Effects of Acute Beetroot Juice and Sodium Nitrate on Selected Blood Metabolites and Response to Transient Ischemia: A Crossover Randomized Clinical Trial.

BACKGROUND: Modification of the nitrate (NO3)-nitrite (NO2)-nitric oxide (NO) pathway can be induced by oral intake of inorganic NO3 (NIT) or NO3-rich products, such as beetroot juice (BRJ). OBJECTIVES: The primary aim of this study was to evaluate the plasma changes in betaine, choline, trimethylamine (TMA), trimethylamine N-oxide (TMAO), and NO3/NO2 (NOx) concentrations over 4 h after single oral ingestion of NIT or BRJ. The flow-mediated skin fluorescence (FMSF) method was applied to measure the changes in nicotinamide adenine dinucleotide reduced form (NADH) in response to transient ischemia and reperfusion. We hypothesized that various sources of NO3 may differently affect endothelial and mitochondrial functions in healthy human subjects. METHODS: In a randomized crossover trial, 8 healthy young adults ingested 800 mg NO3 from either NIT or BRJ on 2 separate days with ≥3 d apart. Venous blood samples were collected every hour, and FMSF determination was applied bihourly. RESULTS: Plasma betaine and choline concentrations peaked at 1 h after BRJ ingestion, and remained significantly higher than baseline values at all time points (P < 0.001 and P < 0.001, compared to preingestion values). Over time, BRJ was more effective in increasing NOx compared with NIT (fixed-trial effect P < 0.001). Baseline fluorescence decreased after both NIT and BRJ consumption (fixed-time effect P = 0.005). Transient ischemia and reperfusion response increased because of NO3 consumption (fixed-time effect P = 0.003), with no differences between trials (P = 0.451; P = 0.912; P = 0.819 at 0, 2, and 4 h, respectively). CONCLUSIONS: Acute ingestion of BRJ elevated plasma betaine and choline, but not TMA and TMAO. Moreover, plasma NOx levels were higher in the BRJ trial than in the NIT trial. Various sources of NO3 positively affected endothelial and mitochondrial functions. This trial was registered at clinicaltrials.gov as NCT05004935.

Urinary excretion of asymmetric (ADMA) and symmetric (SDMA) dimethylarginine is positively related to nitric oxide level in tissues of normotensive and hypertensive rats.

Nitric oxide (NO) is one of the gaseous transmitters which play a very important role in the regulation of the circulatory system. Decreased NO availability is associated with hypertension, cardiovascular and kidney diseases. Endogenous NO is generated enzymatically by nitric oxide synthase (NOS) depending on the availability of the substrate, cofactors, or presence/absence of inhibitors, such as asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). The objective of this study was to evaluate the potential relationship between NO level in rat tissues (heart and kidneys) and the concentrations of endogenous metabolites related to NO in plasma and urine. The experiment was carried out with 16- and 60-week-old male Wistar Kyoto (WKY) and age-matched male Spontaneously Hypertensive Rats (SHR). NO level in tissue homogenates was determined by the colorimetric method. RT-qPCR was used to verify the expression of the eNOS (endothelial NOS) gene. Plasma and urine concentrations of arginine, ornithine, citrulline, and dimethylarginines were examined by the UPLC-MS/MS method. 16-week-old WKY rats had the highest tissue NO and plasma citrulline levels. Furthermore, 16-week-old WKY rats showed higher urinary excretion of ADMA/SDMA compared to other experimental groups, however, plasma concentrations of arginine, ADMA, and SDMA were comparable between the groups. In conclusion, our research shows that hypertension and aging decrease tissue NO levels and are associated with reduced urinary excretion of NOS inhibitors, i.e., ADMA and SDMA.

Twenty-Four Weeks of L-Carnitine Combined with Leucine Supplementation Does Not Increase the Muscle Carnitine Content in Healthy Active Subjects.

