Neuropeptides 45 (2011) 205–211

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Neuropeptides

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Plasma brain-derived neurotrophic factor concentrations in childrenand adolescents

L. Iughetti a,⇑, E. Casarosa b, B. Predieri a, V. Patianna a, S. Luisi c

a Department of Pediatrics, University of Modena and Reggio Emilia, Modena, Italyb Department of Reproductive Medicine and Child Development, Division of Gynecology and Obstetrics, University of Pisa, Pisa, Italyc Department of Pediatric Obstetrics and Reproductive Medicine, Section of Obstetrics and Gynecology, University of Siena, Siena, Italy

a r t i c l e i n f o a b s t r a c t

Article history:Received 30 September 2010Accepted 20 February 2011Available online 21 March 2011

Keywords:Brain-derived neurotrophic factorChildrenSex hormonesPubertyBody mass index

0143-4179/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.npep.2011.02.002

⇑ Corresponding author. Address: Department of Pedand Reggio Emilia, Via del Pozzo 71, 41124 Modena,fax: +39 059 4224583.

E-mail address: iughetti.lorenzo@unimore.it (L. Iug

Background: Brain-derived neurotrophic factor (BDNF) is a mediator of neuronal plasticity influencinglearning, memory and cognitive behavior. The aim of this study is to assess plasma BDNF variationsaccording to pubertal status.Methods: A total of 110 subjects were included in the study. Blood samples were collected after overnightfasting. Plasma BDNF concentrations were measured by enzyme-linked immunosorbent assay. Gonado-trophins, sex steroids, and IGF-1 were also assessed.Results: BDNF was positively correlated with platelet count and negatively associated with both BMI andage. BDNF levels in pubertal males were significantly lower than prepubertal males and both prepubertaland pubertal females.Conclusions: Plasma BDNF levels seem to be influenced by hormonal status. We demonstrate that param-eters such as age or gender have a specific impact on stored and circulating BDNF blood levels and plate-lets remain the most important predictor of their concentration. Further studies are necessary to betterunderstand the role of this neurotrophin in pubertal development.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Brain-derived neurotrophic factor (BDNF) is a member of theneurotrophin family which also includes nerve growth factor(NGF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5)(Mowla et al., 2001). All neurotrophins have common charac-teristics, including similar molecular weight (13.2–15.9 kDa),isoelectric point (range 9–10), and have a high similarity inprimary structure. Six cysteine residues conserved in the samerelative positions give rise to three intra-chain disulfide bonds(Tapia-Arancibia et al., 2004; Kernie et al., 2000). The neurotro-phins are present in solution as no covalently bound dimers andinteract with cell surface receptors, the low affinity P75 receptorand the tyrosine kinase (Trk) family of high affinity Trk receptors.NGF preferentially binds TrkA, both BDNF and NT4/5 bind TrkB,while NT-3 binds TrkC (Mowla et al., 2001).

BDNF has a wide range of biological activities and is producedby different immune and structural cells. It is involved in neuro-genesis and neuronal plasticity during brain development andadulthood (Henderson, 1996; Tapia-Arancibia et al., 2004) and it

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iatrics, University of ModenaItaly. Tel.: +39 059 4222182;

hetti).

has been shown to induce long-lasting changes in synaptic plastic-ity, neurotransmitter and neuropeptide production and to influ-ence the excitability (Kang and Schuman, 1995; Li et al., 1998;Hall et al., 2000; Carter et al., 2002). Both central and peripheralnervous system synthesize and release this neurotrophin thatcan easily cross the blood–brain barrier (Pan et al., 1998; Karegeet al., 2002a). BDNF is detectable in blood because it is also abun-dantly expressed and secreted in other human non-neuronal tis-sues such as endocrine and salivary glands, urinary tract,respiratory system, ovary, macrophages, lymphocytes, vascularendothelial and smooth muscle cells (Donovan et al., 1995;Moalem et al., 2000; Nakahashi et al., 2000).

