Characterising the urinary acylcarnitine and amino acid profiles of HIV/TB co-infection, using LC-MS metabolomics.

INTRODUCTION: The human immunodeficiency virus (HIV) and tuberculosis (TB) co-infection presents significant challenges due to the complex interplay between these diseases, leading to exacerbated metabolic disturbances. Understanding these metabolic profiles is crucial for improving diagnostic and therapeutic approaches. OBJECTIVE: This study aimed to characterise the urinary acylcarnitine and amino acid profiles, including 5-hydroxyindoleacetic acid (5-HIAA), in patients co-infected with HIV and TB using targeted liquid chromatography mass spectrometry (LC-MS) metabolomics. METHODS: Urine samples, categorised into HIV, TB, HIV/TB co-infected, and healthy controls, were analysed using HPLC-MS/MS. Statistical analyses included one-way ANOVA and a Kruskal-Wallis test to determine significant differences in the acylcarnitine and amino acid profiles between groups. RESULTS: The study revealed significant metabolic alterations, especially in TB and co-infected groups. Elevated levels of medium-chain acylcarnitines indicated increased fatty acid oxidation, commonly associated with cachexia in TB. Altered amino acid profiles suggested disruptions in protein and glucose metabolism, indicating a shift towards diabetes-like metabolic states. Notably, TB was identified as a primary driver of these changes, affecting protein turnover, and impacting energy metabolism in co-infected patients. CONCLUSION: The metabolic profiling of HIV/TB co-infection highlights the profound impact of TB on metabolic pathways, which may exacerbate the clinical complexities of co-infection. Understanding these metabolic disruptions can guide the development of targeted treatments and improve management strategies, ultimately enhancing the clinical outcomes for these patients. Further research is required to validate these findings and explore their implications in larger, diverse populations.

Simultaneous quantification of trimethylamine N-oxide, trimethylamine, choline, betaine, creatinine, and propionyl-, acetyl-, and L-carnitine in clinical and food samples using HILIC-LC-MS.

Trimethylamine-N-oxide (TMAO), a microbiome-derived metabolite from the metabolism of choline, betaine, and carnitines, is associated to adverse cardiovascular outcomes. A method suitable for routine quantification of TMAO and its precursors (trimethylamine (TMA), choline, betaine, creatinine, and propionyl-, acetyl-, and L-carnitine) in clinical and food samples has been developed based on LC-MS. TMA was successfully derivatized using iodoacetonitrile, and no cross-reactions with TMAO or the other methylamines were detected. Extraction from clinical samples (plasma and urine) was performed after protein precipitation using acetonitrile:methanol. For food samples (meatballs and eggs), water extraction was shown to be sufficient, but acid hydrolysis was required to release bound choline before extraction. Baseline separation of the methylamines was achieved using a neutral HILIC column and a mobile phase consisting of 25 mmol/L ammonium formate in water:ACN (30:70). Quantification was performed by MS using external calibration and isotopic labelled internal standards. The assay proved suitable for both clinical and food samples and was linear from ≈ 0.1 up to 200 µmol/L for all methylamines except for TMA and TMAO, which were linear up to 100 µmol/L. Recoveries were 91-107% in clinical samples and 76-98% in food samples. The interday (n=8, four duplicate analysis) CVs were below 9% for all metabolites in clinical and food samples. The method was applied successfully to determine the methylamine concentrations in plasma and urine from the subjects participating in an intervention trial (n=10) to determine the effect of animal food ingestion on methylamine concentrations.

Triangulating evidence from longitudinal and Mendelian randomization studies of metabolomic biomarkers for type 2 diabetes.