INTRODUCTION: To increase the total carnitine (TC) content in muscles, L-carnitine (LC) should be co-ingested with carbohydrates to induce an insulin response. Leucine has an insulin secretagogue effect. Therefore, the primary aim of this study was to examine the effects of 24 weeks of LC and leucine supplementation on the skeletal muscle TC content, muscle mass, and strength in active college-aged subjects. The secondary aim was to determine the activation of the Akt/mTOR signaling pathway in skeletal muscles after supplementation. METHODS: Over the 24 weeks, the participants were supplemented with either 1 g of LC-L-tartrate and 3 g of leucine per day (LC + L group; n = 7) or 4 g of leucine per day (L group; n = 7) as a placebo. Before and 24 weeks after the initiation of the study protocol, the free carnitine (FC) and TC content in plasma and muscle samples, as well as body composition and muscle strength, were measured. In addition, the phosphorylation of the Akt/mTOR pathway proteins in muscles was evaluated. RESULTS: Plasma FC and TC content increased in LC + L group after 24 weeks of supplementation (p = 0.003 and 0.010, respectively). However, the skeletal muscle FC and TC contents were not affected by the supplementation protocol. No changes were noted in the body mass and composition; serum insulin-like growth factor-1 concentration; and phosphorylation of the signaling pathway proteins Akt, mTOR, and p70S6K. CONCLUSION: LC supplementation may have the potential to exert beneficial effects in muscle atrophy. Therefore, additional research is necessary to investigate the effect of various LC supplementation protocols.

Trimethylamine, a gut bacteria metabolite and air pollutant, increases blood pressure and markers of kidney damage including proteinuria and KIM-1 in rats.

BACKGROUND: Trimethylamine oxide (TMAO) is a biomarker in cardiovascular and renal diseases. TMAO originates from the oxidation of trimethylamine (TMA), a product of gut microbiota and manufacturing industries-derived pollutant, by flavin monooxygenases (FMOs). The effect of chronic exposure to TMA on cardiovascular and renal systems is undetermined. METHODS: Metabolic, hemodynamic, echocardiographic, biochemical and histopathological evaluations were performed in 12-week-old male SPRD rats receiving water (controls) or TMA (200 or 500 µM/day) in water for 18 weeks. TMA and TMAO levels, the expression of FMOs and renin-angiotensin system (RAS) genes were evaluated in various tissues. RESULTS: In comparison to controls, rats receiving high dose of TMA had significantly increased arterial systolic blood pressure (126.3 ± 11.4 vs 151.2 ± 19.9 mmHg; P = 0.01), urine protein to creatinine ratio (1.6 (1.5; 2.8) vs 3.4 (3.3; 4.2); P = 0.01), urine KIM-1 levels (2338.3 ± 732.0 vs. 3519.0 ± 953.0 pg/mL; P = 0.01), and hypertrophy of the tunica media of arteries and arterioles (36.61 ± 0.15 vs 45.05 ± 2.90 µm, P = 0.001 and 18.44 ± 0.62 vs 23.79 ± 2.60 µm, P = 0.006; respectively). Mild degeneration of renal bodies with glomerulosclerosis was also observed. There was no significant difference between the three groups in body weight, water-electrolyte balance, echocardiographic parameters and RAS expression. TMA groups had marginally increased 24 h TMA urine excretion, whereas serum levels and 24 h TMAO urine excretion were increased up to 24-fold, and significantly increased TMAO levels in the liver, kidneys and heart. TMA groups had lower FMOs expression in the kidneys. CONCLUSIONS: Chronic exposure to TMA increases blood pressure and increases markers of kidney damage, including proteinuria and KIM-1. TMA is rapidly oxidized to TMAO in rats, which may limit the toxic effects of TMA on other organs.

Heart Failure Disturbs Gut-Blood Barrier and Increases Plasma Trimethylamine, a Toxic Bacterial Metabolite.

Trimethylamine (TMA) is a gut bacteria product oxidized by the liver to trimethylamine-N-oxide (TMAO). Clinical evidence suggests that cardiovascular disease is associated with increased plasma TMAO. However, little headway has been made in understanding this relationship on a mechanistic and molecular level. We investigated the mechanisms affecting plasma levels of TMAO in Spontaneously Hypertensive Heart Failure (SHHF) rats. Healthy Wistar Kyoto (WKY) and SHHF rats underwent metabolic, hemodynamic, histopathological and biochemical measurements, including tight junction proteins analysis. Stool, plasma and urine samples were evaluated for TMA and TMAO using ultra performance liquid chromatography-mass spectrometry. SHHF presented disturbances of the gut-blood barrier including reduced intestinal blood flow, decreased thickness of the colonic mucosa and alterations in tight junctions, such as claudin 1 and 3, and zonula occludens-1. This was associated with significantly higher plasma levels of TMA and TMAO and increased gut-to-blood penetration of TMA in SHHF compared to WKY. There was no difference in kidney function or liver oxidation of TMA to TMAO between WKY and SHHF. In conclusion, increased plasma TMAO in heart failure rats results from a perturbed gut-blood barrier and increased gut-to-blood passage of TMAO precursor, i.e., TMA. Increased gut-to-blood penetration of bacterial metabolites may be a marker and a mediator of cardiovascular pathology.

