Journal of Investigative Surgery, 28, 8–17, 2015 C 2015 Informa Healthcare USA, Inc. Copyright ISSN: 0894-1939 print / 1521-0553 online DOI: 10.3109/08941939.2014.943857
ORIGINAL ARTICLE
Bsm1 Vitamin D Receptor Polymorphism and Calcium Homeostasis Following Bariatric Surgery
J Invest Surg Downloaded from informahealthcare.com by Selcuk Universitesi on 01/22/15 For personal use only.
Andreas Alexandrou, MD, PhD,1 Eleni Armeni, MD, PhD,2 George Kaparos, PhD,3 Demetrios Rizos, PhD,3 Evangelia Tsoka, MD,2 Efthymios Deligeoroglou, MD, PhD,2 Maria Creatsa, MD, PhD,2 Areti Augoulea, MD, PhD,2 Theodoros Diamantis, MD, PhD,1 Irene Lambrinoudaki, MD, PhD2 1
1st Department of Surgery, University of Athens, Laiko Athens General Hospital, Athens, Greece 2 2nd Department of Obstetrics and Gynecology, University of Athens, Aretaieio Hospital, Athens, Greece 3 Biochemical and Hormonal Laboratory, University of Athens, Aretaieio Hospital, Athens, Greece
ABSTRACT Purpose/Aim: To evaluate the association between the Bsm1 vitamin D receptor polymorphism and the calciumvitamin D-parathormone axis following bariatric surgery. Materials and Methods: This cross-sectional study included 86 morbidly obese patients, who underwent either gastric bypass or sleeve gastrectomy, with a mean follow-up of four years. Calcium metabolism indices and bone turnover markers were assessed according to the presence of secondary hyperparathyroidism and the Bsm1 vitamin D receptor genotypes. Results: Secondary hyperparathyroidism (42.2% of sample) was associated with lower levels of 25hydroxyvitamin D and elevated markers of bone turnover. In subjects without secondary hyperparathyroidism, presence of the unfavorable B allele resulted in higher levels of parathormone (Bb and BB vs. bb genotype: 50.3 ± 8.2 pg/dl vs. 44.4 ± 10.7 pg/dl, p = .011, adjusted for weight loss, baseline body mass index, 25hydroxyvitamin D, surgical procedure, and duration after surgery). In the whole sample, patients bearing the unfavorable B allele exhibited lower weight loss, a parameter that was negatively associated with markers of bone resorption. Conclusions: Secondary hyperparathyroidism is highly prevalent after bariatric surgery. Bsm1 vitamin D receptor polymorphism may have an effect in early stages of calcium metabolism imbalance, while no association is detected in patients who have already developed secondary hyperparathyroidism. Moreover, vitamin D receptor polymorphism is associated with post-surgery weight loss, a process related to bone turnover. Keywords: Bsm1 VDR polymorphism; parathormone; calcium metabolism; bariatric surgery; Roux-en-Y Gastric bypass; sleeve gastrectomy
INTRODUCTION
loss is associated with 1–2% bone loss at various sites of the skeleton, especially after procedures with a malabsorptive component, like Roux-en-Y Gastric bypass (RYGB) [3, 7]. Post-operative deficiencies of calcium and vitamin D lead frequently to secondary hyperparathyroidism (SHPT), which further contributes to skeletal abnormalities [3, 5, 8]. Given the high frequency of vitamin D deficiency and SHPT after bariatric surgery [9, 10], we decided to investigate whether the Bsm1 restriction fragment length polymorphism of vitamin D receptor (VDR) is associated with the regulation of the
Bariatric surgery remains the most effective means of treatment of morbid obesity, with benefits ranging from pure weight reduction up to a significant improvement in the overall quality of life [1–4]. However, metabolic as well as microbiological and histological modifications of the remnant stomach have been associated with disturbed calcium homeostasis [1, 3, 5, 6]. In fact, bone loss is a significant metabolic complication of bariatric surgery, requiring appropriate therapeutic interventions [1, 3, 7]. Studies indicate that up to 10% weight Received 17 April 2014; accepted 8 July 2014.
