|Year : 2014 | Volume
| Issue : 3 | Page : 317-320
Association of serum Interlukin-6 and glycolysis in sickle cell disease patients
Purnima Dey Sarkar, Gopinath Agnihotram, Lincy K Skaria
Department of Biochemistry, MGM Medical College, Indore, Madhya Pradesh, India
|Date of Web Publication||18-Mar-2014|
MGM Medical College, Indore - 452 001, Madhya Pradesh
Source of Support: None, Conflict of Interest: None
Background: Glycolysis, a major cytosolic oxidative pathway of glucose, is intended for the supply of energy in RBC and, moreover, for the production of 2,3 Bisphsophoglycerate through the Rapaport-Lubering shunt, which delivers oxygen more readily to the tissues. Interleukin 6 (IL-6) is a pro-inflammatory cytokine, playing a pivotal role in the inflammation process and mediating the acute phase process. Aim: This study aimed to investigate the association of the inflammatory parameter, serum IL-6, and glycolytic metabolism in the erythrocytes of sickle cell disease patients. Materials and Methods: This cross-sectional study was performed using a cohort of patients (90 sickle cell disease patients and 60 healthy age-matched controls) followed at the General Medicine Department of MGM Medical College, Indore. Glucose uptake, Hexokinase-2, pyruvate, lactate and 2,3 Bisphosphoglycerate levels were estimated in the RBC by relevant chemical kit methods on an autoanalyzer and enzyme-linked immunosorbent assay (ELISA), respectively. Serum IL-6 is estimated through the ELISA kit method. Statistical analysis was performed by using the Student's unpaired "t" test and Pearson's correlation test. P < 0.05 was considered statistically significant. Results: All glycolytic parameters were elevated along with IL-6 in sickle cell disease when compared with controls. A positive correlation was observed between the IL-6 level and glucose uptake (r = 0.345, P < 0.001), pyruvate (r = 0.512, P < 0.001) and lactate of RBC. Conclusion: This study shows that increased levels of plasma IL-6 might enhance the rate of glycolysis in RBC through the STAT3 pathway in sickle cell disease patients.
Keywords: Cytokines, glycolysis, inflammation, Rapaport-Lubering shunt, sickle cell disease
|How to cite this article:|
Sarkar PD, Agnihotram G, Skaria LK. Association of serum Interlukin-6 and glycolysis in sickle cell disease patients. Med J DY Patil Univ 2014;7:317-20
|How to cite this URL:|
Sarkar PD, Agnihotram G, Skaria LK. Association of serum Interlukin-6 and glycolysis in sickle cell disease patients. Med J DY Patil Univ [serial online] 2014 [cited 2021 Jan 19];7:317-20. Available from: https://www.mjdrdypu.org/text.asp?2014/7/3/317/128973
| Introduction|| |
Sickle cell disease is a prevalent hemoglobinopathy in the central India population; the haplotype, morphology and morbidity patterns are significantly different compared with the other parts of the world. The fetal hemoglobin levels are high in the Indian sickle cell haplotype, which gives some minor relief from chronic symptoms. But, in our previous studies, we observed that the severity of nociceptive pain and end organ damage is high in this group. Sickle cell disease is causing a chronic inflammation in the diseased population.
Inflammation is a complex defense mechanism in which leucocytes migrate from the vasculature into the damaged tissues to destroy the agents that can potentially cause tissue injury. The cytokines that are produced during the inflammatory processes, and those that participate in them, are stimulators of the production of acute phase proteins. These inflammation-associated cytokines include interleukin (IL)-6, IL-1, tumor necrosis factor, interferon, transforming growth factor and, possibly, IL-8. They are produced by a variety of cell types, but the most important sources are macrophages and monocytes at the inflammatory sites.  IL-6 is also considered a myokine, a cytokine produced from the muscle, and is elevated in response to muscle contraction.
IL-6 is an interlukin that acts as both a pro-inflammatory and an anti-inflammatory cytokine. In humans, it is encoded by the IL-6 gene.  IL-6 is secreted by T-cells and macrophages to stimulate immune response, e.g. during infection and after trauma, especially in patients with burns or other tissue damage leading to inflammation. IL-6 also plays a role in fighting infection, as IL-6 has been shown in mice to be required for resistance against the bacterium Streptococcus pneumoniae.