In mice, BDNF is already present in fetal life and its expressionrises to a maximal level after birth, promoting neuronal growthand differentiation in neonates (Can Over and Yancopoulos,1997). BDNF concentrations are lower in human preterm infantscompared with term ones (Nikolaou et al., 2006). More recently,in preterm infants BDNF levels has been demonstrated to rise be-yond the first week of age, following a transient decline after birth,and significantly to correlate with factors like antenatal/postnatalsteroids and retinopathy of prematurity that impact neurodevelop-ment outcomes (Rao et al., 2009). Excesses of neurotrophins andneuropeptides in sera of newborn infants seem to have predictivevalue in determining those children who later will have autism andmental retardation (Nelson et al., 2001; Miyazaki et al., 2004).

206 L. Iughetti et al. / Neuropeptides 45 (2011) 205–211

In human adults, low BDNFs’ serum and plasma levels charac-terize the more frequent neuropsychiatric diseases, like majordepression (Karege et al., 2002b; Shimizu et al., 2003; Piccininiet al., 2008), schizophrenia (Toyooka et al., 2002; Palomino et al.,2006) and eating disorders such as bulimia and anorexia nervosa(Nakazato et al., 2003; Monteleone et al., 2004, 2005), Parkinson’sand Alzheimer’s diseases (Connor et al., 1997; Parain et al., 1999),epileptic and psychogenic nonepileptic seizures (LaFrance et al.,2010). BDNF is also involved in mental retardation of Down’s syn-drome patients. In these subjects BDNF plasma levels have beendemonstrated to be higher than in healthy ones with a significantage-related increase and to have a protective role against athero-sclerosis risk (Dogliotti et al., 2010). The persistently higher levelsof BDNF frequently associate with autoantibodies, as demonstratedin older children with autism, mental retardation and epilepsy,suggest the interaction between the immune system and this neu-rotrophin (Connolly et al., 2006; Correia et al., 2010).

In addition to the effects observed in the nervous system, a re-cent study have found that children with moderate and severeasthma have higher plasma BDNF levels than mild asthma andcontrols; the authors suggest that this neurotrophin may be con-sidered a potential biomarker for clinical severity in subjects withasthma (Müller et al., 2010).

Aging is a relevant factor affecting BDNFs’ ability to protect neu-ronal activity, but age-related effects on BDNF function in non-neuronal cells still remain unclear.

BDNF in rats has a functional role in the reproductive system(Gibbs, 1999; Scharfman et al., 2003) and in human adults it hasa close relationship with sex hormones, in particular estrogens(Barde, 1999). Recently, BDNF concentrations in women have beendemonstrated to be higher in the luteal phase than in the follicularone (Begliuomini et al., 2007; Cubeddu et al., 2011). In amenorrheaand menopausal status, sex steroids modify the BDNFs’ circulatinglevels; combined oral contraceptives abolish the luteal increase ofthis neurotrophin (Begliuomini et al., 2007), while hormonalreplacement therapy is able to increase the low menopausal BDNFlevels (Cubeddu et al., 2010). Moreover, in women with premen-strual syndrome, plasma BDNF decreases during ovarian cycle, inopposition to the increasing trend that is observed in women with-out premenstrual syndrome and this variation is probably due toan altered hormonal response (Cubeddu et al., 2011).

Considering that platelets represent a major storage site ofBDNF in peripheral blood (Fujimura et al., 2002) its serum levelsare higher than plasma ones. Moreover, BDNF plasma concentra-tions significantly decrease with age or weight gain, whereas plate-let or serum levels do not seem to be altered (Lommatzsch et al.,2005).

The aim of the present study is to assess plasma BDNF concen-trations in healthy children and adolescents according to pubertalstatus. We measure BDNFs’ levels in plasma because they repre-sent the current steady state of biologically active BDNF, whereasserum concentrations mainly reflect the content of platelets.

2. Subjects and methods

2.1. Subjects

One hundred and ten children, adolescents and young adults(39 females, 9.11 ± 4.54 years and 71 males, 8.90 ± 5.47 years)were recruited from the Pediatrics Endocrinology Unit of the Uni-versity of Modena and Reggio Emilia between the outpatients sub-mitted to investigations for auxological evaluation and that finallyresulted healthy. Prior to enrollment, all subjects and/or their par-ents provided their written informed consent and the Ethical Boardof Pediatric Department approved this study. The subjects and/or

their parents were asked to disclose any known chronic diseases,current illnesses, regular medications, allergies and family historyfor endocrinological, psychiatric or neurological diseases. Physicalexamination and laboratory tests were performed in all includedsubjects and they revealed no abnormalities. Moreover, none ofthe subjects was taking psychoactive medications, hormonal ther-apies (including oral contraceptives) or anti-inflammatory drugs.Mood or behavior disturbances were not referred at the momentof the enrollment. Exclusion criteria included known genetic orendocrine diseases, significant medical illnesses, and use ofmedications.

