Impact of preoperative immunonutrition on thrombocyte phagocytic activity and early postoperative outcomes in invasive gastric cancer patients.

Background:The aim was to determine the phagocytic activity of thrombocytes in patients with gastric cancer and to assess the effect of oral and parenteral preoperative glutamine-based immunonutrition on nutritional status, thrombocyte phagocytic activity and early postoperative outcomes. Methods:Patients suffer from invasive gastric cancer had been treated with preoperative immunonutrition with glutamine and they were compared to patients without nutritional treatment. Nutritional status, percentage of weight loss and BMI were assessed. Levels of total protein, albumin, cholesterol, triglycerides, platelets and their phagocytic ability were measured twice. Postsurgical complications were assessed via Claven-Dindo classification. Results:Group I-20 patients with an oral glutamine, Group II-38 patients received an intravenous glutamine. Group III-25 patients did not receive preoperative immunonutrition. 47% patients Group I, 54% patients Group II and 33% patients Group III were malnourished. In Group I, percentage of phagocytizing platelet (%PhP) was 1.1 pre- and 1.2 postoperatively. Phagocytic index (PhI) was 1.0 and 1.1. In Group II, %PhP was 1.1 and 1.2. PhI was 1.0 and 1.1. In Group III %PhP was 1.0 and1.2. PhI was 1.0 and 1.1. An increase in triglyceride level was observed in both immunonutrition groups. There was a fall in total protein, albumin level in Group II. In Group III there was a decline in total protein, albumin and cholesterol level. Total platelet count, and PhI was increased in both immunonutrition groups. There was also a rise in %PhP in Group II. In Group III there was no change in blood plateles level, %PhP and PhI. Complications rate was 53% in Group I, 29 % in Group II, 40% in Group III. Conclusions:In invasive gastric cancer, laboratory nutritional parameters are significantly reduced, causing malnutrition in 45.7% of patients. Oral immunonutrition on phagocytic activity of blood platelets in patients with gastric cancer. Effects of both, intravenous infusion of glutamine and oral supplementation, were assessed.


Background:
Gastric cancer incidence worldwide has declined significantly over the last 30 years. Nevertheless, it still constitutes a significant clinical problem. Only in 2015 stomach cancer caused 754 000 deaths globally, which makes it fourth cancer-related mortality [1,2]. Despite progress in diagnostic and therapeutic management, therapy of invasive gastric cancer is associated with high morbidity rate and often low quality of life. Postoperative complication rate after total gastrectomy ranges from 9 to 47.5% and reported mortality rate varies between 1.1 and 10.8% [3][4][5][6]. Causes of poor outcomes are late diagnosis, pre-operative malnutrition and impaired immune system being one of the most important causes. Even eighty-five percent of patients undergoing surgery for gastric cancer are malnourished [7]. It is a result of tumour-related cachexia and decreased food intake due to anorexia.
Moreover, host immune response is impaired [8]. The outcomes of cancer treatment can be optimised by maintaining an unharmed immune system and using immunogenic therapies to re-establish antitumour immune response. Glutamine plays a major role as a fuel source for macrophages, lymphocytes, and enterocytes. As the energy substrate for intestinal epithelial cells, it also protects the intestinal immune barrier against microbes.
Role of various blood cells in host response to cancer has been thoroughly investigated. Platelets (thrombocytes) are predominantly known for their role in coagulation. What is less commonly known is their ability to phagocytise, first reported by Mustard et al. in1968. These nonnuclear blood cells have the potential for chemotaxis and diapedesis [9]. They are able to phagocytise bacteria, viruses, antibody complexes, collagen and latex particles, working both as single platelets as well as in aggregates [10,11]. Upon activation, the thrombocyte changes its shape from discoid to an irregular one with numerous projections. This morphological change is accompanied by intensification of energetic processes and enhanced protein anabolism inside the activated platelet [12]. Aim: The aim of the study was to determine the impact of preoperative glutamine-based immunonutrition on phagocytic activity of blood platelets in patients with gastric cancer. Effects of both, intravenous infusion of glutamine and oral supplementation, were assessed.

