An Evaluation of the Protein Requirements of Japanese Young Men by Consuming Two Types of Dairy Proteins Using the Indicator Amino Acid Oxidation Method.

The indicator amino acid oxidation method is a relatively new method for determining protein requirements. Our hypothesis was that the protein requirement of the casein-whey protein mixture (70% casein and 30% whey protein) was lower than the protein requirement of plain casein, because casein and whey proteins compensate for the lack of the first-limiting amino acids. The optimal mixing ratio was determined based on the amino acid scoring pattern which is used to calculate the digestible indispensable amino acid score. In this study, digestibility was not considered to determine the optimal mixing ratio because dairy protein is a good source of digestible protein. This study aimed to evaluate the protein requirements of Japanese young men by consuming casein and casein-whey protein mixture. Ten healthy young men (22±0.2 y old) participated in 12 experiments according to a graded protein intake (0.5, 0.7, 0.9, 1.0, 1.2, 1.4 g/kg/d) of casein and casein-whey protein mixture. The mean protein requirement was calculated as the breakpoint of breath 13CO2 enrichment using change-point regression models. The mean protein requirements of Japanese young men by consuming casein and casein-whey protein mixture were estimated to be 1.00 g/kg/d and 0.90 g/kg/d, respectively. These estimated requirements were consistent with the protein quality expected from the amount of the first-limiting amino acids. The indicator amino acid oxidation method may be useful to evaluate protein quality.

Genetic influence of cytokine polymorphisms on the clinical outcome of Japanese gastrointestinal cancer patients in palliative care.

Gastrointestinal cancer is one of the most common causes of mortality globally. The present study examined the influence of cytokine genetic polymorphisms [interleukin (IL)-1B C-31T, IL-1RN VNTR, IL-6 C-634G, IL-8 T-251A, IL-10 T-819C and IL-10 A-1082G] on clinical outcomes in patients with gastrointestinal cancer in palliative care. A total of 59 patients with gastrointestinal cancer who were admitted to Iga City General Hospital were analyzed. Genotyping was conducted using a polymerase chain reaction with confronting two-pair primers. Patients with at least one IL-1RN 2 allele demonstrated a significantly better survival (P=0.0275) while those with IL-6-634 G/G demonstrated a worse survival (P=0.0024). Multivariate analyses using the Cox proportional hazard model revealed that those with at least one IL-1RN 2 allele, IL-6-634 G/G or IL-10-1082 A/G had a significantly elevated adjusted hazard ratio of 9.20 (P=0.014), 41.01 (P=0.001) or 6.49 (P=0.046), respectively, compared with those with each homozygous wild-type polymorphism. In addition, the evaluation of weight loss by genotype revealed the potential influence of IL-10 T-819C genotype (P=0.072). IL-1RN, IL-6 and IL-10 polymorphisms were associated with the survival of patients with gastrointestinal cancer, suggesting the clinical feasibility of genetic testing in patients with gastrointestinal cancer in palliative care.

Polymorphisms in folic acid metabolism genes do not associate with cancer cachexia in Japanese gastrointestinal patients.

We used clinical data from Iga General Hospital to examine the association between polymorphisms in MTR (methionine synthase) A2756G (rs1805087), MTRR (methionine synthase reductase) His595Tyr (rs10380), MTHFR (methylenetetrahydrofolate reductase) C677T (rs1801133), MTHFR A1298C (rs1801131) and SHMT (serine hydroxymethyltransferase) C1420T (rs1979277), which are genes involved in folate metabolism, and the risk of weight loss in patients with gastrointestinal cancers, with the aim of establishing personalized palliative care for each patient based on genetic information. The data from 59 patients (37 males and 22 females) with gastrointestinal cancers who visited the outpatient clinic for cancer chemotherapy and palliative care at Iga General Hospital from December 2011 to August 2015 were analyzed. There was no significant association between the single nucleotide polymorphisms (SNPs) in the folate metabolizing genes examined and weight loss defined as weight loss of more than 5 percent or more than 10 percent during the first 6 months after initiation of chemotherapy. We did not detect any significant association between any of the SNPs examined and overall survival of patients. The present study indicated that these SNPs have relatively limited or no roles in the genesis of cachexia in patients with gastrointestinal cancers; however, further investigations into the roles of these folate metabolizing genes in the context of cancer palliative care, from clinical, biological and epidemiological viewpoints are warranted.

Intra-embryo Gene Cassette Knockin by CRISPR/Cas9-Mediated Genome Editing with Adeno-Associated Viral Vector.