Butyric acid, a gut bacteria metabolite, lowers arterial blood pressure via colon-vagus nerve signaling and GPR41/43 receptors.

Butyric acid (BA) is a short-chain fatty acid (SCFA) produced by gut bacteria in the colon. We hypothesized that colon-derived BA may affect hemodynamics. Arterial blood pressure (BP) and heart rate (HR) were recorded in anesthetized, male, 14-week-old Wistar rats. A vehicle, BA, or 3-hydroxybutyrate, an antagonist of SCFA receptors GPR41/43 (ANT) were administered intravenously (IV) or into the colon (IC). Reactivity of mesenteric (MA) and gracilis muscle (GMA) arteries was tested ex vivo. The concentration of BA in stools, urine, portal, and systemic blood was measured with liquid chromatography coupled with mass spectrometry. BA administered IV decreased BP with no significant effect on HR. The ANT reduced, whereas L-NAME, a nitric oxide synthase inhibitor, did not affect the hypotensive effect of BA. In comparison to BA administered intravenously, BA administered into the colon produced a significantly longer decrease in BP and a decrease in HR, which was associated with a 2-3-fold increase in BA colon content. Subphrenic vagotomy and IC pretreatment with the ANT significantly reduced the hypotensive effect. Ex vivo, BA dilated MA and GMA. In conclusion, an increase in the concentration of BA in the colon produces a significant hypotensive effect which depends on the afferent colonic vagus nerve signaling and GPR41/43 receptors. BA seems to be one of mediators between gut microbiota and the circulatory system.

Chronic, low-dose TMAO treatment reduces diastolic dysfunction and heart fibrosis in hypertensive rats.

Several studies have suggested negative effects of trimethylamine oxide (TMAO) on the circulatory system. However, a number of studies have shown protective functions of TMAO, a piezolyte and osmolyte, in animals exposed to high hydrostatic and/or osmotic stress. We evaluated the effects of TMAO treatment on the development of hypertension and its complications in male spontaneously hypertensive rats (SHRs) maintained on water (SHR-Water) and SHRs drinking TMAO water solution from weaning (SHR-TMAO). Wistar-Kyoto (WKY) rats were used as normotensive controls to discriminate between age-dependent and hypertension-dependent changes. Telemetry measurements of blood pressure were performed in rats between the 7th and 16th weeks of life. Anesthetized rats underwent echocardiographic, electrocardiographic, and direct left ventricular end-diastolic pressure (LVEDP) measurements. Hematoxylin and eosin as well as van Gieson staining for histopathological evaluation were performed. Plasma TMAO measured by chromatography coupled with mass spectrometry was significantly higher in the SHR-Water group compared with the WKY group (~20%). TMAO treatment increased plasma TMAO by four- to fivefold and did not affect the development of hypertension in SHRs. Sixteen-week-old rats in the SHR-Water and SHR-TMAO groups (12-wk TMAO treatment) showed similar blood pressures, angiopathy, and cardiac hypertrophy. However, the SHR-TMAO group had lower plasma NH2-terminal pro-B-type natriuretic peptide, LVEDP, and cardiac fibrosis. In contrast to age-matched WKY rats, 60-wk-old SHRs showed hypertensive angiopathy and heart failure with preserved ejection fraction. Compared with the SHR-Water group, the SHR-TMAO group (56-wk TMAO treatment) showed significantly lower plasma NH2-terminal pro-B-type natriuretic peptide and vasopressin, significantly lower LVEDP, and cardiac fibrosis. In conclusion, a four- to fivefold increase in plasma TMAO does not exert negative effects on the circulatory system. In contrast, increased dietary TMAO seems to reduce diastolic dysfunction in pressure-overloaded hearts in rats. NEW & NOTEWORTHY Chronic, low-dose trimethylamine oxide (TMAO) treatment that increases plasma TMAO by four- to fivefold reduces plasma NH2-terminal pro-B-type natriuretic peptide and vasopressin, left ventricular end-diastolic pressure, and cardiac fibrosis in pressure-overloaded hearts in hypertensive rats. Our study provides evidence that a moderate increase in plasma TMAO does not have a negative effect on the circulatory system. In contrast, increased dietary TMAO seems to reduce diastolic dysfunction in the pressure-overloaded heart.

Enalapril decreases rat plasma concentration of TMAO, a gut bacteria-derived cardiovascular marker.

