46 POPOVYCH, Igor, GOZHENKO, Anatoliy, BOMBUSHKAR, Igor, ANCHEV, Anatoliy & ZUKOW, Walery. Sexual dimophism in plasma nitrogenous metabolites levels and some psycho-neuro-endocrine parameters. Quality in Sport. 2022;8(4):46-57. eISSN 2450- 3118. DOI http://dx.doi.org/10.12775/QS.2022.08.04.005 https://apcz.umk.pl/QS/article/view/41503 The journal has had 20 points in Ministry of Education and Science of Poland parametric evaluation. Annex to the announcement of the Minister of Education and Science of December 21, 2021. No. 32582. Has a Journal's Unique Identifier: 201398. Scientific disciplines assigned: Economics and finance (Field of social sciences); Management and Quality Sciences (Field of social sciences). Punkty Ministerialne z 2019 - aktualny rok 20 punktów. Załącznik do komunikatu Ministra Edukacji i Nauki z dnia 21 grudnia 2021 r. Lp. 32582. Posiada Unikatowy Identyfikator Czasopisma: 201398. Przypisane dyscypliny naukowe: Ekonomia i finanse (Dziedzina nauk społecznych); Nauki o zarządzaniu i jakości (Dziedzina nauk społecznych). © The Authors 2022; This article is published with open access at Licensee Open Journal Systems of Nicolaus Copernicus University in Torun, Poland Open Access. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author (s) and source are credited. This is an open access article licensed under the terms of the Creative Commons Attribution Non commercial license Share alike. (http://creativecommons.org/licenses/by-nc-sa/4.0/) which permits unrestricted, non commercial use, distribution and reproduction in any medium, provided the work is properly cited. The authors declare that there is no conflict of interests regarding the publication of this paper. Received: 16.11.2022. Revised: 19.11.2022. Accepted: 19.12.2022. SEXUAL DIMOPHISM IN PLASMA NITROGENOUS METABOLITES LEVELS AND SOME PSYCHO-NEURO-ENDOCRINE PARAMETERS Igor L. Popovych1,4, Anatoliy I. Gozhenko1, Igor S. Bombushkar1,2, Anatoliy S. Anchev2, Walery Zukow3 1Ukrainian Scientific Research Institute of Medicine of Transport, Odesa, Ukraine prof.gozhenko@gmail.com 2Donets’kian National Medical University, Kropyvnyts’kyi, Ukraine bombuchkar@gmail.com doctorjiraha@gmail.com 3Nicolaus Copernicus University, Torun, Poland w.zukow@wp.pl 4OO Bohomolets’ Institute of Physiology, Kyїv, Ukraine i.popovych@biph.kiev.ua Abstract Background. We previously showed that the factor structure of the relationships between the plasma levels of nitrogenous metabolites (uric acid, bilirubin, urea, and creatinine), on the one hand, and the parameters of anxiety and neuro-endocrine status, on the other hand, is significantly different in men and women of reproductive age and postmenopausal. The purpose of this study is to identify the metabolic and psycho-neuro-endocrine features of these three cohorts of people. Materials and methods. The object of observation were 31 males (24÷69 y) and 30 females, from among them 18 postmenopausal (45÷76 y) and 12 of reproductive age (30÷42 y), with dysfunction of neuro-endocrine-immune complex. In basal conditions we determined plasma levels of nitrogenous metabolites and adaptation hormones, estimated the severity of the trait and reactive anxiety, recorded the ongoing HRV and