The number of people affected by Type 2 diabetes mellitus (T2DM) is close to half a billion and is on a sharp rise, representing a major and growing public health burden. Given its mild initial symptoms, T2DM is often diagnosed several years after its onset, leaving half of diabetic individuals undiagnosed. While several classical clinical and genetic biomarkers have been identified, improving early diagnosis by exploring other kinds of omics data remains crucial. In this study, we have combined longitudinal data from two population-based cohorts CoLaus and DESIR (comprising in total 493 incident cases vs. 1360 controls) to identify new or confirm previously implicated metabolomic biomarkers predicting T2DM incidence more than 5 years ahead of clinical diagnosis. Our longitudinal data have shown robust evidence for valine, leucine, carnitine and glutamic acid being predictive of future conversion to T2DM. We confirmed the causality of such association for leucine by 2-sample Mendelian randomisation (MR) based on independent data. Our MR approach further identified new metabolites potentially playing a causal role on T2D, including betaine, lysine and mannose. Interestingly, for valine and leucine a strong reverse causal effect was detected, indicating that the genetic predisposition to T2DM may trigger early changes of these metabolites, which appear well-before any clinical symptoms. In addition, our study revealed a reverse causal effect of metabolites such as glutamic acid and alanine. Collectively, these findings indicate that molecular traits linked to the genetic basis of T2DM may be particularly promising early biomarkers.

Urinary 3-hydroxyisovaleryl carnitine excretion, protein energy malnutrition and risk of all-cause mortality in kidney transplant recipients: Results from the TransplantLines cohort studies.

BACKGROUND: Leucine is an essential amino acid and a potent stimulator of muscle protein synthesis. Since muscle wasting is a major risk factor for mortality in kidney transplant recipients (KTR), dietary leucine intake might be linked to long-term mortality. Urinary 3-hydroxyisovaleryl carnitine (3-HIC) excretion, a functional marker of marginal biotin deficiency, may also serve as a marker for dietary leucine intake. OBJECTIVE: In this study we aimed to investigate the cross-sectional determinants of urinary 3-HIC excretion and to prospectively investigate the association of urinary 3-HIC excretion with all-cause mortality in KTR. DESIGN: Urinary 3-HIC excretion and plasma biotin were measured in a longitudinal cohort of 694 stable KTR. Cross-sectional and prospective analyses were performed using ordinary least squares linear regression analyses and Cox regression analyses, respectively. RESULTS: In KTR (57% male, 53 ± 13 years, estimated glomerular filtration rate 45 ± 19 mL/min/1.73 m2), urinary 3-HIC excretion (0.80 [0.57-1.16] µmol/24 h) was significantly associated with plasma biotin (std. ß = -0.17; P < 0.001). Subsequent adjustment for potential covariates revealed urinary creatinine excretion (std. ß = 0.24; P < 0.001) and urinary urea excretion (std. ß = 0.53; P < 0.001) as the primary determinant of urinary 3-HIC excretion. Whereas plasma biotin explained only 1% of the variance in urinary 3-HIC excretion, urinary urea excretion explained >45%. During median follow-up for 5.4 [4.8-6.1] years, 150 (22%) patients died. Log2-transformed urinary 3-HIC excretion was inversely associated with all-cause mortality (HR: 0.52 [0.43-0.63]; P < 0.001). This association was independent of potential confounders. CONCLUSIONS: Urinary 3-HIC excretion more strongly serves as a marker of leucine intake than of biotin status. A higher urinary 3-HIC excretion is associated with a lower risk of all-cause mortality. Future studies are warranted to explore the underlying mechanism. TRIAL REGISTRATION ID: NCT02811835. TRIAL REGISTRATION URL: https://clinicaltrials.gov/ct2/show/NCT02811835.

Determination of urinary carnitine levels as a potential indicator of uterine fibroids caused by nonylphenol exposure.