INTRODUCTION: Increased plasma level of trimethylamine N-oxide (TMAO), a bacterial metabolite of choline, is associated with an increased cardiovascular risk. Indoxyl sulfate, a bacterial metabolite of tryptophan, is thought to be associated with higher mortality in cardiorenal syndrome. We hypothesized that enalapril, a well-established drug reducing cardiovascular mortality, may affect the plasma level of gut bacteria-derived metabolites and gut bacteria composition. MATERIALS AND METHODS: 14-16-week-old Wistar rats were maintained either on water (controls) or water solution of enalapril for two weeks (5.3 or 12.6 mg/kg b.w.). Blood plasma and urine were analyzed for the concentration of TMAO and indoxyl sulfate using liquid chromatography coupled with triple-quadrupole mass spectrometry. Gut bacteria composition was analyzed with 16S rRNA gene sequence analysis. RESULTS: Rats treated with enalapril showed a significantly lower plasma TMAO level and a trend towards higher 24 h urine excretion of TMA and TMAO. Plasma indoxyl level was similar between the groups. There was no significant difference between the groups in gut bacteria composition. CONCLUSIONS: Enalapril decreases rat plasma TMAO, but does not affect the plasma level of indoxyl sulfate and gut bacteria composition. The enalapril-induced decrease in plasma TMAO level may be of therapeutic and diagnostic importance.

Total testosterone to dihydrotestosterone ratio assessed by LC-MS/MS predicts a worse metabolic profile not only in PCOS patients.

OBJECTIVES: Total testosterone/dihydrotestosterone ratio (TT/DHT) was found to determine metabolic risk in polycystic ovary syndrome (PCOS). The aim of this study was to analyze whether (TT/DHT) may be helpful in predicting metabolic risk not only in PCOS patients but also in healthy women. MATERIAL AND METHODS: Total testosterone (TT), dihydrotestosterone (DHT), androstendione and dehydroepiandrosterone sulphate (DHEA-S) were measured by LC-MS/MS in 36 women with PCOS and in 29 age-matched controls without clinical hyperandrogenism. In all participants, anthropometric data, lipids, adipose tissue percent (%fat), HOMA-IR were also assessed. RESULTS: The studied groups were not different in terms of age, BMI, waist circumference, %fat and HOMA-IR. In the patients group, mean TT and androstendione levels were significantly higher as compared to controls (1.4 nmol/L vs. 1.0 nmol/L, P < 0.001) and (6.6 nmol/L vs. 4.9 nmol/L, P < 0.01), respectively. In the patients group, mean TT/DHT ratio was significantly higher compared to controls (3.6 vs. 2.7, P < 0.01) and correlated with BMI (r = 0.37, P < 0.05), waist circumference (r = 0.44, P < 0.01), %fat (r = 0.30, P < 0.05), as well as with insulin levels (r = 0.38, P < 0.05) and HOMA-IR (r = 0.44, P < 0.05). The association between TT/DHT ratio and unfavorable metabolic parameters was also seen in controls. CONCLUSION: Total testosterone/dihydrotestosterone ratio assessed by LC-MS/MS correlates with a worse metabolic profile not only in PCOS patients, but also in healthy women.

The Potential Role of SCFAs in Modulating Cardiometabolic Risk by Interacting with Adiposity Parameters and Diet.

The main objective of this cross-sectional study was to analyze the influence of lifestyle factors (diet, physical activity, sleep) that can affect the concentration of fecal short-chain fatty acids (SCFAs) and SCFAs' potential role in modulating cardiometabolic disease risk by interacting with biochemical and body composition parameters. The study comprised 77 healthy, non-obese individuals aged 30-45 years who were assessed for the concentration of SCFAs in stool, diet, physical activity level, and sleep duration. Moreover, body composition measurement and patients' biochemical parameters were included in the analysis. We have indicated a significant negative correlation between several SCFAs (especially acetic acid (AA), isobutyric acid (IBA), butyric acid (BA), propionic acid (PA), isovaleric acid (IVA) and valeric acid (VA)) with BMI, VAT/SAT ratio (visceral to subcutaneous fat ratio), and percentage of fat mass in a group of females enrolled in the study as well as with waist circumference (WC) in case of both sexes included in the study. Moreover, the results of our study acknowledged the importance of a diet in shaping the SCFA profile-we noticed significant negative associations between energy and fat intake and some SCFAs in males (IBA, IVA, VA, isocaproic acid (ICA)). Further, we indicated that a high intake of fiber (insoluble and soluble) in both males and females results in an elevated concentration of the vast majority of SCFAs and the amount of SCFAs in total. This effect was particularly noticeable in the case of the soluble fraction of fiber. These correlations reflect the fact that diet shapes the composition of the gut microbiota and SCFAs (main microbial metabolites) are synthesized from dietary fiber. In addition, we noticed that in a group of women, the concentration of AA, PA, and ICA as well as the total concentration of SCFAs showed a significant positive association with their sleep duration. We concluded that SCFAs can have a potential role in modulating cardiometabolic disease risk by interacting with adiposity parameters and diet. In addition, this potential direct link between diet and SCFAs may at least partly contribute to sleep improvement.

