Bone metabolism changes after laparoscopic greater curvature plication . A one-year study

In addition to the positive effect of weight loosing after bariatric surgery, the post-operative period is often associated with signifi cant changes in bone metabolism. Aim. The aim of this study was to examine changes in bone turnover markers and indicators of dual-energy X-ray absorptiometry (DXA) within 12 months after laparoscopic greater curvature plication (LGCP). Materials and methods. We studied 54 patients from class III obesity who underwent LGCP. The average preoperative weight of the patients was 125.5±19.1 kg, and BMI of 43.0±4.9 kg/m2. The gender distribution of the cohort was 35.2% man and 64.8% women. At three time points after the operation (at 3, 6 and 12 months) control examination was conducted that included DXA and markers of bone metabolism. Results. After the surgical treatment we registered signifi cant reduction of all the anthropometric indicators (ρ<0.05; EWL12 45.8±18.8%; EBL12 55.1±23.4%). According to the controlled studies on the 12th months, a signifi cant increase in bone turnover markers was revealed (ρ<0.05; CTX, P1NP, OC, OPG, VTD) and reducing of PTH (ρ<0.05). The measure of DXA on the 12th month of observation showed no signifi cant changes in bone metabolism. Strong positive correlation was uncovered between markers of bone metabolism: CTX / P1NP (r=0.790, p=0.000), CTX/ OC, P1NP/OC (r=0.7, p=0.000), minor correlation between PTH/OC (r=0.249, p=0.027). We also report a minor and weak negative correlation between the markers of bone metabolism (CTX, P1NP, OC and VTD) and anthropometric data. Conclusions. Twelve months post LGCP markers of bone turnover (CTX, P1NP, OC, OPG, VTD) were signifi cantly increased. The increased level of VTD is associated with the loss of FAT. Also the change in DXA was detected.

Висновки.Через  D espite the progress in the prevention and treatment obe- sity, in 2014 more than 1.9 billion adults (18 years and older) were overweight, 600 million of whom were obese [1].According to the literature review, the incidence of bariatric surgery has plateaued at approximately 200,000 cases per year since 2002 [2].
Nowadays, the most common bariatric operations are laparoscopic adjustable gastric banding, laparoscopic Roux-en-Y gastric bypass, and laparoscopic sleeve gastrectomy.Such operations are quite effective at signifi cantly reducing the body mass and are associated with low rates of surgical complications [3,4].Laparoscopic greater curvature plication (LGCP) is a new method of surgical treatment for obesity and was proposed in 2007 by Talebpour et al. [5].It belongs to the «safe organ preserving surgery», has a high effi ciency rate and is commonly chosen for treating extreme cases of obesity.However, issues with changes in bone metabolism have recently been brought to light in association to bariatric surgery [6][7][8][9].
Multiple groups reported that after bariatric surgeries most patients achieved a 50% weight loss with a proportionately greater loss of bone mass [10,11].The mechanisms of bone loss after bariatric surgery are currently unknown, but are likely multifactorial.Secondary hyperparathyroidism due to vitamin D defi ciency and mechanical unloading due to weight loss are among the most commonly cited potential mechanisms underlying bone loss after bariatric surgery.Yet, evidence from the literature does not support these hypotheses.New hypotheses involving crosstalk between the skeleton and gastrointestinal, adipocytic and neurohormonal systems are now being explored [6].In contrast, other authors attribute changes in bone metabolism to the effects of anatomical changes in the stomach, suggesting that anatomic changes in the gastrointestinal tract lead to malabsorption of nutrients, including calcium and vitamin D [12][13][14].
Cong Liu et al. (2015) conducted a meta-analysis of bone changes following bariatric surgery, and identifi ed signifi cant decrease in serum calcium, increase in serum parathyroid hormone (PTH), but no difference in serum vitamin D 25(OH)D (VTD).In addition, they reported signifi cant increase in serum or urinary N-telopeptide and in bone -specifi c alkaline phosphatase, accompanied by a decrease in bone mineral density (BMD) [8].
This time the most informative method of determining BMD is Dual-energy X-ray absorptiometry (DXA).The advantages of DXA are its high sensitivity to osteoporosis.In most studies dedicated to bone metabolism, investigations of BMD were made by DXA, and also by special markers of bone metabolism [7,8,15].
The aim of this study was to analyze possible postoperative changes in blood markers of bone metabolism (including vitamin D (25-OH-D), PTH) and DXA result during a 1-year period (3-6-12 month) after LGCP.