Intra-embryo genome editing by CRISPR/Cas9 enables easy generation of gene-modified animals by non-homologous end joining (NHEJ)-mediated frameshift mutations or homology-directed repair (HDR)-mediated point mutations. However, large modifications, such as gene replacement or gene fusions, are still difficult to introduce in embryos without costly micromanipulators. Moreover, micromanipulation techniques for intra-embryo genome editing have been established in only a small set of animals. To overcome these issues, we developed a method of large-fragment DNA knockin without micromanipulation. In this study, we successfully delivered the knockin donor DNA into zygotes by adeno-associated virus (AAV) without removing the zona pellucida, and we succeeded in both large-DNA fragment knockin and whole exon exchange with electroporation of CRISPR/Cas9 ribonucleoprotein. By this method, we can exchange large DNA fragments conveniently in various animal species without micromanipulation.

Fish oil-enriched nutrition combined with systemic chemotherapy for gastrointestinal cancer patients with cancer cachexia.

Despite recent advances in chemotherapy for gastrointestinal cancer, a crucial factor related to poor prognosis is reduced tolerance to chemotherapy induced by cancer cachexia. Fish oil (FO)-derived eicosapentaenoic acid (EPA) modulates inflammation in patients with various malignancies; however, the impact of FO-enriched nutrition as a combined modality therapy on clinical outcomes remains controversial. We systemically analysed chronological changes in biochemical and physiological status using bioelectrical impedance analysis in 128 gastrointestinal cancer patients provided with or without FO-enriched nutrition during chemotherapy. Furthermore, we evaluated the clinical significance of FO-enriched nutrition and clarified appropriate patient groups that receive prognostic benefits from FO-enriched nutrition during treatment of gastrointestinal cancer. The control group showed significant up-regulation of serum CRP) levels and no significant difference in both skeletal muscle mass and lean body mass. In contrast, the FO-enriched nutrition group showed no changes in serum CRP concentration and significantly increased skeletal muscle mass and lean body mass over time. Furthermore, high CRP levels significantly correlated with reduced tolerance to chemotherapy, and FO-enriched nutrition improved chemotherapy tolerance and prognosis, particularly in gastrointestinal cancer patients with a modified Glasgow prognostic score (mGPS) of 1 or 2. We conclude that FO-enriched nutrition may improve the prognosis of patients with cancer cachexia and systemic inflammation (i.e., those with a mGPS of 1 or 2).

A Simple Evaluation Method for the Quality of Dietary Protein in Rats Using an Indicator Amino Acid Oxidation Technique.

We demonstrated that the indicator amino acid oxidation (IAAO) method could be employed for the evaluation of quality of dietary protein by comparing the protein intakes required to meet metabolic demand in rats fed different proteins. The objective of this study was to validate a simple evaluation method for determining the quality of dietary protein using the IAAO technique. Male Sprague-Dawley rats (5-6 wk old) were fed meals composed of graded protein, using either casein, wheat gluten (WG), soy protein isolate (SPI), or egg white protein (EW), every 3 h from 09:00 to 18:00. Administration of L-[1-(13)C]phenylalanine was performed hourly from 15:00 to 18:00. The (13)CO2 level in breath CO2 was measured at 18:30. The protein intake values required to meet the metabolic demand based on the breath (13)CO2 data for the dietary casein, WG, SPI, and EW intake were 18.0, 22.2, 17.5, and 10.1 g/kg BW/d, respectively. The breath (13)CO2 concentrations corresponding to the protein intake of 7.5 g/kg BW/d for casein, WG, SPI, and EW were 9.8, 10.9, 10.3, and 8.9 (‰)/100 g BW, respectively. A significant correlation was demonstrated between the protein intake required to meet the metabolic demands and the (13)CO2 concentration in the breath for a protein intake of 7.5 g/kg BW/d (r=0.967; p<0.05). These results demonstrated that the protein intake required to meet metabolic demand could be estimated and that the quality of the dietary protein could be evaluated using the (13)CO2 concentration in the breath with a protein intake of 7.5 g/kg BW/d.

Supplementation with branched-chain amino acids attenuates hepatic apoptosis in rats with chronic liver disease.

Branched-chain amino acids (BCAA) can function as pharmacologic nutrients for patients with decompensated cirrhosis. However, the effects of BCAA at the early stage of chronic liver disease are not clear. We hypothesized that early BCAA supplementation would attenuate the progression of chronic liver disease. The present study examined the effects of BCAA supplementation on the progression of chronic liver disease in rats caused by injected carbon tetrachloride (CCl4). Sprague-Dawley rats were fed with a casein diet (control group) or the same diet supplemented with BCAA (BCAA group) for 11 weeks, and all rats were repeatedly injected with CCl4. Food intake did not significantly differ between control and BCAA groups during the experimental period. Plasma alanine aminotransferase activities gradually increased during the experimental period in both groups but peaked later in the BCAA group. Liver fibrosis was more evident in the control group. Levels of connective tissue growth factor messenger RNA were significantly lower in the livers of rats in the BCAA group than in the control group. Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end labeling assays found considerably more hepatic apoptosis in the control group. Liver cytosolic cytochrome c levels and expression of the proapoptotic Bax protein in the mitochondrial fraction were significantly lower in the BCAA group than in the control group. These results suggest that supplementation with BCAA delays the progression of chronic liver disease caused by CCl4 in rats by attenuating hepatic apoptosis.

