ESTABLISHMENT OF REFERENCE INTERVAL FOR SOME BONE TURNOVER MARKERS IN HEALTHY, YOUNG, PREMENOPAUSAL EGYPTIAN FEMALES

* Rania Mohamed El Sharkawy 1 , Noha S. Kandil 1 and MoatazaM. Abdel Wahab 2 . 1. Medical Research Institute, Alexandria University, 165 El-Horreya Avenue, El-Hadara, Alexandria, Egypt. 2. Department of Biostatistics, High Institute of Public Health, Alexandria University & FAMCO at UoD. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


ISSN: 2320-5407
Int. J. Adv. Res. 5 (3), 1522-1531 1523 The biochemical markerslevels varies rapidly and correspondingly to bone turnover, thus could be used to assess response and efficacy of pharmacological agents used in treatment of bone diseases such as osteoporosis. 17 There are so many causes of high BTM levels other thanosteoporosis, such as hyperparathyroidism, myeloma or thyrotoxicosis. 18,19 Vitamin D insufficiency as defined by 25(OH)D levels between 30 and 50 nmol/L is associated with an increase in parathyroid hormone (PTH) levels that is attributed to decreased calcium absorption. This may indicate secondary hyperparathyroidism in postmenopausal women. 15 The aim of antiresorptive treatments is to reduce BTMs to the lower part of the healthy premenopausal reference interval. 20 Thusthe lower limits of BTMs becamemore important than previously, as not only has it been questioned if decreases of BTMs below the reference interval could harm microdamage repair of the bone, but they also may be helpful in taking decisions in clinical practice, such as whether or not to give the next dose of a potent antiresorptive drug. 21,22 Therefore accurate and robust reference intervals for bone turn over markers become mandatory. 23 Unfortunately the available reference ranges are released through commercial laboratory kits, which lack standardization and don't take in consideration preanalytical variables. 24 As a result establishment of population based reference intervals that takes in consideration the preanalytical variables including age is considered a priority.
Several BTM reference intervals for healthy premenopausal women have previously been established. 17,25,26,27 In the last few years, reference intervals in premenopausalwomen from different countries in Europe (UK, France,Belgium and Denmark) 27,17 and the US 28 , havebeen reported. In addition, data from Germany on menand women have been recently published. 29 In Spain,there are data on Procollagen I intact N-Terminal (PINP) and Carboxy-terminal collagen cross links (CTX-I) in older men and inpostmenopausal women 30,31,32 as well as on CTX-I in asubset of 50 premenopausal women. 33 To our knowledge up till this moment there is no published data regarding reference values in premenopausal Egyptian females.Thus establishment of a valid and robust reference interval will allow comparing BTMs among different studied groups, differentiate between normal and osteoporotic patients 34 also predict the likelihood of fracture in osteoporotic patients and monitoring the suggested effective treatment response which cannot be done by bone mineral density as the latter can't detect minor and acute changes caused by therapeutic agents. 17,35 It is reported that nearly 70-80% of adult BMD is attained by the age of 18 years, One study indicated that there was no significant bone loss in 30-45 year-old women who were calcium balanced and of optimal bone health; we chose women aged 30-45 yrs for reference interval calculations because these women have achieved peak bone mass and are not yet perimenopausal. , 36 This study aims to establish robust reference intervals for some bone turnover markers in healthy premenopausal Egyptian females between the age of 30-45 yrs. This cross sectional study was conducted in Alexandria governorate. The specimens used in this study were obtained from an age stratified random sample. All subjects provided written consent.

