Brain & Development xxx (2013) xxx–xxx www.elsevier.com/locate/braindev

Original article

Assessment of metabolic changes within normal appearing gray and white matter in children with growth hormone deficiency: Magnetic resonance spectroscopy and hormonal correlation _ b, Anna Zimny a, Anna Zacharzewska-Gondek c, Joanna Bladowska a,⇑, Teresa Zak c Tomasz Maciej Gondek , Paweł Szewczyk a, Leszek Noga d, Anna Noczyn´ska b, Marek J. Sa˛siadek a a

Department of General Radiology, Interventional Radiology and Neuroradiology, Wroclaw Medical University, Wroclaw, Poland b Department of Endocrinology and Diabetology for Children and Adolescents, Wroclaw Medical University, Wroclaw, Poland c Department of General Radiology, Interventional Radiology and Neuroradiology, University Hospital, Wroclaw, Poland d Department of Pathophysiology, Wroclaw Medical University, Wroclaw, Poland Received 14 September 2013; received in revised form 19 November 2013; accepted 22 November 2013

Abstract The pathogenesis of idiopathic growth hormone deficiency (GHD) in children, including possible cerebral metabolic alterations, remains unclear. The aim of the study was to evaluate metabolic changes within the normal appearing brain in children with GHD using MR spectroscopy (MRS) and to correlate MRS measurements with hormonal concentrations and with pituitary gland size. Methods: Seventy children with GHD (mean age 7.8 yrs) and 11 healthy controls (mean age 8.4 yrs) were enrolled in the study. The MRS examinations were performed on a 1.5T scanner. Voxels were located in the posterior cingulate gyrus (PCG) and the left parietal white matter (PWM). The NAA/Cr, Cho/Cr and mI/Cr ratios were analyzed. The metabolite ratios, pituitary gland size and hormonal concentrations: growth hormone (GH) in two stimulation tests and GH during the night, as well as IGF-1 (insulin-like growth factor) and IGFBP3 (insulin-like growth factor-binding protein) levels were also correlated. Results: There was a significant (p < 0.05) decrease of the NAA/Cr ratios in PCG and PWM in children with GHD compared to the normal subjects. Other metabolite ratios showed no significant differences. We also found significant positive correlations between NAA/Cr ratio in PWM and IGFBP3 level, as well as with GH concentration in a stimulation test with glucagon. Conclusions: The reduction of NAA/Cr ratios may suggest loss of neuronal activity within normal appearing gray and white matters in children with GHD. MRS could be a sensitive marker of cerebral metabolic disturbances associated with GHD and maybe used as an additional indicator for therapy with recombinant GH. Ó 2013 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved. Keywords: Growth hormone deficiency; Brain metabolism; Magnetic resonance spectroscopy; Insulin-like growth factor 1; Cognitive function

1. Introduction

⇑ Corresponding author. Address: Department of General Radiology, Interventional Radiology and Neuroradiology, Wroclaw Medical University, Borowska 213 Str., 50-556 Wroclaw, Poland. Tel.: +48 (71) 733 1660; fax: +48 (71) 733 1689. E-mail address: [email protected] (J. Bladowska).

Growth hormone (GH) is synthesized in the anterior part of the pituitary gland and, to a markedly lesser degree, by other tissues in the brain [1,2]. The production of GH is modulated by two hypothalamic hormones, growth hormone-releasing hormone

0387-7604/$ - see front matter Ó 2013 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.braindev.2013.11.008

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(GHRH), which stimulates both the synthesis and secretion of GH and somatostatin which inhibits GH release in response to GHRH. GH also feeds back to inhibit GHRH secretion [3]. After excreting from the pituitary gland GH reaches the liver and due to this hormone action, the liver produces insulin-like growth factor-1 (IGF-1), which is the main anabolic mediator of GH activity. IGF-1 enters the blood stream where about 99% of IGF-1 is bound to the specific binding proteins and acid labile subunit (ALS) that significantly prolong the half-life of the peptide. The most important binding protein is the insulin-like growth factor-binding protein 3 (IGF-BP3), which bands up to 90% of IGF-1. Then the complex IGF-1/IGF-BP3 reaches the bones and muscles, which are the main final target organs for IGF-1 action [3,4]. For a long time, GH and IGF-1 have been recognized mainly for their critical role in achieving normal height. Recently, it has been proved that the GH/IGF-1 axis is involved not only in brain growth, development and myelination, but also in brain plasticity as rendered by neurogenesis. This may be associated with various cognitive effects of GH/IGF-1 [2,5–11]. However, little is known about the influence of growth hormone deficiency (GHD) on brain structure and possible metabolic alterations, especially in children [1,5,12]. Magnetic resonance spectroscopy (MRS) has enabled the in vivo evaluation of certain metabolites in a variety of pathologic processes affecting the central nervous system (CNS). MRS is capable of detecting the changes in metabolite profiles in normal appearing white matter (NAWM) and normal appearing gray matter (NAGM) [13–15]. Therefore, this advanced MR technique may offer a potentially unique insight into the pathophysiology of CNS changes associated with GHD. Moreover, it should be stressed that in the available world literature there are no articles concerning the analysis of metabolic alterations within NAWM and NAGM in children with GHD. The aim of the study was to evaluate the metabolic changes within the normal appearing brain in children with GHD using MRS, as well as to correlate MRS measurements with hormonal concentrations and the size of the pituitary gland. To the best of our knowledge this is the first study focusing on the role of MRS in assessment of metabolic disturbances associated with GHD in children.

