Comparing isolated soy protein with flaxseed oil vs isolated soy protein with corn oil and wheat flour with corn oil consumption on muscle catabolism, liver function, blood lipid, and sugar in burn patients: a randomized clinical trial

As part of our former study [23], we collected information of burn patients from the Institute of Burn Research of Qutb al-Din hospital, affiliated with Shiraz University of Medical Sciences. Eligible patients were required to have the following criteria: aged 15–60 years; a body mass index (BMI) of 18.0–30 kg/m² for individuals aged > 18 years and – 2 SD to + 2 SD percentile for those aged < 18 years [24]; total burn surface area (TBSA) of 20–50%; and the ability to understand the study protocol and provide written informed consent. Exclusion criteria included: renal or hepatic failure; severe inhalation injury; excessive hemorrhaging; allergy to soy; use of omega-3 fatty acids in the previous month; in need of parenteral or enteral nutritional support before randomization; and > 3 kg weight change over the preceding three months. The ethics board of Shiraz University of Medical Sciences, Shiraz, Iran approved the study protocol (reference no. CT-92-6878). All procedures were followed in accordance with the ethical standards laid down in the Declaration of Helsinki and its later amendments [25]. All patients or their close relatives provided written, informed consent before enrollment and we took consent to participate in the study from their parent or legal guardian in the case of patients aged < 16 years. This trial was registered at http://​en.​irct.​ir/​trial/​16230 as IRCT2014051817740N1.

In this double-blind RCT, allocation of participants to treatment and control groups was performed by permuted block randomization. Each eligible patient received a randomization number which was determined by a computer-generated schedule. The investigator and patients were blinded to the treatment condition, then a randomization table was generated by the method of random permuted blocks. People who were operationally independent from the study investigator performed the study randomization. Patients’ data collected during this trial was kept confidential and locked in a secure area. Randomization codes of the study were opened only after all participants complete the study protocol. To maintain and guarantee blinding, treatment products and control products were identical in appearance. Using simple randomization [26], participants were assigned in a 1:1:1 fashion to three equal groups, labeled as ISP + FO (Group A), ISP + corn oil (CO) (Group B), and wheat flour (WF) + CO (Control group) for three weeks. Because of the large perturbation in water compartments of burn body patients that occurred after admission time, fasting venous blood samples were taken on the morning of day 4 and day 25. During the study, from the 188 patients who were initially screened, 73 met the inclusion criteria and entered the intervention. The 25 patients in Group A received snacks with FO and ISP daily for three weeks. The 24 patients in Group B received snacks with CO and ISP daily for three weeks. The 24 patients in Group C, the control group, received snacks with CO and WF every day for three weeks. Participants were asked to avoid nutritional supplements, isoflavones, or phytate-rich foods during the treatment period. All patients received conventional wound management and fluid replacement. The intervention lasted for 21 days, from day 4 after admission until day 25. The study was performed from June 2014 through December 2014.

Snacks were provided in the form of cookies. Cookies were prepared with 50 g ISP powder (SUPRO® Isolated Soy Protein, DANISCO, Denmark) and 30 g FO (Abkar Golestan Cultivate & Industry Co, Iran) (Group A), 50 g ISP powder and 30 g CO (Group B), and 50 g WF and 30 g CO (Group C, control product). In the control group, CO and WF were selected as substitutes for FO and ISP, respectively. The doses of ISP [27] and FO were determined according to previous studies [28]. Other materials including milk, yogurt, rice flour, sugar, and water were used in similar quantities in cookies of the three groups and were also similar in protein and energy (Table 1). To improve the appearance and acceptance of the cookies, three different types of decoration were used. These decorations contained honey, concentrated date syrup, and chocolate. To ensure blinding, the cookies given to all three groups were of similar appearance each day. The amounts of energy, fat, and carbohydrates were almost similar in the cookies of the three groups. Since this study was a double-blind RCT, cookies were packed in similar packaging for all three groups. All patients received the conventional nutritional support of the Qutb al-Din hospital. This included 40 kcal/kg/day energy average (35–45 kcal/kg/day based on burn extent, number of surgeries, and body weight) and 1.5 g/kg/day protein average (1.2–1.5 g/kg/day) according to previous studies [29, 30]. There was no difference in the average daily calorie and nitrogen intake between individuals of the three groups. The diet of the Qutb al-Din hospital contained on average 24% protein, 27% fat, and 49% carbohydrate for all patients.

Knee height is correlated with stature and, until recently, was the preferred method for estimating height in bedridden patients Knee height was measured as a surrogate measure of height. Knee height was measured in the recumbent position from the top of the patella to the bottom of the heel pad with the knee flexed at 90°. Measurements were recorded to the nearest 0.1 cm. A nomogram was used to convert the knee height to the height with a precision of ± 6 cm (90% confidence limits) [31, 32]. Weight was measured using an electronic scale (Beurer PS160, Germany) to the nearest 0.1 kg. All anthropometric measurements were performed twice and the average of two measurements was considered. If the difference between two measurements was > 1.0 cm or 0.1 kg, a third measurement was taken and the two closest values were averaged.

