Clinical Hemorheology and Microcirculation 60 (2015) 241–251 DOI 10.3233/CH-141865 IOS Press
241
Cell-derived microparticles after exercise in individuals with G6PD Viangchan Makamas Chandaa , Duangdao Nantakomola , Daroonwan Suksomb and Attakorn Palasuwana,∗ a
Molecular Hematology Research Unit, Department of Clinical Microscopy, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand b Faculty of Sports Science, Chulalongkorn University, Bangkok, Thailand
Abstract. Glucose-6-phospate dehydrogenase (G6PD) deficient cells are sensitive to oxidative damage leading to the formation of microparticles (MPs). Therefore, we examined the concentration of MPs and changes in the antioxidant balance after an acute strenuous exercise (SEx) and moderate-intensity exercise (MEx). Eighteen healthy females (18–24 years) with G6PD normal and eighteen age-matched females with G6PD Viangchan (871G>A) were tested by running on a treadmill at their maximal oxygen uptake for SEx and at 75% of their maximal heart rate for MEx. It was found that SEx triggered the release of total microparticles (TTMPs) above baseline levels and remained significantly higher 45 minutes after the exercise in G6PD normal individuals. However, SEx-induced increase in TTMPs was significantly higher in G6PD Viangchan as compared to G6PD normal. In contrast, MEx did not to alter the release of TTMPs in both G6PD normal and Viangchan. Moreover, TTMPs concentrations were inversely correlated with G6PD activity (r = −0.82, P < 0.05) but positively correlated with MDA concentrations (r = 0.74, P < 0.05). Using cell specific antibodies, we determined that MPs were mainly derived from platelets and erythrocytes. Altogether, the present study indicates that G6PD Viangchan may participate in MEx without higher MPs concentration and oxidative stress compared with G6PD normal. Keywords: G6PD, antioxidant status, oxidative stress
1. Introduction Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the rate-determining step in the pentose phosphate pathway results in production of nicotinamide adenine dinucleotide phosphate (NADPH) and protects cells from oxidative stress [2, 5, 32]. Individuals affected by G6PD deficiency are unable to regenerate reduced glutathione (GSH) and are susceptible to damages caused by oxidative stress. G6PD deficiency is the most common human enzyme deficiency affecting over 400 million people worldwide. Genetic analysis showed over 140 types of G6PD mutations [5]. However, mutations in the G6PD gene differentially affect protein stability and enzyme activity [2, 12]. Previous studies characterized molecular mechanisms responsible for G6PD deficiency in several ethnic groups in Southeast Asia. In Thailand, the most common G6PD mutation is G6PD Viangchan, a substitution mutation of G6PD gene at position 871 from Guanine to Alanine (871G>A) [17, 25]. Unfortunately, most individuals with G6PD deficiency ∗
Corresponding author: Attakorn Palasuwan, Ph.D., Department of Clinical Microscopy, Faculty of Allied Health Sciences, Chulalongkorn University, Phayathai road, Pathumwan, Bangkok 10330, Thailand. Tel.: +66 2 218 1069; Fax: +66 2 218 3771; E-mails: [email protected], [email protected]. 1386-0291/15/$35.00 © 2015 – IOS Press and the authors. All rights reserved
242
M. Chanda et al. / MPs after exercise in G6PD Viangchan
are asymptomatic, but exposure to oxidative stressors can induce hemolysis and can be life threatening [5, 12]. It is well accepted that strenuous exercise increases oxygen consumption and perturb intracellular pro-oxidant/antioxidant homeostasis resulting in exercise-induced oxidative damages [8, 15]. Therefore, individuals with G6PD gene mutations are at a higher risk of oxidative stress induced erythrocyte damages, resulting in perturbation of hemostasis. The oxidative stress induces externalization of phosphatidylserine (PS) in the plasma membrane, a marker of cellular senescence and a marker for macrophages recognition signal for removal of damages cells from circulation [13, 16, 18]. Due to an imbalance of phospholipid asymmetry and externalization of PS upon apoptosis or cellular activation, cells release small membrane fragments with diameter less than 1.5 micron called cell-derived microparticles (MPs) [37]. MPs externalize anionic phospholipids, PS, and carry different set of proteins that specific for their originating cells on the surface [21, 34]. Recent studies showed that exercise increases the production of MPs [6, 29, 30]. Physical exercises affect clotting and fibrinolytic factors as well as platelet functions [9]. Therefore, MPs are a potential marker of cell damage and/or activation in individuals during exercise, and play a role in the pathophysiological changes caused by physical exercises. Little is known about how strenuous or moderate intensity exercise affects G6PD antioxidant responses, and formation of MPs from oxidative stress induced changes in cell membrane in individuals with G6PD Viangchan. Therefore, the purpose of our study is to investigate whether the concentration of MPs and prooxidant/antioxidant balance could be affected by strenuous exercise in individuals with G6PD Viangchan. We investigated concentration of circulating MPs and prooxidant/antioxidant status of acute strenuous exercise (SEx) and moderate-intensity exercise (MEx) on before, immediately after, and 45 minutes after exercises in both groups. 2. Materials and methods 2.1. Subjects Eighteen healthy females (18–24 years) with no mutation of G6PD gene (G6PD normal) and eighteen age-matched females with G6PD Viangchan (871G>A) were included in this study. Characteristics of the subjects were shown in Table 1. All subjects have been evaluated for general information, health status, habitual dietary intake, social demography, and general physical activities. All subjects were students with uniform lifestyle and had no routine physical exercise regimen for at least 3 months. Subjects with smoking, alcoholic, hypertension, renal, hepatic, asthma, cancer, diabetes, or any heart diseases were excluded. All subjects had not taken any antioxidant or food supplements for at least 6 months prior to the study. All subjects were normal weight, body fat, and body mass index. Informed consents were signed by all subjects included in this study. The protocol was approved by the Ethic Review Committee for Researches involving human subjects, health sciences group of Faculties, Colleges and Institutes, Chulalongkorn University, Thailand, in accordance with the International Conference on Harmonization - Good Clinical Practice (ICH-GCP). 2.2. Anthropometric data All subjects were investigated for their physical fitness as shown in Table 1. Resting heart rate was measured by a heart rate monitor (Polar Electro S810i, Finland). Body composition including body weight and percentage of body fat mass were measured using bioelectrical-impedance analyzer (Tanita, Model
M. Chanda et al. / MPs after exercise in G6PD Viangchan
243
Table 1 Characteristics of individuals with G6PD normal and G6PD Viangchan Variables Age (yrs) Height (m) Weight (kg) Body fat (%) BMI (kg/m2 ) HR (beats/min) HRmax (beats/min) ˙ 2max (kg/ml/min) VO
G6PD normal
G6PD Viangchan
21.1 ± 0.2 1.6 ± 0.0 51.2 ± 1.8 25.1 ± 1.1 20.2 ± 0.6 87.8 ± 3.2 171.0 ± 4.6 38.2 ± 3.3
20.7 ± 0.2 1.6 ± 0.0 50.4 ± 1.5 24.9 ± 1.1 19.4 ± 0.6 83.3 ± 3.0 166.8 ± 5.6 29.4 ± 1.8#
Values are expressed as means ± SE. # P < 0.05.
Inner Scan BC533, Japan). Each anthropometric reading has been made by a well-trained technician. For baseline measurement before exercise, all subjects’ blood sample were assayed for full blood counts for hematological analysis including erythrocyte count, leukocyte count, platelet count, hemoglobin (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) in all subjects were examined. 2.3. DNA extraction and identification of G6PD Viangchan (871G>A) mutations DNA was extracted from EDTA blood samples by using Qiaquick® Blood DNA extraction kit (Qiagen, Germany) according to manufacturer’s recommendations. For DNA analysis, we adopted previously reported screening strategy for the five known G6PD variants Thai population, including G6PD Mahidol 487, G>A; G6PD Viangchan 871, G>A; G6PD Kaiping 1388, G>A; G6PD Canton 1376, G>T; G6PD Union 1360, C>T. All samples were analysed with polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) as previously described [25]. Mutations in exons 6, 9, 11, 12, 13 of G6PD gene were confirmed by direct DNA sequencing. 2.4. The exercise protocol ˙ 2max ). SEx is an acute single bout strenuous exercise, designed to measure maximum oxygen uptake (VO ˙ 2max [4, 24] by increased speed and In this study, we used modified Bruce exercise protocol to measure VO slope level of treadmill (h/p/cosmos mercury® running machine, h/p/cosmos sports & medical GmbH, Germany) every three minute until exhaustion. Ventilation and gas exchanges were measured using a breath-by-breath gas exchange system (Cortex Metamax 3B, Germany). Heart rate (HR) during exercise was measured by the heart rate monitor (Polar S810i, Finland) to verify that the intensity of each exercise protocol had reached to maximal heart rate (HRmax ). Rating of Perceived Exertion (RPE) was measured every 3 minute to indicate fatigue by used Borg scale (1–10 score) [3]. For MEx, two weeks after the completion of completed SEx exercise protocol, all subjects were required to exercise for 45 minutes by running on treadmill at constant level of 75% of HRmax [14]. For the first ten minutes of exercise, we adjusted speed and slope level of treadmill to reach a constant level of 75% HRmax . Similar to SEx protocol, HR was measured every minute by the heart rate monitor (Polar S810i, Finland) and RPE was measured every 5 minute.
