Edited by: Dmitry A. Fedosov, Forschungszentrum Jülich, Germany
Reviewed by: Gregory Barshtein, The Hebrew University of Jerusalem, Israel; Giovanna Tomaiuolo, University of Naples Federico II, Italy; Jean-Frédéric Brun, INSERM U1046 Physiologie et Médecine Expérimentale du Coeur et des Muscles, France
This article was submitted to Red Blood Cell Physiology, a section of the journal Frontiers in Physiology
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Red blood cell (RBC) deformability is an important determinant of the microcirculation. It is influenced by various hematological parameters but also by nitric oxide (NO) which is produced in RBC from
Red blood cell deformability describes the ability of the cells to reversibly change their shape in response to applied forces. This relevant characteristic enables the passage of RBC through blood vessels with diameters smaller than their own and thus, the supply of oxygen and nutrient to the surroundings (
Reduced deformability levels have been described for patients suffering from cardiovascular diseases, diabetes mellitus or sickle cell anemia (
The recent literature suggests that the regulation of RBC deformability is far more complex than previously believed and additional information on the regulation of RBC function is needed. All cited studies represent cross-sectional studies while knowledge regarding variation of RBC deformability and associated NO levels from longitudinal studies are missing but would help to understand the stability of RBC NO parameters and RBC deformability. Also, a possible effect of hormonal contraceptives on blood parameters, RBC deformability and related parameters is less known. Respective information would be beneficial to assess the possible impact of HC on RBC functional parameters which might help to further understand the regulation of RBC function. Thus, the present study aimed to examine RBC deformability, RBC NO species (nitrite and RxNO) and RBC
A series of experiments was conducted to investigate the study purposes. Experiment 1 aimed to investigate RBC function and related parameters during a 20-week study period in males and females ± HC. Experiment 2 aimed to outline a possible relation between estradiol levels and RBC deformability in females ± HC during one menstruation cycle. The protocols used in this study were approved by the ethics committee of the German Sport University Cologne. These protocols align with the Declaration of Helsinki and all participants gave written informed consent to participate in this study.
Anthropometric data of the study groups.
Female + HC | Female - HC | Male | |
---|---|---|---|
Age [years] | 26.0 (4.3) | 27.3 (7.3) | 24.5 (2.9) |
Height [m] | 1.71 (0.07) | 1.65 (0.04) | 1.83 (0.06) |
Weight [kg] | 67.3 (12.8) | 60.5 (7.9) | 82.2 (8.5) |
Twenty-seven healthy moderately trained subjects (15m/12f: 6 f + HC and 6 f-HC) were recruited. All subjects had a west European background, were non-smokers, and non-blood donors. Hormonal contraceptives used by the female volunteers were oral contraceptive pills consisting of a combination of ethinylestradiol and a member of the gestagen family:
Yasminelle [(Jenapharm GmbH & Co., KG, Jena, Germany): Drospirenon 3 mg/Ethinylestradiol 0.02 mg per pill/]; Chariva [(Gedeon Richter Pharma GmbH, Cologne, Germany): Chlormadinone acetate 2 mg/Ethinylestradiol 0.03 mg per pill]; Daylette [(EMRA-MED Arzneimittel GmbH, Trittau, Germany): Drospirenon 3 mg/Ethinylestradiol 0.02 mg per pill]; MAXIM [(Jenapharm GmbH & Co., KG, Jena, Germany): Dienogest 2 mg/Ethinylestradiol 0.03 mg per pill]; Valette (Jenapharm GmbH & Co., KG, Jena, Germany): Diogenest 2 mg/Ethinylestradiol 0.03 mg per pill. Length of menstrual cycle of Female + HC was 28 ± 0 days and of Female - HC was 28 ± 3.5 days.
Basal anthropometric parameters of the subjects are presented in Table
Training volume of participants during study period. Total training volume was recorded on a weekly basis and included both, resistance and endurance training.
Capillary blood was sampled form the finger pulp into sodium heparinized capillaries (EKF Diagnostic, Germany) once per week for a total of 20 weeks to measure RBC deformability as described below. Additionally, venous blood was sampled weeks 1, 4, 7, 11, 14, 17, and 20 to measure blood parameters, RBC nitrite, RxNO, and RBC
Numbers of red blood cells (RBC) [∗106/μl], white blood cells (WBC) [∗103/μl] and platelets (PLT) [103/μl], hematocrit [%], hemoglobin concentration [g/dl] and mean cellular volume (MCV) [fl] were determined using hematology analyzer Sysmex Digitana KX-21N (Sysmex, Switzerland).
Red blood cell deformability was measured using the Laser-assisted-optical-rotational cell analyser (LORCA; RR Mechatronics, Netherlands) as previously described (
Measurements of NO species were performed according to published protocols (
For nitrite measurements, RBC were placed on ice and methanol (VWR international, Germany) was added to the frozen samples and thoroughly mixed. Samples were centrifuged at 21,000
Twenty healthy moderately trained females (12 Female – HC and 8 Female + HC) were recruited. All females had a west European background, were non-smokers, and non-blood donors. Hormonal contraceptives used by the female volunteers were oral contraceptive pills and similar to the ones described in Experiment 1 “Participants.” Length of menstrual cycle of Female + HC was 28 ± 0 days and of Female - HC was 26.4 ± 3.2 days. Basal anthropometric parameters of Females - HC were: 24.5 ± 4.8 years; 1.7 ± 0.07 m; 62.14 ± 6.6 kg and of Females + HC were: 26.4 ± 3.9 years; 1.71 ± 0.08 m; 67.1 ± 3.8 kg. Average training volume per week was 3.0 ± 1.7 h/week of Female + HC and 2.2 ± 0.9 h/week Female - HC.