Glycolysis is a major oxidative pathway of glucose, which supplies energy to the normal maintenance of RBCs and provides 2,3 Bisphosphoglycerate through the Rapaport-Lubering shunt for ready dissociation of oxygen from oxyhemoglobin for the needs of the tissues. Very few studies have reported glycolysis and glycolytic parameters in sickle cell disease humans,  but no study has explained the relationship between the process of inflammation and glycolysis as well as the mechanism behind and the role of IL-6 in RBC glycolysis. In this study, we intend to study the enzymes, intermediates and products of glycolysis in the RBC of sickle cell disease patients. Although 2,3 Bisphosphoglycerate levels are high due to increased glycolytic metabolism in the sickle cell disease population, it is not very useful in managing the hypoxic conditions. Moreover, increasing the intracellular pH favors more sickling and polymerization, and this further increases the RBC anaerobic glycolysis in sickle cell disease. Therefore, this study was aimed at evaluating the role of IL-6, a chronic inflammatory marker with relation to the glycolytic markers of RBC metabolism in sickle cell disease patients.
| Materials and Methods|| |
Ninety sickle cell disease patients and 60 age-matched healthy controls (who visited the General Medicine Department in the MGM Medical College and Teaching Hospital at Indore, MP) were included in the present cross-sectional study. This work has been carried out between June 2012 and March 2013, and was approved by the Institutional Ethical Committee. Detailed explanation about the study was given to each individual and informed consent was taken from the population under study.
Sickle cell disease is diagnosed and confirmed by cellulose gel electrophoresis. Blood samples were collected from subjects under study in heparinized tubes by venipuncture. Blood was centrifuged at 3000 rpm for 10 min to separate plasma. Plasma was separated from the remaining cellular portion and used for the analysis of IL-6 estimation using an enzyme-linked immunosorbent assay (ELISA) kit  (Anogen Inc, Toronto, Canada). Then, the buffy coat was removed by aspiration. RBCs were subjected to washing two to three times and resuspended in normal saline and resuspended in autologous plasma, adjusting the packed cell volume to 30%. Each blood sample was divided into two parts. In one part, the 0-h glucose, pyruvate, lactate, 2,3 Bisphosphoglycerate and Hexokinase-2 were measured. The second part was incubated at 37 o C for 3 h. After incubation, estimation of glucose, pyruvate, lactate, Hexokinase-2 and 2,3 Bisphosphoglycerate were performed in the second sample. Glucose was estimated by the glucose oxidase-peroxidase (GOD-POD) method.  Pyruvate estimation was carried out by the Friedmann and Haugen method  and lactate estimation was performed by the Barker and Summerson method  using an autoanalyzer. 2,3 Bisphosphoglycerate was estimated using an ELISA kit  (Abnova Inc.; Catalogue no: KA1177).
Hexokinase was estimated essentially according to the method of Sharma et al. (1963), as modified by Gumaa and McLean (1972). For the estimation of Type-II activity, a portion of each fraction was heated at 45°C for 1 h  (Katzen and Schimke, 1965). The reaction mixture contained the following components: Tris-HCl buffer, 20 mM (pH 7.4); MgCl 2 , 8 mM (pH 7.0); NADP, 0.4 mM; ATP/Mg 2+ , 8 mM/2 mM (pH 7.2); and glucose, 5 mM and one unit of purified glucose-6-phosphate dehydrogenase. One unit of activity of Hexokinase was defined as the amount required to form 1 μmol of NADPH per minute at 25°C.
Analysis was performed using SSPS version 11. Glycolytic parameters of sickle cell disease patients and controls were compared by applying Student's unpaired "t"test. Correlations between plasma Levels of IL-6 and glucose uptake, pyruvate and lactate of RBCs were analyzed by applying Pearson's coefficient. P < 0.05 was considered statistically significant.
| Results|| |
The results of the study are expressed as mean ± SD. The glycolytic parameters (glucose uptake, pyruvate, lactate and 2,3 Bisphosphoglycerate levels) were increased along with the pro-inflammatory cytokine, IL-6, in sickle cell disease when compared with age-matched healthy controls [Table 1], [Figure 1] and [Figure 2].There was a positive correlation observed between the plasma levels of IL-6 and glucose uptake (r = 0.345, P < 0.001), pyruvate (r = 0.512, P < 0.001) and lactate (r = 0.438, P < 0.001) of RBC. The Hexokinase-2 isoenzyme measured was very minimal in normal human subjects; however, sickle cell disease patients showed an approximately two-fold increase in the levels.
