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Abstract. – OBJECTIVE: Fat-bone relation-ship involves the interaction among endocrine,inflammatory, immune processes and boneturnover. We tried to assess the association be-tween Leptin and bone turnover markers (OCN,β-CTx, ALP), calciotropic hormones PTH and25(OH)D in obese Saudi children.

PATIENTS AND METHODS: A cross-sectionalstudy performed with 60 obese children and 36lean children. For all subjects, OCN, ALP, β-CTx,PTH, 25(OH)D, leptin, Ca and Pi were investigat-ed. Levels of leptin were measured by [ELISA]method, and OCN, β-CTx, PTH and 25(OH)D byan electrochemiluminesce immunoassay.

RESULTS: Sixty obese Saudi children hadmeans weight (38.3 vs. 13.8 kg), height (121.0 vs.91.8 cm) leptin (23.04 vs.16.88 ng/ml), PTH (31.5vs. 14.7 pg/ml), Pi (1.67 vs. 1.54 mmol/l) weresignificantly higher and 25(OH)D (21.02 vs. 29.45ng/ml) was significantly lower than controls.There was no difference in serum OCN, β-CTx,ALP and calcium between groups (p > 0.05). Inthe correlation study, OCN were significantlypositively correlated with height, ALP, age, PTH,and β-CTx (r = 0.347, 0.32, p < 0.05), (r = 0.35,0.51, 0.66, p < 0.01 respectively), while serum25(OH)D was negatively correlated with PTH,weight, height and BMI (r = -0.45, -0.55, -0.55, -0.47, p < 0.01 respectively). PTH was positivelycorrelated with leptin and β-CTx (r = 0.41, 0.44, p< 0.01), but not to ALP and BMI percentile. β-CTxcorrelated significantly positive with Pi (r = 0.34p < 0.05) and ALP with BMI percentile (r = 0.42, p< 0.05). Multiple regression analysis demonstrat-ed that PTH was predicted by leptin and β- CTx(R2 = 0.55); β-CTx by leptin and OCN (R2 = 0.498);OCN by PTH and β-CTx (R2 = 0.47); and 25(OH)Dby PTH (R2 = 0.21)

CONCLUSIONS: The obese children had in-creased levels of leptin and PTH with strong as-

Interrelationship between bone turnovermarkers, calciotropic hormones and leptin in obese Saudi children

L.M. SABER1, H.N.F. MAHRAN2, H.H. BAGHDADI3, Z.M.H. AL HAWSAWI4

1Department of Medical Laboratories Technology, Faculty of Applied Medical Sciences, Taibah University, Al-Madina Al Mounwara, Kingdom Saudi Arabia2Department of Medical Laboratories Technology, Faculty of Applied Medical Sciences, TaibahUniversity, Al-Madina Al Mounwara, Kingdom Saudi ArabiaFellow of Chemical laboratory, Forensic Medicine Organization, Ministry of Justice, Alexandria, Egypt3Department of Medical Biochemistry, College of medicine, Taibah University, Al-Madinah Al Mounwara, Kingdom Saudi Arabia4Department of Pediatrics, Medina Maternity and Children Hospital, King Abdullah Medical City, Al-Madina Al Mounwara, Kingdom Saudi Arabia

Corresponding Author: Lobna Mohamed Saber, Ph.D; e-mail: lobnahasan@yahoo.com

sociated with bone turn over markers OCN, β-CTx and deficiency of 25(OH)D which may beplaying an important role in the pathogenesis ofobesity and related bone metabolic risk dis-eases as osteoporosis and fractures.

Key Words:Obesity, Children, Leptin, PTH, Osteocalcin, β-CTx,

25(OH)D.

Introduction

Obesity is currently a worldwide pathologicalepidemic and a major public health concern.Medical disorders, health risks, increased risk ofadult obesity, with its subsequent effects on mor-bidity and mortality rates are all related to child-hood and adolescent overweight and obesity. Theetiology is multifactorial, with influences ofmany factors included genetic, environmental,socioeconomic, and behavioral or psychological.Comorbid diseases as cardiovascular disease, In-sulin resistance, type 2 diabetes, orthopedic prob-lems, and several other chronic diseases are high-ly associated with obesity. The prevalence ofobesity in childhood and adolescence observablyincreased in many countries. In 2010, evaluationof 144 studies in several countries consideredoverweight and obese preschool children to bearound 43 million and around 35 million of themin developing countries. Furthermore around 92million stood to be susceptible to be overweight.The global prevalence of childhood overweightand obesity rose from 4.2% in 1990 to 6.7% in

2015; 19: 4332-4343

2010. This trend is anticipated to become 9.1%which is around 60 million, in 20201.

Increased body weight with a higher fat massappears to have a detrimental effect on boneleading to increase incident of fragility and frac-tures. How body fat has a negative effect on bonemetabolism remains, however, unexplained.There are cumulative proofs of a cross-talk be-tween adipose tissue and bone2.

