INTRODUCTION:

Beta thalassemia trait (BTT), is one of the common monogenic disorders of hemoglobin synthesis characterized by decrease in beta chains with concomitant increased synthesis of alpha and gamma chains. The developing erythrocytes become more fragile due to increased alpha chains resulting in early membrane damage and ineffective erythropoiesis. These patients will be mostly clinically asymptomatic with persistent microcytosis and hypochromia. In addition to mild anemia, some of the patients may exhibit impaired immune status, mild inflammation and endothelial dysfunction¹. Morphological abnormalities of erythrocytes are common in peripheral blood smears of patients with BTT².

Membrane bound acetylcholinesterase (AChE) helps in the maintenance of the shape and size of RBCs. The enzyme activity decreases with any change in size and shape of the cells. Therefore,erythrocyte AChE activity is used as an index of membrane integrity³. Plasma butrylcholinesterase (BChE) is considered to be an indicator of low grade systemic inflammation⁴. Inflammatory stimuli not only decrease plasma BChE but also enhance the expression of intracellular adhesion molecule (ICAM-1:CD54). ICAM -1is a transmembrane glycoprotein present in the various cell types like vascular endothelial cells, whose expression is regulated by inflammatory cytokines⁵. Further, changes inplasma ICAM points to endothelial dysfunction. Chronic hemolysis, a hallmark of BTTis believed to cause endothelial dysfunction because products of hemolysis increase nitric oxide degradation and reduce nitric oxide synthesis⁶. Research on cholinesterases activity in BTT is scarce, hence the present study attempts to establish BChE and AChEashemolytic and inflammatory markers in BTT.

MATERIALS AND METHODS:

This study was approved by the Institutional Ethics Committee and informed consent was obtained from all the subjects. The study population consisted of 30 normal and 30 BTT patients of both sex aged between 20 to 50 years. Random samples were collected in EDTA vacuum tubes and centrifuged at 3500rpm for 10mins, Plasma BChEanderythrocyte AChE were determined by Ellman’s method in which cholinesteraseshydrolyse acetyl thiocholine into thiocholinethat reacts with dithionitrobenzoate, to form a yellow color, which is measured spectrphotometrically⁷. Plasma ICAM-1 level was estimated by ELISA based on sandwich principle⁸. Oxidative hemolysis of erythrocytes was determined by incubating RBC suspension with distilled water and saline for two hours⁹. Plasma total bilirubin andglobulin were estimated spectrophotometrically using fully automated autoanalyser Cobas 6000 c501. Hb variant analysis was performed in blood samples using cation –exchange HPLC in BIO RAD D-10.Patients were categorized as BTT only if they had HbA₂ >3.5 and/HbF>2. Patients withdiabetes mellitus, nutritional anemia and systemic illness were excluded from the study. Data were analyzed statistically by student-t- test using software IBM SPSS version 20.0. Pearson’s coefficient was calculated for correlation studies.p value<0.05 was considered as significant.

RESULTS:

Percentage hemolysis was significantly higher in patients with BTT compared to normal. Plasma bilirubin and globulinswere significantly elevated in BTT patients. When compared to controls an apparent decrease of acetylcholinesterase and HbA1C was observed in BTT patients. Further decrease in plasma butrylcholinesterasewas statisticallysignificant in BTT (p<0.0001) (Table1). In patients with BTT mean ICAM-1 wasslightly above that of normal individuals. (Table2) AChE andHbA1C showed a significant negative correlation with % Hemolysis (Table 3). A statisticallysignificant negative correlation was seen between cholinesterases and ICAM -1.Plasma globulins correlated negatively with cholinesterases (Table 4).