Mice, rats, and guinea pigs differ in FMOs expression and tissue concentration of TMAO, a gut bacteria-derived biomarker of cardiovascular and metabolic diseases.

INTRODUCTION: Increased plasma trimethylamine oxide (TMAO) is observed in cardiovascular and metabolic diseases, originating from the gut microbiota product, trimethylamine (TMA), via flavin-containing monooxygenases (FMOs)-dependent oxidation. Numerous studies have investigated the association between plasma TMAO and various pathologies, yet limited knowledge exists regarding tissue concentrations of TMAO, TMAO precursors, and interspecies variability. METHODS: Chromatography coupled with mass spectrometry was employed to evaluate tissue concentrations of TMAO and its precursors in adult male mice, rats, and guinea pigs. FMO mRNA and protein levels were assessed through PCR and Western blot, respectively. RESULTS: Plasma TMAO levels were similar among the studied species. However, significant differences in tissue concentrations of TMAO were observed between mice, rats, and guinea pigs. The rat renal medulla exhibited the highest TMAO concentration, while the lowest was found in the mouse liver. Mice demonstrated significantly higher plasma TMA concentrations compared to rats and guinea pigs, with the highest TMA concentration found in the mouse renal medulla and the lowest in the rat lungs. FMO5 exhibited the highest expression in mouse liver, while FMO3 was highly expressed in rats. Guinea pigs displayed low expression of FMOs in this tissue. CONCLUSION: Despite similar plasma TMAO levels, mice, rats, and guinea pigs exhibited significant differences in tissue concentrations of TMA, TMAO, and FMO expression. These interspecies variations should be considered in the design and interpretation of experimental studies. Furthermore, these findings may suggest a diverse importance of the TMAO pathway in the physiology of the evaluated species.

Spontaneously hypertensive rats exhibit increased liver flavin monooxygenase expression and elevated plasma TMAO levels compared to normotensive and Ang II-dependent hypertensive rats.

Background: Flavin monooxygenases (FMOs) are enzymes responsible for the oxidation of a broad spectrum of exogenous and endogenous amines. There is increasing evidence that trimethylamine (TMA), a compound produced by gut bacteria and also recognized as an industrial pollutant, contributes to cardiovascular diseases. FMOs convert TMA into trimethylamine oxide (TMAO), which is an emerging marker of cardiovascular risk. This study hypothesized that blood pressure phenotypes in rats might be associated with variations in the expression of FMOs. Methods: The expression of FMO1, FMO3, and FMO5 was evaluated in the kidneys, liver, lungs, small intestine, and large intestine of normotensive male Wistar-Kyoto rats (WKY) and two distinct hypertensive rat models: spontaneously hypertensive rats (SHRs) and WKY rats with angiotensin II-induced hypertension (WKY-ANG). Plasma concentrations of TMA and TMAO were measured at baseline and after intravenous administration of TMA using liquid chromatography-mass spectrometry (LC-MS). Results: We found that the expression of FMOs in WKY, SHR, and WKY-ANG rats was in the descending order of FMO3 > FMO1 >> FMO5. The highest expression of FMOs was observed in the liver. Notably, SHRs exhibited a significantly elevated expression of FMO3 in the liver compared to WKY and WKY-ANG rats. Additionally, the plasma TMAO/TMA ratio was significantly higher in SHRs than in WKY rats. Conclusion: SHRs demonstrate enhanced expression of FMO3 and a higher plasma TMAO/TMA ratio. The variability in the expression of FMOs and the metabolism of amines might contribute to the hypertensive phenotype observed in SHRs.

Neutrophil functions in patients with neutropenia due to glycogen storage disease type 1b treated with empagliflozin.