Either formation or resorption of bone can beassessed by biochemical measurements of boneturnover markers. Frequently used markers ofbone formation are osteocalcin (OCN), alkalinephosphatase (ALP), bone specific alkaline phos-phatase (BAP), and procollagen type I propep-tides, PICP (carboxy terminal) and PINP (aminoterminal), all indicative of various phases of os-teoblast proliferation and differentiation. Markersof bone resorption are collagen crosslinks type Icarboxyterminal telopeptide (β-CTx) measured inserum and the urinary marker deoxypyridinoline(DPD), both degradation products of type 1 colla-gen3. Markers of Ca and Pi metabolism, such asthe calciotropic hormones, parathyroid hormone[PTH] and vitamin D metabolites have a role toplay in evaluating pathophysiological changes inthe processes of resorption following physicalsedentariness. Vitamin D act directly on entero-cytes to stimulate calcium (Ca) and inorganicphosphorus (Pi) absorption, in so doing protectionbone from resorption. Calciotropic hormones mayalso influence OCN levels as revealed by in vitroand in vivo researches. OCN gene transcription ispartially controlled by levels of 1,25(OH)2D. Alsolower OCN levels have been stated among obesechildren and adolescents who were deficient in vi-tamin D4.

Osteocalcin, is an osteoblast produced proteinrecognized as “bone gamma-carboxyglutamicacid (Gla) protein (BGP),” It is formed by ma-ture osteoblasts, hypertrophic chondrocytes andodontoblasts through the course of formation ofbone. OCN is the most abundant noncollagenousprotein of bone matrix. Though, there was no ev-ident association of OCN serum concentration tobone mass. OCN exhibits numerous characteris-tics of a hormone: cells specific molecule, dis-charge in a circadian pattern, formed as a pre-pro-molecule and it is liberating into the circula-tion, however till now its receptor is not recog-nized. Modification of OCN occurs by carboxy-lation on three glutamic acid residues (17, 21,and 24) posttranslational depending on vitamin Kby the action of the enzyme γ glutamyl carboxy-

lase. This carboxylation is essential for hydrox-yapatite binding and deposition in the extracellu-lar matrix of bone. Uncarboxylated fraction ofOCN in adults is a fairly large fraction (20-30%)of circulating OCN, and this fraction is evenhigher (up to 60%) in children5. Uncarboxylatedfraction of OCN may be accountable for vital en-docrine function controlling glucose and lipidhomeostasis and appears to exhibit a clear conse-quence on energy metabolism and probably alsothe production of testosterone by the testes. OCNdeficient mice were obese and reported to have adecreased insulin levels with elevated blood glu-cose levels6. The processes connecting OCN toobesity and insulin resistance are not clear. OCNlevels are proposed to be modified by leptin.Leptin is an important modulator of weight con-dition via well- recognized pathways in the hy-pothalamus7.

Leptin, a marker of adipocyte activity is a 16KD neurohormone and is important in the controlof body weight and nutrition through decreasingfood intake and stimulating thermogenesis. It reg-ulate food intake via its receptors in the hypothala-mus by decreasing the production of the neu-ropeptide Y (NPY) which stimulate food intake7.Leptin is a major regulator of bone remodelingthrough changes in local factors controlling osteo-clastogenesis and its alterations in obese childrenpredispose them to low bone mass and fracture.Leptin play its role via the central nervous systemto decrease osteogenesis. In animals models in-tracerebroventricular administration of leptin pro-duces not only weight reduction but also osteoly-sis. Leptin action facilitated by the sympathic ner-vous system; the β2 adrenergic receptor found onthe osteoblasts accelerates osteoclast differentia-tion through the receptor activator of NF-κB(RANK) – RANK ligand pathway8. Leptin re-duce the formation of serotonin in the brain.Serotonin inhibits the activity of the sympatheticnervous system. Leptin signaling therefore raisesthe sympathetic tone and decrease bioactivity ofOCN and consequently bone resorption. Howev-er, leptin decrease osteoclast formation and in-crease osteoblast differentiation in vitro and it isalso reported to decrease osteoclast differentia-tion through the central cocaine- and ampheta-mine-related transcript [CART] pathway8. Theeffect of leptin on bone is complex, but it appearsto exert antiosteogenic effects via a hypothalamicrelay system (primarily affecting the axial skele-ton), while it may have direct proosteogenic ef-fects at appendicular sites9.

