Clinical Nutrition xxx (2014) 1e8

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Original article

Haematological parameters and serum trace elements in “healthy” and “unhealthy” morbidly obese patients before and after gastric bypass Roser Ferrer d,1, Eva Pardina a,1, Joana Rossell a, Juan Antonio Baena-Fustegueras b, Albert Lecube c, José María Balibrea e, Enric Caubet e, Oscar González e, Ramón Vilallonga e, Jose Manuel Fort e, Julia Peinado-Onsurbe a, * a

Biochemistry and Molecular Biology Department, Biology Faculty, Barcelona University, Diagonal 643, 08028 Barcelona, Spain Arnau de Vilanova University Hospital (UdL), Surgery Unit, Spain c Endocrinology and Nutrition Department at Arnau de Vilanova University Hospital (UdL) and Diabetes and Metabolism Research Unit (VHIR, UAB) and CIBER de Diabetes y Enfermedades Metabólicas (CIBERDEM) at the Instituto de Salud Carlos III, Spain d Biochemistry Department, Hospital Universitari Vall D’Hebron, Universitat Autònoma De Barcelona, Spain e Endocrinology Surgery Unit, Hospital Universitari Vall D’Hebron, Universitat Autònoma De Barcelona, Spain b

a r t i c l e i n f o

s u m m a r y

Article history: Received 19 March 2014 Accepted 6 April 2014

Background & aims: We have investigated the differences in plasma parameters and serum trace elements between “healthy” and unhealthy morbidly obese patients before and after Roux-en-Y gastric bypass surgery. Methods: A group of 32 morbidly obese patients undergoing bariatric surgery were divided into three groups. Group 1 subjects were free of dyslipidemia and type II diabetes mellitus (defined as “healthy” obese, DMDL); Group 2 subjects had only the presence of dyslipidemia (DMDLþ), while group 3 patients demonstrated the presence of both (DM þ DLþ). In all patients, we studied haematological, haemostasis, anaemia, coagulation plasma and trace elements parameters before and 1, 6 and 12 months after gastric bypass surgery. Results: We found significant differences in some haematological parameters, including haemostasis (e.g., T-Quick, p ¼ 0.0048) and coagulation (e.g., ATIII and PAI-1, p ¼ 0.001 and p < 0.0001, respectively) and in anaemia parameters (e.g., folate, cobalamin and transferrin, p ¼ 0.0002, p < 0.0001 and p ¼ 0.0001, respectively) but also in serum trace elements between the groups. However, the response to bariatric surgery was similar in the three groups. Conclusion: Any healthy morbid obese subject is really metabolically “unhealthy” because he or she has many other haematologic or serum abnormalities that are often not included in the criteria for the definition of “healthy” in these obese subjects. Ó 2014 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Keywords: Healthy obese Dyslipidemia Diabetes Haematology Haemostasis Bariatric surgery

1. Introduction For the last decade scientists have been trying to discover the mechanism that causes some obese people to develop insulin Abbreviations: RYGBP, Roux-en-Y gastric bypass; HOMA-IR, homeostasis model assessment of insulin resistance; NEFA, non-esterified fatty acid; TAG, triglycerides; TC, total cholesterol; SBP, systolic arterial pressure; DBP, diastolic arterial pressure; DM, diabetes mellitus; DL, dyslipidemia; HTA, arterial hypertension; PAI-1, Plasminogen activator inhibitor-1; ATIII, antithrombin; SAT, subcutaneous adipose tissue; VAT, visceral adipose tissue. * Corresponding author. Tel.: þ34 93 4021524/48; fax: þ34 93 4021559. E-mail address: [email protected] (J. Peinado-Onsurbe). 1 These authors contributed equally to this study.

