Th e utility of procollagen type 1 N-terminal propeptide for the bone status assessment in postmenopausal women

Th e utility of procollagen type  N-terminal propeptide (PNP) in the management of metabolic bone diseases remains a subject of debate since the reference ranges are not rigorously established and fail to account for many of the preanalytical variables. We aimed to establish reference intervals for PNP level in healthy and osteoporotic postmenopausal females stratifi ed by age, body mass index and menopausal duration. We also aimed to assess the relationship between PNP and BMD. Th is cross-sectional study enrolled  postmenopausal females who were divided in osteoporosis group (N=) and control group (N=) with preserved bone mass based on BMD assessed by DXA. In the osteoporosis group median PNP was signifi cantly higher (. ng / mL; CI .-.) compared to control group (. ng/mL; CI .-.)(p<.). After controlling for age, BMI and years since menopause, there was signifi cant inverse association between BMD and PNP at the femoral neck (r=-.), total hip (r=-.) and lumbar spine (r=-.). Th ere was no signifi cant diff erence in PNP concentration across quartiles of age in postmenopausal females. PNP was signifi cantly lower in obese postmenopausal females with preserved bone mass compared to normal weight and overweight females in control and in osteoporosis group. In conclusion, we showed that PNP is inversely associated with BMD even after controlling for age, BMI and years since menopause. Although, PNP is signifi cantly higher in postmenopausal females with osteoporosis compared to postmenopausal females with preserved bone mass its low specifi city does not warrant its utility is diagnosing osteoporosis. ©  Association of Basic Medical Sciences of FB&H. All rights reserved


INTRODUCTION
Bone mineral density (BMD) assessed by dual-energy X-ray absorptiometry (DXA) is the most commonly used measure in the assessment of osteoporotic status, risk for fragility fracture and in the management of postmenopausal osteoporosis [].BMD by DXA provides a static measure of skeletal status: a snapshot of the cumulative effects of diff erent factors on the assessed skeletal site over the time, but does not provide a dynamic estimate of skeletal activity, which could provide insight into the changes the skeleton may undergo in the future [].Bone is a dynamically and metabolically active organ that is continuously subjected to two processes: resorption and formation, collectively called bone turnover or bone remodeling.Bone turnover mark-ers (BMTs), released into bloodstream during remodeling process can aid in the management of postmenopausal osteoporosis as they provide dynamic information regarding skeletal status that is independent from, and often complementary to, BMD measurements [].Formation and resorption are usually tightly coupled in time and space; thus, any such marker refl ects the overall rate of bone turnover.Several studies have looked at various BTMs and their contribution to fracture risk, but the results of these studies have been inconsistent, not the least due to the use of different markers and different methodologies for their assessment [].Current recommendation for the standardization of BTM measurements in future studies is to use serum carboxy terminal telopeptide of collagen type I (s-CTX) as the standard bone resorption marker and serum procollagen type I N-terminal propeptide (PNP) as the standard bone formation marker [].Automated assays for BTMs are now commercially available and are a relatively non-invasive means of assessing bone remodeling activity in routine clinical practice.Traditionally most laboratory results are reported with a reference interval.Th e use of reference intervals with BTMs is limited as BTMs consistently demonstrate large degrees of variability between individuals and between age and physiological maturity [,].Th ese intervals are even wider in the postmenopausal group, which limits the use of a normal reference interval in the interpretation of BTMs in this population [].


Very few studies are available with the reference ranges for the newer, automated assays of bone turnover such as procollagen type I N-terminal propeptide (PNP).Type I collagen is the main protein of bone matrix and is cleaved to N-terminal (PNP) and C-terminal (PICP) propeptides of Type I collagen during bone formation.PNP is released into circulation and off er several practical advantages including its low diurnal variability and stability at room temperature.Its circulating levels are not signifi cantly infl uenced by food intake and, consequently, patients do not need to be fasting [].Th e utility of PNP in the management of metabolic bone diseases in individual patients remains a subject of much debate [] since the reference ranges currently reported by commercial labs are often not rigorously established and fail to account for many of the preanalytical variable such as age, duration of postmenopausal period and sessional variations.Th erefore, it is essential to establish a valid reference range stratifi ed by the known determinants of BTMs, such as age, and body mass index (BMI) for clinical use.Once reference ranges for BTMs are established, comparisons of BTMs between healthy individuals and osteoporotic patients can be performed [].
In this study, we aimed to establish reference intervals for PNP level in healthy and osteoporotic women stratified by age, body mass index and menopausal duration and investigate the utility of PNP measurement as a marker of osteoporosis in postmenopausal women.We also aimed to assess the relationship between BTMs and BMD in postmenopausal women.All participants informed written consent after the explanation of the study procedure.All procedures in this study were conducted in accordance with the guidelines of The Declaration of Helsinki.