Effect of branched-chain amino acid supplementation on the oxidized/reduced state of plasma albumin in rats with chronic liver disease.

We examined whether continuous supplementation with branched-chain amino acids phosphorylates ribosomal protein S6, a downstream effector of mammalian target of rapamycin, and improves hypoalbuminemia of rats with chronic liver disease. Sprague-Dawley rats were fed a casein diet (control group) or a branched-chain amino acid-supplemented casein diet (branched-chain amino acid group) for 11 weeks with repeated injections of carbon tetrachloride. Throughout this experimental period, no significant difference in plasma albumin concentration was seen between groups. The percentage of reduced albumin within total plasma albumin gradually decreased in both control and branched-chain amino acid groups. After 11 weeks with supplementation, phosphorylation of ribosomal protein S6 was significantly increased in the liver of rats in the branched-chain amino acid group compared with the control group. Furthermore, the percentage of reduced albumin within total albumin was significantly higher in the branched-chain amino acid group than in the control group. These results indicate that continuous supplementation with branched-chain amino acids in rats with chronic liver disease induces phosphorylation of hepatic ribosomal protein S6 and attenuates decreases in the percentage of reduced albumin, although levels of plasma albumin are not increased.

Dietary medium-chain triglycerides attenuate hepatic lipid deposition in growing rats with protein malnutrition.

The objective of this study was to investigate the effects of dietary medium-chain triglycerides (MCT) on hepatic lipid accumulation in growing rats with protein malnutrition. Weaning rats were fed either a low-protein diet (3%, LP) or control protein diet (20%, CP), in combination with or without MCT. The four groups were as follows: CP-MCT, CP+MCT, LP-MCT, and LP+MCT. Rats in the CP-MCT, CP+MCT and LP+MCT groups were pair-fed their respective diets based on the amount of diet consumed by the LP-MCT group. Rats were fed each experimental diet for 30 d. Four weeks later, the respiratory quotient was higher in the LP-MCT group than those in the other groups during the fasting period. Hepatic triglyceride content increased in the LP groups compared with the CP groups. Hepatic triglyceride content in the LP+MCT group, however, was significantly decreased compared with that in the LP-MCT group. Levels of carnitine palmitoyltransferase (CPT) 1a mRNA and CPT2 mRNA were significantly decreased in the livers of the LP-MCT group, as compared with corresponding mRNA levels of the other groups. These results suggest that ingestion of a low-protein diet caused fatty liver in growing rats. However, when rats were fed the low-protein diet with MCT, hepatic triglyceride deposition was attenuated, and mRNA levels encoding CPT1a and CPT2 were preserved at the levels of rats fed control protein diets.

An evaluation of protein intake for metabolic demands and the quality of dietary protein in rats using an indicator amino acid oxidation method.

Currently, protein requirements are generally determined based on nitrogen balance studies, but there are a variety of limitations associated with this method. The indicator amino acid oxidation (IAAO) method, with a theoretical base that differs widely from the nitrogen balance method, was developed as an alternative method for humans. The objective of the present study was to evaluate protein intakes for metabolic demands and protein quality, using protein itself, in rats employing the IAAO technique with L-[1-(13)C]phenylalanine. Male Wistar/ST rats (5-6 wk old) received a graded casein (4.3, 8.6, 12.9, 17.2, 21.5, 25.8%), or a wheat gluten (7.2, 10.8, 14.4, 18.0, 21.6, 25.2%) diet, along with L-[1-(13)C]phenylalanine. An isotopic plateau in breath was achieved 210 min after the start of the (13)C ingestion. The protein intakes for metabolic demands were calculated by applying a mixed-effect change-point regression model to breath (13)CO(2) data, which identified a breakpoint at minimal breath (13)CO(2) in response to graded protein intake. The protein intakes for metabolic demands determined by the IAAO method were 13.1 g/kg BW/d for casein and 18.1 g/kg BW/d for wheat gluten, showing a tendency similar to that determined by the nitrogen balance method. These results demonstrated that the IAAO method could be employed to evaluate not only the protein intakes for metabolic demands, but the dietary protein quality in freely living rats, suggesting that this method might be viable in a clinical setting.