Study population
Patients with gastric cancer were enrolled in the study. The only inclusion criterion was an operable gastric cancer. The stage of gastric cancer was based on the TMN classification. Patients were divided into three randomized research groups. Group I and II was received preoperative glutamine-based immunonutrition, Group III did not receive preoperative immunonutrition. Patients receiving preoperative nutritional treatment were divided into two groups depending on the route of glutamine supply. Patients in Group I were fed a regular hospital diet enriched with an oral glutamine solution twice a day. Additionally, an infusion of three-chamber parenteral feeding bag (1447 mls) daily plus one ampoule of vitamins was administered. Group II was also on a regular diet supplemented with three-chamber parenteral feeding bag Smofkabiven Peripheral (1447 mls) fortified with one ampoule of vitamins but also with an intravenous solution of glutamine -100 ml of -N(2)-L-alanyl-L-glutamine dipeptide (20 g N(2)-L-alanyl-L-glutamine, 8.2 g L -alanyl and 13.46 g L-glutamine) with 0.2 g/ml of the medicinal product. Group III was fed only by natural regular hospital diet supplemented with three-chamber parenteral feeding bag Smofkabiven Peripheral (1447 mls) fortified with one ampoule of vitamins.
Exclusion criteria were inoperable gastric cancer and impaired gastric empting in the oral glutamine supply group. Due to the small number of early gastric cancer patients in clinical stage I were excluded from further analysis. Figure 1 shows Data collection On admission to hospital, each patient was evaluated for nutritional status using SGA. The percentage of unintentional weight loss and BMI were calculated. Each patient had total protein, albumin, total cholesterol, triglyceride, platelet count and phagocytic activity assessed. Laboratory tests and phagocytic activity were reassessed 12 days after the surgery. Similar examination was performed in patients without preoperative immunonutrition.
In both groups, the duration of preoperative immunonutrition was 8 to 14 days (mean 12).
Postoperative artificial nutrition without glutamine (three chamber bag + early postoperative enteral nutrition) was continued for 5-6 days after the surgery (mean 5.4) and it was the same in each group.
Operation included stomach resection with excise of regional lymph nodes in the D2 range or above D2. Nasojejunal feeding tube was placed intraoperatively. Early postoperative enteral nutrition with semi-elemental, normocaloric, low fat diet was started 20 hours post-surgery. Three patients with type 2 diabetes mellitus received a special diet normalizing glycaemia.
Imaging technique and imaging analysis Phagocytic activity of blood platelets was determined against Staphylococcus aureus ATCC 653P bacterial strain, according to Mantur`s et al. method [13].

Statistical analysis
Results were compared to a control group with was consisted of 30 healthy individuals. The results were analysed using the Statistica 13.1 program, p < 0.05. Distribution of clinical stages in particular groups is presented in Figure. In Group I the percentage of invasive gastric cancer is 92%, in Group II -85%, in Group III -66%.
Early Gastric Cancer were detected in 3 patients of Group I, in 3 of Group II, and in 10 of Group III.

Preoperatively
Group I included 20 patients (7 women, 13 men) aged from 34 to 82 years (65.2 ± 11.9). Unintentional weight loss above 10% was demonstrated in patients 7 (35%). Percentage of body weight ranged from 10 to 25% (15.3% ± 6). Body mass index was from 16 to 28 (26.3 ± 7.29). Low protein level was found in 9 patients, in 16 patients a low albumin level. Lower triglyceride value was observed in 1 patient as well lower cholesterol level.
A preoperative level of total protein, albumin and triglyceride were significantly lover in comparison to control group. There was no statistically differences in total cholesterol level. Preoperative serum levels of total protein, albumin, cholesterol and triglyceride in patients of Group I are shown in Table 1. Malnutrition was found in 47% patients. No statistically differences of a total count of blood platelets in Group I and control group was observed. A preoperative phagocytic activity of blood platelets and phagocytic index were relevantly lover in comparison to healthy individuals. Preoperative phagocytic activity and phagocytic index are shown in Table 2. Table 2 The number of blood platelets, the number of phagocytizing platelets, and phagocytic index in Group I before surgery in comparison to control group.  Table 3. Malnutrition was observed in 54% patients. No statistically differences of a total count of blood platelets in Group II and control group was observed. A preoperative phagocytic activity of blood platelets and phagocytic index were relevantly lover in comparison to healthy individuals.
Preoperative number of total count of platelets, their phagocytic activity and phagocytic index are shown in Table 4. Table 4 The number of blood platelets, the number of phagocytizing platelets, and phagocytic index in Group II before surgery in comparison to control group. and triglyceride level. As in previous groups the total protein, albumin and triglyceride level was relevantly lover in comparison to control group. There was also a statistical difference in cholesterol level. In 33% patients malnutrition was observed. Preoperative serum levels of total protein, albumin, cholesterol and triglyceride in patients of Group III are shown in Table 5. No statistically differences of a total count of blood platelets in Group III and control group was observed. A preoperative phagocytic activity of blood platelets and phagocytic index were relevantly lover in comparison to healthy individuals. The number of blood platelets, the number of phagocytizing platelets and phagocytic index in Group III before surgery are presented in Table 6.  Table 6 The number of blood platelets, the number of phagocytizing platelets, and phagocytic index in Group III before surgery in comparison to control group.

