Effects of Late-evening Snacks on Nutritional Status and Metabolic Pattern of Patients With Primary Hepatocellular Carcinoma After Hepatectomy
Phase: NA
Conditions: Liver Neoplasms
Sponsor: DU Yao

Version: v2.0 
Date: December 21, 2023 
Effects of Late-evening Snacks on Nutritional 
Status and Metabolic Pattern of Patients With 
Primary Hepatocellular Carcinoma After 
Hepatectomy 
Source：Drum Tower Hospital, Medical School of Nanjing University 
Subject No.: None 
Person in charge: Xiaojie Bian, Yao Du 
Department: Pharmacy 
Contact: Xiaojie Bian, Yao Du 
TEL: 86-18061678828, 86-18351887978 
Study start date: February 2024 
Version number: V2.0 
Version date: December 22, 2023 
Produced by Drum Tower Hospital, Medical 
School of Nanjing University  

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Formula Case Abstract Summary 
Project 
name 
Effects of Late-evening Snacks on Nutritional Status and Metabolic Pattern 
of Patients with Primary Hepatocellular Carcinoma After Hepatectomy 
Research 
purpose 
To observe the effects of Late-evening Snacks on the nutritional status, 
liver function recovery, complication rate and long-term prognosis of 
hepatocellular carcinoma patients who underwent hepatectomy, and to 
further explore the effects of Late-evening Snacks on the metabolic pattern 
of the patients, and to identify the serum differential metabolites, in order to 
initially investigate the potential mechanism of Late-evening Snacks to 
improve the nutritional status of the patients, and to provide theoretical basis 
for the development of an effective nutritional regimen in the clinic. 
Research 
design 
Single-center prospective cohort study 
Total 
Number of 
Cases 
106 cases 
Case 
selection 
Inclusion Criteria: 
1. Age 18-75 years; 
2. Meet the diagnostic criteria of China's "Guidelines for Diagnosis and 
Treatment of Primary Liver Cancer (2022 Edition)", clinically diagnosed with 
primary liver cancer, hospitalized with radical hepatectomy as the main 
surgical treatment, no indication of metastasis of the tumor to extra-hepatic 
organs in preoperative tests and examinations, no absolute contraindications 
to surgery, complete resection of the liver tumor in the operation, and 
hepatocellular carcinoma confirmed by postoperative pathological diagnosis；
3. Child-Pugh grades A and B; 
4. Preoperative Eastern Cooperative Oncology Group Physical Status Score 

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(ECOG-PS) of 0 to 2; 
5. The patient is conscious, has normal verbal communication, and is able to 
cooperate with the relevant examinations; 
6. Fully informed about the study and voluntarily signed an informed consent 
form. 
Exclusion Criteria: 
1. Failure to meet selection criteria; 
2. Nutritional assessment as cachexia; 
3. Presence of contraindications to enteral nutrition (EN) or EN intolerance, 
such as acute gastrointestinal bleeding, intestinal obstruction.(≥ grade 3, 
National Cancer Institute-Common Terminology Criteria for Adverse Events 
[NCINCI-CTCAE v 5.0]); 
4. Simultaneous combination of malignant tumors in other parts of the body; 
5. Combined hepatic encephalopathy or definite infection on admission; 
6. Known refractory metabolic diseases (e.g., poorly controlled diabetes 
mellitus or fasting glucose ≥10 mmol/L, hyperthyroidism, hypothyroidism, 
metabolic acidosis);  
7. Decreased renal function (defined as serum creatinine Cr level ≥176.8 
μmol/L); 
8. Intravenous or oral nutritional supplements, such as proteins, amino acids, 
etc., applied within one month prior to admission to the hospital; 
9. Patients with severe stress or severe complications such as respiratory 
failure with severe cardiac, hepatic, renal and other insufficiencies； 
10. Persons with mental and neurological disorders who are unable to 
cooperate with a physician; 
11. Alzheimer's disease, cerebral atrophy, acute stage or sequelae of 
cerebrovascular disease, cognitive impairment; 
12. Previously poor adherence to medication and nutritional counseling; 
13. Critically ill and difficult to assess; 

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14. On the liver transplant waiting list or under consideration for liver 
transplantation, as such patients may discontinue follow-up before the end of 
the study; 
15. Less than 12 months since last localized treatment (TACE or HAIC or 
ablative therapy); 
16. Other circumstances that the researcher considers inappropriate for 
participation in the study. 
Treatment 
plan 
Patients who were admitted to the Department of Hepatobiliary 
Pancreatic Surgery of Drum Tower Hospital affiliated to Nanjing University 
School of Medicine from February 2024 to January 2025 were selected and 
screened for enrollment according to the admission criteria. The grouping was 
not randomized and blinded, and all patients were enrolled according to their 
individual wishes and divided into a test group (53 cases) and a control group 
(53 cases). The purpose of the study, the content of the investigation were 
explained to the patients or their families at the time of admission, the 
principle of confidentiality was informed to reduce the concerns of the 
respondents, and the subjects were included in the study after signing an 
informed consent form after obtaining their consent. 
1. Treatment programs 
 All patients were screened for nutritional risk within 24h of admission 
using NRS 2002, nutritional assessment using PG-SGA, and malnutrition 
diagnosis using GLIM malnutrition assessment (diagnosis) criteria. A target 
daily amount of 25-30 kcal/kg was set for the patients. Nutritional 
management was standardized to ensure a balanced dietary intake in both 
groups. Nutritional cards were issued to each patient according to his/her 
intake, and were explained and emphasized at the time of admission and at 
the time of discharge. Patients were asked to choose different food items on 
the cards according to their own food preferences and actual situation. The 
experimental group was required to have late-evening snacks (LES) within 

