Research in Veterinary Science 93 (2012) 918–920

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Research in Veterinary Science

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Short Communication

The influence of sex on biochemical markers of bone turnover in dogs

Maja Belic a,⇑, Vesna Kušec b, Ante Svetina a, Juraj Grizelj a, Mirna Robic a, Zoran Vrbanac a,Marijan Benic c, Romana Turk a

a Faculty of Veterinary Medicine, University of Zagreb, Heinzelova 55, Zagreb, Croatiab Clinical Institute of Laboratory Diagnosis, Clinical Hospital Centre, Zagreb, Kišpaticeva 12, Croatiac Sanatio Ltd., Bolnicka cesta 34, Zagreb, Croatia

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

Article history:Received 20 July 2011Accepted 9 January 2012

Keywords:Bone turnoverDogsSex differences

0034-5288/$ - see front matter � 2012 Elsevier Ltd. Adoi:10.1016/j.rvsc.2012.01.008

⇑ Corresponding author. Address: Department ofVeterinary Medicine, University of Zagreb, HeinzeloTel.: +385 1 2390 180; fax: +385 1 2390 184.

E-mail address: mbelic@vef.hr (M. Belic).

Biochemical markers of bone turnover have been shown to be useful as inexpensive and noninvasivetools for monitoring skeletal health. The reference range for bone markers in dogs has been set by differ-ent age groups. However, other sources of biological variations were not fully investigated in dogs. Toexplore whether sex influences the interpretation of bone marker data we examined serum bone markersin 33 male and 25 female dogs. The bone markers selected for this study were: bone alkaline phosphatase(BALP) and osteocalcin (OC) as indicators of bone formation, and C-terminal telopeptide (CTx) of type Icollagen as marker of bone resorption. All concentrations of bone markers were lower, but still withinthe reference range reported for dogs. We found statistically significant differences of the median OCand CTx serum concentrations between males and females. The results of this study suggest that thereare sex differences in biochemical markers of bone turnover in dogs which should be considered in inter-pretation of bone marker data.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

The main process in skeleton is bone turnover which couplesbone formation by osteoblasts and bone resorption by osteoclasts.In healthy subjects these incidences are balanced and there is nonet change in bone mass (Allen, 2003). During formation andresorption of the bone, the bone cells produce and excrete enzymesand organic substances of bone matrix which are released into theblood and excreted by urine and therefore can be measured as bio-markers in blood or urine by variety of methods (Allen et al., 2000).Markers of bone formation include bone alkaline phosphatase(BALP), osteocalcin (OC) and amino and carboxy propeptides of col-lagen type I (PINP and PICP). Markers of bone resorption includeenzyme taratrate resistant acid phosphatase (TRAP), pyridinoline(PYD), deoxypyridinoline (DPD) and amino and carboxy telopep-tides of collagen type I (NTx and CTx). Biochemical markers of boneturnover are widely used in human clinical practice mainly fornoninvasive monitoring of bone metabolism and response to ther-apy of certain musculoskeletal and bone disorders (Deftos et al.,1991; Cosman et al., 1996; Watts, 1999; Watts et al., 2001; Swami-nathan, 2001; Kanakis et al., 2004).

ll rights reserved.

Pathophysiology, Faculty ofva 55, 1000 Zagreb, Croatia.

In veterinary medicine bone biomarkers are mostly used in pre-clinical and clinical studies as a rapid and sensitive method forassessment of bone response to medical treatment and surgicalinterventions (Allen, 2003). Animals used for these studies aremainly dogs (Allen et al., 1998; Breur et al., 2004), horses (Blacket al., 1999; Lepage et al., 2001) and cats (DeLaurier et al., 2002,2004). The reference values of bone markers in these species ofvarious ages have been established. The main deficiency in utiliza-tion of bone markers is their biological variability. Factors affectingbiological variability are age, sex, nutrition, exercise and systemicdisease (Watts, 1999; Souberbielle et al., 1997). Beside that, diurnaland seasonal variations have also been detected in animals as wellas in humans (Arens et al., 2007; Banfi et al., 2010). In humanpractice the influence of variability factors on bone turnover andresults of the measurements are fully investigated. In dogs, theinfluence of age (Allen et al., 1998), diurnal variability (Lieseganget al., 1999; Ladlow et al., 2002; Allen, 2003) as well as possiblebreed influence (Breur et al., 2004) on bone turnover biomarkershave been studied.

In this study the goal was to investigate another potentialsource of biological variation, namely variation introduced by dif-ferent gender. In human medicine the sex differences in bonemarkers are manifested mainly due to a different skeleton size inhealthy men and women (Steinberg and Rogers, 1987; Purduie,2004). However, these differences are not statistically significantand are age related in both genders (Khosla et al., 1997, 1998;Debono et al., 2011). In veterinary medicine the effect of sex on

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)lm/gn(CO)L/U(PLAB

MALES FEMALES

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Fig. 1. Differences in median values of bone formation markers (BALP and OC) inmales and females. For each parameter bars with different letters (a, b) aresignificantly different (p < 0.05).

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CTx (ng/ml)

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Fig. 2. Differences in median values of CTx in males and females. Bars with differentletters (a, b) are significantly different (p < 0.05).