Our previous studies have shown that uterine fibroids are associated with nonylphenol (NP) exposure, and the changes of carnitines in critical reproductive tissues and body fluids could be used to indicate the female reproductive toxicity caused by NP exposure. In this work, on the basis of further clarifying the correlation between NP exposure level and uterine fibroids, the possibility of the urinary carnitine levels as a potential indicator of uterine fibroids caused by NP exposure was discussed. The urine samples were collected from 84 female volunteers: the control group of 34 healthy women without gynecological disease and 50 uterine fibroids patients, respectively. Methods were respectively established for the determination of NP and eight carnitines in human urine samples by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The results showed that the NP level of uterine fibroids group was significantly higher than that of control group (P = 0.002), indicating that NP exposure was an important environmental factor in the occurrence of uterine fibroids. It was further found that in urine samples of the uterine fibroids group, the levels of L-Carnitine (C0), L-Acetyl-carnitine (C2), L-Octanoyl-carnitine (C8), Tetradecanoyl-carnitine (C14), Oleoyl-carnitine (C18:1) and Linoleoyl-carnitine (C18:2) had obviously increased compared with those in the control group (P < 0.001; < 0.001; < 0.001; = 0.003; < 0.001; = 0.010). The concentrations of L-Hexanoyl-carnitine (C6) and L-Palmitoyl-carnitine (C16) in the uterine fibroids group were also higher than those in the control group, although the difference was not statistically significant (P > 0.05). The results suggested that the changes in urinary carnitine levels might be a potential indicator to help to warn of the risk of uterine fibroids caused by NP exposure at the early stage.

A metabolomic study of red and processed meat intake and acylcarnitine concentrations in human urine and blood.

BACKGROUND: Acylcarnitines (ACs) play a major role in fatty acid metabolism and are potential markers of metabolic dysfunction with higher blood concentrations reported in obese and diabetic individuals. Diet, and in particular red and processed meat intake, has been shown to influence AC concentrations but data on the effect of meat consumption on AC concentrations is limited. OBJECTIVES: To investigate the effect of red and processed meat intake on AC concentrations in plasma and urine using a randomized controlled trial with replication in an observational cohort. METHODS: In the randomized crossover trial, 12 volunteers successively consumed 2 different diets containing either pork or tofu for 3 d each. A panel of 44 ACs including several oxidized ACs was analyzed by LC-MS in plasma and urine samples collected after the 3-d period. ACs that were associated with pork intake were then measured in urine (n = 474) and serum samples (n = 451) from the European Prospective Investigation into Cancer and nutrition (EPIC) study and tested for associations with habitual red and processed meat intake derived from dietary questionnaires. RESULTS: In urine samples from the intervention study, pork intake was positively associated with concentrations of 18 short- and medium-chain ACs. Eleven of these were also positively associated with habitual red and processed meat intake in the EPIC cross-sectional study. In blood, C18:0 was positively associated with red meat intake in both the intervention study (q = 0.004, Student's t-test) and the cross-sectional study (q = 0.033, linear regression). CONCLUSIONS: AC concentrations in urine and blood were associated with red meat intake in both a highly controlled intervention study and in subjects of a cross-sectional study. Our data on the role of meat intake on this important pathway of fatty acid and energy metabolism may help understanding the role of red meat consumption in the etiology of some chronic diseases. This trial was registered at Clinicaltrials.gov as NCT03354130.

Carnitine insufficiency is associated with fatigue during lenvatinib treatment in patients with hepatocellular carcinoma.

BACKGROUND: Fatigue is a common adverse event during lenvatinib treatment in patients with hepatocellular carcinoma. One mechanism contributing to development of fatigue might involve abnormal adenosine triphosphate synthesis that is caused by carnitine deficiency. To address this possibility, we examined the relationship between carnitine levels and fatigue during lenvatinib treatment. METHODS: This prospective study evaluated 20 patients with hepatocellular carcinoma who underwent lenvatinib treatment. Both blood and urine samples were collected from the patients before starting lenvatinib therapy (day 0), and on days 3, 7, 14, and 28 thereafter. Plasma and urine concentrations of free and acyl carnitine (AC) were assessed at each time point. The changes in daily fatigue were evaluated using the Brief Fatigue Inventory (BFI). RESULTS: Plasma levels of free carnitine (FC) at days 3 and 7 were significantly higher compared with baseline (p = 0.005, p = 0.005, respectively). The urine FC level at day 3 was significantly higher compared with baseline (p = 0.030) and that of day 7 tended to be higher compared with baseline (p = 0.057). The plasma AC concentration at days 14 and 28 was significantly higher compared with that of baseline (p = 0.002, p = 0.005, respectively). The plasma AC-to-FC (AC/FC) ratio on days 14 and 28 was significantly higher compared with baseline (p = 0.001, p = 0.003, respectively). There were significant correlations between the plasma AC/FC ratio and the change in the BFI score at days 14 and 28 (r = 0.461, p = 0.041; r = 0.770, p = 0.002, respectively). CONCLUSIONS: Longitudinal assessments of carnitine and fatigue in patients with hepatocellular carcinoma suggest that lenvatinib affects the carnitine system in patients undergoing lenvatinib therapy and that carnitine insufficiency increases fatigue. The occurrence of carnitine insufficiency may be a common cause of fatigue during the treatment.

