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The relationship between serum ghrelin and body composition with bone mineral density and QUS parameters in subjects with Rett syndrome
Bone 50 (2012) 830–835
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Original Full Length Article
The relationship between serum ghrelin and body composition with bone mineral density and QUS parameters in subjects with Rett syndrome C. Caffarelli a,⁎, S. Gonnelli a, L. Tanzilli a, J. Hayek b, V. Vichi a, M.B. Franci a, B. Lucani a, R. Nuti a a b
Department of Internal Medicine, Endocrine-Metabolic Science and Biochemistry, University of Siena, Italy Paediatrics Neuropsychiatry Unit, Azienda Ospedaliera Universitaria Senese, Siena, Italy
a r t i c l e
i n f o
Article history: Received 7 September 2011 Revised 16 January 2012 Accepted 20 January 2012 Available online 27 January 2012 Edited by: R. Baron Keywords: Rett's syndrome Ghrelin Lean mass Fat mass BMD-WB Quantitative ultrasound
a b s t r a c t Several studies have reported that females with Rett's syndrome frequently have marked decreases in bone mineral density (BMD). However, the pathogenesis of impaired bone status in RTT girls remains controversial. This study aimed to investigate whether ghrelin, an orexigenic peptide secreted by the stomach, was associated with body composition parameters, bone mineral density and quantitative ultrasound (QUS) in girls with Rett's syndrome. In 123 Rett girls (13.6 ± 8.2 years) and in 55 similar age range controls we evaluated ghrelin serum levels, 25OHD, quantitative ultrasound parameters at phalanxes by Bone Profiler-IGEA (amplitude dependent speed of sound: AD-SoS and bone transmission time: BTT), total body bone mineral density (BMD-WB) by Hologic QDR 4500. Whole body mineral content (BMC-WB), BMC-WB/height, fat mass (FM), fat percentage and lean mass (LM) were determined by using the same DXA device. We found that serum ghrelin levels were significantly higher in the Rett patients with respect to the control group (p b 0.05). In Rett girls ghrelin serum levels were inversely correlated with both age (R2 = 0.17, p b 0.001) and BMI (R2 = 0.14, p b 0.001). Moreover, in Rett subjects the values of BMD-WB, BMC-WB, BMCWB/height and QUS parameters were significantly lower than in control subjects. Fat mass and lean mass were lower in Rett subjects than in controls, but the difference reached the statistical significance only for lean mass. In Rett girls ghrelin serum levels were not predictors of bone status. Instead, we found that in Rett subjects, lean mass, age and 25OHD were significant independent predictors of BMC-WB/h, whereas both age and height were independent predictors of BMD-WB. Moreover, AD-SoS was predicted by age, fat percentage and height; while BTT was predicted only by height. In conclusion, our findings indicate that ghrelin levels were higher in Rett girls with respect to healthy controls, and negatively associated with both DXA and QUS parameters. However, in our study ghrelin was not found to be an independent predictor of bone mass, so supporting the hypothesis that ghrelin is elevated in Rett subjects in a compensatory manner. © 2012 Elsevier Inc. All rights reserved.
Introduction Rett's syndrome is a progressive neurodevelopmental disorder and one of the most common causes of mental retardation in females. About 96% of classic Rett's syndrome cases have mutations in the gene that encodes MeCP2, whereas other forms are largely associated with other genetic mutations, such as CDKL5 in the early onset seizure variant and FOXG1 mutations in the congenital variant. The MeCP2 is a multifunctional nuclear protein, with potentially important roles in chromatin architecture, regulation of RNA splicing and active transcription [1].
⁎ Corresponding author at: Department of Internal Medicine, Endocrine-Metabolic Science and Biochemistry, University of Siena, Policlinico Le Scotte, Viale Bracci 2, 53100 Siena, Italy. Fax: + 39 0577 233446. E-mail addresses: [email protected] (C. Caffarelli), [email protected] (J. Hayek). 8756-3282/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2012.01.017
Rett's syndrome is characterized by apparently normal development for the first 6–18 months of life, followed by a period of regression in language and motor skills. The patients lose purposeful hand uses and replace them with repetitive stereotyped hand movements. They usually have normal head circumference at birth followed by postnatal deceleration of head growth. Social withdrawal, communication dysfunction, loss of acquired speech and cognitive impairment are also characteristics of Rett patients. The impairment of locomotion is also very common. Additional characteristics include autistic features, panic-like attacks, respiratory dysfunctions (episodic apnea or hyperpnea), bruxism, impairment of sleeping patterns, progressive kyphosis or scoliosis, decreased somatic growth and feet and hands are hypotrophic, small and cold [1,2]. In recent years, in literature, there has been a growing interest in bone status in Rett patients. Clinical data show that, along with neurological defects, females with Rett's syndrome frequently have marked decreases in bone mineral density (BMD) [3–11]. As a consequence of the low bone mass
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Rett girls are at an increased risk of fragility fractures and it has been reported that 25–40% of Rett girls have fracture at some time during their lives [3,12]. However, the pathogenesis of impaired bone status in Rett subjects remains controversial. Motility limitation and underweight condition are commonly considered to play a crucial role in determining reduced BMD. In fact, it is generally agreed that a low body weight is associated with lower bone mineral density and increased fracture risk. However, the mechanisms which explain these relationships are still not completely understood. Weight, body composition, and particularly lean mass are among the strongest determinants of bone mass throughout life [13], whereas other studies have established a positive relationship between fat mass and BMD [14]. Moreover, several hormones may contribute to the relationships between fat and bone, those associated with nutrition being prime candidates [15]. The discovery of ghrelin has represented an interesting tool in the study of links between body composition and bone. Ghrelin is a 28-amino acid polypeptide mainly secreted by neuroendocrine cells in the stomach, which has been reported to participate in the release of growth hormone, in energy balance, in food intake, in adipogenesis, in the long-term regulation of body weight and in cortisol release [16–18], all of which potentially affect bone metabolism directly or indirectly. Literature data have reported that circulating ghrelin levels reflect changes in body weight and fat mass, and that ghrelin levels are decreased with obesity and increased after diet-induced weight loss [19]. The ghrelin receptor, known as the growth hormone secretagogue receptor, is found in many organs including the stomach, heart, lung, pancreas, intestine, kidney, testes, and ovary, as well as in the hypothalamus and in the adipose tissues. The wide distribution of this receptor indicates that ghrelin may have a variety of regulatory functions both in the brain and peripheral tissues. Recent studies indicate that ghrelin is involved in the regulation of bone growth and metabolism [20,21]. In particular, a positive effect of ghrelin on osteoblast proliferation and differentiation has been observed in some in vitro and experimental studies [21]. Moreover, the exogenous administration of ghrelin stimulates GH secretion and increases caloric intake and GI motility [22]. Currently, however, no clear evidence is present in literature about the relationship of circulating total ghrelin with bone mineral density and body composition in Rett subjects. The aim of our study was twofold: 1) to evaluate whether serum levels of ghrelin differ with respect to the similar age range healthy controls; 2) to assess whether in Rett subjects there is a relationship of ghrelin serum levels with body composition and bone mineral density. Materials and methods Study population We studied 123 patients (age range 4–33 years; mean age 13.6 ± 8.2) affected by Rett's syndrome, referred to the Department of Paediatric Neuropsychiatry of Siena from June 2009 to December 2010. This Department has a long history of research on Rett syndrome and many patients from different parts of Italy undergo a routine annual follow-up examination in Siena. The diagnosis of Rett syndrome was made according to the internationally accepted diagnostic criteria [23,24]. The patients who had experienced a fragility fracture or who had been treated with antiresorptive drugs in the previous 12 months were excluded. The patients with severe cardiac or pulmonary complications or with a life expectancy of less than 24 months were also excluded. Also Rett subjects on parenteral nutrition or with feeding tube were excluded. Fifty-five similar age range healthy subjects were used as controls. The study was approved by the Ethics Committee for human investigation of our Institution and informed consent was obtained according to the rules of the Ethics Committee.
