Impact of Extracorporeal Detoxification on the Serum Levels of Microbial Aromatic Acid Metabolites in Sepsis

Материалы и методы. Проспективно проведено обследование и лечение 10 больных с острой или хро нической (терминальная стадия) почечной недостаточностью, развившейся на фоне тяжелого сепсиса, ин фекционно токсического шока, длительного искусственного кровообращения, постреанимационной болез ни и др. Всем больным проводили экстракорпоральную детоксикацию, выбор метода определяли, исходя из анамнеза и характера интоксикации. Оценивали органную дисфункцию по шкале SOFA, температуру тела, лейкоцитоз, лейкоцитарный индекс интоксикации, результаты теста на прокальцитонин. Гемодиафильтра ция проводилась по «внепочечным» показаниям, для устранения системного воспалительного ответа, при септическом шоке — с использованием сверхвысокопроницаемого диализатора EMiC2. Для гемодиализа применяли низкопоточные диализаторы Diacap LO PS. Непосредственно до и сразу после операции экстра корпоральной детоксикации забирались образцы крови для оценки в динамике методом ГХ ПИД сыворо точных концентраций фенилкарбоновых кислот БК (бензойной кислоты), ФПК (3 фенилпропионовой кислоты), ФМК (фенилмолочной кислоты), п ГФУК (пара гидроксифенилуксусной кислоты), п ГФМК (пара гидроксифенилмолочной кислоты).

A search for low molecular weight biomarkers to objectively evaluate the efficiency of extracorporeal detoxification methods is extremely relevant.For this purpose, the investigation is to verify whether metabolites, the production of which from aromatic amino acids in the human body can be of microbial ori gin, may be used.
Objective: to evaluate the efficiency of extracorporeal detoxification methods on the serum level of phenyl carboxylic acids in patients with sepsis associated renal failure.

Subjects and methods.
Ten patients with acute or chronic (end stage) renal failure that had developed in the presence of severe sepsis, infective and toxic shock, long term extracorporeal circulation, postresuscitation disease, etc. were prospectively examined and treated.All the patients underwent extracorporeal detoxification; the choice of its technique was determined from their past medical history and intoxication patterns.The investigators eval uated organ dysfunctions using the Sequential Organ Failure Assessment (SOFA) scale, estimated body tempera ture, leukocyte count, and leukocyte index of intoxication, and assessed the results of a procalcitonin test.Hemodiafiltration was done as extrarenally indicated to ameliorate a systemic inflammatory response in septic shock, by applying an EMiC 2 superhigh permeability dialyzer.Low flux Diacap LO PS dialyzers were employed for hemodialysis.Blood samples were taken to estimate changes in the serum concentrations of phenylcarboxylic acid, benzoic acid, 3 phenylpropionic acid, phenyllactic acid, para hydroxyphenylacetic acid (p HPAA), and para hydroxyphenyllactic acid (p HPLA) directly before and immediately after extracorporeal detoxification.
Results.The severity of organ dysfunctions by SOFA score was 10-22 (mean 16 scores); 10 day mortality rates were 40%.In all the patients, the baseline serum levels of some phenylcarboxylic acids were considerably above normal.After hemodiafiltration, the serum concentrations of p HPAA and p HPLA decreased (by an aver age of 1.7 and 1.85 fold, respectively).

Conclusion.
In sepsis associated renal failure, the clearance rate of microbial aromatic acid metabolites (p HPAA and p HPLA) is promising potential biomarker to evaluate the efficiency of extracorporeal detoxification methods.

