The clinical utility of bone marker measurements in osteoporosis

Bone turnover shows a circadian rhythm, this is more obvious in the serum and urinary markers of bone resorption. βCTX for example is highest between 01:30 and 04:30 hours and may be more than twice that at the nadir between 11:00 and 15:00 hours [27], this may be attenuated by several factors such as; age, gender, ethnicity, menopausal status, osteoporotic stage and anti-resorptive agents or calcium supplementation [19], but the disparity is diminished with fasting [25]. All bone markers are significantly lower in the fed state with the exception of BSAP, this may be due to several factors including the clearance rate of individual markers or food composition [16] and may be partly explained by variation in serum insulin [25]. Osteocalcin and P1CP follow the same diurnal pattern but show only twenty percent difference and BSAP has two peaks at 14:00 and 23:30 hours with a nadir thirty percent reduced at 06:30 [19]. Therefore timing of the sample collection and fasting status should be tightly controlled.

The existence of intra-individual low-frequency biological rhythms, imply that biomarkers can also vary between consecutive days, this is more noticeable in the urinary resorption markers [14]. There is a degree of controversy regarding seasonal variation with some researchers suggesting that overall seasonal changes are insignificant [56], whilst others have found a substantial wintertime increase [57], which may be due in part to reduced levels of vitamin D. Physical activity is also significant, TRAP and to a lesser extent BSAP and CTX are reduced immediately after plyometrics, but return to pre concentrations within two hours. Interestingly similar changes were found in PTH [29]. Details of exercise in the previous twenty-four hours should therefore be recorded.

Bone turnover varies with the menstrual cycle, research suggests that osteoblastic activity is higher during the luteal period [58] and bone resorption is increased during the follicular phase [59]. Pregnancy affects all BTMs due in part to the calcium requirements of the foetus, but also to changes in maternal glomerular filtration rate (GFR) affecting renal clearance. However the time change is contentious, one study following ten women at regular intervals reported an increase in urinary resorption markers throughout pregnancy with a significant increase in bone formation in the third trimester [60]. A more recent study measured serum OPG, RANKL, osteocalcin and CTX in twenty six different women at each trimester. The study found increased bone formation in the first trimester and increased resorption in the second which surprisingly decreased again in the third trimester [61]. Postpartum, levels gradually start to decrease but may still be higher than pre-pregnancy levels for up to a year [62].

A comprehensive drug history should also be taken into account when interpreting bone marker results. Anti-resorptive drugs such as bisphosphonates [26] and hormone replacement therapy (HRT) [63] have a major effect on markers of bone resorption and long-term corticosteroid therapy is known to suppress bone formation [64].

Inflammatory conditions are major precipitators for bone loss, especially rheumatoid arthritis (RA) which is further aggravated by decreased functional activity and the use of glucocorticoids [65]. In a prior study, we found that B-cell depletion increases bone formation and decreases bone resorption in RA patients. This may be a direct effect on osteoblasts and osteoclasts respectively and be at least partially explained by the decreased inflammation and disease activity [66]. In diabetes serum osteocalcin is negatively correlated with glucose levels and advanced glycation end products (AGEs) are known to have a negative impact on bone [67]. Thyroid disorders such as thyrotoxicosis are well known to affect bone turnover. Thyroid stimulating hormone (TSH) receptors are present in both osteoblasts and osteoclasts and the low TSH levels observed in thyroidectomised patients on L-thyroxine are associated with an increase in OPG and decrease in RANKL and are significantly correlated with vertebral fractures [32]. Bone markers are cleared through the liver or kidneys and are also influenced by diseases affecting these systems, decreased GFR for example will decrease the urinary excretion of CTX and therefore increase serum levels. They are also affected by any disease states leading to increased periods of bed rest and immobility. Research has shown that microgravity induces significant and progressive bone loss, a consequence of increased bone resorption and retardation of bone formation [68]. Certainly levels of all bone markers increase significantly in the first few weeks after fracture and may remain elevated for up to a year. The rate of increase is dependent on the location, severity and size of the fracture and the age of the patient. BTM’s can be elevated for up to 6 months after minor fractures e.g. forearm fractures but up to one year after a hip fracture and needs to be taken into consideration when measuring them [19, 69]. However, they fall gradually over time and using a reduction of 50% in bone resorption when using anti-resorptives as a good indicator of response would be greater than any reduction that might otherwise occur.