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Questionnaires completed by parents provided information on clinical data, level of mobility, use of anticonvulsants or calcium/Vitamin D supplements, history of fracture and dietary calcium intake of the Rett patients. In our subjects, MECP2 mutations were present in 109 patients (88.6%), CDKL5 mutations were present in 4 subjects (4.2%), no information about genetic status was available for the other 10 subjects. At the time of the evaluation 86 (69.9%) patients were non ambulatory or presented a severe ambulatory impairment, whereas the other 37 (30.1%) were ambulatory. Densitometric and ultrasonographic measurements Whole body scans were carried out in all subjects by dual-energy X-ray absorptiometry (DXA) (Hologic QDR 4500A, Waltham, MA, USA) using standardized scan protocols. Whole body mineral content (BMC-WB), whole bone mineral density (BMD-WB), fat mass (FM), fat percentage and lean mass (LM) were determined by using the same DXA device. All scans were performed by the same operator while the subjects were wearing light indoor clothing and no removable metal objects. Quality control was performed weekly with a whole body phantom. In our Institution, the precision error for bone mineral density and bone mineral content (BMC) measurements is less than 2.5% for the whole body phantom. Previous studies reported that skull size confounds whole body bone data in young children [25]; therefore, all whole body DXA results shown in this study represent values excluding the skull. Whole body scans were analyzed to generate whole body BMC (in g) and areal BMD (in g/cm2). Moreover, whole body BMC data were presented as BMC/height, because of uncertainty of the accuracy of the total body reference volume prediction equation of Katzman et al. [26]. Also estimates of fat mass, fat mass percentage and lean mass were obtained from the whole body DXA scan excluding the skull. The Rett subjects with severe involuntary muscle contractions or uncontrollable movements were lightly sedated with midazolam (0.2 mg/kg/dose) before the scan to prevent repetitive involuntary movements which could invalidate the analysis. Moreover, in all subjects QUS parameters were evaluated at phalanxes by using a QUS device (Bone Profiler, IGEA, Italy). The device used is based on the transmission of ultrasound through the distal end of the first phalangeal diaphysis in the proximity of the condyles of the last four fingers of the hand. Bone Profiler measures the amplitude-dependent speed of sound (AD-SoS, m/s) and some parameters derived from the analysis of the graphic trace of the QUS signal [27,28]. AD-SoS depends on the signal amplitude because it is calculated by considering the time when the electrical signal, generated by the ultrasound mechanical wave at the receiving probe, reaches an amplitude of 2 mV [27]. Among the parameters derived from the analysis of the QUS graphic trace we have considered the bone transmission time (BTT, μs) which is the difference between the time when the first peak of the signal received attains its maximum and the time that would have been measured if only soft tissue and not bone was present between the transducers. Therefore BTT, unlike AD-SoS, is largely independent of ultrasound attenuation and soft tissue bias, and it depends almost exclusively on bone properties [29]. AD-SoS and BTT were measured in the non-dominant hand, and the final result is the average AD-SoS and BTT of the last four fingers. The AD-SoS and BTT values of Rett patients and controls were converted to Z-scores using the normative data obtained from a reference paediatric Italian population [30]. In our Institution the precision of AD-SoS and BTT evaluated in children was 0.7% and 0.8% respectively. In addition, the standardized coefficient of variation (sCV) was calculated for each QUS parameter according to the formula: sCV = CV%/range/mean, where range was the difference between the 5th and the 95th percentile of the population. The sCV were 3.7% for AD-SoS, and 2.6% for BTT. The precision
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assessed in 5 Rett patients measured five times on 1 day by the same operator (C.C.) by repositioning has given similar results (CV = 0.5% and 0.8% for AD-SoS and BTT respectively). Biochemical parameters In Rett subjects and controls, blood samples were also collected under fasting conditions to evaluate total serum ghrelin levels, serum calcium (Ca), phosphate (P), intact parathyroid hormone (PTH) and 25-Hydroxyvitam D (25OHD). Total serum ghrelin levels were measured by radioimmunoassay using a double antibody/PEG technique (Ghrelin total RIA kit, Linco Research, MO, USA). The results of ghrelin were expressed in pg/ml and the intra and interassay coefficients of variation, calculated at concentrations of between 500 and 2000 pg/ml, were 4.5% and 8.9% respectively. Serum PTH was assessed by an immunoradiometric assay using two goat polyclonal antibodies against the human PTH molecule (DiaSorin, Saluggia, Italy). The results were expressed in picograms per milliliter, and the intra- and inter-assay coefficients of variation were 3.6% and 4.9%, respectively. Serum 25OHD was determined by a radioimmunometric method (25-Hydroxyvitam D, DiaSorin, MN, USA). In our Institution the intra- and inter-assay coefficients of variation for 25OHD were 6.8% and 9.2%, respectively.
ghrelin levels were significantly higher in the Rett patients with respect to the control group (p b 0.05). Moreover, no difference in the age of menarche between Rett subjects and controls was observed. Densitometric, ultrasonographic and body composition parameters of the Rett patients and of the control group are reported in Table 2. In Rett subjects the values of BMD-WB, BMC-WB and BMCWB/height were significantly lower than in control subjects. Fat mass and lean mass were lower in Rett subjects than in controls, but the difference reached a statistical significance only for lean mass. However, the percentage of fat was higher in Rett subjects with respect to controls, even though the difference did not reach any statistical significance. Moreover, both AD-SoS and BTT were significantly lower in the Rett patients than in controls (p b 0.05). As expected, the Z-score values of BMD-WB, AD-SoS and BTT were significantly lower in the Rett patients than in controls. The differences in body composition, BMD-WB, BMC-WB, QUS parameters and ghrelin levels between Rett patients and controls were similar in prepubertal and pubertal subjects (i.e. ghrelin: 1251.3 ± 472.5 pg/ml and 1033.6 ± 335.2 pg/ml before puberty and 925.6 ± 331.4 pg/m and 786.6 ± 185.7 pg/ml after puberty, respectively). Moreover, in Rett subjects ghrelin serum levels were inversely correlated with both age (R 2 = 0.17, p b 0.001) and BMI (R 2 = 0.14, p b 0.001) (Fig. 1). Ghrelin and body composition parameters
Statistical analysis The variables normally distributed were expressed as mean ± SD and the significance between the means was tested using Student's t-test. Instead BMC-WB and fat mass were not distributed normally, therefore, these variables were also expressed as median and the significance between the means was tested using the Mann–Whitney test. The correlations between the groups were analyzed with the Pearson's correlation test and the Spearman's correlation where appropriate. Separate multiple linear regression models (method: Stepwise) were used to assess independent predictors of BMD-WB, BMC-WB/h, AD-SoS and BTT, while age, weight, height, BMI, fat mass, lean mass, percentage fat, movement capacity, 25OHD and ghrelin were included as independent variables in the models. For each model the regression coefficients (b-coefficients) and their 95% confidence intervals were described. All tests were two-sided, and p b 0.05 was considered statistically significant. All statistical tests were performed using SPSS 10.1 statistical software (SPSS 10.1).
The correlations of ghrelin serum levels with body composition and QUS parameters are shown in Table 3. In Rett subjects ghrelin serum levels were negatively correlated with all body composition and QUS parameters. After adjusting for age the correlations remained significant only for BMC-WB, fat mass, lean mass, fat percentage and BTT. When adjusting for BMI, only the correlations of ghrelin with BTT, AD-SoS and lean mass remained significant [Table 3]. The age adjusted relationships of body composition parameters with densitometric and ultrasound parameters are shown in Table 4. In Rett subjects and controls both fat mass and lean mass showed significant correlations with BMD-WB and BMC-WB/height. A positive correlation between weight and QUS parameters was observed in Rett subjects, but not in controls. A significant correlation between lean mass and BTT and a negative correlation between fat percentage and AD-SoS were also observed in both Rett girls and controls. Stepwise regression analyses of predictors of DXA and QUS parameters
Results In Table 5 we reported multiple linear regression analysis of predictors of bone mineral density and QUS parameters in Rett subjects.
Baseline characteristics Clinical characteristics and biochemical data of the Rett subjects and of the control group are reported in Table 1. As expected, the Rett patients were significantly shorter in height and lower in weight than the control group. Also serum 25OHD was lower in the Rett patients without reaching any statistical significance, while serum
Table 2 Densitometric, ultrasonographic and body composition parameters in Rett's subjects and controls.
Table 1 Clinical characteristics of patients with Rett's syndrome and controls.
