ABSTRACT
The central position of methionine (Met) in protein metabolism indicates the importance of this essential amino acid for growth and maintenance of lean body mass. Therefore, Met might be a tempting candidate for supplementation. However, because Met is also the precursor of homocysteine (Hcy), a deficient intake of B vitamins or excessive intake of Met may result in hyperhomocysteinemia (HHcy), which is a risk factor for cardiovascular disease. This review discusses the evidence generated in preclinical and clinical studies on the importance and potentially harmful effects of Met supplementation and elaborates on potential clinical applications of supplemental Met with reference to clinical studies performed over the past 20 y. Recently acquired knowledge about the NOAEL (no observed adverse effect level) of 46.3 mg · kg-1 · d-1 and the LOAEL (lowest observed adverse effect level) of 91 mg · kg-1 · d-1 of supplemented Met will guide the design of future studies to further establish the role of Met as a potential (safe) candidate for nutritional supplementation in clinical applications.
Subject(s)
Body Fluid Compartments/metabolism , Cardiovascular Diseases/etiology , Dietary Supplements , Homocysteine/metabolism , Hyperhomocysteinemia/etiology , Methionine , Vitamin B Deficiency/complications , Animals , Cardiovascular Diseases/metabolism , Female , Humans , Hyperhomocysteinemia/metabolism , Male , Methionine/adverse effects , Methionine/metabolism , Methionine/pharmacology , Methionine/therapeutic use , Proteins/metabolism , Vitamin B Complex/blood , Vitamin B Deficiency/bloodABSTRACT
PURPOSE OF REVIEW: Stable isotope methods have been used for many years to assess whole-body protein and amino acid kinetics in healthy conditions and in response to aging, exercise and (clinically stable) disease states. RECENT FINDINGS: In recent years, tracer research expanded to the anabolic response to feeding in critical illness and its use during acute metabolic stressors. Furthermore, new isotope approaches and tracer insights have been obtained. In the postabsorptive state, the novel tracer pulse approach has several advantages above the established continuous tracer approach because of the metabolic information that can be obtained, easy applicability, and low tracer costs. The use of bolus versus sip-feeding approaches to assess the anabolic response to a meal is dependent on the research question and its feasibility. Promising new tracer approaches have been developed to measure the anabolic capacity, and protein digestibility and absorption. Advances have been made in the field of mass spectrometry in low enrichment analysis. SUMMARY: Novel tracer approaches are available that can more readily be used in critical illness and during acute metabolic stressors. Besides the use of tracer application in various clinical conditions, more research is needed on how to incorporate isotopes on an individual level.
Subject(s)
Amino Acids , Isotope Labeling/methods , Proteins , Amino Acids/blood , Amino Acids/chemistry , Amino Acids/metabolism , Amino Acids/pharmacokinetics , Critical Illness , Humans , Mass Spectrometry , Proteins/chemistry , Proteins/metabolism , Proteins/pharmacokineticsABSTRACT
INTRODUCTION: Short-term (4 weeks) supplementation with n-3 polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) has recently been shown to improve protein metabolism in a dose dependent way in normal weight patients with Chronic Obstructive Pulmonary Disease (COPD). Furthermore, EPA/DHA supplementation was able to increase extremity lean soft tissue but not muscle function. No studies are available combining n-3 PUFAs and the leucine metabolite ß-hydroxy-ß-methylbutyrate (HMB) supplementation in chronic clinical conditions. Whether adding HMB to daily EPA/DHA supplementation for 10 weeks enhances muscle and brain health, daily functional performance, and quality of life of patients with COPD by further improving their protein and amino acid homeostasis remains unknown. METHODS: Patients with COPD (GOLD: II-IV, n = 46) received daily for 10 weeks, according to a randomized double-blind placebo-controlled three-group design, EPA/DHA (n = 16), EPA/DHA to which HMB was added (n = 14), or placebo (n = 16). The daily dose of 2.0 g of EPA/DHA or soy + corn oil as the placebo was provided via gel capsules, and 3.0 g of Ca-HMB or maltodextrin as placebo as powders. At pre- and post-intervention, a pulse mixture of multiple amino acids was administered to measure postabsorptive net protein breakdown (netPB as primary endpoint) and whole body production (WBP) and conversion rates of the amino acids. As secondary endpoints, lean soft tissue and fat mass were assessed by dual-energy X-ray absorptiometry, upper and lower muscle function by handgrip and single leg isokinetic dynamometry, brain (cognitive, wellbeing) health by assessments, daily functional performance by measuring 6-min walk distance, 4-m gait speed, and postural balance, and quality of life by questionnaire. Plasma enrichments and concentrations were analyzed by LC-MS/MS, and systemic inflammatory profile and metabolic hormones by Luminex. RESULTS: HMB + EPA/DHA but not EPA/DHA supplementation increased postabsorptive netPB (p = 0.028), and WBPs of glutamine (p = 0.024), taurine (p = 0.039), and tyrosine (p = 0.036). Both EPA/DHA and HMB + EPA/DHA supplementation resulted in increased WBP of phenylalanine (p < 0.05). EPA/DHA but not HMB + EPA/DHA was able to increase WBP of arginine (p = 0.030), citrulline (p = 0.008), valine (p = 0.038), and conversion of citrulline to arginine (p = 0.009). Whole body and extremity fat mass were reduced after HMB + EPA/DHA supplementation only, whereas lean soft tissue was increased after EPA/DHA (p = 0.049) and HMB + EPA/DHA (p = 0.073). No other significant findings were observed. Reductions in several proinflammatory cytokines were observed in the HMB + EPA/DHA group including IL-2, IL-17, IL-6, IL-12P40, and TNF-ß (p < 0.05). CONCLUSIONS: Ten weeks of supplementation with 2 g of EPA/DHA daily is sufficient to induce muscle gain in COPD but HMB is needed to induce fat loss. Whether HMB is solely responsible for the fat mass loss or has a synergistic effect with EPA/DHA remains unclear. The increase in net protein breakdown observed with HMB + EPA/DHA supplementation may indicate a beneficial enhanced protein turnover cycling associated with increased lean soft tissue. CLINICAL TRIAL REGISTRY: ClinicalTrials.gov; NCT03796455.
