ALA Group Research Articles

α-Linolenic acid promotes testosterone synthesis by improving mitochondrial function in primary rooster Leydig cells

The present study aimed to investigate the direct effects of α-Linolenic acid (ALA) on the in vitro production of testosterone and the expression of key enzymes and proteins related to steroidogenesis in Leydig cells of roosters. MethodsPurified primary Leydig cells isolated from 65-week-old roosters were purified and treated with different concentrations of ALA treatments: (0 μm/L [control], solvent control group (DMSO), 20 μM/L, 40 μM/L, and 80 μM/L) and cell counting-8 (CCK-8) for cell viability assay, Enzyme-linked immunosorbent assay (ELISA) kit for the determination of testosterone in cell supernatants, quantitative (real-time) PCR, and analysis of activities of antioxidants catalase (CAT), superoxide dismutase (SOD) and malondialdehyde (MDA), evaluation of mitochondrial membrane potential, pro- and anti-apoptotic proteins/genes Bcl-2, Bcl-2-associated X protein (Bax), apoptosis-inducing factor (AIF) were done respectively. ResultsOur results showed that ALA significantly increased testosterone secretion in primary rooster Leydig cells (P < 0.05), and 40 μM/L is the optimal dose. Leydig cells supplemented with ALA (20, 40, 80 μM) increased the expression of key enzymes and proteins 3β-hydroxysteroid dehydrogenase (3β-HSD), steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc) concerning steroidogenesis, enhanced antioxidant capability, improved mitochondrial biogenesis, and markedly improved the mitochondrial membrane potential (P < 0.05). Furthermore, the expression of the apoptosis-suppressive gene Bcl-2 was significantly increased, but Bax and AIF expression was decreased in the ALA group compared to that in the control group (P < 0.05). ConclusionALA promoted testosterone production, enhanced steroidogenic enzyme expression, improved mitochondrial function, and antioxidant capacity, and reduced apoptosis in primary rooster Leydig cells, with 40 μM/L identified as the optimal concentration.

The beneficial effect of α-lipoic acid on spinal cord injury repair in rats is mediated through inhibition of oxidative stress: A transcriptomic analysis

Background Oxidative stress is a crucial factor contributing to the occurrence and development of secondary damage in spinal cord injuries (SCI), ultimately impacting the recovery process. α-lipoic acid (ALA) exhibits potent antioxidant properties, effectively reducing secondary damage and providing neuroprotective benefits. However, the precise mechanism by which ALA plays its antioxidant role remains unknown. Methods We established a model of moderate spinal cord contusion in rats. Experimental rats were randomly divided into 3 distinct groups: the sham group, the model control group (SCI_Veh), and the ALA treatment group (SCI_ALA). The sham group rats were exposed only to the SC without contusion injury. Rats belonging to SCI_Veh group were not administered any treatment after SCI. Rats of SCI_ALA group were intraperitoneally injected with the corresponding volume of ALA according to body weight for three consecutive days after the surgery. Subsequently, three days after SCI, spinal cord samples were obtained from three groups of rats: the sham group, model control group, and administration group. Thereafter, total RNA was extracted from the samples and the expression of three sets of differential genes was analyzed by transcriptome sequencing technology. Real-time PCR was used to verify the sequencing results. The impact of ALA on oxidative stress in rats following SCI was assessed by measuring their total antioxidant capacity and hydrogen peroxide (H2O2) content. The effects of ALA on rat recovery following SCI was investigated through Beattie and Bresnahan (BBB) score and footprint analysis. Results The findings from the transcriptome sequencing analysis revealed that the model control group had 2975 genes with altered expression levels when compared to the ALA treatment group. Among these genes, 1583 were found to be upregulated while 1392 were down-regulated. Gene ontology (GO) displayed significant enrichment in terms of functionality, specifically in oxidative phosphorylation, oxidoreductase activity, and signaling receptor activity. The Kyoto encyclopedia of genes and genomes (KEGG) pathway was enriched in oxidative phosphorylation, glutathione metabolism and cell cycle. ALA was found to have multiple benefits for rats after SCI, including increasing their antioxidant capacity and reducing H2O2 levels. Additionally, it was effective in improving motor function (such as 7 days after SCI, the BBB score for SCI_ALA was 8.400 ± 0.937 compared to 7.050 ± 1.141 for SCI_Veh) and promoting histological recovery after SCI (The results of HE demonstrated that the percentage of damage area in was 44.002 ± 6.680 in the SCI_ALA and 57.215 ± 3.964 in the SCI_Veh at the center of injury.). The sequence data from this study has been deposited into Sequence Read Archive (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE242507). Conclusion Overall, the findings of this study confirmed the beneficial effects of ALA on recovery in SCI rats through transcriptome sequencing, behavioral, as well histology analyses.