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The complex effect of leptin on bone indicatethat it could operate in collaboration with the cal-ciotropic hormones accountable for mineralhomeostasis and metabolism, specifically PTHand 1,25(OH)2D. Calcium and phosphate move-ment at the gut, bone and kidney is controlled bythese hormones which form an incorporated sys-tem for regulating transportation and are essen-tial for mineral retention by the skeleton. Of themineral-controlling hormones, PTH is most con-stantly said to be raised in obesity10. PTH is pow-erfully anabolic hormone and if given as daily scinjections, producing increased bone turnoverand enhanced bone mass in patients with osteo-porosis11. In obesity, described serum levels of1,25(OH)2D are contradictory, with some re-searches reporting high levels and others lowlevels12. Hypovitaminosis D can be related to itssequestration by fat due to its liposolubility andwas reported to be associated with low forearmbone mineral density and increased incidents ofdistal forearm fractures13.

Thus, the aims of this study were to examinethe effect of simple obesity in children on theserum levels of leptin, PTH, 25(OH)D, OCN,ALP and β-CTx and to correlate their levels tothe degree of obesity. Also to investigate whethertheir interaction could clarify the impact of obe-sity on bone metabolism and to evaluate the pre-dictors of the bone turn over marker OCN, β-CTx, and the calciotropic hormones 25(OH)Dand PTH.

Patients and Methods

PatientsBetween November and June 2014-2015 we

assessed sixty overweight and obese children(body mass index [BMI] > 85th centile; 34 males,26 females), chronological age 7.55 ± 3.34 yearsranged from 3 to 13 years, who had been attend-ing Medina pediatric outpatient clinic of materni-ty and children hospital (Al-Medina, KSA). Theywere all apparently healthy with no obvious en-docrine disease. BMI were expressed as a stan-dard deviation score (SDS) according to WHOChild Growth Standards14. Patients were classi-fied as prepubertal by physical inspection, ac-cording to the standard Tanner’s criteria. Allchildren were diagnosed with simple obesity;other syndromic, organic, and hormonal causeswere excluded. Thirty-six normal-weight,healthy children (21 males and 15 females) of the

same chronological age acted as controls for thebiochemical parameters and bone markers: Allwere prepubertal chronological age 5.64 ± 4.1years ranged from 2 to 13 years. All subjects ofthe control group were healthy and did somephysical activity appropriate for their age. Fur-thermore, none of them was taking drugs knownto intervene with calcium metabolism. All con-trol subjects were within 2 SD of the means forheight and weight, with a BMI < 85th percentile.They were normal children undertaking a routinecheck.

Measurement of AnthropometricParameters

Height measured using precision Harpendenstadiometer and weight using a Scale Tronixscale to the nearest of 0.1 cm and 0.1 kg, respec-tively.

Reference percentiles for children in SaudiArabia were applied for standardization of heightand weight. Referring to national reference dataattached to every child file, the body mass index(BMI) was standardized. Underweight, normalweight and overweight were defined as BMI be-low the 10th, between 10th and 85th and above the85th percentile, respectively.

Study DesignThis was an observational cross-sectional, a

case-control study carried out after appropriateapproval by the Ethics Committee of the Hospi-tal Institutional Board. All children, 60 patientsand 36 controls, submitted to a full evaluationand serum collection after an overnight fast;sample were divided into two parts, ALP, Ca, al-bumin and Pi levels were done on the same dayof collection and the second serum samples werestored at -20C until assayed for PTH, 25(OH)D,β-CTx, OCN, and leptin.

Assays25(OH)D, PTH, N-MID Osteocalcin, and β-

CTx kits (Roche Diagnostics, GmbH,Mannheim, German) used to estimate the serumlevels of these parameters and were determinedusing the Cobas e 411 immunoassay analyzer(Roch Diagnostics, GmbH, German). All assaysare based on chemiluminescence immunoassaytechnology. Intra-assay CVs in our laboratorywere 3% for β-CTx, 3% OCN and 4.5% forPTH. Interassay CVs were 7% for OCN, β-CTx,and PTH. Serum 25(OH)D was with an intra-as-say CV of 5% and an interassay CV of 7%.

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Serum leptin was identified with commerciallyavailable test kits which were used to measureleptin (enzyme-linked immunosorbent assay[ELISA] method, ASSAYPRO, Saint Charles,MO, USA). Assay Max Human Leptin ELISAKit, Catalog No. EL2001-1 that has a range of0.00-32 ng/mL. The ASSAYPRO Leptin ELISAis an enzyme immunoassay for the measurementof leptin in serum and plasma.

By determining this bone resorption marker,Collagen type I fragments β-isomerized C-termi-nal telopeptides (β-CTx), the activity of osteo-clasts can be detected. These isomerized telopep-tides are highly specific for the degradation oftype I collagen dominant in bone. Elevatedserum levels of isomerized C-terminal telopep-tides of type I collagen have been reported forpatients with increased bone resorption.

ALP, Ca, inorganic phosphorous (Pi) and al-bumin were measured by commercially availablekits using standard procedures on a clinicalchemistry automated machine, Dimension XPand, Siemens Healthcare Diagnostics Ltd.,Frimley, Camberley, UK.

