Thalassemia is a genetic disease that occurs due to a decrease or absence of one or more globulin chains that make up the hemoglobin tetramer. Thalassemia is inherited in an autosomal recessive pattern.¹ There are production defects in various polypeptide chains (alpha, beta, gamma or delta), which differ clinically and biochemically. Beta-thalassemia minor is a carrier form of beta thalassemia with heterozygous genotype and mild anemia.¹ It is common in the Middle East and central Asia countries and Mediterranean countries like Turkey.²

Endothelial damage is an important part of the atherosclerotic process. In beta-thalassemia patients, it is known that an increase in iron accumulation due to increased hemolysis, transfusion and intestinal absorption, leads to a decrease in endothelial nitric oxide (NO) bioavailability, and consequently to endothelial dysfunction.³ The resulting oxygen radicals are bound and neutralized by thiols. Free disulfide bonds appear as a result of the reaction and turn into thiol again, leading to a thiol-disulfide homeostasis. Impaired balance causes endothelial dysfunction and atherosclerosis to begin.

It has been shown that the carotid intima media thickness (C-IMT) measurement, which is an objective indicator of both oxidative stress^{4 , 5} and subclinical atherosclerosis, is increased in patients with thalassemia major.^{6 - 8} The relationship of increased oxidative stress with increased C-IMT is clear in many diseases, including the beta-thalassemia major.^{9 , 10}

However, there are not many studies in the literature evaluating C-IMT or oxidative stress in beta-thalassemia minor patients. Only one study reported that both C-IMT value and oxidative stress levels were increased in a limited number of beta-thalassemia minor patients.^{11 , 12} It has been supported that IMT measurement can be predictive of cardiovascular events caused by atherosclerosis and useful in detecting subclinical atherosclerosis.^{13 - 17} Atherosclerosis is a disease that begins in childhood and primarily increases abdominal aortic IMT (A-IMT). Many diseases have an early A-IMT involvement without affecting the C-IMT.^{18 , 19} In this study, we investigated and the relationship between A-IMT, C-IMT and oxidative stress markers, and whether these parameters are changed in beta-thalassemia minor patients.

Our study was a single-center case-control study. The study was approved by the Ethics Committee of the Faculty of Medicine of the Cukurova University (April 13, 2018, meeting number: 76, decision number: 88). Consent of patients wishing to participate in the study was obtained.

The study data were compared between beta thalassemia minor patients and healthy controls. Cohen kappa were above 90% for all electrocardiographic and echocardiographic measurements – inter-observer and intra-observer variability for electrocardiogram (ECG): 96% and 98%, echocardiography: 97% and 98%, respectively). IMT measurements were successfully taken from all patients included in the study. All demographic and clinical data were found to be similar between the groups, except for heart rate, that was higher in beta-thalassemia minor patients. All biochemical parameters of the two groups were similar except for blood count parameters. Red blood cell count, hemoglobin, hematocrit and mean corpuscular volume were lower in beta thalassemia minor patients, and red blood cell distribution width was higher ( Table 1 ). NT, TT and NT/TT ratio were lower in the beta-thalassemia minor patients, and IMA, and the D/NT and D/TT ratios were higher than the healthy control group, serum D level was not different between the two groups ( Table 2 ). While A-IMT value was significantly higher in beta thalassemia minor patients, all C-IMT values were not different compared to healthy controls. A-IMT negatively correlated with the TT level. Linear regression analysis was performed with parameters significantly related to A-IMT measurement ( Table 3 ). Table 4 shows the correlation of A-IMT measurements with the clinical and laboratory parameters. A-IMT positively correlated with the systolic and diastolic blood pressures, NT, D and IMA levels, and the D/NT and D/TT ratios. In linear regression analyses, A-IMT was found to be independently associated with the IMA, and NT and TT levels. The strongest relationship was found between A-IMT and IMA ( Figure 3 ).