In light of the above evidence it can be seen that to use bone turnover to monitor change can be quite difficult. In order to minimise problems it is best to measure the BTMs in as similar a set of circumstances as possible. Particular attention should be paid to the time of day and hence research studies tend to use early morning fasted samples. One way in which to help overcome within person variability in serial measurements and to monitor therapy is to use the ‘least significant change’ (LSC) model [70]. LSC at a significance level of p=<0.05 is defined as 1.96*√2*√ (CV₁²+CV_A²); where CV₁ is the within-subject coefficient of variation and CV_A is the total analytical imprecision. LSC identifies the true physiological change in the marker. In general a change of more than twenty percent is considered significant for formation markers [71], similarly between twenty-seven to thirty-six percent is significant for markers of bone resorption [72].

Between person variability is much harder to control, e.g. age, gender, and menopausal status, but is equally important to validate results. Bone metabolism rates are higher in infants up to three years of age, they are relatively stable throughout adolescence but sex-specific increases in bone marker levels are evident during the pubertal growth spurt and are reportedly influenced by pubertal stage rather than age [73]. BTMs are higher in men between twenty and thirty years of age then reach their lowest levels during their fifties [14], whereas in females there is a substantial increase in bone turnover corresponding to oestrogen deficiency during the menopause [28]. We checked our local population and found a trend towards higher bone turnover in males during their third decade whilst reaching peak bone mass, although there seemed to be little evidence of any age related change in women possibly due to the lower numbers of postmenopausal females in our cohort (Figure 3). It is important for each laboratory to investigate the transferability of the quoted reference intervals to its own patient population based on equivalent standardised collection conditions. The widespread availability of automated immunoassays now means that harmonisation of method specific reference ranges is possible and studies from well-characterised populations have reported robust BTM ranges in large well defined cohorts [74]. In contrast to the use of reference ranges some researchers have suggested combining a marker of formation and resorption to gain a direct insight into the changes in the balance and rate of bone turnover in relation to a reference value [75], leading factors in estimating fracture risk and prognosis. Given the large observed differences observed between genders, different ages and developmental stages means that care must be taken when comparing populations and in the design of research studies.

Over the last decade many of the traditional BTM immunoassays have been automated, improving technical performance and increasing their availability. Nevertheless, analytical aspects such as within and between batch precision, accuracy and standardisation, remain problematic. Inter-laboratory variation is also crucial; a European study in 2001, measuring pooled samples of serum and urine in seventy-three laboratories concluded that even with identical assays results for the majority of the markers were significantly different [76]. Similarly an American study in 2010 comparing six commercial laboratories over an eight month period concluded that reproducibility varied substantially for urine NTX and serum BSAP [77]. Moreover there is an extensive list of bone markers being offered making it very difficult to compare research evidence. Consequently, the International Osteoporosis Foundation (IOF), the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) [20] and the National Bone Health Alliance (NBHA) [78], have recommended that a marker of bone formation and resorption, namely P1NP and CTX, are used as reference analytes in clinical studies. They go on to stipulate that these markers should be measured by standardised assays to minimise immunochemical heterogeneity and recommend that manufacturers adopt international reference standards and minimise batch to batch variability [20].

Appropriate control of sample collection and preparation is vital for successful BTM measurement. Several BTMs, especially osteocalcin and TRAP5b, are sensitive to thermo degradation and levels can be significantly reduced after only a few hours at room temperature [22, 23]. TRAP5b activity is also reduced during storage, samples must be kept at −70°C or lower and multiple freeze-thaw cycles should be avoided. No significant decrease has been detected in CTX stored at −20°C or lower for up to three years, nevertheless it rapidly decreases in serum at both 4°C and 37°C. The molecular mechanism is unknown but this decrease is minimised by ethylenediaminetetraacetic acid (EDTA) [18]. CTX is reportedly stable in EDTA blood tubes before separation for up to forty-eight hours, likewise osteocalcin becomes stable for up to eight hours at room temperature [21]. Consequently blood should be collected into EDTA tubes and separated as soon as possible, if samples are not analysed immediately they should be stored at −20°C or lower. Both P1NP and BSAP were found to be stable in any sample type [21]. Notably current TRAP5b assays are not affected by haemolysis, but erythrocytes are known to contain proteases which degrade osteocalcin. Grossly haemolysed samples in general should always be avoided.