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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., (© 2021. The Author(s).)

Objective: The objective of this study was to determine whether levels of choline (Ch) differ in women with and without overactive bladder (OAB) symptoms., Methods: New patients were evaluated using the overactive bladder symptom score; Medical, Epidemiologic, and Social Aspects of Aging (MESA) urgency incontinence questionnaire; and Impact Questionnaire 7 and provided a urine sample. Patients were stratified into asymptomatic controls, scoring 0 on overactive bladder symptom score and the MESA questionnaire, and patients with OAB and urgency incontinence (OAB-wet). Patients with conditions predisposing to OAB or had a history of OAB treatment were excluded. Choline detection was accomplished using a commercially available kit. Wilcoxon rank sum test and Fisher exact test were used to express differences between groups. Spearman ρ correlation was used to determine the relationship between Ch and questionnaire scores. Logistic regression was used to identify significant variables associated with OAB., Results: Sixty-three women were included in the final analysis. Patients with OAB-wet were older (P = 0.001), more likely to be obese (P = 0.04), had greater apical descent (P = 0.02), were more likely to be postmenopausal (P = 0.01), and were more likely to have stress incontinence (P = 0.005). Choline was 34.8% lower in OAB compared with the controls (P = 0.014). Lower Ch levels were associated with higher MESA (Spearman ρ = -0.311, P = 0.03). After logistic regression, lower Ch (adjusted odds ratio [aOR], 0.97; 95% confidence interval [CI], 0.96-0.98), age (aOR, 1.12; 95% CI, 1.08-1.18), and body mass index (aOR, 1.09; 95% CI, 1.01-1.18) were significantly associated with OAB-wet., Conclusions: Choline levels are significantly decreased in women complaining of OAB with urgency incontinence, and lower levels are associated with higher MESA scores.

Fortification of human milk (HM) for preterm and very low-birth weight (VLBW) infants is a standard practice in most neonatal intensive care units. The optimal fortification strategy and the most suitable protein source for achieving better tolerance and growth rates for fortified infants are still being investigated. In a previous clinical trial, preterm and VLBW infants receiving supplementation of HM with experimental donkey milk-based fortifiers (D-HMF) showed decreased signs of feeding intolerance, including feeding interruptions, bilious gastric residuals and vomiting, with respect to infants receiving bovine milk-based fortifiers (B-HMF). In the present ancillary study, the urinary metabolome of infants fed B-HMF ( n = 27) and D-HMF ( n = 27) for 21 days was analyzed by 1 H NMR spectroscopy at the beginning (T0) and at the end (T1) of the observation period. Results showed that most temporal changes in the metabolic responses were common in the two groups, providing indications of postnatal adaptation. The significantly higher excretion of galactose in D-HMF and of carnitine, choline, lysine and leucine in B-HMF at T1 were likely due to different formulations. In conclusion, isocaloric and isoproteic HM fortification may result in different metabolic patterns, as a consequence of the different quality of the nutrients provided by the fortifiers.

Aim: To standardize acquisition protocols for 18 F-Choline PET/CT to prevent from urine interference, to determine the best time point for the whole-body study, and to assess whether "dual point" acquisition allows for differentiating malignant vs. benign lesions., Methods: One hundred consecutive patients with prostate cancer were prospectively studied. Immediately after 18 F-Choline injection, a pelvis study was acquired, and a whole-body was subsequently obtained 1 and 2 hours p.i. Mean SUVmax was obtained in regions and for every sequential imaging. Mean analysis (χ 2 ) and SUV percentage change (2/1 hours; 1 hours/0 min) were obtained. Metabolic pattern dynamics were assessed: accumulative vs. clearance. Patient follow-up after therapy and directed classification whenever ethically possible were performed., Results: Fifty-three prostate foci, without disturbing urinary activity was ever found on early images. Accumulative pattern in 42, with percentage increase was: 0 min/1 hour: +16.7% (χ 2 0.94); 1/2 hours: +10,0% (χ 2 0.83). Clearance pattern in 11, with percentage decrease: 0 min/1 hour: -21.4% (χ 2 0.91): -7.7% (χ 2 0.85), corresponding in 7 to initial staging and in 4 post-radiotherapy biochemical recurrence. Every infradiaphragmatic uptake (n: 24) showed accumulative pattern, with percentage increase of +9.1% (χ 2 0.97), all of them depicted on early imaging. As for 12 supradiaphragmantic uptake, 8 of them showed clearance pattern with percentage decrease: -13.0% (χ 2 0.95). Accumulative pattern showed in 4 of them with percentage increase +13.0% (χ 2 0.96), thus being assessed as invasive/malignant. Every bone uptake (n: 18) showed accumulative pattern, with percentage increase: +17.1% (χ 2 0.95), all of them depicted on 1 hour imaging., Conclusions: As for prostate assessment is concerned, dual point at 0 min/1 hour proved to be the best procedure. As for supradiaphragmatic lymph-nodes detection, dual point with 1/2 hours performed best. As for infradiaphragmatic and bone involvement, as well as for inconclusive findings, the 2 hour imaging increased our diagnostic confidence., (Copyright © 2020 Sociedad Española de Medicina Nuclear e Imagen Molecular. Publicado por Elsevier España, S.L.U. All rights reserved.)

