Decrease in Serum Protein Carbonyl Groups Concentration and Maintained Hyperhomocysteinemia in Patients Undergoing Bariatric Surgery
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- Sledzinski, T., Goyke, E., Smolenski, R.T. et al. OBES SURG (2009) 19: 321. doi:10.1007/s11695-008-9691-8
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Human obesity is associated with oxidative stress but the factors contributing to the increase of reactive oxygen species (ROS) production remain unknown. We evaluated the association between serum homocysteine concentration, which may increase ROS production, and serum protein carbonyl groups concentration before and after bariatric surgery.
Serum protein carbonyl groups and serum homocysteine concentrations, as well as obesity markers, were compared in 18 obese patients before and 6 months after bariatric surgery. Ten healthy individuals with normal body mass index (BMI) served as controls.
Before bariatric surgery, obese patients displayed approximately 50% higher serum protein carbonyl groups concentration than control subjects. After surgery, serum protein carbonyl groups concentration decreased and matched values observed in controls. Serum homocysteine concentration was also elevated in obese patients, but in contrast to protein carbonyl groups, did not change after surgery. The body weight, BMI, HOMA-IR, serum leptin, triacylglycerols, LDL/HLD cholesterol ratio, insulin, and glucose concentrations were higher in obese patients as compared to controls, and decreased after bariatric surgery.
This study demonstrates that bariatric surgery has protective effect on oxidative protein damage and improves several laboratory parameters including serum lipid concentration and insulin resistance. However, bariatric surgery does not cause a decrease in serum homocysteine concentration, a risk factor for the development of cardiovascular diseases. Collectively, the results presented in this paper suggest that serum homocysteine concentration is not directly associated with oxidative stress in obese patients after bariatric surgery.
KeywordsOxidative stressHomocysteineProtein carbonyl groupsObesityBariatric surgery
Obesity is a risk factor for the development of cardiovascular diseases, type II diabetes mellitus, and other disorders . One possible mechanism involved in the development of obesity-related co-morbidities is the imbalance between the ROS generation and the antioxidant activity in cells, generally called “oxidative stress” . Excessive ROS production leads to the damage of lipids, DNA, carbohydrates, and proteins . Malondialdehyde (MDA) and thiobarbituric acid reactive species (TBARS) are generally accepted markers of lipids oxidative damage, whereas protein carbonyl groups are markers of proteins oxidative damage. Carbonylation is an irreversible modification of proteins, leading to the alteration of proteins structure and function, and finally to proteins degradation . Alteration of proteins function by ROS may lead to cellular dysfunction and tissue damage . Moreover, protein carbonyl groups determination, as an oxidative stress marker, has some advantages over determination of products of lipids oxidation (MDA, TBARS) because: (1) they are formed early and are more stable than MDA or TBARS ; (2) protein carbonyl groups formation can be induced by almost all types of ROS, and (3) there are sensitive and specific methods for protein carbonyl groups detection .
Obese patients display higher plasma TBARS [2, 5–7], MDA [8, 9], and protein carbonyl groups  concentrations in comparison to control subjects. The increased concentrations of serum protein carbonyl groups in animal model of obesity were also observed . Konukoglu et al. [5, 12] claimed that hyperhomocysteinemia may contribute to the elevation of oxidative stress in obese patients. The later suggestion seems to be important, because hyperhomocysteinemia is a risk factor for atherosclerosis. Moreover, oxidative stress is one of the proposed mechanisms of proatherogenic action of elevated serum homocysteine [13, 14]. However, the results reporting serum homocysteine concentration in obese patients are conflicting. Some studies indicate higher [5, 15, 16], whereas other normal [17, 18] serum homocysteine concentration in obese patients. Thus, this problem requires further elucidation.
The decrease of serum MDA and oxidized LDL in patients after bariatric surgery was observed [19, 20]. Recent study indicated that laparoscopic adjustable gastric binding (LAGB) causes a decrease of plasma protein carbonyls in obese patients . Considering that hyperhomocysteinemia would affect oxidative stress in obese patients [2, 13, 14], it was interesting to elucidate the association between serum protein carbonyl groups and serum homocysteine concentrations in obese patients before and 6 months after bariatric surgery.