244
M. Chanda et al. / MPs after exercise in G6PD Viangchan
2.5. Blood sampling procedures On experimental day (7–14 days after subjects recruitment, samples and all informed consent forms were signed), blood samples were collected an hour before the exercise (Pre), immediately after the exercise (Post0) and 45 minutes after the conclusion of exercise (Post45). Blood samples were collected in tubes containing ethylene diamine tetra acetic acid tube (EDTA), lithium heparin tube (Li-heparin) and sodium citrate as anticoagulant at each time point. Complete blood counts (CBC), G6PD, superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities were determined in blood samples collected in tubes containing EDTA as anticoagulant. Measurement of total antioxidant status (TAS) and malondialdehyde (MDA) were assessed in plasma samples collected in tube containing Li-heparin as anticoagulant. Quantification and identification of MPs cellular origins were determined in blood samples collect in tubes containing 3.2% trisodium citrate as anticoagulant tube. All blood samples were kept at 4◦ C and processed within an hour. 2.6. Biological analyses 2.6.1. Hematological parameters All hematological parameters were determined by an automated hematology analyzer (Beckman Coulter Gen S, USA) within 1 hour after blood sample collection. The within-run of coefficient of variation % were less than 3% for all hematological parameters. 2.6.2. Antioxidant and oxidative stress markers Total antioxidant status (TAS) was measured by radical cation decolorization assay [26] using UV spectrophotometer (Shidmadzu UV-1601, Japan). Lipid peroxidation was assayed by monitoring formation of malondialdehyde (MDA) [27]. Erythrocytes superoxide dismutase (E-SOD), whole blood glutathione peroxidase (Wb-GPx) and erythrocyte glutathione peroxidase (E-GPx) activity were analysed by enzyme kinetic-colorimetric assay by Randox reagent kit (Randox Laboratories, UK). G6PD enzymatic activity was determined by using G6PD Randox kit (Randox Laboratories, UK), an enzyme kinetic-colorimetric assay. G6PD activities were expressed as international units per gram hemoglobin (IU/gHb). The within-run of coefficient of variation % were less than 5% for all parameters. 2.6.3. Quantitation and identification of microparticles (MPs) cellular origins Whole blood containing sodium citrate as anticoagulant was used to determine the level of MPs by flow cytometry as described previously [21]. Briefly, annexin V conjugated with Fluorescein isothioacyanate (AV-FITC) (BD PharminogenTM , Heidelberg, Germany) was used to detect PS on MPs’s surface. All annexin V positive-vesicles were defined as total microparticles (TTMPs). Mouse anti-Human CD41a and anti-glycophorin A conjugated with Phycoerythrin (PE) (BD PharminogenTM , Heidelberg, Germany) were used to differentially distinguish cellular origins of TTMPs that released from platelets and erythrocytes, respectively. Two microliters of citrated whole blood were mixed with 3l of FITC-conjugated annexin-V and either 3l of PE-conjugated glycophorin A or PE-conjugated CD41a. Reactions were incubated with 22l of binding buffer for 15 minutes at room temperature and protected from light. After incubation, 1 ml of annexin V binding buffer was added and quantified with flow cytometer using CELLQuestTM software (Becton Dickinson Biosciences, San Jose, CA) program. Subsequently, the absolute numbers of MPs were analyzed as described previously [21].
M. Chanda et al. / MPs after exercise in G6PD Viangchan
245
2.7. Statistical analysis All data in this study were presented as mean ± SE. One way ANOVA with repeated measurement were used. When significant changed were observed, LSD post-hoc test were applied to locate the source of difference. Statistical analyses were carried out using the SPSS 17.0 statistical program (SPSS Corporation, Chicago, IL). A P-value 0.05) (Table 1). Resting HR and HR max (HRmax ) after SEx ˙ 2max was significant lower in showed no significant different between the two group (P < 0.05) while VO G6PD Viangchan (P < 0.05) (Table 1). G6PD activity was significantly lower in individuals with G6PD Viangchan as compared to individuals with G6PD normal (P < 0.05). Plasma and whole blood volumes were calculated, based on Hb and Hct values, by Beaumont’s formula [33]. Post-exercise (Post0) and recovery (Post45) values of plasma TAS and MDA concentrations and MPs were corrected for change in plasma volume. 3.2. Hematological analysis Hematological parameter values of G6PD normal and Viangchan were summarized in Table 2. Before the start of each experiment, all subjects had normal hematological parameters. Immediately after exercise (Post0), hematological parameters were significantly increased including Hb, Hct, erythrocyte counts, leukocyte count, and platelet counts in all subjects (P < 0.05) and returned to basal levels within 45 minutes (Post45) in both individuals with G6PD normal and Viangchan. In contrast, MEx failed to alter any hematological parameters after the exercise in both groups (P > 0.05). 3.3. Antioxidant status and G6PD activity Table 3 shows results of antioxidant and the levels of G6PD activity. Significantly higher levels of E-SOD and E-GPx were found in individuals with G6PD Viangchan at the baseline and after SEx as compared to individuals with G6PD normal (P < 0.05). In addition, Wb-GPx and MDA levels were significantly increased at immediately after SEx (Post0) in individuals with G6PD Viangchan as compared to G6PD normal, and returned to baseline at 45 minutes after the exercise (Post45). Whereas, resting concentration of G6PD activity in individuals with G6PD Viangchan was significantly lower than G6PD normal and decreased continuously after exercise in SEx. In MEx, G6PD and E-GPX activity were significantly lower at the baseline and in all time points in individuals with G6PD Viangchan as compared to G6PD normal at 45 minutes after exercise (P < 0.05). 3.4. Circulating microparticles (MPs) Numbers of total microparticles (TTMPs) were quantified by using annexin V positive population, a specific marker for PS, with a size less than 1.0 m. Baseline concentrations of PS positive microparticles
246
M. Chanda et al. / MPs after exercise in G6PD Viangchan Table 2 Hematological parameters in individuals with G6PD normal and G6PD Viangchan in SEx and MEx
Variables
Time
G6PD normal SEx
Erythrocytes counts (×1012 L)
Hemoglobin (g/dL)
Hematocrit (%)
Leukocytes counts (×109 L)
Platelets counts (×109 L)
MCV
MCH
MCHC
Pre Post0 Post45 Pre Post0 Post45 Pre Post0 Post45 Pre Post0 Post45 Pre Post0 Post45 Pre Post0 Post45 Pre Post0 Post45 Pre Post0 Post45
4.4 ± 0.1 4.7 ± 0.1* 4.7 ± 0.2 12.9 ± 0.3 13.7 ± 0.4* 13.8 ± 0.6 38.4 ± 0.8 40.9 ± 1.1* 40.7 ± 1.6 6.3 ± 0.5 9.7 ± 0.6* 6.5 ± 0.4 252.2 ± 15 274.2 ± 15.2* 240.9 ± 13.9 86.9 ± 2.0 87.0 ± 2.0 86.7 ± 2.0 29.3 ± 0.8 29.2 ± 0.8 29.4 ± 0.8 33.7 ± 0.3 33.5 ± 0.2 33.9 ± 0.3
G6PD Viangchan MEx
4.4 ± 0.2 4.5 ± 0.1 4.4 ± 0.1 12.6 ± 0.3 12.4 ± 0.3 12.6 ± 0.3 36.7 ± 1.8 37.6 ± 0.8 37.9 ± 0.8 6.8 ± 0.4 7.1 ± 0.4 6.9 ± 0.4 244.2 ± 13.6 269.8 ± 14 261 ± 12.7 84.2 ± 2.4 84.6 ± 2.3 85.7 ± 2.4 28.7 ± 2.5 28.0 ± 0.9 28.5 ± 0.9 33.0 ± 3.7 33.1 ± 0.2 33.2 ± 0.2
SEx
MEx
4.4 ± 0.1 4.8 ± 0.2* 4.5 ± 0.1 12.4 ± 0.2 13.5 ± 0.4* 12.7 ± 0.3 37.2 ± 0.6 40.8 ± 1.2* 38.0 ± 0.8 6.2 ± 0.3 9.6 ± 0.4* 6.2 ± 0.3 245.1 ± 12.3 253.7 ± 12.6* 237.6 ± 11.5 85.9 ± 1.5 86.1 ± 1.4 85.7 ± 1.4 28.7 ± 0.6 28.5 ± 0.6 28.6 ± 0.6 33.4 ± 0.2 33.1 ± 0.2 33.4 ± 1.4
4.3 ± 0.1 4.4 ± 0.1 4.4 ± 0.1 12.1 ± 0.3 12.5 ± 0.4 12.5 ± 0.3 36.4 ± 0.8 37.7 ± 1.3 37.7 ± 0.9 6.1 ± 0.2 6.2 ± 0.2 6.1 ± 0.2 248.3 ± 15.5 240.4 ± 12.6 238.9 ± 11.8 84.4 ± 1.4 84.8 ± 1.5 84.7 ± 1.4 28.0 ± 0.5 28.2 ± 0.6 28.0 ± 0.5 33.1 ± 0.1 33.2 ± 0.1 33.1 ± 0.2
Values are expressed as means ± SE. ∗ Statistically significant differences as compared to pre-exercise (Pre) (P < 0.05).