Address correspondence to Irene Lambrinoudaki, Associate Professor, 2nd Department of Obstetrics and Gynecology, University of Athens, Aretaieio Hospital 27, Themistokleous Street, Dionysos, GR-14578, Athens, Greece. E-mail: [email protected]
8
Bsm1 VDR Polymorphism and Bariatric Surgery calcium-vitamin D-parathormone (PTH) axis and bone turnover after bariatric surgery. More specifically we sought to investigate whether the presence of the unfavorable B allele is associated with higher levels of PTH or lower levels of calcium (1) in patients with SHPT and (2) in patients with normal serum PTH.
J Invest Surg Downloaded from informahealthcare.com by Selcuk Universitesi on 01/22/15 For personal use only.
METHODS This study was a cross-sectional analysis of 87 patients who had undergone gastric bypass or gastric sleeve surgery (SG). Our Department serves as a referral Center for bariatric patients from Central and Southern Greece. Patients who undergo bariatric surgery return to their permanent residence, with recommendations for adherence to multivitamin supplementation and for at least two follow-up visits to their local GP. A phonecall invitation was made to 226 patients who were operated in our Department from 2000 until 2011. Sixty-one patients were lost to follow-up. From the 165 patients who were located by phone, 87 patients consented to participate in the study (response rate 52.7%), consisting a total sample of 69 women and 18 men. Serum PTH was not available in four patients. Eligibility criteria for surgery were a body mass index (BMI) ≥ 40 kg/m2 or a BMI ≥ 35 kg/m2 in patients with obesity-related co-morbidities, failure of previous attempts at weight reduction, and expected adherence to post-operative care (e.g., follow-up visits, use of dietary supplements, etc.). Exclusion criteria were the following: current drug or alcohol abuse, uncontrolled or severe psychiatric illness, reversible endocrine or other disorders that can cause obesity, pre-operative gut malabsorption syndrome, presence of any gastric–kidney–liver disease, and pre-operative use of medication affecting bone metabolism (e.g., calcium and vitamin D supplements, glucocorticoids, diuretics, antiepileptic drugs). Surgical techniques in the bariatric unit of the 1st Department of Surgery of the University of Athens had already been standardized before the beginning of this study and remained the same throughout the whole study period. Gastric bypass was performed laparoscopically by constructing a gastric pouch of approximately 30 ml, an alimentary limb with a length of 150 cm, and a biliopancreatic limb of 100 cm with the use of the harmonic scalpel and the Echelon 60 mm linear stapler (Ethicon). The gastrojejunal anastomosis was constructed in a semi-manual way. The posterior wall was constructed with the use of the linear stapler while the anterior wall was sutured manually. The width of the anastomosis was approximately 1 cm. On the other hand, laparoscopic sleeve gastrectomy was performed with the use of the harmonic scalpel for the devascularization of the greater gastric curvature and the stomach was longitudinally divided with the use of the Echelon 60 mm linear stapler (Ethicon). The cal C