Materials and Methods
Eighteen obese patients (eight males and ten females; BMI = 48 ± 9) aged 28–58 years (mean age 43 ± 9 years) underwent vertical banded gastroplasty (VBG) at the Department of General, Endocrine, and Transplant Surgery (Medical University of Gdansk, Poland). Inclusion criteria were: no clinical evidence of endocrine, cardiac, hepatic, or renal failure diseases. Patients had anthropometric and laboratory parameters checked before surgery and 6 months after VBG. Smokers were excluded from the study. After the surgery, patients were regularly supplemented with vitamins B12, B6, and folate. Ten healthy volunteers (six males and four females; BMI = 25 ± 3) aged 24–66 years (mean age 37 ± 16 years) formed the control group. After overnight fast, blood specimens were obtained for serum protein carbonyl groups, homocysteine, lipids (total cholesterol, HDL cholesterol and triacylglycerols), leptin, insulin, and glucose concentrations assays. The research was carried out in accordance with the Declaration of Helsinki of The World Medical Association, and was approved by the Medical University of Gdansk Ethics Committee. All of the patients signed an informed consent for this investigation.
Serum protein carbonyl groups were assayed by the modified method of Levine et al. . A 0.1-ml volume of 20 mM 2,4-dinitrophenylhydrazine (DNPH) in 2 M HCl was added to 0.1 ml of serum samples in glass tubes. Blank samples contained 0.1 ml 2M HCl added to 0.1 ml of serum samples. After 1 h incubation at 25°C with continuous shaking, serum proteins were precipitated by adding 0.5 ml of 20% trichloroacetic acid. After centrifugation (3,000×g; 10 min), the precipitated proteins were washed three times with ethanol/ethyl acetate (1:1), and finally dissolved in 1 ml of 6 M guanidine (adjusted to pH 6.5 with HCl) at 60°C. Absorbance of samples was measured at 360 nm versus blank. The concentration of protein carbonyl groups was calculated using molar absorption coefficient—22,000.
Serum homocysteine concentration was determined using liquid chromatography/mass spectrometry (LC/MS). An aliquot of plasma (0.4 ml) was deproteinized with 0.4 ml of 10% trichloroacetic acid (TCA). The tubes were centrifuged at 4°C, 12,000×g for 5 min. Supernatant was collected and TCA removed by diethyl ether extraction followed by freeze-drying. Material obtained was dissolved in 0.1 ml of 10 mM nonafluoropentanoic acid in H2O and analyzed with the use of ion-pair high-performance liquid chromatography with mass detection. Chromatographic separation was performed using 3 μm Hypersil BDS 150 × 2.0 mm column. Mobile phase was delivered at 0.2 ml/min in gradient from 0% to 60% acetonitrile in 12 min. The mass detector (Thermo-Finnigan LCQ Advantage, Waltham, MA, USA) with electrospray (ESI) ion source was operating in positive MS2 mode for detection of homocysteine with the collision energy setting at 25%. Electrospray cone voltage was set at 4.5 kV and heated capillary temperature was 275°C. Sheath gas flow was set for 35 arbitrary units. Post-column sheet flow of methanol with 0.05% formic acid at 0.2 ml/min was used to improve ionization efficiency. The identity of homocysteine was confirmed by the similarity of molecular weights, fragmentation pattern and chromatographic retention time.
Serum triacylglycerols, total cholesterol and HDL cholesterol concentrations were analyzed by standard enzymatic procedures (Boehringer Mannheim, Mannheim, Germany). Serum LDL cholesterol concentration was calculated as described previously . Serum leptin and insulin concentrations were analyzed by a radioimmunoassay technique . Serum glucose was measured by enzymatic method (Sigma, St Louis, MO). The homeostasis model assessment score (HOMA) was calculated according to the following formula: fasting glucose (mM) × fasting insulin (μU/L) / 22.5, as described by Matthews et al .
Statistical analysis was performed using Microsoft Excel and Statistica. The statistical significance of the differences between parameters studied in patients before and after bariatric surgery was assessed by paired t test. The statistical significance of the differences between obese patients and controls was assessed by Student’s t test. Linear regression coefficient was calculated to assess correlation between selected parameters in obese subjects before and after surgery.
