Effects of two selected SSRIs on hemorheological parameters in rats

Abstract

BACKGROUND:

Selective Serotonin Reuptake Inhibitors (SSRIs), antidepressants commonly used in cardiovascular diseases (CVDs), inhibit the re-uptake of serotonin not only into neurons but also into platelets. Hence they increase the level of serotonin in plasma.

OBJECTIVE:

This study was aimed to clarify the effects of two selected SSRIs on plasma serotonin level, hemorheological parameters (hematocrit, erythrocyte deformability, erythrocyte aggregation and plasma viscosity) and selected oxidative stress markers (MDA, GSH, GSSG levels in plasma and erythrocytes).

METHODS:

Two different SSRIs (Fluvoxamine and Sertraline) were administered to male Sprague-Dawley rats in acute (5 days) or chronic fashion (21 days) at 20 mg/kg/day dose.

RESULTS:

Aggregation amplitude (AMP) decreased significantly in the chronic sertraline and acute fluvoxamine groups; aggregation half time (t1/2) decreased significantly in the chronic fluvoxamine group. Biochemical parameters indicating oxidative stress significantly increased in the chronic sertraline group.

CONCLUSIONS:

Since SSRI’s are commonly used in patients with CVDs, complementary studies are needed to assess the impact of such changes in hemorheological parameters on the risk for CVD, and to reveal the effects of other SSRIs on hemorheological parameters.

Keywords

SSRI serotonin erythrocyte deformability erythrocyte aggregation plasma viscosity hematocrit blood viscosity hemorheology MDA GSH GSSG SOD catalase glutathion reductase glutathione peroxidase glutathione S-transferase MAO

1 Introduction

Serotonin (5-hydroxytryptamine), a monoamine neurotransmitter derived from tryptophan, is the target molecule in the treatment of major depression. Selective serotonin re-uptake inhibitors (SSRIs), typically used as antidepressants, are suggested to inhibit the re-uptake of serotonin into the presynaptic cell, increasing its level in the synaptic cleft and hence its availability to bind the postsynaptic receptors. In addition to the central nervous system, important amounts of serotonin are found in the gastrointestinal tract and the platelets. After being synthesized in the GI tract serotonin in the blood is taken into the platelets by a high affinity serotonin transporter (SERT) [1]. Majority of serotonin in the circulation is found in the platelets whereas plasma serotonin level encompasses only a small fraction of the whole blood serotonin [2]. Not only the SERT on neurons but also the SERT on the platelets are inhibited by SSRIs. It was reported that by blocking SERT, different SSRIs elevate plasma serotonin level up to 40 to 80 nM [3].

Reports indicating that cardiovascular diseases (CVDs) are more common among people with major depression suggest that depression may be an important risk factor for CVDs [4]. Antidepressants and hence SSRIs are used in CVDs [5]. SSRIs may have other benefits in CVD patients in addition to their antidepressant action. Although serotonin is a weak platelet agonist, it stimulates platelet reactions for the other agonists. Platelets are known for their substantial impact in pathophysiological processes [6]. Since SSRIs have been shown to reduce platelet and hence total blood serotonin concentrations and to exert an inhibitory effect on platelet activation and stabilize platelets, it was proposed that SSRIs could have a protective effect against myocardial infarction [7]. A number of studies support these data by the observation of decreased mortality and morbidity by the treatment with SSRIs in CVD patients, however, controversial results are also present [8, 9]. The protective effect of SSRI’s may be through an improvement in the rheological properties of blood.

The present study aimed to elucidate the effects of two SSRIs: fluvoxamine and sertraline on plasma serotonin level and hemorheological parameters (erythrocyte deformability, erythrocyte aggregation and plasma viscosity). Because oxidative deamination of serum serotonin by the enzyme monoamine oxidase (MAO) leads to formation of an oxidant molecule, hydrogen peroxide (H₂O₂), oxidative stress parameters were also determined.

2 Materials and methods

2.1 Experimental animals

50 male Sprague-Dawley rats of 3 months old weighing 200–250 g were included in the study. Animals were housed individually on a 12 hr light/dark cycle and fed with standard rat pellet food and water provided ad libitum. They were randomly divided into five groups (n = 10 rats for each group) as follows: control, acute fluvoxamine, chronic fluvoxamine, acute sertraline and chronic sertraline.

