Blood cells supposedly implement the connection between lipid peroxidation (LPO) and hemostasis [1, 2]. For the participation degree valuation of thrombocytes, erythrocytes and leucocytes - the cells, the role of which in the relation between LPO and continuous intravascular blood clotting has been established [3, 4], we studied the dynamics of the LPO shifts´ nascence and intensity in these cells in vivo, and found out if the LPO shifts in the cells were connected with the shifts of thrombin-fibrinogen interaction (TFI) markers´ level, practically, the markers of continuous intravascular blood clotting (CIBC) [5-9].
While planning the experiments, we chose the actions promoting a slowly developing effect, that allows solving two problems: 1) to elicit the fact of the change nascence priority order, 2) to evaluate the degree of changes taking place in various cells under the same conditions of the experiment (i.e. in one experimental group of animals). Taking into account the fact that a large volume of blood is needed for the cells´ separation, there were so many rats included into the groups, that the number of laboratory definitions (n) after pulling of individual blood samplings were not less than six: the groups usually including 6 rats were expanded to 30 (in the group of 30 rats the blood of 5 rats was combined into one batch, the value of n was equal to 6). Totally 600 nonlinear white rats (170±10 g) were used.
Table 1. The content of plasmatic markers of CIBC, LPO and AOP in thrombocytes, monocytes, neutrophils and erythrocytes in different terms after 6-MTU administration, 150 mg/kg (n = 6)
|
Factors |
Control |
20th day |
25th day |
30th day |
35th day |
|
Р3, % |
85.2±2.0 |
87.1±2.1 |
85.2±1.4 |
79.1±1.2*+ |
74.7±1.3*+ |
|
Р4, sec |
3.3±0.06 |
3.1±0.03 |
2.8±0.06 |
2.1±0.04*+ |
1.8±0.04*+ |
|
FSP, mg% |
16.8±1.1 |
16.2±0.5 |
15.5±0.07 |
13.0±0.04*+ |
12.1±0.04*+ |
|
SFMC, mcg/ml |
25.4±1.1 |
24.2±1.1 |
23.2±1.0 |
20.1±0.4*+ |
18.3±0.3*+ |
|
D-D, mcg/ml |
0.20±.0.007 |
0.18±0.010 |
0.17±0.007 |
0.15±0.002*+ |
0.13±0.006* |
|
DC, А/mg LP |
0.045±0.004 0.032±0.003 0.027±0.002 0.021±0.002 |
0.044±0.004 0.029±0.003 0.030±0.003 0.022±0.003 |
0.031±0.0005*+ 0.0.27±0.003*+ 0.029±0.002 0.022±0.019 |
0.024±0.0005*+ 0.019±0.0003*+ 0.022±0.0002*+ 0.017±0.0001*+ |
0.020±0.0003*+ 0.016±0.0002*+ 0.019±0.0003*+ 0.016±0.0004*+ |
|
TBA, units/mg LP |
0.76±0.056 0.54±0.004 0.33±0.003 0.29±0.002 |
0.71±0.029 0.55±0.005 0.35±0.005 0.39±0.003 |
0.55±0.031*+ 0.49±0.004*+ 0.30±0.003 0.27±0.003 |
0.48±0.012*+ 0.43±0.007*+ 0.27±0.004*+ 0.20±0.002*+ |
0.39±0.024*+ 0.38±0.025*+ 0.23±0.006*+ 0.19±0.03*+ |
|
IP, min/ml |
48.5±2.3 48.5±2.3 46.4±2.1 47.3±2.0 |
53.9±1.0* 48.1±2.1 47.4±2.2 49.9±2.1 |
59.9±1.3*+ 50.9±1.9*+ 49.4±2.3 51.9±2.3 |
64.8±1.4*+ 56.7±1.5*+ 54.1±2.1*+ 52.8 ±2.0*+ |
67.2±1.3*+ 59.3±1.4*+ 56.1±2.0*+ 53.9 ±1.8*+ |
|
OR, мм3/min |
0.69±0.04 0.64±0.03 0.61±0.04 0.63±0.05 |
0.65±0.03* 0.62±0.05 0.59±0.03 0.60±0.03 |
0.60±0.02* 0.58±0.03* 0.57±0.04 0.59±0.04 |
0.48±0.03*+ 0.56±0.03*+ 0.55±0.04* 0.57±0.04* |
0.39±0.02*+ 0.54±0.03*+ 0.51±0.02*+ 0.56±0.03* |
Symbols: Р3 - P3 factor, FSP - fibrin split products, SFMC - soluble fibrin-monomeric complexes, TBA - products reacting with thiobarbituric acid, IP - induction period, OR - oxidation rate; * - authentic difference from the control, + - authentic difference of the 4th, 5th and 6th columns from the 3rd one. Note: the LPO factors represented in the 2-6th columns were determined in thrombocytes (upper line), monocytes (2nd line), neutrophils (3rd line) and erythrocytes (4th line).