14 yrs) were prospectively enrolled in the study. The diagnosis of GHD in childhood was based on the combination of auxological and hormonal criteria – decreased GH peak (below 10 ng/ml) in 2 stimulating tests – with oral clonidine 0.15 mg/m2 and i.m. glucagon 30 lg/kg i.m. (not exceeding 1 mg). The main inclusion criteria were as follows: short stature (height below 2 standard deviations (SDs) from the population mean), slow height velocity (below 10th centile) and GHD (peak GH concentration below 10 ng/ml in two stimulation tests), as well as delayed bone age as assessed by the Greulich-Pyle method [16]. The main radiological criteria were a normal appearing brain and lack of focal lesions within the pituitary gland. The MR examinations in children with GHD were performed prior to the commencement of growth hormone treatment. The study was conducted in accordance with the guidelines of the local University Ethics Committee for conducting research involving humans. Parents or legal guardians of all patients provided their signed informed consent to participate in the examination. 2.2. Endocrinological assessment The endocrinological evaluation consisted of hormonal concentration measurements as follows: the IGF-1, IGF-BP3 levels, as well as the GH concentrations during the night, GH concentration in two stimulations tests: with glucagon and clonidine. The latter included the GH concentration measurement after i.m. administrations of glucagon in the dosage of 30 lg/kg and oral administration of clonidine in the dosage of 0.15 mg/m2. Blood samples for GH were collected at time 0 and after 90, 120, 150 and 180 min after i.m. administration of glucagon, as well as at time 0 and after 30, 60, 90 and 120 min after oral administration of clonidine. During the night test, GH concentrations were measured at time 0 and after 60, 90, 120, 150 and 180 min after falling asleep. Growth hormone concentrations were measured using hHG IMMULITE and DPC assay, calibrated to WHO IRP/80/505 standard. Serum IGF-1 and IGFBP-3 concentrations were assessed by IMMULITE DPC assay, calibrated to WHO NOBSC 1st IRR/87/518 standard for IGF-1 and WHO NIBSC Reagent 93/560 standard for IGFBP-3. 2.3. The control group

2. Materials and methods 2.1. Patients Seventy children with idiopathic growth hormone deficiency (24 girls and 46 boys; mean age 7.8 yrs, ranging from 3 to 15 yrs), as well as 11 healthy children (7 girls and 4 boys, mean age 8.4 yrs, ranging from 3 to

As MR examination in the youngest children requires sedation, ethical reasons precluded us from recruiting healthy volunteers to make up the control group. Therefore we enrolled in the study only children who were referred for MR examination of the brain due to a variety of clinical reasons which are shown in Table 1. The control subjects fulfilled the following clinical criteria:

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Table 1 Clinical findings and indications for brain imaging in children from the control group. Indication for MR imaging

Final neurologic diagnosis

Number of subjects

Large head size Scalp lesion Vertebral anomalies Poor feeding Otitis media Hypoacusis Hypotonia Suspected nonaccidental injury