Procedures
The health and lifestyle questionnaires were administered to the participants by the investigator.Demographic data and information on health and lifestyle of participants were collected from the questionnaire.Questions included smoking, alcohol consumption, exercise habits, current and past history of diseases, short family history.Details of medication and previous fractures were also indicated on the questionnaire.Additional information such as age at menarche, menopausal status, use of hormone replacement therapy and use of contraceptives were obtained.

Anthropometric measurements
Th e subjects' height and weight were measured while they wore indoor clothes and no shoes.Height was measured to the nearest . centimeters using a wall-mounted stadiometer.Weight was measured to the nearest . kg on a seca digital scale.Body mass index (BMI) was calculated as weight (kg) divided by height (m).BMI values in the range  - kg/m were considered normal weight, while subjects with BMI ≥ kg/m and ≥ kg/m were the cutoff levels for overweight and obese subjects, respectively.

PNP measurement
Blood samples were collected by venipuncture into  mL serum separator tube by the clinical laboratory phlebotomist at the Institute of Nuclear Medicine at CCUS between : and : AM to minimize the eff ects of circadian variation after at least a -h fast.Blood sampling was performed no

RESULTS
The    After controlling for age, BMI and years since menopause, there was signifi cant negative correlation between BMD and s-PNP at the femoral neck (r=-.;p<.), total hip (r=-.;p<.) and lumbar spine (r=-.;p<.)(Table ).s-PNP level in postmenopausal females with and without osteoporosis were within reference range but the median serum PNP concentration in postmenopausal females with osteoporosis was signifi cantly higher (. ng/mL; CI .-.ng/mL) compared to healthy postmenopausal females with preserved bone mass (. ng/mL; CI .-.ng/ mL)(p<.)(Figure).Th e area under the ROC curve was . with CI (.-.),which suggested that changes in the PNP levels might have a direct relation to osteoporosis (Figure ).However, when the cut-off value of PNP was accepted as . ng/mL, the sensitivity and specifi city were poor . and . respectively.On the other hand, when a value of . ng/mL was used as the cut-off point, the sensitivity increased to . while specifi city greatly decreased.
In the osteoporosis group there was no significant difference in median s-PNP values between diff erent BMI values.However, in obese postmenopausal females without osteoporosis, PNP was signifi cantly lower compared to normal and overweight subjects and compared to obese females with osteoporosis (Table ).In the osteoporosis group, PNP during the fi rst decade since menopause (. CI (.-.)ng/mL) was significantly higher compared to the second and third decade in the same group (p<.).Females with osteoporosis had signifi cantly higher levels of s-PNP during their fi rst and the third decade (> years) since menopause compared to females with preserved bone mass (Table ).