Subject(s)
Dietary Supplements , Fatty Acids, Omega-3 , Pulmonary Disease, Chronic Obstructive , Valerates , Humans , Pulmonary Disease, Chronic Obstructive/drug therapy , Valerates/administration & dosage , Valerates/pharmacology , Male , Female , Double-Blind Method , Aged , Middle Aged , Fatty Acids, Omega-3/administration & dosage , Fatty Acids, Omega-3/pharmacology , Quality of Life , Eicosapentaenoic Acid/administration & dosage , Eicosapentaenoic Acid/pharmacology , Docosahexaenoic Acids/administration & dosageABSTRACT
BACKGROUND: The trajectory from healthy to critical illness is influenced by numerous factors, including metabolism, which differs substantially between males and females. Whole body protein breakdown is substantially increased in critically ill patients, but it remains unclear whether there are sex differences that could explain the different health outcomes. Hence, we performed a secondary analysis of a study, where we used a novel pulse isotope method in critically ill and matched healthy males and females. METHODS: In 51 critically ill ICU patients (26 males, 15 females) and 49 healthy controls (36 males and 27 females), we assessed their general and disease characteristics and collected arterial(ized) blood in the postabsorptive state after pulse administration of 8 ml of a solution containing 18 stable AA tracers. In contrast to the original study, we now fitted the decay curves and calculated non-compartmental whole body amino acid production (WBP) and compartmental measurements of metabolism, including intracellular amino acid production. We measured amino acid enrichments and concentrations by LC-MS/MS and derived statistics using AN(C)OVA. RESULTS: Critically ill males and females showed an increase in the WBP of many amino acids, including those related to protein breakdown, but females showed greater elevations, or in the event of a reduction, attenuated reductions. Protein breakdown-independent WBP differences remained between males and females, notably increased glutamine and glutamate WBP. Only severely ill females showed a lower increase in WBP of many amino acids in comparison to moderately ill females, suggesting a suppressed metabolism. Compartmental analysis supported the observations. CONCLUSIONS: The present study shows that females have a different response to critical illness in the production of several amino acids and changes in protein breakdown, observations made possible using our innovative stable tracer pulse approach. CLINICAL TRIAL REGISTRY: Data are from the baseline measurements of study NCT02770092 (URL: https://clinicaltrials.gov/ct2/show/NCT02770092) and NCT03628365 (URL: https://clinicaltrials.gov/ct2/show/NCT03628365).