Exclusion CriteriaA detailed medical and family history was ob-

tained from all subjects. At enrollment, obeseand control children submitted to physical exam-ination including weight, standing height, BMI,and blood pressure measurements. Exclusion cri-teria were: (1) The existence of endocrine disor-ders or genetic syndromes, (2) History of anychronic diseases or chronic medication use orchildren under special diets or use of mineraland/or vitamin supplements were not included inthe study. (3) Taking medications that could in-fluence growth, pubertal development. Pubertalstage was established according to the Tannerscale, and subjects who showed pubertal devel-opment were excluded.

Statistical AnalysisStatistical analysis was performed using SPSS

version 20.0 software (SPSS Inc., Chicago, IL,USA). Quantitative data were normally distrib-uted and expressed as mean ± SD. Differencesbetween groups were evaluated by Student’s t-test. Simple correlations were used to investigatethe association between the different parameters.p < 0.05 indicated statistical significance. Calcu-lation of Pearson correlation coefficients and lin-ear regression analyses were performed to assessrelationships between key variables.

Variables chosen for inclusion in the multipleregression analyses were established on physio-logically expected outcomes. The followingvariables were included to measure to what ex-tent: (1) weight, leptin, Ca, 25(OH)D, β-CTx,OCN and Pi predicted PTH; (2) age and weight,leptin, Ca, PTH, OCN, β-CTx and Pi predicted25(OH)D; (3) leptin, Ca, 25(OH)D, Pi, β-CTx,and PTH predicted ALP; (4) weight, leptin, Ca,PTH, 25(OH)D, Pi and OCN predicted β- CTx;and (5) age, weight, leptin, Ca, PTH, 25(OH)D,β-CTx and Pi predicted OCN. For model selec-tion stepwise and r2 methods were used. ModelR2 and partial R2 of the individual model para-meters are specified, where R2 (the square of r)is the coefficient of determination and repre-sents the portion of total variation deducible tothe variables in the model. Partial R2 quantifiesthe distinctive contribution of each explanatoryvariable to the total R2.

Results

Sixty overweight and obese Saudi children hadmeans BMI percentiles (94.7 vs. 20.3), weight(38.3 vs. 13.8 kg), height (121.0 vs. 91.8 cm)serum leptin (23.04 vs. 16.88 ng/ml), PTH (31.5vs. 14.7 pg/ml), phosphate (Pi) (1.67 vs. 1.54mmol/l) were significantly higher and 25(OH)D(21.02 vs. 31.95 ng/ml) was significantly lowerthan controls (Tables I and II, Figure 1). Therewas no difference in serum OCN, β-CTx, calci-um, and ALP between groups (Table II and Fig-ure 2). Normal weight and obese children wereinsignificantly different regarding age, gender orpubertal stage. Mean BMISDS for OW/obese chil-dren was (2.31 ± 1, all pre-pubertal stage) andMean BMI SDS for control children was (-1.85 ±1.49, all pre-pubertal stage) (Table I).

PTH was significantly correlated with OCN(r = 0.51, p < 0.001; Figure 3), β-CTx (r = 0.44,p < 0.001; Figure 4), leptin (r = 0.41, p < 0.01;Figure 6) and 25(OH)D (r = -0.45, p < 0.01;Figure 5), but not to ALP and BMI percentile.Also a significant positive correlation betweenβ-CTx and PTH, OCN or Pi (r = 0.44, 0.66,0.34 p < 0.05 respectively) was detected. Fur-ther, s25(OH)D showed significant negativecorrelations with PTH, weight, height and BMIpercentile (r = -0.45, -0.55, -0.55, -0.47, p <0.01 respectively, Figures 5, 8) while OCNwere positively correlated to height, ALP, age,PTH, and β-CTx (r = 0.347, 0.32, p < 0.05) and

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L. M. Saber, H.N.F. Mahran, H.H. Baghdadi, Z.M.H. Al Hawsawi

(r = 0.35, 0.51, 0.66, p < 0.01 respectively, Fig-ure 7). In addition, OCN demonstrated no sig-nificant association with leptin or BMI. ALPdemonstrated positive significant correlationswith BMI percentile and OCN (r = 0.42, 0.32, p< 0.05 respectively).

We subdivided further the obese children ac-cording to sex, but no difference was found ex-cept in β-CTx which was lower significantly inobese girls comparing to boys (0.74 ± 0.29 vs.1.04 ± 0.44, p = 0.018). This study did not showa clear linkage of OCN to obesity or any highervalues of OCN in control group. The presentstudy established no significant relationship be-tween serum OCN levels and leptin, neither inthe total study population nor in the subsetsstratified for gender but as anticipated leptinserum levels were higher in over-weight andobese children.