resistance and type 2 diabetes mellitus (DM) and other obese people with the same BMI to be “healthy” and not develop those comorbidities [1e3]. However, in our opinion, the lack of diabetes, hypertension and dyslipidemia is not enough to define “healthy obesity”, at least in the context of the morbidly obese. Regardless of how “metabolically healthy obesity” is defined, questions remain, namely: i) Would these obese patients still be considered to be healthy if they had changes in one of the many other measurable parameters, such as the rheological characteristics of the blood, possibly due to changes in the blood lipid level, ions or the total amount of protein in their plasma? ii) Would these

http://dx.doi.org/10.1016/j.clnu.2014.04.003 0261-5614/Ó 2014 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Please cite this article in press as: Ferrer R, et al., Haematological parameters and serum trace elements in “healthy” and “unhealthy” morbidly obese patients before and after gastric bypass, Clinical Nutrition (2014), http://dx.doi.org/10.1016/j.clnu.2014.04.003

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R. Ferrer et al. / Clinical Nutrition xxx (2014) 1e8

patients be considered healthy if they demonstrated altered coagulation or haemostasis parameters? For example, zinc, an essential trace element and a component of many enzymes, is involved in the synthesis, storage and release of insulin. Trace elements are essential nutrients with regulatory, immunologic, and antioxidant functions resulting from their actions as essential components or enzymatic cofactors in metabolism [4]. Trace elements and minerals influence the pathogenesis of obesity and diabetes and complications thereof, mainly through their involvement in peroxidation and inflammation [5]. Despite major innovations in bariatric surgery over the past two decades, the Roux-en-Y gastric bypass (RYGBP) remains the most effective bariatric procedure and is the most effective current treatment for morbid obesity. This procedure produces significant and sustained weight loss as well as markedly reduced comorbidities [6]. However, this popular procedure is associated with longterm complications including haematological disorders, especially chronic anaemia [7], most commonly due to iron deficiency. Deficiencies in vitamins, folate, and copper have also been reported after surgery. The evaluation of haematologic disorders after gastric bypass surgery must take into account issues unique to the postsurgery setting that influence the development of anaemia and other cytopenias [8]. The aim of our study was to investigate whether there are differences between subjects considered to be “healthy” obese as described in the literature, and those obese subjects with DL or with both DM and DL, before and 1, 6 and 12 months after Roux-enY gastric bypass. We focused on haematology (red and white blood cells, platelets), haemostasis, anaemia, coagulation, trace elements, and other parameters in each of the three groups of morbidly obese patients. 2. Materials and methods 2.1. Patient selection A group of 32 morbidly obese patients (23 women and 9 men) aged between 21 and 61 years who underwent RYGBP surgery were enrolled and followed up at the Hospital de la Vall d’Hebron in Barcelona, Catalonia, as described previously [9e12]. All subjects were free of inflammatory and infectious diseases and none were receiving anti-obesity or anti-inflammatory drugs at the time of the study. Patients were excluded if they had neoplasic, renal, hepatic or active systemic diseases, hypothyroidism or endocrine diseases other than diabetes, or if they had been on a restrictive diet during the week previous to the study. All patients reported that their weight had been stable during the previous three months. None of the diabetic patients were being treated with insulin. All patients were required to complete a 2-week preoperative low carbohydrate, high-protein liquid commercially formulation of 800 kilocalories or less per day, in lieu of their usual food intakes. Those patients considered to be “healthy” morbidly obese patients met the most restrictive criteria of Wildman et al. [13]. Thus, for DM the threshold was for fasting plasma glucose 100 mg/dL or medically diagnosed DM; the criteria for HTA were systolic and diastolic blood pressure (SBP and DBP, respectively), SBP  130 mm Hg and DBP  85 mm Hg; and the criteria for DL were TG  150 mg/ dL, cLDL  110 mg/dL and cHDL < 40 and 50 mg/dL for men and women, respectively or medical diagnosis. According with those criteria, the patients were divided into three groups as follows: 10 patients were DMDL (the “healthy” obese group, without type II diabetes mellitus (DM) or dyslipidemia (DL), comprising 6 women and 4 men), 15 patients were