Subject(s)
Amino Acids , Critical Illness , Female , Humans , Male , Amino Acids/metabolism , Chromatography, Liquid , Tandem Mass SpectrometryABSTRACT
BACKGROUND: Production rates of the short-chain fatty acids (SCFA) acetate, propionate, and butyrate, which are beneficial metabolites of the intestinal microbiota, are difficult to measure in humans due to inaccessibility of the intestine to perform measurements, and the high first-pass metabolism of SCFAs in colonocytes and liver. We developed a stable tracer pulse approach to estimate SCFA whole-body production (WBP) in the accessible pool representing the systemic circulation and interstitial fluid. Compartmental modeling of plasma enrichment data allowed us to additionally calculate SCFA kinetics and pool sizes in the inaccessible pool likely representing the intestine with microbiota. We also studied the effects of aging and the presence of Chronic Obstructive Pulmonary Disease (COPD) on SCFA kinetics. METHODS: In this observational study, we designed a two-compartmental model to determine SCFA kinetics in 31 young (20-29 y) and 71 older (55-87 y) adults, as well as in 33 clinically stable patients with moderate to very severe COPD (mean (SD) FEV1, 46.5 (16.2)% of predicted). Participants received in the fasted state a pulse containing stable tracers of acetate, propionate, and butyrate intravenously and blood was sampled four times over a 30 min period. We measured tracer-tracee ratios by GC-MS and used parameters obtained from two-exponential curve fitting to calculate non-compartmental SCFA WBP and perform compartmental analysis. Statistics were done by ANCOVA. RESULTS: Acetate, propionate, and butyrate WBP and fluxes between the accessible and inaccessible pools were lower in older than young adults (all q < 0.0001). Moreover, older participants had lower acetate (q < 0.0001) and propionate (q = 0.019) production rates in the inaccessible pool as well as smaller sizes of the accessible and inaccessible acetate pools (both q < 0.0001) than young participants. WBP, compartmental SCFA kinetics, and pool sizes did not differ between COPD patients and older adults (all q > 0.05). Overall and independent of the group studied, calculated production rates in the inaccessible pool were on average 7 (acetate), 11 (propionate), and 16 (butyrate) times higher than non-compartmental WBP, and sizes of inaccessible pools were 24 (acetate), 31 (propionate), and 55 (butyrate) times higher than sizes of accessible pools (all p < 0.0001). CONCLUSION: Non-compartmental production measurements of SCFAs in the accessible pool (i.e. systemic circulation) substantially underestimate the SCFA production in the inaccessible pool, which likely represents the intestine with microbiota, as assessed by compartmental analysis.
Subject(s)
Fatty Acids, Volatile , Propionates , Young Adult , Humans , Aged , Acetates/metabolism , Butyrates , AgingABSTRACT
BACKGROUND: There is growing interest in the supplementation of arginine (Arg) and citrulline (Cit) in obesity due to their potential anti-obesogenic and anti-inflammatory properties. However, there is no consensus on the metabolic changes in Arg kinetics in obesity. OBJECTIVES: This exploratory cross-sectional study aimed to investigate the association between obesity, sex, and sex-by-obesity interaction on whole-body Arg kinetics in a large group of human subjects. METHODS: We studied 83 nonobese [BMI (kg/m2) <30] and 80 morbidly obese (BMI >30) middle-aged individuals (40% males) enrolled in the MEDIT (Metabolism of Disease with Isotope Tracers) trial. After body-composition measurement by DXA, we collected arterial(ized) blood samples for amino acid (AA) concentrations, markers of inflammation [high-sensitivity C-reactive protein (hs-CRP)], liver function, and glucose in a postabsorptive state. We administered a pulse of AA stable tracers and measured whole-body production (WBP) of Arg, Cit, ornithine (Orn), phenylalanine, and tyrosine, and calculated their clearance (disposal capacity) and metabolite interconversions [markers for NO and de novo Arg production, systemic Arg hydrolysis, and whole-body protein breakdown (wbPB)]. We measured plasma enrichments by LC-MS/MS and statistics by Fisher's exact test or analysis of (co)variance. Significance was set at P < 0.05. RESULTS: Obese individuals were normoglycemic and characterized by low-grade inflammation (P < 0.0001) and greater wbPB (P = 0.0298). We found lower plasma Cit concentration (P < 0.0001) in the obese group but no differences in the WBP of Arg, Cit, and Orn. Furthermore, we observed overproduction of NO (P < 0.0001) in obesity but lower de novo Arg production (P = 0.0007). The WBP of Arg was lower in females for almost all Arg-related AAs, except for plasma Cit and NO production. CONCLUSIONS: Alterations in Arg metabolism are present in morbid obesity. Further studies are needed to investigate if these changes could be related to factors such as increased Arg requirement in obesity or metabolic adaptation.
Subject(s)
Arginine , Obesity, Morbid , Female , Humans , Male , Middle Aged , Chromatography, Liquid , Citrulline , Cross-Sectional Studies , Inflammation , Nitric Oxide , Tandem Mass SpectrometryABSTRACT
PURPOSE: Muscle wasting deteriorates life quality after critical illness and increases mortality. Wasting starts upon admission to intensive care unit (ICU). We aimed to determine whether ß-hydroxy-ß-methylbutyrate (HMB), a metabolite of leucine, can attenuate this process. METHODS: Prospective randomized, placebo-controlled double blind trial. INCLUSION CRITERIA: ICU patients depending on mechanical ventilation on day 3 having a functional gastrointestinal tract. They were randomized to HMB (3 g/day) or placebo (maltodextrin) from day 4 on for 30 days. PRIMARY OUTCOME: magnitude of loss of skeletal muscle area (SMA) of the quadriceps femoris measured by ultrasound at days 4 and 15. SECONDARY OUTCOMES: body composition, change in protein metabolism assessed by amino acids tracer pulse, and global health at 60 days. Data are mean [95% CI]. Statistics by ANCOVA with correction for confounders sex, age and/or BMI. RESULTS: Thirty patients completed the trial, aged 65 [59, 71] years, SAPS2 score 48 [43, 52] and SOFA 8.5 [7.4, 9.7]. The loss of total SMA was 11% between days 4 and 15 (p < 0.001), but not different between the groups (p = 0.86). In the HMB group, net protein breakdown (Δ Estimate HMB-Placebo: -153 [-242, -63]; p = 0.0021) and production of several amino acid was significantly reduced, while phase angle increased more (0.66 [0.09, 1.24]; p = 0.0247), and SF-12 global health improved more (Δ Estimate HMB-Placebo: 27.39 [1.594, 53.19], p = 0.04). CONCLUSION: HMB treatment did not significantly reduce muscle wasting over 10 days of observation (primary endpoint), but resulted in significantly improved amino acid metabolism, reduced net protein breakdown, a higher phase angle and better global health. CLINICALTRIALS. GOV IDENTIFIER: NCT03628365.
