Discussion
Menopause and aging are two important causes of osteoporosis ( ACOG Committee on Clinical Practice Guidelines—Gynecology ). The peak bone mass of women is inherently lower than that of men, and coupled with the physiological menopause causing a cliff like decline in estrogen levels, postmenopausal women experience faster bone loss and more severe osteoporosis ( Yuan et al., 2019 ). At present, there have been a few studies on both short-term and long-term effects of plasmapheresis on bone metabolism ( Amrein et al., 2010 ; Sun et al., 2019 ; Dao et al., 2011 ; Chu et al., 2010 ; Bialkowski et al., 2016 ), but little attention has been paid on those at high risk for osteoporosis. There are also a few studies on Chinese whole blood donor and platelet apheresis donors, but none on plasmapheresis donors. These results do indeed indicate that exposure to citrate during the apheresis procedure acutely affects mineral and bone metabolism. But long-term effects on BMD are different. As a group of more than 3.67 million people ( People’s Medical Publishing House, 2019 ), the bone health of Chinese plasmapheresis donors, which the main population is also a risk group of osteoporosis, has raised concerns.

This article is the first survey in China to focus on the long-term effects of plasmapheresis on bone metabolism and BMD in high-risk plasmapheresis donors for osteoporosis, namely postmenopausal women and men over 50 years old. Bone turnover biomarkers (BTM) are products of bone tissue’s own metabolism, representing the dynamics of systemic bone metabolism ( Schini et al., 2023 ). BTM includes bone formation (such as P1NP, osteocalcin) and bone resorption (such as β -CTX) ( Eastell & Szulc, 2017 ). The dynamic balance between osteogenic activity and osteoclast activity is the foundation for maintaining stable bone mass in the body. The bone mass is also regulated by various hormones. Among them, vitamin D, as one of the important hormones for regulating bones, can increase the absorption of calcium in the intestine and increase osteogenic activity. Currently, 25OHD is a good indicator of vitamin D levels in the body ( Delrue & Speeckaert, 2023 ). As one of the three major hormones regulating calcium and phosphorus, PTH plays an important role in the regulation of bone metabolism. In our study, there was no statistically significant difference in BMD at all measurement sites between the donors group and the control group. However, 25OHD and β -CTX levels were significantly lower, PTH and P1NP levels were significantly higher in donor group ( p < 0.05).

The trend of changes in 25OHD, PTH, and P1NP is consistent with the research of Amrein et al. (2010) and Chen et al. (2011) . But interestingly, the level of β -CTX shows the opposite. The decrease in 25OHD promotes an increase in PTH levels. Previous studies have reported that increasing level of vitamin D can decrease secretion of PTH in the body ( Babić Leko et al., 2021 ) and intermittent administration of PTH can promote osteogenic activity in the body ( Li et al., 2019 ). Similarly, Boot et al. (2015) investigated postmenopausal female apheresis donors and found that the BMD of the lumbar spine and hip joints in the donor group was higher than that in the whole blood donor group. The authors analyzed that this may be due to the chronic long-term elevation of PTH caused bone resorption in a long-term effect. Additionally, in our study, the interval from the last plasma donation has no correlation with changes in the indicators after adjusting for confounding factors. The result is consistent with the research of Amrein et al. (2010) , in which most of the bone metabolic indicators returned to normal within one day.

For bone metabolism results, it can be seen that the main influencing factor of bone metabolism related indexes was the frequency of recent plasma donation. When the frequency of recent plasma donation increased to a certain extent ( i.e., more than 17 times/nearly 12 months, note: the current upper limit in China is 24 times/nearly 12 months), the bone resorption and bone formation indicators changed significantly. When the interval between plasma donation was longer than 14 days (the current minimum interval in China), the bone resorption and osteogenesis indicators returned to their baseline levels. When the interval between plasma donations exceeded 28 days, the levels of 25OHD and PTH returned to those before donation. With the increase of the total times of plasma donation, the bone resorption indicators were affected first, and the bone formation indicators were further affected after 100 times of plasma donation.These finds indicated that plasma donation behavior and usage of citrate, to a certain extent, promoted osteogenesis and reduce osteoclast of the body. This may be a stress and self-regulation way for the body to cope with the imbalance of calcium homeostasis and calcium loss, thereby ensuring bone health.