EEG. After 4 or 7 days, repeated testing was performed. Results. To achieve the goal, the registered parameters were subjected to discriminant analysis. Along with the quite expected nitrogenous metabolites, age, testosterone and calcitonin (both raw and sex- and age- standardized values), trait anxiety, Kerdö's Vegetative Index, one of the vagal markers (SDNN), as well as 10 EEG parameters emerged as characteristic features of the cohorts. In the information space of two discriminant roots, three cohorts are clearly demarcated. Conclusion. We interpret this as another proof of the existence of connections between nitrogenous metabolites and psycho-neuro-endocrine parameters in line with the concept of the functional-metabolic continuum. Keywords: nitrogenous metabolites, adaptation hormones, anxiety, HRV, EEG, men, women. INTRODUCTION We previously showed that the factor structure of the relationship between the plasma level of nitrogenous metabolites (uric acid, bilirubin, urea, and creatinine), on the one hand, http://dx.doi.org/10.12775/QS.2022.08.04.005 https://apcz.umk.pl/QS/article/view/41503 mailto:prof.gozhenko@gmail.com mailto:bombuchkar@gmail.com mailto:w.zukow@wp.pl mailto:i.popovych@biph.kiev.ua 47 and the parameters of anxiety and neuro-endocrine status, on the other hand, is significantly different in men and women of reproductive age and postmenopausal [4-8,26]. To the long- known sexual and age differences in the metabolism of uric acid, creatinine and urea [21], as well as sex hormones by definition, data on sexual and age differences in HRV and EEG parameters, as well as calcitonin [1,11,12,23-25,29] have been added relatively recently. The purpose of this study is to identify the metabolic and psycho-neuro-endocrine features of these three cohorts of people. MATERIALS AND METHODS The object of observation were employees of the clinical sanatorium "Moldova" and PrJSC “Truskavets’ Spa”: 31 males (24÷69 y) and 30 females, from among them 18 postmenopausal (45÷76 y) and 12 of reproductive age (30÷42 y). The volunteers were considered practically healthy (without a clinical diagnosis), but the initial testing revealed deviations from the norm in a number of parameters of the neuro-endocrine-immune complex (details follow) as a manifestation of maladaptation. Testing was performed twice with an interval of 4 ("Moldova") or 7 (“Truskavets’ Spa”) days. We determined the plasma levels of the Bilirubin (by diazoreaction using the Jedrashik- Kleghorn-Grof method), Uric acid (by uricase method), Urea (by urease method by reaction with phenol hypochlorite) and Creatinine (by Jaffe's color reaction by Popper's method) as well as main adaptation hormones Cortisol, Testosterone, Aldosterone, Triiodothyronine and Calcitonin (by the ELISA with the use of corresponding sets of reagents from “Алкор Био”, XEMA Co. Ltd, and DRG International Inc.). The analyzes were carried out according to the instructions described in the manual [14]. The analyzers “Pointe-180” ("Scientific", USA), “Reflotron” (Boehringer Mannheim, BRD) and “RT-2100C” (PRCh) were used. To assess the parameters of heart rate variability (HRV) we recorded during 7 min electrocardiogram in II lead (software-hardware complex "CardioLab+HRV", KhAI- MEDICA, Kharkiv). For further analysis the following parameters HRV were selected [2,3,19,30]. Temporal parameters (Time Domain Methods): heart rate (HR), the mode (Mo), triangular index (TNN), the standard deviation of all NN intervals (SDNN), the square root of the mean of the sum of the squares of differences between adjacent NN intervals (RMSSD), the percent of interval differences of successive NN intervals greater than 50 msec (pNN50). Spectral parameters (Frequency Domain Methods): power spectrum density (PSD) bands of HRV: high-frequency (HF, range 0,40÷0,15 Hz), low-frequency (LF, range 0,15÷0,04 Hz), very low-frequency (VLF, range 0,04÷0,015 Hz) and ultralow-frequency (ULF, range 0,015÷0,003 Hz). Derived indices were calculated: (VLF+LF)/HF, LF/HF, LFnu as well as Kerdoe’s Vegetative Index [13,20], for which HR and diastolic blood pressure were recorded synchronously with the "Omron M4-I" device (Netherlands). Simultaneosly with ECG EEG recorded a hardware-software complex “NeuroCom Standard” (KhAI MEDICA, Kharkiv) monopolar in 16 loci (Fp1, Fp2, F3, F4, F7, F8, C3, C4, T3, T4, P3, P4, T5, T6, O1, O2) by 10-20 international system, with the reference electrodes A and Ref on tassels the ears. The duration of the epoch was 25 sec. Among the options considered the average EEG amplitude (μV), average frequency (Hz), frequency deviation (Hz) as well as absolute (μV2/Hz) and relative (%) PSD of basic rhythms: β (35÷13 Hz), α (13÷8 Hz), θ (8÷4 Hz) and δ (4÷0,5 Hz) in all loci, according to the instructions of the device. We calculated also for HRV and each locus EEG the Entropy (h) of normalized PSD using Popovych’s IL equations [17,27] based on classic Shannon’s CE [31] equation: hHRV =- [PSDHF•log2PSDHF+PSDLF•log2PSDLF+PSDVLF•log2PSDVLF+PSDULF•log2PSDULF]/log24; hEEG = - [PSDα•log2PSDα+PSDβ•log2PSDβ+PSDθ•log2PSDθ+PSDδ•log2PSDδ]/log24. 48 The levels of the trait and reactive anxiety estimated by STAI of Spielberger ChD [32] in modification of Khanin YL [28]. Results processed by using the software package "Statistica 6.4". RESULTS AND DISCUSSION To achieve the goal, the registered parameters were subjected to discriminant analysis [22] (method forward stepwise). The program included only 22 parameters in the discriminant model. Along with the quite expected nitrogenous metabolites, age, testosterone and calcitonin (both raw and sex- and age-standardized values), trait anxiety, Kerdö's Vegetative Index, one of the vagal markers, as well as 10 EEG parameters emerged as characteristic features of the cohorts (Tables 1 and 2). Table 1. Discriminant Function Analysis Summary Step 22, N of vars in model: 22; Grouping: 3 grps; Wilks' Λ: 0,0251; appr. F(44)=23,7; p<10-6 Variables currently in the model Cohorts (n) and Means±SE Parameters of Wilks' Statistics Reproduc- tive age Wo men (24) Postmeno- pausal Wo men (36) Men (62) Wilks Λ Parti- al Λ F-re- move (2,98) p- level Tole- rancy Age, years 36,6±0,9 57,5±1,6 47,4±1,6 0,042 0,597 33,1 10-6 0,441 Uric acid, µM/L 252±18 261±14 296±8 0,027 0,934 3,46 0,035 0,605 Bilirubin, µM/L 11,4±0,7 11,5±0,7 14,2±0,5 0,026 0,959 2,07 0,131 0,751 Urea, mM/L 5,47±0,18 5,50±0,18 5,76±0,13 0,026 0,948 2,71 0,072 0,783 Testosterone, nM/L 