2.2. Methods

Subjects were categorized into three groups for analysis,according to pubertal staging:

Group 1: Prepuberty (59 subjects); Tanner stage for girls:breast (B) 1 and pubic hair (PH) 1; Tanner stagefor boys: genital development (G) 1, PH 1, and tes-ticular volume <4 ml.

Group 2: Midpuberty (32 subjects); Tanner stage for girls: Band PH 2–3; Tanner stage for boys: G and PH 2–3and testicular volume 4–12 ml.

Group 3: Late puberty (19 subjects); Tanner stage for girls: Band PH 4–5; Tanner stage for boys: G and PH 4–5,testicular volume >12 ml.

A single Pediatric Endocrinologist (L.I.) determined pubertaldevelopment using the grading system of Tanner (Marshall andTanner, 1969). Testicular volume was measured using anorchidometer.

After overnight fasting, a blood sample was drawn from thecubital vein of each subject in EDTA-coated tubes (Vacutest Kimas.r.l., Arzergrande, Italy) between 7.30 and 9.00 a.m. in order tominimize the effects of a possible circadian variation of plasmaBDNF levels, as previously suggested (Solum and Handa, 2002).In menstruating females blood sample was drawn in the follicularphase (days 6–8). The tubes were kept on ice and, after collection,blood samples were immediately centrifuged at 4 �C (2500 g for15 min). Plasma and serum were aliquoted and stored at �80 �Cuntil assay for BDNF, estradiol (E2), testosterone (T), and insulin-like growth factor 1 (IGF-1) levels.

Height (Ht), weight (Wt), and platelets (PTL) count were alsoevaluated in all subjects. Ht was measured to the nearest 0.1-cmwith a wall-mounted stadiometer (Harpenden, Crymych, UK). BodyWt was measured to the nearest 0.1-kg and body mass index (BMI)was obtained from the Wt in kg/Ht in meters squared and ex-pressed as z-score (z-BMI) with respect to chronological age (CA).

2.3. BDNF assay

Plasma levels of BDNF were determined in duplicate with anenzyme-linked immunosorbent assay (ELISA) method (BDNF EmaxImmunoassay System, Promega, USA), after appropriate dilution ofsamples (1:4) using Block and Sample Buffer, according to themanufacturer’s instructions. Briefly, 96-well flat bottom immuno-plates were coated with anti-BDNF monoclonal antibody and incu-bated at 4 �C overnight. After blocking by non-specific binding withBlock and Sample Buffer, standards and samples were added to theplates and incubated shaking for 2 h at room temperature. Subse-quently, after washing with TBST wash buffer, plates were incu-bated for 2 h with anti-Human BDNF polyclonal antibody. Thelast incubation required the addition of anti-immunoglobulin Y-horse-radish peroxidase conjugate. In the last step of the assay,TMB One solution was added in order to develop the color. After

Table 1Anthropometric and laboratory data according to pubertal stage.

Prepuberty (N = 59) Puberty (N = 51) P

Sex (M/F) 45/14 26/25 –Age (years ± SD) 5.76 ± 2.91 12.6 ± 4.67 0.000Ht (cm ± SD) 112.4 ± 19.4 147.6 ± 18.4 0.000Ht z-score (SDS ± SD) 0.31 ± 1.04 0.10 ± 1.29 0.206Wt (kg ± SD) 23.4 ± 12.4 48.8 ± 22.0 0.000BMI (kg/m2 ± SD) 17.4 ± 3.20 21.3 ± 5.35 0.000z-BMI (SDS ± SD) 0.53 ± 1.17 0.89 ± 1.32 0.134PTL (103/mm3 ± SD) 302.6 ± 54.3 267.6 ± 76.5 0.069E2 (pg/ml ± SD) 12.0 ± 9.74 21.4 ± 19.7 0.000T (ng/dl ± SD) 0.04 ± 0.06 0.71 ± 0.90 0.000IGF-1 (ng/ml ± SD) 133.0 ± 120.2 183.6 ± 100.8 0.000BDNF (pg/ml ± SD) 504.0 ± 301.6 413.8 ± 288.8 0.078

M: males; F: females; SD: standard deviation; Ht: height; SDS: standard deviationscore; Wt: weight; BMI: body mass index; PTL: platelets; E2: estradiol; T: testos-terone; IGF: insulin like growth factor; and BDNF: brain-derived neurotrophicfactor.