DMDLþ (the obese group with dyslipidemia, comprising 11 women and 4 men), and 7 patients were DM þ DLþ (the obese group with dyslipidemia and diabetes, consisting of 6 women and 1 man). With respect to hypertension, 2 patients in the first group, 4 patients in the second group and all in the third group were hypertensive. Following their assignment into one of the three groups according to whether they were metabolically healthy or unhealthy (with DL and/or DM), the following parameters were analysed in all patients: haematology (red and white blood cells, platelets), haemostasis, anaemia, coagulation and serum trace elements. The study protocol was reviewed and accepted by the hospital ethics committee conforming to the Declaration of Helsinki, and all subjects gave their written informed consent to participate. 2.2. Anthropometric and body composition measurements Body weight, excess weight, height, and waist and hip circumferences were measured according to standardised procedures [14]. The body fat percentage and the amount of total, subcutaneous and visceral fat were calculated as described previously [11,12]. 2.3. Plasma biochemistry assays Leptin, ghrelin, adiponectin, insulin, glucose, homeostasis model assessment of insulin resistance (HOMA-IR) and lipid levels were determined as previously described [11,12]. 2.4. Haematological parameters Blood samples were taken under fasting conditions between 8:00 A.M. and 10:00 A.M. at the time of surgery (OB in Graphs and Tables) and at 1, 6 and 12 months after the gastric bypass surgery (1M, 6M and 12M in Graphs and Tables). The blood was transferred to vacutainer tubes containing EDTA disodium (anticoagulant) for blood cell and haemostasis evaluation. The plasma was separated immediately by centrifugation and aliquots were frozen at 80  C for subsequent analysis. The blood samples for the anaemia parameters and serum trace elements were collected in vacutainer tubes without additives. The plasma blood cells and platelets were measured using an automatic Beckman Coulter LH 750 analyzer. To quantify haemostasis and blood coagulation, an Amelung CS-400 autoanalyser was used (AMGA, Grifols, Spain). Calcium was measured in an Olympus AU 5400 autoanalyser (Izasa, Spain). ATIII was determined using the chromogenic anti-Xa method (BIOPEP S.A., France). PAI-1, a measure of impaired fibrinolysis, was determined using ELISA (IMUBIND, America Diagnostica Inc. USA). 2.5. Statistical analysis The results are presented as the means  SEM. The significant differences between the mean values for the obese (OB) and 1M, 6M, or 12M after surgery (weight loss) timepoints were assessed using one-way ANOVA, and individual comparisons were made using the Bonferroni post-test. The significant differences between the healthy morbidly obese (DMDL), dyslipidemic (DMDLþ) and diabetic and dyslipidemic (DM þ DLþ) groups at different time points after surgery (weight loss effect) were assessed using a twoway ANOVA and Bonferroni post-tests. The statistical comparisons were considered significant at P < 0.05. All statistical analyses were computed using GraphPad Prism version 5.00 software for Windows (GraphPad Software, San Diego, CA, USA, www.graphpad.com).

Please cite this article in press as: Ferrer R, et al., Haematological parameters and serum trace elements in “healthy” and “unhealthy” morbidly obese patients before and after gastric bypass, Clinical Nutrition (2014), http://dx.doi.org/10.1016/j.clnu.2014.04.003

R. Ferrer et al. / Clinical Nutrition xxx (2014) 1e8

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3. Results

3.4. Trace elements and other plasma parameters

3.1. Anthropometric and obesity parameters (leptin, ghrelin and adiponectin)

Trace elements were not affected by either comorbidities or by surgery (data not shown). However, the effects of comorbidities and surgery (Table 4: Table S4 is provided in the Supplementary File) on inorganic phosphate (p ¼ 0.0288 and p < 0.0001, respectively, Fig. S2 Supplementary File), nitrates (p ¼ 0.0084 and p < 0.0001, respectively), creatinine (p ¼ 0.0395 and p ¼ 0.0248, respectively) and total plasma protein (p ¼ 0.0211 and p ¼ 0.0415, respectively) were observed. Alkaline phosphatase was affected by comorbidity (p ¼ 0.0007), but not by surgery. Finally, the parameters that were affected by the weight loss surgery but not by the various morbidities were urate (p < 0.0001, Fig. S2 Supplementary File), urea (p ¼ 0.0012, Fig. S2 Supplementary File) and esterified and total bilirubin (p ¼ 0.0282 and p ¼ 0.0078, respectively).