Control group was given tap water for 5 days. Both SSRIs were given at 20 mg/kg dose. Animals were administered two different SSRIs (Fluvoxamine and Sertraline, Sigma) in acute or chronic fashion [3]. Acute groups received the SSRI for 5 days whereas chronic groups received for 21 days. Water and drugs (dissolved in tap water) were administered by gastric gavage at 10 ml/kg (v/w). At the end of drug administration protocol, blood was withdrawn from the vena cava caudalis of rats under light anesthesia. Blood was collected into the tubes for the determination of hemorheological parameters, complete blood count and biochemical parameters. In required cases heparin (15 U/ml) was used as the anticoagulant.

2.2 Hemorheological parameters

2.2.1 Erythrocyte deformability

Erythrocyte deformability was measured using Laser-Assisted Optical Rotational Cell Analyzer (LORCA) (Mechatronics, Holland) at shear rates 0.3, 0.53, 0.95, 1.69, 3.00, 5.33, 9.49, 26.87 and 30 Pa. Temperature was set to 37°C. 25μl of blood was mixed gently with 5 ml of polyvinylpyrrolidone (PVP) medium (360,000 MW, 300mOsm/kg). One ml of this suspension was placed into the chamber between two concentric glass cylinders of LORCA. Recording made by the aid of laser beam transversing the suspension fluid in the chamber yields a diffraction pattern which can be calculated as an index of deformation by an ellipse fitting program. Attenuation of this elongation index states decreased deformability [10].

2.2.2 Erythrocyte aggregation

Erythrocyte aggregation was measured by LORCA at 37C. Venous blood samples were oxygenated for 10 minutes before measurement. 1 ml of blood was used for each measurement. A computer program was used to analyze the amplitude (AMP, indicating the total amplitude of aggregation), aggregation index (AI, indicating how great and/or quick aggregation is) and half time (t½, time required for the peak intensity to become half, reflecting the kinetics of aggregation) [10 –12].

2.2.3 Plasma viscosity

Plasma viscosity was measured with a cone-plate viscometer (Brookfield LVDV-II+PRO CP40) at 900 s–1 (120 rpm) shear rate and 37°C temperature. For each measurement, 0.5 ml plasma was used.

2.3 Biochemical measurements

All chemicals and enzymes were obtained from Sigma Chemical Co. (Germany). Spectrophotometric measurements were performed using Shimadzu UV-1700 spectrophotometer. HPLC measurements were performed using Dionex ASI-100 HPLC equipment. Blood samples obtained from rats were divided into three portions. One portion was centrifuged for 5 min at 1000×g and the supernatant was kept as plasma. The next portion was centrifuged at 40C for 5 min at 10,000×g to obtain platelet-rich plasma (PRP) and the erythrocyte lysates were prepared from the last portion. Platelet counts were also determined in PRP diluted in Isoton II and counted on a thrombocounter. Serotonin level was measured both in plasma and PRP. MAO activity was measured in PRP. Lipid peroxidation, glutathione content and anti-oxidant enzyme activities were determined in plasma.

2.3.1 Preparation of erythrocyte lysates

Erythrocyte lysates were prepared according to a previous method [13]. Blood samples were centrifuged at 500xg for 15 min at room temperature. The plasma and buffy coat were then removed, and the erythrocytes were washed twice in 100 ml of saline and stored at – 800°C until used. Lysed erythrocytes were prepared by freezing and thawing twice and by the addition of three volumes of ice-cold distilled water. Cell membranes were removed by centrifugation at 1000xg for 20 min, and the supernatant was used as the erythrocyte lysate for the determination of lipid peroxidation, glutathione content and the antioxidant enzyme activities. Protein contents of erythrocyte lysates were determined according to the method of Bradford [14].