Objective : This study was performed to examine the choline status on term and preterm infants using urinary metabolome analysis. Material and methods: Samples were collected from 19 term and 20 preterm infants between 15 days and 1 month, respectively. The infants were separated into four groups: the term-breast group (TB, n  = 13), the term-formula group (TF, n  = 6), the preterm-breast (PB, n  = 11), and the preterm-mixed group (PM, n  = 9). Urinary metabolome analysis was performed using capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF/MS). We also performed metabolome analysis of the infant formulas. Results: Urinary excretion of choline metabolites (choline, N , N -dimethylglycine, sarcosine, and betaine) was significantly higher in TB than TF infants ( p  < .05). Choline, betaine, and sarcosine excretion was not significantly different between the PB and TB infants. Choline and N,N -dimethylglycine excretion was significantly higher in PM than PB infants. Choline metabolites excretion was also significantly higher in PM than TF infants. Choline and betaine levels were significantly higher in the preterm than term formula used in this study. Conclusions: The type of feeding in early infancy affects choline metabolism. Metabolome analysis is useful for assessing choline metabolism to modify the contents of infant formulas also in preterm infants.

Objective: Preterm infants' body composition at term-corrected age differs from that of term infants but appears to be similar at the age of 3 months. The aim of this study was to compare the metabolomic pattern of preterm infants at term and at 3 months with that of term infants and to determine its association with body composition development., Method: We designed a pilot study. Growth and body composition were evaluated by an air displacement plethysmography system in 13 preterm infants and seven term newborns at term and at 3 months of corrected age. Urine samples were collected at the same time points and analysed by nuclear magnetic resonance., Results: At term-corrected age, preterm infants showed a higher fat mass percentage compared with that of term newborns, whereas at 3 months of corrected age, the body composition parameters were similar between the groups. At the first time point, nuclear magnetic resonance analysis showed a urinary increase in choline/phosphocholine, betaine and glucose in preterm infants. At the second time point, the preterm group exhibited a urinary increase in choline/phosphocholine and a decrease in betaine., Conclusions: The increased urinary excretion of choline, a betaine precursor, could reflect a potential altered metabolism in preterm infants.

Metabolites, the small molecules that underpin life, can act as indicators of the physiological state of the body when their abundance varies, offering routes to diagnosis of many diseases. The ability to assay for multiple metabolites simultaneously will underpin a new generation of precision diagnostic tools. Here, we report the development of a handheld device based on complementary metal oxide semiconductor (CMOS) technology with multiple isolated micro-well reaction zones and integrated optical sensing allowing simultaneous enzyme-based assays of multiple metabolites (choline, xanthine, sarcosine and cholesterol) associated with multiple diseases. These metabolites were measured in clinically relevant concentration range with minimum concentrations measured: 25 μM for choline, 100 μM for xanthine, 1.25 μM for sarcosine and 50 μM for cholesterol. Linking the device to an Android-based user interface allows for quantification of metabolites in serum and urine within 2 min of applying samples to the device. The quantitative performance of the device was validated by comparison to accredited tests for cholesterol and glucose., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

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 (R 2 ) >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., (Copyright © 2017 John Wiley & Sons, Ltd.)