Overweight (n=9)/Normal weight obese children (n =51)

Subject number (n = 36) Total (n =60) p-valuea

Age, years 5.65 ± 4.1 7.55 ± 3.34 0.07Height (cm) 91.8 ± 33.23 121.02 ± 27.7 0.007Weight (kg) 13.83 ± 12.5 38.36 ± 16.68 < 0.001Sex:

Male 21 (58.33%) 34 (6/28) (56.66%) 0.681Female 15 (41.66%) 26 (3/23) (43.33%)

BMI % 13.65 ± 2.64 25.34 ± 5.25 < 0.001BMISDS -1.85 ± 1.49 2.31 ± 1 < 0.001Percentile 20.38 ±19.98 94.74 ± 4.16 < 0.001

Table I. Anthropomorphic data in overweight, obese children and control group.

Results are shown as mean ± SD (percentages). aStudent’s t-test.

Figure 1. Concentration of 25(OH)D, leptin ng/ml andPTH pg/ml in overweight and obese children and controlgroup. *p < 0.05 and **p < 0.01.

Overweight (n = 9)/Normal weight obese children (n = 51)

Subject number (n = 36) Total (n =60) p-valuea

OCN (ng/ml) 63.43 ± 26.96 79.01± 37.37 0.063β-CTx (ng/ml) 0.86 ± 0.31 0.90 ± 0.48 0.64Leptin (ng/ml) 16.88 ± 6.88 23.04 ± 10.5 (n = 30) 0.01425(OH)D (ng/ml) 29.45 ± 6.89 21.02 ± 10.07 (n = 41) 0.044PTH (pg/ml) 14.72 ± 11.3 31.54 ± 26.2 0.001ALP (U/L) 258.64 ± 56.6 301.86 ± 96.87 0.49Ca (mmol/L) 2.32 ± 0.22 2.4 ± 0.14 0.109Pi (mmol/L) 1.54 ± 0.19 1.67± 0.19 0.044

Table II. Serum biochemical variables in overweight and obese children and control group.

Results are shown as mean ± SD. aStudent’s t-test.

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Multiple regression analysis verified that PTHwas predicted by leptin and β-CTx (R2 = 0.55);β-CTx by leptin and OCN (R2 = 0.498); OCN byPTH and β-CTx (R2 = 0.47); 25(OH)D by PTH(R2 = 0.21) (Table III).

Discussion

Many indicators suggest that obesity and bonemetabolism are interdependent. Firstly, a com-mon mesenchymal stem cell is a shared precur-

sor for both adipocytes and osteoblasts and fac-tors decreasing adipogenesis increasing os-teoblast differentiation and also, those restrainingosteoblastogenesis enhanced adipogenesis15.Nevertheless, there are inadequate data obtain-able regarding the interactions between the os-teoblast-derived hormone OCN, bone-resorptionmarker β-CTx, weight status, leptin and hor-mones of minerals metabolism, PTH and vitaminD metabolites in obese humans, particularly inchildren and adolescent.

Figure 2. Concentration of β CTX and osteocalcin ng/ml inoverweight and obese children and control group. p > 0.05.

Figure 3. Correlation between osteocalcin and PTH show-ing significant positive correlations, r = 0.51, p < 0.001.

Figure 4. Correlation between β- CTx and PTH showingsignificant positive correlations, r = 0.44, p < 0.001.

Figure 5. Correlation between PTH and 25(OH)D showingsignificant negative correlations, r = - 0.45, p = 0.006.

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In this study, s25(OH)D was lower in over-weight and obese Saudi children than controlgroup. This result comes to an agreement with anumber of researches stating that s25(OH)D is ata low level in obesity and inversely correlated tothe amount of body fat. Also The correlation be-tween s25(OH)D and sPTH was inverse, sup-portive the commonly supposed concept that sec-ondary hyperparathyroidism is a consequence ofvitamin D insufficiency occurred as s25(OH)Ddecreases less than 30 ng/ml16.

Increased serum PTH concentrations werefound in this study and also are well described in

Figure 6. Correlation between leptin and PTH showingsignificant positive correlations, r = 0.41, p = 0.01.

Figure 7. Correlation between osteocalcin and β-CTx show-ing significant positive correlations, r = 0.66, p < 0.001.

Coefficient Partial R2 p-value Model

PTH (pg/ml)Intercept -4.66Leptin (ng/ml) 0.527 0.747 0.0001 R2 = 0.55β-CTX (ng/ml) 8.79 0.336 0.043 p = 0.000125D (ng/ml)Intercept 32.18PTH (pg/ml) -0.181 -0.452 0.034 R2 = 0.21Calcium (mmol/L) -0.292 -0.328 0.147 p = 0.0001β-CTx (ng/ml)Intercept 0.403Osteocalcin (pg/ml) 0.008 0.646 0.0001 R2 = 0.498Leptin (pg/ml) -0.011 -0.261 0.043 p = 0.000125(OH)D (ng/ml) -0.003 0.057 0.0647Osteocalcin (ng/ml)Intercept 27.85PTH (pg/ml) 0.38 0.28 0.01 R2 = 0.47β-CTx (ng/ml) 40.21 0.53 0.0001 p = 0.0001

Table III. Multiple regression models.