were detected in individuals with G6PD normal and G6PD Viangchan before exercise. Average numbers of TTMPs detected in G6PD normal and G6PD Viangchan individuals were not significantly different [(Mean ± SE); 2587 ± 232 and 2859 ± 436 cells/l, respectively, (P = 0.47)]. Immediately after SEx (Post0), concentrations of TTMPs were significantly increased in individuals with G6PD Viangchan [8012 ± 256 MPs/L] as compared to individuals with G6PD normal [5716 ± 538 MPs/L, P < 0.05] and remained significantly higher 45 minutes after the exercise. In contrast, no significant changes in TTMP concentrations in both groups were detected after MEx (P > 0.05) (Fig. 1). Additionally, concentrations of TTMPs were inversely correlated with G6PD activity (r = −0.82, P < 0.05) while positively correlated with a concentration of lipid peroxidation marker (MDA) (r = 0.74, P < 0.05). In order to identify cellular origins of TTMPs, blood samples were stained simultaneously with FITC-conjugated annexin V along with PE-conjugated platelet integinµIIb (CD41a) or PE-conjugated glycophorin A monoclonal antibody. MPs from individuals with G6PD normal and G6PD Viangchan were mostly derived from platelets (Platelet derived microparticles, PMPs) (37% and 38%, respectively) and erythrocyte (erythrocyte derived microparticles, EMPs) (22% and 25%, respectively). However, cellular origins of MPs subpopulation from both G6PD normal and G6PD Viangchan individuals remained
M. Chanda et al. / MPs after exercise in G6PD Viangchan
247
Table 3 Antioxidants status markers and G6PD activity Variables
SOD (IU/g Hb)
E-GPx(IU/g Hb)
wb-GPx(IU/g Hb)
MDA (mmol/l)
TAS (mM troloxEq)
G6PD activity (IU/g Hb)
Time
Pre Post0 Post45 Pre Post0 Post45 Pre Post0 Post45 Pre Post0 Post45 Pre Post0 Post45 Pre Post0 Post45
G6PD normal
G6PD Viangchan
SEx
MEx
SEx
MEx
3192.2 ± 95.9 2875.0 ± 102.4 2630.5 ± 138.0∗ 43.4 ± 2.0 47.0 ± 3.4 42.0 ± 2.9 67.5 ± 3.7 69.1 ± 3.3 63.0 ± 3.4 5.6 ± 0.4 5.8 ± 0.4 5.4 ± 0.4 2.0 ± 0.0 1.9 ± 0.1 2.0 ± 0.1 7606.1 ± 430.6 7907.0 ± 247.4 7518.9 ± 293.5
3690.5 ± 125.3 3869 ± 90.4 3489 ± 192.5 69.5 ± 4.0 65.2 ± 4.1 63.0 ± 1.6 63.6 ± 5.0 56.4 ± 5.1 59.0 ± 4.9 3.2 ± 0.1 3.1 ± 0.1 3.2 ± 0.1 1.6 ± 0.0 1.5 ± 0.0 1.6 ± 0.0 5967.2 ± 101.8 6950.4 ± 380.1 6723.4 ± 415.3
3538.7 ± 69.7# 3192.1 ± 114.0+ 2847.1 ± 122.2+ 54.7 ± 4.0# 53.5 ± 4.7# 36.1 ± 2.1+ 75.8 ± 5.4 81.5 ± 4.9# 70.4 ± 5.0 5.5 ± 0.4 8.3 ± 0.3# 5.3 ± 0.4 2.0 ± 0.0 1.9 ± 0.1 2.0 ± 0.1 5557.1 ± 573.3# 4718.4 ± 557.2# 4560.5 ± 566.7#
3327.1 ± 136.0 3677.7 ± 48.5 3593.4 ± 143.4 59.3 ± 6.3 61.8 ± 7.3 46.7 ± 4.0# 72.3 ± 4.7 71.5 ± 6.5 71.2 ± 7.2 3.3 ± 0.1 3.2 ± 0.1 3.2 ± 0.1 1.7 ± 0.1 1.7 ± 0.1 1.7 ± 0.1 3937.1 ± 469.4# 4269.0 ± 498.4# 4185.8 ± 610.4#
Values are expressed as means ± SE. ∗ Statistically significant difference comparing preexcercise within the same group and in the same exercise protocol (P < 0.05). # Statistically significant difference comparing control group at the same time points and in the same exercise protocol (P < 0.05). + Statistically significant difference comparing control group at the similar time points and baseline level within the same exercise (P < 0.05).
Fig. 1. Absolute count numbers of total cell derived microparticles (TTMPs) (A), platelet derived microparticles (PMPs) (B), erythrocyte derived microparticles (EMPs) (C), at baseline (Pre; white bar), immediately post exercise (Post 0; gray bar), and 45 minutes post exercise (Post45; black bar) by SEx vs MEx. ∗ Statistically significant difference comparing pre-exercise (Pre) within the same group and in the same exercise protocol (P < 0.05). # Statistically significant difference comparing control group at similar time points and pre-exercise in the same exercise protocol (P < 0.05).
unidentified (41% and 37%, respectively). At resting state, average numbers of MPs in individuals with G6PD normal released from platelets and erythrocytes were 926 ± 132 MPs/l, and 385 ± 98 MPs/l, respectively. Whereas, average numbers of MPs in individuals with G6PD Viangchan released from
248
M. Chanda et al. / MPs after exercise in G6PD Viangchan
platelets and erythrocytes were 1106 ± 345 MPs/L, and 423 ± 221 MPs/l, respectively. Concentrations of PMPs and EMPs significantly increased immediately after exercise and remained significantly higher above baseline levels at 45 minutes post exercise in both G6PD normal and Viangchan (P < 0.05). No significant differences in any of EMPs and PMPs concentration between the two groups over all time points were detected in MEx (Fig. 1).
4. Discussion NADPH, produced by G6PD, is an important molecule for cellular antioxidant system. Many studies have shown that partial or complete inhibition of G6PD activity renders cells extremely sensitive to oxidative stress [1, 31, 36]. Many studies showed that eukaryotic cells shed MPs in response to oxidative stress or apoptosis, and exercise increases the production of circulating MPs [6, 29, 30]. However how strenuous exercise affects antioxidant balance and production of MPs in G6PD Viangchan is still unclear. Using flow cytometry, we found that strenuous exercise triggered the release of TTMPs to above baseline levels right after strenuous exercise and remained elevated 45 minutes after the exercise. An increase in TTMPs concentration was significantly higher in individuals with G6PD Viangchan as compared to G6PD normal. Therefore, activity of G6PD enzyme is likely to be an important contributor to this increase. Chen et al. demonstrated that shear stress is a major contributing factor of MP release during physical exercise [7]. Additionally, an imbalance of antioxidant system induced by oxidative stress during exercise is also a contributing factor for MPs release [15]. Thus, individuals with G6PD mutation are at a higher risk of oxidative stress induced cell damages. After acute bout of exercise, antioxidant parameters including SOD, wb-GPx, E-GPx and G6PD activities were significant decreased in individuals with G6PD Viangchan, while no changes in these parameters in individuals with G6PD normal. Our data suggested that a reduction in several antioxidant parameters could be due to low G6PD activity in individuals with Viangchan. Additionally, G6PD knockout mice were highly sensitive to deleterious effects of oxidative stress as evidence in a decrease in NADPH levels, a decrease in intracellular GSH levels and an increase in marker of lipid peroxidation [36]. Consistent with our data, MDA levels, a lipid peroxidation marker, were significantly higher in individuals with G6PD Viangchan after strenuous exercise [SEx] as compared to G6PD normal. Higher lipid peroxidation could be an important contributor for MPs shedding after acute strenuous exercise, and plasma MDA levels were positively correlated with MPs concentrations. In contrast, no significant difference in MPs concentration and antioxidant parameters were found in individuals with G6PD normal and Viangchan after moderate exercise (MEx). These results suggested that exercise duration and intensity are important determinants of exercise-induced changes in antioxidant status and the release of MPs. According to our data, individuals with G6PD Viangchan should exercise at moderate intensity or at 75% of their maximum heart rate for up to 45 minutes without any alterations in MPs concentrations and redox status. MPs from healthy subjects derived mostly from platelets (37%) and erythrocytes (22%). Analysis of MPs cellular origins from individuals with G6PD Viangchan showed similar cellular origins as individuals with G6PD normal [platelets (38%) and erythrocytes (25%), respectively]. However, cellular origins of up to 50% of circulating MPs remained unknown. In this study, PMPs were detected by using monoclonal antibody to glycoprotein (GP) IIb-IIIa (CD41a). It is possible that some PMPs did not have sufficient CD41a antigen or carried other PMPs surface antigens such as, GPIX (CD42a), GPIb (CD42b), or GPIIa (CD61) [28]. Additional studies will be needed to reveal identities of these unidentified antigens. In contrast to variations of antigens on platelet membrane, the majority of erythrocytes carry Glycophorin A
M. Chanda et al. / MPs after exercise in G6PD Viangchan
249
(CD235a) which is the most established marker for identification of EMPs. However, we did not stained all antigen derived from leukocyte and endothelial cell which may account for additional cell origins for unidentified MPs. Further assessment of cellular origins of these unidentified MPs will be required in future study. Our study demonstrated that strenuous exercise caused an increase in PMPs concentrations. This finding is consistent with a previous study showing that PMPs and EMPs were significantly increased after strenuous exercise [6, 19]. Activation of platelets during exercise has previously been described and could be responsible for an increase in PMPs production [35]. Although how platelet activation occur during exercise remains unknown. It is likely that a significant increase in shear stress during physical exercise may initiate PMPs formation [20]. Additionally, PMPs may have procoagulant activity due to externalization of negatively charged membrane phosphatidylserine phospholipids and may be one of the factors responsible for dissemination of prothrombotic activity [23, 34]. The procoagulant activity of PMPs may contribute to activation of coagulation that usually occurs during exercise [10]. However, how PMPs involve in the pathophysiology of exercise-induced alterations in hemostasis will need to be further investigated. In addition to PMPs, MPs shed from erythrocytes have also been found in individuals with G6PD Viangchan after strenuous exercise (SEx). Our data also showed that there were significantly higher levels of EMPs after strenuous exercise (SEx) in individuals with G6PD Viangchan. No significant differences in levels of PMPs and EMPs from G6PD normal and G6PD Viangchan were detected after moderate exercise (MEx). Erythrocyte membrane undergoes vesiculation under a variety of conditions, including disruption of asymmetric organization of membrane phospholipids, increased lipid peroxidation and increased ROS. All of these conditions eventually lead to loss of erythrocyte membranes and MPs formation which are characteristic of alteration in hemostasis after exercise [7, 22, 29]. Since exercise could induce changes in erythrocyte membrane, individuals with G6PD Viangchan are at a higher risk of oxidative stress induced erythrocyte damages after strenuous exercise. Additionally, EMPs may reflect the severity of G6PD deficiency. Our data demonstrated a significant correlation between G6PD activity and the levels of MPs. However, further studies will be needed to examine EMPs concentration during acute hemolysis episode. ˙ 2max refer to the amount of red blood cells to transport oxygen and Maximum oxygen uptake or VO ˙ 2max is influenced by age, sex, exercise behaviors, and cardiovascular used in cellular metabolism [11]. VO fitness [11]. In this study, all subjects were female students with uniform lifestyle and had not any regular ˙ 2max in subjects with G6PD Viangchan might be refer a lower ability of exercise. Thus, a lower VO red blood cells to supply oxygen during strenuous exercise, however the relevance of different exercise capacities between G6PD normal and G6PD Viangchan needs to be further investigated.