Material and method:-
The clinical and Laboratory Standards Īnsti‫‬ tute (CLSI) released the EP28-A3c document that recommends 120 reference values as an adequate sample size for each reference population or subclass. This study included one hundred and fifty one healthy, non pregnant, regularly menstruating (10 or more periods per year), premenopausal females who were recruited as volunteers from the outpatient clinic of the MRI, Alexandria university, family planning clinics as well as volunteers from the medical, nursing, or secretarial staff of the units or their relatives from April 2015 till November 2015. The sample of this study was age-stratified random sample ,the participants where 30-45 years of age; as women in this age have their bone turnover markers stabilized, making them suitable for calculation of clinically useful reference ranges for BTMs. 21  Women under this range were excluded as they might have elevated BTMs due to skeletal immaturity, while those above 45 were excluded as they tend to have increased markers in the perimenausal period with normal estrogen levels. 25 Exclusion criteria:-None of the subjects taking part in the study had cancer, chronic disease, or any medical condition known to affect bone metabolism. All subjects were not receiving, within a year of starting the study, any bone metabolism affecting medications such as : selective estrogen receptor modulators , phytoestrogens , anticonvulsants , calcitonin , anabolic agents , steroids , vitamin D supplements , hormonal treatment for endocrinopathies , NSAID and depo-prover as well as calcium supplements, serotonin uptake inhibitors, L-troxin , antihypertensive drug and antiresorptive treatments . Subjects with history of alcoholism, smoking, recent clinical fracture, pregnancy, lactation, renal and endocrinal disorders were excluded. All participants were subjected to a detailed questionnaire concerning demographic data, medical history, lifestyle, etc.

Sample collection:-
Venous blood were taken from all subjects on a gel separator tube, as well as urine sample after overnight fasting from 8am till 10 am in the second week after menstruation . Venous samples were kept for 10 minutes at room temperature and centrifuged under standard guidelines (2500 g) for 10 min and serum was separated and stored at -80 C , no freezing and thawing is allowed only once upon analysis.
CTX has significant diurnal rhythm with a peak in the morning and a nadir in the afternoon. Moreover the circulating levels are influenced by food intake. In non fasting conditions, the peak is reached at night, followed by a significant decrease and nadir from 1100 to 1500 h. Therefore all the samples were collected after overnight fast in the morning within a standardized time period ideally from 8 and 10 a.m.thus reducing the day to day variation 12,37 Analytical standardization and biochemical analysis:-Standard Operating Procedures were followed during pre-analytical and analytical phases of the study with two levels of internal quality control included in each run. Corrective actions were done based on Westgurdrules . The following biochemical markers were measured at the same time point using a single lot of reagents and the same batch.

Statistical analysis:-
Statistical analyses were performed using SPSS version 22.0. The data were analyzed using nonparametric statistical techniques.
The initially collected 151 values were tested for outliers (outliers were considered for data point below Q1 -1.5×IQR or above Q3 + 1.5×IQR), outliers were investigated and overall, 20 cases were excluded from all the parameters for the reasons mentioned above.

Results:-
In this study one hundred fifty one females participated, of these women; four were excluded for having a fracture within one year, ten were excluded for medication intake (vitamin D, calcium and oral contraceptive pills), 4 were excluded for having abnormally low calcium levels and 2 were excluded for having abnormally high PTH level. 131 women completed all lab tests and the questionnaire fig (1) 1525

Fig (1)
The age selected was between 30-45, the sample was restricted to this age group and our results showed no statistically significant correlation between age and any of the studied parameters except phosphorus (weak indirect correlation r s =-0.189) and Vit D (intermediate correlation r s =-0.58);thus the norms were established from the whole participants for each of the 6 parameters, furthermore VitD values were presented for the age categories 30-<35yrs (n=45), 35-<40yrs (n=42) and 40-45 yrs(n=44) as the values of VitD varied between the 3 age categories using Kruskal Wallis test. And phosphorous was presented by the age groups <40 (n=87) and 40+ years (n=44) as values differed using Mannwhitney U test.   The new values for Ca and phosphorous did not statistically differ from the manufacturer though accepted higher upper limit (10.4) for Ca and a lower upper limit for phosphorous (4.64).
The new values for ALP as well did not statistically differ from the manufacturer. Concerning CTX and Vit D the new averages were lower than that set by the manufacturer , the differences were statistically significant (t=-10.2, p=0.0001 for CTX, and t=-19.7, p=0.0001 for Vit D) .While, the new average for PTH was higher than that of the manufacturer, the difference was statistically significant (t=10.9, p=0.0001).