ABSTRACT: Neutropenia and neutrophil dysfunction in glycogen storage disease type 1b (GSD1b) are caused by the accumulation of 1,5-anhydroglucitol-6-phosphate in granulocytes. The antidiabetic drug empagliflozin reduces the concentration of 1,5-anhydroglucitol (1,5-AG), thus restoring neutrophil counts and functions, leading to promising results in previous case reports. Here, we present a comprehensive analysis of neutrophil function in 7 patients with GSD1b and 11 healthy donors, aiming to evaluate the immediate (after 3 months) and long-term (after 12 months) efficacy of empagliflozin compared with the reference treatment with granulocyte-colony stimulating factor (G-CSF). We found that most patients receiving G-CSF remained neutropenic with dysfunctional granulocytes, whereas treatment with empagliflozin increased neutrophil counts and improved functionality by inhibiting apoptosis, restoring phagocytosis and the chemotactic response, normalizing the oxidative burst, and stabilizing cellular and plasma levels of defensins and lactotransferrin. These improvements correlated with the decrease in serum 1,5-AG levels. However, neither G-CSF nor empagliflozin overcame deficiencies in the production of cathelicidin/LL-37 and neutrophil extracellular traps. Given the general improvement promoted by empagliflozin treatment, patients were less susceptible to severe infections. G-CSF injections were therefore discontinued in 6 patients (and the dose was reduced in the seventh) without adverse effects. Our systematic analysis, the most extensive reported thus far, has demonstrated the superior efficacy of empagliflozin compared with G-CSF, restoring the neutrophil population and normal immune functions. This trial was registered as EudraCT 2021-000580-78.

Mice, Rats and Guinea Pigs Exhibit Significant Variations in the Plasma, Urine and Tissue Levels of Taurine, Betaine, Sarcosine and Other Osmolyte-Active Amino Acids.

BACKGROUND: Osmolytes are naturally occurring compounds that protect cells from osmotic stress in high-osmolarity tissues, such as the kidney medulla. Some amino acids, including taurine, betaine, glycine, alanine, and sarcosine, are known to act as osmolytes. This study aimed to establish the levels of these amino acids in body fluids and tissues of laboratory animals used as models for human diseases in biomedical research. METHODS: Liquid chromatography coupled with mass spectrometry was used to quantify taurine, glycine, betaine, alanine, beta-alanine, and sarcosine in plasma, urine, and tissues of adult, male mice, rats and guinea pigs. RESULTS: Among the species analyzed, taurine was found to have the highest tissue concentrations across all compounds, with the heart containing the greatest amount. In guinea pigs, betaine levels were higher in the renal medulla than in the renal cortex (p < 0.01), while in rats and mice, there were no significant differences in betaine levels between the kidney cortex and medulla. The urine of guinea pigs had lower levels of sarcosine compared to rats (p < 0.001), while the plasma (p < 0.05; > 0.05), heart (p < 0.05; < 0.05), lungs (p < 0.01; < 0.01), liver (p < 0.001; < 0.05), and kidneys (p < 0.01; < 0.01) of rats exhibited notably higher concentrations of sarcosine compared to both mice and guinea pigs, respectively. CONCLUSIONS: There are pronounced differences in the concentrations of taurine, betaine, and other amino acids across the investigated species. It is important to acknowledge these differences when selecting animal models for preclinical studies and to account for variations in amino acid concentrations when selecting amino acids doses for interventional studies.

Effect of a 3-month L-carnitine supplementation and resistance training program on circulating markers and bone mineral density in postmenopausal women: a randomized controlled trial.

BACKGROUND: Higher circulating levels of trimethylamine N-oxide (TMAO), which is a metabolite that can be produced by the gut microbiota from L-carnitine (LC), have been associated with bone mineral density (BMD). Because LC supplementation can improve bone density and microstructural properties in animal models, this study aimed to examine the effects of 12 weeks of LC supplementation on BMD and selected blood markers involved in bone metabolism of postmenopausal women participating in a resistance training (RT) program. METHODS: Twenty-seven postmenopausal women, who had not been treated for osteoporosis, with a total T-score above - 3.0 and no diet differences completed 12 weeks of RT. The participants' diets were supplemented with either 1 g of LC-L-tartrate and 3 g of leucine per day (LC group) or 4 g of leucine per day as a placebo (PLA group), in a double-blind fashion. RESULTS: After the intervention in the LC group, plasma total carnitine and serum decorin levels were higher than the corresponding preintervention values (p = 0.040 and p = 0.042, respectively). Moreover, plasma TMAO and serum SPARC levels were higher in the LC group than the corresponding postintervention values in the PLA group (p < 0.001 and p = 0.030, respectively). No changes in the BMD were observed after 3 months of the intervention. CONCLUSIONS: Twelve weeks of LC supplementation during RT program increased plasma TMAO levels and appeared to affect signaling molecules, as indicated by the increase in the resting SPARC and decorin levels, with no significant modification in the BMD. TRIAL REGISTRATION: Retrospectively registered at the ClinicalTrials.gov (NCT05120011).