3,76±0,77 3,23±0,23 13,5±0,8 0,072 0,348 91,8 10-6 0,224 Testosterone, Z 1,26±0,70 0,78±0,36 0,01±0,24 0,043 0,582 35,2 10-6 0,247 Calcitonin, ng/L 5,01±0,73 6,16±0,49 10,5±0,9 0,041 0,618 30,2 10-6 0,164 Calcitonin, Z -0,02±0,30 0,45±0,20 -0,50±0,13 0,039 0,640 27,6 10-6 0,169 Cortisol, nM/L 333±26 287±15 298±16 0,027 0,914 4,59 0,012 0,706 Kerdö’s Vegetat Ind -2±6 -20±4 -13±3 0,029 0,860 7,99 0,001 0,607 SDNN HRV, msec 64,5±7,0 42,1±2,5 44,2±2,8 0,026 0,953 2,42 0,094 0,765 Amplitude β, μV 11,2±0,6 14,2±0,8 11,7±0,4 0,028 0,908 4,98 0,009 0,202 F3-β PSD, μV2/Hz 59±5 128±12 64±5 0,028 0,890 6,04 0,003 0,327 F4-β PSD, μV2/Hz 51±4 131±15 73±10 0,028 0,903 5,28 0,007 0,309 T5-β PSD, μV2/Hz 61±6 120±20 65±5 0,026 0,971 1,45 0,241 0,382 T6-β PSD, μV2/Hz 75±9 98±9 55±4 0,026 0,964 1,84 0,164 0,473 O2-β PSD, µV2/Hz 90±11 122±11 77±7 0,027 0,916 4,47 0,014 0,362 Fp1-θ PSD, % 13,5±1,4 9,2±0,7 9,2±0,6 0,027 0,923 4,06 0,020 0,825 C4-θ PSD, μV2/Hz 57±9 84±15 38±3 0,026 0,954 2,35 0,100 0,343 F4-α PSD, µV2/Hz 85±15 167±35 89±11 0,028 0,894 5,80 0,004 0,241 Frequency δ, Hz 1,31±0,09 1,07±0,03 1,08±0,02 0,026 0,972 1,43 0,243 0,738 Trait Anxiety, points 42,8±1,7 44,1±1,3 39,6±1,0 0,027 0,916 4,51 0,013 0,753 49 Table 2. Summary of stepwise analysis of discriminant variables ranked by criterion Λ Variables currently in the model F to enter p- level Λ F-va- lue p- level Testosterone, nM/L 66,1 10-6 0,474 66,1 10-6 Testosterone, Z 58,9 10-6 0,237 62,2 10-6 Age, years 44,2 10-6 0,135 67,1 10-6 F4-β PSD, μV2/Hz 13,5 10-5 0,110 58,6 10-6 Fp1-θ PSD, % 8,29 0,0004 0,096 51,3 10-6 Calcitonin, ng/L 8,08 0,0005 0,084 46,6 10-6 Calcitonin, Z 30,8 10-6 0,054 53,1 10-6 C4-θ PSD, μV2/Hz 6,86 0,002 0,048 49,7 10-6 Trait Anxiety, points 4,39 0,015 0,045 45,9 10-6 Cortisol, nM/L 3,64 0,029 0,042 42,6 10-6 Kerdö’s Vegetative Index 3,65 0,029 0,039 40,0 10-6 SDNN HRV, msec 3,08 0,050 0,037 37,6 10-6 Amplitude β, μV 2,36 0,099 0,036 35,3 10-6 Urea, mM/L 2,49 0,088 0,034 33,4 10-6 Uric acid, µM/L 1,56 0,214 0,033 31,5 10-6 Bilirubin, µM/L 2,04 0,135 0,032 29,9 10-6 F3-β PSD, μV2/Hz 2,06 0,132 0,031 28,6 10-6 O2-β PSD, µV2/Hz 2,14 0,123 0,029 27,4 10-6 F4-α PSD, µV2/Hz 4,08 0,020 0,027 26,9 10-6 Frequency δ, Hz 1,30 0,278 0,027 25,7 10-6 T6-β PSD, μV2/Hz 1,37 0,258 0,026 24,6 10-6 T5-β PSD, μV2/Hz 1,45 0,241 0,025 23,7 10-6 A number of variables, despite their recognizable properties, were outside the discriminant model, apparently due to duplication and/or redundancy of information (Tables 3-6). Table 3. Metabolic and psycho-neuro-endocrine parameters not included in the model Variables Cohorts (n) and Means±SE Parameters of Wilks' Statistics Reproduc- tive age Women (24) Postmeno- pausal Wo men (36) Men (62) Wilks Λ Parti- al Λ F to en- ter p- level Tole- rancy Creatinine, µM/L 80±2 82±2 91±2 0,034 0,996 0,21 0,815 0,533 Triiodothyr., nM/L 2,40±0,24 2,04±0,10 2,01±0,11 0,034 0,990 0,47 0,626 0,486 LFnu, % 72,7±2,5 76,4±2,3 81,8±1,4 0,034 0,996 0,17 0,843 0,783 LF/HF HRV 3,67±0,57 4,71±0,50 7,51±0,88 0,034 0,991 0,46 0,635 0,795 (VLF+LF)/HF 10,7±2,6 11,7±1,3 18,3±1,9 0,034 0,988 0,58 0,561 0,733 TNN HRV, units 13,8±0,8 10,3±0,6 10,8±0,5 0,034 0,997 0,13 0,876 0,341 RMSSD HRV, msec 33,1±3,5 24,0±2,8 22,9±2,0 0,034 