Taibah University

Journal of Taibah University Medical Sciences (2015) 10(3), 300e305

Journal of Taibah University Medical Sciences

www.sciencedirect.com

Original Article

Does type 1 diabetes mellitus affect bone quality in prepubertal

children?

Khalid I. Khoshhal, ABOS a,*, Salah A. Sheweita, PhD b,Mohammad S. Al-Maghamsi, ABP c and Abdelhadi M. Habeb, FRCPCH c

aDepartment of Orthopedic Surgery, College of Medicine, Taibah University, Almadinah Almunawwarah, KSAbDepartment of Biotechnology, Institute of Graduate Studies & Research, Alexandria University, EgyptcEndocrine and Diabetes Unit, Maternity and Children Hospital, Almadinah Almunawwarah, KSA

Received 6 February 2015; revised 12 March 2015; accepted 17 March 2015; Available online 28 April 2015

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عونلانميركسلاءادبنيباصملالافطلأاةساردلاةنيعتمض:ثحبلاقرطةعومجمللةنيعلاتناكو.غولبلاتاملاعروهظنودبرثكأوتاونس٣ةدمللولأاةلاحورمعلاثيحنم،ةساردلاةنيعلنيلثاممسرادملابلاطنمةطباضلاتاملاعلانمةعومجمىوتسمسايقمتو.يركسلاءادبةباصلإانودب،غولبلاماظعلانداعمةفاثكتسيقامك.مدلايفهفاشتراوماظعلانيوكتلةيئايميكويحلا.ةيتوصلاقوفتاجوملازاهجب

٣٩و،لولأاعونلانميركسلاءادباباصملافط٣٦ةساردلاتمض:جئاتنلاءادبنيباصملالافطلأانم)٪٦٦.٧(٢٤/٣٦نأةساردلاترهظأو.اميلسلافطلقأاوناك٥مهنمو،رفصلانملقأمهلماظعلانداعمةفاثكتاسايقتناكيركسلانمطقف)٪٣٠.٨(١٢/٣٩دنعماظعلانداعمةفاثكتاسايقتناكامنيب.١-نمىلإةفاضلإابو.١-نودىلإمهنميألصيملو،رفصلانملقأنيميلسلالافطلأا،ماظعلانيوكتلةيئايميكويحلاتاملاعلاضعبيفضافخنادوجوظحول،كلذ.ماظعلافاشترلاةيئايميكويحلاتاملاعلاضعبيفعافتراو

عونلانميركسلاىضرمىدلماظعلانداعمةفاثكضفخنت:تاجاتنتسلاافاشتراتاملاعضعبديزتامنيب،ماظعلانيوكتتاملاعضعبكلذكو،لولأاركبملافشكلايفناديفيةيمظعلاتاملاعلاوماظعلانداعمةفاثكسايقنإ.ماظعلا

Corresponding address: College of Medicine, Taibah University,

. Box 879, Almadinah Almunawwarah, KSA.

E-mail: kkhoshhal@hotmail.com (K.I. Khoshhal)

r review under responsibility of Taibah University.

Production and hosting by Elsevier

8-3612 � 2015 The Authors.

duction and hosting by Elsevier Ltd on behalf of Taibah Universit

tp://creativecommons.org/licenses/by-nc-nd/4.0/). http://dx.doi.org/10

،لولأاعونلانميركسلاءادبنيباصملادنعماظعلاةيعونيفتارييغتيأنع.ةيمظعلاروسكللمهتيلباقنمللقتدقتجلوعنإاهرودبيتلا

؛ةيمكلاةيتوصلاقوفتاجوملا؛ماظعلانداعمةفاثك:ةيحاتفملاتاملكلادنيماتيف؛ماظعلاةشاشه؛نيسلاكوتيسوأ

Abstract

Objectives: Type 1 diabetes mellitus (T1DM) in children

often starts before the achievement of peak bone mass.

This may constitute a landmark in predicting bone frac-

ture risk later in their lives. This study aims to determine

the serum levels of bone markers in children with T1DM

in combination with their bone mineral density (BMD).

Methods: Children diagnosed with T1DM for 3 years or

more without signs of puberty were included in the diabetic

group.Another groupof age-matched healthy non-diabetic

controls was recruited from a local school. The serum levels

of a group of biochemical markers for bone formation and

resorption were determined in both study groups, and

BMD was measured by ultrasound absorptiometry.