Results are shown as mean ± SD. aStudent’s t-test.

Figure 8. Correlation between 25(OH)D and weight show-ing significant negative correlations, r = -0.55, p = 0.005.

obesity in adults16. Several investigators accredit-ed these elevations to secondary hyperparathy-roidism in reply to decreased s25(OH)D,which inobesity seems to be a noticeable observation.Others reported no correlation betweens25(OH)D and PTH17. The s25(OH)D levels inthis study ranged from 10-56.9 ng/ml in bothgroups the obese children and the control groupjoined, but a negative significant correlation ex-isted between PTH and s25(OH)D in obese chil-dren. In contrast, a significant positive relation-ship existed between PTH and leptin when leptinranged from 6.1 to 44.83 ng/ml in obese chil-dren. Because children with primary or sec-ondary hyperparathyroidism were heavyweightthan others in the control group, it may be pro-posed that raised PTH levels participate in obesi-ty. Conversely, PTH levels declined after reduc-ing weight in one research, in accord with mostinvestigations done on obese adults indicatingthat the raised serum PTH is not a cause of obesi-ty but an outcome16.

Multiple regression analysis showed thatserum leptin and β-CTx were the most importantpredictive variables for PTH while s25(OH)D of-fering no contribution to PTH prediction. Thus,low s25(OH)D did not participate to the in-creased sPTH. Consequently, the results favorthe theory that in obesity the inverse relationshipbetween s25-(OH)D and sPTH is not causal, butthat the change in their levels are a straight effectof obesity per se. The existence of an inversecorrelation between s25(OH)D and sPTH mustnot be used to denote the existence of vitamin Ddeficiency in obesity. In obesity the indepen-dence of sPTH from s25(OH)D levels was re-ported by other studies17. The reason of lows25(OH)D in obesity is uncertain. Several pro-posals have been mentioned, involving removalof 25(OH)D by adipose tissue, causing it to be-come less accessible for transformation tos1,25(OH)2D as well as decreased the duration ofexposure to sunlight18. The inverse relationshipbetween 25(OH)D and PTH obtained in thisstudy, proposing that inhibition of 25(OH)D pro-duction in obesity, overruled any stimulatory in-fluence of PTH. Significantly, intervention studygiving vitamin D supplementation for an 8-wk inobese subjects who were suffering from insuffi-cient levels of vitamin D displayed no result onintact sPTH and an insignificant reduction insPTH (1-84) levels in spite of a significant rise ofs25(OH)D18. In addition to weight in some stud-ies, age was another factor added to the decrease

in s25(OH)D with a well recorded association19.We have reported a significant correlation be-tween s25(OH)D and weight status in term of in-verse correlation, which does not essentially in-dicate a cause-and-effect link. Therefore, thesechanges may just be connected to other influ-ences affecting both weight status and the vita-min D endocrine system.

A possible mechanism of increase PTH couldbe through influence of leptin on PTH produc-tion. As reported in this study and other studies, aconfirmed association between serum leptin andsPTH was demonstrated in obesity15. Moreover,the implication that leptin is a PTH secretagoguebrought about by injections of leptin in the leptin-deficient (ob/ob) mouse significantly raise sPTHlevels20. If leptin influences PTH production di-rectly either through paracrine or endocrinemechanisms, it implied that the relationship be-tween leptin and PTH is causative. Unfortunately,no direct studies were done to confirm or dis-prove the paracrine or the endocrine mechanisms.Nevertheless, primary hyperparathyroidism in pa-tients with obesity proven to have elevated levelof PTH and greater parathyroid tumor weightthan non-obese, proposing that increase parathy-roid cell mass is mediated by leptin. Some studiessuggest that leptin could be functioning as a mito-gen working through paracrine mechanisms tocontrol parathyroid cell mass21.

The direct association between bone mass andbody fat mass could be linked to the direct corre-lations between serum leptin and sPTH22 as PTHcontrols sclerostin production, which is the mainregulator of bone mass23. Hyperparathyroidismpathogenesis in obesity stays unclear. Some re-searches prove that hyperparathyroidism in mor-bid obesity fall back with weight loss supportingthe strong role played by fat by itself and someinvestigations had revealed that it changes anddecreases with weight alterations in healthywomen25, proposing a causal association. How-ever, the analysis in our work was a case controlstudy cross sectional and not longitudinal, ren-dering it not easy to assume causality.