2015 Informa Healthcare USA, Inc.
9
ibration of the gastric sleeve was performed with the use of the 9.1 mm endoscope, instead of the commonly used bougies of various diameter, as we have previously reported [11]. All operations were performed under general endotracheal anesthesia. No analgesics were administered before surgery. Intraoperatively patients received Midazolam 5 mg iv (Dormicum HCl, Roche), Xylocaine 40–60 mg (Astra-Zeneca), Fentanyl 100–300 mcg iv (Fentanyl, Sandoz), Propofol 400 mg iv (Propofol, Fresenius), Suxamethonium HCl 100–150 mg iv (Lycitrope, Cooper), Rocuronium Bromide 100–160 mg iv, (Esmeron, Organon), Omeprazole 40 mg iv (Losec, Astra-Zeneca), Ondasetron 8 mg iv (Onda, Medicus), Apotel 1 g iv (Paracetamol), Parecoxib Sodium 40 mg (Dynastat, Pfizer), Mefoxitin 2 g iv (Mefoxil, Vianex), Morphine 5 mg (Morphine, Lavipharm), Citaloprax HCl 20 mg iv (Seropram, Lumbeck), Tramadol 100 mg iv (Tramal, Medicus), and in case of hypertension Furosemide 10–40 mg iv (Lasix, Sandoz) and Clonidine (Catapresan, Boehringer Ingelheim Greece). The average anesthesia time for a sleeve gastrectomy was 75–90 min while the respective duration of anesthesia for a gastric bypass was 200 min. The patients were regularly transferred to the common surgical ward in the immediate post-operative period having a nasogastric tube for the first two post-op days, while no drains were left in the abdominal cavity. Post-operatively all patients received appropriate doses of analgesics, namely, paracetamol 1 g iv, lornoxicam 4 mg iv (Xefo), or meperidine 4 mg iv (Pethidine), according to individual needs. Low molecular weight heparin was administered to all patients as prophylaxis against thromboembolism, namely, enoxaparine 60 mg (Clexane, Abbott) or fondaparinux 2.5 mg subcutaneously (Arixtra, Glaxo) for one month. Antibiotics were continued for the two first post-op days and were always the same that were administered intraoperatively. If Mefoxitine was given during anaesthesia, it was continued as Mefoxil 2 g three times daily iv, if ampicilline-sulbactame (Begalin 1.2 g, Pfizer) was given, it was continued 1.2 g three times daily. All patients also received inhalations with ipratroprium bromide (Atrovent, Boehringer Ingelheim) four times daily and Budesonide (Pulmicort, Astra-Zeneca) twice daily until they were ready to be discharged. Finally, IV omeprazole 20 mg (Losec, Astra-Zeneca) was administered twice daily iv during the first post-operative days and subsequently 20 mg twice per os daily for one month. An upper GI series was performed on the second post-op day and the nasogastric tube was removed. Liquid oral diet was resumed the next day and the patients were discharged on the fourth post-op day. Patients were allowed only liquids for three weeks and strictly only pureed diet until the completion of the first two post-op months. Upon completion of the first post-op month, during their first appointment in the devoted bariatric outpatient clinic, patients were advised to follow a
J Invest Surg Downloaded from informahealthcare.com by Selcuk Universitesi on 01/22/15 For personal use only.
10
A. Alexandrou et al.
high calcium diet (1000 mg/day), as well as to take chewable tables of 500 mg calcium (500 mg × 2 daily) and vitamin D3 (400 IU × 2 daily) (1). Moreover, patients were asked to present at 6 and 12 months after surgery, for follow-up evaluation (1), while subjects diagnosed with vitamin D deficiency were advised to take 5,000 IU of oral vitamin D daily and to be reassessed three months later [12]. Anthropometric data, demographic, and life-style parameters, as well as a detailed medical history were recorded for every subject, using a questionnaire composed for the present study (supplement 1). Data on pre-surgery weight and on weight loss rate were retrospectively derived from patient files, whereas biochemical data, genotyping as well as up-to-date information on demographic and lifestyle parameters were collected during the study. Anthropometric parameters included waist to hip ratio and BMI, using the equation BMI = weight (kg)/height2 (m2 ). Patients were instructed to abstain from food and smoking for 12 hr, and subsequently, fasting venous blood samples were drawn for biochemical evaluation. According to serum PTH levels, subjects were divided in two subsamples: (1) subjects with SHPT (serum PTH above the high normal limit: 62 pg/ml) and (2) subjects with normal levels of PTH, serving as controls [13]. Vitamin D deficiency was defined as serum levels ≤10 ng/ml, while serum levels of vitamin D ≤20 ng/ml were defined as suboptimal [14]. Weight loss was expressed as percentage (%) of excess weight loss (%EWL). Excess weight was estimated using the middle of the Metropolitan Life Insurance tables [15]. Excess BMI was defined as the difference between the BMI of the subject at followup from the cut-off 25 kg/m2 . All patients signed an informed consent and the study was approved by the Ethics Committee of Aretaieion Hospital, University of Athens.
type 1 amino-terminal propeptide (P1NP) and osteocalcin as well as markers of bone resorption, namely, Ctelopeptide. The reference range was as follows: P1NP 14–76 ng/ml; C-telopeptide 100–1000 pg/ml; osteocalcin 15–46 ng/ml. Homeostasis model assessment of insulin resistance was calculated as HOMA-IR = insulin (μIU/ml)∗ Glucose (mg/dl)/405.
