Abstract
The aim of the present study was to test the effects of Pfaffia paniculata (PP) extract on the red blood cell (RBC) rheological properties of patients with sickle cell disease (SCD) and healthy (AA) individuals. Blood from 7 SCD and 4 AA individuals were collected in EDTA tubes. Washed RBCs were incubated with various concentration of PP extract: 0.0, 0.2 or 0.5 mg/ml of PP solution for 5 hrs at 37°C. RBC deformability was measured by ektacytometry at 9 shear stresses ranging from 0.3 to 30 Pa, and RBC aggregation properties were determined by laser-backscattered techniques. Because RBCs from SCD patients are fragile, a stability test was also performed to test for the fragility of RBC exposed to a constant shear stress (70 Pa) for 10 min. While RBC deformability was not improved by the use of PP extract in AA, we noted an improvement of this parameter in patients with SCD between the 0.0 and 0.5 mg/ml conditions. In contrast to AA RBCs, the fragility of SCD RBCs was not affected by PP extract. In conclusion, this study demonstrates the beneficial effects, in-vitro, of PP extract on the RBC deformability of SCD patients, notably at high shear stress (a shear stress condition usually found in capillaries).
Introduction
Sickle cell disease (SCD) is the most frequent monogenic disease in the world. Patients with sickle cell anemia (SCA) and sickle cell-haemoglobin C disease (SCC) present severe and various medical complications caused, in part, by red blood cell (RBC) rheological alterations such as: reduced RBC deformability and increased RBC aggregates strength [19]. These abnormalities have been shown to modulate the clinical severity of the patients. In SCA, reduced RBC deformability is associated with the presence of leg ulcers [5] and glomerulopathy [12]. Moreover, enhanced haemoglobin S polymerization and RBC sickling further reduce the deformability of RBC, which may further impair blood flow into the microcirculation, hence causing vaso-occlusive crisis [2]. In SCC, Lemonne et al. [14] recently reported an association between reduced RBC deformability and the presence of retinopathy. Although the occurrence of these various complications involve the contribution of other factors [10], improvement of RBC rheology, and more particularly of RBC deformability, could improve the clinical status of SCD patients.
In 2000, Ballas tested the effects of a nutritional supplement (Nutritional Biotonix, Fort Lauderdale, FL, USA) containing Pfaffia paniculata (PP) extract [1]. PP (also known as Brazilian Ginseng) is a perennial wild plant of the Amaranthaceae family that grows in South America. The root powder of PP has long been used by South American Indians for a variety of aliments and has been reported to ameliorate the clinical picture of SCD in Brazil [7, 8]. Although the authors did not give large details about the initial clinical condition of the patients, they mentioned that those who received PP had less vaso-occlusive crises, acute chest syndromes, priapism or leg ulcers, and had improved quality of life [7, 8]. Ballas [1] was able to show that incubation of sickle RBC with nutritional supplement containing PP increased the hydration status of RBC (enhanced mean cell volume), which resulted in an improvement of RBC deformability. However, whether PP extract alone improves the deformability of sickle RBC, as well as the other haemorheological parameters, is unknown. The aim of the present study was to test the effects of PP extract powder isolated from nutritional supplement on the RBC rheological properties of SCD and healthy (AA) individuals.
Materials and methods
Preparation of PP solution and RBC incubation
PP dry powder was extracted from PP nutritional supplement [1] with methanol and ether (6:1). Fifty mg of PP dry powder was dissolved at 37°C in buffered saline solution containing potassium and glucose (BSKG: 134 mmol/l NaCl, 5 mmol/l KCl, 8.6 mmol/l Na2HPO4, 1.4 mmol/l NaH2PO4 and 11 mmol/l glucose, adjusted to 290–295 mosmol/kg and pH 7.4). Whole blood from 7 SCD (3 SCA, 1 S-β0-thalassemia, 3 SCC) and 4 AA individuals, who signed informed written consent, was sampled into EDTA tubes and centrifuged for separation of RBCs and plasma (10 min, 2,100 g, 25°C). SCD patients were at steady state as previously defined (no blood transfusions in the previous three months and absence of acute episodes (infection, vaso-occlusive like events, stroke, priapism) at least two months before inclusion into the study) [14]. Plasma was stored at 4°C and RBC pellet was washed 3 times with BSKG buffer and then re-suspended in the same buffer at haematocrit 12.5%. The RBC suspension was either incubated with 0.0 (CTL condition), 0.2 or 0.5 mg/ml of PP solution for 5 hrs at 37°C [1]. At the end of incubation, 25 μl of each suspension was re-suspended in 2 ml polyvinylpyrrolidone (PVP; viscosity = 30 cP) for RBC deformability and RBC stability measurements (see details below). The remaining part of each RBC suspension was centrifuged (10 min, 2,100 g, 25°C) and supernatant was removed and replaced by autologous plasma at haematocrit 30% for RBC aggregation and haematological measurements (see details below). This study was conducted in accordance with the guidelines set by the Declaration of Helsinki and was approved by our local Ethics Committee.