Postoperatively
In Group I postoperatively a significant increase of triglyceride level was observed. There was no relevant changes in total protein, albumin and cholesterol levels.
Postoperative serum levels of total protein, albumin, cholesterol and triglyceride in patients of Group I are shown in Table 7. The total protein, albumin and triglyceride level did not change in relation to Group III. However a significant reduction in cholesterol level was found. Table 7 Postoperative serum levels of total protein, albumin, cholesterol and triglyceride in patients of Group I. A relevant increase of total count of platelets and index was noticed. There was no significant changes in phagocytic activity of blood platelets but phagocytic index was statistically increased.

Min
Postoperative total count of platelets, their phagocytic activity and phagocytic index are show in Table 8. In Group II a relevant fall in total protein, albumin, and cholesterol level was observed. There was no significant changes in triglyceride level in this group of patients.
Postoperative serum levels of total protein, albumin, cholesterol and triglyceride in patients of Group II are shown in Table 9. The total protein, albumin and cholesterol level did not change in comparison to Group III. There was a relevant increase in triglyceride level. There was a statistical increase in postoperative number of total count of platelets, their phagocytic activity and phagocytic index.
Postoperative number of total count of platelets, their phagocytic activity and phagocytic index are shown in Table 10. Table 9 Postoperative serum levels of total protein, albumin, cholesterol and triglyceride in patients of Group II. Group III -there was a relevant decline in total protein, albumin and cholesterol level in patients without immunonutrition. There was a significant rise in triglyceride level.
Postoperative serum levels of total protein, albumin, cholesterol and triglyceride in patients of Group III are shown in Table 11. Postoperatively, the number of total blood platelets increased. There was no significant changes thrombocytes ability to phagocyte bacteria. Postoperative number of total count of platelets, their phagocytic activity and phagocytic index are shown in Table 12 Table 12

Complications
In Group I overall postoperative complication rate was 53% with major complications of 33%. 30-day mortality was 12%.
In Group II overall postoperative complication rate was 29% with major complications of 40%. 30-day mortality was 6%.