Persistent COVID-19 parosmia and olfactory loss post olfactory training: randomized clinical trial comparing central and peripheral-acting therapeutics

PurposeAlthough COVID-19 anosmia is often transient, patients with persistent olfactory dysfunction (pOD) can experience refractory parosmia and diminished smell. This study evaluated four putative therapies for parosmia in patients with chronic COVID-19 olfactory impairment.MethodsAfter screening nasal endoscopy, 85 patients (49 female, 58%) with pOD and treatment-refractory parosmia were randomized to: (1) ultramicronized palmitoylethanolamide and luteolin + olfactory training (OT) (umPEALUT group, n = 17), (2) alpha-lipoic acid + OT (ALA group, n = 21), (3) umPEALUT + ALA + OT (combination group, n = 28), or 4) olfactory training (OT) alone (control group, n = 23). Olfactory function was assessed at baseline (T0) and 6 months (T1) using a parosmia questionnaire and Sniffin’ Sticks test of odor threshold, detection, and identification (TDI). Analyses included one-way ANOVA for numeric data and Chi-Square analyses for nominal data on parosmia.ResultsThe umPEALUT group had the largest improvement in TDI scores (21.8 ± 9.4 to 29.7 ± 7.5) followed by the combination group (19.6 ± 6.29 to 27.5 ± 2.7), both p < 0.01. The control and ALA groups had no significant change. Patients in the combination and umPEALUT groups had significantly improved TDI scores compared to ALA and control groups (p < 0.001). Rates of parosmia resolution after 6 months were reported at 96% for combination, 65% for control, 53% for umPEALUT and 29% for ALA (p < 0.001). All treatment regimens were well-tolerated.ConclusionsumPEALUT and OT, with or without ALA, was associated with improvement in TDI scores and parosmia, whereas OT alone or OT with ALA were associated with little benefit.

Pulsatile Leucine Administration during Continuous Enteral Feeding Enhances Skeletal Muscle Mechanistic Target of Rapamycin Complex 1 Signaling and Protein Synthesis in a Preterm Piglet Model

BackgroundContinuous feeding does not elicit an optimal anabolic response in skeletal muscle but is required for some preterm infants. We reported previously that intermittent intravenous pulses of leucine (Leu; 800 μmol Leu·kg–1·h–1 every 4 h) to continuously fed pigs born at term promoted mechanistic target of rapamycin complex 1 (mTORC1) activation and protein synthesis in skeletal muscle. ObjectivesThe aim was to determine the extent to which intravenous Leu pulses activate mTORC1 and enhance protein synthesis in the skeletal muscle of continuously fed pigs born preterm. MethodsPigs delivered 10 d preterm was advanced to full oral feeding >4 d and then assigned to 1 of the following 4 treatments for 28 h: 1) ALA (continuous feeding; pulsed with 800 μmol alanine·kg–1·h–1 every 4 h; n = 8); 2) L1× (continuous feeding; pulsed with 800 μmol Leu·kg–1·h–1 every 4 h; n = 7); 3) L2× (continuous feeding; pulsed with 1600 μmol Leu·kg–1·h–1 every 4 h; n = 8); and 4) INT (intermittent feeding every 4 h; supplied with 800 μmol alanine·kg–1 per feeding; n = 7). Muscle protein synthesis rates were determined with L-[2H5-ring]Phenylalanine. The activation of insulin, amino acid, and translation initiation signaling pathways were assessed by Western blot. ResultsPeak plasma Leu concentrations were 134% and 420% greater in the L2× compared to the L1× and ALA groups, respectively (P < 0.01). Protein synthesis was greater in the L2× than in the ALA and L1× groups in both the longissimus dorsi and gastrocnemius muscles (P < 0.05) but not different from the INT group (P > 0.10). Amino acid signaling upstream and translation initiation signaling downstream of mTORC1 largely corresponded to the differences in protein synthesis. ConclusionsIntravenous Leu pulses potentiate mTORC1 activity and protein synthesis in the skeletal muscles of continuously fed preterm pigs, but the amount required is greater than in pigs born at term.