Age (years) Weight (kg) Height (cm) Menarche (yes/no) Menarche age (years) Calcium (mg/dl) Phosphorus (mg/dl) PTH (pg/ml) 25OHD (ng/ml) Ghrelin (pg/ml)
Rett patients (N = 123)
Controls (N = 55)
p
13.57 ± 8.18 32.97 ± 15.63 130.74 ± 19.33 (63/60) 11.3 ± 0.3 9.5 ± 0.54 5.0 ± 0.6 36.35 ± 39.2 37.63 ± 24.23 1088.6 ± 437.23
12.75 ± 6.86 50.21 ± 17.04 149.25 ± 14.33 (27/28) 11.6 ± 1.4 9.3 ± 0.63 4.8 ± 0.7 29.9 ± 20.9 40.30 ± 19.07 921.67 ± 270.17
n.s. b 0.001. b 0.001. – n.s. n.s. n.s. n.s. n.s. b 0.05
Rett patients (n = 123)
Controls (n = 55)
p
BMD-WB (g/cm2) BMD-WB Z-score BMC-WB (g)
0.668 ± 0.231 − 1.35 ± 2.1 703.1 ± 351.3 605.4 [410.4–993.1]#
b 0.001. b 0.001. b 0.001. 0.05#
BMC-WB/height (g/cm) Fat Mass (g)
7.45 ± 2.51 12267.3 ± 8073.8 10601.2 [5579.2–16098.6]# 17828.9 ± 8280.9 38.1 ± 8.45 1882.6 ± 74.2 − 1.95 ± 1.54 0.75 ± 0.34 − 1.69 ± 1.93
0.863 ± 0.148 0.5 ± 0.2 1214.63 ± 434.8 1185.8 [880.1–1555.5]# 10.33 ± 2.68 17462.7 ± 9847.7 14889.0 [10157.7–23098.7]# 30185.9 ± 8242.6 33.9 ± 9.55 1926.1 ± 84.5 − 1.51 ± 1.19 1.07 ± 0.38 − 0.48 ± 1.54
Lean Mass (g) Fat Percentage (%) AD-SoS (m/s) AD-SoS Z-score BTT (μs) BTT Z-score #
Median [Q1; Q3], Mann-Whitney test.
b 0.001 n.s. 0.05# b 0.001 n.s b 0.05 b 0.05 b 0.05 b 0.05
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Fig. 1. Curvilinear regression (power function) of ghrelin serum levels with age (1A) and BMI (1B) in Rett subjects (N = 123).
The analysis was performed by including in the model age, weight, height, BMI, fat mass, lean mass, fat percentage, movement capacity, 25OHD and ghrelin as independent variables. In Rett subjects BMDWB was predicted primarily by age and height, whereas BMC-WB/ height was predicted by lean mass, age and 25OHD. In Rett girls ghrelin serum levels were not independent predictors of BMD-WB and BMC-WB/height. Moreover, AD-SoS was predicted by age, fat percentage and height, while BTT was predicted only by height. When the subjects with Rett syndrome were separated in two groups on the basis of the capacity of movement, the Rett subjects with mobility impairment showed both body composition and ultrasound parameters to be lower than in Rett subjects without mobility impairment. The differences between the two groups reached statistical significance for all parameters apart from ghrelin serum levels and 25OHD (Table 6). Discussion To our knowledge, this study is the first to assess the respective contribution of ghrelin and body composition to bone mineral density and QUS parameters in Rett subjects. Our data have shown that serum levels of ghrelin are higher in Rett subjects than in similar age range healthy controls. Concerning the levels of ghrelin in young subjects, it is important to consider that the exact role of ghrelin during childhood and adolescence has not been fully defined, and that data on normal ghrelin levels in children and adolescents are controversial. However, our Table 3 Simple and age or BMI adjusted correlations of serum ghrelin with body composition parameters and ultrasound parameters at phalanxes in Rett subjects. R
BMD-WB (g/cm2) BMC-WB (g/cm2) BMC-WB/h (g/cm) Fat mass (g) Lean mass (g) Fat percentage (%) AD-SoS (m/s) BTT (μs) §
Spearman's correlation.
− 0.28 − 0.39§ − 0.27 − 0.43§ − 0.44 − 0.17 − 0.26 − 0.35
p
0.001 0.001 0.01 0.001 0.001 0.05 0.01 0.001
Age-adjusted
BMI-adjusted
R
p
R
p
− 0.08 − 0.19 − 0.08 − 0.25 − 0.31 − 0.18 − 0.09 − 0.18
n.s. 0.05 n.s. 0.01 0.01 0.05 n.s. 0.05
− 0.10 − 0.12 − 0.07 − 0.10 − 0.24 0.10 − 0.23 − 0.27
n.s. n.s. n.s. n.s. 0.01 n.s. 0.05 0.01
results seem to be in agreement with previous studies by Misra et al. which reported that serum ghrelin levels were higher in girls with anorexia nervosa than in controls [31,32]. Also the study of Altinkaynak et al. reported that ghrelin levels were significantly higher in children with protein energy malnutrition than in those in good health [33]. Moreover, Camurdan et al. found that serum ghrelin was found to be higher in children with short stature with respect to subjects of normal height [34]. Instead, our findings seem to be in contrast with the recent study by Hara et al. which reported lower ghrelin serum levels in Rett patients with respect to controls. However, in this latter study the majority of Rett's syndrome patients showed important eating difficulties [35]. In agreement with some previous studies [36], we have found a negative association between ghrelin and age. In fact, Chanoine et al. reported that circulating ghrelin levels progressively increase during the first 2 years of life before decreasing during late childhood and adolescence, without gender-specific differences. Moreover, ghrelin levels decrease during puberty with advancing Tanner staging and are 30–50% lower in postpubertal compared to prepubertal subjects [36]. Moreover, Whatmore et al. have reported that prepubertal children had higher ghrelin concentrations than those in puberty with significant negative correlations between ghrelin, age and puberty status [37]. In agreement with previous studies we have also found that in Rett patients ghrelin levels are negatively correlated with BMI [31,32]. To date, no generally acceptable explanation exists for this finding. However, the early deceleration in height, weight and BMI that characterizes the natural history of growth failure and undernutrition in Rett subjects could support the hypothesis that ghrelin is elevated in Rett girls in a compensatory manner. This is because of its two important functions: orexigenic functioning as a response to reduced body weight and a strong GH secretagogue functioning as a response to lower height [34]. Therefore, we can logically assume that ghrelin levels could be elevated in Rett girls as an adaptive mechanism to a reduced body weight, as seen in patients with anorexia nervosa [31,32]. In our study the values of serum ghrelin levels were negatively correlated with BMD-WB, BMC-WB and BMC-WB/height, but the correlation disappear when adjusting for BMI. At present in literature there are few studies, carried out primarily on adults, regarding the association of total ghrelin with BMD; the results of these studies are conflicting, above all because of substantial differences among study populations. Those few studies carried out on children have also
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Table 4 Age adjusted correlations of body composition with densitometric and ultrasonographic parameters in Rett subjects and controls. Rett subjects (N = 123)
Weight (kg) BMI (kg/rn2) Fat mass (g) Lean mass (g) Fat percentage (%)
Controls (N = 55)
BMD-WB
BMC-WB/height
AD-SoS
BTT
BMD-WB
BMC-WB/height
AD-SoS
BTT
0.85** 0.61** 0.26* 0.34** 0.15
0.73** 0.52** 0.31** 0.40** 0.13
0.42** 0.17 − 0.13 0.12 0.30**
0.61** 0.35** 0.10 0.29** − 0.18
0.68** 0.33* 0.28* 0.61** 0.10
0.70** 0.34* 0.30** 0.63** 0.11
0.19 − 0.20 − 0.22 0.20 0.54**
0.24 0.04 0,14 0.29* 0.33*
*p b 005; **p b 001.
