FEATURES OF MICROCIRCULATORY DYSFUNCTION ACCORDING TO WAVLET ANALYSIS LASER DOPPLER FLOWMETRY IN PATIENTS WITH CHRONIC ISCHEMIC HEART DISEASE AND CONCOMITANT DIFFUSE LIVER DISEASES

Aim. To determine the features of microcirculatory dysfunction and in patients with chronic ischemic heart disease, depending on the presence of concomitant diffuse liver disease. Materials and methods. We performed a prospective study on the basis of MC «Doctor Vera», as well as in the diagnostic department of Medbud Clinic between 2009 and 2019. A total of 187 patients were examined. Patients’ blood flow was assessed by transthoracic echocardiography, wavelet analysis laser Doppler flowmetry (LDF). Results. In the analysis of indicators of microcirculatory dysfunction in patients with isch­ emic heart disease, statistically significant deterioration of wavelet analysis laser Doppler flowmetry was determined. In patients with ischemic heart disease and left ventricular ejection fraction < 40 %, indicators of wavelet analysis LDF were significantly lower. We also found that the severity of diffuse liver disease significantly disrupts peripheral blood flow (Р < 0.05). Conclusions. The results of intracardiac hemodynamics, micro­ circulatory dysfunction were first presented. In patients with isolated ischemic heart disease as the left ventricular EF decreased, a significant decrease in capillary blood flow was observed as a result of deterioration of central hemodynamics, development of atherosclerotic vascular changes, as well as an increase in their vascular tone. We have proved that in the presence of DLD in patients with ischemic heart disease and EF < 40 %, the degree of increase in peripheral resistance and impaired venous outflow were more pronounced due to the depletion of vasoactive substances production, impaired their excretion by hepatocytes due to irreversible morpho-functional changes in liver at the stage of decompensation of HF, severe fibrosis, the formation of regeneration nodes. In patients with LC, the most severe disorders of peripheral blood flow were determined according to the data of the digital capillaroscopy, which testifies to the influence of the degree of liver damage on the state of microcirculation.

tients were included in the study: 34 women and 154 men from 18 to 85 years old (average age was 46. 1 ± 17.4). There were three groups in our study.
The second group included 102 patients with DLD, average age was (37.3 ± 15.2) years: 12 patients with LC, average age was (45.1 ± 9.2) years; 43 patients with CVH: 6 women, average age was (31.8 ± 8.9) years, 12 patients with CVHB, 24 patients with CVHC, 4 patients with CVHB + С, 2 patients with viral hepati tis Epstein -Barr, 1 patient with CVH TTV; 21 patients with CTH of mixed etiology, especially alcoholic, average age (42.7 ± 12.6) years; 26 patients with NASH, average age (40.8 ± 10.0) years. We also analysed the data of the patients in the second group according to the stages of liver diseases: subgroup A -48 patients with minimal cytolytic syndrome and cholestasis (elevation of alaninaminotrans ferase (ALT) less than in 5 times; bilirubin was less than 100 mcmol/l); subgroup B -42 patients with moderate syndrome of cytolysis and cholestasis (elevation of ALT in 5-10 times, bilirubin from 100 to 200 mcmol/l); subgroup B -12 pa tients with LC.
Transthoracic echocardiography (echoCG) was conducted on the device HDI 11XE Philips according to the general methodology [13] with measurement in 2D-mode of wall thickness of the right ventricle (Trv), posterior wall of the LV (Tlv), intraventricular septum (Tivs), short-axis size of the left atrium (LA), end diastolic volume (EDV) of LV by Simpson, its EF and also short-axis size of the RV in basal chamber -Drv(s), diameter of vena cava inferior (VCI). Diastolic filling of the LV was evaluated according to the ratio of transmitral flow velocity in the period of rapid filling and in systolic phase of the LA (E/A), time of decreasing of early diastolic filling velocity -deceleration time (DT) and duration of the period of left ventricular isovolumic relaxation time (IVRT). Systolic pres sure of the pulmonic artery (SPpa) was evaluated by the speed of tricuspid regur gitation flow [13].
