Sickle cell disease (SCD) clinical outcomes vary widely from mild to severe and the disease has been associated with multi-organ damage and risk of early mortality [¹,²]. Acute and chronic clinical manifestations of SCD include vaso-occlusive pain episodes (VOE), impaired blood flow as a result of intravascular sickling in capillaries and small vessels, inflammation processes and high susceptibility to infection. Researchers have found a complex network of associations among laboratory analyses and clinical events predicting a probably risk of death [¹,³,⁴].

The sickle cell disease vaso-occlusive phenomenon has been described as a complex event with the participation of stressed reticulocytes, sickled erythrocytes, leukocytes, platelets and endothelium activation [²,^5-8]. Reactive oxygen species (ROS), scavenger molecules and nitric oxide (NO) play important roles as regulators of vascular homeostasis in SCD pathogenesis [⁹].

Several biomarkers have been associated with SCD clinical prognosis; some, such as fetal hemoglobin (HbF) concentration, leukocytes count and reticulocyte count are considered to be classic [²,⁵]. Recently, serum lactate dehydrogenase (LDH), a well-known marker of intravascular hemolysis, was described as a biomarker of prognosis in SCD [¹⁰]. It has been associated with nitric oxide resistance, priapism, leg ulceration, pulmonary hypertension, and death in SCD patients [¹¹].

We conducted a prospective study to investigate high-density lipoprotein cholesterol (HDL-C) levels, including also determination of total cholesterol, low-density lipoprotein cholesterol (LDL-C), very low-density lipoprotein cholesterol (VLDL-C) and triglycerides to test the hypothesis that they can be used as a marker of prognosis among steady-state sickle cell disease children. This potential biomarker and their association with others laboratory determination and medical history were investigated in order to identify sub-phenotypes associated with the disease.

First of all we compared the analyses of markers of intravascular hemolysis, hemolysis and hepatic involvement, leukocyte and platelet counts, renal involvement, lipid metabolism, inflammation and Hb profile in order to establish how much are the difference between those markers between control and patients groups (Table 1).

The present study analyzed levels of HDL-C in steady-state children with SCD. Children with SCD, even in steady-state, have differences in several biomarkers as compared to healthy age-matched children [¹³]. Those differences are related to numerous mechanisms associated with infection, inflammation and VOE in the disease [¹,²]. Several biomarkers associated with hemolysis, inflammation, renal metabolism, hepatic metabolism, and lipid metabolism in children with SCD and healthy subjects were studied, and the findings of normal concentrations of protein and globulin as well as the albumin/globulin ratio among the SCD patients suggest an absence of early severe liver cell damage in the studied group [¹³]. Normal levels of creatinine in the patient group confirm previous observations that an increased rate of creatinine secretion by dysfunctional renal tubules may lead to a falsely normal plasma creatinine and creatinine clearance. A more accurate evaluation of different aspects of SCD nephropathy, emphasizing proteinuria and hyperfiltration, needs to be developed in children in order to detect early renal alteration [^14-16].

Hypocholesterolemia has been described in SCD patients with significantly decreased LDL-C and HDL-C [^17-22] and has been also described for our group as a potential biomarker for SCD clinical severity [²³]. A negative association was found for HDL-C and VLDL-C, which was directly associated with triglycerides. Triglyceride-rich VLDL-C particles availability may play an important role in lipid oxidization in SCD patients. It has been suggested that VLDL-C is an important factor for atherosclerosis development. VLDL-C particles assemble by a complex process that includes an apolipoprotein B (apoB)-containing VLDL precursor and a VLDL-sized lipid droplet lacking apoB. Both particles fuse to produce a mature VLDL particle [²⁴]. The increase of triglycerides probably contributes to an increase in the hepatic production of VLDL-C, increasing the number of receptors for LDL-C that is extensively metabolized, decreasing its serum levels. However, the role of cholesterol and triglycerides and the regulation of assembly and production of VLDL-C are poorly understood.

A negative association was observed between LDH and HDL-C, showing that HDL-C, as measured by its concentration, may function as a prognostic marker of intravascular hemolysis and endothelial dysfunction given its anti-inflammatory, anti-oxidative, anti-aggregation, anti-coagulant and pro-fibrinolytic activities [²⁵,²⁶].