Introduction
Searching of candidate biomarkers contributing to mechanisms of critical illness represents a contem porary challenge in reanimatology [1][2][3][4].Sepsis as a clinical form of the systemic inflammatory response to infectious agent is characterized by a steady increase in occurrence and consistently high mortality in patients at emergency departments worldwide.
Today, the mediator cascade concept is not a perfect theory, but it satisfactorily describes the sep sis pathogenesis.Biologically active substances, such as regulatory and effector cytokines, arachidonic acid derivates, free radical lipid peroxidation prod ucts, nitric oxide etc. are main components of the mediator 'storm' underlying systemic inflammatory response.Microvascular endothelial layer presented in all organs is considered as a main target of inflam matory mediators.Endothelial cells occupy a large surface in the body and possess high metabolic activ ity.Generalized endothelial damage leads to hemo dynamic disturbances and multiple organ failures.Septic shock is the most terrible manifestation of the systemic inflammatory response and is responsible for the extremely high mortality in sepsis.
The current level of extracorporeal technology development allows the elimination substances from the blood in a wide range of molecular weight includ ing pathogenically significant inflammation media w w w .r e a n i m a t o l o g y .c o m DOI:10.15360/18139779 2015 5 6 14 ного воспалительного ответа и определяет крайне высокую летальность при сепсисе.
Clinical effectiveness of efferent methods is determined by the mechanisms of molecular sorption, diffusion and convection of low and medium weight substances through semipermeable membranes of a mass transfer device (dialyzer or hemofilter) and parameters of controlled environments (substitution and dialytic fluids).Technological solutions with dif ferent geometry of mass transfer devices, physical principles for eliminating substances, sorbent materi als, membrane properties etc. are deployed for each method of the efferent treatment.The synthetic bio compatible membranes used for hemofiltration are not highly permeable only for cytokines, but also might absorb significant amount of biologically active substances [7].At the same time, lipopolysaccharides of Gram negative bacteria, the triggers of systemic inflammatory response, are not eliminated through the hemofilter membrane Thus, in clinically signifi cant endotoxemia it is reasonable to perform selective hemosorption [8].
Recently, the attention of researchers world wide has attracted to the aromatic structure metabo lites, in particular, phenylcarboxylic acids (PCAs).These low molecular weight compounds are pro duced by living organisms, representatives of the human microbiota and bacteria causing sepsis that have been proved in the experimental and clinical studies [9,10,11,13].Due to multiple increase of some PCAs in patients with sepsis, they have been suggested as molecular biomarkers of sepsis [12,13], although the exact role of PCAs in the genesis of the systemic inflammatory response has not been clari fied to date.
Identification of objective biomarkers that aid to evaluate the effectiveness of extracorporeal detox ification represents an urgent challenge [14].We consider PCAs as candidate molecules for the devel opment of laboratory monitoring methods [15,16].At the same time, the issues on kinetics of the aro matic microbial metabolites during extracorporeal detoxification remain understudied.
The objective of the study was to assess the impact of extracorporeal detoxification on the level of circulating PCAs in the blood in patients with renal failure associated with sepsis.

Материал и методы
Проведено обследование и лечение 10 больных с си стемным воспалительным ответом инфекционного (сеп сис, тяжелый сепсис, инфекционно токсический шок) или неинфекционного генеза (постреанимационная бо examination we focused on advanced clinical and laboratory symptoms of sepsis allowing to objective ly assess the severity of condition.When determin ing indications and selection of method of detoxifica tion, we took into account the results of the procalcitonin test (PCT), level of endotoxemia as evaluated by the LAL test, and leukocytal intoxica tion index.

Результаты и обсуждение
У всех больных зафиксированы высокие концентрации отдельных фенилкарбоновых кис лот, уровень которых достигал 50 μМ, что значи тельно превышало норму, установленную в пре дыдущих исследованиях (0,5-2,5 μМ) [18,19].Максимальные концентрации ФКК регистриро вались у больных с септическим шоком, то есть с method of detoxification was determined as based on the causes of intoxication.Purulent septic complica tions developed in three patients following initially existing chronic (end stage) renal failure.Low flow Diacap LO PS dialyzers on the basis of α polysulfone membrane with area of 1.5 m 2 in intermittent or pro longed mode were deployed for hemodialysis.Hemodiafiltration was performed for 'extrarenal' indications, to eliminate the systemic inflammatory response without high values of uremic intoxication.In patients with septic shock, HDF was carried out by using super highly permeable EMiC 2 dialyzers with capabilities to perform high diffusive clearance of medium weight molecules.In case of the level of endotoxemia higher than 2 EU/ml with clinical symptoms of septic shock the endotoxin selective sorption was performed during 2-6 hours.
Blood samples for phenolic metabolites determin ing were harvested from various segments of the extra corporeal circuit -arterial and venous circuits and effluent line, if detoxification was carried out by HDF.The serum samples were treated according to previous ly described technique [18].Concentration of metabo lites was determined by using the gas chromatograph Crystal 5000.2 equipped with a flame ionization detec tor.Chromatographic separation of the components was carried out by capillary column CP Sil 5 CB.