The signal reflected from the erythrocytes has a Doppler frequency shift relative to the scanning beam. The depth of optical sensing of tissues depends essentially on the wavelength of the laser source. For red radiation (632 nm), it does not exceed 1mm. Signal recorded during LDF characterizes blood flow in microvessels in a volume of 1-1.5 mm 3 tissue [4,12,14].
In human skin, LDF provides integral information on a very large number of erythrocytes, about 3.4 · 10 4 /mm 3 , which are simultaneously in the area of tissue probing. The patient's blood flow is recorded at rest for 8 minutes after a 15-minute adaptation period. Functional test with occlusion of the brachial artery is performed.
The occlusal test (OT) is realized by compressing for 1-3 min the correspon ding area of the limb with the tonometer cuff so as to cause a stop of blood flow and, accordingly, ischemia in the study area.
After cessation of the occlusion, the blood flow resumes and develops reactive post-occlusive hyperemia, which is manifested in an increase in the microcirculation index to a value that exceeds the initial level of indicator of microcirculation (IM) with subsequent decline to the initial level.
The physiological role of a compression test is manifested in stop blood supply in the shoulder arteries and respectively in changes blood circulation in the tissues. In the majority of cases the blood pressure and the rate of blood circulation in the vessels of microcirculation are changed. At the time of decompression of blood, arterial blood flow and reactive hyperemia appears with maximum blood levels of blood vessels of microcirculation. Change blood pressure is the minimum hour of compression up to the maximum time of the reactive characteristic is the entire range of capacities of the blood pressure.
When a given test is carried out, an estimate of the "biological zero" indicator (indicator of microcirculation for the incidence of arterial congestion) and increased reserve of the microcirculatory channel according to the increase in the indicator of microcirculation is observed during reactive post-inclusive hyperemia.
According to experimental studies, post-exclusion reactive hyperemia is a neu rogenic reaction, which is realized mainly through the release of neuropeptide KGRP (cocalcigenin) and neuronal nitric oxide (NO), secreted by afferent nociceptive C-fibers. These factors induce NO synthesis by the endothelium, which in turn, by vasodilating the vasculature by affecting vascular smooth muscle [3,4,7].
Determined the baseline state of microcirculation by indicator of microcircula tion (IM) and capillary blood flow reserve (CBFR) during OT.
Previously, in this study, all patients with the use of an occlusive test sample identified 4 variants of hemodynamic types of microcirculation (HTM). In particu lar: normocirculatory, hyperemic, spastic, stasic. Normocirculatory HTM is characterized by an initial magnitude of IM = 4.5 -6.5 p.u., with a normoreactive type of response to arterial occlusion, CBFR = 200 -300 %. Hyperemic HTM is characterized by an increase in blood flow to the microcirculatory bed with an initial IM value > 6.5 p. u. in OT, the type of response to arterial occlusion is hyperreactive (reducing IM by more than 3.2 p. u.). CBFR is always below 200 %. Spastic HTM is characterized by a decrease in blood flow to the microcirculatory bed due to spasm of precapillary sphincters. initial IM reduced < 4.5 p. u., and at OT the type of blood flow for arterial occlusion is reactive (de crease in IM during occlusion by less than 1.5 p. u.); CBFR > 300 %. Stasic HTM is noted at decrease in speed and stasis of blood flow at the level of the capillary unit, as well as at the level of the post-capillary unit-venules and postcapillaries. Thus IM < 4.5-6.5 p. u., the degree of decrease in IM depends on the severity of the phenomena of blood stasis and rheological disorders (aggregation of blood ele ments, sweet phenomenon); CBFR < 200 %. Type of blood flow to arterial occlu sion -areactive. This type of microcirculation is noted in paresis of vessels of inflow and disturbance of outflow [4]. In wavelet analysis, the following components were identified: A max C -maximum amplitude of cardiac flaxmotions -oscillations of the capil lary wall caused by a contraction of the heart form a peak in the frequency range by synchronous pulse oscillation. A max R -maximum amplitude of respiratory flux motions -in the frequency range of the respiratory function of the oscillation of the capillary wall form a respiratory peak. A max M -maximal amplitude of myogenic flaxmotions and A max N amplitude of neurogenic flaxmotions -myogenic and -neu rogenic activity of precapillary vasomotors are detected with maximum frequencies in the range of 0.02-0.16 Hz. A max E is the maximum amplitude of endothelial flux motions -the slowest oscillations in the microcirculatory system, which are syn chronous and dependent on the activity of endothelial cells due to the secretion of various compounds, in particular nitric oxide. SI -shunting indicator of AV -shunt that indicates the state of nutritional blood flow. Endothelium dependent vasodilatation (EDV) and endothelium-independent va sodilatation (EIDV) by D. S. Celermajer and co-authors method was conducted with ultrasound system HD 11 (Philips MS) and lineal transducer with frequency 3-12 MHz according to its methodology [11,12].