For the LPO inhibition 6-methylthiouracil (6-MTU) and an antioxidant dimephosphon (DM) were used, for the LPO activation thyroxine (Т4) and lead acetate were used (sampling dosage and terms - in the tables). The TFI markers (factors Р3, Р4 FSP, SFMC and D-dimers) were defined according to the description [10, 11], the LPO and antioxidant potential (AOP) were controlled as it is shown [12, 13]. DM, 6-MTU, thyroxine (Т4) or lead acetate were administered to the rats daily.
From the table 1 it is clear that the LPO activity is gradually decreasing and the antioxidant potential (AOP) is growing in thrombocytes. The TFI markers´ level decrease happens later: only on the 30th and more noticeable - on the 35th day the level of FSP, SFMC and D-dimers fell down. In thrombocytes and monocytes there appeared a tendency to the LPO inhibition and AOP growth on the 20th day; on the 25th day these shifts became authentic; on the 30th day they enhanced and by the 35th day became still more significant.
In neutrophils the changes were of the same directivity (gradual LPO lowering and AOP growth), but authentic shifts appeared by the 30th day only. In erythrocytes, monocytes and neutrophils the shifts are the same, but by the 35th there were no shifts registered.
Similar experiments were carried out with DM - the LPO inhibitor varying from 6-MTU by the action mechanism. On the data of table 2, on the 5th day of the DM administration the AOP (IP elongation and OR reduction) was increased in thrombocytes and to a less extent - in monocytes. In the other cells the factors didn´t differ from the control values. On the 10th day the LPO inhibition and AOP growth were detected in thrombocytes and monocytes. On the 15th day these shifts were enhanced in thrombocytes and monocytes, and in neutrophils and erythrocytes the LPO slowed down and the AOP grew (the DC and TBA level decreased, IP elonged and OR reduced).
Table 2. The content of plasmatic markers of TFI, LPO and AOP in thrombocytes, monocytes, neutrophils and erythrocytes after DM administration, 1.0 g/kg, (n = 6)
|
Factors |
Control |
5th day |
10th day |
15th day |
20th day |
|
Р3, % |
84.9±1.7 |
85.2. ±1.2 |
81.9±1.9 |
77.2±1.1*+ |
72.1±1.2*+ |
|
Р4, sec |
3.3±0.05 |
3.1±0.05 |
2.9±0.02* |
2.0±0.03*+ |
1.6±0.03*+ |
|
FSP, ml% |
15.9±1.1 |
15.8±1.1 |
16.5±0.08 |
13.1±0.02*+ |
11.0±0.03*+ |
|
SFMC, mcg/ml |
26.0±1.2 |
24.7±1.2 |
22.8±1.1 |
19.0±0.3*+ |
16.7±0.3*+ |
|
D-D, kg/ml |
0.18±.0.007 |
0.21±0.012 |
0.17±0.012 |
0.15±0.003*+ |
0.12±0.008* |
|
DC, А/mg LP |
0.044±0.003 0.034±0.003 0.028±0.002 0.020±0.003 |
0.043±0.005 0.034±0.006 0.030±0.003 0.022±0.003 |
0.029±0.0006*+ 0.0.27±0.004*+ 0.026±0.004 0.021±0.019 |
0.019±0.0007*+ 0.021±0.0003*+ 0.020±0.0002*+ 0.016±0.0001*+ |
0.016±0.0003*+ 0.016±.0004*+ 0.018±0.0002*+ 0.015±0.0003*+ |
|
TBA,units/mg LP |
0.73±0.050 0.54±0.003 0.32±0.003 0.31±0.002 |
0.72±0.030 0.52±0.006 0.32±0.004 0.33±0.004 |
0.51±0.032*+ 0.47±0.003*+ 0.28±0.004 0.26±0.004 |
0.46±0.011*+ 0.42±0.004*+ 0.28±0.003*+ 0.21±0.003*+ |
0.36±0.022*+ 0.39±0.026*+ 0.22±0.004*+ 0.18±0.02*+ |
|
IP, min/ml |
48.1±2.2 48.4±2.2 47.8±2.1 47.2±2.3 |
54.6±1.1* 48.4±2.2 47.9±2.3 49.1±2.0 |
61.3±1.2*+ 53.8±1.1*+ 48.9±2.4 50.1±2.2 |
65.9±1.3*+ 56.9±1.3*+ 55.3±2.0*+ 52.9 ±1.9*+ |
69.3±1.4*+ 59.9±1.3*+ 56.5±1.9*+ 54.8 ±1.7*+ |
|
OR, mm3 /min |
0.71±0.03 0.65±0.02 0.60±0.03 0.62±0.04 |
0.63±0.03* 0.57±0.06* 0.58±0.04 0.61±0.04 |
0.58±0.03* 0.56±0.02* 0.57±0.03 0.58±0.05 |
0.48±0.03*+ 0.54±0.02*+ 0.53±0.03* 0.54±0.03* |
0.36±0.03*+ 0.52±0.03*+ 0.50±0.03*+ 0.54±0.04* |
Symbols: like in table 1; * - authentic differences from the control; + - the difference of the 4th, 5th and 6th columns from the 2nd one. Note: like in table 1.