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1hour before bedtime, while the control group did not have any food during 
the 1hour before bedtime. 
Test group: patients will have an additional meal (total calories 200-
275kcal, protein 11.5g-18g, complex carbohydrates 25-55g) 1h before 
bedtime. The dietary regimen of the test group will be subtracted from the 
corresponding late-evening snacks intake. 
(1) Preoperative: ①Patients without nutritional risk: independent 
intake according to the issued diet program card. ②Patients with nutritional 
risk: daily oral whole-protein enteral nutrition on the basis of general diet 
after admission. 
(2) Postoperative: start drinking small amount of water on the 1st 
postoperative day, after ventilation, over to liquid diet, and on the 2nd 
postoperative day, it can be over to autonomous semi-fluid diet + daytime 
oral enteral nutrition of whole protein type + LES regimen. As the patient's 
voluntary intake meets 60% of the body's needs, the daytime oral enteral 
nutrition support can be gradually stopped and the LES regimen can be 
continued. 
3) Discharge: daily intake is required on time according to the LES 
regimen. 
    Control group: no more food 1h before bedtime. 
(1) Preoperative: same as the test group. 
(2) Postoperative: start drinking small amount of water on the 1st 
postoperative day, after ventilation, over to liquid diet, and on the 2nd 
postoperative day, it can be over to autonomous semi-fluid diet + daytime 
oral enteral nutrition of whole protein type. As the patient's voluntary intake 
meets 60% of the body's needs, the daytime oral enteral nutrition support can 
be gradually stopped and the LES regimen can be continued. 
(3) Discharge: the doctor will judge whether the patient needs to 
continue nutritional support according to his/her dietary recovery, and if so, 

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his/her supplementation must be completed before 1h before bedtime.  
2. Biological sample collection: 
The remaining post-test serum samples need to be collected for 
metabolomics analysis from patients on day 1 of admission, postoperative 
day 6, postoperative 1 month, postoperative 3 months, and postoperative 6 
months.  
    3. Clinical data collection: 
1) General information: gender, age, height, weight, BMI, ICG 15min 
retention time, name of surgery, duration of surgery, blood loss, blood 
transfusion, tumor stage, NRS2002, GLIM malnutrition assessment 
(diagnostic) criteria diagnosis. 
2) Nutritional indicators: Nutritional indicators such as serum albumin 
(ALB), prealbumin (PA), transferrin (TRF), and total serum protein (TP) 
were collected from patients on the 1st day of admission, the 6th 
postoperative day, 1 month postoperatively, 3 months postoperatively, and 6 
months postoperatively. 
(3) Liver function indexes: Liver function indexes such as alanine 
aminotransferase(ALT), alanine transaminase(AST), γ-glutamyltransferase 
(GGT), cholinesterase (CHE), and total bilirubin (TBIL) were collected on 
the 1st day of admission, the 6th postoperative day, 1 month postoperatively, 
3 months postoperatively, and 6 months postoperatively. 
(4) Clinical outcome: collect the patients' postoperative clinical outcome 
indicators, such as: postoperative complication rate, recurrence-free survival, 
and quality of survival.  
(5) Medical costs: collect the total cost of the patients' hospitalization, 
the cost of anti-infective drugs, and the cost of albumin during hospitalization. 
Evaluation 
of the 
Indicators of effectiveness evaluation 
Main efficacy indicators: serum albumin (ALB) 
Secondary 
efficacy 
indicators: 
serum 
prealbumin 
(PA), 
alanine 

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efficacy of 
treatment 
aminotransferase (ALT), azelaic transaminase (AST), cholinesterase (CHE), 
total bilirubin (TBIL), total hospitalization costs, relapse-free survival, and 
quality of life 
Safety evaluation indicators 
Incidence of gastrointestinal symptoms such as abdominal distension, 
diarrhea, nausea and vomiting, incidence of postoperative complications, and 
length of postoperative hospitalization 
Statistical 
methods 
Descriptive statistics were performed on the clinical data, and measures 
that conformed to normal distribution were expressed as mean±standard 
deviation (x±s ), and t-test was used between groups, and measures that did 
not conform to normal distribution were expressed as median (interquartile 
spacing) [M(IQR)], and Mann-Whitney U test was used between groups. 
Count data were expressed as percentages [n(%)], and the chi-square test was 
used for between-group comparisons, with P<0.05 considered statistically 
significant. The Kaplan-Meier method in STATA 12.0 was used to calculate 
the recurrence rate and recurrence-free survival in each group, and the log-
rank test was used for intergroup comparison.  
Duration of 
the study 
24 months 
 
 

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catalogs 
1. Research background ...................................................................................................................... 1 
2. Research purpose ............................................................................................................................ 4 
3. Types of Research Designs and Experimental Steps ....................................................................... 4 
4. Case selection .................................................................................................................................. 9 
5. Research Methodology and Technical Approach .......................................................................... 10 
6. Observed items and testing time points ....................................................................................... 12 
7. Criteria for evaluating the efficacy of treatment .......................................................................... 13 
8. Adverse Events Watch ................................................................................................................... 14 
9. Data security monitoring ............................................................................................................... 16 
10. Statistical processing ................................................................................................................... 16 
11. Ethical principles of clinical research ........................................................................................... 17 
12. Reference .................................................................................................................................... 17 
 

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1. Research background 
Primary liver cancer (hereinafter referred to as hepatocellular carcinoma) is one of the common 
malignant tumors of the digestive system worldwide, which can be classified into hepatocellular 
carcinoma 
(HCC), 
intrahepatic 
cholangiocarcinoma 
(ICC), 
and 
combined 
hepatocellular-
cholangiocarcinoma (cHCC-CCA) according to the different pathologic types. According to the Global 
Tumor Epidemiology Statistics 2020 (GLOBOCAN), the number of newly diagnosed cases of 
hepatocellular carcinoma amounted to 906,000, which is the 6th most common type of cancer, and the 
number of deaths amounted to 830,000, which is the 3rd most common type of cancer [1]. And the 
incidence rate of liver cancer in China is located in the 4th place, the mortality rate is located in the 2nd 
place [2], and the 5-year survival rate is 14.1% [3]. The treatments for hepatocellular carcinoma vary 
according to the stage and liver function level, including hepatic resection, liver transplantation, ablation 
therapy, transarterial embolization, radiation therapy, etc. Among them, hepatic resection is considered to 
be the most effective treatment for early-stage hepatocellular carcinoma and is the main treatment at 
present, with a 5-year survival rate of up to 69.0%~86.2% [4]. Research results show that the local 
recurrence rate of selecting surgical resection treatment modality is significantly lower than that of 
radiofrequency ablation for hepatocellular carcinoma patients with good liver function reserve, and the 
long-term efficacy is better [5,6]. 
At the same time, the liver serves as the target organ for the metabolism of most nutrients, and the 
high metabolic status and organ invasion of cancer often expose liver cancer patients to malnutrition, with 
an incidence rate as high as 74.36% according to a survey [7]. In addition, hepatic resection will cause 
hepatocyte damage to a certain extent, resulting in the metabolic balance of sugar, protein, fat and 
hormones in the patient's body being disrupted, which makes the body's catabolism stronger than 
anabolism [8], and it is more likely to have a decrease in the level of albumin synthesis and a significant 
decrease in the intake of nutrients after the operation, which in turn results in the decrease in the patient's 
ability to resist infection, the increase in the risk of complications, and the decrease in the quality of life 
and a series of other poor prognosis [9,10,11,12], therefore, nutritional support and intervention are particularly 
important in this situation. According to our previous study, we found that nutritional intervention by 
clinical pharmacists in perioperative liver cancer hepatectomy patients could reduce the complication rate 
by 69%, as well as correct the inflammatory stress response and improve albumin levels more quickly [13]. 