We assessed the muscle catabolism of patients by nitrogen balance and 24-h urine creatinine as primary outcomes, with BMI, weight changes, and liver function indices as secondary outcomes. Fasting venous blood samples were obtained at the start and end of the study phase. The participants were asked to fast overnight for 12–14 h. Twenty-four–hour urine samples were collected on the fourth day of admission and at the end of the study. Serum was frozen at − 20 °C until the final analysis. Cell blood count (CBC) was measured by using a cell counter Sysmex KX21 machine. The bromocresol green method (Pars Azmoon Co., Tehran, Iran) was used for measuring serum hepatic enzymes (including ALT, AST, and ALP). Serum and urine concentrations of creatinine were measured by the colorimetric methods (Pars Azmoon Co., Tehran, Iran). FBS, TG, and cholesterol were measured by enzymatic methods (Pars Azmoon Co., Tehran, Iran). We calculated the total lymphocyte count (TLC) per cubic millimeter by multiplying WBC by the absolute percentage of total lymphocytes. Nitrogen balance was calculated each day as follows: nitrogen balance (gN/kg) = nitrogen intake (gN/kg) – total nitrogen excretion (gN/kg). The nitrogen intake (gr/N) was estimated as protein intake (gr) divided by 6.25 based on the calculated 24-h dietary recall from each patient [33, 34].We collected 24-h urine by using a plastic bag connected to a urinary catheter for three days. On each day, duplicate samples (each approximately 10 mL) were collected from pooled urine after stirring adequately and storing at − 20 °C for later use. The mean value of duplicates was used for further analysis to reduce measurement errors. The concentration of urinary urea nitrogen was measured using the urease method [35]. Criteria for SIRS were established in 1992 as a part of the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference. The conference concluded that the manifestations of SIRS include, but are not limited to, the following: body temperature < 36 °C or > 38 °C, heart rate > 90 beats per minute, tachypnea (high respiratory rate) with > 20 breaths per minute, an arterial partial pressure of carbon dioxide < 32 mmHg, white blood cell count < 4000 cells per cubic millimeter or > 12,000 cells per cubic millimeter, or the presence of > 10% immature neutrophils (band forms). SIRS is diagnosed when two or more of these criteria are present [36].

McNemar’s test was used to analyze the SIRS binary variable before and after the intervention. Dietary intakes were obtained on the first day to assess the baseline nutritional status of the patients. Dietary intakes were analyzed using NUTRITIONIST 4 software, version 3.5.2 (First Data Bank, San Bruno, CA, USA).

Figure 1 illustrates the patient flow throughout the study. A total of 188 individuals were assessed for eligibility. Of these, 92 did not meet inclusion criteria and 23 declined to participate. Of these, nine (9.6%) discontinued intervention before the end of the intervention period (Group A: n = 4, Group B: n = 3, Control: n = 0). The reasons for the drop-outs were as follow: four patients were discharged from the hospital during the course of the study; two died on day 13 and day 16 due to septicemia and pulmonary embolism, respectively; and one lost the willingness to cooperate after day 5 (Fig. 1). There was no significant difference in drop-out rate among the three groups. Causes of burn included: 29 cases of kerosene fire; 24 cases of gas explosion; 11 cases of flame burns; five cases of electrical currents; and four cases from hot liquid. Sixty-six patients (50 men, 16 women) completed the study. The mean age of the patients was 32.5 ± 10.6 years. The average percentage of burn was 33.0 ± 9.7%. Table 2 shows the baseline demographic and laboratory parameters of the study participants. There were no significant differences in baseline characteristics between the groups. Moreover, no significant differences were observed in terms of baseline characteristics between study completers and those who discontinued intervention prematurely.

Any changes in markers of muscle catabolism during the treatment phase are demonstrated in Table 3.

Nitrogen balance (primary outcome) improved and serum creatinine increased in the two ISP groups and they were significant compared with the control group. However, after controlling for potential covariates in ANCOVA models, changes in these indices were statistically significant compared with the control groups (P = 0.008, P = 0.005 for both nitrogen balance and serum creatinine, respectively). Changes in anthropometric outcomes over the three-week intervention period in ITT populations are also presented in Table 3. At three weeks, there were significant decreases in weight and BMI in each group (all P < 0.001). After controlling for potential covariates in ANCOVA models, we found significant differences between groups in terms of three-week changes in all anthropometric outcomes (all P ≤ 0.001). Weight loss was observed to be higher in Group C (controls) compared to Group A (ISP + FO). All of these markers are associated with muscle catabolism. However, 24-h UUN, TLC, and 24-h UUC as other muscle catabolism markers were not significant between groups (P > 0.05). There was a significant decrease in serum levels of ALT, AST, ALP, TG, cholesterol, and FBS in pairwise comparisons of all three groups but changes of these indices were not statistically significant between groups (P > 0.05) (Table 4).

Concentrations of anemia status indexes at the end of treatment phase are shown in Table 5. No significant differences were found in hemoglobin and other CBC markers in pairwise comparisons of all three groups and three-week changes of these indices between groups (P > 0.05) although these values tended to be higher in the ISP groups. These data showed that burn frequently results in acute anemia, which was seen in most patients until discharge unless modified by blood transfusion practices. There were no serious adverse events that occurred during the study due to the study starting on day 5. Clinical investigations with this patient population had to be performed for several days after admission since the peak inflammation response occurred by about the fourth day.

The study was performed from June 2014 through December 2014.