2.3.2 Determination of serotonin in plasma and PRP

After the platelet count, 1 ml PRP was centrifuged at 2000×g for 10 min. The supernatant was discarded and the pellet was suspended in 1 ml of a mixture containing 4% perchloric acid and 0.15% EDTA. The mixture was centrifuged at 2000×g for 10 min at 4°C. The resulting supernatant was filtered through 0.45-μm Millipore filters (Durapore PVDF membrane, SIGMA) by centrifugation at 2000×g for 5 min at 4°C. The samples were kept frozen – 80°C until used. Plasma and platelet serotonin contents were determined according to a previous method [15]. Plasma and PRP samples were applied to the Dionex HPLC system equipped with a 5-μm C18 column. The elution buffer consisted of 50 mM potassium phosphate, pH 5.0 and 12% methanol, with flow rate of 1 ml min–1 under isocratic conditions. The fluorescence detector (Dionex, RF 2000) was set at 230 nm excitation and 338 nm emission. N-methyl serotonin was used as internal standard. Plasma and platelet serotonin contents were expressed as ng/mL and nmol/10⁹ platelets, respectively [16].

2.3.3 Determination of platelet MAO activity

Platelet MAO-B activity was measured in PRP samples according to a previous method [17]. A chromogenic solution, which consisted of 1 mM vanillic acid, 500μM 4-aminoantipyrine, and 4 U ml–1 peroxidase in 0.2 M potassium phosphate buffer, pH 7.6, was prepared daily. The assay mixture contained 167μl chromogenic solution, 667μl 500μM benzylamine, and 133μl KP buffer, pH 7.6. The mixture was preincubated at 37°C for 10 min before the addition of enzyme. Reaction was initiated by the addition of PRP (100μl) and absorbance increase was monitored at 498 nm at 37°C for 60 min. A molar absorption coefficient of 4654 M–1. cm–1 was used to calculate the initial velocity of the reaction. Results were expressed as nmol/10⁹ platelets.

2.3.4 Determination of lipid peroxidation (LPO)

Lipid peroxidation in erythrocyte lysates was determined by the measurement of malondialdehyde (MDA) levels on the basis that MDA when reacted with thiobarbituric acid absorbs at 532 nm, according to the method of [18]. Values were expressed as nmol.mg ^{− 1} protein.

2.3.5 Determination of reduced and oxidized glutathione (GSH and GSSG) Levels

GSH and oxidized glutathione levels in erythrocyte lysates were determined according to a previous method [19]. Glutathione levels were expressed as μmol.mg protein-1. Redox status of glutathione was determined by the nanomolar ratio of GSH and GSSG (GSH/GSSG).

2.3.6 Determination of antioxidant enzyme activities

Catalase (CAT) activity was determined according to a previous method [20] and the activity was expressed as U.mg ^{− 1}. Glutathione S-transferase (GST) activity was determined spectrophotometrically by monitoring the formation of 1-chloro-2,4-dinitrobenzene (CDNB) and GSH conjugate at 340 nm [21], and activity was expressed as μM.mg ^{− 1}. Glutathione reductase (GR), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were determined by commercial assay kits (Cayman Chemicals, Ann Arbor, USA) and activities were expressed as U.mg protein ^{− 1}.

2.4 Statistical analysis

All statistical analyses were performed using “SPSS 15.0 for Windows”. Comparisons between groups for biochemical data were analyzed with one-way analysis of variance (ANOVA) and post hoc Tukey’s test. Hemorheological results were evaluated using Kruskal-Wallis and Mann-Whitney U tests. Data were presented as mean±SEM. p < 0.05 was accepted as statistically significant.

2.5 Ethical permission

Animal experiments were approved by Animal Ethics Committee of Hacettepe University (31.03.2011, 2011/23-9) and were performed in accordance with Helsinki Declaration.

3 Results

3.1 Erythrocyte deformability

Erythrocyte elongation index results showed no difference between groups at any shear stress as in the 3 and 30 Pa shear stress shown in the Figs. 1 and 2. Also no difference was found for SS1/2 and EIMax values (data not shown).

Fig.1

Erythrocyte elongation index values at 3 Pa for control, acute sertraline, acute fluvoxamine, chronic sertraline and chronic fluvoxamine. Results are presented as erythrocyte elongation index ± SEM. No statistically significant result was found (n = 10 for each groups). C: control, AS: acute sertraline, AF: acute fluvoxamine, CS: chronic sertraline, CF: chronic fluvoxamine.