Subject(s)
Amino Acids/drug effects , Dietary Supplements , Muscular Atrophy/prevention & control , Valerates/administration & dosage , Aged , Amino Acids/blood , Body Composition , Critical Illness/therapy , Double-Blind Method , Electric Impedance , Enteral Nutrition , Female , Humans , Intensive Care Units , Male , Middle Aged , Muscle, Skeletal/diagnostic imaging , Muscle, Skeletal/physiopathology , Muscular Atrophy/etiology , Organ Dysfunction Scores , Prospective Studies , Quadriceps Muscle/diagnostic imaging , Quadriceps Muscle/physiopathology , Ultrasonography/methodsABSTRACT
[This corrects the article DOI: 10.1371/journal.pone.0242926.].
ABSTRACT
Amino acid (AA) metabolism is severely disturbed in critically ill ICU patients. To be able to make a more scientifically based decision on the type of protein or AA nutrition to deliver in ICU patients, comprehensive AA phenotyping with measurements of plasma concentrations and whole body production (WBP) is needed. Therefore, we studied ICU patients and matched control subjects using a novel pulse isotope method to obtain in-depth metabolic analysis. In 51 critically ill ICU patients (SOFA~6.6) and 49 healthy controls, we measured REE and body composition/phase-angle using BIA. In the postabsorptive state, we collected arterial (ized) blood for CRP and AA. Then, we administered an 8 mL solution containing 18 stable AA tracers as a pulse and calculated WBP. Enrichments: LC-MS/MS and statistics: t-test, ANCOVA. Compared to healthy, critically ill ICU patients had lower phase-angle (p < 0.00001), and higher CRP (p < 0.0001). Most AA concentrations were lower in ICU patients (p < 0.0001), except tau-methylhistidine and phenylalanine. WBP of most AA were significantly (p < 0.0001) higher with increases in glutamate (160%), glutamine (46%), and essential AA. Remarkably, net protein breakdown was lower. There were only weak relationships between AA concentrations and WBP. Critically ill ICU patients (SOFA 8-16) had lower values for phase angle (p = 0.0005) and small reductions of most plasma AA concentrations, but higher tau-methylhistidine (p = 0.0223) and hydroxyproline (p = 0.0028). Remarkably, the WBP of glutamate and glutamine were lower (p < 0.05), as was their clearance, but WBP of tau-methylhistidine (p = 0.0215) and hydroxyproline (p = 0.0028) were higher. Our study in critically ill ICU patients shows that comprehensive metabolic phenotyping was able to reveal severe disturbances in specific AA pathways, in a disease severity dependent way. This information may guide improving nutritional compositions to improve the health of the critically ill patient. CLINICAL TRIAL REGISTRY: Data are from the baseline measurements of study NCT02770092 (URL: https://clinicaltrials.gov/ct2/show/NCT02770092) and NCT03628365 (URL: https://clinicaltrials.gov/ct2/show/NCT03628365).
Subject(s)
Amino Acids/blood , Body Composition/physiology , Aged , Basal Metabolism/physiology , Critical Illness , Electric Impedance , Female , Humans , Male , Middle AgedABSTRACT
With the rise in physical inactivity and its related diseases, it is necessary to understand the mechanisms involved in physical activity regulation. Biological factors regulating physical activity are studied to establish a possible target for improving the physical activity level. However, little is known about the role metabolism plays in physical activity regulation. Therefore, we studied protein fractional synthesis rate (FSR) of multiple organ tissues of 12-week-old male mice that were previously established as inherently low-active (n = 15, C3H/HeJ strain) and high-active (n = 15, C57L/J strain). Total body water of each mouse was enriched to 5% deuterium oxide (D2O) via intraperitoneal injection and maintained with D2O enriched drinking water for about 24 h. Blood samples from the jugular vein and tissues (kidney, heart, lung, muscle, fat, jejunum, ileum, liver, brain, skin, and bone) were collected for enrichment analysis of alanine by LC-MS/MS. Protein FSR was calculated as -ln(1-enrichment). Data are mean±SE as fraction/day (unpaired t-test). Kidney protein FSR in the low-active mice was 7.82% higher than in high-active mice (low-active: 0.1863±0.0018, high-active: 0.1754±0.0028, p = 0.0030). No differences were found in any of the other measured organ tissues. However, all tissues resulted in a generally higher protein FSR in the low-activity mice compared to the high-activity mice (e.g. lung LA: 0.0711±0.0015, HA: 0.0643±0.0020, heart LA: 0.0649± 0.0013 HA: 0.0712±0.0073). Our observations suggest that high-active mice in most organ tissues are no more inherently equipped for metabolic adaptation than low-active mice, but there may be a connection between protein metabolism of kidney tissue and physical activity level. In addition, low-active mice have higher organ-specific baseline protein FSR possibly contributing to the inability to achieve higher physical activity levels.