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Therefore, we believe that the use of rational and effective nutritional intervention strategies will bring 
positive benefits to patients. 
The 2022 Guidelines for Perioperative Care in Liver Surgery: the Accelerated Rehabilitation After 
Surgery Society (ERAS) recommends early intake after hepatic resection and oral nutritional supplements 
for malnourished patients [14]. However, for patients with abnormal liver function or cirrhosis their 
perioperative and postoperative nutritional support and nutritional support patterns lack guideline 
recommendations. Abnormal liver function and inadequate intake in postoperative patients cause patients 
to exhibit reduced hepatic glycogen reserves, enhanced gluconeogenesis and fat oxidation, and increased 
protein oxidative catabolism. To mitigate accelerated starvation and associated protein hydrolysis, the 
most critical dietary strategy is to eat meals every 4 to 6 hours with the shortest possible interval between 
meals. Therefore, the concept of Late evening snack (LES), in which a portion of food is moved to bedtime 
while the total daily intake remains unchanged, has been proposed to effectively improve the metabolic 
state of accelerated catabolism and has been recommended by the European Society of Clinical Nutrition 
and Metabolism (ESPEN) for patients with cirrhosis who have a high catabolic metabolism[15] , without 
indicating the specific nutritional composition, while the International Society for Hepatic 
Encephalopathy and Nitrogen Metabolism recommended in its consensus published in 2013[16] that LES 
should contain at least 50 g of complex carbohydrates. In most studies, various combinations of LES 
(200-275 kcal total calories, 11.5g-18g protein, and 25-55g complex carbohydrates) have been shown to 
be significant in improving liver function indices and nutritional indices [17]. Researchers have suggested 
that LES containing complex carbohydrates and proteins may reduce lipid oxidation and improve 
nutritional status, muscle mass, liver function and quality of life in patients [18,19]. 
The results of a study in an outpatient long-term late-evening snacks also confirmed a significant 
improvement in nitrogen balance compared to baseline and an increase in serum albumin levels [20]. In a 
meta-analysis published in 2018 [19], serum albumin, prealbumin, and cholinesterase levels were 
significantly elevated in patients with cirrhosis after implementation of LES. In addition, Chen et al [17] 
found that LES reduced the levels of alanine aminotransferase (ALT), alanine aminotransferase (AST), 
prothrombin time (PT), and blood ammonia in patients with liver disease, and reduced the incidence of 
ascites. With further research, Takeshita et al [21] found that LES prevented the decline of albumin levels 
in hepatocellular carcinoma patients undergoing chemoembolization therapy (TACE) and reduced hepatic 
impairment due to TACE. Meanwhile, Harima et al [22] showed that LES improved prealbumin levels, 

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BCAA/tyrosine, and ALT in patients with advanced hepatocellular carcinoma treated with hepatic artery 
perfusion chemotherapy. In addition, Morihara et al [23] found that LES also significantly improved Child-
Pugh score in hepatocellular carcinoma patients at 4 and 12 weeks after radiofrequency ablation. 
The improvement of nutritional status of LES for liver cancer patients is currently limited to the 
detection of several biochemical biomarkers and anthropometric measurements, which lacks a 
comprehensive understanding of metabolic changes. At the metabolic level, metabolomics is an important 
component of systems biology that allows the analysis of metabolic changes in the organism as a result 
of relevant factors. Pathologic changes in the liver affect the body's basal metabolism and have a more 
pronounced effect on the levels and types of small molecule metabolites, which provides a new 
perspective for applying metabolomic analysis to study the changes in metabolic patterns in patients with 
LES. In recent years, metabolomics has also been increasingly used in dietary intervention studies to 
discover new dietary biomarkers and to explore the metabolic changes associated with nutrients and 
dietary patterns, in order to evaluate the effects of dietary interventions on the disease of the organism. 
Tripodi et al [24] found that methionine supplementation in vitro could inhibit AMP-activated protein 
kinase and up-regulate the tricarboxylic acid cycle, through proteomics and metabolomics to inhibit 
hepatocellular carcinoma invasion. Rong et al [25] investigated the effect of antioxidant active substance 
corn oligopeptides on serum metabolism of hepatocellular carcinoma patients based on metabolomics 
combined with multivariate statistics. Initially, 16 differential metabolites were screened, and it was 
concluded that corn oligopeptides' protection of hepatic function was realized through nicotinic acid and 
nicotinamide metabolism and fatty acid metabolism pathways. However, it is not clear how LES affects 
the metabolic patterns of patients to improve nutritional status and liver function. As a result, 
metabolomics has the potential to make an important contribution to the study of the underlying 
mechanisms of LES to improve the nutritional status of patients and to the search for methods of 
nutritional support in malignant tumors. 
Since, nutritional support with LES for hepatocellular carcinoma patients undergoing hepatectomy 
treatment has rarely been reported, and there are no clinical studies applying metabolomics approaches to 
explore the effects of LES on their metabolic patterns. In view of this background, the present study aimed 
to optimize the management of meal timing and frequency in patients with hepatocellular carcinoma 
undergoing hepatectomy using LES, with the aim of reducing patient complications and improving 
clinical outcomes, and to further study and explore the effects of LES on patients' metabolic patterns to 