Fig.2

Erythrocyte elongation index values at 30 Pa for control, acute sertraline, acute fluvoxamine, chronic sertraline and chronic fluvoxamine. Results are presented as erythrocyte elongation index ± SEM. No statistically significant result was found (n = 10 for each groups). C: control, AS: acute sertraline, AF: acute fluvoxamine, CS: chronic sertraline, CF: chronic fluvoxamine.

3.2 Erythrocyte aggregation

AMP values of all SSRI groups were lower than the controls, with significant difference between the control group and the acute fluvoxamine group as well as between the control and chronic sertraline groups (P < 0.05) (Table 1). In both cases, the AMP values of the SSRI groups were significantly lower than the control value. No significant difference was found in AI values of groups although the values of SSRI groups were higher than that of the control group (Table 1). t ½ was significantly low only in the chronic fluvoxamine group compared to the control, although all values of SSRI groups were lower than that of the controls (P < 0.05) (Table 1).

Table 1
Values for erythrocyte aggregation parameters (AMP, AI and t ¹/₂) for control, acute sertraline, acute fluvoxamine, chronic sertraline and chronic fluvoxamine

Group C AS AF CS CF

AMP au 13.8±0.6 13.0±0.2 12.0±0.4^* 12.0±0.3^* 13.3±0.4

AI % 44.7±2.3 50.8±1.9 47.3±3.2 48.8±1.7 53.0±1.4

t ¹/₂s 5.3±0.7 3.8±0.3 4.9±0.7 4.2±0.3 3.4±0.3^*

Group	C	AS	AF	CS	CF
AMP au	13.8±0.6	13.0±0.2	12.0±0.4^*	12.0±0.3^*	13.3±0.4
AI %	44.7±2.3	50.8±1.9	47.3±3.2	48.8±1.7	53.0±1.4
t ¹/₂s	5.3±0.7	3.8±0.3	4.9±0.7	4.2±0.3	3.4±0.3^*

Results are presented as mean±S.E.M. Data were statistically evaluated with Kruskal-Wallis test. ^*p < 0.05 vs. control group. (n = 10 for each group). C: control, AS: acute sertraline, AF: acute fluvoxamine, CS: chronic sertraline, CF: chronic fluvoxamine. AMP: erythrocyte aggregation amplitude, AI: Erythrocyte aggregation index, t ¹/₂: erythrocyte aggregation half time.

3.3 Plasma viscosity

There was no significant difference between groups in plasma viscosity values although plasma viscosity of AS was found highest among groups (Fig. 3).

3.4 Blood count results

Fig.3

Plasma viscosity results for control, acute sertraline, acute fluvoxamine, chronic sertraline and chronic fluvoxamine groups. Results are presented as plasma viscosity ± S.E.M. No significant difference was found between groups. (n = 10 for each group). C: control, AS: acute sertraline, AF: acute fluvoxamine, CS: chronic sertraline, CF: chronic fluvoxamine.

No statistically significant difference was found between the values of red blood cell count, hemoglobin and hematocrit values which is performed by a Coulter Counter (Table 2).

Table 2

Red blood cell count, hemoglobin and hematocrit values for control, acute sertraline, acute fluvoxamine, chronic sertraline and chronic fluvoxamine groups

	C	AS	AF	CS	CF
RBC 10⁶/cm³	7.3±0.2	7.6±0.1	7.4±0.2	7.1±0.2	7.3±0.1
Hb gr/dl	14.0±0.3	14.3±0.2	13.5±0.3	13.4±0.2	13.8±0.3
Hct %	40.2±0.8	41.2±0.5	39.4±0.6	38.9±0.4	40.1±0.6

Results are presented as mean±S.E.M. No significant difference was found between groups. C: control, AS: acute sertraline, AF: acute fluvoxamine, CS: chronic sertraline, CF: chronic fluvoxamine. RBC: erythrocyte count, Hb: Hemoglobin, Hct: Hematocrit.

