Nucleic Acid Metabolism in Patients with Severe Burn Injury and the Possibility of Its Correction

1 Новосибирский Государственный Медицинский Университет Минздрава России, Россия, 630091, г. Новосибирск, Красный проспект, д. 52 2 Государственная Новосибирская Областная Клиническая больница, Россия, 630087, г. Новосибирск, ул. Немировича-Данченко, д. 130 3 Федеральный исследовательский центр Институт цитологии и генетики Сибирского отделения РАН, Россия, 630090, г. Новосибирск, пр. ак. Лаврентьева, д. 10

DOI:10.15360/1813-9779-2019-1-4-11 compared to the normal limit is typical for survivors in the acute period, and these changes were significantly different from the group of deceased patients starting from day 5. Taking into account the data obtained, the level of serum UA can be recommended in clinical practice for the assessment of alimentary status in burn disease, as well as a criterion for f the prescription of glutamine administration in critically ill patients.In patients with burn disease a decrease in the UA level to less than 100 μmol/l was regarded as an absolute indication for the use of L-glutamine products.The use of glutamine in patients with significant decrease in UA levels was accompanied by a significant increase in the intensity of NA metabolism, which was demonstrated by both an increase in uric acid levels (by 85% by day 10 after the start of glutamine administration, P<0.01) and restoration of repair processes.
Conclusion.Therefore, there was a marked alteration of NA metabolism in both groups of patients with severe burn injury.At the same time, surviving patients are characterized by rapid recovery of NA metabolism.Glutamine was employed as a pharmacological agent that effectively abrogates the depression of NA metabolism.Keywords: thermal injury; intensive care; alimentary status; nucleic acid metabolism; oligonucleotides; uric acid; glutamine DOI:10.15360/1813-9779-2019-1-4-11

Introduction
The intensity of nucleic acid (NA) metabolism in critical conditions is an important factor determining the outcome of the critical state.Synthesis of NA fragments (nucleotide) is one of the most active processes in the cell.Nucleotide reproduction requires a significant amount of plastic substances, including amino acids glutamine and glycine.In terms of energy costs, this process is also extremely stressful.At the same time, a number of «barrier» organs (liver, intestines, lymph nodes, spleen) and bone marrow should increase the synthesis of NA tenfold under critical conditions.It is the rate of cell division in these organs, and therefore the rate of NA synthesis that determines the rate of regeneration and functional reserves of these organs.
At present, there are ways to change the intensity of the synthesis and disintegration of NA, but a reliable way to assess the intensity of NA metabolism is necessary for the effective use of these drugs, otherwise their use will be either ineffective or may lead to deterioration of the disease.
A method for diagnosing NA metabolic disorders proposed previously consists in simultaneous determination of two parameters in the blood serum: serum nucleic acids (SNA) and serum uric acid (SUA) [1].SNA concentrations characterize the level of circulating NA liberating from cells.The effect of cell disintegration on the SNA level appears to be minimal, since NA cell fragments disintegrate rapidly under the action of enzymes, or are eliminated at a greater rate through the gatekeeper receptors TLR3 and TLR9.Indeed, our studies [2] have shown that in patients with severe surgical sepsis, in which there is increased catabolism and accelerated cell death, the SNA concentration can both decrease and increase.Moreover, numerous data [3,4] indicate that normally the SNA concentration is a constant value, and changes in the SNA concentration in one direction or another may be accompanied by pathological changes per se.Therefore, maintaining a certain SNA level of is an active process.It is obvious that NA in extracellular fluid corresponds, first Таким образом, при одновременном определении средней концентрации НК и средней концентрации МК в сыворотке крови можно оценивать как интенсивность синтеза НК, так и интенсивность распада НК во всем организме.На основе полученных данных появляется возможность своевременной диагностики и коррекции обмена НК.Ранее было показано, что своевременная диагностика и восстановление сниженного обмена НК может стать важным фактором в исходе лечения больных с алиментарной недостаточностью при ахалазии или рубцовом сужении пищевода [1].
SUA is known to characterize the rate of disintegration of nucleic acids to purine bases and further to uric acid (UA).However, usually, the determination of the SUA concentration is used for the diagnosis of uric acid diathesis or gout; at that, only the increased values are taken into account.At the same time, in critical conditions, we noted a sharp decrease in UA levels, indicating that most of the purine bases formed are again used in the NA synthesis de novo.Therefore, the SUA is a parameter by which the intensity of the NA metabolism can also be estimated.High UA values o indicate the predominance of NA disintegration over synthesis, while a significant part of purine bases degrades to UA.Low UA values, on the contrary, indicate that the need for NA increased significantly, the need for purine bases prevails over the possibility of their synthesis, and as a result, the disintegration of purine bases is sharply reduced.
Therefore, in the case of simultaneous determination of SNA and SUA, both the intensity of the NA synthesis, and the intensity of the NA disintegration in the body can be estimated.On the basis of the data obtained, it is possible to diagnose and correct the NA metabolism on a timely basis.As it has been shown previously, the timely diagnosis and restoration of reduced NA metabolism can be an important factor in the outcome of treatment of patients with alimentary insufficiency with achalasia or cicatricial narrowing of the esophagus [1].
Therapy of a severe burn injury remains the most important problem of intensive care.Immediately after recovery from shock, there is an extreme tension of metabolic processes associated primarily with marked catabolism.Meanwhile, the monitoring of alimentary status using generally accepted criteria (albumin, Quetelet index, and lymphocyte count) in the acute period is not informative.In addition, the nucleic acid metabolism in severe burn injury has not been studied.Therefore, there are no recommendations for correction of the alimentary status, taking into account the NA metabolism in both acute and subacute (stage of septic complications and exhaustion) periods.
Purpose: to assess changes in NA metabolism in severe burn injury and to develop recommendations for correction of the alimentary status taking into account changes in the NA metabolism.