Humoral marker of endothelial dysfunction Willebrand factor (Wf) was identi fied with agglutination method with ristomycin and control with ELISA method on the device Stat Fax 303.
Laboratory marker of endothelial dysfunction (ED) such as circulating endothe lial cells (CEC) were identified with J. Hladovec, Petrischev, V. V. Syvak In this method adrenalin was used instead of adenosine phospate which reduced the cost of investigation [9].
Statistical analyses was made with Statistica for Windows 7.0 (Statsoft, USA). Distribution of the values was expressed as median, mean and Shapiro-Wilk cri terion. Comparing groups was held with Vilkokson -Mann -Witney criterion and Student's t-test. Reliability of nonparametric data discrepancies in groups (including follow-up period) was made with Chi-square criterion. Correlation analyses was made with Pearson's correlation criterion. The discrepancies were statisti cally significant at Р < 0.05.
Results and discussion. According to the recommendations of the Ukrainian association of cardiologists of diagnosis, treatment and prevention of chronic heart insufficiency (CHI) in adults (2017) we divided our patients into two groups: group 1 with normal EF of the LV systolic function (EFlv > 40 %) and group 2 with re duced EF (EFlv < 40 %). For additional evaluation of the stage of diastolic dysfunc tion of LV we created subgroups with E/A more and less than 1 in every group. We evaluated the fact that Е/А > 1.0 at EFlv < 40 % was related to the pseudonormal/ restricrive type of transmitral flow and in case of EFlv > 40 % Е/А > 1.0 better diastolic filling of LV was performed comparing the patients with Е/А < 1.0 (dia stolic dysfunction of the LV by relaxation disorder type). Part of the patients with concomitant DLD among the patients with EFlv > 40 % was 41 % and among the patients with EFlv < 40-50 %.
The results of the analysis of indicators of intracardiac hemodynamics were presented in our previous scientific publications [16], so in this article we reflect the state of microcirculatory dysfunction according to wavelet analysis of LDF.
In the analysis of wavelet analysis in patients with IHD (Table 1) revealed a decrease in A max E (p. u.), A max N (p. u.), A max M (p. u.), while increasing the value of A max R (p. u.) in the group of patients with an ejection fraction of more than 40 %, with impaired left ventricular relaxation. This indicates a decrease in capillary blood flow, due to an increase in myotonus and neurotonus of the capillaries, as well as a decrease in venular outflow in patients with IHD with EF > 40 % and E/A < 1.0, due to the development of heart failure [4,6].
In this case, the degree of these changes is greater in patients with EF < 40 % compared with patients with preserved systolic function (lower values of A max E, A max R than in EF > 40 % indicate worse peripheral blood flow in patients with IHD with low ejection fraction) and the lowest wavelet analysis rates were observed in the group of patients with reduced EF and E/A less than 1.0.
In the analysis of indicators A max E, A max N, A max M, as well as A max R, a tendency to increase the parameters of microcirculation A max E, A max N, A max M, as well as a significant increase in A max R, indicating "pseudonormalization" of the above indica tors, microcirculation, that there is a cyclical course of changes of microcirculation depending on the state of systolic and diastolic dysfunction of the left ventricle with IHD identical to the changes inherent in the development of diastolic dysfunction of the left ventricle.