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provide a theoretical basis for implementing effective nutritional strategies in clinical practice to promote 
patient recovery. 
2. Research purpose 
2.1 Primary purpose 
To observe the effects of late-evening snacks on the nutritional status, liver function recovery, 
complication rate and long-term prognosis of hepatocellular carcinoma patients who underwent 
hepatectomy, and to further explore the effects of late-evening snacks on the metabolic pattern of the 
patients through metabolomics, and to identify the serum differential metabolites, in order to initially 
investigate the potential mechanism of late-evening snacks to improve the nutritional status of the patients, 
and to provide theoretical basis for the development of an effective nutritional regimen in the clinic.  
2.2 Secondary purpose 
The sample size can be enlarged to continue the population study, to verify and continue to optimize 
the test results obtained from the validation, to shift from a single-center study to a multi-center study, 
and to establish an expandable multi-dimensional database. In order to propose a more reasonable 
nutritional support strategy for the clinic and provide reference for doctors' clinical decision-making. 
2.3 Test hypothesis 
It was hypothesized that there would be significant differences in serum albumin, prealbumin, and 
serum metabolic patterns in subjects in the test group compared to control patients, and potential 
mechanisms by which the LES would improve the nutritional status of patients would be identified. 
3. Types of Research Designs and Experimental Steps 
3.1 Types of Research Designs 
Types of study designs: single-center, prospective cohort studies 
Research Center: Drum Tower Hospital, Medical School of Nanjing University 
Grouping method: non-randomized, open, enrolled according to patients' wishes 
Sample size calculation basis and formula:  
This study was a single-center prospective cohort study with two groups, the test group and the 
control group, and the serum albumin level of the study subjects was the primary endpoint outcome 
indicator observed. Based on the review of previous literature [28] and reference to the results of the pretest, 
it was expected that the albumin level before LES intervention was 34.2±3.9 g/L and the albumin level 

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after LES intervention was 37.5±5.4 g/L, with a two-sided α=0.05 and a degree of certainty of 1-β=90%. 
Using PASS15 software can be calculated to obtain n=88 cases. Based on the 1:1 grouping, 44 subjects 
were needed in each of the test and control groups, and assuming a 20% subject dropout rate, the final 
minimum number of subjects needed was 53 in each of the test and control groups, for a total of at least 
106 subjects included. 
3.2 Experimental Steps 
The specific steps of the study are as follows: 
 

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Pre-procedure preparation: 
According to the literature research, establish the form of patients' clinicopathological data that need 
to be collected, including basic information (age, gender, hospitalization number), pre-surgical laboratory 
examination (nutritional indexes, liver function, renal function, coagulation function, blood routine, tumor 
markers such as alpha-fetoprotein, etc.), surgical records (tumor location, size, number, intra-operative 
bleeding, etc.), post-surgical laboratory examination, tumor recurrence and treatment modality and patient 
survival, etc., as well as the establishment of a dietary matching table, respectively writing the dietary 
education content of the experimental group and the control group, in order to personalize the guidance 
of the patient's postoperative diet and the correct understanding of the importance of dietary therapy. A 
case questionnaire was set up for the enrolled patients, and the relevant data of the enrolled patients were 
recorded in the form. 
Screening and Trial: 
Patients who were admitted to the Department of Hepatobiliary Pancreatic Surgery of Drum Tower 
Hospital affiliated to Nanjing University School of Medicine, from February 2024 to January 2025 and 
met the enrollment criteria were selected for inclusion in this study. The grouping was not randomized 
and blinded, and all patients were enrolled according to their individual wishes and divided into a test 
group (53 cases) and a control group (53 cases). The purpose of the study, the content of the investigation, 
the principle of confidentiality were explained to the patients or their families at the time of their 
admission to the hospital to reduce the concerns of the respondents, and the subjects were included in the 
study after signing the informed consent form with their consent. 
1. Treatment program: 
    All patients were screened for nutritional risk using NRS 2002, nutritional assessment using PG-
SGA, and malnutrition diagnosis using GLIM Malnutrition Assessment (Diagnosis) Criteria within 24h 
of admission. In accordance with the recommendations of ESPEN 2021 Practice Guidelines for Surgical 
Nutrition Therapy and CSPEN 2016 Guidelines for Perioperative Nutritional Support for Adults, the 
patients' daily target amount was set at 25-30 kcal/kg.In order to ensure the balanced dietary nutritional 
intake of the two groups, their standardized management was formulated as follows:① Total calories: 
according to the patients' height, weight, age and activity Total calories: Calculate the daily intake 
according to the patient's height, weight, age and activity intensity, about 1200~2000 kcal. ② 
Carbohydrates: account for 60%~65% of the actual energy supply, calculate the actual supply of 180~325 

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g. The staple food is mainly cereals and potatoes. Protein: 1.2~1.5 g/kg/day according to the guideline, 
choose high quality protein, such as eggs, poultry, soybean products. ④Fat: account for 20% of the actual 
energy supply, calculate the actual supply of about 27~44 g. Distribute the energy of three meals as 30%, 
40%, 30%. According to each patient's required intake, the corresponding nutritional program card is 
issued, and explained and emphasized at the time of admission and discharge, respectively. Patients could 
choose different food items on the cards according to their food preferences and actual situation. The 
experimental group was required to consume LES 1 h before bedtime, while the control group did not 
consume any food 1 h before bedtime. 
Experimental group: patients were given additional meal (total calories 200-275kcal, protein 
11.5g-18g, complex carbohydrates 25-55g) 1h before bedtime. the LES regimen was: ① whole protein 
type enteral nutrition; ② whey protein powder + bread or cookie. The addition of meals was carried out 
1h before bedtime. The dietary regimen of the experimental group will be deducted from the 
corresponding late-evening snacks intake. 
(1) Preoperative: ① Patients without nutritional risk: independent intake according to the issued 
diet program card. ② Patients with nutritional risk: daily oral whole-protein enteral nutrition on the basis 
of general diet after admission. 
(2) Postoperative: start drinking small amount of water on the 1st postoperative day, over to liquid 
diet after ventilation, and may over to autonomous semi-liquid diet + oral whole protein enteral nutrition 
during the day + LES program on the 2nd postoperative day. As the patient's voluntary intake meets 60% 
of the body's needs, the daytime oral enteral nutrition support can be gradually stopped and the LES 
program can be continued. 
3) Discharge: daily intake needs to be on time according to the LES regimen. 
Control group: no more food 1h before bedtime. 
(1) Preoperative: same as the test group. 
(2) Postoperative: start drinking small amount of water on the 1st postoperative day, over to liquid 
diet after ventilation, and can over to autonomous semi-liquid diet + oral whole protein type enteral 
nutrition during the day on the 2nd postoperative day. As the patient's voluntary intake meets 60% of the 
body's needs, enteral nutrition support can be gradually stopped. 
(3) Discharge: Doctors judge whether the patients need to continue nutritional support according to 
their dietary recovery, and if they do, their nutritional supplementation should be completed before 1h 

