Alterations in the Gut Microbiota and Metabolomics of Seafarers after a Six-Month Sea Voyage.

Maintaining the health of seafarers is a difficult task during long-term voyages. Little is known about the corresponding changes in the gut microbiome-host interaction. This study recruited 30 seafarers undertaking a 6-month voyage and analyzed their gut microbiota using 16S rRNA gene sequencing. Fecal untargeted metabolomics analysis was performed using liquid chromatography-mass spectrometry. Significant changes in the composition of the gut microbiota and an increased ratio of Firmicutes/Bacteroidetes at the end (day 180) of the 6-month voyage, relative to the start (day 0), were observed. At the genus level, the abundances of Holdemanella and Plesiomonas were significantly increased, while the abundance of Bacteroides was decreased. Predicted microbial functional analysis revealed significant decreases in folate biosynthesis and biotin metabolism. Furthermore, 20 differential metabolites within six differentially enriched human metabolic pathways (including arginine biosynthesis, lysine degradation, phenylalanine metabolism, sphingolipid metabolism, pentose and glucuronate interconversions, and glycine, serine, and threonine metabolism) were identified by comparing the fecal metabolites at day 0 and day 180. Spearman correlation analysis revealed close relationships between the 14 differential microbiota members and the six differential fecal metabolites that might affect specific human metabolic pathways. This study adopted a multi-omics approach and provides potential targets for maintaining the health of seafarers during long-term voyages. These findings are worthy of more in-depth exploration in future studies. IMPORTANCE Maintaining the health of seafarers undertaking long-term voyages is a difficult task. Apart from the alterations in the gut microbiome and fecal metabolites after a long-term voyage, our study also revealed that 20 differential metabolites within six differentially enriched human metabolic pathways are worthy of attention. Moreover, we found close relationships between the 14 differential microbiota members and the six differential fecal metabolites that might impact specific human metabolic pathways. Accordingly, preventative measures, such as adjusting the gut microbiota by decreasing potential pathobionts or increasing potential probiotics as well as offsetting the decrease in B vitamins and beneficial metabolites (e.g., d-glucuronic acid and citrulline) via dietary adjustment or nutritional supplements, might improve the health of seafarers during long-term sea voyages. These findings provide valuable clues about gut microbiome-host interactions and propose potential targets for maintaining the health of seafarers engaged in long-term sea voyages.

Free Amino Acids and Volatile Aroma Compounds in Watermelon Rind, Flesh, and Three Rind-Flesh Juices.

Watermelon rind is treated as agricultural waste, causing biomass loss and environmental issues. This study aimed to identify free amino acids and volatiles in watermelon rind, flesh, and rind-flesh juice blends with ratios of 10%, 20%, and 30%. Among the 16 free amino acids quantified, watermelon rind alone contained higher total amino acids (165 mg/100 g fresh weight) compared to flesh alone (146 mg/100 g). The rind had significantly higher (1.5×) and dominant amounts of citrulline and arginine (61.4 and 53.8 mg/100 g, respectively) than flesh. The rind, however, contained significantly lower amounts of essential amino acids. Volatile analysis showed that watermelon rind total volatiles (peak area) comprised only 15% of the flesh volatiles. Of the 126 volatiles identified, the rind alone contained 77 compounds; 56 of these presented in all five samples. Aldehydes and alcohols were most prevalent, accounting for >80% of the total volatiles in all samples. Nine-carbon aldehyde and alcohol compounds dominated both the flesh and rind, though the rind lacked the diversity of other aldehydes, alcohols, ketones, terpenes, terpenoids, esters and lactones that were more abundant in the watermelon flesh. Watermelon rind was characterized by the major aroma compounds above their thresholds, including 17 aldehydes and six unsaturated nine-carbon alcohols. This study demonstrated the potential for rind as a food or beverage supplement due to its key features such as concentrated citrulline and arginine, relatively low odor intensity, and valuable volatiles associated with fresh, green, cucumber-like aromas.

Metabolites of the Nitric Oxide (NO) Pathway Are Altered and Indicative of Reduced NO and Arginine Bioavailability in Patients with Cardiometabolic Diseases Complicated with Chronic Wounds of Lower Extremities: Targeted Metabolomics Approach (LC-MS/MS).