yielded contradictory results. In fact, in the study by Misra et al. involving 23 adolescent girls with anorexia nervosa and 21 healthy adolescent girls, total ghrelin secretion predicted bone density at the lumbar spine and the hip independently of body composition, GH– IGF-1 axis, cortisol or estradiol in healthy girls but not in those with anorexia nervosa [21]. Moreover, in the study by Pomerants et al. reporting on 60 healthy non-obese male schoolchildren (aged 10–18 years), serum total ghrelin concentration was inversely related to total BMD, lumbar BMD, and BMAD, but those correlations were not as strong as seen between BMD and testosterone or IGF markers [38]. Our study shows that fat mass and lean mass were lower in Rett subjects than in healthy controls, but the difference reached the statistical significance only for lean mass. These data are in agreement with the recent study by Motil et al. [9] which confirmed that lean body mass, but not body fat, was significantly lower in the Rett's syndrome cohort than in the reference population. However, fat percentage was higher in Rett subjects both in our study and in the study by Motil [9]. Moreover, in our study fat percentage was shown to be significantly greater in Rett patients who were non ambulatory and who presumably were suffering from a more severe disease. Although the cause of the increase of fat percentage in Rett girls is unknown, we can logically assume that the impaired ambulatory ability may play a crucial role. The importance of physical activity on body composition is well known. In fact, children with movement disorders, such as cerebral palsy or spinal cord injury have been shown to have decreased lean mass compared with children who have no motor impairment. Conversely in children, positive associations have been observed between body composition parameters and weight-bearing activity [39]. In fact, the study by McDonald et al. showed that young patients aged between 10 and 21 years with spinal cord injury had significantly reduced lean tissue mass and significantly higher fat mass than gender-, age-, and BMImatched controls [40]. Until now, few studies have assessed the relative contribution of fat mass and lean mass to BMC-WB [40] and BMD-WB [41] in children
and young adults, most of these studies being conducted on girls. Whereas the effect of lean mass was consistent across studies [40,41], the effect of fat mass was reported as being more variable [41]. Our data show that both in Rett subjects and in healthy controls fat mass and lean mass were significantly correlated with bone mineral density. Moreover, similar to that which was previously reported for healthy subjects [40,41], in Rett girls lean mass but not fat mass was an independent predictor of bone mass. In fact, we found that in Rett subjects, lean mass, age and 25OHD were significant independent predictors of BMC-WB/h and age and height were independent predictors of BMD-WB. To date, in children and adolescents, the role of fat mass as an independent predictor of bone mineral content or density remains controversial [42]. In fact, in a study carried out on 5 year old children, total body fat mass has been shown to be an independent predictor of bone area [43], and in another study by Clark et al. [44] it was reported that adipose tissue stimulates bone growth in prepubertal children. Instead, Pollock et al. have demonstrated that fat mass may potentially be negative for adolescent bone, and also that in adolescents, unlike adults, bone mass continued to increase during successful weight loss [45]. To our knowledge, this is the first study to assess the respective contribution of lean mass, fat mass and fat percentage to QUS parameters in a Rett population. We have demonstrated a positive correlation between lean mass and BTT; moreover, it is important to underline that BTT has been reported to mainly reflect the properties of cortical bone. This study, in agreement with others, suggests that BTT is likely to be the more appropriate QUS parameter to estimate bone status in children and adolescents [27,28]. Moreover, in our study a negative correlation between fat percentage and QUS parameters at phalanxes (AD-SoS and BTT) has been observed in both Rett subjects and healthy controls. The fact that in multivariate analysis
Table 6 Clinical characteristics of ambulatory and non ambulatory Rett subjects.
Table 5 Multiple linear regression analysis of predictors of bone mineral density and QUS parameters in Rett subjects. Variable BMD-WB (g/cm2) Height (cm) Age (years) BMC-WB/h (g/cm) Lean mass (g) Age (years) 25OHD (ng/ml) AD-SoS (m/s) Age (years) Fat percentage (%) Height (cm) BTT (μs) Height (cm)
Undestandardized coefficient, b
95% Cl
p
0.006 0.008
0.004 to 0.009 0.00 1 to 0.015
b0.0001 0.024
0.005 0.083 0.013
0.003 to 0.008 0.014 to 0.153 0.001 to 0.025
b0.0001 0.018 0.041
3.412 − 1.935 1.270
0.641 to 6.184 − 3.479 to − 0.390 0,188 to 2.352
0.016 0.015 0.022
0.012
0.009 to 0.015
b0.0001
Whole set of variables included into the models: age, weight, height, BMI, fat mass, lean mass, fat percentage, movement capacity, 25OHD and ghrelin.
Age (years) Weight (kg) Height (cm) 25OHD (ng/ml) Ghrelin (pg/ml) BMD-WB (g/cm2) BMC-WB (g)
BMC-WB/height (g/cm) AD-SoS (m/s) BTT (μs) Fat mass (g)
Lean mass (g) Fat percentage (%)
Ambulatory (n = 37)
Non-ambulatory (n = 86)
p
15.1 ± 1.9 37.6 ± 4.2 136.2 ± 15.5 41.2 ± 15.6 1083.2 ± 573.2 0.748 ± 0.163 849.3 ± 195.7 890.2 [498.3–1132.7]# 8.5 ± 1.4 1900.6 ± 37.5 0.83 ± 0.15 13697.3 ± 21819.2 13972.8 [9187.2–19437.8]# 21557.9 ± 15030.9 36.4 ± 21.9
13.0 ± 4.9 31.2 ± 21.2 128.5 ± 14.5 35.5 ± 29.1 1090.1 ± 386.1 0.643 ± 0.239 649.6 ± 343.8 540.1 [351.2–936.8]# 7.2 ± 2.6 1877.5 ± 75.7 0.71 ± 0.07 11796.3 ± 8983.7 9849.0 [5455.6–14889.0]# 16305.2 ± 8323.6 39.3 ± 8.4
b 0.05 b 0.001. b 0.001. n.s. n.s. b 0.01. b 0.001. 0.01#
Median [Q1; Q3], Mann–Whitney test.
b 0.001 b 0.05 b 0.05 b 0.001 0.05# b 0.001 b 0.05
C. Caffarelli et al. / Bone 50 (2012) 830–835
fat percentage was a negative predictor of AD-SoS in Rett subjects seems to confirm that the quantity of soft tissue surrounding phalanxes can affect the measurements of AD-SoS. Our study confirms the usefulness of QUS in the assessment of bone status in children and adolescents. Moreover, in previous studies carried out on children and adolescents with disturbances of growth, QUS and DXA parameters showed similar results, suggesting that both methods are able to identify a reduced bone mineral status [46]. Our study presents some limitations. Firstly, the cross-sectional nature of the study does not permit the inferring of any causality relationships in Rett subjects. Secondly, bone mass measurements carried out by DXA are based on a two-dimensional projection of a three-dimensional structure and this is a possible limitation when measuring growing subjects. Thirdly, the scarce homogeneity of our Rett population with regard to age, genetic profile and motility impairment. Finally, DXA technique does not directly measure abdominal fat compartments and therefore the separate role of visceral and subcutaneous fat remains uncertain. This study also has some strength. One of these being the large sample size and the fact that it is representative of the Italian population with Rett syndrome. Another strength of the study is the comprehensive evaluation of bone status by DXA and QUS measurements. In conclusion, our findings indicate that ghrelin levels were higher in Rett girls with respect to healthy controls, and negatively associated with both DXA and QUS parameters. However, in our study ghrelin was not found to be an independent predictor of bone mass, so supporting the hypothesis that ghrelin is elevated in Rett subjects in a compensatory manner. Moreover, our results also demonstrate that in Rett girls lean mass and height were the main independent predictors of bone mass. Further studies are warranted in order to better define the factors responsible for skeletal fragility of Rett population.
