat is compatible with partial GH deciency. Peak GH response and the IGF-I, demonstrating the rather poor association between GH secretory capacity and circulating IGF-I in these patients in accordance with the ndings in normal adults and in adult patients with GHD [31]. In this context it is of interest that GH intervention studies in adults with PWS have demonstrated signicant effects on as well fat mass as lean body mass [24,25,32]. Hypogonadism in both women and men is associated with negative metabolic consequences [33]. Although hypogonadism is often seen in PWS women replacement therapy is infrequently used, as also reected in this study. With the limited number in mind benecial effects of sex-steroids on body composition were not observed in the present study. However, studies of larger cohorts and specically designed to examine these issues are needed in order to justify and establish the eventual need for sex-steroid replacement in both sexes of PWS adults. In this observational study a GHD control group was not established, but the large number of studied patients in this rare disease and the use of robust and validated methods in our opinion gives credit to our results and justies conclusions. It can be concluded that in adults with PWS body fat is increased but predominantly located subcutaneously in both

genders, and that the metabolic consequences thereof are limited. PWS women are to a higher degree obese than PWS men. Except from hypogonadism seen in approximately 50% and GHD in 15%, hormonal levels were normal. Sex-steroid replacement did not have obvious benecial effects on body composition. The amount of visceral fat was strongly related to BMI and waist circumference. Body composition parameters and previous functional studies on the GH/IGF axis are indicative of partial GHD, justifying the interest for GH treatment in this particular patient group. Acknowledgments The authors thank patients, parents and caretakers for participating in the study and the study nurses for providing the data. This study was supported by: Novo Nordisk Scandinavia AB, Malm, Sweden and Novo Nordisk, Bagsvrd, Denmark; the A.P. Mller Foundation for the Advancement of Medical Science; Research Initiative of Aarhus University Hospital; Aarhus University Hospital Skejby Research Foundation; Aase and Ejnar Danielsen Foundation; the Danish Prader-Willi Syndrome Association and the Augustinus Foundation. References
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