DOI 10.1007/s10517-015-2811-2 Bulletin of Experimental Biology and Medicine, Vol. 158, No. 4, February, 2015
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GENERAL PATHOLOGY AND PATHOPHYSIOLOGY Effect of Cerebral Ischemia on Redox Status of Plasma Aminothiols A. V. Ivanov, V. V. Aleksandrin, B. P. Luzyanin, and A. A. Kubatiev Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 158, No. 10, pp. 409-412, October, 2014 Original article submitted January 18, 2014 We measured the content of reduced aminothiols (cysteine, homocysteine, and glutathione) after mechanical occlusion of the middle cerebral artery in rats. During acute period of ischemia (3 h), these parameters decreased by ~10 times. In 7 days, the content of reduced aminothiols in some animals remained low, but in the others surpassed the initial levels. These results indicate significant impairment of the redox status of the circulating aminothiol pool apparently caused by oxidative stress of the peripheral vascular endothelial occurring as a response to cerebral ischemia. Thus, the total amount of homocysteine is considered a risk factor for stroke, while its reduced fraction can reflect the post stroke state. Key Words: aminothiols; cerebral ischemia; high performance liquid chromatography; dithionitrobenzoic acid The role of hyperhomocysteinemia as an independent risk factor of acute brain stroke is shown in numerous studies [7]. The proposed mechanisms and targets of the pathological effects of homocysteine on blood vessels aroused interest not only to evaluation of its total content, but also to measurement of other forms of homocysteine and other aminothiols [8,10,12]. The total content and cysteine and homocysteine fractions are increased after stroke, while changes in reduced homocysteine are contradictory [6,15]. On the other hand, acute stroke is a stressful event triggering the mechanisms of metabolic response in peripheral tissues, which can affect the redox balance and metabolism of aminothiols. Thus, early studies on experimental model of cerebral ischemia [14] showed increased plasma levels not only of enzyme markers of brain damage (creatine phosphokinases, glutamate Research Institute of General Pathology and Pathophysiology, Russian Academy of Medical Sciences, Moscow, Russia. Address for correspondence:
[email protected]. A. V. Ivanov
transaminases), but also glucocorticoids, fatty acids, triglycerides, and cholesterol within the first hours of ischemia. Reflex release of corticosterone and hydrocortisol, probably because of activation of the sympathetic nervous system, triggers the release of lipids from peripheral tissues, lipolysis, and steatosis [14]. Another consequence of acute stroke is a sustained and long-term (up to 10 days) increase in BP in 1/3 of patients [13]. The mechanism of this increase is not fully elucidated, but it is suggested that nervous pulses causing dysregulation of the arterial tone also play a triggering role. It was shown that ischemia/reperfusion of the middle cerebral artery reduced acetylcholinesterase and nitroprusside-mediated vasodilation of mesenteric arteries. In this case, elevation of plasma IL-6 and IL-1β, ROS (O2–), and nitrosylation of proteins are also observed. All this phenomena attest to endothelial dysfunction in peripheral vessels [11]. Despite ample data on the role of aminothiols, primarily homocysteine, in oxidative stress and endothelial dysfunction, there are still no data on their
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Bulletin of Experimental Biology and Medicine, Vol. 158, No. 4, February, 2015 GENERAL PATHOLOGY AND PATHOPHYSIOLOGY
involvement in the post-stroke response of peripheral organs. In this regard, it is interesting to explore changes in the total content and redox state of plasma aminothiols caused by cerebral ischemia. Most methods of HPLC-UV and capillary electrophoresis (CE) with UV or mass spectrometric detectors are not sufficiently sensitive to determine reduced homocysteine [1,3,4]. Reduced forms of aminothiols are usually assayed by highly sensitive methods: HPLC and CE with fluorescence detectors and GCspectrometric systems [2,5]. However, some studies showed that these compounds could be assayed with common thiol-specific reagents, e.g. dithiodipyridine [6] and 5,5-dithio-bis-2-nitrobenzoic acid (DTNB) [9]. Such reagents as DTNB are characterized by reversibility of action. In total aminothiol assay, DTNB is added in large excess, therefore undesirable disulfide exchange reaction between formed TNB-thiol derivatives and thionitrobenzoate dianion makes no significant contribution to the analysis. However, during the analysis of reduced aminothiols, this reaction and interactions between thionitrobenzoate dianion with thiols oxidized to disulfides lead to the accumulation of corresponding TNB derivatives and thus distort the results of the analysis. Therefore, to suppress the undesired thiol-disulfide interchange reactions, we used NEM as a blocker, which covalently and irreversibly binds the thionitrobenzoate dianion. In this work, we modified the method of determining reduced plasma aminothiols using DTNB to study the changes in the redox status of cysteine, homocysteine and glutathione associated with local cerebral ischemia in rats.
