In vitro regulation of reactive oxygen species formation in red blood cells of homozygous sickle cell patients using Vitamin C

Statement of the problem

Nigeria has the highest number of people with sickle cell disease (SCD) in the world, with an estimated 91,000 babies born with the condition yearly¹⁸. This number is expected to grow. This means that Nigeria and other countries with a high disease burden will continue to require research to improve policies for prevention and management of sickle cell patients. Catastrophic financial burden has occurred in families of sickle cell patients evaluated in a study in Ekiti State, Nigeria¹⁹. Many of these families spent over 10% of family income to cover hospital admissions of an SCD patient and greater than 90% of these families had no health insurance and had to borrow to meet their financial needs. This study investigated a possible role for Vitamin C, a cheap drug, in reducing ROS formation in sickle cell patients.

Subject recruitment and collection of samples

Institutional approval was given by the Lagos University Teaching Hospital Health Research Ethics Committee (Assigned number: ADM/DCST/HREC/APP/1533). 27 HbSS patients were recruited from the Sickle Cell Clinic of Lagos University Teaching Hospital (LUTH) and LUTH clinic annex at Sickle Cell Foundation of Nigeria, Lagos Office between May 22–25, 2017. These patients were known HbSS patients documented on file and previously confirmed by hemoglobin electrophoresis. Every 3^rd HbSS patient who presented in the clinic and met the inclusion criteria was enrolled in the study after written informed consent was obtained in accordance with the Declaration of Helsinki. Afterwards, a screening questionnaire was applied to obtain each candidate’s demographics followed by weight and height measurements. Four volunteers declined height and weight assessment. 4mls of fresh blood was then drawn from the antecubital vein of each enrolled candidate into EDTA tubes. All samples were analyzed within 4 hours of collection.

Inclusion criteria. Patients of all ages who did not receive blood transfusion within 3 months and had not taken Vitamin C or multivitamin supplements within 3 months of the study were recruited. Patients taking hydroxyurea were included if they had been on a stable dose for at least 3 months.

Exclusion criteria. Blood transfusion within 3 months of study; acutely-ill patients; use of Vitamin C or multivitamin supplements within 3 months.

Materials and reagents

Gibco Phosphate-buffered saline (PBS; catalog number 20012043; Life Technologies, Grand Island, New York). CellRox Orange Flow Cytometry Assay kit (catalog number: C10493; Life Technologies). The kit includes the fluorophore, CellRox Orange reagent; N-acetyl cysteine (NAC; an antioxidant) and tert-butyl hydroperoxide solution (TBHP; to induce ROS). CellRox Orange reagent localizes to the cell cytoplasm and has absorption/emission maxima of 545/565 nm respectively²⁰. L-Ascorbic acid powder (CSPC Weisheng Pharmaceutical, China); a 200uM solution of Vitamin C was prepared in cold PBS and protected from light.

Sample preparation

Sample preparation and analysis were done at the Nigerian Institute of Medical Research - Human Virology Lab (NIMR-HVL), an ISO-certified lab in Yaba, Lagos. Pre-wash red cell counts for all samples were obtained from the hematology analyzer before centrifugation at 500xg for 10 minutes and supernatant removal. Cells were washed thrice in cold PBS and finally re-suspended in 5mls of PBS followed by determination of post-wash red cell count. This step was an adaptation of a previous process²¹.

Cell treatment

The protocol for this step was adapted from Life Technologies standardized protocol²⁰. For each sample, volume corresponding to 5 x 10⁵ cells was pipetted into four different micro-centrifuge tubes. One was immediately incubated with CellRox orange stain at a final concentration of 500nM and ROS quantified on the Partec Cyflow Counter Version 2.4 to determine basal ROS present per sample. Another tube was incubated with PBS and then stressed at final concentration of 200uM TBHP to serve as control. Two other tubes were pre-treated with 80uM and 100uM concentrations of Vitamin C respectively, before stressing with 200uM TBHP to represent the test samples. In a supplemental study, nine samples were non-randomly selected and incubated with 200 uM NAC followed by TBHP stressing. Incubation time per added reagent was 30 minutes.

Cell staining and flow cytometry

CellRox Orange was prepared with DMSO and then added to all the samples described above at a final concentration of 500nM. After 30 minutes of incubation, 800uL of the solution was transferred into Rohren tubes for immediate analysis on the flow cytometer. A total of 5 × 10⁵ cells were analysed and pulsed gating was used to exclude doublets.

Statistical analysis

The primary outcome measure was a comparison of the quantity of ROS formed following stressing of test vs control cells. Secondary analyses evaluated the relationship between gender, age, BMI and hydroxyurea use on basal ROS in sickle red cells. Microsoft Excel Version 14 was used for analysis. Analysis of variance (ANOVA) was used for statistical comparisons (significance defined by P values ≤ 0.05).

Characteristics of the participants

27 participants were recruited into the study. The sample was equally distributed between males and females (Table 1). Blood transfusion rate in this population was lower than among Congolese sickle cell patients²², but higher than for U.S Medicaid patients seen between 2007–2012²³. Hydroxyurea utilization was higher than among Florida Medicaid patients²⁴ and improved over a previous report from a major Nigerian Teaching Hospital²⁵.

Red cell count

Average pre-wash red cell count was 2.89×10¹²/L. The average male red cell count was higher than females (Table 2). Post-wash red cell count was calculated after final cell suspension. Volume equivalent of 5 x 10⁵ red cells was calculated from the post-wash red cell count (Table 2).

