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The Effect of Roux‐en‐Y Gastric Bypass on Bone Marrow Adipose Tissue and Bone Mineral Density in Postmenopausal, Nondiabetic Women

Objectives This study aimed to determine the effect of bariatric surgery‐induced weight loss on bone marrow adipose tissue (BMAT) and bone mineral density (BMD) in postmenopausal, nondiabetic women. Methods A total of 14 postmenopausal, nondiabetic women with obesity who were scheduled for laparoscopic Roux‐en‐Y gastric bypass surgery (RYGB) were included in this study. Vertebral bone marrow fat signal fraction was determined by quantitative chemical shift magnetic resonance imaging, and vertebral volumetric BMD (vBMD) was determined by quantitative computed tomography before surgery and 3 and 12 months after surgery. Data were analyzed by linear mixed model. Results Body weight [mean (SD)] decreased after surgery from 108 (13) kg at baseline to 89 (12) kg at 3 months and 74 (11) kg at 12 months (P < 0.001). BMAT decreased after surgery from 51% (8%) at baseline to 50% (8%) at 3 months and 46% (7%) at 12 months (P = 0.004). vBMD decreased after surgery from 101 (26) mg/cm3 at baseline to 94 (28) mg/cm3 at 3 months (P = 0.003) and 94 (28) mg/cm3 at 12 months (P = 0.035). Changes in BMAT and vBMD were not correlated (ρ = −0.10 and P = 0.75). Calcium and vitamin D concentrations did not change after surgery. Conclusions RYGB decreases both BMAT (after 12 months) and vBMD (both after 3 months and 12 months) in postmenopausal, nondiabetic women. Changes in BMAT and vBMD were not correlated. These findings suggest that BMAT does not contribute to bone loss following RYGB.


Introduction
Obesity is becoming more prevalent in our society, and consequently, there is an increase in the bariatric surgery rate (1). Besides the positive effect of bariatric surgery on obesity and its associated morbidity, bariatric surgery has a negative effect on bone health, decreasing bone mineral density (BMD) and increasing fracture risk (2). Especially in postmenopausal women (who are already at risk of developing osteoporosis and sustaining fractures), the effect of bariatric surgery on bone health needs to be addressed.

Study Importance
What is already known?
► Bariatric surgery is associated with increased fracture risk. ► Bone marrow adipose tissue (BMAT) is associated with low bone mineral density (BMD) and increased fracture risk. ► Studies on the effect of bariatric surgery on BMAT have shown conflicting results, possibly caused by heterogeneous study populations and confounding effects of type 2 diabetes mellitus.
What does this study add?
► We show that, after Roux-en-Y gastric bypass, both BMAT (after 12 months) and volumetric BMD (both after 3 months and 12 months) decrease in postmenopausal, nondiabetic women, and that changes in BMAT and volumetric BMD are not correlated. ► This suggests that BMAT does not contribute to bone loss following gastric bypass surgery in this clinically relevant study population of postmenopausal, nondiabetic women. Obesity www.obesityjournal.org Obesity | VOLUME 29 | NUMBER 7 | JULY 2021 1121 Original Article