Results and Discussion
High concentrations of phenylcarboxylic acids were recorded in all patients.Level of PCAs increased to50 μМ that was significantly more than in norm as had been established by previous studies (0.5-2.5 μM) [18,19].The maximum concentra tions of PCAs were recorded in patients with septic shock, i.e. in patients with the most pronounced manifestations of the systemic inflammatory response.More than ten fold increase of HPAA and HPLA concentrations vs those ones in healthy peo ple reflected a high degree of microbial load and exhibited the most pronounced activity of septic process.It is important to note that, according to available data in the literature, high levels of HPAA and HPLA in patients with sepsis were associated with fatal cases [12,18].
PCAs with molecular weight in the range of 122-182 Da belong to low molecular weight com pounds.Therefore, elimination of these substances must occur due to the diffusion law as described for conventional uremic toxins -urea, potassium and creatinine.In accordance to the diffusion mass trans fer concept in the capillary dialyzer, elimination of low molecular weight compounds is most effective at high blood flow and dialysis fluid flow.The latter is confirmed by estimating the value of the dialyzer clearance for circulating HPAA at the blood flow of 200 ml/min as 157.6±21.2ml/min at a dialysis fluid flow rate of 500 ml/min.Thus, the HPAA elimina tion efficiency in hemodialysis is comparable to the diffusion clearance of urea for this type of dialyzer.
In contrast, in a bypass mode the dialyzer clear ance of HPAA was minimal and amounted to 11.6 ml/min at a blood flow of 200 ml/min (fig.2).A spe cial characteristic of the bypass regimen was the dis connecting the dialytic fluid flow.As a result, the outer side of the capillaries in the dialyzer was not washed by the media free from removable sub stances.The absence of concentration gradient across the membrane allowed to separately evaluate the sorption capacity of the material used in the dia lyzer.Carrying out hemodialysis in a bypass mode did not affect the plasma concentration of phenolic metabolites suggesting the low sorption capacity of polysulfone membranes for the compounds of such nature.Data confirm the leading role of the diffusion mechanism in the elimination of PCAs.
Clearance of the low molecular weight sub stances in hemofiltration depends on the volume of the substitution fluid: the value of clearance is rela  tively small and the therapeutic effect is less efficient at low volumes of substitution fluid.The effective ness of HDF in eliminating PCAs was found to be lower.This can be explained by low diffusion compo nent when performing HDF during the low flow technique: dialytic fluid flow during extended HDF is 5-10 times lower than in the 'artificial kidney' device.The EMiC 2 dialyzer clearance of microbial metabolites was lower versus the dialyzer and aver aged 107.38±15 ml/min for HPAA and 66.32 ml/min for HPLA at a blood flow of 200 ml/min (fig.3).
The large thickness and the structural asymme try of the polysulfone hemofilters as well as hydrophobic physicochemical properties of organic molecules with aromatic ring help explain smaller clearance values for phenolic metabolites compared to hemodialysis.At the same time, performing HDF in extended mode may be more effective due to a greater convection dose.Carrying out non selective hemosorption and lipopolysaccharide sorption was not associated by a significant change in the concen tration of microbial metabolites.

Conclusions
1.There was a significant increase in con centration of the low molecular weight aromatic metabolites (phenylcarboxylic acids) in the serum in all examined patients with renal failure and clinical manifestations of the systemic inflamma tory response.
2. Extracorporeal detoxification possess a sig nificant effect on the serum levels of microbial metabolites.As a result of hemodiafiltration, the concentration of para hydroxyphenylacetic acid and para hydroxyphenyllactic acid decreased 1.7 and 1.85 times, correspondingly.

Infectious complications
3. Dynamics of the microbial metabolites con centration depended on the mode of detoxification and the characteristics of the applied hemodiafilter (penetration coefficient and sieving coefficient).A diffusion mass transfer through the dialyzer or hemofilter membrane plays a leading role in the elim ination of these substances.
4. Non selective hemoperfusion and selective lipopolysaccharide sorption had no significant effect on the level of phenylcarboxylic acids in blood serum.
The article was prepared with the support of the grant No. 15 15 00110 of the Russian Science Foundation.Литература . r e a n i m a t o l o g y .c o m DOI:10.15360/18139779 2015 5 6 14 . r e a n i m a t o l o g y .c o m DOI:10.15360/18139779 2015 5 6 14 время экстракорпоральной детоксикации остают ся малоизученными.
. r e a n i m a t o l o g y .c o m DOI:10.15360/18139779 2015 5 6 14