EFFECT OF S UPPLEMENTATION WITH GLUTAMINE AND HYDROXYMETHYLBUTIRATE OVER THE CELLS INVOLVED IN WOUND HEALING

M en C Alejandra Patricia Salas González 1 , Sevilla González María de la Luz 2 and Fuchs Tarlovsky Vanessa 3 . 1. Hospital General de México/ Instituto Politécnico Nacional. 2. Instituto Politécnico Nacional, Escuela Superior de Medicina. 3. Hospital General de México. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


Material and methods:-
Clin ical, experimental, longitudinal, prospective, comparative, randomized trial where patients older than 18 years, with diagnosis of bloody areas hospitalized in the Plastic and Reconstructive Surgery service during the period August 2016 -June 2018 with negative pressure system and those who signed the informed consent were included. Patients with previous supplementation, smokers, glycosylated hemoglobin> 12%, renal failu re, topical collagen therapy, collagen vascular disease, autoimmune disease, radio or chemotherapy, allergy to any ingredient (glutamine or HM B), d iagnosed with chronic d iseases, pregnancy and lactation were excluded. Those patients who did not have adherence to the treatment, who underwent graft or flap skin procedure before co mpleting the supplementation, voluntary discharges and patients who decided to withdraw their informed consent were eliminated.
Once the patient met the inclusion criteria, was invited to participate in the research protocol, exp lain ing what it consists of, as well as the benefits of Glutamine and HM B supplementation.
The protocol consisted in three stages: First stage: Before the supplementation, under local anesthesia and by a standardized Medical Resident of the Plastic and Reconstructive Surgery Serv ice, a sa mple of the edge of the affected tissue was taken after asepsis or surgical toilet; this is a routine procedure in the Service, the sample was sent to the Pathology Service for analysis (procedure described later). Patients were randomly assigned to control or treatment group. Doctors and laboratory technicians, who worked with the participants in the study, as well as the participants themselves, were blinded to if the patients were assigned to treatment or control group. Second stage: If the patient was part of treat ment group the supplement was delivered (box 1) explaining that it must be taken once a day during 15 days diluted in approximately 250 ml of water. Indiv idualized dietary guidance was given in order to improve nutritional status of participants. In case of being part of control group the patients was under observation during a period of 15 days . Third stage: At the end of the supplementation, a second sample of the affected tissue was taken, by the procedure described above; to be analyzed in the laboratory by an experienced Physician Resident of the Pathology service . The samples were analyzed by Giemsa stain for morphological differentiat ion in order to obtain the total number of units per field: ly mphocytes, macrophages, granulocytes, fibroblasts and angiogenesis. In addition, subjects were asked if they experienced any symptoms, such as stomachache, dizziness, nausea, vomit ing, headache, loss of 102 appetite, general weakness among others. A Kolmogorov-Smirnov test was used to check normality for continuous variables. A t-Student test was used for parametric data and Mann-Whitney U test for non parametric data. The study was conducted under the established terms of the Mexican General Health Law, Title Five and the Regulations of the General Health Law on Health Research (RLGS), and NOM -012-SSA3-2012, which establish the criteria fo r the execution of research projects for human health. This study also counts with the approval of the Co mmittee of Ethics in Research and the Research Co mmittee of the General Hospital of Mexico "Dr. Eduardo Liceaga".

Results: -
A total of 21 patients were invited; 4 were excluded because the treatment was not properly concluded, leaving 17 patients, of whom 9 were assigned to the control group (53% ) and 8 to the treatment group (47%). The mean age of the total sample was 38.65 ± 14.47 years (Tab le 1). The control group had a mean age of 43.30 ± 15.00 years and treat ment group 30.00 ± 10.00 years. 277.88±100 --------Student' s t test for independent samples. Data presented as mean ± standard deviation. BMI = Body mass index, kg = kilograms; kg / m2 = kilograms per square meter.
The mean weight before supplementation was 65.87 ± 11.53 kg and at the end 65.85 ± 11.49 kg, no significant differences were found; BMI 24.42 ± 3.6 kg/ m 2 which is interpreted as a Normal body mass index according to the WHO and at the end 24.39 ± 3.49 kg/m 2 ; no significant differences were found. The mean of the wounds size was 277.88 ± 100 cm 2 , the final measurement was not obtained because some patients underwent a surgical procedure before taking the measurement. The admission diagnosis with mo re frequency was car accident (41.20%) followed by, cold weapon wound (23.50%) and others (23.50%) in wh ich it is included: work accident, fall greater tha n two floors, everyday accident, with lower frequency by fire project ile (11.80%) At the end of the supplementation the control group had a lymphocytes mean of 8.440 ± 3.35 units/field and the treatment group: 12.50 ± 7.69 units/ field, without significant differences (p = 0.330) but with a mediu m effect size (d = 0.68); the macrophages in the control group of 5.44 ± 4.64 units/field and the treatment group 5.00 ± 2.44 units/field, without differences (p = 0.806). A granulocyte mean of 6.44 ± 6.78 units/field was observed in the control group and 11.25 ± 10.25 units/field at treatment group, without significant differences (p = 0.237) with a med iu m effect size (d = 0.55) and an increased at the end in the treatment group. A tendency to increase in the fibroblast count was observed in both groups at the end of the treatment with significant differences between the means (p = 0.015); with a large effect size (d = 1.14). Finally, in the n ew blood vessel formation count 3.50 ± 2.99 units/field were reported in the control group and 7.25 ± 3.10 units/field in the treatment group, with significant differences (p = 0.036), and a large effect size. (d = 1.23) (Graph 1).