CLINICAL TRIALS AND INVESTIGATIONS
High bone marrow adipose tissue (BMAT) is associated with low BMD (3) and vertebral fractures (4), and this latter association exists even independently of bone mass in some studies (5,6). There are several theories on the etiology of the negative association between bone volume and BMAT. First, as bone marrow adipocytes and osteoblasts share a common progenitor, bone marrow stromal cells (7,8), an increase in bone marrow adipocytes would be accompanied by a decrease in osteoblasts and, therefore, bone formation. Second, bone marrow adipocytes secrete fatty acids and adipokines, which could influence bone metabolism in a paracrine manner (9). However, the exact interaction between BMAT and bone in vivo remains elusive.
Recently, interest has risen on the interaction between bone and BMAT during extensive weight loss after bariatric surgery. It was reported that patients suffering from anorexia nervosa have increased BMAT with low bone mass (10), and animal studies have shown more BMAT and less bone during caloric restriction (11,12). So far, studies on the association between vertebral BMAT and bone after bariatric surgery have shown inconsistent results (13)(14)(15). A possible explanation for these inconsistent results could be the heterogeneity of participants included in these studies and the chosen time point for measurement. The studies included pre-and postmenopausal women and patients with and without diabetes. Estrogen has anabolic effects on bone, and estradiol is able to reduce BMAT (16,17). Furthermore, BMAT significantly changes during the menstrual cycle, likely because of effects of sex hormones (17), and BMAT and bone both change substantially after menopause. In addition, patients with type 2 diabetes mellitus have an increased risk of fractures (18), and BMAT seems to be associated with glycemic control (19,20).
Owing to the fact that bariatric surgery affects both the hormonal status and glycemic control of patients, the presence of diabetes before surgery may influence the effect of bariatric surgery on BMAT. Furthermore, in the aforementioned studies, measurements were taken at 6 months after surgery, when patients have achieved approximately 75% of the expected weight loss, whereas other studies performed measurements at 12 months after surgery, when body weight has usually reached its nadir. As the rate of weight loss is the largest within the first 3 months following surgery (i.e., approximately 50% of the expected weight loss), we were also interested in determining changes that occur in a shorter time frame following surgery. Therefore, we aimed to determine the effects of weight loss caused by laparoscopic Roux-en-Y gastric bypass surgery (RYGB) on BMAT and volumetric BMD (vBMD) in postmenopausal, nondiabetic women shortly after RYGB, at 3 months, and after 1 year.

Methods Participants
Participants were recruited from a population of women scheduled for laparoscopic RYGB surgery at the MC Slotervaart in Amsterdam, the Netherlands. All participants were older than 50 years old and were postmenopausal. Exclusion criteria were having diabetes mellitus (defined by either a mentioning of diabetes mellitus in their medical history, taking antidiabetes medication, or having fasting plasma glucose ≥ 7.0 mmol/L or hemoglobin A 1c ≥ 48 mmol/mol) and being on medication known to have an influence on bone metabolism, including estrogens, corticosteroids, and bone-resorption inhibitors. Inclusions took place between June 12, 2015, and July 28, 2017. Four visits to the Amsterdam University Medical Center at the Academic Medical Center location were scheduled for radiological examinations and laboratory tests. Two visits were scheduled before surgery, one between 3 months and 2 weeks before surgery, and one between 2 weeks and 1 day before surgery. Two visits were scheduled after surgery: 3 months after surgery and 1 year after surgery. Primary outcome parameters were changes in vertebral bone marrow fat fraction, which was measured by quantitative chemical shift imaging (QCSI) with magnetic resonance imaging (MRI), and changes in vertebral vBMD, which were measured by quantitative computed tomography (QCT