The anthropometric, obesity (leptin, ghrelin and adiponectin), lipid and insulin resistance parameters for each group of “healthy” (DMDL) and “unhealthy” (DM DLþ and DM þ DLþ) morbidly obese patients are summarised in Table 1 (for the entire Table S1, please see the Supplementary File). It should be noted that the values of leptin and ghrelin are very different in “healthy” obese than for the other two groups. The surgery for weight loss had a very significant effect on all the parameters, and the effects were similar for all groups. It is noteworthy that the HOMA-IR clearly indicates that the DM þ DLþ patients are insulin-resistant; the value is almost twice that of the group DMDLþ (10.19  2.75 vs. 5.72  1.45 a.u.). Although the “healthy” obese group was not diagnosed as DM subjects in this group were insulin resistant as indicated by the HOMA-IR values, which are higher (6.92  1.37 a.u.) than those of the DL group. 3.2. Haematological parameters No significant changes in the red blood cells due to comorbidities were observed (Table 2: the full Table S2 is provided in the Supplementary File). However, the surgery led to very significant changes in the reticulocytes of the different groups (p ¼ 0.0005). For the white blood cells, highly significant differences due to comorbidities were observed (Table 2) only in eosinophils (p ¼ 0.0003), and less significantly, in basophils (p ¼ 0.0184). The surgery affected all types of white blood cells except neutrophils. The number of platelets (Table 2), but not their volume (p ¼ ns), was severely affected by the type of comorbidity (p < 0.0001). However, although the surgery did not affect the number of platelets (p ¼ ns), it did affect their volume (p ¼ 0.0058). As for the white cells, the only group in which the differences were observed at the different timepoints after surgery was the DMDLþ group (p ¼ 0.0240). 3.3. Haemostasis, anaemia, and coagulation parameters In the study of haemostasis (Table 3; the entire Table S3 is provided in the Supplementary File), there were significant differences in the clotting Quick time (p ¼ 0.0098) and the prothrombin time (p ¼ 0.0172) as functions of the type of comorbidity. With the exception of the thromboplastin time, the surgery very significantly affected all parameters studied. There was a clear surgery effect (Table 3) in Quick time and PT in all the groups studied, but for aPTT, the only significant difference (p ¼ 0.0239) was for DL þ DM þ group. The coagulation parameters (Table 3) ATIII and fibrinogen were affected both by comorbidities (p ¼ 0.001 and p < 0.0001, respectively) and surgery (p < 0.0001 and p < 0.0001, respectively). Although PAI-1 was not affected by comorbidities, it was affected by surgery (p ¼ ns and p < 0.0001, respectively). All parameters related to anaemia were affected by bariatric surgery (Table 3) and in some cases also by the type of comorbidity, i.e., folate (p ¼ 0.0002), cobalamin (p < 0.0001), transferrin (p ¼ 0.0001, Fig. S1 Supplementary File) and transferrin saturation (p ¼ 0.0085, Fig. S1 Supplementary File). In the case of haptoglobin, an acute phase protein, surgery-induced differences were observed in all groups of patients, DMDL (p ¼ 0.0007), DMDLþ (p ¼ 0.0496) and DM þ DLþ (p ¼ 0.0045).

