[THE CHROMATOGRAPHY-MASS SPECTROMETRY ANALYSIS OF MICROBIAL FATTY ACIDS IN HUMAN BIOLOGICAL FLUIDS AND THEIR CLINICAL SIGNIFICANCE].

The technique of mass-spectrometric microbial markers is known for almost 20 years. The technique is described in a number of research publications, dissertations and methodological literature. It passed the registration in Roszdravnadzor and is permitted for implementation as a new medical technology in medical institutions on the territory of the Russian Federation ("The evaluation of microecological human status using technique of mass-spectrometry" license FS No 2010/038 of 24.02.2010). The technique of mass-spectrometric microbial markers began to be developed as instrument of clinical routine analysis and monitoring of microecological status, infection and disbiosises in clinical and out-patient practice. The description of technology of mass- spectrometric microbial markers in this aspect requires different than before approach to introduction of clinical laboratory assistants and physicians into technique application. The substantiation given concerning species specificity of composition of fatty acids and (fatty) aldehydes of cellular wall of microorganisms as a basis of their species differentiation in pure culture. The choice is explained concerning molecular markers for their detection in blood and other clinical material with the purpose of further reconstruction of composition of human microbial cenosis (microecology) on blood or calculation of composition of mixed infection in organs om samples of inflammation focus--urine, liquor, phlegm, exudate, drainage, and similar samples containing chemical information about microbes.

[Chromatographic mass spectrometric determination of low-molecular-weight aromatic compounds of microbial origin in the serum from patients with sepsis].

The investigation quantitatively determined the content of low-molecular-weight aromatic compounds of microbial origin in the sera of 34 individuals by chromatographic mass spectrometry. An "Agilent Technogies 6890N" gas chromatograph with a 5973 mass selective detector was applied; chromatographic separation of components was effected on an Hp-5MS quartz capillary column. Aromatic small molecules originating from microbes (SMOM) were determined in the sera of 7 patients with sepsis. The diagnosis of sepsis was documented by the presence of the systemic inflammation syndrome and by that of bacteriemia and/or artificial ventilation-associated pneumonia along with the level of procalcitonin of higher than 10 ng/ml. The levels of aromatic SMOM were compared in 10 healthy donors, 8 preoperative cardiosurgical patients, and 9 patients with different abnormalities without sepsis treated in an intensive care unit (ICU). Serum phenylacetic and 3-phenylpropionic acids were found to be prevalent in the healthy donors and postoperative cardiosurgical patients. In ICU patients with different complications without sepsis, more than half the compounds under study were undetectable, the others were found in very low concentrations, which may be accounted for by antibiotic therapy. At the same time, almost the whole spectrum of the test compounds (other than 3-phenylpropionic acid) with the highest concentrations of 3-phenyllactic, p-hydroxyphenylacetic, 3-(p-hydroxyphenyl)lactic and 2-hydroxybutanic acids, was detectable in septic patients receiving a more intensive therapy. The differences were statistically significant (by the Mann-Whitney U-test; p < 0.05). By taking into account the potentially high biological activity of the test compounds, studies are to be continued in this area.

[Study of volatile fatty acids in the blood of patients with periodic disease].

Microecological "failures" are an important pathogenetic factor of different diseases and, in the authors' opinion, periodic disease (PD) is one of them. PD is a recessive disease characterized by fever attacks and neutrophil-mediated serous inflammation. A genetic factor has been established to be responsible for half the cases of PD, the influence of non-hereditary factors, particularly a role of the host automicroflora in the genesis of an inflammatory process, has been little studied. The authors' early studies indicate that there are changes in the qualitative and quantitative composition of microbial molecules in the blood of patients with PD. The anaerobic bacterial metabolites that are volatile fatty acids (VFAs) represent biologically active substances that affect the growth of the microflora, on the one hand, and the host's immunological responsiveness, on the other. Out of VFAs, only is acetic acid detectable in small quantities in the blood of healthy individuals. The other VFAs, namely propionic, valeric, butyric, and caproic acids and their isomers, are absent. Gas chromatography was used for qualiitative and quantitative determination of the metabolites of anaerobic microorganisms in the blood of patients with PD (n = 13) during an attack and remission and in that of healthy volunteers (Armenians) (n = 5) of a control group from one Yerevan region. The blood samples from all the patients with PD displayed a significantly higher concentration of caproic acid while the latter was absent in the blood of the controls. This finding suggests that there is a specific shift in the structure of the microbiocenosis in patients with PD. It is conceivable that caproic acid plays a certain role in the pathogenesis of the disease under study. Further studies will deal with the association of some microbial molecules with the manifestation of an attack of PD, which may provide the key to the goal-oriented regulation of detected homeostatic disorders and to the management of the frequency of its attacks.

[An unusual type of the chromatography profile of blood serum carbohydrates in the diagnosis of deep mycosis by gas and liquid chromatography]. / Neobychnyi tip khromatograficheskogo profilia uglevodov syvorotki krovi pri diagnostike glubokikh mikozov metodom gazozhidkostnoi khromatografii.

The paper contains a description of a diagnosis method (now under elaboration) applicable to human mycosis, which is based on a high-sensitivity gas-liquid chromatography. According to the study results, despite its promising nature, the method in question is not accurate enough in terms of a precise diagnosis of invasive candidosis in oncology patients. The authors point out that the chromatography-diagnosis results need now a histological confirmation.