On the 20th day all the enumerated shifts in all the cells, especially in thrombocytes, enhanced.
The TFI markers´ level shifts are as follows: on the 10th day only the Р4 factor level decreased, on the 15th day all the other factors decreased, the shift became still more noticeable by the end of the observation.
Hence, as in the experiment with 6-MTU, the LPO and AOP change earlier than the TFI markers´ level shifts appear (according to the CIBC). The fact that according to the ability to react on the LPO changes the investigated cells are ranged in the following succession:
thrombocytes > monocytes > neutrophils > erythrocytes.
Further on, Т4 or lead in the dosages changing the LPO and AOP [14, 15] were used in the experiments, and the following was found out (table 3): on the 5th day of T4 administration the signs of LPO acceleration in thrombocytes, monocytes and erythrocytes appeared (DC and TBA level growth), the AOP decreased (the IP shortening in thrombocytes and neutrophils, and OR growth in thrombocytes, monocytes and neutrophils). All the shifts are little, but authentic (р < 0.05); by the 7th day, and especially by the 9th one, the shifts enhanced (exclusive of neutrophils and erythrocytes, wherein the shifts on the 7th and 9th day were practically the same).
Table 3. The content of plasmatic markers of TFI, LPO and AOP in thrombocytes (upper line), monocytes (2nd line), neutrophils (3rd line) and erythrocytes (4th line) in various terms of T4 administration, 8.0 mg/kg (n = 6).
|
Factors |
Control |
3rd day |
5th day |
7th day |
9th day |
|
Р3, % |
85.4±1.4 |
86.0 ±1.7 |
89.0±1.1 |
91.6±1.0* |
98.2±1.3* |
|
Р4 |
3.2±0.02 |
3.4±0.06 |
3.5±0.05 |
4.1±0.05* |
4.6±0.02* |
|
FSP, mg% |
15.8±1.0 |
16.1±0.8 |
16.9±0.09 |
18.3±0.06* |
23.8±0.06* |
|
SFMC, mcg/ml |
26.4±1.5 |
25.9±1.2 |
28.1±1.3 |
27.9±1.5 |
32.4±0.6* |
|
D-D, mcg/ml |
0.21±.0.005 |
0.22±0.014 |
0.22±0.011 |
0.23±0.010 |
0.29±0.004* |
|
DC, А/mg LP |
0.050±0.002 0.031±0.002 0.024±0.003 0.021±0.003 |
0.051±0.003 0.033±0.004 0.025±0.002 0.020±0.003 |
0.064±0.001* 0.037±0.003* 0.029±0.003* 0.024±0.004 |
0.072±0.003* 0.040±0.004* 0.031±0.003* 0.028±0.003* |
0.081±0.005* 0.044±0.003* 0.036±0.004* 0.032±0.002 |
|
TBA, units/mg LP |
0.75±0.051 0.52±0.004 0.33±0.004 0.28±0.003 |
0.79±0.021 0.50±0.005 0.31±0.002 0.29±0.004 |
0.93±0.031* 0.55±0.002* 0.35±0.005 0.34±0.002* |
0.99±0.039* 0.62±0.005* 0.38±0.002* 0.36±0.003* |
1.21±0.039* 0.68±0.006* 0.40±0.003* 0.37±0.002* |
|
IP, min/ml |
48.1±2.2 48.4±2.2 47.8±2.1 48.2±2.3 |
46.1±1.5 48.0±2.0 49.7±2.2 47.6±2.4 |
42.0±1.3* 47.0±2.4 44.8±1.8* 46.2±2.2 |
40.1±1.2* 44.2±2.0* 43.1±2.2* 44.2±2.3* |
36.0±1.3* 42.5±2.1* 44.1±2.0* 44.9±2.2* |
|
OR, mm3/min |
0.71±0.03 0.64±0.03 0.59±0.03 0.64±0.04 |
0.76±0.07 0.66±0.03 0.61±0.04 0.65±0.03 |
0.82±0.03* 0.68±0.02* 0.66±0.02* 0.67±0.04 |
0.88±0.05* 0.71±0.03* 0.68±0.04* 0.69±0.03* |
0.94±0.04* 0.72±0.02* 0.69±0.04* 0.89±0.03* |
Symbols: like in table 1; * - authentic difference from the control
The TFI markers´ (FSP, SFMC, Р3 and Р4 factors) level increase was detected on the 7th day of the experiments already. On the 9th day the shifts were more noticeable, plus the D-dimers´ level increase.