3.5 Biochemical results

3.5.1 Platelet MAO activity, platelet and plasma serotonin levels

Platelet MAO activity was lower in all of the SSRI groups when compared with that of the control group. However the difference was statistically significant only in the chronic sertraline group (p < 0.05). Platelet serotonin level did not change in the acute SSRI groups, whereas it decreased in the chronic SSRI groups. A significant difference was found between the plasma serotonin levels of the chronic sertraline and the control groups, the level of the chronic sertraline group being higher (p < 0.05). Plasma serotonin level did not change in the acute or chronic fluvoxamine groups. The level was slightly higher in the acute sertraline group and significantly higher in the chronic sertraline group (p < 0.05) (Table 3).

Table 3
Platelet MAO activity and plasma and platelet serotonin levels of the study groups^a

C AS AF CS CF

Platelet MAO activity (nmol/10⁹ platelets) 11.8±1.3 10.7±0.6 11.3±1.0 9.5±0.6 ^*# 11.1±1.0

Plasma serotonin level (ng/ml) 12.4±1.4 13.5±1.3 12.2±1.5 46.5±5.9 ^ 12.3±1.3

Platelet serotonin level (ng/10⁹ platelets) 709.0±15.4 710.9±15.3 704.0±12.9 528.8±57.7 ^ 680.9±34.9

	C	AS	AF	CS	CF
Platelet MAO activity (nmol/10⁹ platelets)	11.8±1.3	10.7±0.6	11.3±1.0	9.5±0.6 ^*#	11.1±1.0
Plasma serotonin level (ng/ml)	12.4±1.4	13.5±1.3	12.2±1.5	46.5±5.9 ^**	12.3±1.3
Platelet serotonin level (ng/10⁹ platelets)	709.0±15.4	710.9±15.3	704.0±12.9	528.8±57.7 ^**	680.9±34.9

^aValues represents the mean±SEM of three independent experiments ^*p < 0.05 compared to control, ^**p < 0.05 compared to all groups, ^#p < 0.05 compared to acute and chronic fluvoxamine groups (n = 10 for each group for each group). MAO: Monoamine oxidase. C: control, AS: acute sertraline, AF: acute fluvoxamine, CS: chronic sertraline, CF: chronic fluvoxamine.

3.5.2 Lipid peroxidation (LPO), glutathione content and antioxidant enzyme activities in erythrocyte lysate

Lipid peroxidation (LPO), reduced and oxidized glutathione (GSH, GSSG) contents and antioxidant enzyme activities in erythrocyte lysates of the study groups are presented at Table 4. LPO was higher, GSH was lower, GSSG was higher, GSH to GSSG ratio (GSH/GSSG) was lower significantly in the chronic sertraline group when compared with the control group (p < 0.05).

Table 4
Lipid peroxidation, glutathione content and antioxidant enzyme activities in erythrocyte lysates of the study groups