VDR Genotyping Blood samples were drawn by atraumatic venipuncture into trisodium circle tubes. Genomic DNA was isolated from leukocyte nuclides using High Pure PCR Template Preparation kit (Roche). Real-time polymerase chain reaction (PCR) was performed in capillaries with a reaction volume of 10 μl, containing 2 μl of DNA (50–100 ng), 0.3 μM sense primer VDR bF (TAG GGG GGA TTC TGA GGA ACT A) and antisense primer VDR bsm A (AGT TTT GTA CCC TGC CCG C), 1.4 μl 25 mM MgCl2 , 1 μl H2 O, 1 μl of Light Cycler DNA Master HybProbe (ready to use reactionmix for PCR containing Taq DNA polymerase, reaction buffer, dNTP mix with dUTP instead of dTTP and 10 mM MgCl2 ), 0.6 μM Sensor b, 3’ modified with fluorescein (AGT ATT GGG AAT GCG CAG GCC-FL) and R Red 0.6 μM Anchor b 5’ labeled with Light Cycler 640 and 3’ terminally blocked by phosphorylation (Red 640-TCT GTG GCC CCA GGA ACC CTG-pH). Sensor b covers the BsmI restriction site. The letters “b” and “B” correspond to the common and the variant allele, respectively. Subsequently, the polymorphism was defined as BB (absence of restriction site on both alleles), Bb (heterozygous), and bb (presence of restriction site on both alleles).
Statistical Analysis Biochemical Assays Serum glucose, total cholesterol, triglycerides, and HDL-cholesterol were assessed enzymatically by an autoanalyzer (ARCHITECT-ci8200, Abbott Diagnostics Laboratories, Abbott Park, IL 60064 USA, Abbott 65205, Wiesbaden, Germany). The Friedewald equation (LDL-cholesterol = total cholesterol − triglycerides/5 − HDL-cholesterol) was used to estimate LDL-cholesterol. Creatinine levels were determined by an enzymatic method. Insulin was measured on an Abbott Architect i1000 analyzer. The total CV% ranged from 1.9% to 5.2%, and the analytical sensitivity was 1 μU/ml. Serum levels of total calcium, 25hydroxyvitamin D, and parathormone (PTH) were evaluated using standard commercially available methodologies. Electrochemiluminescence immunoassay method (Elecsys2010 analyzer, Roche) was used to evaluate markers of bone formation, namely, serum levels of procollagen
Data analysis was performed using SPSS version 17.0. (SPSS, Chicago, IL, USA). Data on qualitative characteristics are expressed as percent values; while data on quantitative characteristics are expressed as means ± SD. Non-parametric tests were used in cases of deviation from normal distribution. Differences between continuous variables across the VDR genotype were assessed using analysis of variance (ANOVA) and Mann–Whitney U test as well as Chi-square (X2 ) analysis and analysis of covariance (ANCOVA). Potential correlations between quantitative parameters were evaluated using Spearman’s correlation coefficient, unadjusted as well as adjusted for potential confounding factors. Subsequently, we analyzed the potential effect of the presence of the unfavorable B allele, in subjects with SHPT and in controls, separately. Statistical significance was set at the 0.05 level. The predefined sample size (N = 87) provided statistical power of 70% for detecting differences within the Journal of Investigative Surgery
Bsm1 VDR Polymorphism and Bariatric Surgery different genotypes of the Bsm1 VDR polymorphism. Type I error was predefined at 0.05 for all power calculations. GPower (version 3.0.10) was used for power estimation [16].
J Invest Surg Downloaded from informahealthcare.com by Selcuk Universitesi on 01/22/15 For personal use only.