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before bedtime. 
2. biological sample collection:  
Serum samples remaining after testing were collected from patients for metabolomics analysis. 
3.Clinical information collection: 
(1) General information collection: gender, age, height, weight, BMI, ICG15min retention time, 
name of surgery, duration of surgery, blood loss, blood transfusion, tumor stage, NRS2002, PG-SGA, 
GLIM malnutrition assessment (diagnosis) criteria diagnosis; 
(2) Nutritional indicators collection: collect nutritional indicators such as serum albumin (ALB), 
prealbumin (PA), transferrin (TRF), and total serum protein (TP) on the 1st day of the subject's admission 
to the hospital, on the 6th day of the postoperative period, at 1 month after the operation, at 3 months after 
the operation, and at 6 months after the operation. 
(3) Collection of liver function indexes: Liver function indexes such as alanine aminotransferase 
(ALT), alanine grass aminotransferase (AST), γ-glutamyltransferase (GGT), cholinesterase (CHE), and 
total bilirubin (TBIL) were collected on the first day of hospitalization, the sixth postoperative day, the 
first postoperative month, the third postoperative month and the sixth postoperative month of the subjects. 
(4) Clinical outcome collection: to collect clinical outcome indicators of subjects after surgery, such 
as: postoperative complication rate, recurrence rate, recurrence-free survival, quality of life. 
5) Medical cost collection: to collect subjects' total hospitalization cost, cost of anti-infective drugs, 
cost of albumin during hospitalization. 
Data analysis: 
    Descriptive statistics were performed on the clinical data, and the measures that conformed to normal 
distribution were expressed as mean ± standard deviation (x±s ), and the t-test was used between the 
groups, and the measures that did not conform to normal distribution were expressed as median 
(interquartile spacing) [M(IQR)], and the Mann-Whitney U test was used between the groups. Count data 
were expressed as percentages [n(%)], and the chi-square test was used for between-group comparisons, 
with P<0.05 considered statistically significant. The Kaplan-Meier method in STATA 12.0 was used to 
calculate the recurrence rate and recurrence-free survival in each group, and the log-rank test was used 
for intergroup comparison. 

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4. Case selection 
4.1 Number of cases 
A total of 106 patients needed to be enrolled. 
4.2. Inclusion Criteria 
1) Age 18-75 years; 
2) Meet the diagnostic criteria of China's "Guidelines for Diagnosis and Treatment of Primary Liver 
Cancer (2022 Edition)", clinically diagnosed with primary liver cancer, hospitalized with radical 
hepatectomy as the main surgical treatment, no indication of metastasis of the tumor to extra-hepatic 
organs in preoperative tests and examinations, no absolute contraindications to surgery, complete 
resection of the liver tumor in the operation, and hepatocellular carcinoma confirmed by postoperative 
pathological diagnosis；3. Child-Pugh grades A and B; 
4) Preoperative Eastern Cooperative Oncology Group Physical Status Score (ECOG-PS) of 0 to 2; 
5) The patient is conscious, has normal verbal communication, and is able to cooperate with the 
relevant examinations; 
6) Fully informed about the study and voluntarily signed an informed consent form. 
4.3 Exclusion Criteria 
1) Failure to meet selection criteria; 
2) Nutritional assessment as cachexia; 
3) Presence of contraindications to enteral nutrition (EN) or EN intolerance, such as acute 
gastrointestinal bleeding, intestinal obstruction. (≥grade 3, National Cancer Institute-Common 
Terminology Criteria for Adverse Events [NCINCI-CTCAE v 5.0]); 
4) Simultaneous combination of malignant tumors in other parts of the body; 
5) Combined hepatic encephalopathy or definite infection on admission; 
6) Known refractory metabolic diseases (e.g., poorly controlled diabetes mellitus or fasting glucose 
≥10 mmol/L, hyperthyroidism, hypothyroidism, metabolic acidosis);  
7) Decreased renal function (defined as serum creatinine Cr level ≥176.8 μmol/L); 
8) Intravenous or oral nutritional supplements, such as proteins, amino acids, etc., applied within one 
month prior to admission to the hospital; 
9) Patients with severe stress or severe complications such as respiratory failure with severe cardiac, 

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hepatic, renal and other insufficiencies； 
10) Persons with mental and neurological disorders who are unable to cooperate with a physician; 
11) Alzheimer's disease, cerebral atrophy, acute stage or sequelae of cerebrovascular disease, 
cognitive impairment; 
12) Previously poor adherence to medication and nutritional counseling; 
13) Critically ill and difficult to assess; 
14) On the liver transplant waiting list or under consideration for liver transplantation, as such 
patients may discontinue follow-up before the end of the study; 
15) Less than 12 months since last localized treatment (TACE or HAIC or ablative therapy); 
16) Other circumstances that the researcher considers inappropriate for participation in the study. 
4.4 Exclusion criteria 
Enrolled cases but meeting one of the following should be excluded:  
1) Intraoperative exploration of the tumor has undergone dissemination or is unresectable; 
2) Serious intraoperative or postoperative complications of surgical technique within 24h: bleeding 
or gastrointestinal leakage leading to shock; 
3) Serious cardiac, pulmonary, cerebral or renal vital organ accidents or insufficiency during the 
operation or within 24h after the operation; 
4) Postoperative pathology suggesting non-primary or metastatic hepatocellular carcinoma; 
5) Patients in the control group who had more than 7 additional meals before bedtime; 
6) Patients in the test group who did not have more than 7 bedtime refills during the follow-up period; 
7) Those who voluntarily withdrew. 
5. Research Methodology and Technical Approach 
5.1 Research Methodology 
1) Nutritional Assessment 
All patients were screened for nutritional risk using the NRS 2002 scale within 24 h of admission, 
and a score of ≥3 was considered to be at nutritional risk; patients at nutritional risk were assessed for 
nutrition using the PG-SGA scale and diagnosed with malnutrition according to the GLIM Malnutrition 
Assessment (Diagnosis) Criteria. 
2) Nutritional support therapy 