5. Conclusion Our study demonstrated that strenuous exercise (SEx) triggered the release of TTMPs above baseline levels right after exercise and remained elevated 45 minutes after the exercise. An increase in this TTMPs concentration was significantly higher in individuals with G6PD Viangchan as compared to G6PD normal. No significant differences in MPs concentrations in individuals with G6PD normal and G6PD Viangchan was detected after moderate exercise (MEx). These MPs were found to be originated mostly from platelets and erythrocytes. In addition, concentrations of circulating MPs are associated with lipid peroxidation and G6PD activity. Our data showed that individuals with G6PD Viangchan could perform moderate
250
M. Chanda et al. / MPs after exercise in G6PD Viangchan
exercise with intensity at 75% of their maximum heart rate for up to 45 minutes without experiencing an increase in MPs concentrations or alterations in redox status. Acknowledgments This work was supported financially by Ratchadapiseksomphot Endowment Fund of Chulalongkorn University (RES560530154-HR) and the 90th Anniversary of Chulalongkorn University fund (Ratchadaphiseksomphot Endowment Fund) of 2012 academic year. References [1] K.N. Bangchang, W. Songsaeng, A. Thanavibul, P. Choroenlarp and J. Karbwang, Pharmacokinetics of primaquine in G6PD deficient and G6PD normal patients with vivax malaria, Trans R Soc Trop Med Hyg 88(2) (1994), 220–222. [2] E. Beutler, G6PD deficiency, Blood 84(11) (1994), 3613–3636. [3] G.A. Borg, Psychophysical bases of perceived exertion, Med Sci Sports Exerc 14(5) (1982), 377–381. [4] R.A. Bruce, F. Kusumi and D. Hosmer, Maximal oxygen intake and nomographic assessment of functional aerobic impairment in cardiovascular disease, Am Heart J 85(4) (1973), 546–562. [5] M.D. Cappellini and G. Fiorelli, Glucose-6-phosphate dehydrogenase deficiency, Lancet 371(9606) (2008), 64–74. [6] V. Chaar, M. Romana, J. Tripette, C. Broquere, M.G. Huisse, O. Hue, M.D. Hardy-Dessources and P. Connes, Effect of strenuous physical exercise on circulating cell-derived microparticles, Clin Hemorheol Microcirc 47(1) (2011), 15–25. [7] Y.W. Chen, J.K. Chen and J.S. Wang, Strenuous exercise promotes shear-induced thrombin generation by increasing the shedding of procoagulant microparticles from platelets, Thromb Haemost 104(2) (2010), 293–301. [8] K.J. Davies, A.T. Quintanilha, G.A. Brooks and L. Packer, Free radicals and tissue damage produced by exercise, Biochem Biophys Res Commun 107(4) (1982), 1198–1205. [9] M.S. El-Sayed, A.Z. El-Sayed and S. Ahmadizad, Exercise and training effects on blood haemostasis in health and disease: An update, Sports Med 34 (2004), 181–200. [10] M.S. El-Sayed, P.G. Jones and C. Sale, Exercise induces a change in plasma fibrinogen concentration: Fact or fiction? Thrombosis Res 96 (1999), 467–472. [11] G.F. Fletcher, G.J. Balady, E.A. Amsterdam, B. Chaitman, R. Eckel, J. Fleg and T. Bazzarre, Exercise standards for testing and training: A statement for healthcare professionals from the American Heart Association, Circulation 104(14) (2001), 1694–1740. [12] J.E. Frank, Diagnosis and management of G6PD deficiency, Am Fam Physician 72(7) (2005), 1277–1282. [13] I. Freikman, I. Ringel and E. Fibach, Oxidative stress-induced membrane shedding from RBCs is Ca flux-mediated and affects membrane lipid composition, J Membr Biol 240(2) (2011), 73–82. [14] C.E. Garber, B. Blissmer, M.R. Deschenes, B.A. Franklin, M.J. Lamonte, I.M. Lee, D.C. Nieman and D.P. Swain, American College of Sports Medicine, American College of Sports Medicine position stand. Quantity and quality of exercise for developing and maintaining cardiorespiratory, musculoskeletal, and neuromotor fitness in apparently healthy adults: Guidance for prescribing exercise, Med Sci Sports Exerc 43(7) (2011), 1334–1359. [15] L.L. Ji, Antioxidants and oxidative stress in exercise, Proc Soc Exp Biol Med 222 (1999), 283–292. [16] V.E. Kagan, B. Gleiss, Y.Y. Tyurina, V.A. Tyurin, C. Elenstr¨om-Magnusson, S.X. Liu, F.B. Serinkan, A. Arroyo, J. Chandra, S. Orrenius and B. Fadeel, A role for oxidative stress in apoptosis: Oxidation and externalization of phosphatidylserine is required for macrophage clearance of cells undergoing Fas-mediated apoptosis, J Immunol 169(1) (2002), 487–499. [17] V. Laosombat, B. Sattayasevana, W. Janejindamai, V. Viprakasit, T. Shirakawa, K. Nishiyama, and M. Matsuo, Molecular heterogeneity of glucose-6-phosphate dehydrogenase (G6PD) variants in the south of Thailand and identification of a novel variant (G6PD Songklanagarind), Blood Cells Mol Dis 34(2) (2005), 191–196. [18] A. Marczak and Z. Jozwiak, Damage to the cell antioxidative system in human erythrocytes incubated with idarubicin and glutaraldehyde, Toxicol In Vitro 23(6) (2009), 1188–1194. [19] K. Maruyama, T. Kadono and E. Morishita, Plasma levels of platelet-derived microparticles are increased after anaerobic exercise in healthy subjects, J Atheroscler Thromb 19(6) (2012), 585–587.
M. Chanda et al. / MPs after exercise in G6PD Viangchan
251
[20] Y. Miyazaki, S. Nomura, T. Miyake, H. Kagawa, C. Kitada, H. Taniguchi, Y. Komiyama, Y. Fujimura, Y. Ikeda and S. Fukuhara, High shear stress can initiate both platelet aggregation and shedding of procoagulant containing microparticles, Blood 88 (1996), 3456–3464. [21] D. Nantakomol, M. Imwong, I. Soontarawirat, D. Kotjanya, C. Khakhai, J. Ohashi and P. Nuchnoi, The absolute counting of red cell-derived microparticles with red cell bead by flow rate based assay, Cytometry B Clin Cytom 76(3) (2009), 191–198. [22] M.G. Nikolaidis, A.Z. Jamurtas, V. Paschalis, I.A. Kostaropoulos, A. Kladi-Skandali, V. Balamitsi, Y. Koutedakis and D. Kouretas, Exercise-induced oxidative stress in G6PD-deficient individuals, Med Sci Sports Exerc 38(8) (2006), 1443. [23] S. Nomura, Function and clinical significance of platelet-derived microparticles, Int J Hematol 74 (2001), 397–404. [24] V. Noonan and E. Dean, Submaximal exercise testing: Clinical application and interpretation, Phys Ther 80(8) (2000), 782–807. [25] I. Nuchprayoon, S. Sanpavat and S. Nuchprayoon, Glucose-6-phosphate dehydrogenase (G6PD) mutations in Thailand: G6PD Viangchan (871G>A) is the most common deficiency variant in the Thai population, Hum Mutat 19(2) (2002), 185. [26] R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang and C. Rice-Evans, Antioxidant activity applying an improved ABTS radical cation decolorization assay, Free Radic Biol Med 26(9-10) (1999), 1231–1237. [27] K. Satoh, Serum lipid peroxide in cerebrovascular disorders determined by a new colorimetric method, Clin Chim Acta 90(1) (1978), 37–43. [28] J. Simak and M.P. Gelderman, Cell membrane microparticles in blood and blood products: Potentially pathogenic agents and diagnostic markers, Transfus Med Rev 20 (2006), 1–26. [29] M. Sossdorf, G.P. Otto, R.A. Claus, H.H. Gabriel and W. Losche, Cell-derived microparticles promote coagulation after moderate exercise, Med Sci Sports Exer 43(7) (2011), 1169–1176. [30] K. Sumikawa, Z. Mu, T. Inoue, T. Okochi, T. Yoshida and K. Adachi, Changes in erythrocyte membrane phospholipid composition induced by physical training and physical exercise, Eur J Appl Physiol Occup Physiol 67(2) (1993), 132–137. [31] W.N. Tian, L.D. Braunstein, K. Apse, J. Pang, M. Rose, X. Tian and R.C. Stanton, Importance of glucose-6-phosphate dehydrogenase activity in cell death, Am J Physiol 276(5) (1999), C1121-C1131. [32] K.J. Tsai, I.J. Hung, C.K. Chow, A. Stern, S.S. Chao and D.T. Chiu, Impaired production of nitric oxide, superoxide, and hydrogen peroxide in glucose 6-phosphate-dehydrogenase-deficient granulocytes, FEBS lett 436(3) (1998), 411–414. [33] W. Van Beaumont, J.E. Greenleaf and L. Juhos, Disproportional changes in hematocrit, plasma volume, and proteins during exercise and bed rest, J Appl Physiol 33(1) (1972), 55–61. [34] M.J. Van Wijk, E. Van Bavel, A. Sturk and R. Nieuwland, Microparticles in cardiovascular diseases, Cardiovascular Research 59(2) (2003), 277–287. [35] N.H. Wallen, A.H. Goodall, N. Li and P. Hjemdahl, Activation of haemostasis by exercise, mental stress and adrenaline: Effects on platelet sensitivity to thrombin and thrombin generation, Clin Sci (Lond) 97(1) (1999), 27–35. [36] Y. Xu, Z. Zhang, J. Hu, I.E. Stillman, J.A. Leopold, D.E. Handy, J. Loscalzo and R.C. Stanton, Glucose-6-phosphate dehydrogenase-deficient mice have increased renal oxidative stress and increased albuminuria, FASEB J 24(2) (2010), 609–616. [37] R.F. Zwaal, P. Comfurius and E.M. Bevers, Surface exposure of phosphatidylserine in pathological cells, Cell Mol Life Sci 62(9) (2005), 971–988.