Quantitation of Urinary Acylcarnitines by DMS-MS/MS Uncovers the Effects of Total Body Irradiation in Cancer Patients.

Acylcarnitines have been identified in human and animal metabolomic-profiling studies as urinary markers of radiation exposure, a result which is consistent with their cytoprotective effects and roles in energy metabolism. In the present work, a rapid method for quantitation of the more abundant acylcarnitines in human urine is developed using a valuable set of samples from cancer patients who received total body irradiation (TBI) at Memorial Sloan Kettering Cancer Center. The method uses solid-phase extraction (SPE) processing followed by differential mobility spectrometry (DMS with ethyl acetate modifier) tandem mass spectrometry (ESI-DMS-MS/MS) with deuterated internal standards. The analyzed human urine samples were collected from 38 individual patients at three time points over 24 h during and after the course of radiation treatment, a design allowing each patient to act as their own control and creatinine normalization. Creatinine-normalized concentrations for nine urinary acylcarnitine (acyl-CN) species are reported. Six acyl-CN species were reduced at the 6 h point. Acetylcarnitine (C2:0-CN) and valerylcarnitine (C5:0-CN) showed recovery at 24 h, but none of the other acyl-CN species showed recovery at that point. Levels of three acyl-CN species were not significantly altered by radiation. This rapid quantitative method for clinical samples covers the short- and medium-chain acylcarnitines and has the flexibility to be expanded to cover additional radiation-linked metabolites. The human data presented here indicates the utility of the current approach as a rapid, quantitative technique with potential applications by the medical community, by space research laboratories concerned with radiation exposure, and by disaster response groups.

Carnitine Profile Changes in Pediatric Hematopoietic Stem Cell Transplant: New Role for Carnitine?

Carnitine is an essential cofactor for mitochondrial import and oxidation of fatty acids. High-dose chemotherapy and radiation, often required for hematopoietic stem cell transplant (HSCT), leads to tissue damage, mitochondrial dysfunction, and alterations in carnitine metabolism. The aim of this pilot cohort study was to describe plasma and urinary carnitine profiles during pediatric HSCT and their relationships with clinical outcomes. Plasma and urinary carnitine samples were collected from 22 pediatric patients before and through day 180 post-HSCT. Associations were observed between graft-versus-host disease and an elevated plasma total carnitine (P=0.019), and also increased plasma acyl:free carnitine ratio with veno-occlusive disease (P=0.016). Mortality was observed in those with their highest urinary total carnitine losses on day 0 (P=0.005), and in those with an abnormal day 28 plasma ratio either above or below the reference range (P=0.007). Changes in carnitine profiles were more reflective of metabolic stress and negative outcomes than of inadequate dietary intake. Associations observed direct larger studies to assess the validity of carnitine profiles as a prognostic indicator and also to assess whether prophylactic carnitine supplementation pre-HSCT could reduce mitochondrial injury and urinary losses and help mitigate inflammatory and metabolic comorbidities of HSCT.

Mass Spectrometry Fingerprints of Small-Molecule Metabolites in Biofluids: Building a Spectral Library of Recurrent Spectra for Urine Analysis.

