Sickle cell disease (SCD) is an inherited disorder of haemoglobin synthesis that is determined by homozygosity of haemoglobin S (HbSS) and characterized by recurring episodes of vaso-occlusion, chronic inflammatory state, chronic haemolysis and progressive vasculopathy with resultant imbalance in the signalling mediated by nitric oxide (NO) (Sullivan et al, 2010).

NO is a powerful vasodilator that acts by preventing the adhesion of leucocytes to the endothelium, inhibiting the expression of adhesion molecules and acting as an anti-inflammatory substance (Kato et al 2007). In sickle cell anaemia (SCA), arginase – an enzyme present inside red blood cells, is released during haemolysis, catalysing the hydrolysis of L-arginine, the substrate for the production of NO in ornithine and urea, reducing the bioavailability of NO (Kato et al, 2007). The reduction of L-arginine also occurs following the increased consumption of NO as a result of the increase in reactive oxygen species (ROS), generated by the presence of free haemoglobin, ischaemic injury of recurrent reperfusion, pro-inflammatory state, and the high autoxidation of haemoglobin S (HbS) (Kato et al, 2007).

Hydroxycarbamide (HC; also known as hydroxyurea) is a cytotoxic, mutagenic, recombinogenic and antineoplastic agent. It has been used to treat SCA by increasing the synthesis of HbF and total haemoglobin and reducing haemolysis (-Morris et al, 2008). HC also acts to reduce the expression of adhesion molecules, with anti-inflammatory and anti-aggregating properties, contributing to the decrease of vaso-occlusive episodes and reducing the need for blood transfusions, the frequency of hospitalizations, and mortality rate (-Morris et al, 2008). HC has also been attributed to affect NO metabolism, increasing production thereof via the cGMP cycle and consequently increasing HbF (Morris et al, 2003). HC therapy increases utilization of the arginine substrate, for the production of NO, by the activity of NOS (Nahavandi et al, 2000).

L-arginine, a semi-essential amino acid, is a substrate for the endothelial nitric oxide synthase (eNOS) enzyme for the production of NO, and is reduced in SCA patients, thereby limiting the effectiveness of HC (Sullivan et al, 2010). The reduced overall bioavailability of arginine, measured by low plasma levels of L-arginine or L-ornithine, or L-ornithine and L-citrulline, is associated with increased mortality in SCA patients (Morris et al, 2005). The reduction of arginine levels in patients with SCA was demonstrated to be associated with endothelial damage, multiple organ injury, increased haemolysis and pulmonary hypertension, contributing to high mortality of these patients (Morris et al, 2008).

The present study aimed to evaluate a therapeutic proposal for treating SCA, including supplementation with L-arginine as an adjuvant drug for treatment with HC.

This was a randomized clinical trial that included 21 adult patients (9 men and 12 women, aged 20–40 years) with a clinical and laboratory diagnosis of SCD, confirmed by molecular biology, undergoing treatment with HC for more than one year at a referral university hospital in Fortaleza, Ceará, Brazil. After informed consent, the selected patients were randomly divided into Group I (Control group: HC only therapy, n = 09) and Group II (Study group; therapy with HC + L-arginine, n = 12), matched for age and sex. The patients selected for Group II were prescribed Reforgan® (L-Arginine 250 mg), with a dose of one pill daily for 90 d, as a supplement to Hydréia® (HC, dosage ranged from 500 to 1500 mg/day).

Group II patients (HC + L-arginine) showed a significant increase in nitrite levels, HbF and reticulocytes, when compared to both baseline values and the Group I (HC only) at the various time points tested, demonstrating that supplementation with L-arginine increased the bioavailability of this substrate, leading to a better response to treatment with HC, as seen in Fig 1 (Table 1).

Table 1. Evolution of haematological and oxidative stress parameters in SCA patients (n = 23)
 Group I (HC only)N = 9Group II (HC + L-arginine) N = 12 
ParametersBaselineWeek 4Week 8Week 12P-value
  1. Results are expressed as mean ± standard error of mean (SEM).

  2. a

    Friedman test.