4. Discussion A recently published report indicated that there is a group of morbidly obese subjects that fits the characteristics of a “metabolically healthy” but obese population [15]. To conduct our study, we used the most restrictive criteria of Wildman et al. [13], mentioned in the Methods section, to define which patients were considered “healthy”. These patients, also, met the definition of metabolic health proposed by Aguilar-Salinas [16], Karelis [17] and Meigs [18]. However, it should be noted that although they met the abovementioned criteria, our “healthy” morbidly obese patients had steatosis (20e70%). Moreover, in the other two groups, between 71 and 73% of the patients had varying degrees of steatosis. However, it is not only the steatosis that was of concern. 4.1. Anthropometric and obesity parameters (leptin, ghrelin and adiponectin) Based on the anthropometric data observed in these three groups, these patients had what Tchernof and Despres [19] call a “hypertriglyceridemic waist,” a simple clinical phenotype that is predictive of excess visceral fat and metabolic abnormalities associated with elevated insulin and glucose and an altered lipid profile. From that point of view, none of the groups are “healthy obese”. Our results did not agree with those observed by some other authors for leptin, although these data are not consistent. For instance, in some studies, leptin was decreased in the unhealthy obese [16,17], while in other cases, it was increased [18,13]. The three groups of obese patients in our study had adiponectin values within the range reported by other authors for metabolically healthy patients with BMIs of 20e24.9 kg/m2 [16], but many other parameters, such as insulin, glucose and HOMA-IR, were altered in our study. All 3 groups were insulin-resistant with HOMA-IR values of 6.9 (in the “healthy” obese), 5.7 (in DMDLþ) and 10.2 (in DM þ DLþ). One year after bariatric surgery, the insulin concentrations decreased by 60e70% compared to obese patients before surgery, independently of whether the patient was “unhealthy” (diabetic or/and dyslipidemic) or “healthy.” The “unhealthy” patients (DMDLþ and DM þ DLþ) were all dyslipidemic, and all lipid parameters were higher than in healthy individuals (DMDL). The hypertriglyceridemia of varying degrees that usually accompanies morbid obesity may cause changes in the rheology of blood, causing small alterations in capillary endothelial cells, and some of the parameters involved in these changes are the PAI-1, ATIII and fibrinogen, which we described previously [20].

Please cite this article in press as: Ferrer R, et al., Haematological parameters and serum trace elements in “healthy” and “unhealthy” morbidly obese patients before and after gastric bypass, Clinical Nutrition (2014), http://dx.doi.org/10.1016/j.clnu.2014.04.003

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R. Ferrer et al. / Clinical Nutrition xxx (2014) 1e8

Table 1 Anthropometrics, obesity, lipids and the insulin resistance parameters of morbidly obese patients in each group before and after bariatric surgery. Parameters

Anthropometrics Body weight (kg) BMI (kg/m2)

Total fat (kg)

SAT (kg)

VAT (kg)

Waist (cm)

Obesity Leptin (ng/mL)

Ghrelin (pg/mL)

Adiponectin (mg/mL) Lipid metabolism TG (mg/dL plasma) NEFA (mM)

Chol (mg/dL)

cLDL (mg/dL)

cHDL (mg/dL)

Insulin resistant Insulin (mUI/L)

Glucose (mg/dL)

HOMA-IR (a.u.)

DMDL

DM þ DLþ

DMDLþ

Anova-2, p value

OB

12M

OB-

12M-

OBþ

12Mþ

Comorbidities effect

Surgery effect

136.70  6.44 DMDL p < 0.0001 49.38  1.93 DMDL p < 0.0001 81.21  5.80 DMDL p < 0.0001 62.43  5.12 DMDL p ¼ 0.0008 21.29  2.40 DMDL p ¼ 0.0004 134.80  3.92 DMDL p < 0.0001

83.83  4.90 ooo

131.80  4.68 DMDLþ p < 0.0001 49.79  1.27 DMDLþ p < 0.0001 82.25  4.64 DMDLþ p < 0.0001 60.49  4.30 DMDLþ p ¼ 0.0001 22.56  2.41 DMDLþ p ¼ 0.0008 136.70  3.71 DMDLþ p < 0.0001

84.81 ooo

118.00  7.36 DM þ DLþ p < 0.0001 47.37  1.24 DM þ DLþ p < 0.0001 70.96  4.57 DM þ DLþ p < 0.0001 51.50  4.00 DM þ DLþ p ¼ 0.0151 19.44  2.49 DM þ DLþ p ¼ 0.0491 138.10  5.04 DM þ DLþ p ¼ 0068

74.43  4.35 ooo

0.0004

Haematological parameters and serum trace elements in "healthy" and "unhealthy" morbidly obese patients before and after gastric bypass.

We have investigated the differences in plasma parameters and serum trace elements between "healthy" and unhealthy morbidly obese patients before and ...
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