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The target intake for all patients was 25-30 kcal/kg/d. Nutritional supportive therapy was given to 
patients who were at nutritional risk and could not achieve the target intake. Oral nutritional supplements 
were preferred, and enteral nutritional supplementation was used in patients who still could not achieve 
60% of the target amount. Supplemental parenteral nutrition was considered if patients could not achieve 
60% of the target amount for 7 days after admission to the hospital.  
3) Interventions 
From the total daily intake, patients in the test group were divided into approximately 250kcal for late-
evening snacks 1h before bedtime (200-275kcal total, 11.5g-18g protein, 25-55g complex carbohydrates); 
patients in the control group did not eat any more food 1h before bedtime. 
Preparations used: 
① Enteral nutrition powder: 450kcal/15.9g/100g; 
② Whole protein type enteral nutrition suspension, energy density 1kcal/ml, protein 0.05g/ml, complex 
carbohydrate 0.126g/ml; 
③ Whey protein powder, protein content >80g/100g. 
4) Biological sample collection 
   Post-test residual serum samples from patients on day 1 of admission, day 6 postoperatively, 1 month 
postoperatively, 3 months postoperatively, and 6 months postoperatively were collected for metabolomic 
analysis to see if the metabolic pattern of the patients was altered in LES mode. 
5) Clinical outcomes 
The postoperative complication rate, tumor recurrence rate, recurrence-free survival, postoperative 
hospitalization time, total hospitalization cost, and cost of anti-infective drugs of the subjects after 
admission to the hospital were counted, and the chi-square test and independent samples t-test were 
performed to compare the differences, in order to find out whether the clinical outcomes had been 
improved under the LES model. 
5.2 Technical Approach 

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6. Observed items and testing time points 
6.1 
Intake 
Daily intake and time of intake were recorded during the perioperative period, and patients were 
followed up at 1 month postoperatively, 3 months postoperatively, and 6 months postoperatively for intake 
and time of intake.  
6.2 
Nutrition indicators 
Serum albumin (ALB), prealbumin (PA), total serum protein (TP), and transferrin were collected 
from subjects on day 1 of admission, day 6 postoperatively, 1 month postoperatively, 3 months 
postoperatively, and 6 months postoperatively. 
6.3 
Liver function indicators 
The patients' alanine aminotransferase (ALT), alanine transaminase (AST), γ-glutamyl transferase 
(GGT), cholinesterase (CHE), and total bilirubin (TBIL) were collected on the 1st day of the patients' 

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admission, on the 6th day of the postoperative period, at 1 month postoperatively, at 3 months 
postoperatively, and at 6 months postoperatively. 
6.4 
Medical costs 
After the patients were discharged from the hospital, the patients' postoperative length of stay, total 
hospitalization costs, and drug costs were recorded; 
6.5 
Clinical outcomes 
The time of occurrence of postoperative complications and symptoms of the subjects were recorded 
daily during the patients' hospitalization, and the recurrence-free survival and quality of survival were 
followed up at 1 month, 3 months, and 6 months postoperatively. 
6.6 
Metabolomics testing 
Post-test residual serum samples were collected from subjects on day 1 after admission, day 6 
postoperatively, 1 month postoperatively, 3 months postoperatively, and 6 months postoperatively for 
metabolomics testing. 
7. Criteria for evaluating the efficacy of treatment 
7.1 
Nutritional indicators 
Primary Efficacy Indicator: 
Serum albumin (ALB), recording ALB data from patient biochemical tests. 
Secondary efficacy indicators: 
Prealbumin (PA), alanine aminotransferase (ALT), glutamate aminotransferase (AST), serum 
gamma-glutamyl transpeptidase (GGT), cholinesterase (CHE), total bilirubin (TBIL), and C-reactive 
protein (CRP) were recorded, and the corresponding data in the patient's biochemical tests were recorded; 
7.2 
Clinical outcomes 
1) Post-operative complications 
a) 
Postoperative infections: surgical site infections (SSI), remote infections (RI) and bacteremia. 
SSI: Shallow/deep incision SSI and organ/space SSI were defined as redness, swelling, heat, pain, 
and purulent discharge observed in any incision or space operated on intraoperatively, or the need for a 
new drain within 30 days postoperatively. 
Distant infections: including cholangitis, enteritis, pneumonia, catheter-related infections, and 
urinary tract infections, defined as fever greater than 38.5°C, leukocytosis, and bacteria detected in 

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sputum, catheter tips, and urine and feces. 
Bacteremia: Bacteremia is diagnosed when a strain is cultured from a single blood culture. And when 
the strain is identified as coagulase-negative staphylococci, it is considered contaminating; coagulase-
negative staphylococci may be considered the causative agent when isolated from two or more blood 
cultures. 
b) Bile leakage 
Fluid with a bilirubin concentration of at least three times the serum bilirubin concentration drained 
through an abdominal drain on or after postoperative day 3. Regardless of the initial bilirubin 
concentration of the drain placed through surgery, the need for radiologic (i.e., interventional drainage) 
or surgical intervention (i.e., relative to cesarean section) for biliary collection or biliary peritonitis is also 
part of the diagnosis of bile leakage. 
c) 
Liver failure after hepatectomy 
Elevated serum bilirubin concentration and elevated INR on or after postoperative day 5 compared 
to the previous day; or coagulation factors such as fresh frozen plasma (FFP) are required to maintain 
normal INR values on or after postoperative day 5 in combination with hyperbilirubinemia. 
2) Total hospitalization cost: the total cost of the patient from admission to discharge; 
3) Recurrence-free survival: the time from the start of the patient's hepatectomy to the first detection of 
tumor recurrence; 
4) Quality of Survival: Quality of Survival was evaluated by the EORTC QLQ - HCC18 scale 
(Appendix 1); 
5) Safety, diarrhea, vomiting: complaints of gastrointestinal distress that occur after the patient has 
initiated nutritional support therapy, with diarrhea defined as unformed stools more than three times 
per day. 
8. Adverse Events Watch 
Adverse events (AEs), all adverse medical events that occur after a subject receives a late-night 
snack, which can be manifested as symptoms signs, illnesses, or abnormal laboratory tests, but are not 
necessarily causally related to the late-night snacks. 
Serious Adverse Event (SAE), an adverse medical event such as death, life-threatening, permanent 
or severe disability or loss of function, subject requiring hospitalization or prolonged hospitalization, and 