241
Cell-derived microparticles after exercise in individuals with G6PD Viangchan Makamas Chandaa , Duangdao Nantakomola , Daroonwan Suksomb and Attakorn Palasuwana,∗ a
Molecular Hematology Research Unit, Department of Clinical Microscopy, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand b Faculty of Sports Science, Chulalongkorn University, Bangkok, Thailand
Abstract. Glucose-6-phospate dehydrogenase (G6PD) deficient cells are sensitive to oxidative damage leading to the formation of microparticles (MPs). Therefore, we examined the concentration of MPs and changes in the antioxidant balance after an acute strenuous exercise (SEx) and moderate-intensity exercise (MEx). Eighteen healthy females (18–24 years) with G6PD normal and eighteen age-matched females with G6PD Viangchan (871G>A) were tested by running on a treadmill at their maximal oxygen uptake for SEx and at 75% of their maximal heart rate for MEx. It was found that SEx triggered the release of total microparticles (TTMPs) above baseline levels and remained significantly higher 45 minutes after the exercise in G6PD normal individuals. However, SEx-induced increase in TTMPs was significantly higher in G6PD Viangchan as compared to G6PD normal. In contrast, MEx did not to alter the release of TTMPs in both G6PD normal and Viangchan. Moreover, TTMPs concentrations were inversely correlated with G6PD activity (r = −0.82, P < 0.05) but positively correlated with MDA concentrations (r = 0.74, P < 0.05). Using cell specific antibodies, we determined that MPs were mainly derived from platelets and erythrocytes. Altogether, the present study indicates that G6PD Viangchan may participate in MEx without higher MPs concentration and oxidative stress compared with G6PD normal. Keywords: G6PD, antioxidant status, oxidative stress
1. Introduction Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the rate-determining step in the pentose phosphate pathway results in production of nicotinamide adenine dinucleotide phosphate (NADPH) and protects cells from oxidative stress [2, 5, 32]. Individuals affected by G6PD deficiency are unable to regenerate reduced glutathione (GSH) and are susceptible to damages caused by oxidative stress. G6PD deficiency is the most common human enzyme deficiency affecting over 400 million people worldwide. Genetic analysis showed over 140 types of G6PD mutations [5]. However, mutations in the G6PD gene differentially affect protein stability and enzyme activity [2, 12]. Previous studies characterized molecular mechanisms responsible for G6PD deficiency in several ethnic groups in Southeast Asia. In Thailand, the most common G6PD mutation is G6PD Viangchan, a substitution mutation of G6PD gene at position 871 from Guanine to Alanine (871G>A) [17, 25]. Unfortunately, most individuals with G6PD deficiency ∗
Corresponding author: Attakorn Palasuwan, Ph.D., Department of Clinical Microscopy, Faculty of Allied Health Sciences, Chulalongkorn University, Phayathai road, Pathumwan, Bangkok 10330, Thailand. Tel.: +66 2 218 1069; Fax: +66 2 218 3771; E-mails: [email protected], [email protected]. 1386-0291/15/$35.00 © 2015 – IOS Press and the authors. All rights reserved
242
M. Chanda et al. / MPs after exercise in G6PD Viangchan
are asymptomatic, but exposure to oxidative stressors can induce hemolysis and can be life threatening [5, 12]. It is well accepted that strenuous exercise increases oxygen consumption and perturb intracellular pro-oxidant/antioxidant homeostasis resulting in exercise-induced oxidative damages [8, 15]. Therefore, individuals with G6PD gene mutations are at a higher risk of oxidative stress induced erythrocyte damages, resulting in perturbation of hemostasis. The oxidative stress induces externalization of phosphatidylserine (PS) in the plasma membrane, a marker of cellular senescence and a marker for macrophages recognition signal for removal of damages cells from circulation [13, 16, 18]. Due to an imbalance of phospholipid asymmetry and externalization of PS upon apoptosis or cellular activation, cells release small membrane fragments with diameter less than 1.5 micron called cell-derived microparticles (MPs) [37]. MPs externalize anionic phospholipids, PS, and carry different set of proteins that specific for their originating cells on the surface [21, 34]. Recent studies showed that exercise increases the production of MPs [6, 29, 30]. Physical exercises affect clotting and fibrinolytic factors as well as platelet functions [9]. Therefore, MPs are a potential marker of cell damage and/or activation in individuals during exercise, and play a role in the pathophysiological changes caused by physical exercises. Little is known about how strenuous or moderate intensity exercise affects G6PD antioxidant responses, and formation of MPs from oxidative stress induced changes in cell membrane in individuals with G6PD Viangchan. Therefore, the purpose of our study is to investigate whether the concentration of MPs and prooxidant/antioxidant balance could be affected by strenuous exercise in individuals with G6PD Viangchan. We investigated concentration of circulating MPs and prooxidant/antioxidant status of acute strenuous exercise (SEx) and moderate-intensity exercise (MEx) on before, immediately after, and 45 minutes after exercises in both groups. 2. Materials and methods 2.1. Subjects Eighteen healthy females (18–24 years) with no mutation of G6PD gene (G6PD normal) and eighteen age-matched females with G6PD Viangchan (871G>A) were included in this study. Characteristics of the subjects were shown in Table 1. All subjects have been evaluated for general information, health status, habitual dietary intake, social demography, and general physical activities. All subjects were students with uniform lifestyle and had no routine physical exercise regimen for at least 3 months. Subjects with smoking, alcoholic, hypertension, renal, hepatic, asthma, cancer, diabetes, or any heart diseases were excluded. All subjects had not taken any antioxidant or food supplements for at least 6 months prior to the study. All subjects were normal weight, body fat, and body mass index. Informed consents were signed by all subjects included in this study. The protocol was approved by the Ethic Review Committee for Researches involving human subjects, health sciences group of Faculties, Colleges and Institutes, Chulalongkorn University, Thailand, in accordance with the International Conference on Harmonization - Good Clinical Practice (ICH-GCP). 2.2. Anthropometric data All subjects were investigated for their physical fitness as shown in Table 1. Resting heart rate was measured by a heart rate monitor (Polar Electro S810i, Finland). Body composition including body weight and percentage of body fat mass were measured using bioelectrical-impedance analyzer (Tanita, Model
M. Chanda et al. / MPs after exercise in G6PD Viangchan
243
Table 1 Characteristics of individuals with G6PD normal and G6PD Viangchan Variables Age (yrs) Height (m) Weight (kg) Body fat (%) BMI (kg/m2 ) HR (beats/min) HRmax (beats/min) ˙ 2max (kg/ml/min) VO
G6PD normal
G6PD Viangchan
21.1 ± 0.2 1.6 ± 0.0 51.2 ± 1.8 25.1 ± 1.1 20.2 ± 0.6 87.8 ± 3.2 171.0 ± 4.6 38.2 ± 3.3
20.7 ± 0.2 1.6 ± 0.0 50.4 ± 1.5 24.9 ± 1.1 19.4 ± 0.6 83.3 ± 3.0 166.8 ± 5.6 29.4 ± 1.8#
Values are expressed as means ± SE. # P < 0.05.
Inner Scan BC533, Japan). Each anthropometric reading has been made by a well-trained technician. For baseline measurement before exercise, all subjects’ blood sample were assayed for full blood counts for hematological analysis including erythrocyte count, leukocyte count, platelet count, hemoglobin (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) in all subjects were examined. 2.3. DNA extraction and identification of G6PD Viangchan (871G>A) mutations DNA was extracted from EDTA blood samples by using Qiaquick® Blood DNA extraction kit (Qiagen, Germany) according to manufacturer’s recommendations. For DNA analysis, we adopted previously reported screening strategy for the five known G6PD variants Thai population, including G6PD Mahidol 487, G>A; G6PD Viangchan 871, G>A; G6PD Kaiping 1388, G>A; G6PD Canton 1376, G>T; G6PD Union 1360, C>T. All samples were analysed with polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) as previously described [25]. Mutations in exons 6, 9, 11, 12, 13 of G6PD gene were confirmed by direct DNA sequencing. 2.4. The exercise protocol ˙ 2max ). SEx is an acute single bout strenuous exercise, designed to measure maximum oxygen uptake (VO ˙ 2max [4, 24] by increased speed and In this study, we used modified Bruce exercise protocol to measure VO slope level of treadmill (h/p/cosmos mercury® running machine, h/p/cosmos sports & medical GmbH, Germany) every three minute until exhaustion. Ventilation and gas exchanges were measured using a breath-by-breath gas exchange system (Cortex Metamax 3B, Germany). Heart rate (HR) during exercise was measured by the heart rate monitor (Polar S810i, Finland) to verify that the intensity of each exercise protocol had reached to maximal heart rate (HRmax ). Rating of Perceived Exertion (RPE) was measured every 3 minute to indicate fatigue by used Borg scale (1–10 score) [3]. For MEx, two weeks after the completion of completed SEx exercise protocol, all subjects were required to exercise for 45 minutes by running on treadmill at constant level of 75% of HRmax [14]. For the first ten minutes of exercise, we adjusted speed and slope level of treadmill to reach a constant level of 75% HRmax . Similar to SEx protocol, HR was measured every minute by the heart rate monitor (Polar S810i, Finland) and RPE was measured every 5 minute.