0,993 0,34 0,713 0,436 VLF PSD, mcec2 1573±239 807±97 1020±119 0,034 0,986 0,70 0,499 0,457 LF PSD, mcec2 1479±228 751±109 944±135 0,034 0,996 0,19 0,828 0,282 HF PSD, mcec2 596±137 243±50 249±51 0,034 0,987 0,66 0,519 0,473 HF PSD, % 15,4±2,1 11,6±1,5 8,0±0,8 0,034 0,990 0,33 0,710 0,430 Reactive Anxiety, ps 23,2±1,5 23,0±1,2 25,2±1,2 0,034 0,999 0,03 0,968 0,083 50 Table 4. Parameters of beta-rhythm not included in the model Variables Cohorts (n) and Means±SE Parameters of Wilks' Statistics Reproduc- tive age Women (24) Postmeno- pausal Wo men (36) Men (62) Wilks Λ Parti- al Λ F to en- ter p- level Tole- rancy Fp1-β PSD, µV2/Hz 42±4 102±12 55±4 0,033 0,984 0,81 0,446 0,219 Fp2-β PSD, µV2/Hz 63±17 100±12 53±3 0,034 1,000 0,01 0,989 0,408 F7-β PSD, µV2/Hz 42±6 81±12 49±7 0,034 0,983 0,82 0,445 0,476 F8-β PSD, µV2/Hz 31±5 64±12 46±5 0,034 0,995 0,26 0,769 0,408 T3-β PSD, μV2/Hz 71±10 122±19 65±5 0,034 0,991 0,43 0,650 0,343 T4-β PSD, μV2/Hz 55±5 117±18 72±8 0,034 0,998 0,08 0,922 0,151 C3-β PSD, μV2/Hz 76±7 153±17 75±6 0,034 0,998 0,10 0,907 0,246 C4-β PSD, μV2/Hz 71±6 149±17 81±7 0,034 0,991 0,43 0,654 0,210 P3-β PSD, μV2/Hz 86±12 150±18 78±6 0,034 0,995 0,26 0,769 0,215 P4-β PSD, μV2/Hz 82±10 127±14 74±6 0,034 0,984 0,81 0,449 0,164 O1-β PSD, µV2/Hz 92±10 137±15 91±7 0,034 0,998 0,07 0,929 0,207 Table 5. Parameters of theta-rhythm not included in the model Variables Cohorts (n) and Means±SE Parameters of Wilks' Statistics Reproduc- tive age Women (24) Postmeno- pausal Wo men (36) Men (62) Wilks Λ Parti- al Λ F to en- ter p- level Tole- rancy Amplitude θ, μV 8,6±0,6 10,9±1,0 8,2±0,4 0,034 0,984 0,77 0,467 0,442 Fp2-θ PSD, % 11,8±0,9 8,9±0,6 9,0±0,7 0,034 0,999 0,05 0,949 0,552 F3-θ PSD, μV2/Hz 49±8 78±12 35±3 0,034 0,991 0,45 0,639 0,358 F4-θ PSD, % 12,0±0,9 9,7±0,8 9,6±0,7 0,034 0,984 0,78 0,462 0,496 F7-θ PSD, % 10,9±0,6 10,5±0,8 8,3±0,6 0,034 0,994 0,27 0,761 0,532 F7-θ PSD, μV2/Hz 23±4 66±12 25±5 0,034 0,994 0,31 0,731 0,727 F8-θ PSD, % 12,5±1,2 9,6±0,8 8,9±0,6 0,033 0,980 0,99 0,374 0,476 T3-θ PSD, μV2/Hz 36±4 68±12 26±2 0,034 0,989 0,52 0,598 0,390 C3-θ PSD, μV2/Hz 54±7 78±13 37±3 0,034 0,997 0,13 0,877 0,155 T5-θ PSD, μV2/Hz 36±6 63±12 31±3 0,034 0,997 0,17 0,846 0,295 Table 6. Parameters of delta- and alpha-rhythm and entropy not included in the model Variables Cohorts (n) and Means±SE Parameters of Wilks' Statistics Reproduc- tive age Women (24) Postmeno- pausal Wo men (36) Men (62) Wilks Λ Parti- al Λ F to en- ter p- level Tole- rancy Amplitude δ, μV 19±2 27±4 25±3 0,034 1,000 0,01 0,991 0,602 F8-δ PSD, µV2/Hz 105±31 495±201 552±206 0,034 0,998 0,08 0,927 0,716 T4-δ PSD, µV2/Hz 141±33 567±194 380±93 0,034 0,986 0,70 0,498 0,809 O2-δ PSD, µV2/Hz 122±22 233±63 328±91 0,034 0,985 0,72 0,488 0,715 Fp1-α PSD, µV2/Hz 61±11 152±33 82±9 0,034 0,983 0,82 0,444 0,051 F3-α PSD, µV2/Hz 84±14 188±39 96±12 0,034 0,999 0,03 0,968 0,083 F7-α PSD, µV2/Hz 42±6 140±39 44±5 0,034 0,991 0,43 0,649 0,213 T3-α PSD, µV2/Hz 72±11 156±34 75±10 0,034 0,998 0,10 0,909 0,208 T4-α PSD, µV2/Hz 73±12 133±29 64±7 0,034 0,997 0,16 0,851 0,130 Entropy F7 0,85±0,02 0,80±0,03 0,71±0,03 0,034 0,995 0,23 0,795 0,679 Entropy F8 0,85±0,03 0,76±0,04 0,72±0,03 0,034 0,998 0,10 0,909 0,208 Entropy T6 0,85±0,02 