Lipoic acid abates testis lead accumulation, sperm-endocrine deficits, testicular oxidative inflammation and apoptosis and modulates gene expression of Bax and Bcl-2 in rats

The current study explored whether ALA could prevent testis Pb accumulation and Pb-induced oxidative testicular toxicity in rats. Adult male rats were randomly divided and administered ALA (25 mg/kg/day) and/or Pb (Pb acetate, 20 mg/kg/day) in Control group, ALA group, Pb group, and ALA + Pb group for 4 weeks consecutively. Pb exposure significantly increased Pb accumulation in the testes and blood of Pb-exposed rats compared to the control. Pb caused marked deficits in sperm count, motility along with elevated abnormal sperm cells, while serum testosterone, follicle stimulating hormone (FSH) and leutenizing hormone (LH) levels reduced prominently in comparison to the control. It remarkably depressed the testicular activities of catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), as well as levels of interleukin-4 (IL-4) and interleukin-10 (IL-10), whereas malondialdehyde (MDA), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) increased significantly compared to the control. The levels of caspase-3, capsase-9, and the PCR gene expression of Bax and Bcl2 were adversely altered with alterations in testis histology. Interestingly, the co-treatment with ALA blocks Pb accumulation in the testis and blood. It inhibited the Pb-induced alterations in reproductive indices and reversed the oxidative stress-mediated inflammatory stress, apoptotic signaling and histopathological abrasions. The findings reveal that ALA could abrogate Pb-induced testicular toxicity through blocking testicular oxidative inflammation and apoptosis in rats.

Alpha-linolenic acid alleviates the detrimental effects of lipopolysaccharide during in vitro ovine oocyte development

During the transition period and early lactation of ruminants with higher production, the reproductive organs are exposed to various stressors, like inflammation stimulators such as lipopolysaccharides (LPS), as a consequence of high concentrate consumption. In this study, we aimed to determine the probable potential of α-linolenic acid (ALA) in alleviating LPS-induced effects in ovine oocytes in vitro as well as the underlying controlling mechanisms. Different concentrations of LPS (0, 0.01, 0.1, 1, and 10 μg/mL) were added to the oocyte maturation medium to evaluate its effect on oocyte developmental competence. Likewise, different concentrations of ALA (0, 10, 50, 100, and 200 μM/mL) were added to the maturation medium to define its effects on oocyte developmental competence. Accordingly, a combination of ALA and LPS in a dose-dependent manner was added to the maturation medium to elucidate their effect on oocyte developmental competence and uncover any possible potential of ALA to alleviate the detrimental effect induced by the presence of LPS. The expressions of candidate genes were measured in mature oocytes treated either with ALA, LPS, or ALA plus LPS. Adding LPS to the maturation medium decreased the cleavage rate of the treated oocytes, and those oocytes reached the blastocyst stage at a lower rate. Adding ALA to the maturation medium in the presence of LPS alleviated the detrimental effects of LPS in a dose-dependent manner, which ultimately led to higher cleavage and blastocyst formation. A higher expression of Trim26, GRHPR, NDUFA, PGC-1α, SOD, CS, SDH, p53, and CAT was observed in LPS-treated oocytes compared with the ALA and control groups. Additionally, CS and CAT transcripts were down-regulated in oocytes in LPS plus ALA-treated group compared to that of the LPS-treated group. These findings revealed that ALA has the potential to alleviate the detrimental effects induced by LPS on in ovine oocytes during maturation in vitro. Thus, LPS-detrimental effect and ALA-preventing mechanisms seem to be regulated through the expression of genes involved in mitochondrial biogenesis and function, oxidative stress, and antioxidant systems.

Efficacy and Safety of 5-Aminolevulinic Acid Combined with Iron on Skeletal Muscle Mass Index and Physical Performance of Patients with Sarcopenia: A Multicenter, Double-Blinded, Randomized-Controlled Trial (ALADDIN Study).

Sarcopenia is a geriatric syndrome characterized by decreased physical performance, muscle mass, and strength. Since the intake of 5-aminolevulinic acid (ALA) with iron can increase muscle mass and mitochondria in mice and elevate physical exercise performance in humans, the beneficial effects of ALA in patients with sarcopenia are expected, but this remains unexplored in the literature. This study aimed to assess the efficacy and dose dependency of ALA combined with iron in sarcopenia by measuring skeletal muscle mass index (SMI). Subjects with sarcopenia were enrolled and randomized into the ALA and sodium ferrous citrate (SFC) intake groups (ALA50/SFC29, ALA100/SFC29, ALA150/SFC29, ALA 100/SFC57, and ALA0/SFC29 placebo) and ingested the assigned study food for 12 weeks. The primary endpoint, the change in SMI from baseline to week 12, did not differ significantly between the groups. Hand grip significantly increased or tended to increase from baseline after 12 weeks with all doses of ALA or SFC compared with the placebo group. No consistent changes were observed in the other endpoints, including calf circumference, physical function, or quality of life (QOL). Although this study suggests safe administration and the possibility of ALA improving hand grip strength in patients with sarcopenia, further investigation is required.