  3. b

    anova Repeated Measures test. *P < 0·05.

  4. c

    Statistically significant difference between Group II at Week 12 compared with baseline (where the patient had not started Arginine therapy) and Group I.

  5. d

    Statistically significant difference between Week 12 and baseline.

  6. Bold values indicate statistical significance.

Oxidative stress
Nitrite (μ/mol)a3·0 ± 1·05c3·8 ± 1·07c6·4 ± 1·997·3 ± 1·911·0 ± 4·16c0·0365
Red blood cells (x 1012/l)b2·5 ± 0·142·6 ± 0·142·6 ± 0·132·7 ± 0·122·7 ± 0·120·3597
Haemoglobin (g/l)b91 ± 3·792 ± 4·196 ± 4·694 ± 3·495 ± 3·30·4730
Haematocrit (%)b26·5 ± 1·1426·4 ± 1·1127·2 ± 1·1927·6 ± 1·0027·5 ± 0·750·2167
MCV (fl)b103·0 ± 3·12102·4 ± 3·75102·7 ± 3·08102·8 ± 3·18102·0 ± 3·650·9446
White blood cells (x 109/l)b8·1 ± 0·678·0 ± 0·709·3 ± 0·872·4 ± 0·718·4 ± 0·490·2755
Neutrophils (x 109/l)b3·9 ± 0·484·1 ± 0·664·5 ± 0·693·9 ± 0·443·7 ± 0·360·6848
Platelets (x 109/l)b340·7 ± 32·2336·6 ± 22·72339·7 ± 24·39346·1 ± 20·85345·3 ± 23·290·9661
HbF (%)b14·9 ± 2·5414·9 ± 2·54d16·0 ± 2·4616·3 ± 2·4516·8 ± 2·59d0·0375
Reticulocytes (x 109/l)b179·7 ± 12·2c155·2 ± 21·73c201·1 ± 19·86201·2 ± 27·64249·6 ± 20·91c 0·0165

Figure 1. (A) Nitrite levels in sickle cell patients (SCD) treated with hydrocarbamide (HC), with or without supplementation of L-arginine for 0, 4, 8 and 12 weeks. (B) HbF levels in SCD patients treated with HC with or without supplementation of L-arginine for 0, 4, 8 and 12. (C) Reticulocyte count in SCD patients treated with HC, with or without supplementation of L-arginine for 0, 4, 8 and 12 weeks.

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The increase in the levels of nitrite and HbF in patients after 12 weeks of use of HC + L-arginine, in relation to the baseline, demonstrates that such association induces a better therapeutic response to HC and confirms that the action of HC involves an NO-dependent pathway causing increased consumption of L-arginine, which is reduced in SCA patients and limits their response to HC. Our results support those of previous studies, which demonstrated that the in vitro induction of HF in progenitor cells occurs via NOSand soluble guanylate cyclase (Strouse et al, 2008), that there is chronic depletion of arginine levels in knockout sickle cell mice, and when treated with L-arginine, there is an increase in NO levels (Dasgupta et al, 2006).

Patients using HC + L-arginine also showed an increase in reticulocyte counts at 12 weeks compared to the baseline, indicating that the arginine acts by stimulating erythropoiesis. Baliga et al (2010) evaluated the effect of the association of HC + L-arginine on the synthesis of fetal haemoglobin by erythroid colony-forming units, demonstrating excellent synthesis of HbF and minimal cytotoxicity induced by HC + L-arginine at doses of (0, 15, 25, 100 μ/mol) of HC and (0, 25, 50 and 100 μ/mol) of Arginine. Our results agree with those of Baliga et al (2010), suggesting that the induction of HbF synthesis is dependent upon the action of NO on the erythroid progenitor cells.

Our results demonstrate that arginine supplementation leads to a better response to treatment with HC, and is an important adjuvant drug that should be included in the therapeutic protocol of treatment of SCA.


  1. Top of page
  2. Acknowledgements
  3. References

This work was supported by National Council of Technological and Scientific Development (CNPq).


  1. Top of page
  2. Acknowledgements
  3. References
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