|Year : 2020 | Volume
| Issue : 1 | Page : 222-225
Nitrate assessment in patients with thalassemia and sickle cell anemia
Mohamed A Soliman1, Rasha I Noreldin1, Doaa M Elian2, Hamdia S Mohamed3
1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Pediatric, Faculty of Medicine, Menoufia University, Menoufia, Egypt
3 Department of Clinical Pathology, El-Shohada Central Hospital, Menoufia, Egypt
|Date of Submission||16-May-2018|
|Date of Decision||21-Jun-2018|
|Date of Acceptance||24-Jun-2018|
|Date of Web Publication||25-Mar-2020|
Hamdia S Mohamed
El-Shohada, Menoufia 32717
Source of Support: None, Conflict of Interest: None
To evaluate the plasma levels of nitrate in patients with thalassemia and sickle cell anemia.
The bioavailability of nitric oxide (NO) in patients with thalassemia and sickle cell anemia is decreased due to chronic hemolysis and consequently endothelial dysfunction that can result in the development of certain complications in both the diseases. Plasma concentration of nitrate is considered as an important reservoir for NO whose level can be a landmark for the bioavailability of NO.
Patients and methods
Our case–control study involved 93 patients aged 10–18 years divided into two groups, a case group composed of 73 patients; 44 patients with thalassemia and 29 patients with sickle cell anemia and 20 age-matched apparently healthy persons as a control group. Plasma levels of nitrate were evaluated by enzyme-linked immunosorbent assay.
The results of our study have revealed that the plasma levels of nitrate are significantly lower in the case group than the control group.
An evidence of decreased NO bioavailability proved by a decreased plasma nitrate is present in patients with thalassemia and sickle cell anemia that can be implicated for multiple complications in both the diseases.
Keywords: anemia, hypoxia, nitric oxide, sickle cell, thalassemia
|How to cite this article:|
Soliman MA, Noreldin RI, Elian DM, Mohamed HS. Nitrate assessment in patients with thalassemia and sickle cell anemia. Menoufia Med J 2020;33:222-5
|How to cite this URL:|
Soliman MA, Noreldin RI, Elian DM, Mohamed HS. Nitrate assessment in patients with thalassemia and sickle cell anemia. Menoufia Med J [serial online] 2020 [cited 2020 Mar 30];33:222-5. Available from: http://www.mmj.eg.net/text.asp?2020/33/1/222/281266
| Introduction|| |
Thalassemia is an autosomal recessive inherited blood disorder that can result in abnormal formation of hemoglobin. It is the most common genetic disorder worldwide and ∼7% of the world's populations are carriers of that hemoglobin disorder. Thalassemias usually result in underproduction of normal globin proteins, often through mutations in regulatory genes. Thalassemia syndrome can be classified into three subgroups according to the severity of clinical presentation: thalassemia major, thalassemia intermedia, and thalassemia minor. Sickle cell anemia is another autosomal recessive inherited blood disease characterized by the presence of homozygous hemoglobin, known as sickle hemoglobin. During hypoxic conditions, the red blood cell (RBC) becomes sickled and the resulting change in structure restricts circulation causing obstruction of the blood flow within the capillaries and early destruction of the cell. The two major characteristics are chronic hemolytic anemia and intermittent vaso-occlusion that results in tissue ischemia causing acute, severe pain episodes. Nitric oxide (NO) is a free radical molecule that was originally referred to the endothelium-derived relaxing factor because it acted as a potent vasodilator by elevating the amount of cyclic GMP in blood vessel walls resulting in a vasorelaxant effect. NO bioavailability in thalassemia and sickle cell anemia patients wasreported to be decreased as chronic hemolysis. Such diseases could elevate plasma cell free hemoglobin, which reacts rapidly with bioactive NO and nitrite to form nitrate ion. The hemolysis also causes a release of arginase from erythrocytes, causing degradation of the arginine substrate for the endothelial NO synthase. The depletion of NO and its metabolites could lead to the abnormal vasoconstriction and platelet activation, and be associated with the thalassemia and sickle cell anemia complications, such as platelet hyperactivity, thrombosis, pulmonary arterial hypertension, severe ischemic pain attacks, and priapism.
| Patients and Methods|| |
We designed a case–control study, which included 93 patients aged 10–18 years. Patients were divided into two groups, a patient group composed of 73 patients: 44 patients with thalassemia (group I), 29 patients with sickle cell anemia (group II), and 20 age-matched apparently healthy persons as a control group (group III). This work was approved by the scientific committee of Menoufia University and a written consent was obtained from everyone before rolling in this study. The study was held between December 2015 and December 2017. All patients in the patient group were subjected to full history taking, proper general examination, and routine laboratory investigations in the form of liver function tests like alanine transaminase and fractionated bilirubin, renal function tests like serum creatinine, blood urea, and complete blood picture, which includes RBC count, evaluation of the size and shape of the RBCs (red cell indices), and measurement of the type and relative amounts of hemoglobin present in RBC. Plasma levels of nitrate were estimated by enzyme-linked immunosorbent assay for every patient in both groups. The essential reagents required for enzyme-linked immunosorbent assay are high affinity and specificity antibodies (enzyme conjugated and immobilized) with different and distinct epitope recognition, in excess and native antigen. In this procedure, the immobilization takes place during the assay at the surface of a microplate well through the interaction of streptavidin coated on the well and exogenously added biotinylated monoclonal anti-antibody (product code: EK-048-56, Human Kisspeptin; Phoenix Pharmaceuticals Inc., Burlingame, California, USA). An interaction was achieved between a labeled antibody and a serum containing the native antigen in the form of a globule sandwich complex.