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congenital anomalies or birth defects that occur after a subject receives late-evening snacks. 
The investigator will closely observe or follow up the various reactions of the patients after 
performing the LES in order to detect adverse events or serious adverse events in a timely manner, and 
all adverse events such as bloating, diarrhea, etc., reported by the subjects or observed by the investigator 
during the period from enrollment to the end of the study or early withdrawal from the study must be 
documented in the medical record or CRF, including laboratory and ancillary investigations, and should 
include, at a minimum, the name of the AE, its occurrence time (to the nearest minute), end time (to the 
nearest minute), degree and frequency of episodes, whether or not treatment was corrected, the course of 
treatment and regression if treatment was documented, and the causal relationship to the study. All 
medical documentation related to the adverse event must be recorded in the original documentation, 
including copies of requests for laboratory tests and reports of test results. The name of the investigator 
must be signed and dated (to the nearest minute) on the original documents and written reports. 
Changes in the subject's vital signs, physical examination, clinical manifestations, and laboratory 
tests should be evaluated during the study. At the time of signing the informed consent form, the name 
and telephone number of an investigator whom the subject can contact in case of an emergency, or report 
of any medical symptom, or occurrence of an AE related to the subject must be provided. The investigator 
should follow up all adverse events and serious adverse events, decide the duration of follow-up according 
to the condition, and give necessary treatment and therapeutic measures during follow-up until proper 
resolution or stabilization of the condition, and in case of abnormal laboratory tests should be followed 
up until the return visit is normal to ensure that the damage to the subject is minimized. Detailed records 
of the follow-up visits and treatment results should be kept. 
Toxic reactions or AEs that existed prior to the subject's participation in the clinical study were 
recorded as AEs only if the grading was found to have increased by one or more grades from the baseline 
assessment during the study. 
CTCAE 
classification 
equivalent to 
definition 
Level 1 
mildly 
Mild discomfort that does not affect daily life or 
functioning 
Level 2 
moderately 
Unwell, interfering with daily life and function, 

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no indication for treatment 
Level 3 
severe 
Symptomatic, affecting daily life and function, 
with indication for treatment 
Level 4 
Life 
threatening/disability/loss 
of function 
Life-threatening conditions, indications for 
emergency treatment, physically or mentally 
handicapped conditions 
Level 5 
death 
Adverse events leading to death. 
AE and study correlation analysis table 
 
Definitely Likely 
Possible Suspicious unlikely 
Reasonable timing with 
medications/interventions 
+ 
+ 
+ 
+ 
- 
Known types of drugs/reactions 
+ 
+ 
+ 
+ 
- 
Reduction or disappearance of symptoms 
with cessation of intervention 
+ 
+ 
± 
± 
- 
Response recurs after another intervention 
+ 
？ 
？ 
？ 
- 
Cannot be explained by disease, co-
morbidities 
+ 
+ 
- 
± 
- 
9. Data security monitoring 
A data safety monitoring program will be established for clinical studies according to the level of 
risk. All adverse events will be recorded in detail, appropriately handled and followed up until properly 
resolved or stabilized, and serious adverse events and unintended events, etc., will be reported to the 
Ethics Committee, the competent authority, the sponsor, and the drug regulatory authority in a timely 
manner according to the regulations; the principal investigator will conduct a cumulative review of all 
adverse events on a regular basis, and convene a meeting of the investigators to assess the risks and 
benefits of the study, if necessary; the injuries or discomfort is no more likely and no greater in magnitude 
than that encountered in daily life or than that of a routine examination, and that data and safety 
monitoring can be accomplished by the principal investigator. 
10. Statistical processing 
 应用SPSS26.0 统计软件对临床数据进行描述性统计，符合正态分布的计量资料以均数±标

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准差(x±s ) )表示，组间采用t 检验，不符合正态分布的计量资料以中位数（四分位间距）[M(IQR)]
表示，组间采用Mann-Whitney U 检验。计数资料用百分比[n(%)]表示，组间比较采用卡方检验，
P<0.05 为有显著性统计学意义。采用STATA12.0 中的 Kaplan-Meier 法计算各组复发率及无复发
生存期，log-rank 检验进行组间比较。SPSS26.0 statistical software was applied to carry out descriptive 
statistics of the clinical data, and the measurements that conformed to normal distribution were expressed 
as mean ± standard deviation (x±s )), and t-test was used between the groups, and the measurements that 
did not conform to normal distribution were expressed as median (inter-quartile spacing) [M(IQR)], and 
Mann-Whitney U-test was used between the groups. Count data were expressed as percentages [n(%)], 
and the chi-square test was used for between-group comparisons, with P<0.05 considered statistically 
significant. The Kaplan-Meier method in STATA 12.0 was used to calculate the recurrence rate and 
recurrence-free survival in each group, and the log-rank test was used for between-group comparisons. 
11. Ethical principles of clinical research 
The clinical study will follow the relevant regulations such as the Declaration of Helsinki of the 
World Medical Assembly. The clinical study will be implemented only after approval of the trial protocol 
by the Ethics Committee prior to the commencement of the study. Before each subject is enrolled in the 
study, it is the responsibility of the investigator to provide the subject or his/her agent with a complete 
and comprehensive description of the purpose, procedures, and possible risks of the study, and to sign a 
written informed consent form, which should let subjects know that they have the right to withdraw from 
the study at any time, and that the informed consent should be retained as a clinical study document for 
review. Subjects' privacy and data confidentiality will be protected during the study. 
12. Reference 
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Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209-
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[2] Rongshou Zheng, Siwei Zhang, Kexin Sun et al. Analysis of malignant tumor prevalence in China 
in 2016[J]. Chinese Journal of Oncology, 2023; 45(3):212-220. 
[3] Allemani C, Matsuda T, Di Carlo V, et al. Global surveillance of trends in cancer survival 2000-14 
(CONCORD-3): analysis of individual records for 37 513 025 patients diagnosed with one of 18 cancers 
from 322 population-based registries in 71 countries. Lancet. 2018;391(10125):1023-1075.  