C AS AF CS CF

LPO nmol MDA/mg 0.21±0.02 0.25±0.06 0.22±0.04 0.43±0.08 ^ 0.25±0.05

GSH μmol/mg 202.3±18.0 192.2±15.5 206.7±14.9 151.4±24.7 ^ 189.7±18.5

GSSG μmol/mg 19.06±1.64 22.56±2.57 18.87±1.77 41.14±6.65 ^ 18.82±1.61

GSH/GSSG 10.63±0.80 8.67±1.63 11.03±1.26 3.34±1.15 ^^ψ 10.16±1.47

CAT U/mg 228.4±34.8 219.0±30.5 221.5±29.1 212.5±29.5 216.3±27.5

GR nmol/mg 12.89±1.34 12.58±1.36 12.74±1.45 11.97±1.40 12.32±2.32

SOD U/mg 2.87±0.30 2.92±0.42 2.81±0.31 2.58±0.36 2.81±0.31

GPX U/mg 0.60±0.11 0.59±0.12 0.60±0.06 0.59±0.10 0.60±0.10

GST μmol/mg 0.22±0.06 0.21±0.06 0.22±0.06 0.22±0.07 0.22±0.05

	C	AS	AF	CS	CF
LPO nmol MDA/mg	0.21±0.02	0.25±0.06	0.22±0.04	0.43±0.08 ^**	0.25±0.05
GSH μmol/mg	202.3±18.0	192.2±15.5	206.7±14.9	151.4±24.7 ^**	189.7±18.5
GSSG μmol/mg	19.06±1.64	22.56±2.57	18.87±1.77	41.14±6.65 ^**	18.82±1.61
GSH/GSSG	10.63±0.80	8.67±1.63	11.03±1.26	3.34±1.15 ^**^ψ	10.16±1.47
CAT U/mg	228.4±34.8	219.0±30.5	221.5±29.1	212.5±29.5	216.3±27.5
GR nmol/mg	12.89±1.34	12.58±1.36	12.74±1.45	11.97±1.40	12.32±2.32
SOD U/mg	2.87±0.30	2.92±0.42	2.81±0.31	2.58±0.36	2.81±0.31
GPX U/mg	0.60±0.11	0.59±0.12	0.60±0.06	0.59±0.10	0.60±0.10
GST μmol/mg	0.22±0.06	0.21±0.06	0.22±0.06	0.22±0.07	0.22±0.05

Results are presented as mean± S.E.M. p < 0.05 vs. control group. (n = 10 for each group). LPO: lipid peroxidation, MDA: malondialdehyde, GSH: glutathione (reduced), GSSG: glutathione (oxidized), CAT: Catalase, GR: glutathione reductase, SOD: superoxide dismutase, GPx: glutathione peroxidase, GST: glutathione S-transferase. C: control, AS: acute sertraline, AF: acute fluvoxamine, CS: chronic sertraline, CF: chronic fluvoxamine. ^**p < 0.05 compared to all groups, ^ψp < 0.05 compared to acute fluvoxamine group.

Similarly with the data obtained for erythrocyte lyzates, LPO was higher, GSH was lower, GSSG was higher, GSH to GSSG ratio (GSH/GSSG) was lower significantly in the chronic sertraline group in the plasma (p < 0.05) (Table 5) suggesting oxidative damage. Antioxidant enzyme activities did not alter significantly in the erythrocyte lysates of the study groups. In apart with erythrocyte lysate results, plasma SOD value of the chronic sertraline group also decreased significantly (p < 0.05).

Table 5

Lipid peroxidation, glutathione content and antioxidant enzyme activities in plasma samples of the study groups

	C	AS	AF	CS	CF
LPO nmol MDA/mg	0.33±0.08	0.34±0.07	0.31±0.07	0.89±0.11 ^**	0.33±0.06
GSH μmol/mg	1.27±0.09	1.26±0.08	1.30±0.05	1.10±0.03 ^**	1.30±0.03
GSSG μmol/mg	0.12±0.01	0.13±0.01	0.12±0.01	0.16±0.03 ^*	0.12±0.01
GSH/GSSG	10.66±0.84	9.40±0.78	11.08±1.31	7.07±1.35 ^**	10.87±0.82
CAT U/mg	9.13±1.00	8.97±0.51	9.27±0.82	9.17±0.97	9.22±0.87
GR nmol/mg	0.07±0.02	0.07±0.03	0.07±0.01	0.07±0.02	0.07±0.02
SOD U/mg	1.20±0.06	1.20±0.04	1.20±0.04	1.15±0.03 ^**	1.21±0.05
GPX U/mg	0.95±0.10	0.93±0.12	0.93±0.18	0.93±0.14	0.95±0.13
GST μmol/mg	1.08±0.13	1.12±0.15	1.12±0.10	1.12±0.11	1.09±0.10

Results are presented as mean ± S.E.M. p < 0.05 vs. the control group. (n = 10 for each group). LPO: lipid peroxidation, MDA: malondialdehyde, GSH: glutathione (reduced), GSSG: glutathione (oxidized), CAT: Catalase, GR: glutathione reductase, SOD: superoxide dismutase, GPx: glutathione peroxidase, GST: glutathione S-transferase. C: control, AS: acute sertraline, AF: acute fluvoxamine, CS: chronic sertraline, CF: chronic fluvoxamine. *p < 0.05 compared to control, **p < 0.05 compared to all groups.