Clinical effectiveness of α-lipoic acid, myo-inositol and propolis supplementation on metabolic profiles and liver function in obese patients with NAFLD: A randomized controlled clinical trial

To compare the effects of α-lipoic acid (ALA), myo-inositol (MI) and propolis supplementation on metabolic parameters and liver function in obese patients with non-alcoholic fatty liver disease (NAFLD) METHODS: Ninety-two obese patients with NAFLD were randomly allocated into one of the four groups (ALA, MI, propolis, and control groups) for 8 weeks. At pre-and post-intervention, anthropometric measures, metabolic parameters and liver function were assessed. Clinical effectiveness was assessed using Absolute Risk Reduction (ARR) and Number Needed to Treat (NNT). After 8 weeks, apart from waist-to-hip ratio, all studied anthropometric measures decreased significantly in each of the groups over the trial. Although the greatest improvements in glycemic indices were observed in MI group (p<0.05), the differences among the groups were not significant. Control group showed the greatest reduction in serum triglyceride level (p=0.026) while the greatest improvements in serum total cholesterol, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were observed in MI group (p=0.043, p=0.019 and p=0.041, respectively). Alanine aminotransferase (ALT) levels reduced significantly in all groups, particularly in propolis group (p=0.012). The greatest reduction in serum aspartate transaminase (AST) level was observed in control group (p<0.001), however, the difference among the groups was statistically marginal (p=0.058). The estimated NNTs for one grade reduction in liver steatosis for MI, ALA and propolis supplementation compared with control group were 1.5, 2.2 and 3, respectively. Dietary recommendation for weight loss accompanied by MI and then ALA supplementation improved metabolic parameters and liver steatosis. "Registered under ClinicalTrials.gov Identifier no: IRCT20100209003320N22".

Effect of Supplemental Dietary α-linolenic Acid (18:3n-3) on the Growth Performance, Body Composition, and Fatty Acid Profile of Coho Salmon (Oncorhynchus kisutch) Alevins Cultured in Freshwater

The present study evaluated the effects of supplemental dietary α-linolenic acid (ALA, 18:3n-3) on growth performance, body composition, hepatic fat metabolizing enzymes, and fatty acid profiles of coho salmon (Oncorhynchus kisutch) alevins cultured in freshwater. Six isonitrogenous and isolipidic experimental diets were formulated to attain different levels of ALA (0.09, 0.41, 0.76, 1.03, 1.32, and 1.68% dry weight) by adding linseed oil in a basal diet, respectively. Each diet was fed to triplicate groups of alevins (50 fish in each tank) with initial mean body weight of 0.352 ± 0.002 g. Fish were reared in a freshwater flow-through rearing system and fed to apparent satiation 4 times daily for 12 weeks. The specific growth rate (SGR) increased with increasing dietary ALA level, which reached the peak at 1.03% ALA group ( p &lt; 0.05 ). The final body weight (FBW) and SGR showed linear, quadratic, and cubic responses with the dietary ALA level. The whole-body crude protein increased with increasing dietary ALA level while lipid content was the opposite trend ( p &lt; 0.05 ). However, the content of crude lipid in the whole-body of fish showed quadratic and cubic responses with the dietary ALA level. In addition, except for fatty acid synthase (FAS), the hepatic enzymes activities of lipoprotein lipase (LPL), hepatic lipase (HL), and malate dehydrogenase (MDH) showed linear, quadratic, and cubic response with the dietary ALA level ( p &lt; 0.05 ) The activities of hepatic LPL and HL in 1.32% ALA group were significantly higher than other groups ( p &lt; 0.05 ). The activities of hepatic MDH and FAS in 1.32% ALA group were the lowest, which were significantly lower than those in ALA groups (form 0.09% to1.03%) ( p &lt; 0.05 ). Except for the content of MUFA, the contents of other fatty acids showed linear, quadratic, and cubic response with the dietary ALA level. The ALA and ∑n-3 PUFA content significantly increased with increasing dietary ALA levels ( p &lt; 0.05 ). These results indicated that dietary ALA actively impacted fish growth performance, hepatic lipid metabolizing enzymes, and muscle fatty acid profile. The cubic regression analysis based on SGR as evaluation indices indicated that the optimal dietary ALA content was 1.33% dry weight of diet for alevins.

Alamandine alleviates methotrexate-induced nephrotoxicity in rats by targeting oxidative stress and inflammation