0,77±0,03 0,76±0,03 0,034 1,000 0,02 0,984 0,630 51 The identifying information contained in the 22 discriminant variables is condensed into two roots. The major root contains 86,4% of discriminatory opportunities (r*=0,962; Wilks' Λ=0,025; χ2(44)=400; p<10-6), while minor root – 14,6% only (r*=0,814; Wilks' Λ=0,338; χ2(21)=118; p<10-6). Calculating the values of discriminant roots for each patient as the sum of the products of raw coefficients for individual values of discriminant variables together with the constant (Table 7) allows visualization of each patient in the information space of roots. Table 7. Standardized and raw coefficients and constants for discriminant variables Coefficients Standardized Raw Variables Root 1 Root 2 Root 1 Root 2 Testosterone, nM/L 1,749 0,343 0,362 0,071 Testosterone, Z -1,286 -0,492 -0,553 -0,212 Age, years 0,796 0,703 0,076 0,067 F4-β PSD, μV2/Hz -0,428 0,468 -0,0057 0,0063 Fp1-θ PSD, % -0,174 -0,313 -0,035 -0,063 Calcitonin, ng/L 1,555 -0,363 0,294 -0,069 Calcitonin, Z -1,371 0,769 -1,216 0,682 C4-θ PSD, μV2/Hz -0,376 0,059 -0,0068 0,0011 Trait Anxiety, points -0,346 -0,039 -0,044 -0,005 Cortisol, nM/L -0,008 -0,428 -0,0001 -0,0037 Kerdö’s Vegetat Index -0,224 -0,528 -0,009 -0,021 SDNN HRV, msec -0,131 -0,262 -0,006 -0,011 Amplitude β, μV 0,570 -0,485 0,157 -0,133 Urea, mM/L 0,197 0,216 0,205 0,224 Uric acid, µM/L -0,213 0,318 -0,0030 0,0046 Bilirubin, µM/L 0,154 -0,220 0,038 -0,054 F3-β PSD, μV2/Hz -0,141 0,693 -0,0029 0,0141 O2-β PSD, µV2/Hz -0,045 0,588 -0,0008 0,0102 F4-α PSD, µV2/Hz -0,020 -0,815 -0,0002 -0,0061 Frequency δ, Hz 0,015 -0,240 0,057 -0,923 T6-β PSD, μV2/Hz 0,027 0,338 0,0006 0,0081 T5-β PSD, μV2/Hz -0,182 -0,259 -0,0025 -0,0035 Constants -8,129 -2,337 Eigenvalues 12,47 1,963 Cumulative Proportions 0,864 1 Reference (R) values of HRV parameters are taken from the instructions for "CardioLab+HRV", hormones - from the instructions for the kits, nitrogenous metabolites - from the handbook [21], EEG – from the data base of Truskavetsian Scientific School of Balneology [27]. In order to make a correct comparison, the individual actual values of the Variables (V) were transformed into Z-scores according to the classical equations [17,18,27]: Z = (V/R-1)/Cv = (V - R)/SD = 4•(V-R)/(Max - Min). Following the algorithm of Truskavetsian Scientific School of Balneology [17,18,27], Table 8 shows the Z-scores of variables both included in the discriminant model and worthy of attention in view of their identifying information. 52 Table 8. Correlations between variables and roots, centroids of cohorts and Z-scores of variables and their clusters Variables Correlations Variables-Roots Reproductive age Women (24) Postmenopausal Women (36) Men (62) Root 1 (86,4 %) Root 1 Root 2 -4,24 -3,04 +3,41 Bilirubin 0,093 -0,010 -0,06±0,16 -0,05±0,18 +0,60±0,13 LFnu HRV +0,73±0,21 +0,80±0,16 +1,26±0,10 LF/HF HRV +0,56±0,32 +0,86±0,24 +2,28±0,42 (VLF+LF)/HF HRV +1,73±0,86 +1,22±0,35 +2,96±0,45 Mean +0,74±0,37 +0,71±0,27 +1,78±0,52 Creatinine +0,49±0,17 +0,47±0,14 0,00±0,11 Testosterone -0,062 -0,033 +1,26±0,70 +0,78±0,36 +0,01±0,24 Trait Anxiety -0,071 0,061 +1,38±0,49 +1,74±0,37 +0,45±0,28 Mean +1,04±0,28 +1,00±0,38 +0,15±0,15 Uric acid -0,34±0,18 -0,66±0,19 -1,30±0,12 HF band PSDr -0,08±0,14 -0,07±0,19 -0,57±0,06 Mean -0,21±0,13 -0,37±0,30 -0,94±0,37 Root 2 (13,6 %) Root 1 Root 2 -2,23 +1,77 -0,16 Urea +0,49±0,20 0,00±0,19 +0,11±0,13 SDNN HRV -0,053 -0,228 +0,14±0,24 -0,42±0,08 -0,39±0,09 TNN HRV +1,05±0,8 -0,37±0,24 -0,15±0,22 VLF band PSDa +0,16±0,33 -0,55±0,15 -0,37±0,17 Frequency δ -0,056 -0,217 +0,69±0,34 -0,18±0,10 -0,13±0,09 Fp1-θ PSDr -0,060 -0,197 +0,51±0,22 -0,19±0,12 -0,20±0,10 Kerdö’s Vegetative Index 0,010 -0,183 +0,85±0,22 +0,12±0,12 +0,41±0,11 Cortisol -0,012 -0,101 -0,33±0,23 -0,76±0,13 -0,65±0,14 Triiodothyronine +0,39±0,24 -0,32±0,20 -0,38±0,23 Mean +0,44±0,14 -0,30±0,09 -0,19±0,10 Age -0,001 0,499 -0,92±0,07 +0,73±0,12 -0,07±0,13 F3-β PSDa -0,088 0,375 -0,37±0,09 +0,90±0,23 -0,28±0,09 F4-β PSDa -0,037 0,278 -0,45±0,06 +0,67±0,21 -0,15±0,14 Amplitude β -0,042 0,219 -0,23±0,18 +0,61±0,22 -0,10±0,11 T5-β PSDa -0,050 0,216 -0,20±0,13 +1,02±0,42 -0,12±0,11 T6-β PSDa -0,107 0,166 +0,02±0,13 +0,33±0,12 -0,26±0,06 F4-α PSDa -0,040 0,164 -0,22±0,18 +0,73±0,41 -0,18±0,13 O2-β PSDa -0,070 0,161 0,00±0,23 +0,64±0,23 -0,25±0,13 C4-θ PSDa -0,083 0,145 +0,27±0,22 +0,93±0,37 -0,19±0,09 Calcitonin -0,081 0,139 -0,02±0,30 +0,45±0,20 -0,50±0,13 Mean -0,21±0,10 +0,70±0,07 -0,21±0,04 Further, the variables were grouped into several clusters. The first cluster (Fig. 1) reflects a situation in which men have an upper limit level of bilirubin accompanied by increased levels of HRV markers of sympathetic tone, while women of both age groups have a completely normal level of bilirubin, and the levels of sympathetic markers are equally upper limit. The second cluster shows that in men a completely normal level of creatinine is accompanied by a completely normal level of testosterone and an upper borderline level of trait anxiety, while in women of both age groups creatinineemia is upper borderline, and the levels of testosterone and trait anxiety are moderately elevated. The third cluster illustrates the combination of hypouricemia in men with the lower limit level of HRV marker of vagal tone, while in women the level of uric acid is in the lower zone of the age norm, and the vagal tone fully corresponds to the age norm. 53 Fig. 1. Z-scores of bilirubin (circles), creatinine (squares), and urate (triangles) cluster variables (Y axis) and the centroids of the first root (X axis) of cohorts of women of reproductive age (green icons), postmenopausal women (red icons), and men (blue icons) The other two clusters reflect differences between women of different ages (Fig. 2). Fig. 2. Z-scores of urea (squares) and age (circles) cluster variables (Y axis) and the second root centroids (X axis) of cohorts of women of reproductive age (green icons), men (blue icons), and postmenopausal women (red icons) In particular, in women of reproductive age, the upper-limit level of urea is accompanied by upper-limit or normal levels of vagal markers, delta-rhythm frequency, PSD of theta- rhythm in Fp1 locus and triiodothyronine, as well as the lower-limit level of cortisol. At the same time, in postmenopausal women, the level of urea is completely normal, other parameters are normal or at the lower limit, and cortisol level is reduced. On the other hand, upper limit levels of calcitonin and a number of EEG parameters were found in postmenopausal women, while in younger women similar parameters are quite normal or lower limit. According to these parameters, men generally occupy an intermediate position. In the information space of two discriminant roots, three cohorts are clearly demarcated (Fig. 3). 