L. Iughetti et al. / Neuropeptides 45 (2011) 205–211 207

stopping the reaction with HCl 1 N, the absorbance was read at450 nm on a microplate reader and BDNF concentrations weredetermined automatically according to the BDNF standard curve(ranging from 7.8 to 500 pg/ml purified BDNF). The entire proce-dure was performed by using a semi-automated Basic RadimImmunoassay Operator (BRIO-Radim, Italy) equipped with amicroplate reader of optical density. A computer system linkedto the BRIO analyzed the final results and expressed them in pico-gram per milliliter. The sensitivity of the assay was 15 pg/ml andthe intra- and inter assay CVs were 6.1% and 8.0%, respectively.

2.4. Estradiol assay

Serum E2 was determined, after extraction and chromato-graphic partition on C18 Sep-Pak cartridge, by RIA using a commer-cially available kit (Radim, Pomezia, Italy). The sensitivity of theassay was 10 pg/ml and the intra- and inter assay CVs were 3.7%and 5.8%, respectively.

2.5. Testosterone assay

Serum T was determined, after extraction and chromatographicpartition on C18 Sep-Pak cartridge, by RIA using a commerciallyavailable kit (Radim, Pomezia, Italy). The sensitivity of the assaywas 0.01 ng/dl and the intra- and inter assay CVs were 3.4% and4.8%, respectively.

2.6. IGF-1 assay

Serum IGF-1 levels were measured using immunometric testsprovided by Siemens (Immulite 2000 IGF-1, Siemens Medical Solu-tions Diagnostics). The limit of sensitivity was below 20 ng/ml. Theintra- and inter assay coefficients of variation were 4.1% and 5.2%,respectively. The reference values considered for IGF-1 were trea-ted according to gender and age of the subjects.

2.7. Statistical analysis

All results are reported as the mean ± standard deviation (SD).Data were checked for normal distribution using the Kolmogo-rov–Smirnov statistics, so nonparametric statistical analysis (STAT-ISTICA™ software, StatSoft Inc., Tulsa, OK, USA) was performed.Between-group comparisons were evaluated using Mann–Whit-ney’s U-test and Kruskal–Wallis ANOVA when appropriate. Spear-man’s correlation analysis was performed to assess the relationbetween indices. The association between potential predictorsand BDNF values was evaluated considering the following multi-variate logistic regression model including gender, CA, Ht z-score,

Table 2Anthropometric and laboratory data according to gender and pubertal stage.

Prepuberty Puber

Males Females Males

Age (years ± SD) 5.73 ± 2.93 5.82 ± 2.94 14.1Ht (cm ± SD) 112.5 ± 18.7 112.3 ± 22.2 153.1Ht z-score (SDS ± SD) 0.33 ± 1.03 0.26 ± 1.09 - 0.20Wt (kg ± SD) 23.1 ± 10.2 24.6 ± 18.2 53.1BMI (kg/m2 ± SD) 17.2 ± 2.30 17.8 ± 5.14 21.6z-BMI (SDS ± SD) 0.55 ± 1.15 0.50 ± 1.29 0.79PTL (103/mm3 ± SD) 305.0 ± 54.7 293.8 ± 54.8 274.9E2 (pg/ml ± SD) 10.8 ± 7.90 16.0 ± 13.6 19.4T (ng/dl ± SD) 0.05 ± 0.06 0.05 ± 0.06 1.07IGF-1 (ng/ml ± SD) 140.7 ± 131.5 108.7 ± 72.5 180.5BDNF (pg/ml ± SD) 497.9 ± 264.4 522.9 ± 406.7 318.9

SD: standard deviation; Ht: height; SDS: standard deviation score; Wt: weight; BMI: bodfactor; and BDNF: brain-derived neurotrophic factor.

z-BMI, pubertal status (no/yes), PTL, E2, and T. Statistical signifi-cance was set at p < 0.05.