OBJECTIVE: The status of metabolites of the nitric oxide (NO) pathway in patients with chronic wounds in the course of cardiometabolic diseases is largely unknown. Yet arginine supplementation and citrulline supplementation as novel therapeutic modalities aimed at increasing NO are tested. MATERIAL AND METHODS: Targeted metabolomics approach (LC-MS/MS) was applied to determine the concentrations of L-arginine, L-citrulline, asymmetric and symmetric dimethylarginines (ADMA and SDMA), and arginine/ADMA and arginine/SDMA ratios as surrogate markers of NO and arginine availability in ulnar and femoral veins, representing systemic and local levels of metabolites, in patients with chronic wounds in the course of cardiometabolic diseases (n = 59) as compared to patients without chronic wounds but with similar cardiometabolic burden (n = 55) and healthy individuals (n = 88). RESULTS: Patients with chronic wounds had significantly lower systemic L-citrulline and higher ADMA and SDMA concentrations and lower L-arginine/ADMA and L-arginine/SDMA as compared to healthy controls. The presence of chronic wounds in patients with cardiometabolic diseases was associated with decreased L-arginine but with increased L-citrulline, ADMA, and SDMA concentrations and decreased L-arginine/ADMA and L-arginine/SDMA. Serum obtained from the ulnar and femoral veins of patients with chronic wounds differed by L-arginine concentrations and L-arginine/SDMA ratio, both lower in the femoral vein. Wound etiology affected L-citrulline and SDMA concentrations, lower and higher, respectively, in patients with venous stasis, and the L-arginine/SDMA ratio-lower in venous stasis. The wound type affected L-arginine/ADMA and citrulline-lower in patients with ulcerations or gangrene. IL-6 was an independent predictor of L-arginine/ADMA, VEGF-A of ADMA, G-CSF of L-arginine/SDMA, and GM-CSF of L-citrulline and SDMA. CONCLUSION: Chronic wounds in the course of cardiometabolic diseases are associated with reduced NO and arginine availability due to ADMA and SDMA accumulation rather than arginine deficiency, not supporting its supplementation. Wound character seems to affect NO bioavailability and wound etiology-arginine bioavailability. Arginine concentration and its availability are more markedly reduced at the local level than the systemic level.

[Protein carbamylation: what it is and why it concerns nephrologists].

Spontaneous urea dissociation in water solution is a prominent source of protein carbamylation in our body. Protein carbamylation is a well-known phenomenon since early seventies. Some years ago, much interest in the diagnostic power of carbamylated protein arouse. Recently the target of the researches focused on its potential cardiovascular pathogenicity. Some authors claimed that this could be a reason for higher cardiovascular mortality in uremic patients. Nutritional therapy, amino acids supplementation and intensive dialysis regimen are some of the therapeutic tools tested to lower the carbamylation burst in this population.

Watermelon extract reduces blood pressure but does not change sympathovagal balance in prehypertensive and hypertensive subjects.

Previous studies have shown that watermelon extract reduces blood pressure through vasodilation. However, those studies have not verified whether sympathetic nervous activity is influenced by watermelon extract. This study aimed to evaluate the effect of supplementation with watermelon extract for 6 weeks on blood pressure and sympathovagal balance of prehypertensive and hypertensive individuals. Forty volunteers participated in a randomized, double-blind, experimental and placebo-controlled study. They consumed 6 g of watermelon extract daily (n = 20; age 48.7 ± 1.9 years, 10 men) or a placebo (n = 20; age 47.4 ± 1.2 years, 11 men) for 6 weeks. Blood pressure and cardiac autonomic modulation were measured. Watermelon extract promoted a significant reduction in systolic (137.8 ± 3.9 to 126.0 ± 4.0 mmHg, p < 0.0001) and diastolic (79.2 ± 2.2 to 72.3 ± 2.0 mmHg, p < 0.001) blood pressure, but showed no differences compared to the placebo group. This significant reduction in blood pressure occurred without a significant change in sympathovagal balance from the beginning (1.7 ± 0.1) to the end of the study (1.7 ± 0.4). In conclusion, supplementation with watermelon extract reduces systolic and diastolic blood pressure in prehypertensive and hypertensive individuals, but does not alter the cardiac autonomic modulation of these individuals.

Peptidylarginine deiminase inhibition reduces vascular damage and modulates innate immune responses in murine models of atherosclerosis.