Subject(s)
Muscles/metabolism , Protein Biosynthesis/genetics , Proteins/genetics , Sedentary Behavior , Animals , Chromatography, Liquid , Humans , Injections, Intraperitoneal , Jejunum/metabolism , Liver/metabolism , Mice , Mice, Inbred C3H , Organ Specificity/genetics , Physical Conditioning, Animal/methods , Proteins/isolation & purification , Tandem Mass Spectrometry , Tissue Distribution/geneticsABSTRACT
Our previous studies suggest that physical activity (PA) levels are potentially regulated by endogenous metabolic mechanisms such as the vasodilatory roles of nitric oxide (NO) production via the precursor arginine (ARG) and ARG-related pathways. We assessed ARG metabolism and its precursors [citrulline (CIT), glutamine (GLN), glutamate (GLU), ornithine (ORN), and phenylalanine (PHE)] by measuring plasma concentration, whole-body production (WBP), de novo ARG and NO production, and clearance rates in previously classified low-active (LA) or high-active (HA) mice. We assessed LA (n = 23) and HA (n = 20) male mice by administering a stable isotope tracer pulse via jugular catheterization. We measured plasma enrichments via liquid chromatography tandem mass spectrometry (LC-MS/MS) and body compostion by echo-MRI. WBP, clearance rates, and de novo ARG and NO were calculated. Compared to LA mice, HA mice had lower plasma concentrations of GLU (71.1%; 36.8 ± 2.9 vs. 17.5 ± 1.7µM; p<0.0001), CIT (21%; 57.3 ± 2.3 vs. 46.4 ± 1.5µM; p = 0.0003), and ORN (40.1%; 55.4 ± 7.3 vs. 36.9 ± 2.6µM; p = 0.0241), but no differences for GLN, PHE, and ARG. However, HA mice had higher estimated NO production ratio (0.64 ± 0.08; p = 0.0197), higher WBP for CIT (21.8%, 8.6 ± 0.2 vs. 10.7 ± 0.3 nmol/g-lbm/min; p<0.0001), ARG (21.4%, 35.0 ± 0.6 vs. 43.4 ± 0.7 nmol/g-lbm/min; p<0.0001), PHE (7.6%, 23.8 ± 0.5 vs. 25.6 ± 0.5 nmol/g-lbm/min; p<0.0100), and lower GLU (78.5%; 9.4 ± 1.1 vs. 4.1 ± 1.6 nmol/g lbm/min; p = 0.0161). We observed no significant differences in WBP for GLN, ORN, PHE, or de novo ARG. We concluded that HA mice have an activated whole-body ARG pathway, which may be associated with regulating PA levels via increased NO production.
Subject(s)
Arginine/blood , Motor Activity , Nitric Oxide/blood , Animals , Chromatography, Liquid/methods , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Physical Conditioning, Animal , Tandem Mass Spectrometry/methodsABSTRACT
BACKGROUND & AIMS: Metabolic characterization of a well-defined group of patients could be a powerful tool in revealing metabolic signatures to explain limb muscle weakness in chronic diseases. Studies are currently limited in Chronic Obstructive Pulmonary Disease (COPD) to the identification of differential amino acid concentrations but lack comprehensive analysis of the flux through relevant muscle function related metabolic pathways. METHODS: In 23 stable patients with moderate to very severe COPD and 19 healthy controls, a comprehensive metabolic flux analysis was conducted by administering an intravenous pulse and primed constant infusion of multiple stable tracers of amino acids known to play a role in muscle health. Blood samples were obtained to calculate production (WBP) and interconversion rates, and plasma concentrations of these amino acids. Lower and upper limb muscle strength, muscle mass, lung function, physical activity level, and disease history and characteristics were assessed. RESULTS: The COPD group was characterized by lower and upper limb muscle weakness (P < 0.01) despite preserved muscle mass. Higher values were found in COPD for plasma glutamine, WBP of leucine (P < 0.001), 3-methylhistidine (P < 0.01) (marker of enhanced myofibrillar protein breakdown), citrulline (P < 0.05), and arginine to citrulline conversion (P < 0.05) (reflecting enhanced nitric oxide synthesis). Plasma concentration of ß-hydroxy ß-methylbutyrate (HMB with anticatabolic, anabolic and contractile properties), WBP of glycine (precursor of creatine and glutathione), and transcutaneous O2 saturation explained up to 79% and 65% of the variation in strength of the lower and upper limb muscles, respectively, in COPD. CONCLUSIONS: Comprehensive metabolic flux analysis revealed a homogenous metabolic signature in stable patients with COPD and a specific metabolic profile in those with skeletal muscle weakness. CLINICAL TRIAL REGISTRY: ClinicalTrials.gov; No. NCT01787682; URL: www.clinicaltrials.gov.