The interplay between gingival crevicular fluid microbiome and metabolomic profile in intensively treated people with type 1 diabetes - a combined metagenomic/metabolomic approach cross-sectional study.

Aims: This study aimed to assess the gingival crevicular fluid (GCF) microbiome and metabolome of adults with type 1 diabetes (T1D) treated with continuous subcutaneous insulin infusion (CSII). Methods: In this cross-sectional study, the GCF of adults with T1D treated with CSII and non-diabetic controls were sampled, and metagenomic/metabolomic analyses were performed. Results: In total, 65 participants with T1D and 45 healthy controls with a mean age of 27.05 ± 5.95 years were investigated. There were 22 cases of mild gingivitis (G) in the T1D group. There were no differences considering the Shannon and Chao indices and ß-diversity between people with T1D and G, with T1D without G, and healthy controls. Differential taxa were identified, which were mainly enriched in people with T1D and G. Acetic acid concentration was higher in people with T1D, regardless of the presence of G, than in healthy controls. Propionic acid was higher in people with T1D and G than in healthy controls. Isobutyric and isovaleric acid levels were higher in individuals with T1D and G than in the other two subgroups. The concentration of valeric acid was lower and that of caproic acid was higher in people with T1D (regardless of gingival status) than in healthy controls. Conclusions: The identification of early changes in periodontal tissues by targeting the microbiome and metabolome could potentially enable effective prevention and initial treatment of periodontal disease in people with T1D.

Relationship between Bone Mineral Density and Selected Parameters of Calcium-Phosphate Economy with Dietary Management and Metabolic Control in Polish Pediatric Patients with Classical Homocystinuria-A Preliminary Study.

BACKGROUND: Classical homocystinuria (HCU) is an inborn defect of methionine metabolism caused by a deficiency of the enzyme cystathionine ß-synthase (CBS). The main symptoms of classical homocystinuria are lens subluxation, bone lesions, vascular disease and developmental delay/intellectual disability. The treatment method for HCU is a methionine-poor diet supplemented with amino acid preparations. The aim of the study was to examine the relationship of dietary factors, metabolic compensation and selected skeletal parameters in patients with HCU. METHODS: Bone mineral density measurements (DXA) were performed in pediatric patients with HCU, and blood levels of selected amino acids, minerals and vitamins, as well as dietary nutritional value, were analyzed. RESULTS: A total of 11 patients with HCU whose median age was 9.3 years were enrolled in the study. The median DXA total body less head of HCU patients was -0.4 z-score, and the lumbar spine was -1.4 z-score. Despite supplementation, calcium intake was below the age norm. Average vitamin D3 intake was in line with recommendations, but 36% of patients had reduced blood levels. Bone mineral density depended on blood levels of 25-hydroxyvitamin D, homocysteine and methionine, as well as on BMI, age and intake of natural protein (R2 = 98.5%, p = 0.015; R2 = 86.7%, p = 0.0049) and protein from an amino acid preparation (r = 0.69, p = 0.026). CONCLUSION: The results of the study indicate the need for regular densitometry in patients with HCU and also the use of additional calcium and vitamin D3 supplementation. It is also necessary to perform a comprehensive analysis of the diet and metabolic controls.

Serum Metabolite Biomarkers for Pancreatic Tumors: Neuroendocrine and Pancreatic Ductal Adenocarcinomas-A Preliminary Study.

BACKGROUND: Pancreatic cancer is the most common pancreatic solid malignancy with an aggressive clinical course and low survival rate. There are a limited number of reliable prognostic biomarkers and a need to understand the pathogenesis of pancreatic tumors; neuroendocrine (PNET) and pancreatic ductal adenocarcinomas (PDAC) encouraged us to analyze the serum metabolome of pancreatic tumors and disturbances in the metabolism of PDAC and PNET. METHODS: Using the AbsoluteIDQ® p180 kit (Biocrates Life Sciences AG, Innsbruck, Austria) with liquid chromatography-mass spectrometry (LC-MS), we identified changes in metabolite profiles and disrupted metabolic pathways serum of NET and PDAC patients. RESULTS: The concentration of six metabolites showed statistically significant differences between the control group and PDAC patients (p.adj < 0.05). Glutamine (Gln), acetylcarnitine (C2), and citrulline (Cit) presented a lower concentration in the serum of PDAC patients, while phosphatidylcholine aa C32:0 (PC aa C32:0), sphingomyelin C26:1 (SM C26:1), and glutamic acid (Glu) achieved higher concentrations compared to serum samples from healthy individuals. Five of the tested metabolites: C2 (FC = 8.67), and serotonin (FC = 2.68) reached higher concentration values in the PNET serum samples compared to PDAC, while phosphatidylcholine aa C34:1 (PC aa C34:1) (FC = -1.46 (0.68)) had a higher concentration in the PDAC samples. The area under the curves (AUC) of the receiver operating characteristic (ROC) curves presented diagnostic power to discriminate pancreatic tumor patients, which were highest for acylcarnitines: C2 with AUC = 0.93, serotonin with AUC = 0.85, and PC aa C34:1 with AUC = 0.86. CONCLUSIONS: The observations presented provide better insight into the metabolism of pancreatic tumors, and improve the diagnosis and classification of tumors. Serum-circulating metabolites can be easily monitored without invasive procedures and show the present clinical patients' condition, helping with pharmacological treatment or dietary strategies.