Normal Benign scalp lesion Scoliosis Normal Otitis media Normal Normal Normal

2 2 2 1 1 1 1 1

(1) normal neurologic assessment both before MR examination and at the time of discharge, with no evidence of developmental delay; (2) no clinical, radiological or laboratory evidence of neurologic damage; (3) normal neurologic follow-up results, if available; (4) normal height of the child. 2.4. MR imaging and MRS protocols MR imaging was performed on a 1.5 T Signa Hdx scanner (GE Healthcare, Medical System) using a 16channel coil dedicated for head and spine imaging. MR examinations were performed under general anesthetic in 53 children with GHD (76%) and 7 healthy children (64%) due to their young age (ranging from 3 to 7 yrs). The conventional sequences included coronal and sagittal T1 FSE, coronal and sagittal T2 FSE, and post-contrast T1-weighted images in coronal and sagittal planes (slice thickness 3 mm) of the pituitary gland, as well as axial T2-weighted images covering the whole brain. The MRS examinations were performed using the Single Voxel Spectroscopy (SVS) technique (PRESS

sequence). The data acquisition parameters were as follows: TR = 1500 ms, TE = 35 ms, number of acquisitions – 128, time of acquisition – 3 min 45 s. Using the localizing axial T2-weighted images of the brain, two voxels of 2  2  2 cm (8 cm3) were placed in the gray and white matter. Voxels were located in the following 2 regions: posterior cingulate gyrus – PCG (Fig. 1A) and left parietal white matter – PWM (Fig. 1B). The total acquisition time was 3 min 45 s for each voxel. MRS data was post-processed using algorithms provided by the manufacturer (GE workstation, ADW 4.4). Each spectrum was automatically fitted to four peaks corresponding to the levels of N-acetylaspartate (NAA) (2.02 ppm), total creatine (Cr) (3.03 ppm), choline-containing compounds (Cho) (3.23 ppm) and myo-inositol (mI) (3.56 ppm). Ratios of NAA, Cho and mI to creatine (NAA/Cr, Cho/Cr, mI/Cr, respectively) were calculated and analyzed. Additionally, the transverse (TR), cranio-caudal (CC) as well as anterior-posterior (AP) diameters of the pituitary gland were measured in children with GHD as well as in the control group. We also calculated the volume of the pituitary gland according to the equation: 4/3p  (TR/2  CC/2  AP/2) [17].

Fig. 1. Representative T2-weighted images (transverse cross-section) indicating voxel locations. Single voxel magnetic resonance spectroscopy was performed on two regions: in the posterior cingulate gyrus (PCG) (A); in the white matter of the left parietal region (PWM) (B).

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2.5. Statistical analysis

3.3. Comparison of the pituitary gland size

The MRS measurements as well as the size of the pituitary gland in children with GHD and healthy subjects were compared using the Student t-test. Additionally, in order to assess sensitivity, specificity and accuracy of MRS in distinguishing children with GHD and normal controls, the receiver-operating characteristic (ROC) analysis was performed for metabolite ratios showing the most significant differences between these two groups. The rate of accuracy was based on the area under the ROC curve. Associations between hormonal concentrations and MRS measurements as well as the pituitary gland size in children with GHD were assessed using Pearson’s correlation coefficient. Statistical computations were performed using the Statistica PL software package version 10.0, and the p < 0.05 was set as a significant level.

We found no statistically significant differences between children with GHD and the normal control subjects in the pituitary gland size, regarding the transverse (p = 0.115), cranio-caudal (p = 0.428), anterior– posterior (p = 0.595) diameters as well as total volume of the pituitary gland (p = 0.998).

3. Results 3.1. The size of the pituitary gland in children with GHD The mean transverse diameter of the pituitary gland in the analyzed group was 10.4 mm (ranging from 6.2 to 15.7 mm), the mean cranio-caudal diameter was 3.5 mm (ranging from 2.1 to 5.9 mm), while the mean anterior–posterior diameter was 6.5 mm (ranging from 4.3 to 9.7 mm). The mean volume of the pituitary gland was 127.3 mm3 (ranging from 30.66 to 270.17 mm3).

3.4. Comparison of MRS measurements Table 2 shows the mean values and standard deviations of the metabolite/creatine ratios in each brain region in children with GHD and normal control subjects. Compared to the control group (CG), subjects with GHD revealed a statistically significant decrease (p < 0.05) of the NAA/Cr ratios in both PCG and PWM regions. ROC analysis was performed separately for the PCG and PWM region using NAA/Cr ratios. Both ROC curves demonstrated good diagnostic accuracy with the area under the curve 0.83 for the PCG region and 0.74 for the PWM region (Fig. 2). When a cut off value for PCG NAA/Cr ratio was fixed at

Assessment of metabolic changes within normal appearing gray and white matter in children with growth hormone deficiency: magnetic resonance spectroscopy and hormonal correlation.

The pathogenesis of idiopathic growth hormone deficiency (GHD) in children, including possible cerebral metabolic alterations, remains unclear. The ai...
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