Subject(s)
Metabolome , Muscle Strength , Muscle Weakness/metabolism , Muscle, Skeletal/metabolism , Pulmonary Disease, Chronic Obstructive/metabolism , Aged , Biomarkers/blood , Case-Control Studies , Clinical Trials as Topic , Female , Humans , Male , Metabolomics , Middle Aged , Muscle Weakness/diagnosis , Muscle Weakness/physiopathology , Muscle, Skeletal/physiopathology , Pulmonary Disease, Chronic Obstructive/diagnosis , Pulmonary Disease, Chronic Obstructive/physiopathologyABSTRACT
BACKGROUND: Metabolomics, petroleum and biodiesel chemistry, biomarker discovery, and other fields which rely on high-resolution profiling of complex chemical mixtures generate datasets which contain millions of detector intensity readings, each uniquely addressed along dimensions of time (e.g., retention time of chemicals on a chromatographic column), a spectral value (e.g., mass-to-charge ratio of ions derived from chemicals), and the analytical run number. They also must rely on data preprocessing techniques. In particular, inter-run variance in the retention time of chemical species poses a significant hurdle that must be cleared before feature extraction, data reduction, and knowledge discovery can ensue. Alignment methods, for calibrating retention reportedly (and in our experience) can misalign matching chemicals, falsely align distinct ones, be unduly sensitive to chosen values of input parameters, and result in distortions of peak shape and area. RESULTS: We present an iterative block-shifting approach for retention-time calibration that detects chromatographic features and qualifies them by retention time, spectrum, and the effect of their inclusion on the quality of alignment itself. Mass chromatograms are aligned pairwise to one selected as a reference. In tests using a 45-run GC-MS experiment, block-shifting reduced the absolute deviation of retention by greater than 30-fold. It compared favourably to COW and XCMS with respect to alignment, and was markedly superior in preservation of peak area. CONCLUSION: Iterative block-shifting is an attractive method to align GC-MS mass chromatograms that is also generalizable to other two-dimensional techniques such as HPLC-MS.
Subject(s)
Algorithms , Artificial Intelligence , Biopolymers/chemistry , Gas Chromatography-Mass Spectrometry/methods , Pattern Recognition, Automated/methods , Sequence Alignment/methods , Biopolymers/analysisABSTRACT
BACKGROUND: The aging process is often associated with the presence of sarcopenia. Although changes in the plasma concentration of several amino acids have been observed in older adults, it remains unclear whether these changes are related to disturbances in whole body production and/or interconversions. METHODS: We studied 10 healthy young (~22.7y) and 17 older adults (~64.8y) by administering a mixture of stable amino acid tracers in a pulse and in a primed constant infusion. We calculated whole body production (WBP) and metabolite to metabolite interconversions. In addition, we measured body composition, muscle function, and provided questionnaires to assess daily dietary intake, physical activity, mood (anxiety, depression) and markers of cognitive function. Plasma enrichments and metabolite concentrations were measured by GC- and LC-MS/MS and statistics were performed by student t-test. RESULTS: Older adults had a 11% higher body mass index (p=0.04) and 27% reduced peak leg extension force (p=0.02) than the younger group, but comparable values for muscle mass, mood and cognitive function. Although small differences in several plasma amino acid concentrations were observed, we found older adults had about 40% higher values of WBP for glutamine (221±27 vs. 305±21µmol/kgffm/h, p=0.03) and tau-methylhistidine (0.15±0.01 vs. 0.21±0.02µmol/kgffm/h, p=0.04), 26% lower WBP value for arginine (59±4 vs. 44±4µmol/kgffm/h, p=0.02) and a reduction in WBP (50%; 1.23±0.15 vs. 0.69±0.06µmol/kgffm/h, p=0.001) and concentration (25%; 3.5±0.3µmol/l vs. 2.6±0.2µmol/l, p=0.01) for ß-Hydroxy ß-Methylbutyrate. No differences were observed in protein catabolism. Clearance of arginine was decreased (27%, p=0.03) and clearance of glutamine (58%, p=0.01), leucine (67%, p=0.001) and KIC (76%, p=0.004) were increased in older adults. CONCLUSIONS: Specific differences exist between young and older adults in amino acid metabolism.