244
M. Chanda et al. / MPs after exercise in G6PD Viangchan
2.5. Blood sampling procedures On experimental day (7–14 days after subjects recruitment, samples and all informed consent forms were signed), blood samples were collected an hour before the exercise (Pre), immediately after the exercise (Post0) and 45 minutes after the conclusion of exercise (Post45). Blood samples were collected in tubes containing ethylene diamine tetra acetic acid tube (EDTA), lithium heparin tube (Li-heparin) and sodium citrate as anticoagulant at each time point. Complete blood counts (CBC), G6PD, superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities were determined in blood samples collected in tubes containing EDTA as anticoagulant. Measurement of total antioxidant status (TAS) and malondialdehyde (MDA) were assessed in plasma samples collected in tube containing Li-heparin as anticoagulant. Quantification and identification of MPs cellular origins were determined in blood samples collect in tubes containing 3.2% trisodium citrate as anticoagulant tube. All blood samples were kept at 4◦ C and processed within an hour. 2.6. Biological analyses 2.6.1. Hematological parameters All hematological parameters were determined by an automated hematology analyzer (Beckman Coulter Gen S, USA) within 1 hour after blood sample collection. The within-run of coefficient of variation % were less than 3% for all hematological parameters. 2.6.2. Antioxidant and oxidative stress markers Total antioxidant status (TAS) was measured by radical cation decolorization assay [26] using UV spectrophotometer (Shidmadzu UV-1601, Japan). Lipid peroxidation was assayed by monitoring formation of malondialdehyde (MDA) [27]. Erythrocytes superoxide dismutase (E-SOD), whole blood glutathione peroxidase (Wb-GPx) and erythrocyte glutathione peroxidase (E-GPx) activity were analysed by enzyme kinetic-colorimetric assay by Randox reagent kit (Randox Laboratories, UK). G6PD enzymatic activity was determined by using G6PD Randox kit (Randox Laboratories, UK), an enzyme kinetic-colorimetric assay. G6PD activities were expressed as international units per gram hemoglobin (IU/gHb). The within-run of coefficient of variation % were less than 5% for all parameters. 2.6.3. Quantitation and identification of microparticles (MPs) cellular origins Whole blood containing sodium citrate as anticoagulant was used to determine the level of MPs by flow cytometry as described previously [21]. Briefly, annexin V conjugated with Fluorescein isothioacyanate (AV-FITC) (BD PharminogenTM , Heidelberg, Germany) was used to detect PS on MPs’s surface. All annexin V positive-vesicles were defined as total microparticles (TTMPs). Mouse anti-Human CD41a and anti-glycophorin A conjugated with Phycoerythrin (PE) (BD PharminogenTM , Heidelberg, Germany) were used to differentially distinguish cellular origins of TTMPs that released from platelets and erythrocytes, respectively. Two microliters of citrated whole blood were mixed with 3l of FITC-conjugated annexin-V and either 3l of PE-conjugated glycophorin A or PE-conjugated CD41a. Reactions were incubated with 22l of binding buffer for 15 minutes at room temperature and protected from light. After incubation, 1 ml of annexin V binding buffer was added and quantified with flow cytometer using CELLQuestTM software (Becton Dickinson Biosciences, San Jose, CA) program. Subsequently, the absolute numbers of MPs were analyzed as described previously [21].
M. Chanda et al. / MPs after exercise in G6PD Viangchan
245
2.7. Statistical analysis All data in this study were presented as mean ± SE. One way ANOVA with repeated measurement were used. When significant changed were observed, LSD post-hoc test were applied to locate the source of difference. Statistical analyses were carried out using the SPSS 17.0 statistical program (SPSS Corporation, Chicago, IL). A P-value 0.05) (Table 1). Resting HR and HR max (HRmax ) after SEx ˙ 2max was significant lower in showed no significant different between the two group (P < 0.05) while VO G6PD Viangchan (P < 0.05) (Table 1). G6PD activity was significantly lower in individuals with G6PD Viangchan as compared to individuals with G6PD normal (P < 0.05). Plasma and whole blood volumes were calculated, based on Hb and Hct values, by Beaumont’s formula [33]. Post-exercise (Post0) and recovery (Post45) values of plasma TAS and MDA concentrations and MPs were corrected for change in plasma volume. 3.2. Hematological analysis Hematological parameter values of G6PD normal and Viangchan were summarized in Table 2. Before the start of each experiment, all subjects had normal hematological parameters. Immediately after exercise (Post0), hematological parameters were significantly increased including Hb, Hct, erythrocyte counts, leukocyte count, and platelet counts in all subjects (P < 0.05) and returned to basal levels within 45 minutes (Post45) in both individuals with G6PD normal and Viangchan. In contrast, MEx failed to alter any hematological parameters after the exercise in both groups (P > 0.05). 3.3. Antioxidant status and G6PD activity Table 3 shows results of antioxidant and the levels of G6PD activity. Significantly higher levels of E-SOD and E-GPx were found in individuals with G6PD Viangchan at the baseline and after SEx as compared to individuals with G6PD normal (P < 0.05). In addition, Wb-GPx and MDA levels were significantly increased at immediately after SEx (Post0) in individuals with G6PD Viangchan as compared to G6PD normal, and returned to baseline at 45 minutes after the exercise (Post45). Whereas, resting concentration of G6PD activity in individuals with G6PD Viangchan was significantly lower than G6PD normal and decreased continuously after exercise in SEx. In MEx, G6PD and E-GPX activity were significantly lower at the baseline and in all time points in individuals with G6PD Viangchan as compared to G6PD normal at 45 minutes after exercise (P < 0.05). 3.4. Circulating microparticles (MPs) Numbers of total microparticles (TTMPs) were quantified by using annexin V positive population, a specific marker for PS, with a size less than 1.0 m. Baseline concentrations of PS positive microparticles
246
M. Chanda et al. / MPs after exercise in G6PD Viangchan Table 2 Hematological parameters in individuals with G6PD normal and G6PD Viangchan in SEx and MEx
Variables
Time
G6PD normal SEx
Erythrocytes counts (×1012 L)
Hemoglobin (g/dL)
Hematocrit (%)
Leukocytes counts (×109 L)
Platelets counts (×109 L)
MCV
MCH
MCHC
Pre Post0 Post45 Pre Post0 Post45 Pre Post0 Post45 Pre Post0 Post45 Pre Post0 Post45 Pre Post0 Post45 Pre Post0 Post45 Pre Post0 Post45
4.4 ± 0.1 4.7 ± 0.1* 4.7 ± 0.2 12.9 ± 0.3 13.7 ± 0.4* 13.8 ± 0.6 38.4 ± 0.8 40.9 ± 1.1* 40.7 ± 1.6 6.3 ± 0.5 9.7 ± 0.6* 6.5 ± 0.4 252.2 ± 15 274.2 ± 15.2* 240.9 ± 13.9 86.9 ± 2.0 87.0 ± 2.0 86.7 ± 2.0 29.3 ± 0.8 29.2 ± 0.8 29.4 ± 0.8 33.7 ± 0.3 33.5 ± 0.2 33.9 ± 0.3
G6PD Viangchan MEx
4.4 ± 0.2 4.5 ± 0.1 4.4 ± 0.1 12.6 ± 0.3 12.4 ± 0.3 12.6 ± 0.3 36.7 ± 1.8 37.6 ± 0.8 37.9 ± 0.8 6.8 ± 0.4 7.1 ± 0.4 6.9 ± 0.4 244.2 ± 13.6 269.8 ± 14 261 ± 12.7 84.2 ± 2.4 84.6 ± 2.3 85.7 ± 2.4 28.7 ± 2.5 28.0 ± 0.9 28.5 ± 0.9 33.0 ± 3.7 33.1 ± 0.2 33.2 ± 0.2
SEx
MEx
4.4 ± 0.1 4.8 ± 0.2* 4.5 ± 0.1 12.4 ± 0.2 13.5 ± 0.4* 12.7 ± 0.3 37.2 ± 0.6 40.8 ± 1.2* 38.0 ± 0.8 6.2 ± 0.3 9.6 ± 0.4* 6.2 ± 0.3 245.1 ± 12.3 253.7 ± 12.6* 237.6 ± 11.5 85.9 ± 1.5 86.1 ± 1.4 85.7 ± 1.4 28.7 ± 0.6 28.5 ± 0.6 28.6 ± 0.6 33.4 ± 0.2 33.1 ± 0.2 33.4 ± 1.4
4.3 ± 0.1 4.4 ± 0.1 4.4 ± 0.1 12.1 ± 0.3 12.5 ± 0.4 12.5 ± 0.3 36.4 ± 0.8 37.7 ± 1.3 37.7 ± 0.9 6.1 ± 0.2 6.2 ± 0.2 6.1 ± 0.2 248.3 ± 15.5 240.4 ± 12.6 238.9 ± 11.8 84.4 ± 1.4 84.8 ± 1.5 84.7 ± 1.4 28.0 ± 0.5 28.2 ± 0.6 28.0 ± 0.5 33.1 ± 0.1 33.2 ± 0.1 33.1 ± 0.2
Values are expressed as means ± SE. ∗ Statistically significant differences as compared to pre-exercise (Pre) (P < 0.05).