RESULTS At the time of the study, the mean follow-up was 3.9 years, median was 3.0 years, and the range was 1–12 years. 42.2% of the total sample had PTH values above the high normal limit (62 pg/ml) and were classified as having SHPT. 91.8% of the whole sample had suboptimal levels of 25hydroxyvitamin D (
ORIGINAL ARTICLE
Bsm1 Vitamin D Receptor Polymorphism and Calcium Homeostasis Following Bariatric Surgery
J Invest Surg Downloaded from informahealthcare.com by Selcuk Universitesi on 01/22/15 For personal use only.
Andreas Alexandrou, MD, PhD,1 Eleni Armeni, MD, PhD,2 George Kaparos, PhD,3 Demetrios Rizos, PhD,3 Evangelia Tsoka, MD,2 Efthymios Deligeoroglou, MD, PhD,2 Maria Creatsa, MD, PhD,2 Areti Augoulea, MD, PhD,2 Theodoros Diamantis, MD, PhD,1 Irene Lambrinoudaki, MD, PhD2 1
1st Department of Surgery, University of Athens, Laiko Athens General Hospital, Athens, Greece 2 2nd Department of Obstetrics and Gynecology, University of Athens, Aretaieio Hospital, Athens, Greece 3 Biochemical and Hormonal Laboratory, University of Athens, Aretaieio Hospital, Athens, Greece
ABSTRACT Purpose/Aim: To evaluate the association between the Bsm1 vitamin D receptor polymorphism and the calciumvitamin D-parathormone axis following bariatric surgery. Materials and Methods: This cross-sectional study included 86 morbidly obese patients, who underwent either gastric bypass or sleeve gastrectomy, with a mean follow-up of four years. Calcium metabolism indices and bone turnover markers were assessed according to the presence of secondary hyperparathyroidism and the Bsm1 vitamin D receptor genotypes. Results: Secondary hyperparathyroidism (42.2% of sample) was associated with lower levels of 25hydroxyvitamin D and elevated markers of bone turnover. In subjects without secondary hyperparathyroidism, presence of the unfavorable B allele resulted in higher levels of parathormone (Bb and BB vs. bb genotype: 50.3 ± 8.2 pg/dl vs. 44.4 ± 10.7 pg/dl, p = .011, adjusted for weight loss, baseline body mass index, 25hydroxyvitamin D, surgical procedure, and duration after surgery). In the whole sample, patients bearing the unfavorable B allele exhibited lower weight loss, a parameter that was negatively associated with markers of bone resorption. Conclusions: Secondary hyperparathyroidism is highly prevalent after bariatric surgery. Bsm1 vitamin D receptor polymorphism may have an effect in early stages of calcium metabolism imbalance, while no association is detected in patients who have already developed secondary hyperparathyroidism. Moreover, vitamin D receptor polymorphism is associated with post-surgery weight loss, a process related to bone turnover. Keywords: Bsm1 VDR polymorphism; parathormone; calcium metabolism; bariatric surgery; Roux-en-Y Gastric bypass; sleeve gastrectomy
INTRODUCTION
loss is associated with 1–2% bone loss at various sites of the skeleton, especially after procedures with a malabsorptive component, like Roux-en-Y Gastric bypass (RYGB) [3, 7]. Post-operative deficiencies of calcium and vitamin D lead frequently to secondary hyperparathyroidism (SHPT), which further contributes to skeletal abnormalities [3, 5, 8]. Given the high frequency of vitamin D deficiency and SHPT after bariatric surgery [9, 10], we decided to investigate whether the Bsm1 restriction fragment length polymorphism of vitamin D receptor (VDR) is associated with the regulation of the
Bariatric surgery remains the most effective means of treatment of morbid obesity, with benefits ranging from pure weight reduction up to a significant improvement in the overall quality of life [1–4]. However, metabolic as well as microbiological and histological modifications of the remnant stomach have been associated with disturbed calcium homeostasis [1, 3, 5, 6]. In fact, bone loss is a significant metabolic complication of bariatric surgery, requiring appropriate therapeutic interventions [1, 3, 7]. Studies indicate that up to 10% weight Received 17 April 2014; accepted 8 July 2014.