Subject(s)
Aging/metabolism , Amino Acids/blood , Adult , Affect/physiology , Aging/blood , Body Composition/physiology , Body Mass Index , Cognition/physiology , Dietary Supplements , Exercise/physiology , Female , Humans , Kinetics , Male , Middle Aged , Muscle, Skeletal/metabolism , Sarcopenia/blood , Sarcopenia/metabolism , Young AdultABSTRACT
Our objective was to develop a quick and simplified method for the determination of ß-Hydroxy-ß-methylbutyrate (HMB) and É-ketoisocaproic acid (KIC) concentrations and enrichments by GC/MS/MS to determine the turnover rate of HMB in humans. In experiment 1, we provided a pulse of L-[5,5,5-2H3]leucine to younger adults in the postabsorptive state then collected blood samples over a 4h time period. In experiment 2, we provided a pulse of [3,4,methyl-13C3]HMB to older adults in the postabsorptive state then collected blood samples over a 3h time period. Plasma concentrations of KIC and HMB and MPE of KIC and HMB were determined by GC/MS/MS. Plasma enrichment of leucine was determined by LC/MS/MS. To determine plasma enrichment of [5,5,5-2H3]HMB and [3,4,methyl-13C3]HMB, samples were derivatized using pentafluorobenzyl bromide and analyzed using chemical ionization mode. The final methods used included multiple reaction monitoring of transitions 117.3>59.3 for M+0 and 120.3>59.3 for M+3. In experiment 1, peak MPE of Leu peaked at 9.76% generating a peak MPE of KIC at 2.67% and a peak HMB MPE of 0.3%. In experiment 2, the rate of appearance for HMB was 0.66µmol/kg ffm/h. We calculated that production of HMB in humans accounts for 0.66% of total leucine turnover.
Subject(s)
Gas Chromatography-Mass Spectrometry/methods , Keto Acids/blood , Tandem Mass Spectrometry/methods , Valerates/blood , Adult , Aged , Gas Chromatography-Mass Spectrometry/economics , Humans , Limit of Detection , Middle Aged , Tandem Mass Spectrometry/economics , Young AdultABSTRACT
Quantitative trait loci (QTL) can implicate an unbiased sampling of genes underlying a complex, polygenic phenotype. QTL affecting longevity in Caenorhabditis elegans were mapped using a CL2a x Bergerac-BO recombinant-inbred population. Genotypes were compared at 30 transposon-specific markers for two paired sample sets totaling 171 young controls and 172 longevity-selected worms (the last-surviving 1%) from a synchronously aged population. A third sample set, totaling 161 worms from an independent culture, was analyzed for confirmation of loci. At least six highly significant QTL affecting life span were detected both by single-marker (chi(2)) analysis and by two interval-mapping procedures--one intended for nonparametric traits and another developed specifically for mapping of categorical traits. These life-span QTL were located on chromosomes I (near the hP4 locus), III (near stP127), IV (near stP44), V (a cluster of three peaks, near stP192, stP23, and stP6), and X (two distinct peaks, near stP129 and stP2). Epistatic effects on longevity were also analyzed by Fisher's exact test, which indicated a significant life-span interaction between markers on chromosomes V (stP128) and III (stP127). Several further interactions were significant in the initial unselected population; two of these, between distal loci on chromosome V, were completely eliminated in the long-lived subset. Allelic longevity effects for two QTL, on chromosomes IV and V, were confirmed in backcrossed congenic lines and were highly significant in two very different environments-growth on solid agar medium and in liquid suspension culture.
Subject(s)
Caenorhabditis elegans/genetics , Chromosome Mapping , Longevity/genetics , Quantitative Trait Loci , Animals , Animals, Congenic , Animals, Inbred Strains , Caenorhabditis elegans/physiology , Genetic Markers , Longevity/physiologyABSTRACT
Incorporation of deuterium from deuterium oxide ((2) H2 O) into biological components is a commonly used approach in metabolic studies. Determining the dilution of deuterium in the body water (BW) pool can be used to estimate body composition. We describe three sensitive GC/MS/MS methods to measure water enrichment in BW. Samples were reacted with NaOH and U-(13) C3 -acetone in an autosampler vial to promote deuterium exchange with U-(13) C3 -acetone hydrogens. Headspace injections were made of U-(13) C3 -acetone-saturated air onto a 30-m DB-1MS column in electron impact-mode. Subjects ingested 30 ml (2) H2 O, and plasma samples were collected. BW was determined by standard equation. Dual-energy X-ray absorptiometry scans were performed to calculate body mass, body volume and bone mineral content. A four-compartmental model was used to estimate body composition (fat and fat free mass). Full-scan experiments generated an m/z 45 peak and to a lesser extent an m/z 61 peak. Product fragment ions further monitored included 45 and 46 using selected ion monitoring (Method1), the 61 > 45 and 62 > 46 transition using multiple reaction monitoring (MRM; Method2) and the neutral loss, 62 > 45, transition (Method3). MRM methods were optimized for collision energy (CE) and collision-induced dissociation (CID) argon gas pressure with 6 eV CE and 1.5 mTorr CID gas being optimal. Method2 was used for final determination of (2) H2 O enrichment of subjects because of lower natural background. We have developed a sensitive method to determine (2) H2 O enrichment in BW to enable measurement of FM and FFM.