4 Discussion

The present study was aimed to investigate the effects of two selective serotonin re-uptake inhibitors (SSRIs) (sertraline and fluvoxamine) on hemorheological parameters (namely erythrocyte deformability, erythrocyte aggregation, hematocrit and plasma viscosity), on plasma and platelet serotonin levels, and on selected oxidative stress parameters.

Aggregation amplitude (AMP) was lower in all SSRI groups. The decrease in AMP was significant in the chronic sertraline and the acute fluvoxamine groups. Aggregation half time (t½) decreased in all groups reaching statistically significant level in the chronic fluvoxamine group (indicating faster aggregation) and aggregation index (AI) increased in all groups compared with the controls.

Plasma serotonin level increased slightly in the acute sertraline group and significantly in the chronic sertraline group. Platelet serotonin level, decreased slightly in the chronic fluvoxamine group and significantly in the chronic sertraline group. Platelet MAO activity was significantly low in the chronic sertraline group.

Indicators of oxidative stress in both plasma and erythrocytes were significantly different in the chronic sertraline group. These included increased malonyldialdehyde (MDA, which is an indicator of lipid peroxidation), decreased reduced glutathione (GSH), increased oxidized glutathione (GSSG) and decreased GSH/GSSG ratio in both plasma and erythrocytes. Among the oxidative enzymes studied in both erythrocytes and plasma (catalase, superoxide dismutase, glutathione reductase, glutathione peroxidase and glutathione S-transferase) only significant difference was the decrease in plasma SOD activity in the chronic sertraline group.

Lower AMP value states an attenuated aggregation amplitude. Lower aggregation half time (t½) states an increase in the speed of aggregation. Cumulative effect of AMP and t ½ property of aggregation can be judged with the help of AI which integrates these two parameters. In the present study AMP values of the SSRI groups were low indicating smaller aggregation amplitude. The low levels of t½ can be due to smaller aggregation that is completed in a shorter period of time. Low t½ can in turn be the cause of high AI. LORCA calculates the AI using the first 10 seconds of the syllectogram. With a lower AMP and hence quicker aggregation, it could be better to calculate the AI during a shorter period, for instance during the first 7 seconds. However the special computer program is not able to give us the results of such a calculation. Therefore we believe that the important finding of the present study is the decrease in AMP.

Although erythrocytes have no serotonin receptors [22], simply due to the fact that SSRI’s raise the plasma serotonin level, one may assume that their effect on aggregation is due to an increase in plasma serotonin. Our literature search revealed no articles on the effects of SSRI’s on erythrocyte aggregation or deformability. Previous research is basically on the hemorheological effects of serotonin and its antagonists. Kirsten et al. [23] showed that natridrofuryl affect in vitro and ex vivo platelet aggregation which is also supported by study of Wiernsperger et al. [24]. Nordt et al. in an in vitro study [22] treated erythrocytes with the serotonin antagonist naftidrofuryl, which resulted in a 30% decrease in erythrocyte aggregation. Their results are not in accordance with ours and so do not support the idea that the aggregation effect in the present study is through plasma serotonin. On the other hand Jung et al. [25] showed that acute Natridrofuryl administration increased erythrocyte deformability and exerted positive effects on microcirculation.

Hematocrit did not vary among the groups, which eliminated the possibility of a hematocrit effect on erythrocyte aggregation. Dormandy [26] has reported a decrease in blood viscosity measured at low shear rates after treatment with serotonin antagonist ketanserin. His results are in accordance with that of Ding et al. [27] but not with ours.

In the present study AMP was low in the chronic sertraline group and this finding was accompanied by a higher plasma and lower platelet serotonin level. However the low AMP level in the acute fluvoxamine group was not accompanied by a similar change in these serotonin levels. There was a slight decrease in platelet serotonin level in the chronic fluvoxamine group, which made us doubt that the effect of these SSRI’s on erythrocyte aggregation was through the plasma level of serotonin. Different SSRI’s may have different timings in their effects of lowering the platelet serotonin level. Sertraline probably decreases the platelet serotonin content more quickly and this may as well be the cause of a rise in plasma serotonin level. Fluvoxamine seems to decrease the platelet level slowly, which may be the reason why the plasma level does not rise due to slow release from the platelets and accompanying elimination from the plasma.