Address correspondence to Irene Lambrinoudaki, Associate Professor, 2nd Department of Obstetrics and Gynecology, University of Athens, Aretaieio Hospital 27, Themistokleous Street, Dionysos, GR-14578, Athens, Greece. E-mail: [email protected]
8
Bsm1 VDR Polymorphism and Bariatric Surgery calcium-vitamin D-parathormone (PTH) axis and bone turnover after bariatric surgery. More specifically we sought to investigate whether the presence of the unfavorable B allele is associated with higher levels of PTH or lower levels of calcium (1) in patients with SHPT and (2) in patients with normal serum PTH.
J Invest Surg Downloaded from informahealthcare.com by Selcuk Universitesi on 01/22/15 For personal use only.
METHODS This study was a cross-sectional analysis of 87 patients who had undergone gastric bypass or gastric sleeve surgery (SG). Our Department serves as a referral Center for bariatric patients from Central and Southern Greece. Patients who undergo bariatric surgery return to their permanent residence, with recommendations for adherence to multivitamin supplementation and for at least two follow-up visits to their local GP. A phonecall invitation was made to 226 patients who were operated in our Department from 2000 until 2011. Sixty-one patients were lost to follow-up. From the 165 patients who were located by phone, 87 patients consented to participate in the study (response rate 52.7%), consisting a total sample of 69 women and 18 men. Serum PTH was not available in four patients. Eligibility criteria for surgery were a body mass index (BMI) ≥ 40 kg/m2 or a BMI ≥ 35 kg/m2 in patients with obesity-related co-morbidities, failure of previous attempts at weight reduction, and expected adherence to post-operative care (e.g., follow-up visits, use of dietary supplements, etc.). Exclusion criteria were the following: current drug or alcohol abuse, uncontrolled or severe psychiatric illness, reversible endocrine or other disorders that can cause obesity, pre-operative gut malabsorption syndrome, presence of any gastric–kidney–liver disease, and pre-operative use of medication affecting bone metabolism (e.g., calcium and vitamin D supplements, glucocorticoids, diuretics, antiepileptic drugs). Surgical techniques in the bariatric unit of the 1st Department of Surgery of the University of Athens had already been standardized before the beginning of this study and remained the same throughout the whole study period. Gastric bypass was performed laparoscopically by constructing a gastric pouch of approximately 30 ml, an alimentary limb with a length of 150 cm, and a biliopancreatic limb of 100 cm with the use of the harmonic scalpel and the Echelon 60 mm linear stapler (Ethicon). The gastrojejunal anastomosis was constructed in a semi-manual way. The posterior wall was constructed with the use of the linear stapler while the anterior wall was sutured manually. The width of the anastomosis was approximately 1 cm. On the other hand, laparoscopic sleeve gastrectomy was performed with the use of the harmonic scalpel for the devascularization of the greater gastric curvature and the stomach was longitudinally divided with the use of the Echelon 60 mm linear stapler (Ethicon). The cal C