This study showed the association betweenobesity and the changed serum levels of vitaminD and PTH, the calciotropic hormones control-ling calcium and phosphate homeostasis. Therewas no change in Ca levels denoting no notice-able influence of PTH on Ca, proposing thatPTH changes is not big enough to give a re-sponse on this objective variable or that addition-al controlling circumstances stand out. In our re-

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port, Pi level was found to be higher in obesechildren but investigating renal tubular reabsorp-tion of Pi directly was not determined, the find-ing is similar to the results of Lenders et al25.Other study found that no significant alteration inPi although the hormone FGF23 regulating phos-phate homeostasis was increased. FGF23 is pro-duced by bone cells and cause down-regulationof phosphate reabsorption and suppressing renal1,25-(OH)2D formation26. Marked 25(OH)D defi-ciency happens at s25(OH)D concentration con-stantly less than 10 ng/ml and lead to significantdeficiency in calcium absorption, decrease calci-um levels, secondary increase in PTH, stimulatedbone turnover, and rickets in children and osteo-malacia in adults. Alternatively, vitamin D insuf-ficiency arises when s25(OH)D is persistentlylower than 30 ng/ml but goes above deficiencyconcentration of 20 ng/ml which was the case forsome of the overweight and obese children in ourresearch. While there is common concept thatmild secondary hyperparathyroidism occurs as abiochemical manifestation resulting from vitaminD insufficiency. The consequences of vitamin Dinsufficiency remain controversial regardingmineral homeostasis. Cardiovascular disease, hy-pertension, insulin resistance, type 2 diabetes,and osteoarthritis all are comorbid condition as-sociated with obesity in which vitamin D insuffi-ciency may have a role to play in their pathogen-esis.

In our study no significant changes in circulat-ing OCN and β-CTx were found between obeseand lean children but the effect of leptin on boneturn over markers was found by multiple regres-sion analysis as β-CTx was predicted by leptinand OCN but not s25(OH)D suggesting a director indirect effect of leptin on osteoclasts. AlsoPTH predicted OCN, be indicative of its influ-ence on osteoblasts. Bone is an active organ thatconstantly undertakes considerable turnover, anactivity called modeling/remodeling includingbone production and growth by osteoblasts andbone resorption by osteoclasts27. The process ofbone remodeling preserves the quality of theskeleton by constantly substituting old bone withnew one. Therefore, the balance between boneproduction and destruction at any specific timehas an effect on bone mass. As others havedemonstrated earlier, in obesity bone turnovermarkers were increased16. Serum β-CTx is apreferable marker than other resorption markersas urine desoxypyridinoline/creatinine (DPD/cre)because it is creatinine-independent. A positive

correlation existed between a oesteogenesismarker, OCN and a osteolysis marker, β-CTxand OCN levels demonstrated positive correla-tion with age (r = 0.347 p < 0.01). In the humanlife cycle, OCN concentrations are at their peakduring adolescence28. Others reported decreaselevels of OCN in obese children29 but elevatedOCN values are described in patients sufferingfrom increased bone formation as primary hyper-parathyroidism. These findings in children wereemphasized through an investigation showingthat substantial decrease in weight was also cor-related with a rise in OCN29. As OCN concentra-tions may vary with age, gender, height, growthvelocity, puberty, and time at blood draw30 Themajority of these investigations showed a con-nection between OCN and obesity. Individuals atTanner stage II-III were more likely to havehigher OCN levels and those with 25(OH)D defi-ciency were more likely to have lower OCN us-ing a sample of obese adolescents of mixedrace/ethnicity25.

In some researches in the morbid obese pa-tients, markers of both osteogenesis and osteoly-sis were raised. Weight was responsible for therise in the bone formation marker, bone alkalinephosphatase which may imply that adipokineshave an effect on bone formation directly. Fur-thermore, low bone mineral density and contentwith reduced femora length was observed in theleptin-deficient (ob/ob) mouse. Also in vitrostudies found leptin to be responsible for the re-duction of bone fragility and the boosting of os-teoblast differentiation and proliferation21. Thera-peutically, providing leptin can inhibit osteogen-esis working via the central nervous system orincrease bone formation acting peripherally32.The role for leptin in bone formation can be en-dorsed by the presence of profuse functional lep-tin receptors in osteoblasts. In consequence,many evidences support that bone turnover is di-rectly affected by leptin. Leptin effects on bonemay be mediated through harmoniously interac-tion with calciotropic hormones.

