In this prospective interventional study, we investigated changes in the circulating levels of TRAP 5a and TRAP 5b derived from patients with severe AN during rapid weight gain therapy for 12 weeks. Markers of bone turnover, including TRAP 5b, have previously been studied and assessed for their potential to predict the uncoupling of bone formation and resorption in patients with AN [23, 24]. The present study constitutes, to our knowledge, the first effort to address changes in both serum TRAP 5a and TRAP 5b during large metabolic changes over short period of time in patients that initially are in a late stage of severe AN and return to a more normalized BMI. Moreover, this is also the first study to investigate the potential use of TRAP 5a and TRAP 5b, and the 5a/5b ratio, as markers to reflect restoration of bone remodeling in patients with AN during nutritional therapy resulting in a weight increase mainly due to substantial net gain in fat mass. Patients were served six meals daily according to the described nutritional plan, which resulted in a large change in body composition. Fat mass percentage was highly increased from 11.4 to 26.7%, demonstrating that the BMI increase was mainly due to increased fat mass and not lean mass. Hence, this therapy provides a possible clinical model to study changes of the different forms of TRAP within a rather short period of time during large changes in energy metabolism.
Serum TRAP 5a was upregulated after 12 weeks and has previously been shown to be associated with adipose parameters [12, 30] and bone tissue . Serum TRAP 5a reached levels that were in the same magnitude that has been reported for healthy individuals , which also is in concurrence with bone and weight assessments reaching a near-healthy state . The lack of association between TRAP 5a or TRAP 5a/5b and anthropometric fat parameters, e.g., BMI, fat mass and fat content does, however, not support a role for TRAP 5a in expansion of the adipose tissue in this particular situation, in contrast to the development of obesity . A major contributor may be related to whether the accumulation of adipose tissue occurs as a result of going from underweight to normal as in this cohort or pathological, i.e., from normal to obese weight gain . Weight gain in AN versus obesity reflects different states with different components such as adipose inflammation and metabolic aberrations that could affect the involvement of TRAP 5a. On the other hand, the observed downregulation of TRAP 5b is in accordance with weight restoration and increased bone mass after weight gain therapy in the AN group . Thus, the increased bone mass is most likely due to a combination of increased bone formation, as reflected by the increase in bone anabolic parameters, i.e., levels of BALP induced by the 12-week therapy , as well as decreased bone resorption and lower numbers of osteoclasts, as reflected by decreased serum levels of TRAP 5b.
The observed changes of TRAP 5a and TRAP 5b (in opposite directions) agree with that of total TRAP (i.e., the sum of TRAP 5a and 5b was not significantly changed). The TRAP 5a/TRAP 5b ratio was also significantly altered with approximately a 4-fold increase). The abundance of TRAP 5a, that is, 4-fold more than TRAP 5b, has previously been reported in healthy individuals where TRAP 5a has been in the range of 4-8 ng/ml [33, 34], while other studies have reported TRAP 5b levels in the range of 1–3 ng/ml [23, 24]. Thus far, there are no studies that have investigated the ratio between TRAP 5a and 5b directly in the same serum sample from humans. Previously, it has been reported that TRAP 5a is secreted by macrophages from adipose tissue ; thus, the observed inverse correlation between TRAP 5a and weight at baseline, but not at week 12, could be due to exceptionally low amounts of adipose tissue and increased inflammation at the study start.
Interestingly, TRAP 5a was negatively associated with bone parameters such as lumbar spine BMD, which further implicate the involvement of TRAP 5a in bone regulation since it has been proposed to function as both positive and negative regulators of, e.g., osteoblasts . However, TRAP 5a did not correlate to most of the bone parameters measured in this study. The role of TRAP 5a as a bone marker is unknown and this study is limited by a rather small number of participants; therefore, it is difficult to conclude the reason and implication for this. Future studies including larger cohorts are necessary to elucidate the possible role of TRAP 5a as a bone marker in patients with AN or other pathologies affecting bone.
TRAP 5b was associated with bone parameters such as CTX, calcaneal BMD and BMC, as well as cortical and trabecular density, in accordance with previous studies , thus further demonstrating that serum TRAP 5b is predominantly derived from bone tissue and reflects the number and activity of osteoclasts. The correlation of ΔTRAP 5b to ΔpQCT trabecular density is additionally consistent with more rapid turnover in trabecular compared to cortical bone. It is difficult to reason why TRAP 5b is correlated to, e.g., calcaneal BMD but not spinal or total BMD. One reason might be the small number of participants and other that there are site-specific differences in osteoclast metabolism. As for TRAP 5a, future studies including larger cohorts are necessary to elucidate the precise role of TRAP 5b as a bone marker in AN or other bone pathologies.
Additionally, this study further suggests that it is important to calculate the TRAP 5a/5b ratio. TRAP 5a/5b ratio correlated negatively to total BMC and calcaneal BMD/BMC. This strengthens the idea that TRAP correlation to BMD and BMC in spine vs calcaneal bone might be a consequence of site-specific differences between spine and calcaneal bone since TRAP 5a/5b ratio correlates with calcaneal but not spine BMC and BMD at week 1. TRAP 5a/5b ratio also correlates to both cortical (positive) and trabecular (negative) density at week 1 implying that TRAP as a bone marker might not only be site specific between different bones entities but also site specific within a bone. Together, the increased number of bone parameters correlating to TRAP 5a/5b ratio compared to TRAP 5b alone suggests that the TRAP 5a/5b ratio could be a more sensitive parameter for bone resorption than TRAP 5b alone indicating that a low TRAP 5a/5b ratio could predict a pathological bone phenotype.
The observed delta values in serum TRAP 5a, between baseline and 12 weeks and TRAP 5a/5b ratio at week 1, were correlated to changes in serum insulin indicating that TRAP 5a is involved in metabolic changes, which is in conjunction with previous reports [33, 36]. However, the exact mechanism by which TRAP 5a responds to metabolic changes and insulin sensitivity/resistance remains to be elucidated. Finally, Δ decrease in TRAP 5b was shown to be correlated to Δ increase in BALP, further suggesting that coupling between bone formation and bone resorption is restored during weight gain treatment of the AN cohort.
The rather small number of participants is a limitation, which makes our results difficult to generalize. However, this 12-week nutrition therapy for AN is highly staff-intensive (with 24-h surveillance), which made it difficult to include further AN patients. The lack of a control group is also a limitation; however, controls were not included because we found it unethical to recruit normal-weight young women that would agree to a diet with the objective of gaining weight in a hospitalized environment for 12 weeks.
In conclusion, patients with AN, who received the described intensive weight gain therapy and increased their BMI and body weight, demonstrated also changes, in opposite directions, for serum TRAP 5a and TRAP 5b toward normal levels. The observed decrease for TRAP 5b was linked to increased bone formation and decreased bone resorption, leading to improved bone mass. The increase for TRAP 5a seems to derive from overall systemic changes in bone as well as metabolic changes but is not directly correlated to expansion of adipose tissue. The combination of serum TRAP 5a and TRAP 5b, as well as the restoration of TRAP 5a/TRAP 5b ratio, could be further indicative of reduced bone resorption and overall improved bone homeostasis after 12-week nutrition therapy. Future studies should aim to verify these results in larger cohorts of AN patients as well as other adipose tissue and bone pathologies to fully understand the role of TRAP isoforms as biomarkers.