Materials and Methods
This study was conducted in the burn center of the regional clinical hospital of Novosibirsk (Russia) in 2016-2018.In total, the study included 37 patients of both sexes aged 15-70 years with severe burn injury: II-III degree burn with an area of more than 40%, or II-IV degree burn with an area of more than 20%, or II-III burn with an area of more than 20% + burns of the upper respiratory tract; patients were in the combustiological ICU for more than 3 DOI:10.15360/1813-9779-2019-1-4-11
The exclusion criteria were as follows: age less than 14 and more than 70 years, decompensated comorbidities, as well as diseases accompanied by pronounced changes in the metabolism of nucleic acids (cancer, autoimmune diseases and gout).Patients with renal failure requiring renal replacement therapy were also excluded.
Intensive therapy of thermal injury in acute and subacute period was carried out according to accepted clinical recommendations [5].Nutritional support was provided according to the recommendations of the European Society of Clinical Nutrition and Metabolism [6].
Monitoring of biochemical parameters of blood serum, including uric acid level, was carried out using biochemical analyzer «AU-480»(«Beckman Coulter», UK).The oligonucleotides serum level was estimated by the Schmidt-Thanhauser method.Pre-preparation of samples was carried out as follows.Add 11.6 M HClO 4 to 0.4 ml of blood plasma up to a final concentration of 0.5 M and boil in a water bath for 30 min.Cool hydrolysates and centrifuge them for 5 minutes at 12000-14000 g.After oligonucleotide precipitation, spectrophotometry at wavelengths of 270 and 290 nm against 0.5 M HClO 4 reference solution was carried out; spectrophotometer «Genesys 10 uv»(«Thermo Spectronic», Germany) was used.The content (ng/ml) of nucleic acids was calculated using the following formula: C = (А 270 -А 290 ) 10.3/0.19,where 0.19 is the (А 270 -А 290 ) value of the hydrolysate of nucleic acids containing 1 ng of nucleic phosphorus in 1 ml of the solution; 10.3 is the average conversion factor for conversion of the amount of nucleic phosphorus to the amount of nucleic acids.
In order to correct the decreased level of NA synthesis in some patients (19 subjects), a glutamine solution, pharmaconutrient L-glutamine, was used intravenously at a dose of 0.4 g/kg/day for 7-10 days.Reduction of uric acid concentration below 100 μmol / l (i.e. 2 times lower than normal values) was taken as an indication for glutamine administration.
A two-way repeated measures ANOVA (Repeated ANOVA) was carried out.Statistical processing of dara was performed using the STATISTICA 8.0 package.

Results and Discussion
23 of 37 patients with severe burn injury survived; the mortality rate was 38%.Purulent-septic complications that developed on days 10-28 after burn injury were the cause of death in all 14 cases.The mean age of survivors was 49.2±2.9years; in Group II, it was 42.4±3.6years.Therefore, no significant age differences between groups were observed (P>0.167).
The analysis of UA dynamics in the survived patients (Group I) showed 3 stages of changes in UA serum concentration (table 1).As a rule, normal UA values were observed at the first stage (burn shock, days 1-3).The average UA level was 344.1±32.1 on the first day (normal limits: 200-415 μmol/l for men and 200-350 μmol/l for women).
In group II, there is a similar dynamics (table 1), however, starting from day 7, there are significantly lower UA values as compared to Group I (P<0.01).In addition, there was no significant increase in uric acid concentrations; and on day 20, the UA concentration was 112.1±6.9 (P<0.001 vs. Group 1).
Changes in SNA in the groups were also multidirectional (table 2).In the case of burn shock (days 1-3), the SNA values within the normal range (10-50 ng/ml) were observed.The subsequent 2-fold increase is typical for survivors; at that, this dynamics was observed as early as on the 5th day (P<0.001).The most significant intergroup differences were observed later starting from day 10.Particularly, the oligonucleotide serum levels in Group I patients on day 14 was 2.4 times higher (P<0.0001) as compared to Group II.
Analysis of results demonstrated that in group I intravenous glutamine was used 1.6 times more often than in the group of the deceased (53 and 33%, respectively).The use of glutamine was accompanied by a rapid increase in the UA level by 25-84%.For comparison, in the group of patients where glutamine was not used, the increase in UA levels on day 7 was 18% (P< 0.01) (fig.).