Material and Methods
We studied 54 patients from class III obesity [16] who underwent LGCP.We classifi ed participants according to IFSO 2005 guidelines [17].Thus, patients with a body mass index (BMI) > 40 kg/m 2 or BMI > 35 kg/m 2 with associated co-morbidities were included into the study.The study included patients, aged 20 to 58 years, referred for laparoscopic gastric plication to the old surgical ward Care Center digestive tract Vitkovice Hospital in Ostrava and the Center for Endoscopy Clinic of Internal Medicine University Hospital Ostrava.LGCP was performed according to the usual protocol -general anesthesia and a laparoscopic approach with fi ve ports.The ports were placed in the abdomen according to a standard schema.The plication was standard with endoscopic observation.The percent of excess weight loss (EWL) was calculated using the formula described by Deitel and Greenstein [18].
At time points of 3, 6 and 12 months after the surgery, supervisory examinations were performed on all study participants.Control examination included: specialized surgical examination at the surgical clinic, DXA, bioimmunoassay (BIA) and blood sampling on basic and specialized examinations (C-terminal telopeptide of collagen I (CTX), parathyroid hormone (PTH), the serum receptor-activator of nuclear factor-kB ligand (sRANKL), N-terminal procollagen type 1 (P1NP), Osteocalcin (OC), Osteoprotegerin (OPG) and vitamin D (25-OH-D) (VTD) levels).Therefore, to assess bone metabolism, the entire cluster parameters of bone remodeling were evaluated.These parameters included both parameters of osteoresorption (bone resorption), CTX, PTH, and sRANKL and parameters of bone osteoformation (bone formation), P1NP, OC, and OPG.Also, the level of hormones Ghrelin, Leptin, and Adiponectin were studied before and after operation at 3, 6 and 12 months.
Method DXA was performed on Discovery apparatus (Hollogic, Inc., USA) and a whole-body scan was done on body composition density and bone tissue in the femoral neck and   vertebral segments L1-L5 within the sagittal projection in the laboratory of the Institute of Clinical Physiology, Medical Physiology OU.BMD in g/cm 2 and then the value of T and the Z score were assessed.The measured bone mineral density was automatically compared to the average value in young healthy individuals of the same sex (ie.T-score) and the average for the same age category (ie.Z-score).This comparison was expressed in standard deviations from the mean.Osteoporosis was defi ned as the condition where bone loss exceeds -2.5 standard deviations (as determined by the World Health Organization).Decrease of -1 to -2.5 standard deviations is considered osteopenia, which is not yet a disease, but signifi es a greater risk of osteoporosis in the future.Also, using the DXA we have received the results of fat mass -Fat DXA in % and in kg and lean body mass (LBM) -it is a total lean mass -in kg.Statistical Analysis All analyses were performed using the Statistica program STATISTICA 7.0 for Windows (StatSoft.Inc., США).The distribution of analyzed data was checked by the Shapiro-Wilk test.Values were expressed as means ± standard deviation (SD) and for not normally distributed represented by Me (median) and lower/upper quartile (25-75%).Upon the comparison of the groups, before and after the treatment (3-6-12 months), we used parametric statistics, t-test for dependent variables, and nonparametric statistics, Wilcoxon singed-ranked test.Pearson test was performed to evaluate associations between variables.The estimated parameters were considered as signifi cant at r<0.05.
The average age of the patients was 41.3±7.9 years (men 46.5±8.7,women 38.5±5.9).The average preoperative weight of the patients was 125.5±19.1 kg, and BMI of 43.0±4.9kg/m 2 .This is consistent with the BMI of the III class obesity patient [17].During the study period, patients didn't take any specifi c therapy to improve their bone metabolism.
Based on DXA analysis, initial average weight of the patients was 125.5±19.1 kg.Under dynamic observation the weight went down to -16.7 kg over the period of 3 months, to 22.1 kg -over the 6 th months and to 25.0 kg over the 12 months respectively.Table 1 presents the dynamic of weight loss.This result implies that on the 3 rd month, the average weight of the participants was within class II of obesity, and on the 6th and the 12th months -within class I.The fi nal EWL was 45.8%, which signifi es the effectiveness of surgical treatment based on the results of the 12-month period.As the «Fat DXA» and LBM assays indicated, there was a signifi cant loss of the fat mass, nearly 19.4 kg and the muscle loss of 5.2 kg during the fi rst three months, with a signifi cant decrease during the following 12th months.