Aim: Nephrotoxicity due to the use of methotrexate (Mtx) is one of the most important problems associated with chemotherapy. Oxidative stress and inflammation are the major pathomechanisms of Mtx-induced nephrotoxicity. Alamandine (Ala), a new member of the renin-angiotensin system (RAS), is an important peptide with antioxidant and anti-inflammatory capacities. In this study, it was investigated whether Ala ameliorates Mtx-induced kidney damage by reducing oxidative stress and inflammation. Materials and Methods: Male Wistar albino rats were assigned into three groups: control group, Mtx group, and Mtx+Ala group. At the end of the experiment, kidney tissues were quickly removed. Glutathione (GSH) and malondialdehyde (MDA) levels were measured to determine the oxidative state in kidney tissues. In addition, tissue samples were assessed as histopathological and immunohistochemical for heat shock protein 60 (HSP60), caspase-3, tumor necrosis factor-α (TNF-α), and receptor-interacting protein kinase-3 (RIPK3). Results: Mtx treatment resulted in reduced GSH content, elevated MDA level, increased heat shock protein 60 (HSP60), and caspase-3 expression. These changes in kidney tissues of rats treated with Mtx triggered oxidative stress characterized by apoptosis and kidney damage. Mtx also markedly increased the expression of TNF-α, an inflammation marker, and RIPK3, a marker of necroptosis. However, Ala administration significantly alleviated Mtx-induced kidney damage by reducing apoptosis and necroptosis by suppressing oxidative stress and inflammation. Conclusion: Taken together, our results support that Ala treatment can serve as a new and promising therapeutic strategy against Mtx-induced nephrotoxicity. Keywords: Alamandine; methotrexate; oxidative stress; inflammation; apoptosis; necroptosis; nephrotoxicity

Receptivity, autophagy, and implantation in endometriosis; does antioxidant work? An experimental study.

Implantation is a key point in pathological processes associated with infertility, especially in endometriosis. In this complex process, there is limited evidence to demonstrate the role of receptivity and autophagy. The present study aimed to evaluate LC3A/B, P62/SQSTM1, Beclin 1, and integrin expressions and receptivity and autophagy processes in endometriosis in pregnant rats with healthy endometriosis on day 6 of the process. Pregnancy was observed in all rats in the control group (8/8), while the pregnancy was 4/8 in the endometriosis group, and 6/8 in the endometriosis + ALA group. LC3A/B and P62/SQSTM1 expression increased significantly in the endometriosis + ALA group, compared with endometriosis groups (p < .05). The effect of ALA on autophagy and receptivity in endometriosis was shown for the first time. Antioxidant and anti-inflammatory treatments in endometriosis should be investigated as new treatment modalities for implantation problems. PRACTICAL APPLICATIONS: Endometriosis, the etiology of which remains unknown, is an important cause of infertility. Implantation is the key point in pathological processes associated with infertility. In this complex process, there is limited evidence to demonstrate the role of receptivity and autophagy. The present study aimed to evaluate LC3A/B, P62/SQSTM1, Beclin 1, and integrin expressions and receptivity and autophagy processes in endometriosis in pregnant rats with healthy endometriosis on day 6 of the process. Oral alpha-lipoic acid was administered to one group and the effect of this powerful antioxidant on the process was evaluated.

α-Lipoic Acid Supplementation Salivary Glands in the Hyperglycaemic Rats.

The aim of the present study was to investigate whether α-lipoic acid (ALA) could reverse/prevent high fat diet (HFD) -induced salivary gland dysfunction and oxidative damage in the salivary glands of rats, and strengthen their antioxidant defense. The enzymatic and non-enzymatic antioxidants as well as their redox status, oxidative damage products and salivary flow rate were investigated in the parotid (PG) and submandibular (SMG) glands of Wistar rats exposed to a high-fat diet and then supplemented with ALA for a period of 4 weeks. The rats in the study were divided into 4 groups of 10 animals each: C (control), HFD, C + ALA, HFD + ALA. The HFD + ALA group in comparison to the HFD group showed normalization of the activity of antioxidant enzymes to the levels observed in the C group only in the case of the SMG. Additionally, ALA supplementation was more effective in reducing the value of oxidative damage products in the PG compared to the SMG. ALA supplementation in the HFD group was not able to restore the disturbed total antioxidant capacity (TAC) of the salivary glands to the level observed in the C group. In the group of HFD + ALA rats, both unstimulated and stimulated salivation and the protein concentration in the SMG did not differ significantly from the parameters recorded in the group fed with HFD. ALA supplementation by rats fed the HFD diet prevents/reverses oxidative damage in the PG to a greater extent than in the SMG and is unable to completely restore disturbed TAC to the levels seen in C rats. Moreover, we observed that ALA supplementation did not improve the impaired secretory function of the salivary glands.

Does α-lipoic acid therapeutically effective against experimentally induced-acute pulpitis in rats?