Quantification of BMAT by QCSI MRI
In order to quantify the bone marrow fat signal fraction (i.e., BMAT), an in-house-developed turbo spin echo-Dixon sequence was used as described previously (17,21). MRI scans were made on a 1.5-T MAGNETOM Avanto (Siemens Healthineers AG, Erlangen, Germany). Five participants had their last QCSI measurement on the upgraded 1.5-T MAGNETOM Avanto fit (Siemens Healthineers AG). The upgraded version of the MRI was cross calibrated by scanning three healthy volunteers (intraclass correlation coefficient = 0.89; P = 0.12). Robustness of the QCSI method was previously tested over four scanner changes (22), and exclusion of the scans made on the upgraded Avanto fit did not change the results. BMAT was measured at the level of the L3-L5 vertebrea in a coronal plane. The Dixon technique is based on the different resonance frequencies of the water and fat protons within the bone marrow, and it measures the fat signal fraction (i.e., the fat signal divided by the sum of the fat and the water signal). The in-phase (IP) image is acquired when the water and fat signal are in phase, and the out-of-phase (OP) image is acquired when the water and fat signals are out of phase. Water-only and fat-only images can be reconstructed from the IP and OP images. Imaging parameters were as follows: repetition time (TR) = 2,500 millseconds, echo time (TE) = 22.3 millseconds, slice thickness = 4 mm, matrix size = 256 × 256, number of excitations = 1, and field of view = 350 × 350 mm 2 with a turbo factor of 4, resulting in an acquisition time of 5 minutes and 20 seconds. Paracoronal IP, OP, and magnitude images were acquired of a single slice through the dorsal third of the vertebral bodies of L3-L5. A midsagittal localizer image was used for slice positioning of the measurement. A small flex coil was used to detect the MR signal for all participants to avoid motion and respiration artifacts. Regions of interest were drawn on the individual vertebral bodies ( Figure 1A), and three erosions were applied to ensure that only the trabecular bone area within the vertebral bodies was included in the regions of interest ( Figure 1B). Complex data and a region-growing algorithm were used to construct a fat signal fraction map ( Figure 1C).

QCT
QCT was used to quantify vBMD within the vertebral bodies (L3-L4) using Mindways QCT Pro Bone Investigational Toolkit (BIT; Mindways Software Inc., Austin, Texas). Images were acquired on a Brilliance CT 64-slice (Philips, Best, the Netherlands), and the CT scanner was calibrated prior to the QCT measurements.

Visceral adipose tissue
Visceral adipose tissue (VAT) was quantified in a single slice at the level of L4, using the CT images acquired for the vBMD assessment. Semiautomated prototype software was used to quantify VAT (Cardiac Risk Assessment package, syngo.via, Siemens Healthineers AG). The internal abdominal wall was traced in a single slice, at the center of L4, and a threshold was set at −50 to −200 Hounsfield units to detect pixels with a value in the adipose tissue range (23).

Biochemical measurements
Morning fasted blood samples were drawn to determine the boneformation marker P1NP and bone-resorption marker CTx. Additionally, calcium, phosphorus, 25OH-vitD, and PTH concentrations were determined. FSH, hemoglobin A 1c , and fasted glucose were determined at baseline. We measured serum concentrations of calcium, phosphate, and PTH on a Hitachi Modular 8000 (C602 and C702; Roche Diagnostics, Mannheim, Germany), and 25OH-vitD was measured using an automated immunoassay (LIAISON analyzer; DiaSorin Inc., Stillwater, Minnesota). Serum concentrations of CTx and P1NP were measured using an immunoassay (Modular Analytics E 170, Roche Diagnostics and Aidian, Espoo, Finland, respectively). Interassay coefficients of variation for CTx and P1NP were 3% and 8%, respectively.

Statistics
This study was powered to detect a difference in bone marrow fat signal fraction of 5% with a variance of 2.4%. A 5% difference is likely to represent a clinically relevant change, owing to the fact that we previously showed that BMAT decreases by 5% after 2 weeks of 17β-estradiol treatment in postmenopausal women (17). In addition, BMAT increases with age by an estimated 7% for each decade (24). The variance is based on our previous study in postmenopausal women (17). The sample size was calculated using nQuery Advisor (version 7.0; GraphPad Software, San Diego, California), and the power analysis was approved by the ethics committee of the Amsterdam UMC. Outlier analysis was performed; significant outliers were excluded from further analysis, but this had no effect on the results. SPSS Statistics for Windows (version 22; IBM Corp., Armonk, New York) was used for statistical analysis. Graphs were constructed using GraphPad Prism (version 7.0). Mean and standard deviations (SD) or median and interquartile ranges (IQR) are reported, depending on the distribution of the data. A linear mixed model was used to determine the changes in the primary outcome parameters over time and to account for missing data. Covariance structure was chosen based on the Akaike information criterion and the Schwarz's Bayesian criterion. We used the Spearman rank correlation test to determine correlations between variables.