Selected laboratory parameters of controls and obese patients before and 6 month after bariatric surgery
Control, mean ± SD
Obese patients before surgery, mean ± SD
Obese patients 6 months after surgery, mean ± SD
Number of control subjects/ patients
37 ± 16
43 ± 9
43 ± 9
Body weight (kg)
76 ± 14
134 ± 24
99 ± 22
128 ± 13
206 ± 87
130 ± 49
Total cholesterol (mg/dl)
203 ± 34
226 ± 48
202 ± 25
HDL cholesterol (mg/dl)
57 ± 17
39 ± 8
50 ± 10
LDL cholesterol (mg/dl)
124 ± 29
131 ± 31
126 ± 21
2.2 ± 0.7
3.4 ± 1.1
2.5 ± 0.7
11 ± 4.5
42 ± 27
23 ± 14
4.8 ± 0.7
6.6 ± 1.8
5.2 ± 1.2
2.4 ± 1
13.5 ± 10
6.1 ± 5
6.8 ± 2.9
22.3 ± 13
8.1 ± 4.2
Among factors which theoretically may increase ROS production in obese patients (like BMI, HOMA-IR, serum leptin, and homocysteine concentrations), tested by univariate analysis, serum carbonyl groups concentration was found to be positively correlated with BMI (r = 0.40; p < 0.05) and HOMA-IR (r = 0.41; p < 0.05) but not with serum leptin and homocysteine concentrations.
This study demonstrated that bariatric surgery had protective effect on oxidative damage of proteins and improved several laboratory parameters including serum lipid concentration and insulin resistance. These results are consistent with previously reported data indicating that obesity is associated with the increase in oxidative stress as measured by serum carbonyl groups concentration , and with the increase in serum homocysteine concentration [5, 12, 26–30]. Moreover, our results showed a significant decrease of serum protein carbonyl groups concentration 6 months after bariatric surgery. It should be noted that 6–12 h after either laparoscopic or open Swedish adjustable gastric binding (SAGB), plasma MDA level increased significantly together with markers of systemic inflammation (CRP and IL-6) . Moreover, the increase in plasma CRP, IL-6, and MDA levels was more pronounced after open than laparoscopic SAGB . These data suggest that surgical trauma and its intensity are mainly contributing to the systemic inflammatory response and to the increase of plasma MDA concentration few hours after surgery .
Serum homocysteine concentration, significantly higher in obese patients than in controls, did not decrease 6 months after bariatric surgery. Thus, the novel finding of the present report is a lack of association between changes in serum homocysteine and serum protein carbonyl groups concentration after bariatric surgery. Bariatric surgery also causes a decrease in serum triacylglycerols, leptin, glucose, insulin concentrations as well as in HOMA index and LDL/HDL ratio. After surgery, serum HDL cholesterol concentration significantly increased (Table 1). Collectively, the decrease of protein oxidative damage, but not serum homocysteine concentration, can be added to the list of beneficial effects of bariatric surgery, beside the weight reduction, increase of HDL cholesterol concentration and decrease of serum triacylglycerols concentration (Table 1). One can suggest that elevated serum homocysteine concentration after bariatric surgery could minimize the beneficial effect of weight loss on the cardiovascular system in obese subjects.
The decrease of protein oxidative damage has also been observed after dietary restriction. Dandona et al.  demonstrated approximately 15% decrease of protein carbonyls after 4 weeks of dietary restriction. During preparation of this manuscript, Uzun et al. , reported reduction of plasma protein carbonyls caused by LAGB in obese patients. Collectively, our data and those reported previously [21, 32] indicate that body-weight reduction, induced either surgically or not surgically (by dietary restriction), is associated with a decrease of oxidative stress in humans. It seems, however, that the effect of bariatric surgery on serum protein carbonyl groups is stronger than the effect of dietary restriction. The question arises what factors contribute to the decrease of oxidative stress in obese patients after bariatric surgery. In the light of the results reported previously, the following factors can contribute to the increase of oxidative stress in obese patients: (1) elevated serum homocysteine concentration [5, 33]; (2) insulin resistance , and (3) increased serum leptin concentration . Consequently, the decrease in any of these parameters would lead to the decrease in serum carbonyl groups concentration.