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[27] Wuster C, Albanese C, De Aloysio D, Duboeuf F, Gambacciani M, Gonnelli S, et al. Phalangeal osteosonogrammetry study: age-related changes, diagnostic sensitivity, and discrimination power. J Bone Miner Res 2000;15:1603–14. [28] Montagnani A, Gonnelli S, Cepollaro C, Bruni D, Franci MB, Lucani B, et al. Graphic trace analysis of quantitative ultrasound at phalanxes seems to improve the diagnosis of primary hyperparathyroidism among patients with low bone mass. Osteoporos Int 2002;13:222–7. [29] Barkmann R, Lusse S, Stampa B, Sakata S, Heller M, Gluer CC. Assessment of the geometry of human finger phalanges using quantitative ultrasound in vivo. Osteoporos Int 2000;11:745–55. [30] Baroncelli GI, Federico G, Vignolo M, Valerio G, Del Puente A, Maghnie M, et al. Cross-sectional reference data for phalangeal quantitative ultrasound from early childhood to young-adulthood according to gender, age, skeletal growth, and pubertal development. Bone 2006;39:17159–317. 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Ghrelin in growth and development. Horm Res 2005;63:129–38. [37] Whatmore AJ, Hall CM, Jones J, Westwood M, Clayton PE. Ghrelin concentrations in healthy children and adolescents. Clin Endocrinol (Oxf) 2003;59:649–54. [38] Pomerants T, Tillmann V, Jürimäe J, Jürimäe T. The influence of serum ghrelin, IGF axis and testosterone on bone mineral density in boys at different stages of sexual maturity. J Bone Miner Metab 2007;25:193–7. [39] Farr JN, Chen Z, Lisse JR, Lohman TG, Going SB. Relationship of total body fat mass to weight-bearing bone volumetric density, geometry, and strength in young girls. Bone 2010;46:977–84. [40] McDonald CM, Abresch-Meyer AL, Nelson MD, Widman LM. Body Mass Index and Body Composition Measures by Dual X-Ray Absorptiometry in Patients Aged 10 to 21 Years With Spinal Cord Injury. J Spinal Cord Med 2007;30:S97–S104. [41] Arabi A, Tamim H, Nabulsi M, Maalouf J, Khalifé H, Choucair M, et al. 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Original Full Length Article
The relationship between serum ghrelin and body composition with bone mineral density and QUS parameters in subjects with Rett syndrome C. Caffarelli a,⁎, S. Gonnelli a, L. Tanzilli a, J. Hayek b, V. Vichi a, M.B. Franci a, B. Lucani a, R. Nuti a a b
Department of Internal Medicine, Endocrine-Metabolic Science and Biochemistry, University of Siena, Italy Paediatrics Neuropsychiatry Unit, Azienda Ospedaliera Universitaria Senese, Siena, Italy
a r t i c l e
i n f o
Article history: Received 7 September 2011 Revised 16 January 2012 Accepted 20 January 2012 Available online 27 January 2012 Edited by: R. Baron Keywords: Rett's syndrome Ghrelin Lean mass Fat mass BMD-WB Quantitative ultrasound
a b s t r a c t Several studies have reported that females with Rett's syndrome frequently have marked decreases in bone mineral density (BMD). However, the pathogenesis of impaired bone status in RTT girls remains controversial. This study aimed to investigate whether ghrelin, an orexigenic peptide secreted by the stomach, was associated with body composition parameters, bone mineral density and quantitative ultrasound (QUS) in girls with Rett's syndrome. In 123 Rett girls (13.6 ± 8.2 years) and in 55 similar age range controls we evaluated ghrelin serum levels, 25OHD, quantitative ultrasound parameters at phalanxes by Bone Profiler-IGEA (amplitude dependent speed of sound: AD-SoS and bone transmission time: BTT), total body bone mineral density (BMD-WB) by Hologic QDR 4500. Whole body mineral content (BMC-WB), BMC-WB/height, fat mass (FM), fat percentage and lean mass (LM) were determined by using the same DXA device. We found that serum ghrelin levels were significantly higher in the Rett patients with respect to the control group (p b 0.05). In Rett girls ghrelin serum levels were inversely correlated with both age (R2 = 0.17, p b 0.001) and BMI (R2 = 0.14, p b 0.001). Moreover, in Rett subjects the values of BMD-WB, BMC-WB, BMCWB/height and QUS parameters were significantly lower than in control subjects. Fat mass and lean mass were lower in Rett subjects than in controls, but the difference reached the statistical significance only for lean mass. In Rett girls ghrelin serum levels were not predictors of bone status. Instead, we found that in Rett subjects, lean mass, age and 25OHD were significant independent predictors of BMC-WB/h, whereas both age and height were independent predictors of BMD-WB. Moreover, AD-SoS was predicted by age, fat percentage and height; while BTT was predicted only by height. In conclusion, our findings indicate that ghrelin levels were higher in Rett girls with respect to healthy controls, and negatively associated with both DXA and QUS parameters. However, in our study ghrelin was not found to be an independent predictor of bone mass, so supporting the hypothesis that ghrelin is elevated in Rett subjects in a compensatory manner. © 2012 Elsevier Inc. All rights reserved.
Introduction Rett's syndrome is a progressive neurodevelopmental disorder and one of the most common causes of mental retardation in females. About 96% of classic Rett's syndrome cases have mutations in the gene that encodes MeCP2, whereas other forms are largely associated with other genetic mutations, such as CDKL5 in the early onset seizure variant and FOXG1 mutations in the congenital variant. The MeCP2 is a multifunctional nuclear protein, with potentially important roles in chromatin architecture, regulation of RNA splicing and active transcription [1].
⁎ Corresponding author at: Department of Internal Medicine, Endocrine-Metabolic Science and Biochemistry, University of Siena, Policlinico Le Scotte, Viale Bracci 2, 53100 Siena, Italy. Fax: + 39 0577 233446. E-mail addresses: [email protected] (C. Caffarelli), [email protected] (J. Hayek). 8756-3282/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2012.01.017
Rett's syndrome is characterized by apparently normal development for the first 6–18 months of life, followed by a period of regression in language and motor skills. The patients lose purposeful hand uses and replace them with repetitive stereotyped hand movements. They usually have normal head circumference at birth followed by postnatal deceleration of head growth. Social withdrawal, communication dysfunction, loss of acquired speech and cognitive impairment are also characteristics of Rett patients. The impairment of locomotion is also very common. Additional characteristics include autistic features, panic-like attacks, respiratory dysfunctions (episodic apnea or hyperpnea), bruxism, impairment of sleeping patterns, progressive kyphosis or scoliosis, decreased somatic growth and feet and hands are hypotrophic, small and cold [1,2]. In recent years, in literature, there has been a growing interest in bone status in Rett patients. Clinical data show that, along with neurological defects, females with Rett's syndrome frequently have marked decreases in bone mineral density (BMD) [3–11]. As a consequence of the low bone mass
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Rett girls are at an increased risk of fragility fractures and it has been reported that 25–40% of Rett girls have fracture at some time during their lives [3,12]. However, the pathogenesis of impaired bone status in Rett subjects remains controversial. Motility limitation and underweight condition are commonly considered to play a crucial role in determining reduced BMD. In fact, it is generally agreed that a low body weight is associated with lower bone mineral density and increased fracture risk. However, the mechanisms which explain these relationships are still not completely understood. Weight, body composition, and particularly lean mass are among the strongest determinants of bone mass throughout life [13], whereas other studies have established a positive relationship between fat mass and BMD [14]. Moreover, several hormones may contribute to the relationships between fat and bone, those associated with nutrition being prime candidates [15]. The discovery of ghrelin has represented an interesting tool in the study of links between body composition and bone. Ghrelin is a 28-amino acid polypeptide mainly secreted by neuroendocrine cells in the stomach, which has been reported to participate in the release of growth hormone, in energy balance, in food intake, in adipogenesis, in the long-term regulation of body weight and in cortisol release [16–18], all of which potentially affect bone metabolism directly or indirectly. Literature data have reported that circulating ghrelin levels reflect changes in body weight and fat mass, and that ghrelin levels are decreased with obesity and increased after diet-induced weight loss [19]. The ghrelin receptor, known as the growth hormone secretagogue receptor, is found in many organs including the stomach, heart, lung, pancreas, intestine, kidney, testes, and ovary, as well as in the hypothalamus and in the adipose tissues. The wide distribution of this receptor indicates that ghrelin may have a variety of regulatory functions both in the brain and peripheral tissues. Recent studies indicate that ghrelin is involved in the regulation of bone growth and metabolism [20,21]. In particular, a positive effect of ghrelin on osteoblast proliferation and differentiation has been observed in some in vitro and experimental studies [21]. Moreover, the exogenous administration of ghrelin stimulates GH secretion and increases caloric intake and GI motility [22]. Currently, however, no clear evidence is present in literature about the relationship of circulating total ghrelin with bone mineral density and body composition in Rett subjects. The aim of our study was twofold: 1) to evaluate whether serum levels of ghrelin differ with respect to the similar age range healthy controls; 2) to assess whether in Rett subjects there is a relationship of ghrelin serum levels with body composition and bone mineral density. Materials and methods Study population We studied 123 patients (age range 4–33 years; mean age 13.6 ± 8.2) affected by Rett's syndrome, referred to the Department of Paediatric Neuropsychiatry of Siena from June 2009 to December 2010. This Department has a long history of research on Rett syndrome and many patients from different parts of Italy undergo a routine annual follow-up examination in Siena. The diagnosis of Rett syndrome was made according to the internationally accepted diagnostic criteria [23,24]. The patients who had experienced a fragility fracture or who had been treated with antiresorptive drugs in the previous 12 months were excluded. The patients with severe cardiac or pulmonary complications or with a life expectancy of less than 24 months were also excluded. Also Rett subjects on parenteral nutrition or with feeding tube were excluded. Fifty-five similar age range healthy subjects were used as controls. The study was approved by the Ethics Committee for human investigation of our Institution and informed consent was obtained according to the rules of the Ethics Committee.