RATIONALE: Neutrophil extracellular trap (NET) formation promotes vascular damage, thrombosis, and activation of interferon-α-producing plasmacytoid dendritic cells in diseased arteries. Peptidylarginine deiminase inhibition is a strategy that can decrease in vivo NET formation. OBJECTIVE: To test whether peptidylarginine deiminase inhibition, a novel approach to targeting arterial disease, can reduce vascular damage and inhibit innate immune responses in murine models of atherosclerosis. METHODS AND RESULTS: Apolipoprotein-E (Apoe)(-/-) mice demonstrated enhanced NET formation, developed autoantibodies to NETs, and expressed high levels of interferon-α in diseased arteries. Apoe(-/-) mice were treated for 11 weeks with daily injections of Cl-amidine, a peptidylarginine deiminase inhibitor. Peptidylarginine deiminase inhibition blocked NET formation, reduced atherosclerotic lesion area, and delayed time to carotid artery thrombosis in a photochemical injury model. Decreases in atherosclerosis burden were accompanied by reduced recruitment of netting neutrophils and macrophages to arteries, as well as by reduced arterial interferon-α expression. CONCLUSIONS: Pharmacological interventions that block NET formation can reduce atherosclerosis burden and arterial thrombosis in murine systems. These results support a role for aberrant NET formation in the pathogenesis of atherosclerosis through modulation of innate immune responses.

Bioactive compounds from flesh and by-product of fresh-cut watermelon cultivars.

BACKGROUND: The fresh-cut industry produces thousands of tons of waste in non-edible portions that present an environmental and management problem. These by-products could be reused, in particular, to obtain bioactive compounds. In this study, five different fresh-cut watermelon cultivars were assessed for their flesh and by-product bioactive contents. RESULTS: The amount of by-product varied between 31.27 and 40.61% of initial fresh weight (f.w.) depending on the cultivar. Watermelon cultivars were poor sources of total antioxidant, and the content was similar between rind and flesh samples (46.96 vs 43.46 mg ascorbic acid equivalent antioxidant capacity kg(-1) f.w.). However, the rind had a moderate total phenolic content higher than that of the flesh (458 vs 389 mg chlorogenic acid equivalent kg(-1) f.w.) and a much higher content of the amino acid citrulline (3.34 vs 2.33 g kg(-1) f.w.), which has potential bioactive properties. CONCLUSION: Watermelon rind offers quantitative interest as a natural source of citrulline, particularly Fashion, a dark-skinned, seedless cultivar. More research is required on the efficient extraction of citrulline from watermelon rind and its suitability as an additive to drinks, juices or others products to produce new functional food products with valid health claims.

Simultaneous bioanalysis of L-arginine, L-citrulline, and dimethylarginines by LC-MS/MS.

PURPOSE: L-Arginine (ARG) is converted to nitric oxide (NO) and L-citrulline (CIT) by endothelial nitric oxide synthase which is competitively inhibited by asymmetric dimethylarginine (ADMA). We have developed a liquid chromatography-mass spectrometric method for the simultaneous determination of endogenous ARG, labeled ARG (¹5N4-ARG), CIT, ADMA, and its inactive isomer, symmetric dimethylarginine (SDMA) in biological samples. METHODS: Concentrations of unlabeled ARG, ¹5N4-ARG, CIT, ADMA, and SDMA in EA.hy926 human endothelial cell lysate, cell incubation media, rat plasma or rat urine were measured by hydrophilic-interaction liquid chromatography electrospray tandem mass spectrometry. ¹³C6-ARG, D4-CIT and D7-ADMA were used as internal standards for ARG and ¹5N4-ARG, CIT, and dimethylarginines, respectively. RESULTS: The calibration curves of ARG, ¹5N4-ARG, CIT, ADMA, and SDMA were linear and independent of several sample matrices. Intra- and inter-day variabilities for the quantification of all the compounds were below 15% in quality control samples. Application of this method to determine the uptake as well as efflux of these compounds was illustrated through in vitro cell study by exposing human endothelial cells to ¹5N4-ARG, which allowed the observation of generation of ¹5N3-CIT and ¹5N3-ARG in the cell lyate. Use of these isotopes adds insights into the cellular handling of endogenous vs. exogenous ARG. Application of this method for rat plasma and rat urine assays was demonstrated after ARG oral supplementation in rats. CONCLUSION: An LC-MS/MS method was developed to quantify 6 ARG-related compounds simultaneously, utilizing 3 separate internal standards. This assay allows concurrent monitoring of uptake, efflux and metabolic processes when isotope-labeled ARG and CIT are measured, and can be applied for determination of these compounds in rat plasma and rat urine.

Effects of maternal L-citrulline supplementation on renal function and blood pressure in offspring exposed to maternal caloric restriction: the impact of nitric oxide pathway.