|Figure 1: Showing the concentration of RBC glycolytic parameters in sickle cell disease and healthy age-matched control subjects|
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|Figure 2: Showing the plasma Interlukin-6 concentration with concentration of pyruvate formation and 2,3 Bisphosphoglycerate in sickle cell disease patients and controls|
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| Discussion|| |
IL-6, an acute phase protein, elevated markedly in the sickle cell disease population, showing that chronic inflammation had a relation with glycolysis. There is an ambiguity in studies related to the rapid RBC glycolysis process and altered hexose metabolism in sickle cell disease populations. There is no metabolic study carried out yet to reveal the association of IL-6 and glycolysis, particularly in this population in which the disease is mainly related to the RBC. 2,3 Bisphosphoglycerate concentrations are increased due to increased glycolsyis through the Rapaport-Lubering shunt. 
Previous studies in cancer patients with relation to the above mechanism revealed that there is a P53 gene induced by IL-6 that regulates the glycolytic metabolism in humans through an NF-kB-mediated mechanism,  which increases the Glut2 and Glut4 receptors on the cells and increases glycolysis. Recently, Ando et al.  proved that the IL-6-STAT3 pathway induces the glycolytic pathway and expression of glycolytic enzymes in cancer patients. Based on this, we conducted a study in the sickle cell disease population, and we strongly believe that IL-6 activates STAT3, which induces HIF1-α that is the major factor behind the elevation of Hexokinase-2.
In this study, we intended to fill the gaps in the basic metabolic mechanism study of RBC in a sickle cell disease population and tries to answer the ambiguity in the levels of glucose and altered hexose sugar metabolism. Overexpression of Hexokinase enzyme, which is a main inducer and regulator of the glycolysis pathway, was observed mainly in the population that had pulmonary hypertension with sickle cell disease.  Elevated glycolysis in the RBC causing elevated lactic acid and increased pH inside the RBC may have a relationship with dehydration of the RBC in the sickle cell disease population. Intracellular lactic acid elevated the levels further, elevating the Jacobs and Steward mechanism operating in the RBC and rapidly increasing the dehydration mechanism in the RBC. Elevated 2,3 Bisphosphoglycerate levels are further evidence for the rapid glycolysis in the RBC. But, we observed that elevated 2,3 Bisphosphoglycerate is highly elevated the pH of the RBC, creating a positive environment for polymerization of the sickled erythrocytes  [Figure 3]. Increasing the intracellular acidic environment further stimulated the rapid breakdown of RBC glucose through the anaerobin glycolytic pathway and lactic acid production. Increased glycolysis also reduced the availability of glucose-6-phosphate for the pentose phosphate pathway and further reduced the NADPH concentration in the RBC, which produced elevated levels of oxidized Glutathoine and severely affected the membrane integrity of the RBCs. In our study, we observed that end organ damage is rapid in people who had rapid glycolysis. Therefore, we are suggesting that further research is needed to investigate the role of therapeutic agents like 2-deoxyglucoseandarsenate compounds that reduce the 2,3 Biphosphoglycerate levels. This might improve the dehydration conditions of sickle cell disease. Regarding the role of IL-6 in glycolysis and its management by using anti-inflammatory agents, future studies are required to resolve the issue.
|Figure 3: Showing the relation between the HbS polymerization and intracellular acidic pH as the main reason for the RBC dehydration in sickle cell disease as per our proposed theory. Along with the Jacobs and Steward mechanism (J-S), elevated intracellular lactic acid also plays a key role in the depletion of K+ ion from the RBC and further causes rapid anaerobic glycolysis due to acidic pH (which again polymerize sickled RBC) in sickle cell disease (Virgilio and Robert, 2005)|
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| Conclusion|| |
This study highlights that patients with sickle cell disease had elevated plasma IL-6 levels, which could increase the rate of glycolysis in the RBC. Hence, these patients needed an effective therapeutic strategy for dehydration and deranged membrane integrity. Further, this association could be a causative factor for rapid extravascular hemolytic and end organ damage in this population.
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[Figure 1], [Figure 2], [Figure 3]