Results: Thirty six children with T1DM and 39 normal

children were included in this study. The results showed

that 24/36 (66.7%) diabetic children had a Z score below

zero. Of these, five scored below �1. In contrast, 12/39

(30.8%) children from the control group had a Z score

below zero, but none had a score below �1. Significantly

lower levels of osteocalcin and procollagen N-terminal

peptide were detected in the diabetic group. The serum

levels of bone resorption markers were significantly

higher in the diabetic group.

y. This is an open access article under the CC BY-NC-ND license

.1016/j.jtumed.2015.03.004

K.I. Khoshhal et al. 301

Conclusion: T1DM decreases BMD and some bone for-

mation and increases some bone resorption biomarkers.

BMD and bone markers are useful diagnostic tools for

the early detection of alterations in the bone quality of

children with T1DM. This, if treated in a timely manner,

may decrease future bone fracture susceptibility.

Keywords: Bone mineral density; Osteoporosis; Osteocalcin;

Quantitative ultrasound; Vitamin D

� 2015 The Authors.

Production and hosting by Elsevier Ltd on behalf of Taibah

University. This is an open access article under the CC BY-

NC-ND license (http://creativecommons.org/licenses/by-nc-

nd/4.0/).

Introduction

Osteoporosis has become an alarming health problem

throughout the entire world, and approximately 200 millionpeople in the world are threatened by this deleterious dis-ease.1,2 Osteoporosis is often described as a silent diseasebecause it is typically asymptomatic until a fracture

occurs.3 Like osteoporosis, diabetes mellitus (DM) is apandemic and a chronic metabolic disorder. In fact, 374million people in the world may suffer from diabetic

complications.3e6 As a chronic condition, DM adverselyaffects many different parts of the body including bones,nerves, muscles, eyes, and kidneys.4 As increased rates of

bone fractures and osteoporosis have been found amongpatients with type 1 DM (T1DM),5,7e11 in bothchildren12,13 and adults,14 and because the mechanisms of

diabetic effects on bone cells are very complex, severalresearchers have described different mechanisms thatshowed how DM induces osteoporosis and bone fracturesthrough multiple pathways.6,14,15

Bone marrow-derived endothelial progenitor cells (EPCs)play a significant role in bone healing.5,16 DM was found todown-regulate the expression of EPCs through different

mechanisms and thereby decrease bone formation at fracturesites.5,17,18 DM is also responsible for the deposition of lipidin the bone marrow, thereby leading to the expansion of the

marrow cavity and a decrease in the rate of blood flow to thebone, which is required for the transfer of nutrients.5 Thetransformation of osteoblast to adipocyte leads to thereduction of osteoblasts available for bone formation.5,19 It

is known that DM is responsible for the over expression ofadvanced glycation end products (AGE) and has roles inbone rigidity.20,21 Pancreatic b cells also produce other

osteoporotic factors including amylin and preptin. Amylinand preptin induce bone formation and sequester boneresorption and reduce the apoptosis of osteoblasts.4

Osteocalcin is a peptide that positively regulatesosteogenesis. DM limits the production of osteocalcinthrough the negative regulation of osteoblasts by decreased

synthesis of insulin, amylin and preptin.Dual energy X-ray absorptiometry and peripheral quan-

titative computed tomography may be used to measure bonemineral density (BMD) in children who are exposed to an

increased risk of osteoporosis in their adulthood, but bothexpose children to unnecessarily ionizing radiation, which is

a limiting factor for preventive studies in children. Therefore,in recent years, quantitative ultrasound (QUS) methods havebeen developed to assess bone mineral status in some pe-

ripheral skeletal sites such as hand phalanges, tibia andcalcaneus. QUS techniques are safe, easy to use, radiation-free, and come with portable devices so they are particu-

larly suitable and indicated to assess bone mineral status inchildren.

The optimal use of bone marker measurement withassessment of BMD using QUS of the calcaneum in pre-

dicting osteoporosis has not yet been established. Therefore,the present study aims to investigate the levels of bonemarkers in plasma of children with T1DM in combination

with their BMD.

Materials and Methods

Setting

This study was conducted in the Maternity and Children

Hospital (MCH), Almadinah Almunawwarah, Kingdom ofSaudi Arabia and was approved by the MCH research andethics committee. The diabetes unit at MCH provides

comprehensive services for children with DM up to the age of12 years, and the city has one of the highest incidences ofchildhood T1DM in the world.22

Inclusion and exclusion criteria

Patients diagnosed with T1DM for 3 years or more

without signs of puberty were included.We excluded patientswith a history of fractures of less than 1 year and those withassociated bone/joint problems, liver disease or patients on

long-term steroid therapy. Patients with incomplete datawere also excluded.

Control and diabetic groups

Informed written consent was obtained from all of the

families of the participating children. All 36 T1DM patientswere included from the Endocrine and Diabetes Unit, MCH,from February to May 2013. 39 age-matched healthy non-

diabetic controls were recruited from a local school. Thestudy included 75 children (age 4e12 years), with 49 malesand 26 females. Biochemical markers for bone formation andresorption and QUS for BMD were utilized for all patients

and the control group.