2015 Informa Healthcare USA, Inc.
9
ibration of the gastric sleeve was performed with the use of the 9.1 mm endoscope, instead of the commonly used bougies of various diameter, as we have previously reported [11]. All operations were performed under general endotracheal anesthesia. No analgesics were administered before surgery. Intraoperatively patients received Midazolam 5 mg iv (Dormicum HCl, Roche), Xylocaine 40–60 mg (Astra-Zeneca), Fentanyl 100–300 mcg iv (Fentanyl, Sandoz), Propofol 400 mg iv (Propofol, Fresenius), Suxamethonium HCl 100–150 mg iv (Lycitrope, Cooper), Rocuronium Bromide 100–160 mg iv, (Esmeron, Organon), Omeprazole 40 mg iv (Losec, Astra-Zeneca), Ondasetron 8 mg iv (Onda, Medicus), Apotel 1 g iv (Paracetamol), Parecoxib Sodium 40 mg (Dynastat, Pfizer), Mefoxitin 2 g iv (Mefoxil, Vianex), Morphine 5 mg (Morphine, Lavipharm), Citaloprax HCl 20 mg iv (Seropram, Lumbeck), Tramadol 100 mg iv (Tramal, Medicus), and in case of hypertension Furosemide 10–40 mg iv (Lasix, Sandoz) and Clonidine (Catapresan, Boehringer Ingelheim Greece). The average anesthesia time for a sleeve gastrectomy was 75–90 min while the respective duration of anesthesia for a gastric bypass was 200 min. The patients were regularly transferred to the common surgical ward in the immediate post-operative period having a nasogastric tube for the first two post-op days, while no drains were left in the abdominal cavity. Post-operatively all patients received appropriate doses of analgesics, namely, paracetamol 1 g iv, lornoxicam 4 mg iv (Xefo), or meperidine 4 mg iv (Pethidine), according to individual needs. Low molecular weight heparin was administered to all patients as prophylaxis against thromboembolism, namely, enoxaparine 60 mg (Clexane, Abbott) or fondaparinux 2.5 mg subcutaneously (Arixtra, Glaxo) for one month. Antibiotics were continued for the two first post-op days and were always the same that were administered intraoperatively. If Mefoxitine was given during anaesthesia, it was continued as Mefoxil 2 g three times daily iv, if ampicilline-sulbactame (Begalin 1.2 g, Pfizer) was given, it was continued 1.2 g three times daily. All patients also received inhalations with ipratroprium bromide (Atrovent, Boehringer Ingelheim) four times daily and Budesonide (Pulmicort, Astra-Zeneca) twice daily until they were ready to be discharged. Finally, IV omeprazole 20 mg (Losec, Astra-Zeneca) was administered twice daily iv during the first post-operative days and subsequently 20 mg twice per os daily for one month. An upper GI series was performed on the second post-op day and the nasogastric tube was removed. Liquid oral diet was resumed the next day and the patients were discharged on the fourth post-op day. Patients were allowed only liquids for three weeks and strictly only pureed diet until the completion of the first two post-op months. Upon completion of the first post-op month, during their first appointment in the devoted bariatric outpatient clinic, patients were advised to follow a
J Invest Surg Downloaded from informahealthcare.com by Selcuk Universitesi on 01/22/15 For personal use only.
10
A. Alexandrou et al.
high calcium diet (1000 mg/day), as well as to take chewable tables of 500 mg calcium (500 mg × 2 daily) and vitamin D3 (400 IU × 2 daily) (1). Moreover, patients were asked to present at 6 and 12 months after surgery, for follow-up evaluation (1), while subjects diagnosed with vitamin D deficiency were advised to take 5,000 IU of oral vitamin D daily and to be reassessed three months later [12]. Anthropometric data, demographic, and life-style parameters, as well as a detailed medical history were recorded for every subject, using a questionnaire composed for the present study (supplement 1). Data on pre-surgery weight and on weight loss rate were retrospectively derived from patient files, whereas biochemical data, genotyping as well as up-to-date information on demographic and lifestyle parameters were collected during the study. Anthropometric parameters included waist to hip ratio and BMI, using the equation BMI = weight (kg)/height2 (m2 ). Patients were instructed to abstain from food and smoking for 12 hr, and subsequently, fasting venous blood samples were drawn for biochemical evaluation. According to serum PTH levels, subjects were divided in two subsamples: (1) subjects with SHPT (serum PTH above the high normal limit: 62 pg/ml) and (2) subjects with normal levels of PTH, serving as controls [13]. Vitamin D deficiency was defined as serum levels ≤10 ng/ml, while serum levels of vitamin D ≤20 ng/ml were defined as suboptimal [14]. Weight loss was expressed as percentage (%) of excess weight loss (%EWL). Excess weight was estimated using the middle of the Metropolitan Life Insurance tables [15]. Excess BMI was defined as the difference between the BMI of the subject at followup from the cut-off 25 kg/m2 . All patients signed an informed consent and the study was approved by the Ethics Committee of Aretaieion Hospital, University of Athens.
type 1 amino-terminal propeptide (P1NP) and osteocalcin as well as markers of bone resorption, namely, Ctelopeptide. The reference range was as follows: P1NP 14–76 ng/ml; C-telopeptide 100–1000 pg/ml; osteocalcin 15–46 ng/ml. Homeostasis model assessment of insulin resistance was calculated as HOMA-IR = insulin (μIU/ml)∗ Glucose (mg/dl)/405.