Table 1

	Beta thalassemia minor n=80	Healthy control group n=50	p
Age (years)	38.5 ± 13.9	38.4 ± 13.9	0.957
Female gender n (%)	53 (%66.2)	33 (%66)	0.977
Systolic blood pressure (mmHg)	116 ± 6.0	117 ± 6.8	0.399
Diastolic blood pressure (mmHg)	73.2 ± 3.6	74.3 ± 5.4	0.182
Heart rate (pulse/minute)	81.4 ± 11.3	67.3 ± 3.6	<0.001
Body mass index (kg/m2)	28.0 ± 2.5	27.4 ± 1.6	0.154
Urea (mg/dL)	23.4 ± 5.40	23.2 ± 5.3	0.800
Creatinine (mg/dL)	0.56 ± 0.16	0.55 ± 0.16	0.867
Glucose (mg/dL)	93.5 ±9.86	92.8 ± 10.5	0.677
Aspartate aminotransferase (u/L)	19 (12.8 – 21.7)	19.5 (12.4 – 21.7)	0.961
Alanine aminotransferase (u/L)	16.6 (12.1 – 19.1)	16.6 (12.1 – 19.1)	0.940
Triglyceride (mg/dL)	94 (82 – 167)	102 (82 – 171)	0.858
Low density lipoprotein cholesterol (mg/dL)	114 ± 27	115 ± 27	0.852
Thyroid stimulating hormone (uIU/dL)	1.74 ± 0.88	1.67 ± 0.92	0.679
High sensitive C reactive protein (mg/dL)	0.60 (0.30 – 0.90)	0.55 (0.30 – 0.90)	0.799
White Blood Cell Count (x10^6/μl)	7.54 ± 1.48	7.57 ± 1.51	0.903
Red Blood Cell Count (x10^6/μl)	5.51 ± 0.86	4.55 ± 0.41	< 0.001
Hemoglobin (g/dL)	11.6 ± 1.45	12.8 ± 1.22	< 0.001
Hematocrit (%)	35.8 ± 4.82	38.6 ± 3.75	< 0.001
Mean Corpuscular Volume (fL)	65.2 ± 8.1	84.8 ± 7.1	< 0.001
Red Blood Cell Distribution Width	16.8 ± 2.9	13.3 ± 1.5	< 0.001
Thrombocyte Count (x10^3/μl)	249 ± 69	249 ± 71	0.990

Table 2

	Beta thalassemia minor n=80	Healthy control group n=50	p
Native thiol (μmoL)	337 ± 52	384 ± 38	<0.001
Total thiol (μmoL)	350 ± 42	417 ± 35	<0.001
Disulfide (μmoL)	18.2 ± 7.40	16.6 ± 3.72	0.106
Disulfide / Native thiol ratio	6.08 ± 2.73	5.16 ± 1.23	0.010
Disulfide / total thiol ratio	5.32 ± 2.06	4.64 ± 1.21	0.020
Native thiol / total thiol ratio	89 ± 4.13	91 ± 2.25	0.026
Ischemia modified Albumin (absorbance unit)	0.70 ± 0.14	0.59 ± 0.06	<0.001

Table 3

	Talassemia menor n=80	Indivíduos sadios n=50	p
Aortic IMT(mm)	1.46 ± 0.37	1.23 ± 0.22	<0.001
Right common carotid IMT (mm)	0.57 ± 0.11	0.56 ± 0.11	0.943
Right internal carotid IMT (mm)	0.56 ± 0.13	0.56 ± 0.12	0.941
Left common carotid IMT (mm)	0.59 ± 0.12	0.58 ± 0.12	0.948
Left internal carotid IMT (mm)	0.56 ± 0.11	0.56 ± 0.10	0.940

IMT: intima-media thickness.

Table 4

	Correlation Analysis		Regression Analysis
	p	R	p	β
Systolic Blood Pressure (mmHg)	<0.001	0.701	0.127	0.113
Diastolic Blood Pressure (mmHg)	<0.001	0.720	0.127	0.089
Native thiol levels (μmoL)	<0.001	– 0.435	0.002	– 0.173
Total thiol levels (μmoL)	<0.001	– 0.721	<0.001	– 0.296
Disulfide levels (μmoL)	<0.001	0.609	0.223	0.118
Disulfide / Native thiol ratio	<0.001	0.621	0.455	0.021
Disulfide / total thiol ratio	<0.001	0.645	0.372	0.026
Native thiol / total thiol ratio	<0.001	– 0.670	0.787	– 0.270
Ischemia Modified Albumin levels (absorbance unit)	<0.001	0.784	<0.001	0.491

R²_adjusted= 0.666.

Figure 3

Our study gave a lot of new information to the literature about beta thalassemia minor. The first and the main finding was that A-IMT but not C-IMT values were increased in individuals with beta-thalassemia minor. This is the first study to evaluate and to demonstrate the increase in A-IMT in these patients. We also evaluated the thiol-disulfide balance and IMA levels for oxidative stress status and showed that it was increased in beta-thalassemia minor patients. In addition, increased A-IMT was positively correlated with IMA, one of the oxidative stress parameters, and negatively and closely related to TT and NT. Although the relationship between increased oxidative stress and increased IMT is known for many diseases other than beta-thalassemia minor, this is the first time that this association was shown in this group of individuals.