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Questionnaires completed by parents provided information on clinical data, level of mobility, use of anticonvulsants or calcium/Vitamin D supplements, history of fracture and dietary calcium intake of the Rett patients. In our subjects, MECP2 mutations were present in 109 patients (88.6%), CDKL5 mutations were present in 4 subjects (4.2%), no information about genetic status was available for the other 10 subjects. At the time of the evaluation 86 (69.9%) patients were non ambulatory or presented a severe ambulatory impairment, whereas the other 37 (30.1%) were ambulatory. Densitometric and ultrasonographic measurements Whole body scans were carried out in all subjects by dual-energy X-ray absorptiometry (DXA) (Hologic QDR 4500A, Waltham, MA, USA) using standardized scan protocols. Whole body mineral content (BMC-WB), whole bone mineral density (BMD-WB), fat mass (FM), fat percentage and lean mass (LM) were determined by using the same DXA device. All scans were performed by the same operator while the subjects were wearing light indoor clothing and no removable metal objects. Quality control was performed weekly with a whole body phantom. In our Institution, the precision error for bone mineral density and bone mineral content (BMC) measurements is less than 2.5% for the whole body phantom. Previous studies reported that skull size confounds whole body bone data in young children [25]; therefore, all whole body DXA results shown in this study represent values excluding the skull. Whole body scans were analyzed to generate whole body BMC (in g) and areal BMD (in g/cm2). Moreover, whole body BMC data were presented as BMC/height, because of uncertainty of the accuracy of the total body reference volume prediction equation of Katzman et al. [26]. Also estimates of fat mass, fat mass percentage and lean mass were obtained from the whole body DXA scan excluding the skull. The Rett subjects with severe involuntary muscle contractions or uncontrollable movements were lightly sedated with midazolam (0.2 mg/kg/dose) before the scan to prevent repetitive involuntary movements which could invalidate the analysis. Moreover, in all subjects QUS parameters were evaluated at phalanxes by using a QUS device (Bone Profiler, IGEA, Italy). The device used is based on the transmission of ultrasound through the distal end of the first phalangeal diaphysis in the proximity of the condyles of the last four fingers of the hand. Bone Profiler measures the amplitude-dependent speed of sound (AD-SoS, m/s) and some parameters derived from the analysis of the graphic trace of the QUS signal [27,28]. AD-SoS depends on the signal amplitude because it is calculated by considering the time when the electrical signal, generated by the ultrasound mechanical wave at the receiving probe, reaches an amplitude of 2 mV [27]. Among the parameters derived from the analysis of the QUS graphic trace we have considered the bone transmission time (BTT, μs) which is the difference between the time when the first peak of the signal received attains its maximum and the time that would have been measured if only soft tissue and not bone was present between the transducers. Therefore BTT, unlike AD-SoS, is largely independent of ultrasound attenuation and soft tissue bias, and it depends almost exclusively on bone properties [29]. AD-SoS and BTT were measured in the non-dominant hand, and the final result is the average AD-SoS and BTT of the last four fingers. The AD-SoS and BTT values of Rett patients and controls were converted to Z-scores using the normative data obtained from a reference paediatric Italian population [30]. In our Institution the precision of AD-SoS and BTT evaluated in children was 0.7% and 0.8% respectively. In addition, the standardized coefficient of variation (sCV) was calculated for each QUS parameter according to the formula: sCV = CV%/range/mean, where range was the difference between the 5th and the 95th percentile of the population. The sCV were 3.7% for AD-SoS, and 2.6% for BTT. The precision
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assessed in 5 Rett patients measured five times on 1 day by the same operator (C.C.) by repositioning has given similar results (CV = 0.5% and 0.8% for AD-SoS and BTT respectively). Biochemical parameters In Rett subjects and controls, blood samples were also collected under fasting conditions to evaluate total serum ghrelin levels, serum calcium (Ca), phosphate (P), intact parathyroid hormone (PTH) and 25-Hydroxyvitam D (25OHD). Total serum ghrelin levels were measured by radioimmunoassay using a double antibody/PEG technique (Ghrelin total RIA kit, Linco Research, MO, USA). The results of ghrelin were expressed in pg/ml and the intra and interassay coefficients of variation, calculated at concentrations of between 500 and 2000 pg/ml, were 4.5% and 8.9% respectively. Serum PTH was assessed by an immunoradiometric assay using two goat polyclonal antibodies against the human PTH molecule (DiaSorin, Saluggia, Italy). The results were expressed in picograms per milliliter, and the intra- and inter-assay coefficients of variation were 3.6% and 4.9%, respectively. Serum 25OHD was determined by a radioimmunometric method (25-Hydroxyvitam D, DiaSorin, MN, USA). In our Institution the intra- and inter-assay coefficients of variation for 25OHD were 6.8% and 9.2%, respectively.
ghrelin levels were significantly higher in the Rett patients with respect to the control group (p b 0.05). Moreover, no difference in the age of menarche between Rett subjects and controls was observed. Densitometric, ultrasonographic and body composition parameters of the Rett patients and of the control group are reported in Table 2. In Rett subjects the values of BMD-WB, BMC-WB and BMCWB/height were significantly lower than in control subjects. Fat mass and lean mass were lower in Rett subjects than in controls, but the difference reached a statistical significance only for lean mass. However, the percentage of fat was higher in Rett subjects with respect to controls, even though the difference did not reach any statistical significance. Moreover, both AD-SoS and BTT were significantly lower in the Rett patients than in controls (p b 0.05). As expected, the Z-score values of BMD-WB, AD-SoS and BTT were significantly lower in the Rett patients than in controls. The differences in body composition, BMD-WB, BMC-WB, QUS parameters and ghrelin levels between Rett patients and controls were similar in prepubertal and pubertal subjects (i.e. ghrelin: 1251.3 ± 472.5 pg/ml and 1033.6 ± 335.2 pg/ml before puberty and 925.6 ± 331.4 pg/m and 786.6 ± 185.7 pg/ml after puberty, respectively). Moreover, in Rett subjects ghrelin serum levels were inversely correlated with both age (R 2 = 0.17, p b 0.001) and BMI (R 2 = 0.14, p b 0.001) (Fig. 1). Ghrelin and body composition parameters
Statistical analysis The variables normally distributed were expressed as mean ± SD and the significance between the means was tested using Student's t-test. Instead BMC-WB and fat mass were not distributed normally, therefore, these variables were also expressed as median and the significance between the means was tested using the Mann–Whitney test. The correlations between the groups were analyzed with the Pearson's correlation test and the Spearman's correlation where appropriate. Separate multiple linear regression models (method: Stepwise) were used to assess independent predictors of BMD-WB, BMC-WB/h, AD-SoS and BTT, while age, weight, height, BMI, fat mass, lean mass, percentage fat, movement capacity, 25OHD and ghrelin were included as independent variables in the models. For each model the regression coefficients (b-coefficients) and their 95% confidence intervals were described. All tests were two-sided, and p b 0.05 was considered statistically significant. All statistical tests were performed using SPSS 10.1 statistical software (SPSS 10.1).
The correlations of ghrelin serum levels with body composition and QUS parameters are shown in Table 3. In Rett subjects ghrelin serum levels were negatively correlated with all body composition and QUS parameters. After adjusting for age the correlations remained significant only for BMC-WB, fat mass, lean mass, fat percentage and BTT. When adjusting for BMI, only the correlations of ghrelin with BTT, AD-SoS and lean mass remained significant [Table 3]. The age adjusted relationships of body composition parameters with densitometric and ultrasound parameters are shown in Table 4. In Rett subjects and controls both fat mass and lean mass showed significant correlations with BMD-WB and BMC-WB/height. A positive correlation between weight and QUS parameters was observed in Rett subjects, but not in controls. A significant correlation between lean mass and BTT and a negative correlation between fat percentage and AD-SoS were also observed in both Rett girls and controls. Stepwise regression analyses of predictors of DXA and QUS parameters
Results In Table 5 we reported multiple linear regression analysis of predictors of bone mineral density and QUS parameters in Rett subjects.