Measurement of bone mineral density (BMD)

BMD in the current study was measured using The LunarAchilles (InSight, GE Healthcare, USA), which is a proven

bone ultrasonometer to avoid exposing the children to un-necessarily ionizing radiation. In this study, BMD wasmeasured using QUS of left calcaneum according to the

method of Valerio et al.23 All measurements were convertedto Z-scores using a data bank for age-matched speed ofsound values supplied by the manufacturer.

Figure 1: Bone mineral density (Z score) versus age for children

with type 1 diabetes mellitus compared to the non-diabetic control

group.

Does T1DM affect bone quality in prepubertal children?302

Biochemical bone markers

Human osteocalcin assay was performed on a multiwell

plate.24 In addition, the levels of the bone formation markersprocollagen N-terminal peptide (PINP), andcarboxyterminalpropeptide of type I procollagen (PICP),bone resorption markers [tartrate resistant acid

phosphatase (TRAP), pyridinoline (PYD), cross-linked car-boxy-terminal telopeptide of type I collagen (CTX-I) anddeoxypridinoline (DPD)] and vitamin D were measured us-

ing appropriate commercial ELISA kits (MyBioSourceIncorporation, San Diego, CA, USA).

Assay of bone alkaline phosphatase activity

Alkaline phosphatase (ALP) (AlP; EC 3.1.3.1) catalysesthe transfer of the phosphate group from para-nitrophenylphosphate and liberating para-nitrophenol in an alkaline

medium. The incubation mixture contained 2-amino-2-methyl-1-propanol (0.35 mol/L), zinc sulphate (1 mmol/L),magnesium acetate (2 mmol/L), pH 10.4, 4-nitro-phenylphosphate (12 mmol/L), and 20 microliters of plasma.

The liberated para-nitrophenol was measured calorimetri-cally at 405 nm every minute for three minutes, and theaverage absorbance per minute was calculated.25

Statistical analyses

Statistical analyses were performed using the SPSS sta-tistical software package. Normality and homogeneity of the

data were confirmed before ANOVA; differences among thestudy groups were assessed by one-way ANOVA followed byDuncan’s multiple range tests.

Results

There is no significant difference between the mean age ofboth groups of the control and diabetic children. As ex-

pected, all diabetic children showed higher levels of glycatedhaemoglobin (HgA1C) than those of control group (Table 1).Figure 1 shows the distribution of Z score versus age among

control and diabetic children. It shows that the majority ofdiabetic children have Z scores below zero (24/36, 66.7%with a mean Z score of �0.593 � 0.230), and five of them

(13.9%) actually have Z scores below �1 (low BMD),whereas only 12/39 (30.8%) of the children in the controlgroup are below zero and no one was below �1 (mean of

Table 1: Characteristics of children with type 1 diabetes mel-

litus and control subjects.

Variables Non-diabetic

control

(n ¼ 39)

Group with

Type 1 diabetes

mellitus (n ¼ 36)

P value

BMD (Z score) 0.495 � 0.169 �0.593 � 0.230 <0.05

Age (Years) 7.89 7.99 >0.05

Hg A1C Normal 10.69 � 0.392 <0.05

Duration of

diabetes (Years)

0.0 4.51 � 0.298 <0.05

Z score �0.495 � 0.169), which indicates that diabetesplays a role in decreasing BMD.

Among bone formation markers, osteocalcin and PINP

levels were significantly lower in diabetic children than in thecontrol subjects. Conversely, PICP did not differ signifi-cantly (Table 2). Among bone resorption markers in diabetic

children, TRAP, PYD and CTX-1 levels were found to besignificantly higher in diabetics (Table 3). On the other hand,the level of vitamin D (non-diabetic 23.94 � 0.98, diabetics

26.79 � 1.05) did not differ significantly (P > 0.05)between the two groups, although the level in diabeticchildren was slightly higher.

Discussion

Many studies have confirmed that T1DM is associatedwith decreased BMD.26e28 In agreement with theseobservations, the present study showed that BMD (Z

score) was lower in T1DM patients than in the controlgroup. The score in most children was not low enough tobe labelled as high risk for fractures at the time of the

study, which explains why they did not experience frequentfractures. Conversely, 13.9% of the diabetic children had Z

Table 2: Changes in bone formation markers in diabetic

children.