Blood sampling was scheduled daily at the same time of day for a whole menstruation cycle. The sampling started on the first day of menstruation. Upon arrival to the laboratory, females were asked to rest in supine position for 15 min prior to blood sampling. Capillary blood was sampled from the fingertip and anticoagulated using sodium heparin. For RBC deformability measurement, blood was diluted with PVP, transferred to the LORCA device and measured as described above. For 17β-estradiol measurement, 110 μl blood was sampled from the fingertip into a heparinized capillary, sealed and centrifuged at 1000
Statistical analyses of data and representation of data were performed using GraphPad Prism 6 software (United States). Data are expressed as mean ± SD and presented for each investigation day. Experiment 1: Whiskers plot comprising all data of the whole study period was additionally presented. Data were analyzed for differences between the three tested groups and during intervention period using Friedman-Test or Kruskal–Wallis-Test where appropriate. Linear regression analysis was performed to test for a relation between training hours and RBC deformability. For Experiment 2, paired
The tested parameters were presented for all investigation days of the study period and additionally, data of all investigation days were pooled and presented as Whiskers plot.
Red blood cell count was significantly higher in male participants compared to the two tested female groups for all investigation days (Figure
Differences in blood cell count of participants during study period.
Differences in RBC parameters between participants during intervention.
Maximum deformability variation of male and female subjects during investigation period. Individual values were presented along with mean values for
Individual RBC deformability data and mean values of the three tested groups were presented in Figure
Red blood cell deformability measured during a whole menstruation cycle suggest higher values in Female - HC compared to Female + HC. Significantly higher values were observed in Female - HC on days 2, 7, 9–16, 18, 20, and 22 (Figure
Hormone concentration of Female + HC was in the range between 8 and 30 pg/ml throughout the whole menstruation cycle while Female - HC showed high estradiol variation ranging from 22 to 40 pg/ml during follicular phase (days 1–10), between 90 and 100 pg/ml during ovulation (days 14–16) and between 60 and 70 pg/ml during mid luteal phase (day 21).
Female - HC showed higher 17β-estradiol levels on days 8–28 compared to Female + HC (Figure
Red blood cell nitrite concentration was highest in Female - HC (Figure
Measured
The hematological profiles of males and females are well known while only few studies reported gender differences of RBC deformability. Also, less is known about possible variations of blood profile, RBC deformability and related parameters over a longer period of time with a special regard on the influence of hormonal contraceptives on RBC hematological and hemorheological parameters which was thus, aim of the present study.
The key findings of the recent study indicate that blood parameters, RBC deformability and related parameters, such as NO and
Differences of the blood profile have been already reported between males and females. For instance,
Differences between men and women were also shown for RBC parameters, i.e., RBC count, hematocrit and hemoglobin concentration. Males show higher hematocrit levels than females (m: 41–50% vs. f: 35–45%) and higher hemoglobin concentrations (m: 13.5–17 g/dl vs. f: 12–16 g/dl) which was associated to higher testosterone levels in men which facilitates erythropoiesis, but also genetic differences in the erythropoietin gene and of its receptor have been reported between males and females (
Week to week comparison of the hematological profile indicate that the described gender differences were consistent throughout the study period thus representing general gender differences.
Maximum deformability and 17β-estradiol levels of Females – HC and Females + HC during a menstruation cycle.
Red blood cell deformability was described to be of major importance to transit the smallest capillaries for oxygen supply to the tissues, organs or working muscles. To the best of our knowledge, studies on long-term monitoring of RBC deformability are lacking. Thus, the recent investigation is the first to show low week-to-week intra-group variation of RBC deformability, thus suggesting preserved stability in constant situations. Also, variation in RBC deformability of Female - HC and Male during course of study was low while in Female + HC, RBC deformability increased over time. This increase was highly related to increasing training volume observed in this study group. While training hours of Female - HC and Male remained constant throughout the study period, training hours of Female + HC increased from 2.25 ± 0.61 h/week at week 1 to 5.46 ± 2.05 h/week at week 20 (
Differences of RBC nitrite and RBC RxNO levels between study subjects during study period.
Red blood cell (RBC)
Comparing RBC deformability of the recent investigation between the study groups revealed that values obtained for Females - HC were highest compared to male and Females + HC while values of males and Females + HC show similar results. A recent study by
The recent data also indicate an influence of hormonal contraception on RBC deformability. Female participants are often excluded from investigations because hormonal variations during the menstrual cycle were thought to impede the interpretation of the data and might explain varying conclusions from different studies. Serum estradiol is the most potent endogenous estrogen and estradiol levels of women taking hormonal contraceptives was shown to be in the range of 20–30 pg/ml throughout the month (
The results of the present study suggest long-term stability of hematological and hemorheological parameters at constant conditions. Alterations of the regular conditions, such as an increase of weekly training hours, leads to an increase in RBC deformability. Gender differences were observed for RBC dependent blood parameters but also contraceptive use of women was shown to affect the blood profile. Highest RBC deformability data were reported for Female - HC and most likely associated to higher RBC NO production. Differences between the female groups were highly probably related to differing estradiol levels suggested to affect NO synthesizing pathways. Thus, the present results add information on the complex regulation of RBC deformability and recommend to consider gender, training status and hormone levels to interpret basal blood parameters, RBC deformability and related NO data.
MG designed the study, collected and analyzed the data, performed statistics, and wrote the manuscript. JC, MK, and SS collected and analyzed the data. WB contributed to study design, interpretation of the data, and the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors would like to thank Anika Voß, Anke Schmitz, and Bianca Collins for excellent technical assistance.