Baseline characteristics Clinical characteristics and biochemical data of the Rett subjects and of the control group are reported in Table 1. As expected, the Rett patients were significantly shorter in height and lower in weight than the control group. Also serum 25OHD was lower in the Rett patients without reaching any statistical significance, while serum
Table 2 Densitometric, ultrasonographic and body composition parameters in Rett's subjects and controls.
Table 1 Clinical characteristics of patients with Rett's syndrome and controls.
Age (years) Weight (kg) Height (cm) Menarche (yes/no) Menarche age (years) Calcium (mg/dl) Phosphorus (mg/dl) PTH (pg/ml) 25OHD (ng/ml) Ghrelin (pg/ml)
Rett patients (N = 123)
Controls (N = 55)
p
13.57 ± 8.18 32.97 ± 15.63 130.74 ± 19.33 (63/60) 11.3 ± 0.3 9.5 ± 0.54 5.0 ± 0.6 36.35 ± 39.2 37.63 ± 24.23 1088.6 ± 437.23
12.75 ± 6.86 50.21 ± 17.04 149.25 ± 14.33 (27/28) 11.6 ± 1.4 9.3 ± 0.63 4.8 ± 0.7 29.9 ± 20.9 40.30 ± 19.07 921.67 ± 270.17
n.s. b 0.001. b 0.001. – n.s. n.s. n.s. n.s. n.s. b 0.05
Rett patients (n = 123)
Controls (n = 55)
p
BMD-WB (g/cm2) BMD-WB Z-score BMC-WB (g)
0.668 ± 0.231 − 1.35 ± 2.1 703.1 ± 351.3 605.4 [410.4–993.1]#
b 0.001. b 0.001. b 0.001. 0.05#
BMC-WB/height (g/cm) Fat Mass (g)
7.45 ± 2.51 12267.3 ± 8073.8 10601.2 [5579.2–16098.6]# 17828.9 ± 8280.9 38.1 ± 8.45 1882.6 ± 74.2 − 1.95 ± 1.54 0.75 ± 0.34 − 1.69 ± 1.93
0.863 ± 0.148 0.5 ± 0.2 1214.63 ± 434.8 1185.8 [880.1–1555.5]# 10.33 ± 2.68 17462.7 ± 9847.7 14889.0 [10157.7–23098.7]# 30185.9 ± 8242.6 33.9 ± 9.55 1926.1 ± 84.5 − 1.51 ± 1.19 1.07 ± 0.38 − 0.48 ± 1.54
Lean Mass (g) Fat Percentage (%) AD-SoS (m/s) AD-SoS Z-score BTT (μs) BTT Z-score #
Median [Q1; Q3], Mann-Whitney test.
b 0.001 n.s. 0.05# b 0.001 n.s b 0.05 b 0.05 b 0.05 b 0.05
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833
Fig. 1. Curvilinear regression (power function) of ghrelin serum levels with age (1A) and BMI (1B) in Rett subjects (N = 123).
The analysis was performed by including in the model age, weight, height, BMI, fat mass, lean mass, fat percentage, movement capacity, 25OHD and ghrelin as independent variables. In Rett subjects BMDWB was predicted primarily by age and height, whereas BMC-WB/ height was predicted by lean mass, age and 25OHD. In Rett girls ghrelin serum levels were not independent predictors of BMD-WB and BMC-WB/height. Moreover, AD-SoS was predicted by age, fat percentage and height, while BTT was predicted only by height. When the subjects with Rett syndrome were separated in two groups on the basis of the capacity of movement, the Rett subjects with mobility impairment showed both body composition and ultrasound parameters to be lower than in Rett subjects without mobility impairment. The differences between the two groups reached statistical significance for all parameters apart from ghrelin serum levels and 25OHD (Table 6). Discussion To our knowledge, this study is the first to assess the respective contribution of ghrelin and body composition to bone mineral density and QUS parameters in Rett subjects. Our data have shown that serum levels of ghrelin are higher in Rett subjects than in similar age range healthy controls. Concerning the levels of ghrelin in young subjects, it is important to consider that the exact role of ghrelin during childhood and adolescence has not been fully defined, and that data on normal ghrelin levels in children and adolescents are controversial. However, our Table 3 Simple and age or BMI adjusted correlations of serum ghrelin with body composition parameters and ultrasound parameters at phalanxes in Rett subjects. R
BMD-WB (g/cm2) BMC-WB (g/cm2) BMC-WB/h (g/cm) Fat mass (g) Lean mass (g) Fat percentage (%) AD-SoS (m/s) BTT (μs) §
Spearman's correlation.
− 0.28 − 0.39§ − 0.27 − 0.43§ − 0.44 − 0.17 − 0.26 − 0.35
p
0.001 0.001 0.01 0.001 0.001 0.05 0.01 0.001
Age-adjusted
BMI-adjusted
R
p
R
p
− 0.08 − 0.19 − 0.08 − 0.25 − 0.31 − 0.18 − 0.09 − 0.18
n.s. 0.05 n.s. 0.01 0.01 0.05 n.s. 0.05
− 0.10 − 0.12 − 0.07 − 0.10 − 0.24 0.10 − 0.23 − 0.27
n.s. n.s. n.s. n.s. 0.01 n.s. 0.05 0.01
results seem to be in agreement with previous studies by Misra et al. which reported that serum ghrelin levels were higher in girls with anorexia nervosa than in controls [31,32]. Also the study of Altinkaynak et al. reported that ghrelin levels were significantly higher in children with protein energy malnutrition than in those in good health [33]. Moreover, Camurdan et al. found that serum ghrelin was found to be higher in children with short stature with respect to subjects of normal height [34]. Instead, our findings seem to be in contrast with the recent study by Hara et al. which reported lower ghrelin serum levels in Rett patients with respect to controls. However, in this latter study the majority of Rett's syndrome patients showed important eating difficulties [35]. In agreement with some previous studies [36], we have found a negative association between ghrelin and age. In fact, Chanoine et al. reported that circulating ghrelin levels progressively increase during the first 2 years of life before decreasing during late childhood and adolescence, without gender-specific differences. Moreover, ghrelin levels decrease during puberty with advancing Tanner staging and are 30–50% lower in postpubertal compared to prepubertal subjects [36]. Moreover, Whatmore et al. have reported that prepubertal children had higher ghrelin concentrations than those in puberty with significant negative correlations between ghrelin, age and puberty status [37]. In agreement with previous studies we have also found that in Rett patients ghrelin levels are negatively correlated with BMI [31,32]. To date, no generally acceptable explanation exists for this finding. However, the early deceleration in height, weight and BMI that characterizes the natural history of growth failure and undernutrition in Rett subjects could support the hypothesis that ghrelin is elevated in Rett girls in a compensatory manner. This is because of its two important functions: orexigenic functioning as a response to reduced body weight and a strong GH secretagogue functioning as a response to lower height [34]. Therefore, we can logically assume that ghrelin levels could be elevated in Rett girls as an adaptive mechanism to a reduced body weight, as seen in patients with anorexia nervosa [31,32]. In our study the values of serum ghrelin levels were negatively correlated with BMD-WB, BMC-WB and BMC-WB/height, but the correlation disappear when adjusting for BMI. At present in literature there are few studies, carried out primarily on adults, regarding the association of total ghrelin with BMD; the results of these studies are conflicting, above all because of substantial differences among study populations. Those few studies carried out on children have also
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C. Caffarelli et al. / Bone 50 (2012) 830–835
Table 4 Age adjusted correlations of body composition with densitometric and ultrasonographic parameters in Rett subjects and controls. Rett subjects (N = 123)
Weight (kg) BMI (kg/rn2) Fat mass (g) Lean mass (g) Fat percentage (%)
Controls (N = 55)
BMD-WB
BMC-WB/height
AD-SoS
BTT
BMD-WB
BMC-WB/height
AD-SoS
BTT
0.85** 0.61** 0.26* 0.34** 0.15
0.73** 0.52** 0.31** 0.40** 0.13
0.42** 0.17 − 0.13 0.12 0.30**
0.61** 0.35** 0.10 0.29** − 0.18
0.68** 0.33* 0.28* 0.61** 0.10
0.70** 0.34* 0.30** 0.63** 0.11
0.19 − 0.20 − 0.22 0.20 0.54**
0.24 0.04 0,14 0.29* 0.33*
*p b 005; **p b 001.