In the experiments with the administration of lead - a pro-oxidant, (table 4) differing from T4 on the action mechanism, the following was found out: on the 10th day already the LPO and AOP shifts were detected (the TBA and DC growth in thrombocytes and monocytes, and DC growth in neutrophils as well), the IP shortening in all the cells (excepting erythrocytes) and OR increase (exclusive of neutrophils). By the 12th day the shifts became more significant and spread onto all the cells, enhancing by 15th and 18th days.
The content of TFI markers didn´t change on the 10th and 12th days of the lead administration, but somehow increased (excepting SFMC and D-dimers) by the 12th day. On the 18th day the content of all the TFI markers was increased.
Thus, while being administrated in comparatively small doses, lead and T4 cause the progressing growth LPO intensity and the AOP reduction. These shifts are ahead of the TFI markers´ level change in time.
The data got allow coming to the following conclusions:
- The LPO activation or inhibition and the conforming to them decrease (or increase) of the AOP are attended by a decrease (increase accordingly) of the TFI intensity, i.e. the intensity of the continuous intravascular blood clotting.
- The LPO and AOP changes in thrombocytes preface the changes of continuous intravascular blood clotting intensity.
- Simultaneously with the LPO and AOP changes in thrombocytes, while affected with pro- and antioxidants, the LPO and AOP in monocytes, heterophilic leukocytes and erythrocytes change in the same direction.
In connection with this it seems to be advisable to control the content of plasmatic TFI markers at the diseases attended with the activation of free-radical processes.
Naturally, the investigations aimed at the specification of the character of the relation between the LPO intensity in blood cells, especially in thrombocytes, and the CIBC markers´ content make sense.
Table 4. The content of CIBC markers in plasma, LPO and AOP in thrombocytes, monocytes, neutrophils and erythrocytes (1st, 2nd, 3rd and 4th line accordingly) in various terms of lead acetate administration in the dosage of 50 mg/kg (n = 6 in every stage).
|
Factors |
Control |
10th day |
12th day |
15th day |
18th day |
|
Р3, % |
85.3±1.4 |
86±.1.5 |
87.8±1.1 |
101±1.4* |
122±1.7* |
|
Р4, sec |
3.1±0.02 |
3.3±0.03 |
3.5±0.05 |
4.1±0.03* |
5.0±0.04* |
|
FSP,mg % |
17.0±1.0 |
16.9±0.8 |
16.8±0.1.0 |
19.9±0.7* |
24.2±0.06* |
|
SFMC, mcg/ml |
25.4±1.3 |
25.9±1.2 |
26.4±1.3 |
29.1±1.4 |
36.8±0.7* |
|
D-D, mcg/ml |
0.18±.0.003 |
0.20±0.014 |
0.19±0.011 |
0.27±0.034 |
0.35±0.011* |
|
DC, А/mg LP |
0.048±0.003 0.033±0.002 0.023±0.002 0.021±0.002 |
0.057±0.003* 0.039±0.001* 0.029±0.002* 0.024±0.004 |
0.064±0.004* 0.042±0.002* 0.032±0.004* 0.027±0.004* |
0.072±0.003* 0.048±0.005* 0.037±0.003* 0.028±0.003* |
0.083±0.006* 0.05±±0.002* 0.040±0.005* 0.029±0.002* |
|
TBA, units/mg LP |
0.76±0.052 0.51±0.003 0.32±0.003 0.28±0.003 |
0.83±0.022* 0.56±0.002* 0.37±0.001 0.29±0.004 |
0.94±0.033* 0.59±0.002* 0.39±0.003* 0.34±0.002* |
0.98±0.032* 0.64±0.005* 0.39±0.002* 0.36±0.002* |
1.25±0.041* 0.69±0.005* 0.40±0.003* 0.38±0.002* |
|
IP, min/ml |
48.5±2.1 47.1±1.9 46.3±1.2 48.3±2.3 |
44.1±1.1* 45.0±0.4* 45.1±1.8* 47.4±2.5 |
41.1±1.1* 44.1±2.0* 44.2±1.2* 45.3±2.1* |
39.6±1.2* 42.1±1.2* 43.1±2.0* 43.9±2.1* |
35.2±1.3* 41.3±2.0* 42.9±2.1* 43.5±2.3* |
|
OR, mm3/min
|
0.70±0.03 0.65±0.02 0.60±0.04 0.63±0.03 |
0.76±0.03* 0.69±0.01* 0.62±0.05 0.68±0.02* |
0.83±0.04* 0.67±0.02* 0.67±0.02* 0.69±0.03* |
0.89±0.04* 0.72±0.04* 0.69±0.03* 0.71±0.03* |
0.96±0.04* 0.73±0.03* 0.70±0.05* 0.71±0.03* |
Symbols: like in table 1; * - authentic difference from the control.