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[4] Tsilimigras DI, Bagante F, Sahara K, et al. Prognosis After Resection of Barcelona Clinic Liver 
Cancer (BCLC) Stage 0, A, and B Hepatocellular Carcinoma: A Comprehensive Assessment of the 
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Unresectable Hepatocellular Carcinoma. J Clin Oncol. 2020;38(26):2960-2970.  
[6] Liu PH, Hsu CY, Hsia CY, et al. Surgical Resection Versus Radiofrequency Ablation for Single 
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Nutrition,2015;2(03):73-76. 
[8] Min Weng, Zhengyan Dai, Kunhua Wang. Nutritional therapy for hepatocellular carcinoma patients 
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complications following hepatectomy in patients with hepatocellular carcinoma. HPB (Oxford). 
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[12] Puri P, Dhiman RK, Taneja S, et al. Nutrition in Chronic Liver Disease: Consensus Statement of the 
Indian National Association for Study of the Liver. J Clin Exp Hepatol. 2021;11(1):97-143. 
[13] Dayu Chen, Xiaojie Bian, Jinchun Chen et al. A study of standardized nutritional drug therapy 
service model in the perioperative period of liver resection for hepatocellular carcinoma[J]. Pharmacy 
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[14] Joliat GR, Kobayashi K, Hasegawa K, et al. Guidelines for Perioperative Care for Liver Surgery: 
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[15] Plauth M, Bernal W, Dasarathy S, et al. ESPEN guideline on clinical nutrition in liver disease. Clin 
Nutr. 2019;38(2):485-521. 
[16] Amodio P, Bemeur C, Butterworth R, et al. The nutritional management of hepatic encephalopathy 
in patients with cirrhosis: International Society for Hepatic Encephalopathy and Nitrogen Metabolism 
Consensus. Hepatology. 2013;58(1):325-336. 
[17] Leoni L, Valoriani F, Barbieri R, et al. Unlocking the Power of Late-Evening Snacks: Practical 
Ready-to-Prescribe Chart Menu for Patients with Cirrhosis. Nutrients. 2023;15(15):3471. 
[18] Guo YJ, Tian ZB, Jiang N, et al. Effects of Late Evening Snack on Cirrhotic Patients: A Systematic 
Review and Meta-Analysis. Gastroenterol Res Pract. 2018;2018:9189062. 
[19] Chen CJ, Wang LC, Kuo HT, et al. Significant effects of late evening snack on liver functions in 
patients with liver cirrhosis: A meta-analysis of randomized controlled trials. J Gastroenterol Hepatol. 
2019;34(7):1143-1152. 
[20] Nakaya Y, Okita K, Suzuki K, et al. BCAA-enriched snack improves nutritional state of cirrhosis. 
Nutrition. 2007;23(2):113-120. 
[21] Takeshita S, Ichikawa T, Nakao K, et al. A snack enriched with oral branched-chain amino acids 
prevents a fall in albumin in patients with liver cirrhosis undergoing chemoembolization for 
hepatocellular carcinoma. Nutr Res. 2009;29(2):89-93. 
[22] Harima Y, Yamasaki T, Hamabe S, et al. Effect of a late evening snack using branched-chain amino 
acid-enriched nutrients in patients undergoing hepatic arterial infusion chemotherapy for advanced 
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[23] Morihara D, Iwata K, Hanano T, et al. Late-evening snack with branched-chain amino acids 
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[24] Tripodi F, Badone B, Vescovi M, et al. Methionine Supplementation Affects Metabolism and 
Reduces Tumor Aggressiveness in Liver Cancer Cells. Cells. 2020;9(11):2491. 
[25] Rong W, Xia H, Zhang K, et al. Serum metabolic effects of corn oligopeptides with 7-day 
supplementation on early post-surgery primary liver cancer patients: a double-blind randomized 
controlled trial. Hepatobiliary Surg Nutr. 2022;11(6):834-847. 
[26] Koreeda C, Seki T, Okazaki K, et al. Effects of late evening snack including branched-chain amino 
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2011;41(5):417-422. 
[27] Weimann A, Braga M, Carli F, et al. ESPEN practical guideline: Clinical nutrition in surgery. Clin 
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nutritional support in adults[J]. Chinese Journal of Surgery,2016;54(9):641-657. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Program signature page 
Investigator Statement: 
I agree to comply with the review of the Ethics Committee and to start the execution of the 
clinical trial after approval, to report any changes in the clinical trial activities and 
unanticipated problems involving risks to the subjects or others to the Ethics Committee in a 
timely manner, and to execute the study after reapproval of the ethical review. Comply with the 
requirements of the Ethics Committee for follow-up review and end-of-study review during the 
study. 
I agree to conduct the clinical trial in strict accordance with the design and specifications of the 
protocol. 
I understand that I may interrupt or terminate this clinical trial at any time if it is necessary to 
ensure the best interests of the subjects.  
I agree that I will personally perform or supervise this clinical trial and that I will ensure that all 
investigators in my organization assisting me in the performance of this clinical trial 
understand their responsibilities in this clinical trial. 
I will strictly adhere to current GCP and the Declaration of Helsinki during the conduct of this 
clinical trial. I am committed to the moral, ethical and scientific principles of the trial. 
 During the execution of the clinical trial, I will strictly comply with all laws and regulations 
related to the clinical trial and protect the rights and interests of patients. 
I agree to maintain adequate and accurate medical records and to ensure that these medical 
records are readily available for audit and inspection in accordance with relevant laws and 
regulations. 
 
 
 
 ______________       ________________   ___________________ 
Name (in block letters)       Signature               Date   
  
 
 
 

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Appendix 1