The mechanism by which PTH affects bone for-mation and resorbtion is by upregulation of the ex-pression of and to elevate levels of RANKL, animportant cytokine for osteoclastogenesis33. PTHis also known to negatively regulate the produc-tion of sclerostin by osteocytes. Sclerostin inhibitboth Wnts and several members of the bone mor-phogenetic protein (BMPs) actions which arecritical for osteoblastogenesis and synchronizethe activity of mature osteoblasts. PTH may in-

L. M. Saber, H.N.F. Mahran, H.H. Baghdadi, Z.M.H. Al Hawsawi

crease bone formation through direct influenceson osteocytes by decreasing the expression of theosteocyte specific gene Sost (producing scle-rostin)34. Rises osteoblast number and bone con-struction occurs also as a result of chronic PTHelevation. Increase osteoblast formation happensas a consequence of increase release of growthfactors embedded in the bone matrix as conse-quent of stimulation of bone resorption. Alsostrong anti-apoptotic effects on osteoblasts havebeen related to PTH35.

Many processes have been suggested to clarifythe interaction between obesity and bone metab-olism, involving raised proinflammatory cy-tokines and increased leptin production. Thereare many new evidences suggesting that at bothyoung and old age, the infiltration of bone mar-row by fat has harmful effect on bone remodel-ing unit affecting its function and also decreasingbone strength and density. Significantly,adipocytes in bone marrow inhibit osteoblastoge-nesis affecting bone formation as well as secreteinflammatory cytokines which have the capabili-ty to recruit osteoclasts. Adipocytes through theamplified production of leptin and/or reducedformation of adiponectin, influence bone forma-tion directly or influence bone resorption indi-rectly. Additionally through the increased pro-duction of the proinflammatory cytokine as IL-6,IL-7, IL-1 a, IL-1 β, and TNF-a which are inparticular raised in obesity and recognized tostimulate osteoclastogenesis, through altering thereceptor activator of NF-κB (RANK)/RANK lig-and/osteoprotegerin pathway36.

The strength of the research is being a crosssectional case control study in overweight andobese children that cover a wide range of BMI. Itverifies our assumption that serum leptin is oneof the factors which have a direct effect on theprocesses leading to alterations in the mineral-regulating hormones. Thought, there was no ma-jor correlation between OCN and markers ofbody weight. However, we cannot eliminate asmall influence of OCN on the very obese chil-dren.

This study has a small number of hypotheticalshortcomings. BMI percentiles for age andweight were applied to categorize overweightand obese children, but BMI is considered to beonly one of many markers of fat mass. Second, across sectional study cannot detect causal rela-tionship, longitudinal researches are desirable fora clear understanding and recognition of assumedconnections between these markers and fat tissue

throughout the change from normal weight toobesity, to determine the reasons that create thealteration in the mineral-regulating hormones,and how these change with decrease in fat mass.Finally, we have no data on gender-specific hor-mone measurements which could of help in un-derstanding the gender differences we noticedwith β-CTx. Another possible weakness is thesample size which may have limited the ability toidentify a meaningful connection between leptinand osteocalcin.

In summary, this cross sectional case controlstudy of obese children reveals that they have el-evated PTH levels and that these alteration clear-ly associate with changes in leptin and not due tos25(OH)D levels considered to be in the insuffi-ciency and deficiency ranges. Our finding pro-poses that leptin has an endocrine or paracrineinfluence on PTH secretion. Serum 25(OH)Dwas decreased and seemed to be the consequenceof boosted regulatory mechanisms. The associat-ed bone turn over markers with PTH, shown inobesity could be a result of direct effect of leptinwhich support our hypothesis that that leptin canparticipate in the processes causing alteration incalcitropic hormones and bone turnover.

Conclusions

The obese children could be at a bigger risk ofproducing increased level of leptin and PTH withstrong associated with bone turn over markerOCN and β-CTx and also deficiency of25(OH)D which could have an important role toplay in the pathogenesis of obesity and linkedbone metabolic risk diseases as osteoporosis andfractures. While calciotropic hormones are finelytuned to the control of mineral metabolism, theyalso impact and are influenced by hormonal fac-tors directly involved in the modulation of ener-gy metabolism.

These studies provide clear evidence of the in-terrelationship among leptin, calciotropic hor-mones and bone turn over markers showing theintegrative role of fat on skeleton and bone meta-bolic homeostasis.

––––––––––––––––––––AcknowledgementsThe research was supported by grants provided by Deanshipof Scientific Research, project number 4103/2014, Taibah-University Al Madina Al Mounawara, Saudi Arabia. The in-stitution review committee of ethical research in the Madi-nah maternity and children’s hospital approved the study.

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Interrelationship between bone turnover markers, calciotropic hormones and leptin in obese

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L. M. Saber, H.N.F. Mahran, H.H. Baghdadi, Z.M.H. Al Hawsawi

The authors thank the staff of outpatient clinics in Madinahmaternity and children’s hospital for their collaboration insampling and for their support of this study, also our pa-tients who participated in the study. We also thank Mr AdelAhmed A. Mowafy and Fahad Al-Sehli for assisting withthe technical work and data collection.

–––––––––––––––––-––––Conflict of InterestThe Authors declare that there are no conflicts of interest.

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