A large fraction of ions observed in electrospray liquid chromatography-mass spectrometry (LC-ESI-MS) experiments of biological samples remain unidentified. One of the main reasons for this is that spectral libraries of pure compounds fail to account for the complexity of the metabolite profiling of complex materials. Recently, the NIST Mass Spectrometry Data Center has been developing a novel type of searchable mass spectral library that includes all recurrent unidentified spectra found in the sample profile. These libraries, in conjunction with the NIST tandem mass spectral library, allow analysts to explore most of the chemical space accessible to LC-MS analysis. In this work, we demonstrate how these libraries can provide a reliable fingerprint of the material by applying them to a variety of urine samples, including an extremely altered urine from cancer patients undergoing total body irradiation. The same workflow is applicable to any other biological fluid. The selected class of acylcarnitines is examined in detail, and derived libraries and related software are freely available. They are intended to serve as online resources for continuing community review and improvement.

Liquid Chromatography-Mass Spectrometry-Based Metabolomics of Nonhuman Primates after 4 Gy Total Body Radiation Exposure: Global Effects and Targeted Panels.

Rapid assessment of radiation signatures in noninvasive biofluids may aid in assigning proper medical treatments for acute radiation syndrome (ARS) and delegating limited resources after a nuclear disaster. Metabolomic platforms allow for rapid screening of biofluid signatures and show promise in differentiating radiation quality and time postexposure. Here, we use global metabolomics to differentiate temporal effects (1-60 d) found in nonhuman primate (NHP) urine and serum small molecule signatures after a 4 Gy total body irradiation. Random Forests analysis differentially classifies biofluid signatures according to days post 4 Gy exposure. Eight compounds involved in protein metabolism, fatty acid ß oxidation, DNA base deamination, and general energy metabolism were identified in each urine and serum sample and validated through tandem MS. The greatest perturbations were seen at 1 d in urine and 1-21 d in serum. Furthermore, we developed a targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM) method to quantify a six compound panel (hypoxanthine, carnitine, acetylcarnitine, proline, taurine, and citrulline) identified in a previous training cohort at 7 d after a 4 Gy exposure. The highest sensitivity and specificity for classifying exposure at 7 d after a 4 Gy exposure included carnitine and acetylcarnitine in urine and taurine, carnitine, and hypoxanthine in serum. Receiver operator characteristic (ROC) curve analysis using combined compounds show excellent sensitivity and specificity in urine (area under the curve [AUC] = 0.99) and serum (AUC = 0.95). These results highlight the utility of MS platforms to differentiate time postexposure and acquire reliable quantitative biomarker panels for classifying exposed individuals.

Development of a metabolic pathway-based pseudo-targeted metabolomics method using liquid chromatography coupled with mass spectrometry.

The pseudo-targeted metabolomics approach was developed recently which combined the advantages of untargeted and targeted analysis. However, the current pseudo-targeted analysis method has limitations due to the technical characteristics. In this study, a novel metabolic pathway-based pseudo-targeted approach was proposed for urine metabolomics analysis using an ultra-high-performance liquid chromatography (UPLC)-MS/MS system operated in the multiple reaction monitoring (MRM) mode. MRM ion pairs were acquired from urine samples through untargeted analysis using UPLC-HRMS, as well as by searching for metabolites in related pathways in relevant databases and from previous relevant research, including amino acids, fatty acids, nucleosides, carnitines, glycolysis metabolites, and steroids. This improved pseudo-targeted method exhibited good repeatability and precision, and no complicated peak alignment was required. As a proof of concept, the developed novel method was applied to the discovery of urine biomarkers for patients with esophageal squamous cell carcinoma (ESCC). The results showed that ESCC patients had altered acylcarnitines, amino acids, nucleosides, and steroid derivative levels et al. compared to those of healthy controls. The novelty of this study lies in the fact that it provides an approach for acquiring MRM ion pairs not only from untargeted MS analysis but also from targeted searching for metabolites in related metabolic pathways. By improving the detection limit of low-abundance metabolites, it enlarges the range for the discovery of potential biomarkers. Our work provides a foundation for achieving pseudo-targeted metabolomics analysis on the widely used LC-MS/MS MRM platform.