A worse condition of A max E in patients with low EF and E/A greater than 1.0 is associated with a deterioration of the microcirculation state than in relatively preserved EF due to a number of researchers [2,5,6,10] with a decrease in cardiac output and blood volume per capillary, as well as with increased peripheral resistance due to the activation of SAS and RAAS, as well as hyperproduction of TNF-α, endothelin, angiotensin II, thromboxane and other vasoconstrictors [3,4,7,14].
Among patients with IHD with EF > 40 % at E/A < 1.0, higher values of A max M, A max C, A max R were observed, which indicates a more pronounced violation of venular outflow in patients with impaired LV relaxation.
Thus, in the complex analysis of indicators of intracardiac hemodynamics, micro circulation and endothelial function in patients with IHD revealed their dependence not only on the magnitude of EF (more/less than 40 %), but also on the state of its dias tolic filling. In this case, the dependence of indicators (A max E, A max N, A max M) on the nature of the filling of the LV was observed already at EF more than 40 %.
The results of the study of the microcirculation state by the LDF method in patients with IHD and in patients with DLD, as well as in their combination are given in table 2. In patients with IHD without DLD, the lowest values of A max E were observed, when compared with healthy and patients of all groups, which is associated with a decrease in production of nitric oxide on the one hand, as well as activation of the sympatho-adrenal system and renin angiotensin aldosterone system (RAAS) of pa tients with IHD on the other [1,18,19].
A max N and A max M were also lower compared with healthy, indicating an increase in neurotonus and myotonus in patients with IHD.
IS was larger in the group of patients with IHD, indicating a decrease in nutri tional blood flow when compared with healthy, which is consistent with the results of other studies [16]. In patients with DLD, a moderate decrease in A max E and A max M is observed, with an increase in IS, indicating moderate endothelial dysfunction, an increase in the myotonic tone of precapillary sphincters, and a slight decrease in nutritional blood flow when compared with healthy volunteers.
The combination of IHD and DLD showed the greatest decrease in A max M, A max R, indicating a deterioration of capillary blood flow due to increased myotonus of the precapillary sphincters, increased venular outflow, due to the production of proin flammatory cytokines (IL 2, 6, 8, TNFa) violation of their utilization by the af fected liver -on the other (which explains the absence of a pronounced peri pheral spasm, as well as violation of the venular outflow). The moderate decrease in A max E (similar to the A max E value in patients with DLD) was observed, possibly asso ciated with a decrease in the activity of the L-arginine NO system [7,8,10].
In patients with DLD compared with healthy volunteers there was a significant decrease in A max E, A max M, as well as an increase in SI. Endothelial dysfunction is less pronounced than in patients with IHD, and similar in value to patients with IHD + DLD, the precapillary myotonus was greater than in healthy but less than in patients with IHD, as well as IHD + DLD. Passive mechanisms of regulation of microcirculation did not differ from the control group, but the nutritional blood flow was worse than in healthy volunteers.
At EF > 40 %, in patients with IHD in the presence of DLD, a smaller value of A max M, A max R, A max C was observed, indicating an increase in myotonic tone of the arterioles and a slight (relative) narrowing of the precapillary sphincters (table 3). This may be due to changes in liver function with increased concentrations of vasoconstric tors (thromboxane A2, endothelin I, angiotensin II), and decreased utilization of their liver.  Among patients with IHD with EF < 40 % in the presence of concomitant DLD, lower values of A max R and A max C are noted -a decrease in the influence of passive mechanisms of regulation of microcirculation, with activation of active mechanisms (a significant increase in A max E, as well as a tendency to increase A max N, A max M).
That is, the relative expansion of precapillary sphincters occurs due to a decrease in myotonus and neurotonus, caused on the one hand by a vasoconstrictor effect (absence/reduction of release of nitric oxide in response to acetylcholine release), depletion of cytokine production with vasoconstrictor effect (thromboxane A2, en dothelin 1, etc.), increased production of liver inflammatory factors with vasodilat ing effect (TNFα, IL1, 6, 8, Endothelin 1, etc.), on the other hand, increasing the concentration of asymmetric dimethylarginine (ADMA) [3].