Haematological and RBC deformability measurements
Mean cell volume (MCV) and mean corpuscular haemoglobin concentration (MCHC) were determined using haematology analyzer (Max M-Retic, Coulter, USA).
RBC deformability was first determined at 37°C and at 9 shear stresses ranging from 0.3 to 30 Pa by laser diffraction analysis (ecktacytometry), using the Laser-assisted Optical Rotational Cell Analyzer (LORCA, RR Mechatronics, Hoorn, The Netherlands). The system has been described elsewhere in detail [3]. The RBC-PVP suspension was sheared into a Couette system made of glass. Recently, Rabai et al. [17] demonstrated that while changing the diffraction pattern size by altering the aperture within the camera lens of the LORCA did not affect the RBC deformability values in healthy individuals, it had a significant effect in SCD patients. In our computer system the longitudinal size of the diffraction pattern before running the incremental shear stress protocol was standardized at 4 cm on the monitor [17]. Then, each diffraction pattern obtained at the different shear stresses was analysed by the computer and an elongation index was calculated. An increase of the elongation index indicates greater RBC deformability. RBCs from SCD patients are known to be more fragile than those from healthy individuals [6]. Indeed, each RBC-PVP suspension was submitted to a stability test with RBC being exposed to a constant shear stress (70 Pa) for 10 min: RBC elongation index was measured every minute and a decrease indicated RBC fragmentation [3, 11]. This type of cell stability test has already been used in previous study [11].
The remaining part of each RBC-PVP suspension was used to re-assess RBC deformability at 9 higher shear stresses than in the previous condition (0.5 to 50 Pa), in SCD patients only. The size of the diffraction pattern before running the incremental shear stress protocol was standardized, at first 5.4 cm, where the pattern had a reliable shape presenting all of the RBC populations (i.e. rigid and deformable), and then, during a second measurement, at 3.8 cm [17]. A Lineweaver-Burk approach was used to estimate the maximum RBC deformability at infinite shear stress [4]. The difference between the two maximum RBC deformability obtained with the two standardized diffraction pattern size conditions has been recently shown to be highly correlated with the percentage of irreversibly sickle cells in SCD patients [17].
RBC aggregation
RBC aggregation properties were determined at 37°C by laser backscatter method, using the Laser-assisted Optical Rotational Cell Analyzer (LORCA, RR Mechatronics, Hoorn, The Netherlands), after adjustment of the Hct to 30% with autologous plasma [3]. Blood was inserted into the Couette system of the LORCA and after a short period of high shearing (800 s–1) to dissociate pre-existing RBC aggregates, shearing was stopped abruptly. The changes in laser backscatter intensity was monitored for 2 minutes (syllectogram) by a photodiode sensor incorporated into the LORCA. The amplitude and the half-time of the syllectogram are then used to calculate the RBC aggregation index. Then, RBC aggregates strength was determined using a re-iteration procedure [3]: 7 separate pre-defined shear rate between 7.5 s–1 and 800 s–1 were applied on the RBC suspension, with or without alternating disaggregation shear rate, to locate the minimal shear rate needed to prevent RBC aggregation.
The guidelines for international standardization in blood rheology techniques/measurements were strictly followed and RBC suspensions were pre-oxygenated for 10 min before measurements [3].
Statistical analysis
Analysis of variance (ANOVA) with and without repeated measurements (with Newman-Keulspost-hoc test to locate the differences) was used to test the effects of PP on the different haematological and haemorheological parameters in the two groups and between the two groups. Significance level was defined as p < 0.05. Analyses were conducted using SPSS (v. 20, IBM SPSS Statistics, Chicago, IL).
Results
Haematological and RBC deformability parameters
As reported in the Table 1, both MCV and MCHC were not significantly different between the two groups and were not affected by PP, whatever the concentration used.