Clinical, Biochemical, and Genetic Findings of Cystinuria in Chinese Children.

BACKGROUND: Cystinuria is a rare inherited renal stone disease caused by mutations in the SLC3A1 and SLC7A9 genes. The Chinese cystinuria phenotype and genotype have rarely been reported in the literature. METHODS: For this research, the clinical features and genetic etiology were analyzed in seven children, and the clinical characteristics were summarized. The blood and urine amino acids and acylcarnitine were analyzed. Additionally, the whole coding sequence and exon-intron junctions of the SLC3A1 and SLC7A9 genes were analyzed. RESULTS: These seven patients with cystinuria were from seven unrelated Chinese families, and they were diagnosed between the ages of 1 month and 16 years old. The urinary amino acids, including ornithine, arginine, and threonine, were elevated in these patients. A homozygous c.325G>A mutation in SLC7A9 was identified in two patients, and six SLC3A1 mutations were found in five patients. CONCLUSIONS: The core pedigree analysis showed that most of the parents carried mutations; however, there was no association between the clinical course and the genotype.

Changes to trimethylamine-N-oxide and its precursors in nascent metabolic syndrome.

Background Metabolic syndrome (MetS), a cardio-metabolic cluster afflicting 35% of American adults, increases cardiovascular disease (CVD) and type-2 diabetes (T2DM) risk. Increased levels of trimethylamine N-oxide (TMAO), a metabolite derived from choline and L-carnitine, correlates with CVD and T2DM. However, the precise role of TMAO and its precursors in MetS remains unclear. We tested the hypothesis that choline, L-carnitine and TMAO in MetS patients without CVD or T2DM would be altered and correlate with inflammatory markers. Materials and methods This was an exploratory study of 30 patients with nascent MetS (without CVD or T2DM) and 20 matched controls. MetS was defined by the Adult Treatment Panel III criteria. TMAO and its precursors were evaluated from each patient's frozen early morning urine samples and quantified using liquid chromatography/mass spectrometry (LC-MS). These amines were correlated with a detailed repertoire of biomarkers of inflammation and adipokines. Results L-carnitine was significantly increased (p = 0.0002) compared to controls. There was a trend for a significant increase in TMAO levels (p = 0.08). Choline was not significantly altered in MetS. L-carnitine correlated significantly with soluble tumor necrosis factor 1 (sTNFR1) and leptin, and inversely to adiponectin. TMAO correlated with IL-6, endotoxin and chemerin. Neither choline, nor L-carnitine significantly correlated with TMAO. Conclusion L-carnitine is directly correlated with markers of inflammation in nascent MetS. Cellular L-carnitine could be a biomediator or marker of inflammation in the pathogenesis of MetS, and the sequelae of CVD and T2DM.

Characterization of metabolic responses to healthy diets and association with blood pressure: application to the Optimal Macronutrient Intake Trial for Heart Health (OmniHeart), a randomized controlled study.