3. Results

Our study population was 8.98 ± 5.13 years old (range 1.17–23.8 years) and the main clinical characteristics were the follow-ing: Ht z-score 0.21 ± 1.17 SDS, BMI 19.3 ± 4.77 kg/m2, and z-BMI0.71 ± 1.25 SDS. Laboratory data were: PTL 293.8 ± 62.0 103/mm3,E2 16.4 ± 15.8 pg/ml, T 0.35 ± 0.70 ng/dl, IGF-1 156.9 ± 113.4 ng/ml, and BDNF 460.2 ± 296.9 pg/ml.

According to gender, females and males were not significantlydifferent for CA (9.11 ± 4.54 vs. 8.90 ± 5.47 years, respectively;p = 0.552), Ht z-score (0.36 ± 1.22 vs. 0.12 ± 1.13 SDS, respectively;p = 0.904), BMI (19.9 ± 5.51 vs. 18.9 ± 4.28 kg/m2, respectively;p = 0.400), z-BMI (0.82 ± 1.33 vs. 0.64 ± 1.21 SDS, respectively;p = 0.637), PTL (279.8 ± 62.0 vs. 298.1 ± 61.9 103/mm3, respec-tively; p = 0.279), IGF-1 (158.8 ± 90.8 vs. 155.8 ± 124.6 ng/ml,respectively; p = 0.353), and BDNF (518.9 ± 353.8 vs. 428.3 ± 258.1pg/ml, respectively; p = 0.291) levels. As expected, E2 concentra-tions resulted significantly higher in females than males (20.9 ±18.5 and 13.9 ± 13.5 pg/ml, respectively; p = 0.019), while T levelswere lower (0.23 ± 0.54 vs. 0.42 ± 0.77 ng/dl, respectively; p = 0.552).

Considering the pubertal stage, as expected we found that pre-pubertal subjects (n = 59) had significantly lower CA, Ht, Wt, BMI,E2, T, and IGF-1 values than the pubertal ones (n = 51), while nosignificant difference was demonstrated in Ht z-score, z-BMI, andPTL (Table 1).

ty Kruskal–Wallis ANOVA

Females Test x2 p

± 4.61 10.9 ± 4.24 57.3 44.5 0.000± 18.0 141.8 ± 17.4 54.0 48.6 0.000± 1.23 0.42 ± 1.30 4.24 4.08 0.236± 23.9 44.4 ± 19.4 51.8 52.4 0.000± 5.35 21.1 ± 5.44 23.0 11.5 0.000± 1.30 0.99 ± 1.35 2.75 3.28 0.430± 80.6 251.8 ± 72.5 4.20 0.17 0.240± 19.0 23.6 ± 20.5 25.6 20.5 0.000± 0.97 0.34 ± 0.65 54.1 38.3 0.000± 112.7 186.8 ± 89.0 13.5 11.8 0.003± 211.5 516.6 ± 328.5 8.52 9.74 0.036

y mass index; PTL: platelets; E2: estradiol; T: testosterone; IGF: insulin like growth

±SD

±SE

Mean

Group

BD

NF

(pg

/ml)

0

200

400

600

800

1000

1200

PPM PM PPF PF

p = 0.014

p = 0.036

p = 0.017

Fig. 1. BDNF levels according to gender and pubertal status. BDNF: brain-derivedneurotrophic factor; PP: prepubertal; P: pubertal; M: males; F: females; SD:standard deviation; and SE: standard error.

208 L. Iughetti et al. / Neuropeptides 45 (2011) 205–211

Plasma BDNF levels were lower in pubertal subjects than in pre-pubertal ones, without attending statistical significance.

When we analyzed data of four sub-groups according to bothgender and pubertal stage (Table 2), we showed statistically signif-icant differences in the levels of BDNF (x2 = 9.74; Kruskal–Wallistest = 8.52; p = 0.036), which resulted lower in pubertal boys thanpre-pubertal ones and both prepubertal and pubertal girls (Fig. 1).

Considering both gender and pubertal stage we also subdividedour subjects into six sub-groups according to prepuberty, midpu-berty, and late puberty stage. E2, T, IGF-1, and BDNF values re-sulted to be significantly different (Table 3).