Clinical case
Patient S. was admitted to the intensive care unit with II-III degree thermal injury and the affected area of more than 60%.On the 58th day of burn disease, which corresponds to the phase of complications and exhaustion, rapid rejection of the skin flap was noted after another plastic surgery.The UA serum level was determined; marked decrease to 75 μmol/l was observed.Low UA level was considered an absolute indication for intravenous administration of L-glutamine at a dose of 0.4 g/day for 7 days.SUA was 125 mmol/day 5 days later.Another skin plastic was successful; 7 days later, the patient was transferred from the ICU in a state of moderate severity.Thus, it was the low level (75 μmol/l) of uric acid that was the indication for the use of L-glutamine.In turn, glutamine contributed to the При анализе результатов отметили, что в группе I препарат глютамина использовали внутривенно в 1,6 раза чаще в сравнении с группой умерших (53 и 33% соответственно).Использование глютамина сопровождалось быстрым увеличением уровня МК на 25-84%.Для сравнения, в группе пациентов, где глютамин не использовали, прирост МК на 7-е сутки составил 18%, (р<0,01) (рис.).
Анализ изменений НКСК также позволяет сделать несколько выводов.Во-первых, в период шока содержание НКСК снижается по сравнению с нормой, что ставит под сомнение некро/апоптотическую природу олигонуклеотидов в сыворотке крови.Эти дан-Along with clinical observations, where hypoglutaminemia in critically ill patients was associated with a negative outcome, there are experimental studies confirming the negative prognosis in the case of glutamine deficiency, in the first place, due to suppression of the immune system and damage of the intestinal mucosa [7].Therefore, (1) glutamine administration in critical illness, as a rule, has a positive effect despite the baseline serum glutamine content, and (2) parenteral administration mainly has an advantage over the enteral one [8,9].However, the absence of strictly defined indications for the use of glutamine is the main limitation of its use in clinical practice [10].
A number of studies have shown that it is not correct to consider the glutamine plasma concentration with no focus on its tissue content [12][13][14].Normal serum glutamine levels may be associated with low muscle levels, and vice versa.At the same time, it should be taken into account that glutamine synthesis and accumulation occurs in muscle tissue, and bone marrow, intestinal mucosa, lymphoid tissue belong to tissues most sensitive to glutamine deficiency [15].
Therefore, the blood glutamine level cannot be used as an indication for administration of glutamine in critically ill patients.More reliable criteria are needed to determine the indications for glutamine administration in critically ill patients.Presumably, the UA might serve as one such criterion.
The results obtained from the use of glutamine solution when the concentrations of UA is low demonstrate the effectiveness of the proposed method of correction of altered nucleic acid metabolism.Indeed, the use of glutamine in all cases was accompanied by an increase in the SUA levels, and clinically it was expressed in the way that may be considered as a result of a restoration of repair processes.In a number of patients, the timely use of glutamine resulted in radical changes of the situation toward favorable outcome.Thus, UA can be recommended as a criterion for assessing the alimentary status in burn disease, as well as it may be used to determine the indications for glutamine administration.According to our analysis, a decrease in the UA acid below 100 μmol/l was an absolute indication for administration of glutamine.On the contrary, the use of glutamine at high UA values (more than 400 μmol/l) may be associated with negative consequences, since the metabolic conversion of purine bases to UA is known to be accompanied by the generation of reactive oxygen species.
Analysis of changes in the SNA levels also leads to several conclusions.First, during the period of shock, the SNA content decreases in comparison with the normal limits, which dispute the necrotic/apoptotic nature of oligonucleotides in the blood serum.From our point of view, these data favors the concept of active secretion of oligonucleotides into the extracellular space, and active maintenance of their level in the blood.Second, support of a high level of SNA (2-fold above the normal limits) appears to be a factor that influences the survival.This is confirmed by a significant increase in the SNA level by 2-3-fold in the acute period in patients of Group I as compared to the normal limits and Group II.