Table 1: Comparison of hemolytic parameters in normal and beta thalassemia trait patients

	Normal N=30	BTT N=30
% Hemolysis	1.813 ± 0.93	2.523 ± 1.42 *
AChE(U/L)	3305 ± 537	3114 ± 544
BChE (U/L)	1137 ± 89	970 ± 211 **
Bilirubin (mg/dl)	0.48 ± 0.14	0.64 ± 0.37 *
HbA1c (%)	5.47 ± 0.37	5.37 ± 0.39

* p<0.03, ** p<0.0001 different from normal

Table 2: Comparison of inflammatory parameters in normal and beta thalassemia trait patients

	Normal N=30	BTT N=30
BChE (U/L)	1137 ± 89	970 ± 211 **
AChE(U/L)	3305 ± 537	3114 ± 544
ICAM-1 (ng/ml)	999 ± 229	1002 ± 254
Globulins (g/dl)	2.8 ± 0.12	3.4 ± 0.24 **

** p=0.0001 different from normal

Table 3:Correlation of % hemolysis with other hemolytic markers in BTT

	% Hemolysis
	r value	p value
AChE	-0.337	0.049*
BChE	-0.46	0.014*
HbA1c	-0.252	0.195
Bilirubin	0.139	0.480

*statisticallysignificant

Table 4:Correlation of cholinesterases with inflammatory markers in BTT

	BChE		AChE
	r	P	r	p
ICAM-1 (ng/ml)	-0.097	0.622	-0.469	0.012*
Globulins (g/dl)	-0.100	0.614	-0.082	0.679

*statisticallysignificant

DISCUSSION:

β-thalassemia trait, is a clinically silent condition, characterized by mild anemia, hypochromia, microcytosis, anisocytosis, poikilocytosis and increased percentage of hemoglobin A₂¹⁰. Morphological abnormalities of erythrocytes in BTT may also include ovalocytes, elliptocytes, cells with basophilic stippling and irregularly contracted cells. Acetylcholinesterase, an enzyme located on the outer leaflet of erythrocyte membrane is a biomarker of membrane integrity and cell aging. It helps in the maintenance of the shape and size of RBCs. Several studieshave reported decreased AChE activity in newborn infants affected with ABO hemolytic disease, G6PD deficiencypatients with thalassemia major and autoimmune hemolytic anemia^{11, 12}. Hence it is logical to observe low erythrocyte AChE in BTT patients. However, Najjia¹³et al have reported significant increase in the activity of AChE in beta thalassemia major patients. AChE was also found to be lower in paroxysmal nocturnal hemoglobinuria and megaloblastic anemia^12,3In contrast to this, erythrocyte cholinesterase activity was found to be slightly elevated in spherocytosis, acquired hemolytic anemia, sickle cell disease, other microcytic and macrocytic anemias¹⁴. Decreased AChE observed in the current study is in confirmation with anegative correlation of AChE with HbF in one of the earlier studies¹⁵. Significantly high serum bilirubin corroborates increased hemolysis in BTT. Hemolysis is also known to cause falsely low HbA1c, which is substantiated in the present study. Both AchE and HbA1c correlated negatively with percentage hemolysis in patients with BTT. Hence, AchE may be considered as a hemolytic marker along with HbA1c. Further, AChE is the enzyme that executes acetylcholine breakdown which is a key anti-inflammatory molecule of cholinergic signaling. Peripheral inflammation is controlled by the cholinergic anti-inflammatory pathway which involves elevation of acetylcholine by the reduction of AChE activity as seen in BTT.BChE is seen to be dramatically reduced during systemic inflammation¹⁶. A significant decrease of plasma BChE supports the development of the inflammatory condition in BTT.