Spearman’s correlation demonstrated a significant positive cor-relation between the values of BDNF and PTL (r = 0.333; p = 0.007).Significant negative correlations between BDNF and CA(r = �0.225; p = 0.019), Ht (r = �0.231; p = 0.016), and T(r = �0.261; p = 0.006) (Fig. 2) were also found. Moreover, inpubertal subjects BDNF and z-BMI resulted significantly correlated(r = 0.293; p = 0.038) (Fig. 3). No significant correlation was foundbetween BDNF and IGF-1 concentrations.

Logistic regression analysis (Table 4) showed that the main pre-dictor of BDNF was PTL count (b = 0.357, p = 0.008).

4. Discussion

The aim of this pilot study was to investigate the physiologicchanges in plasma BDNF circulating levels in healthy children andadolescents over the pubertal development and the importance

Table 3Anthropometric and laboratory data according to gender and pubertal stage.

Prepuberty Midpuberty

Males Females Males Fem

Age (years ± SD) 5.73 ± 2.93 5.82 ± 2.94 11.4 ± 1.75 8Ht (cm ± SD) 112.5 ± 18.7 112.3 ± 22.2 141.7 ± 8.69 13Ht z-score (SDS ± SD) 0.33 ± 1.03 0.26 ± 1.09 - 0.44 ± 1.05 0Wt (kg ± SD) 23.1 ± 10.2 24.6 ± 18.2 39.9 ± 9.61 7BMI (kg/m2 ± SD) 17.2 ± 2.30 17.8 ± 5.14 19.6 ± 3.39 1z-BMI (SDS ± SD) 0.55 ± 1.15 0.50 ± 1.29 0.80 ± 1.16 0PTL (103/mm3 ± SD) 305.0 ± 54.7 293.8 ± 54.8 267.8 ± 88.2 26E2 (pg/ml ± SD) 10.8 ± 7.90 16.0 ± 13.6 13.4 ± 3.22 1T (ng/dl ± SD) 0.05 ± 0.06 0.05 ± 0.06 0.51 ± 0.52 0IGF-1 (ng/ml ± SD) 140.7 ± 131.5 108.7 ± 72.5 168. 7 ± 100.8 17BDNF (pg/ml ± SD) 497.9 ± 264.4 522.9 ± 406.7 270.4 ± 156.0 65

SD: standard deviation; Ht: height; SDS: standard deviation score; Wt: weight; BMI: bodfactor; and BDNF: brain-derived neurotrophic factor.

of auxologic/laboratory factors as main predictors. One point ofinterest of our study is the hypothesized association between thevariations of BDNF concentrations related to pubertal stages whichhave never been investigated in detail.

BDNF, a member of the neurotrophin family, plays critical rolesin the survival, growth, and maintenance of brain and peripheralneurons. BDNF is well known for its trophic functions and more re-cently it has been implicated in synaptic modulation, induction oflong-term potentiation, learning, and memory (Croll et al., 1998;Mu et al., 1999). BDNF is a relevant factor of neuronal plasticity/activity in adulthood and it is known to be stored in human plate-lets and to circulate in plasma, but its regulation and function inperipheral blood is still unclear.

In most published studies BDNFs’ assay is performed on serum,considering it as the index of peripheral levels. Nevertheless, in ourapproach to study the BDNF levels, we measure this neurotrophinin plasma, because platelets represent its major reservoir inperipheral blood (Fujimura et al., 2002). In particular, we choseto measure plasma concentrations because its content may be con-sidered the current steady state of biologically active BDNF,whereas serum levels, reflecting the content of platelets to a higherextent, are always substantially higher than plasma ones.

Published studies have shown abnormal levels of BDNF in avariety of childhood diseases but, to our best knowledge, untilnow its plasma levels in healthy children/adolescents and itschanges throughout pubertal development have not yet beenreported.

In our study, the statistical analysis of the variables consideringgender groups showed that only the levels of sex steroids were dif-ferent. BDNF concentrations were higher in females compared tomale peers, but this data was not significantly different. In females,BDNF levels were similar in prepuberty and midpuberty while theysignificantly decreased from midpubertal to late pubertal stage.Midpubertal females had BDNF levels higher than other studiedsubjects. Interesting, E2 concentrations were not significantly dif-ferent between midpuberty and late puberty. Prepubertal maleshad significantly higher BDNF and lower T levels respect to midpu-bertal ones. All these data could be linked to the timing whenblood sample was carried out and the characteristics of the studiedsubjects. In fact, most of our females were not yet menstruated andblood sample from menstruating ones was collected in the firstdays following the menstrual cycle. The rise in BDNF concentra-tions induced by estrogen, which levels surge and fall rapidly, arelong lasting during the periovulatory and perimenstrual periods(Scharfman and MacLusky, 2008). In the early follicular phaseestrogen levels are at lowest concentrations and it has been dem-onstrated that fertile women have significantly higher plasmaBDNF levels during the luteal phase than in the follicular one