The purpose of the study was to investigate the therapeutic effects of α-lipoic acid (ALA) on an induced-acute pulpitis model in rats. Twenty-four Wistar albino rats were randomly divided into three groups: control, induced-acute pulpitis (PULP) and PULP+ALA groups. In the PULP and PULP+ALA groups, the crowns of the maxillary left incisors were removed horizontally. All exposed pulp tissues were treated with 5µL LPS solution. In the PULP+ALA group, the rats were treated intraperitoneally with a single dose of ALA (100mg/kg). The rats were sacrificed 24h after pulp injury, and the trunk blood and pulp samples were collected and then determined using ELISA assay kits. TNF-α, IL-1β, MMP-1 and MMP-2levels in the serum and pulp tissues were considerably higher in the PULP group than the control group (p<0.01-0.001). In the PULP+ALA group, TNF-α, IL-1β, MMP-1 and MMP-2levels in the serum and pulp tissues decreased significantly compared to the PULP group (p<0.05-0.001). ALA decreases pro-inflammatory mediators and proteolytic enzymes, which might relieve acute inflammation.

Biohydrogenation Pathway of α-Linolenic Acid in Rumen of Dairy Cow In Vitro.

Simple SummaryFew studies have investigated a relationship between t9,c12,c15-C18:3 and ALA, an α-Linolenic acid c9,c12,c15-C18:3 in the rumen. These results indicated that t9,c12,c15-C18:3 was an intermediate of the α-linolenic acid shifted rumen biohydrogenation pathway. This study hypothesized a pathway for α-linolenic acid biohydrogenation in rumen.The t9,c12,c15-C18:3 as an isomer of α-linolenic acid (c9,c12,c15-C18:3; ALA), has been recently detected in milk, but has not been found in the rumen. This study hypothesized that it may be a biohydrogenation product of ALA in rumen and aimed to explore whether it was present in the rumen and help to understand the rumen biohydrogenation mechanisms of ALA. The in vitro experiment included two treatments, a control check (CK group) with 50 µL ethanol added, and ALA group with 50 µL ethanol and 2.6 mg ALA (ALA addition calculated by 1.30% of dry matter base of diet); each sample of fermentation fluid had the composition of C18 fatty acids analyzed at 0, 0.5, 1, 2, 3, 4, 5, and 6 h. The results showed that no t9,c12,c15-C18:3 was detected in the CK group, but ALA addition increased the concentration of t9,c12,c15-C18:3 in fermentation fluid. The content of t9,c12,c15-C18:3 peaked 1 h after fermentation, then declined gradually. At 1 h, no t9c12c15-C18:3 was detected in the fermentation fluid of the CK treatment. The results suggested that ALA converted to the isomer t9,c12,c15-C18:3 through biohydrogenation in the rumen. The addition of ALA can also increase the concentration of t9,c12-C18:2, c9,t11-C18:2, c12-C18:1, t11-C18:1, t9-C18:1, and c6-C18:1 in fermentation fluid. It was concluded using an in vitro experiment that t9,c12,c15-C18:3 was a product of rumen biohydrogenation of ALA.

Assignment of Ala, Ile, LeuproS, Met, and ValproS methyl groups of the protruding domain of murine norovirus capsid protein VP1 using methyl–methyl NOEs, site directed mutagenesis, and pseudocontact shifts

The protruding domain (P-domain) of the murine norovirus (MNV) capsid protein VP1 is essential for infection. It mediates receptor binding and attachment of neutralizing antibodies. Protein NMR studies into interactions of the P-domain with ligands will yield insights not easily available from other biophysical techniques and will extend our understanding of MNV attachment to host cells. Such studies require at least partial NMR assignments. Here, we describe the assignment of about 70% of the Ala, Ile, LeuproS, Met, and ValproS methyl groups. An unfavorable distribution of methyl group resonance signals prevents complete assignment based exclusively on 4D HMQC-NOESY-HMQC experiments, yielding assignment of only 55 out of 100 methyl groups. Therefore, we created point mutants and measured pseudo contact shifts, extending and validating assignments based on methyl-methyl NOEs. Of note, the P-domains are present in two different forms caused by an approximate equal distribution of trans- and cis-configured proline residues in position 361.

Effects of omega fatty acids on the short-term postprandial satiety related peptides in rats.

We aimed to assess the effects of omega fatty acids on time depending on responses of satiety hormones. Sixty adult rats were randomly divided into 4 groups; linoleic acid (LA), α-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) groups. For each fatty acid, the dose of 400 mg/kg was applied by oral gavage. Blood samples were taken after the 15, 30, 60 and 120 minutes. Ghrelin, cholecystokinin (CCK), glucagon-like peptide-1 (GLP-1), peptide YY (PYY), leptin and insulin hormones were analyzed by ELISA. We observed the significant increases (p<0.05) of the levels of CCK between n-3 (ALA, at 60th min; EPA, at 30th and 60th min and DHA, at 60 min) and n-6 (LA) supplemented rats. The highest GLP-1 levels were in ALA (0.70 ng/mL) and DHA (0.67 ng/mL) supplemented groups at 60th and 120th min indicating n-3 fatty acids efficiency on satiety compared to LA. It seems that ALA at 60th min and EPA at 120th min could provide the highest satiety effect with the highest insulin response, while the efficiency of LA supplementation on insulin-induced satiety diminished. The only significant change in AUC values among all hormones was in the CCK of the ALA group (p=0.004). The level of leptin increased in DHA and EPA supplemented rats (p=0.140). Our results showed that dietary omega fatty acids influenced the releasing of hormones in different ways possibly depending on chain length or saturation degree. Comprehensive studies need to be addressed for each fatty acid on satiety-related peptide hormones.