The excess of unpaired alpha chains in BTT leads to its denaturation and auto oxidation resulting in oxidative damage to RBCs. Denaturation and autooxidation of excess unpaired alpha chains result in oxidative damage of RBCs. Products of hemolysis subsequently, cause endothelial dysfunction and vasculopathy. Ferric hemoglobin released from hemolysed cellsinduces expression of ICAM-1, a pro-inflammatory molecule¹⁷. An apparent increase inICAM observed in the current studymay be due to enhanced hemolysis in BTT. Further, BTTpatients have increased number of circulating erythroblasts rich in ICAM-1 which is an endothelial activation molecule. Increased endothelial activation further enhances production of ICAM and VCAM production, culminating in vasculopathy. Interaction of ICAM with leukocyte function antigen is thought to play a major role in inflammation and pathogenesis of several diseases like asthma, psoriasis. ICAM inhibitors have shown to be therapeutic molecules for surmounting inflammation in these diseases¹⁸. Accumulating experimental evidence indicates increase in adhesion molecules inthalassemia major, thalassemia intermedia and sickle cell disease¹⁹. However, one of the earlier studies showed no correlation between ICAM and other inflammatory molecules in thalassemia patients²⁰. A marked decrease in cholinesterases with elevation in ICAM-1 in BTT supports the hypothesis that inflammation and endothelial dysfunction underlie the pathology of BTT also as in thalassemia major²¹. A negative correlation between globulins,the key inflammatory marker and cholinesterases in BTT, highlights the involvement of cholinesterases in inflammation. Further, cholinesterasesmay be considered novel hemolytic markers as they show a significant negative correlation with percentage hemolysis. On the basis of our findings, we conclude that cholinesterases play a key role in inflammation and can be considered as biomarkers of hemolysis in BTT.

ACKNOWLEDGEMENT:

We express our gratitude to the patients, authorities and the technicians of the institution and the associated hospitals for permitting us to undertake this research project.

REFERENCES:

1. Galanello R, Origa R. Beta-thalassemia. Orphanet J Rare Dis. 2010 May 21;5:11. doi: 10.1186/1750-1172-5-11.

2. Carolin K,Albert W, Manfred N, Christoph R. Red blood cell morphology in patients with beta thalassemia minor. Laboratoriums Medizin. 2017;41(1):49-52.

3. Saldanha C. Human Erythrocyte Acetylcholinesterase in Health and Disease. Molecules. 2017 Sep 8;22(9):1499. doi: 10.3390/molecules22091499.

4. Lampón N, Hermida-Cadahia EF, Riveiro A, Tutor JC. Association between butyrylcholinesterase activity and low-grade systemic inflammation. Ann Hepatol. 2012;11(3):356-63.

5. Figenschau SL, Knutsen E, Urbarova I, Fenton C, Elston B, Perander M, Mortensen ES, Fenton KA. ICAM1 expression is induced by proinflammatory cytokines and associated with TLS formation in aggressive breast cancer subtypes. Sci Rep. 2018 Aug 6;8(1):11720. doi: 10.1038/s41598-018-29604-2.

6. Triantafyllou AI, Farmakis DT, Lampropoulos KM, Karkalousos PL, Triantafyllou EA, Papingiotis G, Megalou A, Karpanou EA. Impact of β-thalassemia trait carrier state on inflammatory status in patients with newly diagnosed hypertension. J Cardiovasc Med (Hagerstown). 2019;20(5):284-289. doi: 10.2459/JCM.0000000000000787.

7. Poojary TL, Sudha K, Sowndarya K, Kumarachandra R, Durgarao Y. Biochemical role of zinc in dengue fever. J Nat ScBiol Med 2021;12:131 4.

8. Profumo E, Buttari B, D'Arcangelo D, Tinaburri L, Dettori MA, Fabbri D et al. The NutraceuticalDehydrozingerone and Its Dimer Counteract Inflammation- and Oxidative Stress-Induced Dysfunction of In Vitro Cultured Human Endothelial Cells: A Novel Perspective for the Prevention and Therapy of Atherosclerosis. Oxid Med Cell Longev. 2016;2016:1246485. doi: 10.1155/2016/1246485.

9. Sudha K, Rao AV, Rao S, Rao A. Lipid peroxidation, hemolysis and antioxidant enzymes of erythrocytes in stroke. Indian J PhysiolPharmacol. 2004;48(2):199-205.