Data were fed to the computer and analyzed using IBM SPSS software package (version 11; IBM Corp., Armonk, New York, USA). Qualitative data were described using number and percent. Analysis of variance test and pairwise comparison were used to verify the normality of distribution. Quantitative data were described using range (minimum and maximum), mean, SD, and median. Significance of the obtained results was judged at the 5% level.
| Results|| |
Comparison between the studied groups according to demographic data (sex and age in years) is listed in [Table 1], which indicated that there are no significant differences between the age of all groups (mean age of group I is 13.41 ± 2.71 years; mean age of group II is 13.55 ± 2.38 years; mean age of group III is 13.70 ± 2.81 years; P = 0.916).
|Table 1: Comparison between the studied groups according to the demographic data|
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The study shows that the nitrate concentration in normal persons is 29.95 ± 5.81 (mean ± SD). When a normal person is compared with an individual with thalassemia, we found that concentration of nitrate in thalassemia is lower than 22.11 ± 7.40 μm/ml (mean ± SD). When compared thalassemia groups with sickle groups we found that concentration of nitrate in sickle groups is lower than thalassemia, which is 15.19 ± 4.98 μm/ml. All this indicates that the higher concentration of nitrate is present in normal individuals and the lower concentration is in sickle groups [Table 2].
|Table 2: Comparison between the studied groups according to the concentration of nitrate|
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Comparison between the studied groups is done according to renal function and liver function as shown in [Table 3]. It is not statistically significant in the three groups.
|Table 3: Comparison between the studied groups according to different parameters|
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Comparison between the studied groups according to direct bilirubin and total bilirubin as shown in [Table 3], which is statistically significant (mean concentration of direct bilirubin group I is 1.11 ± 0.44 mg/dl; mean concentration of direct bilirubin group II is 1.22 ± 0.49 mg/dl; mean concentration of direct bilirubin group III is 0.21 ± 0.11 mg/dl; P > 0.001). The mean concentration of total bilirubin group I is 1.75 ± 0.35 mg/dl; mean concentration of total bilirubin group II is 2.10 ± 0.30 mg/dl; mean concentration of total bilirubin group III is 0.93 ± 0.18 mg/dl; and P value is less than 0.001.
[Figure 1] indicates comparison between the studied groups according to the concentration of nitrate (μm/ml).
|Figure 1: Indicates comparison between the studied groups according to the concentration of nitrate.|
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| Discussion|| |
Thalassemia and sickle cell anemia are considered to be very common autosomal recessive inherited blood disorders that can result in formation of abnormal hemoglobin and subsequently chronic anemia,,,,,,,,. One of the most important neurotransmitters whose defective bioavailability can be implicated for the development of various complication, mainly vaso-occlusive ones, in these hemolytic syndromes is NO. NO is a free radical molecule with a variety of physiological effects as smooth muscle relaxation, regulation of kidney function, inflammation, and endocrine secretion. Nitrite and nitrate are not only the products of the metabolism of NO but also act as a reservoir. Hypoxia is known to reduce endothelial NO synthesis activity and expression. The endothelial dysfunction could contribute to a reduction in NO bioavailability and vascular complications in thalassemia; such as pulmonary arterial hypertension, platelet hyperactivity, and thromboembolism. Previous work has shown that NO decreases sickle RBC adhesion most likely by decreasing the action of endothelial cell P-selectin. We hypothesized that the plasma concentrations of nitrate, which is an indicator for the bioavailability of NO, might be decreased in patients with thalassemia and sickle cell anemia compared to the normal individuals. To this end, we designed our case–control study to compare between the plasma concentrations of nitrate in these patients (case group) and their age-matched normal ones (control group). The results of our study indicated that plasma concentrations of nitrates in the patients with sickle cell anemia and thalassemia are significantly lower in the case groups than in the control group. Our results indicate that under hypoxic, ischemic, and acidic conditions there is acceleration of reduction of nitrite to NO in blood, which catalyzes the negative feedback inhibition of nitrate synthesis from nitrite oxidation. Decreased endothelial NO synthesis and degradation of arginine substrate for NO synthesis in thalassemia stimulates synthesis of NO from reduction of nitrite and nitrate that leads to rapid consumption of them and reduction of plasma level of nitrite and nitrate in comparison to normal healthy individuals Another study for the aim showed that there are no significant differences regarding the concentrations of plasma nitrate between patients with thalassemia and their age-matched control group. When thalassemia was compared with sickle patients it was observed that nitrate concentration decreased in sickle patients, which explained that occlusive crisis are more common in sickle patients. Another important factor not evaluated in this study is the blood transfusion in the patient group, more commonly in thalassemia patients. This improves the concentrations of nitrate thereby improving the bioavailability of NO. However, in agreement with our results another study showed the baseline nitrate levels in erythrocytes of the moderate and severe thalassemia patients were lower than those of the control. The nitrate levels in whole blood decreased in the thalassemia and sickle groups compared with those in the control group. Our results showed that during NO synthesis, there is a key regulator, which determine the entry of nitrate into reduction to form NO or not, and determine the balance of action of hemoglobin to be NO scavenger or producer.
| Conclusion|| |
An evidence of decreased NO bioavailability proved by a decreased plasma nitrate is present in patients with thalassemia and sickle cell anemia that can be implicated for multiple complications in both diseases, so medication that increase concentration of nitrate can help in reduction of complications in these patients. However, further study and research are recommended.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]