Figure 1 shows the results for RBC deformability obtained with standardized pre-diffraction pattern size of 4 cm. RBC deformability was higher in AA compared to SCD individuals at every shear stress and whatever the PP concentration (p < 0.001). PP treatment did not affect RBC deformability in the AA group. In contrast, we observed higher RBC deformability in the SCD group when PP was used at 0.5 mg/ml in comparison with the CTL condition (i.e., 0 mg/ml) at shear stress of 9.49, 16.87 and 30 Pa (p < 0.05). Below 5.33 Pa, PP had no significant effect on RBC deformability in SCD patients.
The Fig. 2 shows the effects of PP on the maximum RBC deformability difference calculated from the method described above [17] in SCD patients. No significant difference was observed between the three PP concentrations.
RBC stability
The RBC deformability measured for 10 min under high shear stress was higher in AA subjects than in SCD patients at 0 and 0.2 mg/ml of PP (p ranging from <0.05 to 0.001; Fig. 3). However, at 0.5 mg/ml of PP, RBC deformability of AA was higher than the one of SCD patients from the beginning of the stability test until 300 s (p ranging from <0.05 to <0.01). Then, the two groups were not further different. This finding may be explained by the different effect of PP between the two groups. While PP used at 0 or 0.2 mg/ml decreased AA RBC deformability below baseline level (i.e., below the first value of the stability test) at 540 and 600 s only (p < 0.05 and p < 0.01, respectively), 0.5 mg/ml of PP decreased AA RBC deformability below baseline from 300 to 600 s (p ranging from <0.05 to 0.01). In contrast, PP had no effect on the evolution of RBC deformability during the stability test in the SCD group and, when used at 0.5 mg/ml, it increased RBC deformability at all time points in comparison with the 0 mg/ml condition (p < 0.05).
RBC aggregation
As shown in the Table 1, RBC aggregation index was not different between the two groups but the strength of RBC aggregates was higher in the SCD group compared to the AA group (p < 0.05). PP had no effect on the RBC aggregation properties of the two groups.
Discussion
The present study demonstrates the beneficial effects of PP on the RBC deformability of SCD patients, notably at high shear stress, without any changes in MCV and MCHC. The later finding contrasts with the previous study of Ballas [1] but the major difference is that, in the present study we used PP extract while in the experiment done by Ballas [1], a nutritional supplement containing PP was utilized. Nevertheless, in both cases, an improvement of RBC deformability was observed in SCD patients. De Araujo et al. [7, 8] showed that PP powder extract reversed the sickling effect of methabisulfide in-vitro. Moreover, the improvement of clinical status in SCD patients receiving PP was not accompanied by MCV or MCHC changes [7, 8]. Indeed, it can be suspected that PP could modulate RBC deformability in SCD patients by other mechanisms than RBC re-hydration. Besides, PP had no effect on the difference in maximum RBC deformability obtained with the method of Rabai et al. [17], which reflects no changes in the percentages of irreversibly sickle cells. Several compounds have been isolated from the roots of PP, including pfaffic acid (C29H44O3) and various derivatives as pfaffosides and saponins [15, 18]. Very few properties of PP have been described. However, it was shown that pfaffosides have remarkable inhibitory effects on the growth of cultured cell melanomas [16]. Moreover, Leal et al. [13] reported antioxidant activity in PP extracts. Whether antioxidant activity of PP is responsible for the improvement of RBC deformability in SCD patients require further studies. However, this antioxidant activity could be of value for SCD patients since we recently demonstrated that RBC from SCD patients have impaired anti-oxidant defense, increased reactive oxygen species content and, when submitted to in-vitro oxidative stress, the RBC rheological alterations, particularly RBC deformability and RBC disaggregation threshold, are of greater magnitude than in RBC from healthy individuals [9].
While PP at 0.5 mg/ml improved RBC deformability and did not affect RBC stability in SCD patients, we observed a deleterious effect of PP used at this concentration in the RBC of AA during the stability test. The important RBC fragmentation induced by the use of 0.5 mg/ml of PP in AA leads this group to reach the same RBC deformability of SCD patients at the end of stability test. The reasons of this adverse effect in AA individuals are unknown. Ballas [1] previously reported a decrease of RBC deformability in healthy individuals when PP was used at high concentration and this was attributed to an over-hydration of RBCs. However, in the present study, MCV and MCHC remained unchanged by PP treatment. Additional studies are needed to better understand this finding.
In conclusion, this study demonstrates the beneficial effects, in-vitro, of PP on the RBC deformability of SCD patients, notably at high shear stress; a shear stress condition usually found in capillaries. Future studies should investigate the effects of PP on SCD mouse models to test whether hypoxia induced vaso-occlusive events can be prevented by the use of PP.
Conflict of interest statement
The authors claim no conflict of interest.