Effect of Immune-enhancing Enteral Nutrition Formula Enriched With Vegetable Derived n-3 Fatty Acids on NK Cell Activity in Hospitalized Patients for Rehabilitation
Phase: NA
Conditions: Enteral Nutrition
Sponsor: Yonsei University

Effect of immune-enhancing enteral nutrition formula 
enriched with vegetable derived n-3 fatty acids on NK cell 
activity in hospitalized patients for rehabilitation 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
NCT number : In Progress (not assigned) 
Date of the document : March, 2018. 

Materials and Methods 
Participants 
From August 2015 to March 2018, ninety-four patients were enrolled in this study after 
admission to the department of rehabilitation at Yonsei University Severance Hospital. Yet, 
thirty-four subjects discontinued the study for personal reasons (withdrawn consent for clinical 
trial, move to another department or hospital, other disease treatment, Nil Per Os, etc.). There 
were no serious adverse effects for the withdrawal reasons. Results of this study were analyzed 
with sixty subjects, except for the dropouts. Majority of patients were forty-three quadriplegia 
patients and five hemiplegia patients with cerebellar pathophysiological origin. Eleven patients 
with ataxic quadriparesis and one patient with lateral medullary syndrome trauma were also 
enrolled. There was no difference between two groups regarding diagnostic distribution 
(p=0.100, chi-square test). Informed consent was provided by patients or their close family 
member. This investigation was approved by the Institutional Review Board at Yonsei 
University Severance Hospital, Seoul, Korea (Approval number: 2015-0907). All 
comorbidities and histories of the study participants were recorded under IRB tracking number. 
 
Randomization and Intervention 
Using computer-generated randomization lists, ninety-four patients were randomized 
to receive one of two types of enteral nutrition: soybean enteral nutrition (control), vegetable 
(canola, flaxseed) derived n-3 fatty acid enriched enteral nutrition (n3EN). The ready-to-use 
soybean used formula and n3EN formula had identical packaging with no differences in 
appearance, texture, or smell. Patients assigned to the control group received a soybean used 
formula by tube feeding. Those assigned to the n3EN group received vegetable n-3 fatty acid 

enriched formula by tube feeding (product; Yonsei Dairy Co., Seoul, South Korea). The 
composition of both control and test group enteral formula shown in Table 1. The nutrient (fatty 
acids) composition of Canola, Flaxseed and Soybean oil by United States Department of 
Agriculture database (25) attached as supplemental data (Supplemental table 1). 
Enteral nutrition was initiated within 24 h of admission. Enteral feeding was delivered 
at a constant rate to achieve a minimum of 50% basal energy expenditure (BEE; determined 
using the Harris–Benedict equation) × 1.2 within the first 12 h. If well tolerated, enteral 
nutrition was advanced to achieve a BEE × 1.2 within 48 h. Complementary feeding with 
enteral or parenteral nutrition was allowed for an initial 48 h. From the third day, the patient 
received a minimum of 75% of BEE × 1.2. The daily enteral intake was recorded to obtain the 
total volume and calories delivered to the patients. The data on daily total calorie intake were 
abstracted from the medical records.  
 
Anthropometric Parameters and Blood Collection 
The subjects’ gender, age, height (cm), weight (kg), weight change were tracked. Body 
weight during hospital stay was derived from the medical records on the first day and 14 days 
after consumption of each formula and was used to calculate BMI (Body Mass Index = body 
weight (kg) / height (m2)) (26). Venous blood specimens were collected in EDTA-treated and 
plain tubes and centrifuged to obtain plasma and serum. The collected blood samples were 
stored at −70 °C until analysis.  
 
Hematology and biochemical analysis 
Serum glucose concentrations were measured according to the hexokinase method on 

a Hitachi 7600 autoanalyzer. Serum albumin concentrations were analyzed through the BCG 
method using an ALB kit (Siemens, Tarrytown, NY, USA) with an ADVIA 2400 auto-analyzer 
(Siemens, Tarrytown, NY, USA). Leukocyte count was determined using the HORIBA ABX 
diagnostic analyzer (HORIBA ABX SAS, ParcEuromedicine, Montpellier, France). Serum 
high-sensitivity C-reactive protein (hs-CRP) levels were measured with a kit from the N-Assay 
LA CRP-S D-TYPE (Nittobo, Tokyo, Japan) with a Hitachi 7600 autoanalyzer. 
 
Cytokine Assay in Serum  
Interferon (IFN)-γ was measured with a kit from an IFN gamma High-Sensitivity 
Human ELISA Kit (Abcam plc-Cambridge Science Park, Cambridge, UK) according to the 
manufacturer’s instructions. Interleukin (IL)-12 levels were analyzed by a High-Sensitivity 
Human IL-12 (P70) ELISA kit (Genway Biotech Inc., San Diego, CA, USA) using a Victor ×5 
2030 multi label plate reader (PerkinElmer, Hopkinton, MA, USA) at 450 nm. IL-6, IL-1β, and 
tumor necrosis factor (TNF)-α levels in serum were measured using the Bio-Plex™Reagent 
Kit (Bio-Rad Laboratories, Hercules, CA, USA). 
 
NK Cell Activity 
Isolated PBMCs (peripheral blood mononuclear cells) from the whole blood samples 
were incubated with K562 cells to analyze the cytotoxic activity of NK cells. A whole blood 
sample was mixed with the same volume of RPMI medium 1640 (Gibco, Thermo Fisher 
Scientific, Waltham, MA), then gently overlaid on Histopaque®1077 (Sigma-Aldrich, Irvine, 
UK) and centrifuged for 20 min at 1800 rpm at 15 °C. After separation, a buffer coat layer was 
isolated, washed once with RPMI 1640 medium, and then resuspended in 1 mL of 10% fetal 

bovine serum. The isolated PBMCs (effector cell, E) were seeded into 96-well plates at ratios 
of 10:1, 5:1, 2.5:1, 1.25:1 and 0.625:1 with the K562 cells (target cell, T) and then incubated 
at 37 °C under 5% CO2 for more than 4 h. The cytolytic activities of NK cells were analyzed 
via the CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit (Promega Co., Fitchburg, WI, 
USA) according to the manufacturer’s instructions. The color reactions were read at 490 nm 
using a Victor ×5 2030 multi label plate reader (PerkinElmer, Hopkinton, MA, USA), and the 
results were calculated by the following formula: 
% Cytotoxicity = (Experimental-Effector Spontaneous-Target Spontaneous) / (Target 
Maximum-Target Spontaneous) ×100