References:
- Byshevsky A.Sh. Blood cells in lipid peroxidation and hemostasis relation implementation / Byshevsky A.Sh., Galyan S.L., Dementyeva I.A. and others) // Problems of physiology and hemostasis pathology (works of problem committee at Interdepartmental Research Council on hematology and transfusiology of RAMS. - Barnaul. - 2000 - pp. 156-163.
- Galyan S.L. Influence of levonorgestrel on continuous intravascular blood clotting depending on lipid peroxidation intensity / Galyan S.L., Aptekar I.A., Alborov R.G. and others // Scientific Reporter of TSMA, 2003. - 2. - p. 31.
- Alborov R.G. Blood cells - factor of lipid peroxidation and continuous intravascular blood clotting relation / Alborov R.G. // Materials of interregional conference of biochemists of Ural, Western Siberia and Volga Region "Biochemistry: from investigation of molecular mechanisms to introduction into clinical practice and production", Orenburg, 2003, pp. 152-154.
- Gawaz M.P. Blood Platelets / M.P.Gawaz // Stuttg.; New York: Time. - 2001. - p. 190.
- Zubairov D.M. Über die Thrombinzirculation in Blut / D.M. Zubairov // Folia Haematol. - 1962. - 79. - 1. - S. 62-75
- Zwaal R.F.A. Blood, membranes and haemostasis / R.F.A.Zwaal // Haemostasis. - 1982. -11. - Pp. 12-39.
- Bokarev I.N. Differential diagnostics and treatment of internal diseases. Bleeding sickness, or hemorrhagic syndrome. Differential diagnostics / Bokarev I.N. // Moscow. - 2002. - p. 75.
- Zubairov D.M. Molecular fundamentals of blood clotting and thrombosis. Kazan: FES AST. - 2000. - p. 367.
- Byshevsky A.Sh. Vitamins, intravascular blood clotting and lipid peroxidation / Byshevsky A.Sh., Galyan S.L., Shapovalov P.Ya. // Moscow: Medicine. - 2006. - p. 105.
- Baluda V.P. Laboratory methods of hemostasis system investigation / Baluda V.P., Barkagan Z.S., Goldberg Ye.D. and others. - Tomsk. - 1980. - p. 310.
- Barkagan Z.S. Introduction into clinical hemostasiology / Barkagan Z.S. // M.: Newmediamed-JSC. - 1988. - p. 45.
- Ushkalova V.N. Complex analysis of blood lipids by spectrophotometric, fluorometric and kinetic methods / Ushkalova V.N., Ioanidis N.V., Deyeva Z.M. and others // Laboratory work. - 1987. - 6. - pp. 446-460.
- Ushkalova V.N. Free-radical lipid peroxidation in experiment and clinic. - Tyumen: TSU Publishing House - 1997. - pp. 5-21.
- Venditti P. Antioxidant-sensitive shortening of ventricular action potential in hyperthyroid rats is independent of lipid peroxidation / P.Venditti, De T.Leo, Di S.Meo // Mol. Cell.Endocrinol. - 1998. - 142. - 1.- 2. P. 15-23
- Venditti P. Thyroid hormone-induced oxidative stress / P.Venditti, Di Meo S. P. Venditti, S. Di Meo // Cell. Mol. Life Sci. - 2006. - 63(4). - Р. 414-434.