Simvastatin does not alleviate muscle pathology in a mouse model of Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is an incurable disease, caused by the mutations in the DMD gene, encoding dystrophin, an actin-binding cytoskeletal protein. Lack of functional dystrophin results in muscle weakness, degeneration, and as an outcome cardiac and respiratory failure. As there is still no cure for affected individuals, the pharmacological compounds with the potential to treat or at least attenuate the symptoms of the disease are under constant evaluation. The pleiotropic agents, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, known as statins, have been suggested to exert beneficial effects in the mouse model of DMD. On the other hand, they were also reported to induce skeletal-muscle myopathy. Therefore, we decided to verify the hypothesis that simvastatin may be considered a potential therapeutic agent in DMD. METHODS: Several methods including functional assessment of muscle function via grip strength measurement, treadmill test, and single-muscle force estimation, enzymatic assays, histological analysis of muscle damage, gene expression evaluation, and immunofluorescence staining were conducted to study simvastatin-related alterations in the mdx mouse model of DMD. RESULTS: In our study, simvastatin treatment of mdx mice did not result in improved running performance, grip strength, or specific force of the single muscle. Creatine kinase and lactate dehydrogenase activity, markers of muscle injury, were also unaffected by simvastatin delivery in mdx mice. Furthermore, no significant changes in inflammation, fibrosis, and angiogenesis were noted. Despite the decreased percentage of centrally nucleated myofibers in gastrocnemius muscle after simvastatin delivery, no changes were noticed in other regeneration-related parameters. Of note, even an increased rate of necrosis was found in simvastatin-treated mdx mice. CONCLUSION: In conclusion, our study revealed that simvastatin does not ameliorate DMD pathology.

Clinical Implications of Intestinal Barrier Damage in Psoriasis.

BACKGROUND: An increasing amount of evidence suggests an association between increased intestinal permeability and the pathogenesis of chronic inflammatory diseases. However, the clinical significance of gut barrier dysfunction in psoriasis remains to be established. OBJECTIVE: To evaluate whether there are differences in disease activity, the severity of gastrointestinal symptoms and the blood concentration of bacterial metabolites in psoriatic patients with a normal and altered intestinal barrier. PATIENTS AND METHODS: Gut barrier integrity was assessed with the serum concentrations of claudin-3, a modulator of intestinal tight junctions and an intestinal fatty acid-binding protein, a marker of enterocyte damage. Gastrointestinal symptoms were evaluated with a validated questionnaire. The concentration of trimethylamine N-oxide (TMAO), a gut microbiota-associated metabolite, was measured with high-performance liquid chromatography. RESULTS: One hundred and fourteen patients with psoriasis were finally enrolled in the study - 68 with an altered gut barrier and 46 with a properly functioning intestinal barrier. Patients with an altered gut barrier showed a significantly higher score in the Gastrointestinal Symptom Rating Scale (3.20 vs 1.46, p<0.001). Moreover, patients with psoriasis and a disrupted intestinal barrier demonstrated a higher disease activity (PASI: 19.7 vs 10.3, p<0.001) and systemic inflammatory parameters (neutrophil-to-lymphocyte ratio: 2.86 vs 1.71, p<0.001; C-reactive protein 3.76 vs 1.92; p<0.05). The marker of bacterial translocation was significantly higher in psoriatic patients with damaged gut integrity (TMAO: 375.7±51.9 vs 119.4±27.5 ng/mL; p<0.05). CONCLUSION: The altered gut barrier in psoriasis is associated with gastrointestinal symptoms, systemic inflammatory profile and the increased blood concentration of gut microbiota-derived metabolite - TMAO. Intestinal barrier modulation represents a new promising therapeutic approach.