M. Belic et al. / Research in Veterinary Science 93 (2012) 918–920 919

bone turnover and bone markers was investigated in several spe-cies, and differences between males and females were found,mainly for osteocalcin (Cahoon et al., 1996; Chiappe et al., 1999;Fletcher et al., 2000; Jackson et al., 2003; Havill et al., 2004). There-fore, we hypothesize that BALP, OC and CTx concentrations in maleand female dogs are different but within the same range reportedfor dogs of various age. Serum BALP, OC and CTx concentrationswere measured in 25 female with a median age 3 years (range 1–10) and 33 male dogs with a median age 6 years (range 1.5–10).Dogs were treated in outpatient clinic and in Clinic for obstetricand reproduction, Veterinary Faculty in Zagreb. All dogs werenon neutered, randomly chosen and of different breed. Onlyhealthy dogs were included in the study. Dogs with a history ofbone fracture in the past 6 months, dogs with ortophedic prob-lems, pregnant and ones in lactation were excluded from the study.The blood samples were collected in vacutainer tubes and centri-fuged at 3000 pm for 15 min. The serum samples were stored at�70 �C until assayed.

The activities of BALP were measured with a commercial humanenzyme immunoassay kit (Metra™ BAP EIA kit). The concentra-tions of OC and CTx were measured by use of commercial ELISA as-says (N-MID™ Osteocalcin One Step ELISA; Serum CrossLaps™ OneStep ELISA). All three human assays were proved to have a goodcross-reactivity with canine BALP, OC and CTx (Allen et al., 2000;Breur et al., 2004).

Kolmogorov–Smirnov test and Leven’s test were used for test-ing data for normality and equal variance. Differences betweenstudy groups were tested by non-parametric Mann–Whitney test.Spearman correlation coefficients were used to evaluate the corre-lation between measured biochemical markers of bone turnover aswell as their relationship to clinical data. SigmaStat 3.0 for Win-dows (Jandel Corporation, San Rafael, CA, USA) was used for statis-tical analysis. Statistical significance between values was set atp < 0.05.

There was no statistically significant difference (p = 0.51)between the median BALP values (U/L) of males 1.00 (range 0.7–12.0) and females 0.8 (range 0.7–4.2), but there was significantdifference (p < 0.05) between the median OC concentration (ng/ml) of males 4.00 (range 2–19) and females 1.00 (range 0.5–12)(Fig. 1). The median CTx concentration (ng/ml) also showed statis-tically significant differences (p < 0.05) between males 0.38 (range0.11–2.85) and females 0.55 (range 0.11–1.83) (Fig. 2).

In this investigation median values of bone turnover markerswere lower than the previously reported values (Allen, 2003;Sanecki et al., 1993; Allen et al., 1998) but still within the reference

range determined for the marker’s activities in dogs of certain age(Allen et al., 1998). We do not have a logical explanation for thelower results since the commercial human assays used in the studyhave good cross reactivity with canine markers (Allen et al., 2000;Breur et al., 2004). In studies performed on human population, thedifferences between men and women in bone turnover markerswere more distinct in young people (Riggs et al., 2002), especiallyafter skeleton maturation. Nevertheless, differences were alsofound in adults and elderly people (Gundberg et al., 2002).

In veterinary medicine studies on horses showed differences inOC values between genders, but only during exercise (Fletcheret al., 2000; Jackson et al., 2003). In adult baboons also, significantsex and age effects were determined for BALP and OC (Havill et al.,2004). In this study statistically significant differences in bonemarkers were found for OC and CTx, but not for BALP. We believethat a lack of statistical difference for BALP is a consequence of het-erogenous study group and that in homogenous group of dogs theeffect of sex would have been expressed in BALP too. However, thedifferences in OC and CTx between genders indicate that sex effectsbone turnover. OC, as a bone formation marker was significantlyhigher in males than in females, while CTx, as a bone resorptionmarker, was higher in females than in males. This result shows thatmales have higher bone formation, whilst females have higherbone resorption. The results are expected and in agreement withformer investigations in human as well as in veterinary medicine.Generally, males have larger and stronger skeleton than females,thus greater bone formation in males is understandable, especiallyduring growth and puberty, when skeleton gains size and volume.In human medicine, the difference in skeleton between genders isexplained firstly by influence of sex hormones on skeleton’sgrowth and development. In animals, the sex hormones also influ-ence the bone growth, because male animals have stronger and lar-ger skeleton than females.

Statistically significant and positive correlation between BALPand OC (r = 0.32, p < 0.05) found in this investigation was expectedsince both enzymes are osteoblasts’ synthesis products (Parthemoreet al., 1993; Stein and Lian, 1993). The result indicates connectionbetween bone markers during bone formation. In humans the sig-nificant correlation was found in children especially during puberty,and in patients with increased bone turnover (Yang and Grey, 2006;Ross and Knowlton, 1998).

Results show that markers of bone formation and resorption aredifferent in male and female dogs and this variability should beconsidered in interpretation of bone marker data.

920 M. Belic et al. / Research in Veterinary Science 93 (2012) 918–920

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