Further, a high rate of vitamin D inadequacy was observed in our study,, similar to national epidemiological data ( Liao et al., 2014 ). Multiple linear regression analysis demonstrated a significant negative correlation between 25OHD levels and recent donation frequency, consistent with Figure 2B . The main form of vitamin D in the blood is its binding to α globulins ( Khazai, Judd & Tangpricha, 2008 ). Due to the loss of plasma protein caused by donation, 25OHD levels reduced as a result. It can also be seen from Figs. 1A and 1B that the level of 25OHD was inversely proportional to the change in PTH. Therefore, it is recommended to provide extensive health education to plasmapheresis donors, and long-term plasmapheresis donors should increase their outdoor activity time and supplement vitamin D appropriately since plasma donation can further increase vitamin D deficiency.

The theory of iron accumulation is one of the mechanisms underlying osteoporosis in recent years. Clinical research observations have shown that the BMD of iron accumulating individuals is lower than that of the general population, and there is a statistically significant difference between the two. Using the Bootstrap method to study the mediating effect, it was found that SF levels partially mediate the relationship between plasma donation behavior and P1NP, with a mediating effect ratio of 21.8%. This suggests that plasma donors can directly affect P1NP through their plasma donation behavior, and can indirectly affect P1NP by affecting SF levels. So we made a hypothesis that plasma donation might improve iron accumulation for individuals at high risk of osteoporosis, thereby improving bone metabolism and alleviating the progression of osteoporosis due to SF’s effect of mediation. However, no evidence was found in our study.

Since plasma donation was found to be significantly associated with changes in bone metabolism parameters in our study, we had high expectations for its further effect on BMD. However, no significant difference in BMD found finally between the two groups. Our results are consistent with the studies of Bialkowski et al. (2019) , Sun et al. (2019) , Xu et al. (2012) , that is, plasmapheresis will not damage the BMD of donors. A prospective study by Grau et al. (2017) also showed that frequent plasmapheresis donation does not increase the risk of fractures. However, Amrein et al. (2010) found that compared to the control group, the lumbar spine BMD in the donor group was lower with statistical differences. This study cannot exclude the influence of genetic, nutritional, or lifestyle risk factors on the results, and there is heterogeneity in donation frequency among the study population, with over 80% of researchers rarely participating in donations (less than once a month).

The above findings, including ours, indicated that several underlying mechanisms may have contributed alone or acted in concert to maintain stable bone mass for donors: repetitive stress to calcium homeostasis induced by citrate exposure, and/or recurring biological factor changes including specific factors potentially important for bone and mineral homeostasis (such as chronic long-term elevation of PTH). The use of anticoagulant in the process of collection and saline infusion for hemodilution may cause hypocalcemia and short-term alterations in bone metabolism, but fortunately, the body can basically return to normal levels in a short time. However, after long-term and frequent donation, even elderly donors at high risk for osteoporosis may cause self-regulation of the body to promote osteogenesis and reduce osteoclast, and ultimately maintaining stable bone mass of donors despite vitamin D level reduction ( Figure 4 ).

This study recruited long-term and highly active plasmapheresis donors at high risk for osteoporosis as study participants ( i.e., postmenopausal women and men over 50 years old, donation times ranged 54.5–196.5), and explored the possible effects of plasma donation behavior indicators on bone metabolism biomarkers from three dimensions: long-term, medium-term and short-term. We also used IOF Osteoporosis Risk 1-Minute Test Questions and appropriate statistical methods to minimize the effect of background confounding. These are complementary and innovative to previous similar studies. However, this article still has the following shortcomings. (1) This article only investigates two plasmapheresis stations and cannot comprehensively reflect the overall situation of the Chinese plasmapheresis population. Further multicenter and larger sample studies are needed. (2) This article is a cross-sectional study, which can only preliminarily explore the correlation between plasmapheresis donation and bone metabolism markers, and cannot conduct further mechanism research, and cannot strictly control the confounding factors, and the evidence for causal inference is low, so further prospective research is needed.

In summary, long-term and frequent plasmapheresis donation does not affect the bone mass of even elderly donors at high risk for osteoporosis under the existing collection standards and anticoagulant use in China. However, as a self-regulator way, it does increase the osteogenic activity of the body. It also recommended that middle-aged and elderly plasmapheresis donors should be appropriately supplemented with vitamin D.