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 (r 2 ≥ 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., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Polybrominated diphenyl ethers (PBDEs) are commonly used to retard the combustion of materials such as foam padding, textiles, or plastics, and numerous studies have confirmed the accumulation thereof in the environment and in fish, mammals, and humans. In this study, we used metabolomics to conduct an environmental risk assessment of the PBDE-209. We profiled the urinary metabolites of control and PBDE-treated rats (exposed to PBDE-209) using nuclear magnetic resonance (NMR) and mass spectrometry (MS). Global metabolic profiling indicated that the effects of PBDE-209 on the urinary metabolic profile were not significant. However, targeted metabolic profiling revealed progressive effects of PBDE-209 over a 7-day PBDE-209 treatment. Moreover, despite the weak PBDE-209 effects, we observed that choline, acetylcholine, 3-indoxylsulfate, creatinine, urea, and dimethyl sulfone levels were decreased, whereas that of pyruvate was significantly increased. Furthermore, we suggest that the increased pyruvate level and decreased levels of choline, acetylcholine, and uremic toxins were suggestive of endocrine disruption and neurodevelopmental toxicity caused by PBDEs. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1262-1272, 2017., (© 2016 Wiley Periodicals, Inc.)

Background: Several compounds in the choline oxidation pathway are associated with insulin resistance and prevalent diabetes; however, prospective data are scarce.We explored the relationships between systemic and urinary choline-related metabolites and incident type 2 diabetes in an observational prospective study among Norwegian patients., Methods: We explored risk associations by logistic regression among 3621 nondiabetic individuals with suspected stable angina pectoris, of whom 3242 provided urine samples. Reclassification of patients was investigated according to continuous net reclassification improvement (NRI >0)., Results: After median (25th to 75th percentile) follow-up of 7.5 (6.4-8.7) years, 233 patients (6.4%) were registered with incident type 2 diabetes. In models adjusted for age, sex, and fasting status, plasma betaine was inversely related to new-onset disease [odds ratio (OR) per 1 SD, 0.72; 95% CI, 0.62-0.83; P < 0.00001], whereas positive associations were observed for urine betaine (1.25; 1.09-1.43; P = 0.001), dimethylglycine (1.22; 1.06-1.40; P = 0.007), and sarcosine (1.30; 1.13-1.49; P < 0.001). The associations were maintained in a multivariable model adjusting for body mass index, hemoglobin A1c, urine albumin-to-creatinine ratio, estimated glomerular filtration rate, C-reactive protein, HDL cholesterol, and medications. Plasma betaine and urine sarcosine, the indices most strongly related to incident type 2 diabetes, improved reclassification [NRI >0 (95% CI) 0.33 (0.19-0.47) and 0.16 (0.01-0.31), respectively] and showed good within-person reproducibility., Conclusions: Systemic and urinary concentrations of several choline metabolites were associated with risk of incident type 2 diabetes, and relevant biomarkers may improve risk prediction., (© 2016 American Association for Clinical Chemistry.)

A simple, sensitive, and precise ultra-high performance liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of trimethylamine N-oxide, choline, and betaine in human plasma and urine. Sample preparation involved protein precipitation with methanol containing internal standards. Chromatographic separation was achieved using an Acquity BEH Amide (2.1mm×50mm, 1.7μm) analytical column with gradient elution of solvent A (10mM ammonium formate, pH 3.5) and solvent B (acetonitrile). The flow rate was 0.4mL/min and the total run time was 5min. Detection of analytes was performed using heated electrospray ionization (positive mode) and selected reaction monitoring. Excellent linearity was observed over the standard curve concentration ranges of 0.010-5.00μg/mL (plasma) and 1.00-150μg/mL (urine) for all analytes. The intra- and inter-day accuracy and precision for all quality controls were within ±10%. Excellent recovery was observed. The method is rapid, accurate and reproducible, and was successfully applied to a pilot study of markers of atherosclerosis in patients with kidney disease who underwent successful kidney transplantation., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Plasma betaine concentrations and urinary betaine excretions have high test-retest reliability. Abnormal betaine excretion is common in diabetes. We aimed to confirm the individuality of plasma betaine and urinary betaine excretion in an overweight population with type 2 diabetes and compare this with the individuality of other osmolytes, one-carbon metabolites and trimethylamine-N-oxide (TMAO), thus assessing their potential usefulness as disease markers., Methods: Urine and plasma were collected from overweight subjects with type 2 diabetes at four time points over a two-year period. We measured the concentrations of the osmolytes: betaine, glycerophosphorylcholine (GPC) and taurine, as well as TMAO, and the one-carbon metabolites, N,N-dimethylglycine (DMG) and free choline. Samples were measured using tandem mass spectrometry (LC-MS/MS)., Results: Betaine showed a high degree of individuality (or test-retest reliability) in the plasma (index of individuality = 0.52) and urine (index of individuality = 0.45). Betaine in the plasma had positive and negative log-normal reference change values (RCVs) of 54% and -35%, respectively. The other osmolytes, taurine and GPC were more variable in the plasma of individuals compared to the urine. DMG and choline showed high individuality in the plasma and urine. TMAO was highly variable in the plasma and urine (log-normal RCVs ranging from 403% to -80% in plasma)., Conclusions: Betaine is highly individual in overweight people with diabetes. Betaine, its metabolite DMG, and precursor choline showed more reliability than the osmolytes, GPC and taurine. The low reliability of TMAO suggests that a single TMAO measurement has low diagnostic value., (© The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.)