Long-term survival of patients with stage II and III gastric cancer who underwent gastrectomy with inadequate nodal assessment.

BACKGROUNDGastric cancer is an aggressive disease with frequent lymph node (LN) involvement. The NCCN recommends a D2 lymphadenectomy and the harvesting of at least 16 LNs. This threshold has been the subject of great debate, not only for the extent of surgery but also for more appropriate staging. The reclassification of stage IIB through IIIC based on N3b nodal staging in the eighth edition of the American Joint Committee on Cancer (AJCC) staging system highlights the efforts to more accurately discriminate survival expectancy based on nodal number. Furthermore, studies have suggested that pathologic assessment of 30 or more LNs improve prognostic accuracy and is required for proper staging of gastric cancer.AIMTo evaluate the long-term survival of advanced gastric cancer patients who deviated from expected survival curves because of inadequate nodal evaluation.METHODSEligible patients were identified from the Surveillance, Epidemiology, and End Results database. Those with stage II–III gastric cancer were considered for inclusion. Three groups were compared based on the number of analyzed LNs. They were inadequate LN assessment (ILA, < 16 LNs), adequate LN assessment (ALA, 16-29 LNs), and optimal LN assessment (OLA, ≥ 30 LNs). The main outcomes were overall survival (OS) and cancer-specific survival. Data were analyzed by the Kaplan-Meier product-limit method, log-rank test, hazard risk, and Cox proportional univariate and multivariate models. Propensity score matching (PSM) was used to compare the ALA and OLA groups.RESULTSThe analysis included 11607 patients. Most had advanced T stages (T3 = 48%; T4 = 42%). The pathological AJCC stage distribution was IIA = 22%, IIB = 18%, IIIA = 26%, IIIB = 22%, and IIIC = 12%. The overall sample divided by the study objective included ILA (50%), ALA (35%), and OLA (15%). Median OS was 24 mo for the ILA group, 29 mo for the ALA group, and 34 mo for the OLA group (P < 0.001). Univariate analysis showed that the ALA and OLA groups had better OS than the ILA group [ALA hazard ratio (HR) = 0.84, 95% confidence interval (CI): 0.79–0.88, P < 0.001 and OLA HR = 0.73, 95%CI: 0.68–0.79, P < 0.001]. The OS outcome was confirmed by multivariate analysis (ALA HR = 0.68, 95%CI: 0.64–0.71, P < 0.001 and OLA: HR = 0.48, 95%CI: 0.44–0.52, P < 0.001). A 1:1 PSM analysis in 3428 patients found that the OLA group had better survival than the ALA group (OS: OLA median = 34 mo vs ALA median = 26 mo, P < 0.001, which was confirmed by univariate analysis (HR = 0.81, 95%CI: 0.75–0.89, P < 0.001) and multivariate analysis: (HR = 0.71, 95%CI: 0.65–0.78, P < 0.001).CONCLUSIONProper nodal staging is a critical issue in gastric cancer. Assessment of an inadequate number of LNs places patients at high risk of adverse long-term survival outcomes.

191 Pulsatile Administration of Leucine Promotes mTOR Signaling and Protein Synthesis in Skeletal Muscle of Continuously Fed Preterm Pigs

Abstract Continuous feeding does not elicit an optimal anabolic response but is required for some premature infants. We reported previously that intermittent intravenous pulses of Leu (800 μmol Leu·kg-1·h-1 every 4 h) to continuously fed pigs born at term enhanced skeletal muscle mTOR signaling and protein synthesis (PS). The objective of this study was to determine if the anabolic response of skeletal muscle to intermittent Leu pulses is altered following premature birth. Pigs delivered 10 d preterm by cesarean section were advanced to full oral feeding over four days (195 kcal·kg-1·d-1; 13.5 g protein·kg-1·d-1). Pigs were assigned to 1 of 4 treatments: 1) ALA (continuous feeding; 800 μmol Ala·kg-1·h-1 every 4 h; n = 7); 2) L1× (continuous feeding; 800 μmol Leu·kg-1·h-1 every 4 h; n = 6); 3) L2× (continuous feeding; 1600 μmol Leu·kg-1·h-1 every 4 h; n = 6); and 4) INT (intermittent feeding every 4 h; n = 5). Pigs received a Phe tracer 30 min following the amino acid pulse or intermittent feeding to measure PS in longissimus dorsi muscle. Anabolic signaling downstream of mTOR was determined by immunoblot.ResultsPS was higher in L2× and INT compared to ALA (P &amp;lt; 0.05) but was not different between L2× and INT; PS in L1× was intermediate and not different from other groups. Phosphorylation of 4EBP1 and S6K1 was higher in INT compared to L1× and ALA groups (P &amp;lt; 0.05) but was not different compared to L2×. Phosphorylation of 4EBP1, but not S6K1, was higher in L2× compared to ALA (P &amp;lt; 0.05); phosphorylation of 4EBP1 and S6K1 was not different between L1× and L2×.ConclusionsThese results demonstrate that intravenous Leu enhances mTOR activation and PS in LD muscle of continuously fed preterm pigs. However, the amount required may be higher than in pigs born at term.Source of Research SupportNIH and USDA.