Biochemical

parameters

Non-diabetic

control

Diabetic

children

P value

Osteocalcin [ng/ml] 48.50 � 1.50 38.50 � 1.06 <0.001

PINP [ng/ml] 914.07 � 142.5 829.30 � 28.25 <0.05

Alkaline

Phosphatase

177.80 � 31.76 171.50 � 21.93 >0.05

PCIP [ng/ml] 33.00 � 0.47 34.96 � 3.86 >0.05

Table 3: Changes in bone resorption markers in diabetic

children.

Biochemical

parameters

Non-diabetic

control

Diabetic

children

P value

TRAP [ng/ml] 27.87 � 1.24 38.34 � 1.48 <0.05

DPD [ng/ml] 107.97 � 2.47 109.35 � 2.51 >0.05

PYD [ng/ml] 29.49 � 1.38 37.20 � 5 <0.05

CTX-1 [ng/ml] 48.18 � 1.67 54.73 � 5.68 <0.05

K.I. Khoshhal et al. 303

scores below �1, whereas no one from the control group had

a Z score below �1. This may explain the higher percentageof osteopenia in older diabetics compared to the generalpopulation. Keeping this in mind, one might suggest

interfering earlier in diabetic children to prevent futureosteopenia and osteoporosis with proper advice regardingexercise and supplementations.

The development of biochemical markers that are specificand sensitive in reflecting the overall rate of bone formationand resorption has markedly improved the non-invasive

evaluation of bone turnover in various metabolic bone dis-eases such as DM.29,30 Serum or plasma bone specific ALP(BSAP), osteocalcin, P1CP and P1NP are the most widelymeasured bone formation markers. The levels of

osteocalcin and P1NP in the present study were lower inthe serum of diabetic children than that of non-diabeticcontrol. Supporting our finding, it has been found that dia-

betic osteopenic patients displayed lower serum levels ofP1NP, which reflects poor bone formation.31 P1NP hasseveral functional advantages and has been recommended

by the one Marker Standards Working Group due to lowinter-individual variability32 and its stability in serum atroom temperature.33 It is also used as a preliminarybiomarker on the effectiveness of a given drug on bone

formation.In the present study, the decreased level of osteocalcin was

correlated with low BMD, whereas the activity of ALP was

not. However, in other studies, BMDwas not correlated witheither osteocalcin or ALP.34,35 A decrease in bone formationin DM might be due to low levels of serum PTH.36,37 In

addition, DM reduces osteocalcin levels through thenegative regulation of osteoblasts by decreased synthesis ofinsulin, amylin and preptin.4

The majority of bone resorption markers are bonecollagen degradation products, with the exception of TRAP.Examples include PYD and DPD, and CTX-1, of whichCTX-1 is considered the marker of choice.38 TRAP in

children with T1DM increased gradually and after 12months was higher than at onset.38,39 In the current study,the levels of TRAP, CTX-1 and PYD increased in the

serum of diabetic children compared to that of normalchildren. In other studies, it has been found that levels ofboth CTX-1 and BMD were lower in T1DM than in the

control group.39,40 It has been suggested that increasedinflammation and induced levels of cytokines areresponsible for the acceleration of bone turnover and bone

loss in DM patients.41 Another possible mechanism is thatDM is associated with oxidative reactions. Many studieshave shown that increases in free oxygen radical levelscould induce tissue damage and abnormalities in

antioxidant defences in DM.42,43 Oxidative stress can

impair the balance between proteolytic enzymes and theirinhibitors that may cause or worsen osteoporosis in DM

patients.44 In vitro studies have shown that oxidative stressinhibits osteoblastic differentiation and induces osteoblastinsults and apoptosis. Thus, oxidative stress may be related

to the pathogenesis of bone disorders.43,45,46

We conclude that children with T1DM have lower BMD(Z score), low bone formation and high bone resorption

markers, which might lead to osteoporosis in the future.Future double-blind studies on the benefits of early inter-vention to reduce the chances of osteoporosis in diabetics areneeded. Moreover, bone marker measurements in combina-

tion with BMD are more useful than measuring BMD alonefor predicting osteoporosis in diabetic children. In addition,we recommend the QUS devices for their simplicity, the lack

of radiation exposure, and the ease of the examination,which can be carried out at a bedside.

Authors contribution

KK and SS designed the study and submitted the grant.MM and AH recruited the patients. SS wrote the first draft

and KK, MM and AH were responsible for the criticalrevision of the manuscript. All authors read and approvedthe final submitted version.

Conflict of interest

All authors of this manuscript declare that there is no

conflict of interest.

Acknowledgements

The authors thank the Deanship of Scientific Research,TaibahUniversity, Almadinah Almunawwarah, Kingdom of

Saudi Arabia for funding this project, (grant number 514 /432). The authors also thank the nursing staff at MCH, Mr.Wael Barakat and Mr. Mohamed Abdel Samad for their

help during the study and data collection.

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