DISCUSSION
Type I collagen, which constitutes  of bone proteins, is synthesized as type I procollagen.During the extracellular processing of type I procollagen, there is cleavage of the amino terminal [N-terminal propeptide of type I collagen (PNP)] and carboxy terminal propeptide (PCP).These propeptides circulate in blood, where they are markers of bone formation.In contrast to serum PCP, which is a single protein, PNP circulates as different forms, including the intact authentic trimeric PNP, a monomer, and several fragments [].Intact PNP is mainly metabolized by the endothelial cells of the liver whereas clearance of mo-  [].Diff erent assays can measure both monomeric and trimeric forms.Diff erent methodologies used to measure PNP (intact, monomers etc.) are the reasons, which make it diffi cult to compare the results from other studies and to understand clinical utility of bone turnover markers in clinical practice [].The lack of assay standardisation is a matter of concern, making difficult the comparison of results obtained by different methods and/or in different laboratories [].Moreover, there is a paucity of published reference ranges particularly using the newer, automated assays of BTMs.Therefore, National Bone Health Alliance (NBHA) is conducting a project to standardize bone turnover marker sample collection procedures in the USA, establish a USA reference range for one bone formation (serum procollagen type I N propeptide, PNP) and one bone resorption (serum C-terminal telopeptide of type I collagen, s-CTX) marker, and standardize bone turnover marker assays used in clinical laboratories and stress the importance of harmonization for future research [].
In this cross-sectional study we included  postmenopausal females in whom we measured PNP level using the new automated assay.Median PNP in postmenopausal females included in our study was within reference range (. ng/mL) as provided by the manufacturer.In a study by Glover et al. [] which included  premenopausal women from four countries (United Kingdom, France, Belgium, United States) reference intervals for bone turnover markers were established.Th e study revealed that the median PNP level in women from France was . ng/mL and higher compared to median PNP levels of women from Belgium (. ng/mL), the United States (. ng/mL), and the United Kingdom (. ng/mL) suggesting that there exist diff erences between the levels of bone turnover in women living in geographically diverse locations.In another recently published, cross-sectional registry study, which included  healthy, premenopausal, European Caucasian women serum PNP reference range was established (.-.ng/mL) [].Th e median PNP concentration in our postmenopausal females was very similar to the median values obtained from premenopausal females from European countries.When we compared the PNP between osteoporosis and healthy postmenopausal females, serum PNP level was signifi cantly higher in females with osteoporosis compared to females with preserved bone mass (. ng/mL CI (.-.) vs. .ng/mL; CI (.-.)).But, when we analyzed sensitivity and specifi city of PNP in discriminating between postmenopausal females with osteoporosis and preserved bone mass, the marker was of poor value.When we set the cut-off value of . ng/mL the sensitivity was . but the specifi city was poor.Garnero et al. [] found that in postmenopausal women with osteoporosis, concentrations of total PNP were  higher than in premenopausal women.In postmenopausal osteoporosis, levels of bone resorption markers above the upper limit of the premenopausal range are associated with an increased risk of hip, vertebral, and nonvertebral fracture, independent of BMD.Th erefore, the combined use of BMD measurement and biochemical markers is helpful in risk assessment, especially in those women who are not identifi ed as at risk by BMD measurement alone [].
Many pre-analytical variables such as age, menopause, gender, body mass index are known to aff ect measures of BTMs [].In addition, medical conditions such as metabolic bone diseases or recent fractures and medications such as antiresorptives, corticosteroids, anticonvulsants, oral contraceptives (OCs) can also affect marker measurements.The median PNP in postmenopausal females below the age of  y was ., in the age group - y PNP was . and in females above  y it was . ng/mL.We did not observe signifi cant diff erence in PNP levels across age quartiles which is also confi rmed in a study by Glover et al. [] who also did not observed the diff erence in bone turnover markers across diff erent ages, but the study included only premenopausal women.Also our results are similar to those obtained by Hu et al. [] who used the same automated assay for PNP measurement.In their study median PnP for the age group - y was ., slightly decreased but not signifi cantly in the older age groups (- y -. and - y -. ng/mL).In their study, PNP was significantly higher in the postmenopausal females compared to premenopausal females.In our study we found that PNP and the BMD of lumbar spine, femoral neck, and total hip were signifi cantly inversely correlated.Th e eff ects remained after being adjusted for age, height, weight, and years since menopause.Our results are in line with the results from Hu et al. [] who also observed negative association between bone turnover markers and BMD.After menopause, an increased bone turnover is re-lated to bone loss by an imbalance between bone formation and bone resorption.During the fi rst three decades of life, bone formation predominates over bone resorption, while after the age of  years, the remodeling process is not in balance and bone resorption predominates over bone formation.Individuals with a high rate of bone loss are at risk of developing osteoporosis and fracture.In our normal weight and overweight postmenopausal females no significant diff erences in PNP values between osteoporosis and control group was observed.However, in obese control subjects PNP was significantly lower compared to obese postmenopausal females with osteoporosis and compared to normal weight and overweight postmenopausal females.
In our previous report we have shown that the levels of bone markers decrease rapidly with antiresorptive therapies [] but we did not measure PNP levels.Considering the increased levels found in patients with osteoporosis, the measurement of PNP before the initiation of the therapy and during the follow up could aid in monitoring the treatment efficiency.Serum PNP show responsiveness to treatment and low within-subject variability.Thus, the PNP measurement usually enables the identifi cation of the majority of responders to treatment [].
The BTMs levels reached after - months of therapy have been shown to be more strongly associated with fracture outcome than changes in BMD [].
Preliminary studies indicate that monitoring changes of bone formation markers could also be useful to monitor anabolic therapies, including intermittent parathyroid hormone administration and, possibly, to improve adherence to treatment.Thus, repeated measurements of bone markers during therapy may help improve the management of osteoporosis in patients.
Our study has several strengths and limitations.Strengths include the large sample size of extensively characterized study participants, measured BMD at hip and spine and stratification of the population by osteoporosis and postmenopausal status.On the other hand our reference ranges are method-sensitive and may not be applicable to other methods of PNP measurement.In addition, our sample is restricted to Caucasian subjects.It may therefore not be appropriate to apply our reference ranges to other ethnicities.

CONCLUSION
In conclusion, we established robust reference intervals for PNP in postmenopausal females.PNP is inversely associated with BMD even after controlling for age, BMI and years since menopause.Although, PNP is signifi cantly higher in postmenopausal females with osteoporosis compared to postmenopausal females with preserved bone mass its low specifi city does not warrant its utility is diagnosing osteoporosis.Our study also showed that PNP is signifi cantly higher in postmenopausal females with osteoporosis compared to healthy subjects during the first decade since menopause.