Background: Interindividual variation in the response to diet is common, but the underlying mechanism for such variation is unclear. Objective: The objective of this study was to use a metabolic profiling approach to identify a panel of urinary metabolites representing individuals demonstrating typical (homogeneous) metabolic responses to healthy diets, and subsequently to define the association of these metabolites with improvement of risk factors for cardiovascular diseases (CVDs). Design: 24-h urine samples from 158 participants with pre-hypertension and stage 1 hypertension, collected at baseline and following the consumption of a carbohydrate-rich, a protein-rich, and a monounsaturated fat-rich healthy diet (6 wk/diet) in a randomized, crossover study, were analyzed by proton (1H) nuclear magnetic resonance (NMR) spectroscopy. Urinary metabolite profiles were interrogated to identify typical and variable responses to each diet. We quantified the differences in absolute excretion of metabolites, distinguishing between dietary comparisons within the typical response groups, and established their associations with CVD risk factors using linear regression. Results: Globally all 3 diets induced a similar pattern of change in the urinary metabolic profiles for the majority of participants (60.1%). Diet-dependent metabolic variation was not significantly associated with total cholesterol or low-density lipoprotein (LDL) cholesterol concentration. However, blood pressure (BP) was found to be significantly associated with 6 urinary metabolites reflecting dietary intake [proline-betaine (inverse), carnitine (direct)], gut microbial co-metabolites [hippurate (direct), 4-cresyl sulfate (inverse), phenylacetylglutamine (inverse)], and tryptophan metabolism [N-methyl-2-pyridone-5-carboxamide (inverse)]. A dampened clinical response was observed in some individuals with variable metabolic responses, which could be attributed to nonadherence to diet (≤25.3%), variation in gut microbiome activity (7.6%), or a combination of both (7.0%). Conclusions: These data indicate interindividual variations in BP in response to dietary change and highlight the potential influence of the gut microbiome in mediating this relation. This approach provides a framework for stratification of individuals undergoing dietary management. The original OmniHeart intervention study and the metabolomics study were registered at www.clinicaltrials.gov as NCT00051350 and NCT03369535, respectively.

Simple determination of betaine, l-carnitine and choline in human urine using self-packed column and column-switching ion chromatography with nonsuppressed conductivity detection.

A sequential online extraction, clean-up and separation system for the determination of betaine, l-carnitine and choline in human urine using column-switching ion chromatography with nonsuppressed conductivity detection was developed in this work. A self-packed pretreatment column (50 × 4.6 mm, i.d.) was used for the extraction and clean-up of betaine, l-carnitine and choline. The separation was achieved using self-packed cationic exchange column (150 × 4.6 mm, i.d.), followed by nonsuppressed conductivity detection. Under optimized experimental conditions, the developed method presented good analytical performance, with excellent linearity in the range of 0.60-100 µg mL-1 for betaine, 0.75-100 µg mL-1 for l-carnitine and 0.50-100 µg mL-1 for choline, with all correlation coefficients (R2 ) >0.99 in urine. The limits of detection were 0.15 µg mL-1 for betaine, 0.20 µg mL-1 for l-carnitine and 0.09 µg mL-1 for choline. The intra- and inter-day accuracy and precision for all quality controls were within ±10.32 and ±9.05%, respectively. Satisfactory recovery was observed between 92.8 and 102.0%. The validated method was successfully applied to the detection of urinary samples from 10 healthy people. The values detected in human urine using the proposed method showed good agreement with the measurement reported previously.

Determination of betaine, l-carnitine, and choline in human urine using a self-packed column and column-switching ion chromatography with nonsuppressed conductivity detection.

A simple method for the determination of betaine, l-carnitine, and choline in human urine was developed based on column-switching ion chromatography coupled with nonsuppressed conductivity detection by using a self-packed column. A pretreatment column (50 mm × 4.6 mm, id) packed with poly(glycidyl methacrylate-divinylbenzene) microspheres was used for the extraction and cleanup of analytes. Chromatographic separation was achieved within 10 min on a cationic exchange column (150 mm × 4.6 mm, id) using maleic anhydride modified poly(glycidyl methacrylate-divinylbenzene) as the particles for packing. The detection was performed by ion chromatography with nonsuppressed conductivity detection. Parameters including column-switching time, eluent type, flow rates of eluent, and interfering effects were optimized. Linearity (r2 ≥ 0.99) was obtained for the concentration range of 0.50-100, 0.75-100, and 0.25-100 µg/mL for betaine, l-carnitine, and choline, respectively. Detection limits were 0.12, 0.20, and 0.05 µg/mL for betaine, l-carnitine, and choline, respectively. The intra- and interday accuracy and precision for all quality controls were within ±10.11%. Satisfactory recovery was observed between 92.5 and 105.0%. The validated method was successfully applied for the determination of betaine, l-carnitine, and choline in urine samples from healthy people.

Dynamics of L-Carnitine in Plasma and Urine in Patients Undergoing Cisplatin Chemotherapy.