Results
A total of 17 postmenopausal, nondiabetic Caucasian women who were scheduled for laparoscopic RYGB were enrolled in this study. Three participants were excluded, as vBMD was incorrectly measured in L1-L2 instead of L3-L4, leaving 14 participants subject to further analysis. One participant retracted informed consent after the first MRI because of physical complaints during the scan (i.e., headache). Two participants missed their second visit because their surgery was rescheduled last minute to an earlier date. One participant had 9 months between her second visit and the surgery, as her RYGB surgery was postponed because of complications of earlier knee replacement surgery.   Figure 2B). Changes in BMAT and changes in body weight at visit 4 compared with visit 1 tended to be positively correlated, possibly suggesting that participants who lost more body weight also lost more BMAT ( Figure 3B; ρ = 0.55 and P = 0.052). BMAT and body weight were not correlated at visit 1 or visit 4 (ρ = 0.43 and P = 0.12; ρ = 0.12 and P = 0.69, respectively).

Discussion
In this prospective longitudinal study, we show that both BMAT and vBMD decrease after RYGB in postmenopausal, nondiabetic women.
BMAT was decreased 12 months after RYGB in this homogenous, clinically relevant population of postmenopausal, nondiabetic female participants. Our results are consistent with a recent study by Blom-Høgestøl and colleagues (25) that showed a decrease in BMAT in iliac crest biopsies of female individuals 1 year after RYGB surgery compared with baseline. However, in the study by Blom-Høgestøl and colleagues, only eight out of eighteen female individuals were postmenopausal, and the study also included individuals with diabetes. Other studies have not shown changes in BMAT following RYGB (13)(14)(15). This lack of change is likely related to the more heterogeneous study populations, including male and female individuals, pre-and postmenopausal women, and individuals both with and without diabetes. Another difference between these studies and our current study is that we used QCSI, whereas other studies (13)(14)(15) used MR spectroscopy to quantify BMAT, which limits direct comparison. MR spectroscopy is considered the gold-standard method for quantification of BMAT; however, good agreement has been shown between QCSI and MR spectroscopy (26)(27)(28).
In addition to a decrease in BMAT, we also show a decrease in vBMD both 3 months and 12 months after RYGB. This implies that the decrease in vBMD precedes the decrease in BMAT. At baseline, BMAT and vBMD showed the well-known inverse relationship that is also present in women with osteoporosis (16,(29)(30)(31). However, 1 year after RYGB, the association between BMAT and vBMD was lost, suggesting that BMAT is not functionally linked to bone mass following RYGB. Weight loss is most rapid within the first 3 months after surgery, which could be a possible explanation for the rapid increase in bone turnover with a negative balance, leading to a decrease in vBMD. The observed increases in bone-turnover markers P1NP and CTx are consistent with previous studies (14,15). Although calcium remained unchanged, we observed an increase in phosphate after RYGB. The latter is most likely related to the catabolic state induced by bariatric surgery and is consistent with previous research (32). Vitamin D and PTH remained unchanged after RYGB, which suggests that the decrease in vBMD is not caused by deficiencies, although we cannot exclude small changes that were not detected in this study that may have had an effect. Interestingly, we did find a positive correlation between changes in BMAT and changes in PTH, whereas BMAT and PTH were not correlated at baseline or after RYGB. Bone marrow adipocytes are responsive to PTH, and intermittent administration of teriparatide decreases BMAT (30,33). However, to our knowledge, we are the first study to report these longitudinal results on changes in BMAT and PTH following RYGB, and further research is necessary to interpret these findings.