Oxidative stress is caused by the unbalance between the production of reactive oxygen species and the antioxidant system. One of the antioxidant mechanisms is the thiol-disulfide balance; the evaluation of this balance is critical for elucidating the effects of oxidative stress on the pathogenesis of diseases and evaluating responses to antioxidant treatments.²⁰ Studies have shown that an abnormal thiol-disulfide balance is involved in the pathogenesis of various diseases such as diabetes mellitus, cardiovascular diseases, malignancies, rheumatoid arthritis, Parkinson’s disease, celiac disease and other inflammatory bowel diseases, Alzheimer’s disease and multiple sclerosis.^{21 - 24} In our study, the dynamic thiol-disulfide balance was compared between beta-thalassemia minor individuals and healthy control group. In addition, the relationship between IMA and C-IMT, previously shown in beta-thalassemia major patients, was evaluated in beta-thalassemia minor individuals. Also, this is the first and only study to evaluate both IMA levels and thiol /D homeostasis in individuals with beta-thalassemia minor. While IMA levels, D/NT, and D/TT ratios were significantly higher in beta-thalassemia minor patients than the control group, NT and TT levels, and NT/ TT ratio were significantly lower than the control group. This may be explained by the presence of excess free alpha globin chains due to β-globin chain deficiency, leading to formation of superoxide and hydroxyl radicals and initiation of oxidative chain reactions.²⁵ Epidemiological studies and clinical trials have shown that C-IMT, determined by high-resolution B-mode ultrasonography, positively correlates with traditional cardiovascular risk factors, and can provide increased risk information. Ultrasonography for C-IMT evaluation is recommended by traditional guidelines on cardiovascular risk classification as a non-invasive screening method for subclinical atherosclerosis.^{14 - 17} In autopsy studies, the first atherosclerotic lesion was shown to start from the dorsal surface of the distal abdominal aorta.²⁶ Although the abdominal aorta is an artery prone to atherosclerosis, A-IMT has not been as extensively investigated as C-IMT. Studies have found a positive correlation between A-IMT and systolic blood pressure, heart rate, creatinine, thyroid stimulating hormone, insulin-like growth factor-1 and growth hormone levels. Examination of abdominal aortic atherosclerosis has the potential to provide important information for cardiovascular risk assessment. Current ultrasound devices and high-resolution probes allow clear visualization of the abdominal aorta and measurement of the A-IMT.^{18 , 19 , 27 - 29} It has been clearly shown that C-IMT measurement is increased in patients with beta-thalassemia major.^{6 - 8} However, as far as we know, IMT evaluation in beta-thalassemia minor patients was performed in a limited number of patients in only one study,¹¹ which reported that this group had increased C-IMT.¹¹ The most important reason for this may be that in beta-thalassemia minor patients, the risk of initiating a subclinical atherosclerotic process is lower than in beta-thalassemia major patients, and current clinical features are not at a level to increase IMT. In our study, C-IMT measurements were made from four different regions – right and left internal and external carotid artery, and it was found that IMT values were not different between beta-thalassemia minor and control groups. In the study by Gullu et al.,¹¹ IMT measurement was taken from the right common carotid artery only, and the number of beta thalassemia minor patients included in the study was half as our study.¹¹ Therefore, our results may be more meaningful than those of the previous report. However, to elucidate the relationship between the pathophysiology of beta-thalassemia minor and C-IMT, further studies are required. It is known that A-IMT is an earlier indicator of atherosclerotic diseases and risk factors for many diseases than C-IMT.^{18 , 19 , 27 - 29} In the literature, there is no study evaluating A-IMT in beta-thalassemia patients. In our study, A-IMT was found to be significantly greater in beta-thalassemia minor patients than in healthy controls. In recent studies on A-IMT as an early indicator of atherosclerosis, A-IMT increase without a C-IMT increase was found in patients with myocardial infarction, hyperparathyroidism, and diabetes mellitus in accordance with our study.^{18 , 19 , 30}

Paraoxonase-1 and oxidative status have been shown to be increased in beta-thalassemia major patients, contributing to the development of coronary artery disease and atherosclerotic plaque formation.³¹

In another study, it was shown that oxidative stress increases with decreased paraoxonase-1 activity in beta-thalassemia minor patients.¹² In addition, the prevalence of metabolic syndrome is relatively high in individuals with beta-thalassemia minor, which is also in accordance with our study, considering the contribution of metabolic syndrome to atherosclerosis.³² Another study also showed that individuals with beta-thalassemia minor are at twice the risk of diabetes and insulin resistance compared to the individuals without the disease.³³

In our study, beta-thalassemia minor patients had increased oxidative stress, with impaired thiol-disulfide hemostasis and increased IMA; and all these oxidative stress parameters were closely related to A-IMT. This finding proved that oxidative stress was associated with increased IMT in beta-thalassemia minor patients as well as in beta-thalassemia major patients.¹⁰

In the present study, we found that A-IMT, which can be evaluated non-invasively and reliably with abdominal ultrasound, was increased in patients with beta thalassemia minor. In addition, the levels of NT and TT were decreased and IMA levels were increased; the antioxidant mechanism and the prooxidant-antioxidant balance were deteriorated in favor of prooxidants. Similarly to the relationship between increased oxidative stress and elevated C-IMT reported in the literature, in our study, A-IMT was found to be closely related to increased oxidative stress. Also, the assessment of A-IMT may be a promising tool in the detection of subclinical atherosclerosis and in the evaluation of the oxidative stress status. Further studies with a long-term follow-up of beta thalassemia minor patients are warranted.