were detected in individuals with G6PD normal and G6PD Viangchan before exercise. Average numbers of TTMPs detected in G6PD normal and G6PD Viangchan individuals were not significantly different [(Mean ± SE); 2587 ± 232 and 2859 ± 436 cells/l, respectively, (P = 0.47)]. Immediately after SEx (Post0), concentrations of TTMPs were significantly increased in individuals with G6PD Viangchan [8012 ± 256 MPs/L] as compared to individuals with G6PD normal [5716 ± 538 MPs/L, P < 0.05] and remained significantly higher 45 minutes after the exercise. In contrast, no significant changes in TTMP concentrations in both groups were detected after MEx (P > 0.05) (Fig. 1). Additionally, concentrations of TTMPs were inversely correlated with G6PD activity (r = −0.82, P < 0.05) while positively correlated with a concentration of lipid peroxidation marker (MDA) (r = 0.74, P < 0.05). In order to identify cellular origins of TTMPs, blood samples were stained simultaneously with FITC-conjugated annexin V along with PE-conjugated platelet integinµIIb (CD41a) or PE-conjugated glycophorin A monoclonal antibody. MPs from individuals with G6PD normal and G6PD Viangchan were mostly derived from platelets (Platelet derived microparticles, PMPs) (37% and 38%, respectively) and erythrocyte (erythrocyte derived microparticles, EMPs) (22% and 25%, respectively). However, cellular origins of MPs subpopulation from both G6PD normal and G6PD Viangchan individuals remained
M. Chanda et al. / MPs after exercise in G6PD Viangchan
247
Table 3 Antioxidants status markers and G6PD activity Variables
SOD (IU/g Hb)
E-GPx(IU/g Hb)
wb-GPx(IU/g Hb)
MDA (mmol/l)
TAS (mM troloxEq)
G6PD activity (IU/g Hb)
Time
Pre Post0 Post45 Pre Post0 Post45 Pre Post0 Post45 Pre Post0 Post45 Pre Post0 Post45 Pre Post0 Post45
G6PD normal
G6PD Viangchan
SEx
MEx
SEx
MEx
3192.2 ± 95.9 2875.0 ± 102.4 2630.5 ± 138.0∗ 43.4 ± 2.0 47.0 ± 3.4 42.0 ± 2.9 67.5 ± 3.7 69.1 ± 3.3 63.0 ± 3.4 5.6 ± 0.4 5.8 ± 0.4 5.4 ± 0.4 2.0 ± 0.0 1.9 ± 0.1 2.0 ± 0.1 7606.1 ± 430.6 7907.0 ± 247.4 7518.9 ± 293.5
3690.5 ± 125.3 3869 ± 90.4 3489 ± 192.5 69.5 ± 4.0 65.2 ± 4.1 63.0 ± 1.6 63.6 ± 5.0 56.4 ± 5.1 59.0 ± 4.9 3.2 ± 0.1 3.1 ± 0.1 3.2 ± 0.1 1.6 ± 0.0 1.5 ± 0.0 1.6 ± 0.0 5967.2 ± 101.8 6950.4 ± 380.1 6723.4 ± 415.3
3538.7 ± 69.7# 3192.1 ± 114.0+ 2847.1 ± 122.2+ 54.7 ± 4.0# 53.5 ± 4.7# 36.1 ± 2.1+ 75.8 ± 5.4 81.5 ± 4.9# 70.4 ± 5.0 5.5 ± 0.4 8.3 ± 0.3# 5.3 ± 0.4 2.0 ± 0.0 1.9 ± 0.1 2.0 ± 0.1 5557.1 ± 573.3# 4718.4 ± 557.2# 4560.5 ± 566.7#
3327.1 ± 136.0 3677.7 ± 48.5 3593.4 ± 143.4 59.3 ± 6.3 61.8 ± 7.3 46.7 ± 4.0# 72.3 ± 4.7 71.5 ± 6.5 71.2 ± 7.2 3.3 ± 0.1 3.2 ± 0.1 3.2 ± 0.1 1.7 ± 0.1 1.7 ± 0.1 1.7 ± 0.1 3937.1 ± 469.4# 4269.0 ± 498.4# 4185.8 ± 610.4#
Values are expressed as means ± SE. ∗ Statistically significant difference comparing preexcercise within the same group and in the same exercise protocol (P < 0.05). # Statistically significant difference comparing control group at the same time points and in the same exercise protocol (P < 0.05). + Statistically significant difference comparing control group at the similar time points and baseline level within the same exercise (P < 0.05).
Fig. 1. Absolute count numbers of total cell derived microparticles (TTMPs) (A), platelet derived microparticles (PMPs) (B), erythrocyte derived microparticles (EMPs) (C), at baseline (Pre; white bar), immediately post exercise (Post 0; gray bar), and 45 minutes post exercise (Post45; black bar) by SEx vs MEx. ∗ Statistically significant difference comparing pre-exercise (Pre) within the same group and in the same exercise protocol (P < 0.05). # Statistically significant difference comparing control group at similar time points and pre-exercise in the same exercise protocol (P < 0.05).
unidentified (41% and 37%, respectively). At resting state, average numbers of MPs in individuals with G6PD normal released from platelets and erythrocytes were 926 ± 132 MPs/l, and 385 ± 98 MPs/l, respectively. Whereas, average numbers of MPs in individuals with G6PD Viangchan released from
248
M. Chanda et al. / MPs after exercise in G6PD Viangchan
platelets and erythrocytes were 1106 ± 345 MPs/L, and 423 ± 221 MPs/l, respectively. Concentrations of PMPs and EMPs significantly increased immediately after exercise and remained significantly higher above baseline levels at 45 minutes post exercise in both G6PD normal and Viangchan (P < 0.05). No significant differences in any of EMPs and PMPs concentration between the two groups over all time points were detected in MEx (Fig. 1).
4. Discussion NADPH, produced by G6PD, is an important molecule for cellular antioxidant system. Many studies have shown that partial or complete inhibition of G6PD activity renders cells extremely sensitive to oxidative stress [1, 31, 36]. Many studies showed that eukaryotic cells shed MPs in response to oxidative stress or apoptosis, and exercise increases the production of circulating MPs [6, 29, 30]. However how strenuous exercise affects antioxidant balance and production of MPs in G6PD Viangchan is still unclear. Using flow cytometry, we found that strenuous exercise triggered the release of TTMPs to above baseline levels right after strenuous exercise and remained elevated 45 minutes after the exercise. An increase in TTMPs concentration was significantly higher in individuals with G6PD Viangchan as compared to G6PD normal. Therefore, activity of G6PD enzyme is likely to be an important contributor to this increase. Chen et al. demonstrated that shear stress is a major contributing factor of MP release during physical exercise [7]. Additionally, an imbalance of antioxidant system induced by oxidative stress during exercise is also a contributing factor for MPs release [15]. Thus, individuals with G6PD mutation are at a higher risk of oxidative stress induced cell damages. After acute bout of exercise, antioxidant parameters including SOD, wb-GPx, E-GPx and G6PD activities were significant decreased in individuals with G6PD Viangchan, while no changes in these parameters in individuals with G6PD normal. Our data suggested that a reduction in several antioxidant parameters could be due to low G6PD activity in individuals with Viangchan. Additionally, G6PD knockout mice were highly sensitive to deleterious effects of oxidative stress as evidence in a decrease in NADPH levels, a decrease in intracellular GSH levels and an increase in marker of lipid peroxidation [36]. Consistent with our data, MDA levels, a lipid peroxidation marker, were significantly higher in individuals with G6PD Viangchan after strenuous exercise [SEx] as compared to G6PD normal. Higher lipid peroxidation could be an important contributor for MPs shedding after acute strenuous exercise, and plasma MDA levels were positively correlated with MPs concentrations. In contrast, no significant difference in MPs concentration and antioxidant parameters were found in individuals with G6PD normal and Viangchan after moderate exercise (MEx). These results suggested that exercise duration and intensity are important determinants of exercise-induced changes in antioxidant status and the release of MPs. According to our data, individuals with G6PD Viangchan should exercise at moderate intensity or at 75% of their maximum heart rate for up to 45 minutes without any alterations in MPs concentrations and redox status. MPs from healthy subjects derived mostly from platelets (37%) and erythrocytes (22%). Analysis of MPs cellular origins from individuals with G6PD Viangchan showed similar cellular origins as individuals with G6PD normal [platelets (38%) and erythrocytes (25%), respectively]. However, cellular origins of up to 50% of circulating MPs remained unknown. In this study, PMPs were detected by using monoclonal antibody to glycoprotein (GP) IIb-IIIa (CD41a). It is possible that some PMPs did not have sufficient CD41a antigen or carried other PMPs surface antigens such as, GPIX (CD42a), GPIb (CD42b), or GPIIa (CD61) [28]. Additional studies will be needed to reveal identities of these unidentified antigens. In contrast to variations of antigens on platelet membrane, the majority of erythrocytes carry Glycophorin A
M. Chanda et al. / MPs after exercise in G6PD Viangchan
249
(CD235a) which is the most established marker for identification of EMPs. However, we did not stained all antigen derived from leukocyte and endothelial cell which may account for additional cell origins for unidentified MPs. Further assessment of cellular origins of these unidentified MPs will be required in future study. Our study demonstrated that strenuous exercise caused an increase in PMPs concentrations. This finding is consistent with a previous study showing that PMPs and EMPs were significantly increased after strenuous exercise [6, 19]. Activation of platelets during exercise has previously been described and could be responsible for an increase in PMPs production [35]. Although how platelet activation occur during exercise remains unknown. It is likely that a significant increase in shear stress during physical exercise may initiate PMPs formation [20]. Additionally, PMPs may have procoagulant activity due to externalization of negatively charged membrane phosphatidylserine phospholipids and may be one of the factors responsible for dissemination of prothrombotic activity [23, 34]. The procoagulant activity of PMPs may contribute to activation of coagulation that usually occurs during exercise [10]. However, how PMPs involve in the pathophysiology of exercise-induced alterations in hemostasis will need to be further investigated. In addition to PMPs, MPs shed from erythrocytes have also been found in individuals with G6PD Viangchan after strenuous exercise (SEx). Our data also showed that there were significantly higher levels of EMPs after strenuous exercise (SEx) in individuals with G6PD Viangchan. No significant differences in levels of PMPs and EMPs from G6PD normal and G6PD Viangchan were detected after moderate exercise (MEx). Erythrocyte membrane undergoes vesiculation under a variety of conditions, including disruption of asymmetric organization of membrane phospholipids, increased lipid peroxidation and increased ROS. All of these conditions eventually lead to loss of erythrocyte membranes and MPs formation which are characteristic of alteration in hemostasis after exercise [7, 22, 29]. Since exercise could induce changes in erythrocyte membrane, individuals with G6PD Viangchan are at a higher risk of oxidative stress induced erythrocyte damages after strenuous exercise. Additionally, EMPs may reflect the severity of G6PD deficiency. Our data demonstrated a significant correlation between G6PD activity and the levels of MPs. However, further studies will be needed to examine EMPs concentration during acute hemolysis episode. ˙ 2max refer to the amount of red blood cells to transport oxygen and Maximum oxygen uptake or VO ˙ 2max is influenced by age, sex, exercise behaviors, and cardiovascular used in cellular metabolism [11]. VO fitness [11]. In this study, all subjects were female students with uniform lifestyle and had not any regular ˙ 2max in subjects with G6PD Viangchan might be refer a lower ability of exercise. Thus, a lower VO red blood cells to supply oxygen during strenuous exercise, however the relevance of different exercise capacities between G6PD normal and G6PD Viangchan needs to be further investigated.