The present study did not reveal any change in the deformability of erythrocytes after acute or chronic administration of the two different SSRIs. Previous studies that have examined the effect of serotonin on erythrocyte deformability are mainly reporting the effects of the serotonin antagonist: ketanserin. Therefore they give an indirect information about the possible effect of serotonin itself. We could identify only one study that examined the direct effect of serotonin. De Cree et al. [28] treated erythrocytes with serotonin under in vitro conditions. The deformability of erythrocytes was found to be lower, only after 16 hours of incubation, but not before. In addition to this, ketanserin treatment reversed this effect. Most of the indirect studies using ketanserin have reported that this serotonin antagonist improved the deformability of erythrocytes [28 –31]. There are only two studies that have reported that ketanserin does not affect erythrocyte deformability [32, 33]. Our results are in accordance with these two. The discrepancy between the erythrocyte deformability results of the present study and the majority of previous studies may have three explanations. One is the methodology, because they have all used the method of filterability, in comparison to the ektacytometry technique used in the present study. The second explanation may lie in the fact that all these studies were performed on subjects with a disease (myocardial infarction, hypertension, claudication, Raynaud) whereas our experimental animals were healthy. The third reason as stated above may be due to the fact that the effect of SSRI’s may not be through plasma serotonin.

No significant difference in plasma viscosity between control and SSRI groups was observed in the present study. Since the major determinant of plasma viscosity is the plasma proteins it was concluded that SSRI administration has no impact on the protein level of blood and no impact on the interaction between plasma proteins. Results of previous experiments with Ketanserin also suggest no difference in this parameter.

In the present study both acute (5 days) and chronic (21 days) administrations of sertraline resulted in increases in plasma serotonin level. The level in the acute sertraline group was approximately 109% and that in the chronic sertraline group was approximately 370% of the controls. However no such effect was observed by fluvoxamine at 5th as well as 21st days. Our blood samples were obtained 1 hour after the administration of the last doses of the SSRI’s. In a study by Ortiz and Artigas [3] a single dose (10 mg/kg) of sertraline or fluvoxamine increased plasma serotonin level to 335% and 776% of the controls respectively, 30 minutes after their i.p. administration. In the same study another SSRI paroxetine, increased plasma serotonin to a maximum level at 30 minutes (358%) and the level dropped back to normal values within 4 hours. In the same study, another SSRI fluoxetine caused a rise in plasma serotonin (520%) in 30 minutes after its single application. However plasma serotonin was unaltered after 14 days of fluoxetine treatment. They explained this situation with possible physiological adaptations. These results all together suggest that plasma serotonin level may be affected differently by different SSRI’s. The duration of application and the time of blood sampling after the last dose also have effects on plasma serotonin level.

It is known that a rise in monoamine level results in a rise in MAO activity. As seen in Table 3, in the present study platelet MAO activity was found to be decreased in the chronic sertraline group when compared to the control, acute and chronic fluvoxamine groups (p < 0.05). This was accompanied by a significant decrease in platelet serotonin level and a significant rise in plasma serotonin level (Table 3) in accordance with the previous reports indicating that SSRIs caused a marked decrease in platelet serotonin level [34].

During the deamination process MAO enzyme is known to produce reactive oxygen species like hydroxyl radical and hydrogen peroxide. In the present study, MDA, GSH, GSSG, GSH to GSSG ratio all indicated oxidative damage in the chronic sertraline group (Table 4, 5). Results of previous studies have shown that the use of SSRIs in depressive patients improved oxidative stress [35]. Further studies with depressed rat groups are needed to clarify the connection of oxidative stress with the medication with SSRIs.

5 Conclusion

The SSRI’s in this study seem to effect the hemorheological parameters positively. Therefore they may be beneficial in CVDs. Complementary studies are needed to assess the hemorheological effects of all SSRI’s and antidepressants in common use, as well as to evaluate the effects of the changes in aggregation on blood viscosity.

Declaration of interest

Authors state no conflict of interest.

Footnotes

Acknowledgments

Authors would like to thank to Hacettepe University, Scientific Research Administration for financial support (Project Number: 011D10101001).

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