Late puberty Kruskal–Wallis ANOVA

ales Males Females Test x2 p

.72 ± 2.68 18.4 ± 4.50 14.9 ± 3.59 69.3 50.3 0.0002.5 ± 14.4 171.3 ± 13.5 158.4 ± 5.54 66.6 52.2 0.000.63 ± 1.47 0.18 ± 1.44 0.05 ± 0.90 7.22 6.90 0.2044.3 ± 24.9 33.6 ± 9.32 63.5 ± 18.0 62.2 55.0 0.0008.8 ± 3.01 24.7 ± 6.56 25.2 ± 6.51 30.8 17.4 0.000.82 ± 1.26 0.79 ± 1.58 1.30 ± 1.51 3.41 3.41 0.6365.2 ± 76.1 306.5 ± 19.0 198.0 ± 10.4 7.31 3.89 0.1987.9 ± 16.6 28.9 ± 28.6 33.7 ± 23.9 37.8 27.4 0.000.10 ± 0.05 1.97 ± 0.85 0.76 ± 0.98 62.5 40.8 0.0008.6 ± 81.6 199.3 ± 133.0 201.4 ± 104.5 13.9 11.9 0.0165.4 ± 336.8 396.5 ± 269.9 285.2 ± 123.9 17.5 19.5 0.003

y mass index; PTL: platelets; E2: estradiol; T: testosterone; IGF: insulin like growth

CA (yr)

BD

NF

(pg

/ml)

0

200

400

600

800

1000

1200

1400

1600

0 2 4 6 8 10 12 14 16 18 20 22 24

r = - 0.225; p = 0.019

Ht (cm)

BD

NF

(pg

/ml)

0

200

400

600

800

1000

1200

1400

1600

60 80 100 120 140 160 180 200

r = - 0.231; p = 0.016

PTL (103/mm3)

BD

NF

(pg

/ml)

0

200

400

600

800

1000

1200

1400

1600

150 200 250 300 350 400 450 500

r = 0.333; p = 0.007

T (ng/dl)

BD

NF

(pg

/ml)

0

200

400

600

800

1000

1200

1400

1600

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

r = - 0.261; p = 0.006

Fig. 2. Spearman’s significant correlations between plasma BDNF levels and anthropometric and laboratory data. BDNF: brain-derived neurotrophic factor; CA: chronologicalage; Ht: height; PTL: platelets; and T: testosterone.

Pubertal Subjects

z-BMI

BD

NF

(pg

/ml)

0

200

400

600

800

1000

1200

1400

1600

-2.5 -1.5 -0.5 0.5 1.5 2.5 3.5 4.5

r = 0.293; p = 0.038

Fig. 3. Spearman’s significant correlation between plasma BDNF levels and z-BMI inpubertal subjects. BDNF: brain-derived neurotrophic factor and BMI: body massindex.

L. Iughetti et al. / Neuropeptides 45 (2011) 205–211 209

(Begliuomini et al., 2007). The close relationship between levels ofBDNF and sex hormones, in particular E2, according to menstrualcycle phase suggests how the fluctuations of both are extremelysimilar: they are very low in the first days of the cycle, reachinga peak just before ovulation. In normally menstruating womenplasma BDNF levels rise in the luteal phase, together with the in-crease of E2 and progesterone, while in women with premenstrualsyndrome E2 and BDNF concentrations significantly decrease(Cubeddu et al., 2011). These data confirm that sex hormones dee-ply influence BDNF production, in particular it is well establishedthat estrogens and BDNF are reciprocally positively involved intheir regulation (Sohrabji and Lewis, 2006). The reciprocal influ-ence between sex steroids and BDNF is probably corroborated byour demonstration of a significant correlation between BNDF and

T, while the lack of correlation with E2 may be probably explainedby previous considerations. Menstruating women display signifi-cantly lower BDNF levels than men and we shown that BDNF’s lev-els were strongly influenced by pubertal status, especially in males.Considering the lack of humans data, it is really difficult to explainthe negative relationship we found between BDNF and T. However,we must probably reflect only on a simply decrease of BDNF due tothe increase of CA.