Graph 1:-Histogram of units/field ± standard deviation between groups after treatment
On a daily basis, a symptomatology questionnaire was applied to the patients in both groups, the frequencies at the end of the supplementation are shown in the following table: 2) 4 (50.0) 0 (0.00) Data presented in frequencies and percentages before and after supplementation referring to the number of patients who presented the symptomatology. n1 = 9 patients in the control group, n2 = 8 patients in the treatment group.
At the end of 15 days the control group showed higher frequencies in sy mptomatology compared to the treat ment group. In first place, there is a decrease in the frequency of patients who showed general weakness in the treatment group from 75% to 12.50%, who reported an increase in the sensation of physical energy. There was a decrease in loss of appetite frequency fro m 4 to 0; to which the patients of this group reported an increase in the appetite sensation. A decrease in gastrointestinal symptoms such as diarrhea fro m 37.5% to 0% was also reported, indicating better consistency in the evacuations. The feeling of nausea decreased in both groups, from 55.5% to 22.2% in the control group and from 37.5% to 0.0% in the treat ment group. These symptoms were reported before the supplementation and patients themselves related nausea and vomitting to anesthesia side effects.

Discussion:-
It is considered, that this is one of the few formal research es studies that have been accomplished with: glutamine (14g/d) and hydroxymethylbutyrate (HMB 3g/d), since the literature review yielded little information about it and arginina has been added to the mixture in other studies. The biggest effect was observed in Fibroblasts and Angiogenesis, both reported significant differences between groups at the end of the supplementation, with a large effect size. In co mparison with another study of similar characteristics where the histology of the wound was evaluated subjectively (inflammation, reepithelialization, granular tissue, collagen and angiogenesis) where no significant differences were found between the parameters attributed to the lack of standardization in the protein supplementation between groups (p> 0.05) 25 .
The tendency to increase found in the results agree with that described by Witte et al. about the difference in appearance time of the cells in the wound during the healing process, macrophages are predominant during inflammat ion taking its maximu m peak between the 4th and 6th day after the wound, wh ile the ly mphocyte count increases to its maximu m peak on the 6th day and a decrease is expected fro m day 10-14, this cells are predominant, along with the fibroblasts, during the proliferative phase with maximu m peaks around day 8-13 rising to the maturation phase, decreasing the degradation of the damaged extracellular matrix and increasing the production of collagen III 26 .
Biochemical parameters were measured in order to monitor the protein metabolism such as albumin, total proteins, blood urea nitrogen and creatinine, no significant differences were observed and levels were mantained within normal range which is why the results are not reported. Results differ fro m reported by Rathmacher where blood urea nitrogen increased in a HM B+arg inina+g lutamine supplemented group unlike the results in this study, main ly because the supplementation was different resulting as extra nit rogen consumed, so it is relevant to evaluate the importance or not of argin ine in supplementation. 26 Regarding the symptomatology, in general a decrease of the symptomatolog y was reported in both groups, it is worth mentioning that, the patients who received the treatment reported imp rovement in: general well-being, increased appetite and better intestinal transit (referred to as a better consistency in the evacuations by those who presented diarrhea and a higher number of evacuations in those who presented constipation); which was not referred by the control group. Benefits of intestinal permeability have been reported when supplemented with glutamine 27 , mainly by decreasing the duration of diarrhea since it helps to maintain the intestinal immunological barrier, increasing the levels of intestinal immunoglobulin A, reduces bacterial translocation and promotes the absorption of sodium and water by functioning as a substrate in the co-transport of sodium; Therefore, it reduces the duration and frequency of gastrointestinal symptoms 28,29 .
It should be mentioned that the sample size could have limited the results of the study, despite this; the results demostrate that the mixture of g lutamine and HMB as daily supplementation can have a positive effect on the cells involved in the healing process. It is considered that the perspective of the study is to continue the research on the benefits of immunonutrition on wound healing in different pathologies, in addition the need to carry out a new study 105 including cluster differentiat ion in order to specifically iden tify the type of cells observed and designing and in which the long-term permanence of the effect can be evaluated.