VDR Genotyping Blood samples were drawn by atraumatic venipuncture into trisodium circle tubes. Genomic DNA was isolated from leukocyte nuclides using High Pure PCR Template Preparation kit (Roche). Real-time polymerase chain reaction (PCR) was performed in capillaries with a reaction volume of 10 μl, containing 2 μl of DNA (50–100 ng), 0.3 μM sense primer VDR bF (TAG GGG GGA TTC TGA GGA ACT A) and antisense primer VDR bsm A (AGT TTT GTA CCC TGC CCG C), 1.4 μl 25 mM MgCl2 , 1 μl H2 O, 1 μl of Light Cycler DNA Master HybProbe (ready to use reactionmix for PCR containing Taq DNA polymerase, reaction buffer, dNTP mix with dUTP instead of dTTP and 10 mM MgCl2 ), 0.6 μM Sensor b, 3’ modified with fluorescein (AGT ATT GGG AAT GCG CAG GCC-FL) and R Red 0.6 μM Anchor b 5’ labeled with Light Cycler 640 and 3’ terminally blocked by phosphorylation (Red 640-TCT GTG GCC CCA GGA ACC CTG-pH). Sensor b covers the BsmI restriction site. The letters “b” and “B” correspond to the common and the variant allele, respectively. Subsequently, the polymorphism was defined as BB (absence of restriction site on both alleles), Bb (heterozygous), and bb (presence of restriction site on both alleles).
Statistical Analysis Biochemical Assays Serum glucose, total cholesterol, triglycerides, and HDL-cholesterol were assessed enzymatically by an autoanalyzer (ARCHITECT-ci8200, Abbott Diagnostics Laboratories, Abbott Park, IL 60064 USA, Abbott 65205, Wiesbaden, Germany). The Friedewald equation (LDL-cholesterol = total cholesterol − triglycerides/5 − HDL-cholesterol) was used to estimate LDL-cholesterol. Creatinine levels were determined by an enzymatic method. Insulin was measured on an Abbott Architect i1000 analyzer. The total CV% ranged from 1.9% to 5.2%, and the analytical sensitivity was 1 μU/ml. Serum levels of total calcium, 25hydroxyvitamin D, and parathormone (PTH) were evaluated using standard commercially available methodologies. Electrochemiluminescence immunoassay method (Elecsys2010 analyzer, Roche) was used to evaluate markers of bone formation, namely, serum levels of procollagen
Data analysis was performed using SPSS version 17.0. (SPSS, Chicago, IL, USA). Data on qualitative characteristics are expressed as percent values; while data on quantitative characteristics are expressed as means ± SD. Non-parametric tests were used in cases of deviation from normal distribution. Differences between continuous variables across the VDR genotype were assessed using analysis of variance (ANOVA) and Mann–Whitney U test as well as Chi-square (X2 ) analysis and analysis of covariance (ANCOVA). Potential correlations between quantitative parameters were evaluated using Spearman’s correlation coefficient, unadjusted as well as adjusted for potential confounding factors. Subsequently, we analyzed the potential effect of the presence of the unfavorable B allele, in subjects with SHPT and in controls, separately. Statistical significance was set at the 0.05 level. The predefined sample size (N = 87) provided statistical power of 70% for detecting differences within the Journal of Investigative Surgery
Bsm1 VDR Polymorphism and Bariatric Surgery different genotypes of the Bsm1 VDR polymorphism. Type I error was predefined at 0.05 for all power calculations. GPower (version 3.0.10) was used for power estimation [16].
J Invest Surg Downloaded from informahealthcare.com by Selcuk Universitesi on 01/22/15 For personal use only.
RESULTS At the time of the study, the mean follow-up was 3.9 years, median was 3.0 years, and the range was 1–12 years. 42.2% of the total sample had PTH values above the high normal limit (62 pg/ml) and were classified as having SHPT. 91.8% of the whole sample had suboptimal levels of 25hydroxyvitamin D (