Relationship between baseline ROS count and patient characteristics

Baseline ROS count (BRC) refers to number of ROS per ul before any red cell intervention. Average total BRC was 441.9/ul (Table 2). When BRC was matched in terms of gender, men were found to have a significantly higher count than women, with average ROS count of 583.1/ul (95% CI, 373.1 to 793.1; p-value = 0.03; Table 3). Average BRC for those ≥ 20 years was higher than those <20 years; those with BMI ≥ 18.5 had higher average ROS than those <18.5; and those on hydroxyurea also had higher average basal ROS than those not taking hydroxyurea.

Baseline ROS prediction score

A score of 1 each was given to the male subjects, subjects on hydroxyurea at time of recruitment, age ≥20 years and for BMI ≥ 18.5. A score of 0 was applied in each case for women, those not on hydroxyurea, aged <20 years and BMI < 18.5 (Table 2). Average BRC for subjects with scores ≥ 3 were higher than those with scores 2, though not significant (p-value = 0.11). When each score cohort was matched in terms of gender, the average ROS production for men remained higher than for women without statistical signifance (Table 3).

Vitamin C regulation of ROS formation in stressed red cells

ROS count after TBHP stressing of test and matched controls were compared for each sample (Figure 1). The controls had a higher ROS count than test cells. As shown in Figure 2, at both concentrations of Vitamin C, there were significantly less ROS formation than controls (p-value<0.001). Average ROS count for 80uM test samples was 27.5/ul (95% CI, 17.5 to 72.5) and for 100uM test group, it was 3.9/ul (95% CI, 1.9 to 5.9). No statistical difference existed between Vitamin C pretreatment at 80uM and at 100uM (p-value = 0.31).

Figure 1. ROS count per sample matched for control and at 80uM and 100uM Vitamin C pretreatment.

Figure 2. Average ROS count for control and following 80uM and 100uM Vitamin C pretreatment.

NAC regulation of ROS formation – a supplemental analysis

We compared a subset of controls with NAC-pretreated cells. Samples 1 – 9 were pre-treated with 200uM NAC and the results compared with matched controls pretreated with PBS (Table 4 and Figure 3). NAC at 200uM final concentration reduced ROS formation compared to control. However, no statistical significance was noted (p-value = 0.12).

Figure 3. Average ROS count for control and following 200uM NAC pretreatment.

Individual characteristics and baseline reactive oxygen species count

Individually, age ≥ 20 years, BMI ≥ 18.5, hydroxyurea use and the male gender appear to be associated with increased baseline ROS count (BRC). In normal subjects, a statistically significant correlation was described by Kukovetz et al. between increasing age and generation of ROS²⁶. However, even though our study did not show a statistical significance between increasing age and increased BRC, an appropriate path would be to investigate this relationship further with larger patient cohorts. Furthermore, this study has revealed an association between BMI ≥ 18.5 and increased BRC. Previously, a relationship between increasing obesity and generation of serum ROS Japanese students was noted²⁷, while increased ROS production has been described in obese diabetics, particularly with abdominal obesity²⁸. We also found that patients on hydroxyurea had a higher average BRC than those not taking hydroxyurea. It has been shown that hydroxyurea mediated E. coli cell death by inducing envelope ROS formation leads to membrane damage and cell death^29,30. Elevated ROS formation has also been seen in hydroxyurea-treated yeast cell cytoplasm³¹. Hydroxyurea inhibits ribonucleotide reductase leading to a replication fork arrest³² and accumulation of ROS. Male gender is an independent factor statistically associated with increased BRC (p<0.05) in this study. Previous studies have shown that men have increased oxidative stress levels, more biomarkers of stress, elevated ROS production and lower antioxidant levels^33–36. Among sickle cell patients in a Camerounian Hospital, women had a greater total anti-oxidant capacity compared with males³⁷. Our data showed that, on average, men aged ≥ 20 years whose BMI were ≥ 18.5 and who were also on hydroxyurea produced more ROS. This is equivalent to Score 3 on the baseline ROS score described in the Results section. This group might be a target for an ROS-reducing agent. Further studies are required here.

Vitamin C regulation of ROS formation in stressed cells

A significant reduction in ROS formation for all test samples pretreated with 80uM and 100uM concentrations of Vitamin C prior to tert-butyl hydroperoxide stressing vs matched controls (P-values < 0.05) was noted. It is evident that Vitamin C inhibited formation of ROS formation in test cells and may have served to protect the red cell membrane from damage. Guaiquil et al. found that increased sensitivity of glutathione-depleted human myeloid cells to membrane damage was significantly reversed by preloading with Vitamin C³⁸. As noted earlier, there is a reduction in ROS formation in murine models with pulmonary contusion treated with Vitamin C¹¹. This finding is significant because if we can reduce ROS formation in sickle cell patients by simply administering Vitamin C, we would theoretically reduce cell injury and improve disease-free intervals for patients. Our study did not find a significant difference in ROS formation between cell cohorts pretreated at 80uM vs 100uM concentrations of Vitamin C. One sample pretreated with 80uM Vitamin C did not show as much reduction in ROS in the degrees seen with other samples (Figure 1). This effect was not noted at incubation with 100uM Vitamin C. This might be due to individual variation in response rates at 80uM concentration. It is possible that extending Vitamin C incubation time beyond 30 minutes might have yielded larger changes.

Rate of cell lysis between treated vs control was not compared in this study. It will be interesting to see if the treated cells were more resistant to lysis than matched controls. Additionally, controlled studies to examine the effect on basal ROS formation of administering Vitamin C to sickle cell patient cohorts over time would be appropriate.

Supplemental information

N-acetylcysteine did not significantly reduce ROS. When compared to Vitamin C, there was a significant difference in efficacy, with Vitamin C showing superiority over NAC. It may help to consider other concentrations of NAC in future experiments.