Parameters of bone osteoresorption and bone osteoformation are presented in Table 2.The initial fi gures of CTX were within reference values (N = 0.142-0.55μg/l) and on the 3 rd , 6 th , 12 th months there was a signifi cant increase of CTX with the fi nal fi gure of 0.38 μg/l after the 12 th month of medical supervision.PTH level on the 3 rd month after surgery decreased signifi cantly to 3.8 pmmol/l.Changes of sRANKL (N=300-380 mmol/l) during the observation period were not authentic and after surgery ranged within 96.9 -122.0 pmmol/l.Before the surgery sRANKL results were below normal values.It is noteworthy   to mention a signifi cant increase in P1NP on the 3 rd , 6 th and 12 th post-surgery months, however the values stayed within the reference range (N=5-58.6 μg/l).Level of osteocalcin (OC) on the 12 th month of supervision was 19.2 μg/l, which was a signifi cant increase with respect to the preoperative period, and was within normal parameters (11-42.5 μg/l).There were no notable changes in the level of OPG, but from the 3 rd month there was a signifi cant decrease, and at the 12 th month an increase, the fi nal result was 5.2 pmmol/l at the reference value of 1.8-6.4pmmol/l.VTD level has grown signifi cantly by the end of the supervision period and reached 30.5 nmol/l.All patients included in the study underwent a densitometry testing and DXA at the femoral neck and lumbar spine of the bone (Table 3).Based on the statistical analysis (Mann-Whitney U test and t-test) and the comparisons of DXA studies results, there were no clinically signifi cant changes in DXA before and after surgical treatment of patients.
Thus, our study revealed signifi cant changes in the following markers of bone metabolism: CTX, PTH, P1NP, OC, OPG and VTD, and also anthropometric indicators.To establish the dependence of changes we conducted a correlation between the markers of bone metabolism and anthropometry (Table 4).Positive correlation was observed between the OC and CTX/PTH/ P1NP, between CTX and P1NP as well as between the OS and BMI/FAT.In other cases, there was a negative correlation: VTD and PTH, BMI/FAT; between P1NP and FAT; between CTX and BMI/FAT.There was no correlation between other parameters.

Discussion
In the current study all LGCP patients attained effective weight loss -EWL on the 12 th month after surgery was 45.8±18.8%,EBL -55.1±23.4%.These fi gures confi rm the effectiveness of the operation.There were no complications or mortalities after the surgery.
However, we found signifi cant changes in bone metabolism according to the performed assays -serum bone turnover markers.Currently, serum-CTX and P1NP assays are recommended osteoporosis diagnostics, and to the level of bone turnover determination [19,20].CTX and P1NP assays are specifi c markers of bone metabolism and their increasing levels can denote osteoporosis or osteopenia.In our study, we found a signifi cant increase of CTX and P1NP levels from the 3 rd postoperative month, but it was within the reference values.According to the analysis of correlation a strong positive dependence of these parameters on each other was revealed (r=0.790,p=0.000).The increase of P1NP and CTX levels strongly correlated with increased content of OC in serum.CTX and P1NP showed slightly negative correlation with BMI and FAT.This proved that the decrease in body weight was accompanied by increase in blood levels of CTX and P1NP.This pattern was more signifi cant between CTX and FAT -during the loss of fat mass an increase of CTX occurred.This might indicate a potential development and progression of osteoporosis after 12 months post-surgery.
Most studies have reported that increased levels of OPG and sRANKL are characteristic of osteoporosis [21][22][23].In observing the patients within 12 th month, we have found increased level of OPG (p 12 =0.015)and decreased sRANKL p 12 =0.84.Such changes are typical for osteoresorption.Taken into consideration a signifi cant change in OPG, we didn't fi nd any dependence in its growth and any of the analyzed parameters (no correlation).
OC is synthesized by osteoblast cells in the late maturation stage, and when it enters the blood, OC behaves as a hormone that has effect on various cells including fat cells.OC levels are related to increase in bone turnover [24].Signifi cant OC levels increase were seen at the the 3 rd month of the post-operative observation.According to the result for the correlation analysis, this increase was related to decrease in BMI and FAT (weak negative correlation).
According to the literature, the level of PTH in the blood after bariatric surgery can change in the direction of its decrease or increase [25][26][27].In our study, we note a signifi cant decrease in PTH levels starting from the 3rd month of observation, which is in weak negative correlation with VTD, BMI and FAT.The changes of VTD were signifi cant from the fi rst control month till the end of the observation period.The growth of VTD was remarked.Also, this hypothesis is supported by the works of various authors [28], suggesting that the increase of the VTD is associated with a signifi cant decrease in the volume of adipose tissue in patients, because adipose tissue is a depot for VTD [29].
In our study, we observed slight correlation with the loss of fat mass and with an increase of VTD level, which is confi rmed by the increase in VTD that accompanies weight loss.Thus, the above-revealed changes in serum bone turnover markers are likely to be associated with a significant and clinically apparent loss of total body mass (p 12 =0.0004;EWL 12 % 45.8±18.8;EBL 12 % 55.1±23.4).As the result, there is a reduction of unloading on a skeleton, which leads to a signifi cant, but not clinically important, increase of CTX and P1NP levels.Since the changes in these indicators were not clinically signifi cant in our sample, this has not led to an increase of sRANKL and OPG, and to the changes of DXA results.
Conclusions.Twelve months post LGCP markers of bone turnover (CTX, P1NP, OC, OPG, VTD) were signifi cantly increased.The increased level of VTD is associated with the loss of FAT.Also the change in DXA was detected.
Confl icts of Interest: authors have no confl ict of interests.