It is interesting to notice that even if plasma BDNF concentra-tions were significantly related with T, no correlation with IGF-1was found. To our best knowledge this is the first report evaluatingthe relationship between BDNF and IGF-1 and our data suggest apredominant role of sex steroid hormones in the regulation of neu-rotrophin expression. This assumption should be supported by fur-ther data, considering the importance of this nerve growth factorand its implications.

The relationship between BDNF and body weight is also of greatinterest and seems to be complex to explain, because of the char-acteristics of subjects included in the published study. BDNF andTrkB are expressed in hypothalamic regions believed to be impor-tant for the maintenance of normal body weight (Mowla et al.,2001; Tapia-Arancibia et al., 2004). The haploinsufficiency of BDNFis associated with decrease serum BDNF levels, hyperphagia, andobesity so the BDNF gene has been hypothesized to play a role inthe WAGR syndrome (Wilm’s tumor, aniridia, genitourinary anom-alies, and mental retardation) (Nelson et al., 2001; Han et al., 2008).Moreover, a mutation in TrkB has been described in a child with se-vere obesity and developmental delay (Yeo et al., 2004). BDNF isbelieved to function downstream of leptin–melanocortin signalingand to play an important role in the regulation of energy homeo-stasis (Xu et al., 2003). In our study, we showed a significant posi-tive relationship between z-BMI and plasma BDNF levels, at least in

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210 L. Iughetti et al. / Neuropeptides 45 (2011) 205–211

pubertal subjects. This conclusion appears in agreement with ser-um BDNF data reported in literature. In fact, since conditions oftenrelated with energy restriction like anorexia nervosa, bulimia nerv-osa, and depression have been found to be associated with lowBDNF (Shimizu et al., 2003; Monteleone et al., 2004, 2005). BDNFhas been reported to reflect central BDNF levels in rats (Karegeet al., 2002a; Sartorius et al., 2009) so it has been suggested thatits concentrations would be positively related to BMI or body adi-posity. Nakazato et al. (2003) reported a significant positive rela-tionship between BDNF and BMI in adults but this correlationwas not demonstrated in a group of normal-weight subjects. How-ever, the lack of significant association (Saito et al., 2009) or the in-verse relationship between serum BDNF concentrations and BMIwere also reported. El-Gharbawy et al. (2006) measured the levelsof serum BDNF in a large cohort of normal weight/overweight chil-dren and adolescents and analyzed the link between BDNF andanthropometric features, including the relationship between thisneurotrophin and the immediate food intake. Even if they did notdemonstrated a significant relationship between BMI and serumBDNF levels, extremely overweight subjects showed lower BDNFconcentrations than normal-weight controls. Recently, Han et al.(2010) demonstrated lower serum and plasma BDNF concentra-tions in subjects with Prader-Willi syndrome compared to both ob-ese and lean controls suggesting the potential role of thisneurotrophin in the pathophysiology of the disordered appetiteregulation and neurocognitive abnormalities associate with thissyndrome. Larger studies including individuals with peculiar phe-notypes like childhood-onset obesity in combination with learningor mood disorders are needed to better understand the possiblerole of BDNF in these conditions.

Platelet count is considered the strongest factor associated withserum BDNF concentrations, as already shown by the data of liter-ature (Begliuomini et al., 2007; Saito et al., 2009). In our approachto study BDNF we analyze its plasma concentration in order toavoid possible inter-individual variation due to physiological alter-ation of platelets count. Lommatzsch et al. (2005) showed thatBDNF plasma levels significantly decreased with increasing of ageor weight, whereas platelets did not. In our study, we demon-strated that plasma BDNF levels were positively correlated withplatelets. Thus, plasma BDNF concentrations in children may needto be interpreted with age-specific and platelet count-specific stan-dards. BDNF was mainly explained by platelets, but probably otherparameters affect its concentration.

In conclusion, we demonstrate that parameters such as age orgender have a specific impact on circulating BDNF levels in periph-eral blood and platelets are the most important predictor for plas-ma BDNF.

Conflict of interest statement

All authors have no conflicts of interest.

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