Changes in BMAT and changes in body weight tended to be positively associated in our study, which could indicate that participants who lost more weight also lost more BMAT. However, VAT was not associated with BMAT, and changes in VAT were not associated with changes in BMAT. These results are consistent with previous research which has shown no association between VAT and BMAT in a group of individuals with morbid obesity who were scheduled for bariatric surgery (20) and in a group of postmenopausal, nondiabetic women with obesity (19). However, other studies have shown an association between VAT and BMAT in a group of premenopausal women (34), as well as changes in BMAT and changes in VAT following RYGB (14). Hormonal status and glycemic control in these individuals could possibly explain these differences because both are associated with changes in BMAT, as well as changes in body composition and VAT (35). Furthermore, we did not find a negative association between VAT and vBMD as described previously (36,37). A possible explanation could be the relatively small range in VAT in our participants, as morbid obesity is the indication for Our participant lost some weight prior to surgery, as they were advised to lose weight if possible, with the guidance of a dietician as part of the program and to reduce the risks of surgery. However, we observed no changes in BMAT and BMD between the baseline measurements prior to surgery. Our results are in line with a short-term diet interventional study by Cordes and colleagues that showed no changes in BMAT after 4 weeks of diet intervention (38). However, our results are inconsistent with a longer study of 12-week-long dietinduced weight loss that showed a decrease in BMAT and an increase in BMD (39) but no relationship between changes in BMAT and changes in BMD. The discrepancy with our study is likely related to the shorter interval (59 d) and limited weight loss (3 kg) between the baseline visits in our study.
As aforementioned, the baseline data confirm the well-known inverse association between BMAT and vBMD. However, after RYGB, BMAT and vBMD were no longer associated. Furthermore, we did not find an association between changes in BMAT and changes in vBMD. These findings implicate that changes in BMAT and bone metabolism following RYGB are different from changes following administration of estrogen (16), teriparatide (29,30), or bisphosphonates (31), in which there is an association between decreases in BMAT and increases in vBMD. Decreases in both BMAT and BMD are also described in animal studies on lactating mice (40) and in mice after cold exposure (41). A possible explanation could be that the effects of bariatric surgery on bone and BMAT separately are stronger than the effects of BMAT on bone, and this might also be the case for lactation and cold exposure.
Our study has several strengths and limitations. We used QCSI to measure bone marrow adiposity, whereas other studies have used MR spectroscopy. The QCSI method is well validated and considered the gold standard for quantification of bone marrow fat in patients with Gaucher disease (22). The QCSI method measures the fat signal fraction, as it does not correct for T1 and T2 effects. However, T2 effects are minimized by using a short echo time (23 ms), and T1 bias is minimized by the long repetition time of the sequence (2,500 ms). A limitation of our study is that we did not include a control group. Therefore, we cannot distinguish between the effects of bariatric surgery and weight loss per se. We performed two baseline measurements to show that vBMD and BMAT were stable before surgery. Therefore, the effects observed after surgery are more likely to be caused by surgery and weight loss, and not by other factors. Calcium carbonate was used as calcium supplement. Calcium carbonate is best absorbed in an acidic environment, and absorption is possibly lower after RYGB (42,43), although other studies have shown no difference between calcium carbonate and calcium citrate supplementation on PTH and BMD after RYGB (44,45). Generalizability of our results is limited, as we only included Caucasian women. Finally, our study has a relatively small sample size, but we included only postmenopausal women without diabetes to exclude effects of concurrent changes in estrogen status and glucose homeostasis on BMAT. Other studies on the effects of bariatric surgery on BMAT (13)(14)(15)25) included lower numbers of postmenopausal, nondiabetic participants, often too small for subgroup analysis, which makes our study the first, to our knowledge, to report BMAT and vBMD changes in this clinically relevant study population.

Conclusion
RYGB decreases BMAT 12 months after surgery and decreases vBMD both 3 months and 12 months after surgery in postmenopausal, nondiabetic women, and changes in BMAT and vBMD are not correlated. These findings suggest that BMAT does not contribute to bone loss following gastric bypass surgery.O