Grade Group I Group II
Different surgical techniques have various local and general imbroglios [6]. of patients with gastric cancer [16]. Even prolonged malnutrition during early life may increase the risk of stomach cancer mortality in later life [17]. Hence nutritional status needs to be optimised in the preoperative period. Fukuda et al. analysed 800 gastric cancer patients who had undergone gastrectomy. They classified 19% patients as a malnourished. In multivariate analysis malnutrition was independent factor postoperative fewer infections. Based on the assessment of nutritional status based on biochemical analysis, malnutrition was found in 47% patients in Group I, 54% patients in Group II, 33% patients in Group III. Well-managed preoperative nutritional support decreased the incidence of postoperative surgical site infections [18]. The current ESPEN guidelines recommends about a 10-14 day period of feeding with immunologically active compounds [19]. In the present study, malnutrition has also been reported. Unintentional weight loss above 10% of body weight in last 6 months was found in 34% patients. The main unintentional weight loss was 13.7 kg.
Biochemical analysis shown similar values of examined parameters in all groups of patients. In Group I low protein level was found in 9 patients, in 16 patients a low albumin level. Lower triglyceride value was observed in 1 patient as well lower cholesterol level. In Group II low protein level was found in 17 patients, in 19 patients a low albumin level. Lower triglyceride value was observed in 1 patient. No patient had low cholesterol level. As in previous groups in group without immunonutrition the total protein, albumin and triglyceride level was relevantly lover in comparison to control group. There was also a statistical difference in cholesterol level. It was found that in all patients, body mass index correlated with an unintentional weight loss and serum albumin concentrations. Moreover, the percentage of unintentional weight loss negatively correlated with total preoperative protein and triglyceride levels. Furthermore, total protein concentration was positively correlated with albumin concentration and triglycerides. Liu et al found that preoperative BMI was positively correlated with albumin and triglyceride levels and preoperative albumin levels were positively correlated with triglycerides. Therefore, serum albumin level is not only a window into the patients' nutritional status but is also a useful factor for predicting prognosis [20]. Poor survival was also observed in gastric cancer patients with lower levels of BMI, albumin, and triglyceride [21].
Because low levels of serum albumin are associated with poor outcomes in cancer patients, they can be used as an independent indicator when assessing the need for aggressive nutritional intervention [22].
Glutamine-supplemented perioperative nutrition has been investigated in patients with a variety of diseases but the effects have not been conclusively established [23]. In our study, interesting results in two different models of preoperative immunonutrition were observed.
In Group I the increased triglyceride level was observed. In Group II the total protein, albumin and cholesterol level was declined. In Group III both groups the decreased total protein after the surgery was noticed but albumin level was similar before and after surgery.
In Group I the total protein, albumin and triglyceride level did not change in relation to Group III.
However a significant reduction in cholesterol level was found. In Group II The total protein, albumin and cholesterol level did not change in comparison to Group III. There was a relevant increase in triglyceride level.
We can say that glutamine using during preoperative immunonutrition helped to sustain higher albumin levels whilst on the other hand, a decrease in total protein level was recorded. Tue et al have also observed an improvement in postoperative cumulative nitrogen balance with perioperative parenteral nutrition supplemented with glutamine in patients undergoing abdominal surgery [24]. One explanation for this protein metabolism may be a reduced production of proinflammatory cytokines.
In a previous study of our group, we found elevated levels of interleukin 6 (IL-6) in gastric cancer patients which correlated negatively with the disease stage. Its values were highest in patients with early gastric cancer [25]. Administration of the preoperative parenteral glutamine immunonutrition results in normalization of cholesterol and triglycerides by increasing or decreasing their levels to achieve the normal range. In oral glutamine supplementation model, there were no significant changes in total protein, albumin and cholesterol levels. Glutamine enriched nutrition support in surgical patient's remains controversial. The meta-analysis thirteen randomized controlled trials showed improving immune function, reducing incidence of infectious complications and shortening the length of hospital stay [26]. Jiang et al noticed alanyl-glutamine supplemented parenteral nutrition clinically safe with better nitrogen balance, and maintained intestinal permeability in postoperative patients than patients who received isonitrogenous and isocaloric parenteral nutrition [27]. Furthermore, platelets exert cytotoxic effects on cancer cells by adhering to them via antigenic determinants. Then characteristic structural changes, such as Golgi apparatus hypertrophy, increase in secretory granulations and displacing them towards contact zone with a neoplastic cell are observed. Platelet cytotoxicity ensues from their ability to product and release lytic mediators [29].
Additionally, platelets play an important role in tumour metastasis. Platelet-delivered proteolytic enzymes facilitate the release and migration of tumour cells across the vessel wall [30]. Furthermore, activated platelets release substances which increase vascular permeability, factors stimulating myocytes proliferation, platelet activating factor, prostaglandins, histamine and serotonin. The substances above facilitate an implantation and growth of metastatic tumour [31]. In our last studies we found that the fraction of phagocytizing platelets and their phagocytic index in gastric cancer patients was markedly impaired as compared to healthy individuals [32]. Current work confirms impaired phagocytic activity in patients with gastric cancer. A decreased phagocytic activity can influence inflammatory processes as well as cancer growth [25]. In this study, impact of immunonutrition on phagocytic activity of platelets was evaluated. Glutamine is an essential amino acid for fast-dividing immune cells, epithelial cells of the gastrointestinal tract, fibroblasts and reticulocytes. It is a precursor of protein and nucleotide synthesis, it is also involved in hepatic gluconeogenesis and glutathione synthesis. Its high concentration was found in the intestinal mucosa cells. Glutamine and glutamate are amino acids responsible for the transport of nitrogen and detoxification of ammonia. The consequence of inhibition of glutathione synthesis is mucosal destruction, diarrhoea, and growth inhibition. Parenteral supplementation of glutamine in rats demonstrated its protective role against bacterial translocation [33]. Glutamine impact on blood platelets phagocytic activity is unknown.
In our study in all groups showed severe impairment of thrombocyte phagocytic activity. The significant decline in phagocytic activity of blood platelets and their phagocytic index in all patients was observed. In patients with oral glutamine supplementation the percentage of blood platelets and their phagocytic index was increased. In patients with intravenous glutamine administration both total count of platelets, their phagocytic activity and index was improved. There was no improvement in platelet phagocytic activity in patients without immunonutrition.
Authors had previously reported partial improved thrombocyte phagocytic activity in gastric cancer patients as a result of perioperative immunonutrition enriched with glutamine and ω-3 fatty acids both in local disease and in peritoneal dissemination [32] but this is the first study analysing blood platelets phagocytic activity in gastric cancer patients receiving enteral glutamine diet preoperatively.

Conclusions:
In invasive gastric cancer, laboratory nutritional parameters are significantly reduced, causing Clinical stages of Gastric Cancer in examined particular groups of patients

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