Subject(s)
Body Water/chemistry , Deuterium Oxide/chemistry , Deuterium/blood , Gas Chromatography-Mass Spectrometry/methods , Acetone , Adult , Aged , Body Water/metabolism , Deuterium/metabolism , Deuterium Oxide/metabolism , Humans , Middle Aged , Tandem Mass Spectrometry/methods , Young AdultABSTRACT
By linkage mapping of quantitative trait loci, we previously identified at least 11 natural genetic variants that significantly modulate Caenorhabditis elegans life-span (LS), many of which would have eluded discovery by knock-down or mutation screens. A region on chromosome IV between markers stP13 and stP35 had striking effects on longevity in three inter-strain crosses (each P < 10(-9)). In order to define the limits of that interval, we have now constructed two independent lines by marker-based selection during 20 backcross generations, isolating the stP13-stP35 interval from strain Bergerac-BO in a CL2a background. These congenic lines differed significantly from CL2a in LS, assayed in two environments (each P < 0.001). We then screened for exchange of flanking markers to isolate recombinants that partition this region, because fine-mapping the boundaries for overlapping heteroallelic spans can greatly narrow the implicated interval. Recombinants carrying the CL2a allele at stP35 were consistently long-lived compared to those retaining the Bergerac-BO allele (P < 0.001), and more resistant to temperature elevation and paraquat (each â¼1.7-fold, P < 0.0001), but gained little protection from ultraviolet or peroxide stresses. Two rounds of recombinant screening, followed by fine-mapping of break-points and survival testing, narrowed the interval to 0.18 Mb (13.35-13.53 Mb) containing 26 putative genes and six small-nuclear RNAs - a manageable number of targets for functional assessment.
ABSTRACT
Many lifespan-modulating genes are involved in either generation of oxidative substrates and end-products, or their detoxification and removal. Among such metabolites, only lipoperoxides have the ability to produce free-radical chain reactions. For this study, fatty-acid profiles were compared across a panel of C. elegans mutants that span a tenfold range of longevities in a uniform genetic background. Two lipid structural properties correlated extremely well with lifespan in these worms: fatty-acid chain length and susceptibility to oxidation both decreased sharply in the longest-lived mutants (affecting the insulinlike-signaling pathway). This suggested a functional model in which longevity benefits from a reduction in lipid peroxidation substrates, offset by a coordinate decline in fatty-acid chain length to maintain membrane fluidity. This model was tested by disrupting the underlying steps in lipid biosynthesis, using RNAi knockdown to deplete transcripts of genes involved in fatty-acid metabolism. These interventions produced effects on longevity that were fully consistent with the functions and abundances of their products. Most knockdowns also produced concordant effects on survival of hydrogen peroxide stress, which can trigger lipoperoxide chain reactions.
Subject(s)
Caenorhabditis elegans/genetics , Caenorhabditis elegans/physiology , Lipids/biosynthesis , Longevity/physiology , Oxidative Stress , Animals , Caenorhabditis elegans/drug effects , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Fatty Acids/chemistry , Fatty Acids/metabolism , Hydrogen Peroxide/pharmacology , Oxidants/pharmacology , RNA Interference , Survival Rate , Transcription, GeneticABSTRACT
The great majority of lifespan-augmenting mutations were discovered in the nematode Caenorhabditis elegans. In particular, genetic disruption of insulin-like signaling extends longevity 1.5- to 3-fold in the nematode, and to lesser degrees in other taxa, including fruit flies and mice. C. elegans strains bearing homozygous nonsense mutations in the age-1 gene, which encodes the class-I phosphatidylinositol 3-kinase catalytic subunit (PI3K(CS)), produce progeny that were thought to undergo obligatory developmental arrest. We now find that, after prolonged developmental times at 15-20 degrees C, they mature into extremely long-lived adults with near-normal feeding rates and motility. They survive to a median of 145-190 days at 20 degrees C, with nearly 10-fold extension of both median and maximum adult lifespan relative to N2DRM, a long-lived wild-type stock into which the null mutant was outcrossed. PI3K-null adults, although a little less thermotolerant, are considerably more resistant to oxidative and electrophilic stresses than worms bearing normal or less long-lived alleles. Their unprecedented factorial gains in survival, under both normal and toxic environments, are attributed to elimination of residual and maternally contributed PI3K(CS) or its products, and consequent modification of kinase signaling cascades.