BACKGROUND AND AIMS: Several studies have indicated that cisplatin (cis-diamminedichloroplatinum II; CDDP) causes urinary excretion of L-carnitine (LC). However, the underlying cofactors affecting the increased urinary excretion remain unclear. The present study was performed to evaluate the dynamics of LC in plasma and urine after CDDP chemotherapy and to examine the relations with clinical parameters, such as gender, body mass index (BMI), and renal function. METHODS: Twenty-two patients treated with CDDP therapy were selected. Blood and urine samples were taken from patients before starting CDDP treatment (day 0), on the next day (day 1), and on the seventh day (day 7). We measured plasma and urine concentrations of total, free, and acyl-LC, and examined the relationships with gender, age, treatment cycle, skeletal muscle mass, BMI, glomerular filtration rate, and change in creatinine concentration after CDDP administration. RESULTS: Both urinary and plasma concentrations of 3 types of LC increased markedly on day 1 and subsequently reverted to the pre-CDDP level on day 7. There was a positive correlation between the % changes in plasma and urine LC (correlation coefficient 0.59, p = 0.003) on day 1, but no significant relations were seen in other clinical parameters. CONCLUSIONS: CDDP transiently increased plasma LC levels. The mechanism seemed to involve recruitment for marked urinary loss of LC. However, these changes in plasma and urinary LC levels were not related to clinical factors, suggesting that the dynamics of LC were independent of preexisting physical parameters.

Relationship between Carnitine Pharmacokinetics and Fatigue in Patients Treated with Cisplatin-Containing Chemotherapy.

BACKGROUND: Approximately 70% of the patients who receive chemotherapy suffer from fatigue, which lowers their quality of life and also has a negative influence on therapeutic efficacy. Previous studies have suggested a relationship between blood carnitine levels and fatigue. We conducted a prospective observational study to examine the relationship between carnitine pharmacokinetics and chemotherapy-induced fatigue in patients receiving cancer chemotherapy regimens that include cisplatin. PATIENTS AND METHODS: 11 patients receiving chemotherapy including cisplatin (60-80 mg/m2) were included in the study. We performed 24-h urine collections and took blood samples on day 1 (before the initiation of chemotherapy) and days 2, 3, 4, and 8 in order to measure the carnitine concentrations in the serum and urine. These were compared with measures of self-reported fatigue. The primary endpoint was the change in self-reported fatigue subscales from baseline to day 8. RESULTS: Urinary carnitine concentrations differed significantly on days 2 and 3 (p = 0.003). The Functional Assessment of Chronic Illness Therapy-Fatigue scale version 4A score on day 8 indicated significantly higher levels of fatigue as compared to day 1 (p = 0.013). CONCLUSION: This study suggests that there is an association between urinary carnitine levels and self-reported fatigue.

Quantitative acylcarnitine determination by UHPLC-MS/MS--Going beyond tandem MS acylcarnitine "profiles".

Tandem MS "profiling" of acylcarnitines and amino acids was conceived as a first-tier screening method, and its application to expanded newborn screening has been enormously successful. However, unlike amino acid screening (which uses amino acid analysis as its second-tier validation of screening results), acylcarnitine "profiling" also assumed the role of second-tier validation, due to the lack of a generally accepted second-tier acylcarnitine determination method. In this report, we present results from the application of our validated UHPLC-MS/MS second-tier method for the quantification of total carnitine, free carnitine, butyrobetaine, and acylcarnitines to patient samples with known diagnoses: malonic acidemia, short-chain acyl-CoA dehydrogenase deficiency (SCADD) or isobutyryl-CoA dehydrogenase deficiency (IBD), 3-methyl-crotonyl carboxylase deficiency (3-MCC) or ß-ketothiolase deficiency (BKT), and methylmalonic acidemia (MMA). We demonstrate the assay's ability to separate constitutional isomers and diastereomeric acylcarnitines and generate values with a high level of accuracy and precision. These capabilities are unavailable when using tandem MS "profiles". We also show examples of research interest, where separation of acylcarnitine species and accurate and precise acylcarnitine quantification is necessary.