The analysis of LDF indices in patients of the third group confirms the signifi cant effect of DLD on microcirculation. When comparing LDF data in patients with DLD of different etiology, changes in the value of wavelet analysis are less pro nounced in CTH than in viral hepatitis and NASH and in LC (table 4). The data obtained indicate a less pronounced violation of cytomicroarchitectonics (by A max M), as well as blood flow velocity in the arterial and venous capillar ies, with toxic hepatitis, compared with viral hepatitis NASH and LC. Patients with CTH experience an increase in myotonic tone more than with CVH and NASH, but less than in patients with LC.
Patients with viral hepatitis showed a greater impairment of endothelial function by A max E, when compared with controls and other patients (except LC group), with marked deterioration of venular outflow (by A max R), an increase in myotone, slight ly less than in patients with CTH. In patients with NASH there is an increase in the value of A max R, as well as a decrease in IS, this figure was the lowest in gr. NASH. When compared to the CTH group, more A max M was observed.
Decreased endothelial function and increased peripheral resistance due to pare sis of precapillary sphincters (by indicators A max E, A max M and A max C) in patients with LC compared with patients with CTH and CVH. IS was the highest in the LC group, that is, the nutritional blood flow was the worst in the LC patients.
In the CTH and NASH groups, the A max E was the highest, probably on the one hand, due to the overproduction of nitric oxide, as well as the proinflammatory cytokines with vasodilating activity (Il1, Il6, Il8) on the one hand, which causes vasodilation of the precapillaries, which in turn on the other hand with impaired liver utilization of substances such as glucagon, estrogens, adrenomedulin, prosta glandins, adenosine, bile acids and the like [3,7,8].
In general, by most indicators, the degree of hemodynamic abnormalities was highest in liver cirrhosis (significant changes in wavelet analysis).
At DLD of minimum gravity changes of values of active and passive mechanisms of regulation of microcirculation are less expressed, than at LC (table 5). This indicates less impaired venular outflow, decreased tone of the precapillary sphincters, and a slight increase in capillary blood flow with minimal severity of liver damage compared with liver cirrhosis.
In patients with moderate syndrome of cytolysis and/or cholestasis revealed a moderate disturbance of venular outflow by A max R and a decrease in peripheral re sistance of the precapillaries due to a decrease in myotonic tone by indicators (A max M and A max C). This is due to the overproduction of proinflammatory cytokines (IL 2, 6, 8, TNFα) in DLD patients with moderate and minimal cytolysis and cholestasis syndrome [7,8,17].
The phase changes of A max E, A max N, A max M, A max R and IS depending on the severity of the liver lesions were interesting. With minimal liver damage, a decrease in the indicators of active mechanisms of regulation of microcirculation was ob served, as well as an increase in the values of passive mechanisms of regulation of microcirculation. In moderate syndrome of cytolysis and cholestasis increased the values of A max E, IS and A max N (increased intensification of the system L-arginine NO, decreased neurotonus, decreased nutritional blood flow), as well as A max R and A max C (deterioration of the venular outflow and enlargement of ma xima) started to decline (slight increase in myotonic tone) [3,5,6]. In severe course, a moderate decrease in all indicators (both active and passive mechanisms of regulation of microcirculation) was observed. In cirrhosis of the liver there is a slight increase in all parameters except A max N, in cirrhosis of the liver the system (L-arginine-NO) of the reticulo-endothelial system of the liver is depleted, which causes deterioration of microcirculation [10,16,19].
In general, the hepatic impairment was the highest in liver cirrhosis in most cases. This is due to hyperproduction and/or delayed utilization of vasoconstrictors, as well as structural and functional alteration of the liver with liver cirrhosis, which is in line with the literature [1,3,8].
The correlation analysis revealed a significant relationship between the values of many wavelet analysis and endothelial function (table 6).