Plasma unesterified eicosapentaenoic acid is converted to docosahexaenoic acid (DHA) in the liver and supplies the brain with DHA in the presence or absence of dietary DHA

Recent meta-analyses suggest that high eicosapentaenoic acid (EPA, 20:5n−3) supplements may be beneficial in managing the symptoms of major depression. However, brain EPA levels are hundreds-fold lower than docosahexaenoic acid (DHA, 22:6n−3), making the potential mechanisms of action of EPA in the brain less clear. Using a kinetic model the goal of this study was to determine how EPA impacts brain DHA levels. Following 8 weeks feeding of a 2% alpha-linolenic acid (ALA, 18:3n−3) or DHA diet (2% ALA + 2% DHA), 11-week-old Long Evans rats were infused with unesterified 13C-EPA at steady-state for 3 h with plasma collected at 30 min intervals and livers and brains collected after 3 h for determining DHA synthesis-accretion kinetics in multiple lipid fractions. Most of the newly synthesized liver 13C-DHA was in phosphatidylethanolamine (PE, 37%–56%), however, 75–80% of plasma 13C-DHA was found in triacylglycerols (TAG) at 14 ± 5 and 46 ± 12 nmol/g/day (p < 0.05) in the ALA and DHA group, respectively. In the brain, PE and phosphatidylserine (PS) accreted the most 13C-DHA, and DHA compared to ALA feeding shortened DHA half-lives in most lipid fractions, resulting in total brain DHA half-lives of 32 ± 6 and 96 ± 24 (days/g ± SEM), respectively (p < 0.05). EPA was predominantly converted and stored as PE-DHA in the liver, secreted to plasma as TAG-DHA and accumulated in brain as PE and PS-DHA. In conclusion, EPA is a substantial source for brain DHA turnover and suggests an important role for EPA in maintaining brain DHA levels.

Stimulation of follicle growth and development during estrus in Ettawa Grade does fed a flushing supplement of different polyunsaturated fatty acids.

Background and Aim:Flushing with the manipulation of fatty acids, particularly polyunsaturated fatty acids, like linoleic and α-linolenic acids in the ration, is a strategy to raise the nutritional status of the female mammals to improve ovarian function and follicle development. This study was designed to investigate the effectiveness of flushing supplementation with different types of polyunsaturated and saturated fatty acids in stimulating follicle growth and development during estrus in Ettawa Grade does with a low initial body condition score (BCS ≤2).Materials and Methods:Eighteen Ettawa Grade does in the second parity, with an average body weight of 32.11±2.19 kg, were divided into three groups according to the fatty acid supplemented to their ration: (i) About 2.8% lauric acid flushing (group); (ii) 2.8% linoleic acid flushing (LA group); and (iii) 2.8% α-linolenic acid flushing (ALA group). The ration was formulated to be isocaloric (total digestible nutrient = 77%) and isonitrogenous (crude protein = 15%). The experiment was conducted for 35 days; that is, 14 days for acclimatization and synchronization of the estrous cycle and 21 days for fatty acid flushing until the appearance of the next estrus. A completely randomized design was applied.Results:According to the results, none of the different fatty acids in the ration affected the nutrient intakes, BCSs, average daily gains, and plasma glucose, cholesterol, and progesterone concentrations of the three groups of does. However, the BCSs (by 0.8-0.9) and the plasma cholesterol concentrations were higher after fatty acid flushing for 21 days than before the flushing period. The ALA group had the highest number of large-sized preovulatory follicles, whereas the LAURIC group had the highest plasma estradiol concentration during estrus. All three groups had similar plasma progesterone concentrations during estrus after fatty acid flushing.Conclusion:Flushing supplementation with 2.8% ALA from flaxseed oil gave the best results in terms of stimulating the highest number of large-sized preovulatory follicles in Ettawa Grade does.