Discussion:-
The importance of establishment of a reliable reference interval is because most medically important decisions are based on comparing the laboratory tests with their reference intervals.
In fact most reference intervals used in many laboratories are those of the manufacturer. These reference intervals don't represent the actual population and thus needs to be verified or established for the relevant population.
Bone turnover markers are convenient tool used to assesssystemic bone turnover rates, both in clinical practice and inclinical trials.
It has been recommended that normal reference ranges forBTMs shall be established in large samples of healthy premenopausalwomen. 17 However, the reference rangescurrently reported by commercial laboratories are often not rigorouslyestablished and fail to account for relevantpreanalytical variables. 12 Some of these variables including fracture, metabolic bone disease, chronic disease (including; malignancies, rheumatoid arthritis, diabetes), abnormal laboratory results of serum calcium, phosphorous. 17 In the last few years many reference intervals in premenopausal women in different countries like UK, France, Belgium, Danmark, US and Germany have been established and to our knowledge this is the first for Egyptian population. 17,27,28,29 The present study aimed to establish robust reference interval of some bone turn over markers in healthypremenopausal females aged from 30 to 45 years with mean age 36.6±4.5 years. This age range was taken because BTM levels were considered to be stable by Glover et al. in their study assessing thereference interval in premenopausal British women which is beneficial in the assessment of response to antiresorptive therapy in postmenopausal osteoporosis . (17) One of the main limiting factors was to include larger number of healthy individuals based on the literature exclusion criteria. 17 The CLSI released EP28-A3c document to help researchers by trying to unify reference interval studies. However bone turn over markers required detailed history due to many factors influencing their level.
The womenwho participated in the current study fulfilled the detailed inclusioncriteria and provided additional information that was used tocharacterize the population as a whole.
The exclusion criteria to the studied group included: vitamin D supplements, history of fracture, smoking, consumption of alcohol, metabolic bone disease and chronic diseases. 17 All these preanalyticalvariables contribute to the BTM markers in premenopausal females.
This study includes 151premenaupausal females who reached 131 after application of the above exclusion criteria. Calcium, phosphorous, Alkaline phosphatase, 25 (OH) vitamin D, PTH and CTX were done to all the candidates of this study.  Lenora etal xl where the median CTx values were 289, 280, 300, 387, 312 ng/L respectively. These discrepancies could be attributed to inter-assay variability of BTM, which represents a significant problem in their clinical application. Variability of collection as well as diurnal variability and intra individual variability have been described and lead to wider reference intervals and less diagnostic certainty. It is known that circadian variation is greater for bone resorption than bone formation. For example serum CTX exhibits a circadian rhythm with an amplitude of 80% of the 24 hour mean. xli Also Figure (2) shows scatter plots for the age range 30-45 years, showing that VitD values decrease by age (intermediate inverse correlation) and phosphorous values as well decrease by age but with a weak inverse correlation. Otherwise the correlation between age and the other 4 parameters was not statistically significant.
Other studies shows negative correlation between BTM and age ,but these studies included women aged from 21-54 , so they included women with skeletal immaturity . 35,xlii Other study included the same age group as ours and reported higher CTX between the age of 30-34 suggesting they haven't yet reached skeletal maturity. 25 This highlights the need for determination of the age at which each bone mass is reached.In our study this problem was unlikely and therefore the selected age group was the optimum for bone turnover markers reference interval establishment.
Comparing the norms resulted from the current research with those set by the manufacturer it was found that: The new values for Ca and phosphorous did not statistically differ from the manufacturer though accepted higher upper limit (10.4) for Ca and a lower upper limit for phosphorous (4.64).
The new values for ALP as well did not statistically differ from the manufacturer. Concerning CTX and Vit D the new averages were lower than that set by the manufacturer , the differences were statistically significant (t=-10.2, p=0.0001 for CTX, and t=-19.7, p=0.0001 for Vit D) .
While, the new average for PTH was higher than that of the manufacturer, the difference was statistically significant (t=10.9, p=0.0001).
The low level of vit D in the Egyptian population may be due to lack of direct sun exposure and reduced intake of vit D supplements as well as reduced calcium , this decreased vit D stimulates the parathyroid gland to secret more parathyroid hormone and this explains the increased level of PTH which exceeds the manufacturer reference range

Conclusion:-
BTM are widely used in therapeutic monitoring of osteoporosis or other bone diseases as well as having a significant impact in research. Our trial for establishment of reference interval in Egyptian population may help in harmonization of reporting by Egyptian laboratories. The strength points of this study are the standardization of the preanalytical and analytical conditions with adherence to the CLSI guidelines recommendations together with considering seasonal variation. The small sample size and the absence of the bone mineral density data with relatively low level of 25OHD were the limitations of this study .For a new or improved assay for BTM it is important to include a larger sample size with for a clinical reliable referencerange to be used in assessment of the efficancy of the antiresorptive therapy.