10. RahamatUnissa, Bayyaram Monica, SowmyaKonakanchi, Rahul Darak, SandagallaLipiKeerthana, SaranyaArun Kumar. Thalassemia: A Review . Asian J. Pharm. Res. 2018; 8(3): 195-202. doi: 10.5958/2231-5691.2018.00034.5

11. J. InsiraSarbeen, GowriSethu. Glucose-6-Phosphate Dehydrogenase Deficiency. Research J. Pharm. and Tech. 8(6): June, 2015; Page 792-795. doi: 10.5958/0974-360X.2015.00127.4

12. Gupta S, Belle VS, KumbarakeriRajashekhar R, Jogi S, Prabhu RK. Correlation of Red Blood Cell Acetylcholinesterase Enzyme Activity with Various RBC Indices. Indian J ClinBiochem. 2018;33(4):445-449. doi: 10.1007/s12291-017-0691-0.

13. Naji NA, Saleh SS, Taher GN. Studying of oxidative stress and some biochemical parameters in patients with beta thalassemia major in Kirkuk city. IJBAR. 2018;42(2):239-251.

14. Castilhos LG, de Oliveira JS, Adefegha SA, Magni LP, Doleski PH, Abdalla FH, de Andrade CM, Leal DBR. Increased oxidative stress alters nucleosides metabolite levels in sickle cell anemia. Redox Rep. 2017;22(6):451-459. doi: 10.1080/13510002.2017.1288973.

15. Choremis C, Nicolopoulos D, Metaxotou K, Moschos A. Erythrocyte Cholinesterase Activity In Hemolytic Anemias. ActaPaediatr Scand. 1965;54:218-24. doi: 10.1111/j.1651-2227.1965.tb06365.x.

16. Zivkovic AR, Schmidt K, Sigl A, Decker SO, Brenner T, Hofer S. Reduced serum butyrylcholinesterase activity indicates severe systemic inflammation in critically ill patients. Mediators Inflamm. 2015;2015:274607. doi: 10.1155/2015/274607.

17. Erdei J, Tóth A, Nagy A, Nyakundi BB, Fejes Z, Nagy B Jr, Novák L, Bognár L, Balogh E, Paragh G, Kappelmayer J, Bácsi A, Jeney V. The Role of Hemoglobin Oxidation Products in Triggering Inflammatory Response UponIntraventricular Hemorrhage in Premature Infants. Front Immunol. 2020;11:228. doi: 10.3389/fimmu.2020.00228.

18. Monika Gaba, PunamGaba, Sarbjot Singh, NeelimaDhingra. Inhibition of LFA-1/ICAM-1 Interaction: A Therapeutic Strategy for Surmounting Inflammation. Asian J. Pharm. Res. 5(1): Jan.-Mar. 2015; Page 37-47. doi: 10.5958/2231-5691.2015.00006.4.

19. Cappellini MD. Coagulation in the pathophysiology of hemolytic anemias. Hematology Am SocHematolEduc Program. 2007:74-8. doi: 10.1182/asheducation-2007.1.74.

20. El-Kinawy NS, Andrawes NG. Endothelial and peripheral blood cell activation in beta thalassemia children. Egypt J Haematol 2012;37:156-61.DOI: 10.7123/01.EJH.0000416545.61549.0e

21. Aggeli C, Antoniades C, Cosma C, Chrysohoou C, Tousoulis D, Ladis V, Karageorga M, Pitsavos C, Stefanadis C. Endothelial dysfunction and inflammatory process in transfusion-dependent patients with beta-thalassemia major. Int J Cardiol. 2005;105(1):80-4. doi: 10.1016/j.ijcard.2004.12.025.

Received on 31.03.2022 Modified on 29.08.2022

Research J. Pharm. and Tech 2023; 16(7):3311-3313.

DOI: 10.52711/0974-360X.2023.00546