Previously reported data suggest that hyperhomocysteinemia may promote the production of hydroxyl radical [5, 33] which could initiate protein carbonylation . Our results show about 50% higher serum homocysteine concentration in obese patients than in controls (Fig. 3). Weight loss observed 6 months after bariatric surgery was not associated with significant changes in serum homocysteine concentration (Fig. 3). Gomez-Ambrosi et al.  also did not observe change in serum homocysteine concentration after gastric bypass. Similar results were also reported by Dixon et al.  in patients who were supplemented with folate, vitamin B12, and vitamin B6. Other studies showed decrease  or increase of serum homocysteine concentration after bariatric surgery [26–29]. At present, it is difficult to explain these discrepancies. It is known that different categories of bariatric surgery are associated with different late complications. For instance, vitamin B12 and folate deficiencies are rare or not found after VBG, whereas are frequently observed after biliopancreatic diversion or roux-en-Y gastric bypass . Since metabolism of homocysteine requires adequate levels of folate, vitamin B12 and vitamin B6 one can speculate that different restriction of supply with these vitamins induced by different types of bariatric surgery might influence plasma homocysteine concentration. This is confirmed by findings of Dixon et al.  who found that regular supplementation of patients with vitamins B12, B6, and folate may prevent the increase of serum homocysteine concentration after bariatric surgery. Our patients were regularly supplemented with vitamins B12, B6, and folate. It explains why we did not observe the increase in serum homocysteine concentration after bariatric surgery.
Elevated serum leptin concentration was postulated to be the source of oxidative stress, because it leads to (1) the increase of ROS formation, (2) the increase of low-grade inflammation and (3) the reduction of paraoxonase activity . Thus, the increase of serum leptin concentration in obese patients (Table 1) may be one of the factors promoting oxidative stress. Consequently, a decrease of serum leptin concentration 6 months after VBG (Table 1) seems to be involved in a decrease of serum protein carbonyl groups concentration (Fig. 2).
Insulin resistance and hyperglycemia could be sources of oxidative stress in obese patients, because they lead to: (1) an increase of advanced glycation end products formation, (2) glucose autooxidation and (3) excessive sorbitol formation . Significant decrease of HOMA index in obese patients 6 months after VBG (Table 1) indicate the improvement of insulin sensitivity and could be another reason of decrease of protein carbonylation, beside the decrease of leptin concentration.
Univariate analysis of serum carbonyl groups concentration demonstrated positive correlation only with BMI and HOMA-IR, but not with serum leptin and homocysteine concentrations. Therefore, it seems unlikely, that hyperleptinemia and hyperhomocysteinemia may affect oxidative stress in obese patients as has been suggested previously [2, 13, 14]. Positive correlation between BMI and serum protein carbonyl groups concentration suggests that body mass plays an important role in ROS production in obese subjects. It cannot be excluded that nutrient excess in obese patients contributes to overproduction of ROS. They are generated during oxidation of excess fatty acids and pyruvate (formed from glucose) in mitochondria. Excessive production of ROS creates oxidative stress, which can damage proteins. After bariatric surgery, when food intake is limited, less ROS is produced by the mitochondria, and consequently, less protein carbonyl groups is generated. There is also another possibility to explain the strong association between BMI and serum protein carbonyl groups concentration in obese patients before and after bariatric surgery. Considering that inflammation is consequent to obesity  and that systemic inflammatory response after laparoscopic and open SAGB is associated with the increased plasma MDA level , one can assume that systemic inflammation associated with obesity could play an important role in ROS production. Before bariatric surgery, excess of fat mass produces proinflammatory substances , leading to systemic inflammation. This phenomenon could be associated with an increase of plasma carbonyl groups concentration. After bariatric surgery, when loss of fat mass occurs, less proinflammatory substances are produced, the systemic inflammatory responses are reduced and serum protein carbonyl groups concentration decreases.
In conclusion, our results show the increased oxidative damage of proteins in obese patients in comparison to controls that reversed after bariatric surgery. Moreover, the data presented in this paper indicate that bariatric surgery did not cause a decrease of serum homocysteine concentration. The lack of association between serum protein carbonyl groups concentration and serum homocysteine concentration after bariatric surgery suggest that hyperhomocysteinemia would not affect oxidative stress in obese patients as it was suggested previously [5, 12]. One can suppose that persistently elevated serum homocysteine concentration after bariatric surgery could minimize the benefit of weight loss on the risk of cardiovascular disease in patients suffering from obesity.
We are indebted to Marcin Lipinski for assistance in the homocysteine determination and Professor M.M. Zydowo, Professor L. Zelewski and Dr. Anna Nogalska for critical reading of this manuscript. This work was supported by a grant from the Ministry of Science and Higher Education (Badania Statutowe St-41).