yielded contradictory results. In fact, in the study by Misra et al. involving 23 adolescent girls with anorexia nervosa and 21 healthy adolescent girls, total ghrelin secretion predicted bone density at the lumbar spine and the hip independently of body composition, GH– IGF-1 axis, cortisol or estradiol in healthy girls but not in those with anorexia nervosa [21]. Moreover, in the study by Pomerants et al. reporting on 60 healthy non-obese male schoolchildren (aged 10–18 years), serum total ghrelin concentration was inversely related to total BMD, lumbar BMD, and BMAD, but those correlations were not as strong as seen between BMD and testosterone or IGF markers [38]. Our study shows that fat mass and lean mass were lower in Rett subjects than in healthy controls, but the difference reached the statistical significance only for lean mass. These data are in agreement with the recent study by Motil et al. [9] which confirmed that lean body mass, but not body fat, was significantly lower in the Rett's syndrome cohort than in the reference population. However, fat percentage was higher in Rett subjects both in our study and in the study by Motil [9]. Moreover, in our study fat percentage was shown to be significantly greater in Rett patients who were non ambulatory and who presumably were suffering from a more severe disease. Although the cause of the increase of fat percentage in Rett girls is unknown, we can logically assume that the impaired ambulatory ability may play a crucial role. The importance of physical activity on body composition is well known. In fact, children with movement disorders, such as cerebral palsy or spinal cord injury have been shown to have decreased lean mass compared with children who have no motor impairment. Conversely in children, positive associations have been observed between body composition parameters and weight-bearing activity [39]. In fact, the study by McDonald et al. showed that young patients aged between 10 and 21 years with spinal cord injury had significantly reduced lean tissue mass and significantly higher fat mass than gender-, age-, and BMImatched controls [40]. Until now, few studies have assessed the relative contribution of fat mass and lean mass to BMC-WB [40] and BMD-WB [41] in children
and young adults, most of these studies being conducted on girls. Whereas the effect of lean mass was consistent across studies [40,41], the effect of fat mass was reported as being more variable [41]. Our data show that both in Rett subjects and in healthy controls fat mass and lean mass were significantly correlated with bone mineral density. Moreover, similar to that which was previously reported for healthy subjects [40,41], in Rett girls lean mass but not fat mass was an independent predictor of bone mass. In fact, we found that in Rett subjects, lean mass, age and 25OHD were significant independent predictors of BMC-WB/h and age and height were independent predictors of BMD-WB. To date, in children and adolescents, the role of fat mass as an independent predictor of bone mineral content or density remains controversial [42]. In fact, in a study carried out on 5 year old children, total body fat mass has been shown to be an independent predictor of bone area [43], and in another study by Clark et al. [44] it was reported that adipose tissue stimulates bone growth in prepubertal children. Instead, Pollock et al. have demonstrated that fat mass may potentially be negative for adolescent bone, and also that in adolescents, unlike adults, bone mass continued to increase during successful weight loss [45]. To our knowledge, this is the first study to assess the respective contribution of lean mass, fat mass and fat percentage to QUS parameters in a Rett population. We have demonstrated a positive correlation between lean mass and BTT; moreover, it is important to underline that BTT has been reported to mainly reflect the properties of cortical bone. This study, in agreement with others, suggests that BTT is likely to be the more appropriate QUS parameter to estimate bone status in children and adolescents [27,28]. Moreover, in our study a negative correlation between fat percentage and QUS parameters at phalanxes (AD-SoS and BTT) has been observed in both Rett subjects and healthy controls. The fact that in multivariate analysis
Table 6 Clinical characteristics of ambulatory and non ambulatory Rett subjects.
Table 5 Multiple linear regression analysis of predictors of bone mineral density and QUS parameters in Rett subjects. Variable BMD-WB (g/cm2) Height (cm) Age (years) BMC-WB/h (g/cm) Lean mass (g) Age (years) 25OHD (ng/ml) AD-SoS (m/s) Age (years) Fat percentage (%) Height (cm) BTT (μs) Height (cm)
Undestandardized coefficient, b
95% Cl
p
0.006 0.008
0.004 to 0.009 0.00 1 to 0.015
b0.0001 0.024
0.005 0.083 0.013
0.003 to 0.008 0.014 to 0.153 0.001 to 0.025
b0.0001 0.018 0.041
3.412 − 1.935 1.270
0.641 to 6.184 − 3.479 to − 0.390 0,188 to 2.352
0.016 0.015 0.022
0.012
0.009 to 0.015
b0.0001
Whole set of variables included into the models: age, weight, height, BMI, fat mass, lean mass, fat percentage, movement capacity, 25OHD and ghrelin.
Age (years) Weight (kg) Height (cm) 25OHD (ng/ml) Ghrelin (pg/ml) BMD-WB (g/cm2) BMC-WB (g)
BMC-WB/height (g/cm) AD-SoS (m/s) BTT (μs) Fat mass (g)
Lean mass (g) Fat percentage (%)
Ambulatory (n = 37)
Non-ambulatory (n = 86)
p
15.1 ± 1.9 37.6 ± 4.2 136.2 ± 15.5 41.2 ± 15.6 1083.2 ± 573.2 0.748 ± 0.163 849.3 ± 195.7 890.2 [498.3–1132.7]# 8.5 ± 1.4 1900.6 ± 37.5 0.83 ± 0.15 13697.3 ± 21819.2 13972.8 [9187.2–19437.8]# 21557.9 ± 15030.9 36.4 ± 21.9
13.0 ± 4.9 31.2 ± 21.2 128.5 ± 14.5 35.5 ± 29.1 1090.1 ± 386.1 0.643 ± 0.239 649.6 ± 343.8 540.1 [351.2–936.8]# 7.2 ± 2.6 1877.5 ± 75.7 0.71 ± 0.07 11796.3 ± 8983.7 9849.0 [5455.6–14889.0]# 16305.2 ± 8323.6 39.3 ± 8.4
b 0.05 b 0.001. b 0.001. n.s. n.s. b 0.01. b 0.001. 0.01#
Median [Q1; Q3], Mann–Whitney test.
b 0.001 b 0.05 b 0.05 b 0.001 0.05# b 0.001 b 0.05
C. Caffarelli et al. / Bone 50 (2012) 830–835
fat percentage was a negative predictor of AD-SoS in Rett subjects seems to confirm that the quantity of soft tissue surrounding phalanxes can affect the measurements of AD-SoS. Our study confirms the usefulness of QUS in the assessment of bone status in children and adolescents. Moreover, in previous studies carried out on children and adolescents with disturbances of growth, QUS and DXA parameters showed similar results, suggesting that both methods are able to identify a reduced bone mineral status [46]. Our study presents some limitations. Firstly, the cross-sectional nature of the study does not permit the inferring of any causality relationships in Rett subjects. Secondly, bone mass measurements carried out by DXA are based on a two-dimensional projection of a three-dimensional structure and this is a possible limitation when measuring growing subjects. Thirdly, the scarce homogeneity of our Rett population with regard to age, genetic profile and motility impairment. Finally, DXA technique does not directly measure abdominal fat compartments and therefore the separate role of visceral and subcutaneous fat remains uncertain. This study also has some strength. One of these being the large sample size and the fact that it is representative of the Italian population with Rett syndrome. Another strength of the study is the comprehensive evaluation of bone status by DXA and QUS measurements. In conclusion, our findings indicate that ghrelin levels were higher in Rett girls with respect to healthy controls, and negatively associated with both DXA and QUS parameters. However, in our study ghrelin was not found to be an independent predictor of bone mass, so supporting the hypothesis that ghrelin is elevated in Rett subjects in a compensatory manner. Moreover, our results also demonstrate that in Rett girls lean mass and height were the main independent predictors of bone mass. Further studies are warranted in order to better define the factors responsible for skeletal fragility of Rett population.
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