Maternal undernutrition can cause reduced nephron number and glomerular hypertrophy, consequently leading to adult kidney disease. We intended to elucidate whether NO deficiency evolves to kidney disease vulnerability in offspring from mothers with caloric restriction diets and whether maternal L-citrulline (L-Cit) supplementation can prevent this. Using a rat model with 50% caloric restriction, four groups of 3-month-old male offspring were sacrificed to determine their renal outcome: control, caloric restriction (CR), control treated with 0.25% L-citrulline solution during the whole period of pregnancy and lactation (Cit), and CR treated in the same way (CR+Cit group). The CR group had low nephron numbers, increased glomerular diameter, and an increased plasma creatinine level compared with the control group. Maternal L-Cit supplementation prevented these effects. The CR+Cit and Cit groups developed hypertension beginning at 4 and 8weeks of age, respectively. Plasma asymmetric and symmetric dimethylarginine (ADMA and SDMA) levels were increased, but L-arginine/ADMA ratios (AAR) were decreased in the CR group vs the control group. This was prevented by maternal L-Cit supplementation. Renal cortical neuronal NOS-alpha (nNOSalpha) protein abundance was significantly decreased in the Cit and CR+Cit groups. Collectively, reduced nephron number, reduced renal nNOSalpha expression, increased ADMA, and decreased AAR contribute to the developmental programming of adult kidney disease and hypertension. Although maternal L-Cit supplementation prevents caloric restriction-induced low nephron number and renal dysfunction, it also induces hypertension.

Aberrations of ammonia metabolism in ornithine carbamoyltransferase-deficient spf-ash mice and their prevention by treatment with urea cycle intermediate amino acids and an ornithine aminotransferase inactivator.

Sparse fur with abnormal skin and hair (spf-ash) mice are deficient in ornithine carbamoyltransferase (OCT) activity, but their OCT protein is kinetically normal. We administered ammonium chloride to spf-ash mice, in order to analyze ammonia metabolism and to find a rationale for the therapy of OCT deficiency. Ammonia concentration in the liver of spf-ash mice increased to a level much higher than in the control. Ammonium chloride injection caused an increase in ornithine (Orn) 5 min after injection and an increase in the sum of Orn, citrulline (Cit) and arginine (Arg) for at least 15 min in the liver of control mice, but no increase in Orn, Cit and Arg in the liver of spf-ash mice. Treatment of spf-ash mice with Arg 5-20 min prior to the injection of ammonium chloride kept the hepatic ammonia concentration at a level comparable to that without the load. A significant reciprocal relationship between ammonia and Orn concentrations in the liver of spf-ash mice 5 min after an ammonium chloride load with or without Arg strongly suggests that ammonia disposal is dependent on the supply of Orn. In spf-ash mice loaded with tryptone as a nitrogen source, Arg supplementation showed a dramatic decrease in urinary orotic acid excretion in a dose-dependent manner. Similar effects were observed with Cit and Orn at the same dose, and a long-lasting effect with an ornithine aminotransferase inactivator, 5-(fluoromethyl)ornithine, at a much lower dose. The rate of urea formation in liver perfused with ammonium chloride was lower in spf-ash mice than in controls, but with the addition of Orn to the medium it increased to a similar level in control and spf-ash mice. These results indicate that OCT is not saturated with Orn in vivo under physiological conditions and that the administration or enrichment of the urea cycle intermediate amino acids enhances the OCT reaction so that the ammonia metabolism of OCT-deficient spf-ash mice is at least partially normalized.

Maternal aggression is reduced in neuronal nitric oxide synthase-deficient mice.

Lactating females express rapid extremes in behavior, ranging from gentle nurturance toward offspring to fiercely protective aggression against intruders. Although males often behave aggressively against intruders, female rodents usually express aggression only when rearing and protecting pups. Nitric oxide (NO) inhibits male aggression; however, its role in maternal aggression is unknown. In the present study, female mice with targeted disruption of the neuronal nitric oxide synthase gene (nNOS-/-) displayed significant deficits in maternal aggression relative to wild-type (WT) mice in terms of percentage displaying aggression, the average number of attacks against a male intruder, and the total time spent attacking the male intruder. The nNOS-/- mice displayed normal pup retrieval behavior. Because the specific deficits in maternal aggression in the nNOS-/- mice suggested a possible role for NO in maternal aggression, we combined behavioral testing of WT mice with immunohistochemistry for citrulline, an indirect marker of NO synthesis, to examine indirectly NO synthesis during maternal aggression. A significant increase in the number of citrulline-positive cells was identified in the medial preoptic nucleus, the suprachiasmatic nucleus, and the subparaventricular zone regions of the hypothalamus in aggressive lactating females relative to control mice. In other regions of the brain, no changes in the number of citrulline-positive cells were observed across either groups or treatments. These results provide two indirect lines of evidence that NO release is associated with maternal aggression.