[Clinical significance of studies of microorganisms of the intestinal mucosa by culture biochemical methods and mass fragmentography]. / Klinicheskoe znachenie issledovaniia mikroorganizmov slizistoi obolochki kishechnika kul'tural'no-biokhimicheskim i khromato-mass-spektrometricheskim metodami.

This research is a continuation of the series of studies of the parietal microbiota of the bowel tissue samplings by the gas chromatography and mass spectrometry (GCMS) method [G.A. Ossipov et al.//Journal of the Society of Russian Gastroenterologists, 2001, 1:54-69]. The purpose was to study a number of new microorganisms in view of new data on the composition of their fatty acids (FA) and aldehydes; to confirm the presence of a number of bacteria, fungi and aerobic actinomycetes revealed earlier by FA markers in the composition of the bowel parietal microbiota by their isolation in a pure culture; to amend the estimation of the clinical value of changes in the composition of the human bowel parietal microflora in case of irritable bowel syndrome (IBS) and antibiotics-associated diarrhea (AAD). We examined 31 patients with IBS with predominating diarrhea, 18 patients with AAD and 3 volunteers (a control group). We studied the blood samples, tissue samplings of the mucous coat of the jejunum, ileum and colon and composition of healthy people's feces. The GCMS method was applied. Morphology of defined strains was controlled by methods of light and scanning electron microscopy. We found a substantial portion of eubacteria among the bowel microorganisms and specific changes of their species in case of IBS and AAD. Taking into account their physiological and biochemical activity, when regulating their concentration one can expect at least the same effect as when regulating the number of bifidobacteria and lactobacilli in treatment of intestinal pathologies and other diseases related to bowel dysbacteriosis. The analysis of the feces microbiota using the GCMS method by FA of parietal microorganisms provides reliable data on their number both in feces and in tissue samplings. We found a substantial portion of eubacteria among other bowel microorganisms (27% in the jejunum and 16% in the colon) and specific changes of their species in case of IBS and AAD. The concentration of streptomycetes, rhodococci and other members of the Actinomycetales order becomes dozens times more and/or reduces in pathological states. The tenfold concentration of markers of lactobacilli and bifidobacteria in some diseases stimulates the differentiated application of widespread probiotics based on these bacteria.

[Minor fatty acids of biological fluids of urogenital organs and their significance in the diagnosis of inflammatory processes]. / Minornye zhirnye kisloty biologicheskikh zhidkostei urogenital'nykh organov i ikh znachimost' v diagnostike vospalitel'nykh protsessov.

Minor lipid components (less than 1%), not occurring in the cells of mammals, have been detected in the profile of fatty acids (FA) in urine and vaginal fluid in females, as well as in ejaculate in males, by the method of gas chromatography and mass spectrometry. Their origin is supposedly attributed to microorganisms colonizing urogenital organs under normal conditions and in infectious pathology. Minor FA in the biological fluids of the urogenital tract (UGT) in man are compared with the FA composition of pure microbial cultures. The statistical analysis of FA concentrations in 500 specimens of UGT biological fluids indicates that lipid markers of microbial origin (in UGT inflammatory diseases) have a range of quantitative changes exceeding the limits of values characteristic of healthy persons and in a number of cases correlate with the diagnosis of the disease or the results of the bacteriological study. The reliability of the interpretation of the results of the marker analysis is confirmed by the difference in the composition of UGT microorganisms under normal conditions and in pathology, correlation with the clinical picture of pathological deviations, a decrease (to zero or normal values) in the concentration of the markers of pathogens as the result of treatment with antibiotics, as well as by the adequate composition of UGT microorganisms, determined by the marker method in comparison with the cultural biochemical method.

[Gas chromatography in express diagnosis of candidiasis and monitoring of antifungal therapy efficacy by D-arabinitol and mannose levels in pediatric patients]. / Gazokhromatograficheskaia ékspress diagnostika kandidoza i monitoring éffektivnosti protivogribkovoi terapii po urovniu D-arabinitola i mannozy u detei.

Seventy three children (40 blood and 43 liquor specimens) were examined with the use of gas chromatography (GC) to detect background concentrations of Candida metabolites. The criterium of the children enrollment to the control group was the absence of the clinical and laboratory signs of the fungal infection. The normal contents of the fungus metabolites were considered to be 0.51 +/- 0.28 microgram/ml for D-arabinitol and 17.7 +/- 10.4 micrograms/ml for mannose in the serum and 7.24 +/- 3.04 micrograms/ml for D-arabinitol and 67.1 +/- 47.4 micrograms/ml for mannose in the liquor. Fifty four children at the age of 1 month to 12 years with the signs of the fungal infection requiring systemic antifungal therapy were also examined. Prior to the use of antifungal drugs the routine microbiological tests and GC detection of the fungus metabolites were performed. The fungus was isolated with the cultural method from the blood in 2 patients (6.3 per cent), from the mucosa in 25 (71.4 per cent) out of 32 patients with fungal complications at the background of cytostatic therapy and neutropenia, from the liquor in 3 (21.4 per cent) out of 14 patients with meningitis and from the urine in 8 (100 per cent) out of 8 patients with urinary infection. The GC examination revealed increased levels of the Candida metabolites in 96 per cent of the children. A favourable time course of the infection at the background of amphotericin B or fluconasol use was recorded by the clinical indices which correlated with a reliable decrease of the contents of D-arabinitol and mannose to the normal. The use of GC is recommended in express diagnosis of candidiasis especially when the results of the cultural tests are negative as well as in monitoring of the fungal therapy efficacy.