Direct correlation
Between А max С and EDVD r = (0.42), P < 0.05 Inverse correlation dependence Between А max Е and EIVD r = (-0.43), P < 0.05 Between А max М and EIVD r = (-0.60), P < 0.05 Between А max R and EIVD r = (-0.57), P < 0.05 Between SI and EDVD r = (-0.73), P < 0.05 Between SI and EIDV r = (-0.37), P < 0.05 This indicates the presence of features of CHF formation in these groups, which is confirmed by the greater number of reliable correlations in the analysis of these patients with IHD and DLD in isolation, compared with patients with the combina tion of IHD and DLD.
In the study of microcirculation in patients with IHD, signs of spasm of the precapillary sphincters were noted, due to increased myotonus, decreased blood cir culation of the microcirculation system, which is consistent with the results of other studies [16,19]. This is due to the overproduction of endothelin, thromboxane A2, angiotensin II, and other pro-inflammatory cytokines with vasoconstrictive effects.
We found some differences depending on changes in the state of microcircula tion from contractility of the left ventricle in patients with coronary heart disease, depending on the presence of concomitant DLD. In patients with isolated IHD as TFlv decreases, there are clear signs of decreased capillary blood flow, probably due to the deterioration of central hemodynamics, atherosclerotic changes in blood vessels, and an increase in their tone.
In patients with IHD and EFlv < 40 % increase in vascular tone -was higher compared to patients with higher EFlv values, which may be due to phase changes in the metabolism of vasoactive substances (proinflammatory cytokines: IL1, IL6, TNF-a, prostacyclin, interferon-gamma etc.) [2,3,7].
Comparison of LDF and wavelet analysis in patients with coronary artery disease with similar EFlv shows marked differences in their values, especially with initial and severe HF..
Thus, at EFlv > 40 % in patients with IHD in the presence of DLD, there are noticeable tendencies to a higher myotonic tone, a decrease in the activity of passive mechanisms of regulation of microcirculation, a lower level of microcirculation compared with patients without DLD, which may be due to some extent related to swelling of the Disse space, as well as star cell contraction. When EFlv < 40 % among patients with IHD, the degree of decrease in the regulation of active and passive mechanisms of regulation of microcirculation is more pronounced in dia stolic dysfunction type pseudonormalization or restrictive type (E/A > 1.0), the nature of the changes characteristic of the smoothed plateau type curve.
In patients with coronary artery disease in the presence of DLD at TFlv > 40 %, there is a decrease in the level of microcirculation by increasing the neuro-and myo tonus, possibly due to the depletion of the production of vasoactive substances. In patients with coronary artery disease with EF < 40 % with con comitant DLD there is an increase in the influence of active mechanisms of regu lation of microcirculation (by indicators A max E, A max N, A max M), while reducing the impact of passive. This is probably explained by the violation of the disposal of vasoactive sub stances, due to irreversible morphofunctional changes in the liver at the stage of decompensation of HF, as well as pronounced fibrosis and the formation of regeneration nodes, which disrupt the cytoarchitectonics of the liver and, as a conse quence, the morphology of deceleration of blood stream in the microcirculatory bed due to stagnation, inflammation, metabolic acidosis.
In patients with coronary heart disease without DLD, lower values of A max E, A max H, and A max M were observed, indicating a more pronounced increase in neu rotonus, myotonus, impaired release of nitric oxide, as opposed to microcirculation in patients with concomitant DLD.
In patients with DLD there is a marked decrease in blood flow of the microcir culatory bed, as well as a significant disturbance of venous outflow. In this case, we detected phase changes in LDF indices in DLD of different severity: with DLD of moderate severity, venous outflow disturbance is more pronounced, neurotonus and myotonus of precapillary sphincters are more reduced compared with patients with both mild and severe liver lesions.. This can be explained by the hyperactivity of the L-arginine-NO system in patients with moderate severity of hepatitis, and in severe hepatitis and, in particular, in liver cirrhosis, the L-arginine-NO system of the reticulo-endothelial system of the liver is depleted, causing blood flow to dete riorate [3,4,7,8,19].