5. Conclusion Our study demonstrated that strenuous exercise (SEx) triggered the release of TTMPs above baseline levels right after exercise and remained elevated 45 minutes after the exercise. An increase in this TTMPs concentration was significantly higher in individuals with G6PD Viangchan as compared to G6PD normal. No significant differences in MPs concentrations in individuals with G6PD normal and G6PD Viangchan was detected after moderate exercise (MEx). These MPs were found to be originated mostly from platelets and erythrocytes. In addition, concentrations of circulating MPs are associated with lipid peroxidation and G6PD activity. Our data showed that individuals with G6PD Viangchan could perform moderate
250
M. Chanda et al. / MPs after exercise in G6PD Viangchan
exercise with intensity at 75% of their maximum heart rate for up to 45 minutes without experiencing an increase in MPs concentrations or alterations in redox status. Acknowledgments This work was supported financially by Ratchadapiseksomphot Endowment Fund of Chulalongkorn University (RES560530154-HR) and the 90th Anniversary of Chulalongkorn University fund (Ratchadaphiseksomphot Endowment Fund) of 2012 academic year. References [1] K.N. Bangchang, W. Songsaeng, A. Thanavibul, P. Choroenlarp and J. Karbwang, Pharmacokinetics of primaquine in G6PD deficient and G6PD normal patients with vivax malaria, Trans R Soc Trop Med Hyg 88(2) (1994), 220–222. [2] E. Beutler, G6PD deficiency, Blood 84(11) (1994), 3613–3636. [3] G.A. Borg, Psychophysical bases of perceived exertion, Med Sci Sports Exerc 14(5) (1982), 377–381. [4] R.A. Bruce, F. Kusumi and D. Hosmer, Maximal oxygen intake and nomographic assessment of functional aerobic impairment in cardiovascular disease, Am Heart J 85(4) (1973), 546–562. [5] M.D. Cappellini and G. Fiorelli, Glucose-6-phosphate dehydrogenase deficiency, Lancet 371(9606) (2008), 64–74. [6] V. Chaar, M. Romana, J. Tripette, C. Broquere, M.G. Huisse, O. Hue, M.D. Hardy-Dessources and P. Connes, Effect of strenuous physical exercise on circulating cell-derived microparticles, Clin Hemorheol Microcirc 47(1) (2011), 15–25. [7] Y.W. Chen, J.K. Chen and J.S. Wang, Strenuous exercise promotes shear-induced thrombin generation by increasing the shedding of procoagulant microparticles from platelets, Thromb Haemost 104(2) (2010), 293–301. [8] K.J. Davies, A.T. Quintanilha, G.A. Brooks and L. Packer, Free radicals and tissue damage produced by exercise, Biochem Biophys Res Commun 107(4) (1982), 1198–1205. [9] M.S. El-Sayed, A.Z. El-Sayed and S. Ahmadizad, Exercise and training effects on blood haemostasis in health and disease: An update, Sports Med 34 (2004), 181–200. [10] M.S. El-Sayed, P.G. Jones and C. Sale, Exercise induces a change in plasma fibrinogen concentration: Fact or fiction? Thrombosis Res 96 (1999), 467–472. [11] G.F. Fletcher, G.J. Balady, E.A. Amsterdam, B. Chaitman, R. Eckel, J. Fleg and T. Bazzarre, Exercise standards for testing and training: A statement for healthcare professionals from the American Heart Association, Circulation 104(14) (2001), 1694–1740. [12] J.E. Frank, Diagnosis and management of G6PD deficiency, Am Fam Physician 72(7) (2005), 1277–1282. [13] I. Freikman, I. Ringel and E. Fibach, Oxidative stress-induced membrane shedding from RBCs is Ca flux-mediated and affects membrane lipid composition, J Membr Biol 240(2) (2011), 73–82. [14] C.E. Garber, B. Blissmer, M.R. Deschenes, B.A. Franklin, M.J. Lamonte, I.M. Lee, D.C. Nieman and D.P. Swain, American College of Sports Medicine, American College of Sports Medicine position stand. Quantity and quality of exercise for developing and maintaining cardiorespiratory, musculoskeletal, and neuromotor fitness in apparently healthy adults: Guidance for prescribing exercise, Med Sci Sports Exerc 43(7) (2011), 1334–1359. [15] L.L. Ji, Antioxidants and oxidative stress in exercise, Proc Soc Exp Biol Med 222 (1999), 283–292. [16] V.E. Kagan, B. Gleiss, Y.Y. Tyurina, V.A. Tyurin, C. Elenstr¨om-Magnusson, S.X. Liu, F.B. Serinkan, A. Arroyo, J. Chandra, S. Orrenius and B. Fadeel, A role for oxidative stress in apoptosis: Oxidation and externalization of phosphatidylserine is required for macrophage clearance of cells undergoing Fas-mediated apoptosis, J Immunol 169(1) (2002), 487–499. [17] V. Laosombat, B. Sattayasevana, W. Janejindamai, V. Viprakasit, T. Shirakawa, K. Nishiyama, and M. Matsuo, Molecular heterogeneity of glucose-6-phosphate dehydrogenase (G6PD) variants in the south of Thailand and identification of a novel variant (G6PD Songklanagarind), Blood Cells Mol Dis 34(2) (2005), 191–196. [18] A. Marczak and Z. Jozwiak, Damage to the cell antioxidative system in human erythrocytes incubated with idarubicin and glutaraldehyde, Toxicol In Vitro 23(6) (2009), 1188–1194. [19] K. Maruyama, T. Kadono and E. Morishita, Plasma levels of platelet-derived microparticles are increased after anaerobic exercise in healthy subjects, J Atheroscler Thromb 19(6) (2012), 585–587.
M. Chanda et al. / MPs after exercise in G6PD Viangchan
251
[20] Y. Miyazaki, S. Nomura, T. Miyake, H. Kagawa, C. Kitada, H. Taniguchi, Y. Komiyama, Y. Fujimura, Y. Ikeda and S. Fukuhara, High shear stress can initiate both platelet aggregation and shedding of procoagulant containing microparticles, Blood 88 (1996), 3456–3464. [21] D. Nantakomol, M. Imwong, I. Soontarawirat, D. Kotjanya, C. Khakhai, J. Ohashi and P. Nuchnoi, The absolute counting of red cell-derived microparticles with red cell bead by flow rate based assay, Cytometry B Clin Cytom 76(3) (2009), 191–198. [22] M.G. Nikolaidis, A.Z. Jamurtas, V. Paschalis, I.A. Kostaropoulos, A. Kladi-Skandali, V. Balamitsi, Y. Koutedakis and D. Kouretas, Exercise-induced oxidative stress in G6PD-deficient individuals, Med Sci Sports Exerc 38(8) (2006), 1443. [23] S. Nomura, Function and clinical significance of platelet-derived microparticles, Int J Hematol 74 (2001), 397–404. [24] V. Noonan and E. Dean, Submaximal exercise testing: Clinical application and interpretation, Phys Ther 80(8) (2000), 782–807. [25] I. Nuchprayoon, S. Sanpavat and S. Nuchprayoon, Glucose-6-phosphate dehydrogenase (G6PD) mutations in Thailand: G6PD Viangchan (871G>A) is the most common deficiency variant in the Thai population, Hum Mutat 19(2) (2002), 185. [26] R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang and C. Rice-Evans, Antioxidant activity applying an improved ABTS radical cation decolorization assay, Free Radic Biol Med 26(9-10) (1999), 1231–1237. [27] K. Satoh, Serum lipid peroxide in cerebrovascular disorders determined by a new colorimetric method, Clin Chim Acta 90(1) (1978), 37–43. [28] J. Simak and M.P. Gelderman, Cell membrane microparticles in blood and blood products: Potentially pathogenic agents and diagnostic markers, Transfus Med Rev 20 (2006), 1–26. [29] M. Sossdorf, G.P. Otto, R.A. Claus, H.H. Gabriel and W. Losche, Cell-derived microparticles promote coagulation after moderate exercise, Med Sci Sports Exer 43(7) (2011), 1169–1176. [30] K. Sumikawa, Z. Mu, T. Inoue, T. Okochi, T. Yoshida and K. Adachi, Changes in erythrocyte membrane phospholipid composition induced by physical training and physical exercise, Eur J Appl Physiol Occup Physiol 67(2) (1993), 132–137. [31] W.N. Tian, L.D. Braunstein, K. Apse, J. Pang, M. Rose, X. Tian and R.C. Stanton, Importance of glucose-6-phosphate dehydrogenase activity in cell death, Am J Physiol 276(5) (1999), C1121-C1131. [32] K.J. Tsai, I.J. Hung, C.K. Chow, A. Stern, S.S. Chao and D.T. Chiu, Impaired production of nitric oxide, superoxide, and hydrogen peroxide in glucose 6-phosphate-dehydrogenase-deficient granulocytes, FEBS lett 436(3) (1998), 411–414. [33] W. Van Beaumont, J.E. Greenleaf and L. Juhos, Disproportional changes in hematocrit, plasma volume, and proteins during exercise and bed rest, J Appl Physiol 33(1) (1972), 55–61. [34] M.J. Van Wijk, E. Van Bavel, A. Sturk and R. Nieuwland, Microparticles in cardiovascular diseases, Cardiovascular Research 59(2) (2003), 277–287. [35] N.H. Wallen, A.H. Goodall, N. Li and P. Hjemdahl, Activation of haemostasis by exercise, mental stress and adrenaline: Effects on platelet sensitivity to thrombin and thrombin generation, Clin Sci (Lond) 97(1) (1999), 27–35. [36] Y. Xu, Z. Zhang, J. Hu, I.E. Stillman, J.A. Leopold, D.E. Handy, J. Loscalzo and R.C. Stanton, Glucose-6-phosphate dehydrogenase-deficient mice have increased renal oxidative stress and increased albuminuria, FASEB J 24(2) (2010), 609–616. [37] R.F. Zwaal, P. Comfurius and E.M. Bevers, Surface exposure of phosphatidylserine in pathological cells, Cell Mol Life Sci 62(9) (2005), 971–988.