DECLARATION OF INTEREST
Authors declare no confl ict of interest Bosn J Basic Med Sci 2013; 13 (4): 260-265 ELMA KUČUKALIĆSELIMOVIĆ ET AL.: THE UTILITY OF PROCOLLAGEN TYPE 1 NTERMINAL PROPEPTIDE FOR THE BONE STATUS ASSESSMENT IN POSTMENOPAUSAL WOMEN median serum PNP concentration measured in  postmenopausal females was . (.-.)ng/mL; rang-ing from .-.ng/mL.Th e referral values in our laboratory for postmenopausal females without HRT are .-.ng/mL with the median of . ng/mL.Serum PNP below lower and over upper reference range was measured in  () and  () postmenopausal females respectively.Th ere was no signifi cant diff erence in PNP concentration across quartiles of age in postmenopausal females (Table).In our study there was significant difference in PNP levels across quartiles of BMD for lumbar spine, total hip and femoral neck.s-PNP level in the th and rd quartile of lumbar spine BMD was significantly lower (.(.-.)and . (.-.)ng/mL, respectively) compared to st and nd quartile (. (.-.)and . (.-.)ng/mL, respectively).Also, s-PNP level was significantly lower in the th quartile (. (.-.)ng/mL) compared to lower three quartiles of total hip BMD (Figure).

FIGURE 1 .
FIGURE 1. s-P1NP concentration across quartiles of lumbar spine, total hip and femoral neck bone mineral density.*-p<0.01 ELMA KUČUKALIĆSELIMOVIĆ ET AL.: THE UTILITY OF PROCOLLAGEN TYPE 1 NTERMINAL PROPEPTIDE FOR THE BONE STATUS ASSESSMENT IN POSTMENOPAUSAL WOMEN longer than two week after the bone mineral density measurement.For the measurement of PNP the tube was immediately put on ice and kept cool until serum separation.Tubes were centrifuged after  hour at room temperature.Serum levels of total PNP were determined using electrochemiluminescence immunoassay "ECLIA" on Elecsys  (Roche Diagnostic GmbH) at the Clinics for Nuclear Medicine at the CCUS.This assay detects both intact mono-and trimetric forms (total PNP).Referral values using this method for the PNP are .-.ng/mL.
ter University of Sarajevo.Values of bone mineral density are expressed as BMC (g) and areal BMD (g/ cm) and then converted into T-scores and Z-scores.The bone mineral density was measured at the lumbar spine (L-L) and all regions of the hip including total hip, femoral neck, trochanter and intertrochanteric shaft.For the lumbar spine measurement, the patient was position supine on the scanner table with arms resting on the tabletop with the knees fl exed over a ° and placed on a support pad.Hip measurements were always performed on the left side, unless there was a previous fracture or joint replacement.Precision coeffi cients of variation of the method for hip and lumbar spine is  and for neck and trochanter .Statistical analysisData were analyzed using SPSS . (SPSS Inc., Chicago, IL, USA).Th e anthropometric characteristics were presented as the means ± SD.PNP was determined to be not normally distributed by the Kolmogorov-Smirnov test.PNP reference range was presented as medians with  BMD, controlling age, BMI and years since menopause.

TABLE 1 .
s-P1NP concentration in postmenopausal females (N=183) across quartiles of age.Data are presented as median and 95%CI (2.5 th and 97.5 th percentile)

TABLE 2 .
Standardized correlation coeffi cients between s-P1NP and BMD in 183 postmenopausal women.

TABLE 3 .
P1NP concentration in postmenopausal females with osteoporosis and in healthy controls presented by diff erent age, BMI and years since menopause ranges.Serum P1NP levels in the osteoporosis and control groups of postmenopausal females.The solid horizontal lines denote the median value, the box represents the 25% and 75% interquartile ranges and the whiskers represent minimum and maximum values.FIGURE 3. Receiver operating characteristic (ROC) curve of serum P1NP level for diff erentiation between postmenopausal females with osteoporosis and healthy controls.ELMA KUČUKALIĆSELIMOVIĆ ET AL.: THE UTILITY OF PROCOLLAGEN TYPE 1 NTERMINAL PROPEPTIDE FOR THE BONE STATUS ASSESSMENT IN POSTMENOPAUSAL WOMEN nomeric PNP depends on kidney function.In infants and children, the concentration is much higher than in adults.Serum PNP is a useful indicator of disease activity in Paget's disease of bone, in bone metastases of osteoblastic nature, and in the follow-up of treatment of osteoporosis.Th e IFCC and IOF recently recommended the use of PNP as a reference marker for bone formation in studies concerning fracture risk assessment and treatment response Data are presented as median and 95%CI (2.5th and 97.5th percentile).* -signifi cant diff erence between osteoporosis and control group ¶ -signifi cant diff erence between normal weight, overweight compared to obese subjects (BMI <25 vs. 25-30 and ≥ 30 ) # -signifi cant diff erence between fi rst compared to second and third decade since menopause FIGURE 2.