Sickle cell disease patients with higher HDL-C levels presented a low risk of hemolysis and endothelial dysfunction, including lower reticulocyte and erythroblast counts as well as a lower HbS concentration and it may be related to the high consumption of cholesterol due to acceleration of blood marrow cell production during hemolysis crisis. Sickle cell disease patients with higher HDL-C levels had lower leukocyte, monocyte and platelet counts as well as a lower concentration of hepatic and hemolytic markers and significantly lower VLDL-C, triglycerides and A1AT concentrations; this may reflect the action of the anti-inflammatory and anti-oxidative properties of this biomarker [²⁵,²⁶]. The high-density lipoprotein cholesterol removes excess cholesterol from peripheral tissues and transports it to the liver for excretion via bile by reverse cholesterol transport. The high-density lipoprotein is made up of several particles with different composition and function [²⁴,²⁷,²⁸].

Further confirmation of these associations came from comparing HDL-C concentrations and patients' clinical records, which revealed a higher occurrence of pneumonia and cardiac abnormalities among those with lower HDL-C levels. The results related to pneumonia risk can be explained by the production of auto-antibodies specific to oxidized phospholipids; these auto-antibodies have been shown to inhibit macrophage uptake of oxidized LDL and to provide protection against virulent pneumococcal infection [²⁹]. Low levels of HDL-C are an important cardiovascular risk factor, and HDL-C and apoA-I have been shown to decrease lesions and improve vascular reactivity in animal models of atherosclerosis and in humans; these changes may be due to the reduction of oxidized lipids and the enhancement of reverse cholesterol transport [³⁰]. The presence of pulmonary hypertension was shown to be associated with several laboratory test alterations [³¹]. Recent study has also demonstrated the important role of the apolipoprotein pathway and its association with endothelial dysfunction in SCD patients with pulmonary hypertension [³¹].

Patients with lower HDL-C levels were also likely to have had more blood transfusions; this can be linked with a more severe clinical course of disease, once that it is a therapeutic strategy used to prevent several clinical symptoms, such as stroke [¹].

It is well known that gallstones in patients with hemolytic anemia are said to be calcium bilirubinate stones. In view our results of correlation of cholesterol and triglycerides with hemolysis, we propose that the stones in SCD patients could be related directly to hemolysis and bilirubin generation, and indirectly to cholesterol and lipids and it could be a novel observation and needs to be confirmed by further studies. The association of acute-phase proteins and cholelithiasis may be explained by the response to stress due to traumatic injury or infection-related mechanisms including hypermetabolism and protein catabolism associated with a cytokine-driven inflammatory response.

In conclusion, we hypothesize that some SCD patients can have a specific dyslipidemic subphenotype characterized by low HDL-C with hypertriglyceridemia and high VLDL-C in association with other biomarkers, including those related to inflammation. This represents an important step toward a more reliable clinical prognosis. Additional studies are warranted to test this hypothesis and the probably mechanisms involved in this complex network of markers and their role in SCD pathogenesis.

SCD: Sickle cell disease; VOE: vaso-occlusive pain episodes; HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol; VLDL-C: very low-density lipoprotein cholesterol; LDH: lactate dehydrogenase; CRP: C-reactive protein; A1AT: alpha 1-antitripsin; ASO: antistreptolysin-O; HB: hemoglobin; HBF: hemoglobin fetal; HPLC: high performance liquid chromatography; RBC: red blood cells; MCV: mean cell volume; MCH: mean cell hemoglobin; MCHC: mean cell hemoglobin concentration; HBS: hemoglobin S; COLELIT: cholelithiasis.

The authors declare that they have no competing interests.

MOS performed experiments and analyzed the results; LR, MBC, JM, RDC, and AMA performed experiments; IML, VMLN and LR performed clinical evaluation of patients; MGR analyzed the results; and MSG was the principal investigator and takes primary responsibility for the paper, designed the research, analyzed the results and wrote the paper. All authors read and approved the final manuscript.

The authors would like to thank the subjects from HEMOBA and PHAR-UFBA who participated in the study. They are also grateful for the expertise provided by the staff of FIOCRUZ, HEMOBA and PHAR-UFBA. This work was supported by grants from the Brazilian National Council of Research (CNPq) (3065427/2007-5 and 484457/2007-1) (M.S.G.); the Foundation of Research and Extension of Bahia (FAPESB) (1431040053063 and 9073/2007) (M.S.G.); and MCD/CNPq/MS-SCTIE-DECIT (409800/2006-6), (M.S.G.). The sponsors of this study are public or nonprofit organizations that support science in general. They had no role in gathering analyzing, or interpreting the data.