MATERIALS AND METHODS We used acetonitrile Ultra Gradient (RCI Labscan) 99.9%, NEM >98% (HPLC) (Sigma-Aldrich), DTNB, dihydrate of 5-sulfosalicylic acid (SSA), phosphoric acid, sodium citrate (dihydrate), trifluoroacetic acid 99.0% (Sigma-Aldrich), NaH2PO4·2H2O 98-100.5% (Panreac), Na2HPO4·2H2O 98-101% (Panreac), NaOH, EDTA disodium salt dihydrate 99% (AppliChem), Dpenicillamine P4875 (PA; Sigma), DL-dithiothreitol >99.5% (Fluka), L-cysteine 97%, glutathione 99% (Sigma-Aldrich), DL-homocysteine >95% (Sigma), ethanol rectified. The experiment was performed on adult outbred white male rats weighing 260-300 g. The operations were performed under general anesthesia (chloral hydrate intraperitoneally at a dose of 300 mg/kg). To measure systemic BP and blood sampling, both femoral arteries were isolated and cannulated (heparin intraarterially, 500 U/kg). The head was fixed in a stereotaxic frame. To register the blood flow with a cylindri-
cal sensor, the parietal bone was trephined (5×3 mm) without impairing dura mater integrity (coordinates: AP, 5 mm; L, 3 mm). Blood flow was recorded and spectral wavelet analysis of blood flow oscillations was performed using blood flow analyzer LAKK-02 (version 2.2.0.507; LAZMA). Recording was started 30 min after surgery completion at the ambient temperature of 20 to 21oC. Control blood samples were taken 7 days before surgery. The blood was collected after 3 hours and 7 days after surgery. Focal cerebral ischemia was reproduced using the middle cerebral artery occlusion. Surgical 4-00 polypropylene thread (22 mm long; Ethicon) treated with silicone and poly-L-lysine was inserted retrogradely into the left external carotid artery and then carried through the bifurcation of the common carotid artery and the internal carotid artery to the mouth of the middle cerebral artery blocking it. Venous blood was collected into tubes with sodium citrate and centrifuged at 3000g for 3 min. The plasma for total thiol assay was collected, frozen at -20oC, and stored until analysis. To determine reduced thiols, 900 μl blood plasma was added to 225 μl 5-sulfosalicylic acid (SSA) solution (230 g/liter) immediately after isolation. The samples were mixed thoroughly, frozen, and stored at -80oC. Before derivatization, the samples were centrifuged for 5 min at 15,000g; 100 μl 10 mM DTNB in 0.4 M Na-phosphate buffer (pH 8.0) supplemented with 5 mM EDTA-Na, 50 μl 25 μM internal standard (TNB-derivative of PA), 500 μl of plasma supernatant and 95 μl 1.5 M NaOH were added sequentially to the test tube. The sample was vigorously shaken for 5 sec, then 50 μl 100 mM NEM was added and after 30-sec stirring, 50 μl 300 g/liter SSA was added. Total thiols were assayed as described previously [3]. The components were separated using an Agilent Eclipse XDB-C18 column (150 mm×4.6 mm, 5 μ) and Agilent Hypersil BDS-C18 (150 mm×4.6 mm, 5 μ) serially connected. The temperature of the columns was maintained at 35oC, that of eluent at 24oC. Injection volume was about 500 μl (injector loop was completely filled). The flow rate of the liquid phase was 0.5 ml/ min. Eluent A: 0.04% TFA in 0.2 M Na-phosphate buffer (pH 6.6); eluent B: acetonitrile. Direct B gradient elution occurred for 14 min from 4.5 to 9.3%, columns were washed for 3 min with 50% B, followed by a linear decrease of B from 50 to 4.5% for 1 min. Then the columns were regenerated for 10 min at 4.5% B. The absorption signal was determined at 330 nm. Primary processing (integration of chromatographic peaks) was performed with Perkin Elmer TotalChrom 6.2; calibration curves were generated by Mi-
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crosoft Excel. To determine the concentration of aminothiols, the ratio of the areas of the analyte and the internal standard was used. The signal was calibrated by the analysis of plasma samples with addition of a known amount of the analytes and regression analysis.
RESULTS Figure 1 shows a blood plasma chromatogram after sequential treatment with DTNB and NEM. Elution time for TNB derivatives of cysteine, homocysteine, glutathione, and PA was 8.1, 11.3, 14.5, and 20.7 min, respectively. Retention time variation was from 3 to 5%. The detection limits of the analytes were 0.1 μM (s/n=4). Cerebral blood flow is reduced by half 3 hours after middle cerebral artery occlusion, on average to 18.0±1.9 perfusion units (p