54 PMPW RAW M -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 Root 1 -4 -3 -2 -1 0 1 2 3 R oo t 2 Fig. 3. Scattering of individual values of the first and second discriminant roots of postmenopausal women (PMPW), reproductive age women (RAW) and men (M) The visual impression of a clear demarcation is documented by calculating the distances of Mahalanobis (Table 9). Table 9. Squares of Mahalanobis distances between clusters (above the diagonal) and F- criteria (df=23,0) and p-levels (below the diagonal) Cohorts PMPW RAW Men Postmenopausal Women (36) 0 17,4 45,3 Reproductive age Women (24) 9,4 10-6 0 62,8 Men (62) 38,6 10-6 40,7 10-6 0 Selected discriminant variables were used to identify the affiliation of a patient to a particular cohort. This goal of discriminant analysis is realized with the help of classification functions (Table 10). 55 Table 10. Coefficients and constants of classification functions Cohorts Postmeno- pausal Women (36) Reproduc- tive age Women (24) Men (62) Variables p=,295 p=,197 p=,508 Testosterone, nM/L 2,557 1,837 4,753 Testosterone, Z -5,773 -4,260 -8,930 Age, years 1,175 0,816 1,534 F4-β PSD, μV2/Hz -0,016 -0,035 -0,065 Fp1-θ PSD, % 0,031 0,323 -0,073 Calcitonin, ng/L 0,940 0,860 2,971 Calcitonin, Z -1,139 -2,402 -10,29 C4-θ PSD, μV2/Hz -0,022 -0,018 -0,068 Trait Anxiety, points 0,607 0,679 0,333 Cortisol, nM/L 0,008 0,023 0,015 Kerdö’s Vegetat Index -0,082 0,013 -0,099 SDNN HRV, msec 0,132 0,185 0,117 Amplitude β, μV 2,195 2,540 3,464 Urea, mM/L 8,789 7,648 9,677 Uric acid, µM/L 0,047 0,032 0,018 Bilirubin, µM/L 0,719 0,888 1,066 F3-β PSD, μV2/Hz 0,026 -0,027 -0,019 O2-β PSD, µV2/Hz 0,096 0,056 0,071 F4-α PSD, µV2/Hz -0,042 -0,017 -0,031 Frequency δ, Hz 19,15 22,77 21,30 T6-β PSD, μV2/Hz 0,017 -0,016 0,006 T5-β PSD, μV2/Hz -0,092 -0,075 -0,102 Constants -115,5 -102,1 -162,5 The classification accuracy is absolute (Table 11). Table 11. Classifications matrix Rows: Observed classifications Columns: Predicted classifications Group Percent Correct PMPW p=,29508 RAW p=,19672 M p=,50820 PMPW RAW M Total 100 36 0 0 100 0 24 0 100 0 0 62 100 36 24 62 СONCLUSION It is significant that one or another nitrogenous metabolite was found in the composition of each cluster. Earlier we discovered peculiarities of relationships between plasma levels of nitrogenous metabolites and EEG&HRV parameters in patients with postradiation encephalopaty [9]. We interpret this as another proof of the existence of connections between nitrogenous metabolites and psycho-neuro-endocrine parameters in line with the concept of the functional-metabolic continuum [15,16]. 56 ACKNOWLEDGMENT We express sincere gratitude to administrations of clinical sanatorium “Moldova” and PrJSC “Truskavets’ Spa” as well as TA Korolyshyn and VV Kikhtan for help in carrying out this investigation. 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RESULTS AND DISCUSSION