Unlabelled: There is no clinically applicable biomarker for surveillance of hepatocellular carcinoma (HCC), because the sensitivity of serum alpha-fetoprotein (AFP) is too low for this purpose. Here, we determined the diagnostic performance of a panel of urinary metabolites of HCC patients from West Africa. Urine samples were collected from Nigerian and Gambian patients recruited on the case-control platform of the Prevention of Liver Fibrosis and Cancer in Africa (PROLIFICA) program. Urinary proton nuclear magnetic resonance ((1) H-NMR) spectroscopy was used to metabolically phenotype 290 subjects: 63 with HCC; 32 with cirrhosis (Cir); 107 with noncirrhotic liver disease (DC); and 88 normal control (NC) healthy volunteers. Urine samples from a further cohort of 463 subjects (141 HCC, 56 Cir, 178 DC, and 88 NC) were analyzed, the results of which validated the initial cohort. The urinary metabotype of patients with HCC was distinct from those with Cir, DC, and NC with areas under the receiver operating characteristic (AUROC) curves of 0.86 (0.78-0.94), 0.93 (0.89-0.97), and 0.89 (0.80-0.98) in the training set and 0.81 (0.73-0.89), 0.96 (0.94-0.99), and 0.90 (0.85-0.96), respectively, in the validation cohort. A urinary metabolite panel, comprising inosine, indole-3-acetate, galactose, and an N-acetylated amino acid (NAA), showed a high sensitivity (86.9% [75.8-94.2]) and specificity (90.3% [74.2-98.0]) in the discrimination of HCC from cirrhosis, a finding that was corroborated in a validation cohort (AUROC: urinary panel = 0.72; AFP =  0.58). Metabolites that were significantly increased in urine of HCC patients, and which correlated with clinical stage of HCC, were NAA, dimethylglycine, 1-methylnicotinamide, methionine, acetylcarnitine, 2-oxoglutarate, choline, and creatine., Conclusion: The urinary metabotyping of this West African cohort identified and validated a metabolite panel that diagnostically outperforms serum AFP., (© 2014 by the American Association for the Study of Liver Diseases.)

Purpose: Betaine deficiency is a probable cardiovascular risk factor and a cause of elevated homocysteine. Urinary betaine excretion is increased by fibrate treatment, and is also often elevated in diabetes. Does fibrate further increase betaine excretion in diabetes, and does it affect the plasma concentrations and excretions of related metabolites and of other osmolytes?, Methods: Samples from a previous study of type 2 diabetes were selected if participants were taking bezafibrate (n = 32). These samples were compared with participants matched for age and gender and not on a fibrate (comparator group, n = 64). Betaine, related metabolites, and osmolytes were measured in plasma and urine samples from these 96 participants., Results: Median urinary betaine excretion in those on bezafibrate was 5-fold higher than in the comparator group (p < 0.001), itself 3.5-fold higher than the median reported for healthy populations. In the bezafibrate group, median dimethylglycine excretion was higher (9-fold, p < 0.001). Excretions of choline, and of the osmolytes myo-inositol, taurine and glycerophosphorylcholine, were not significantly different between groups. Some participants excreted more betaine than usual dietary intakes. Several betaine fractional clearances were >100 %. Betaine excretion correlated with excretions of the osmolytes myo-inositol and glycerophosphorylcholine, and also with the excretion of choline and N,N-dimethylglycine, but it was inconclusive whether these relationships were affected by bezafibrate therapy., Conclusions: Increased urinary betaine excretions in type 2 diabetes are further increased by fibrate treatment, sometimes to more than their dietary intake. Concurrent betaine supplementation may be beneficial.

Background: Cross-sectional data suggest that bezafibrate increases betaine excretion in dyslipidemic patients., Objective: We aimed to demonstrate that fenofibrate induces increased betaine excretion in normal subjects and explore whether other 1-carbon metabolites and osmolytes are similarly affected., Methods: Urine was collected from 26 healthy adults before and after treatment with fenofibrate (145 mg/day for 6 weeks). Excretions of betaine, N,N-dimethylglycine, free choline, myo-inositol, taurine, trimethylamine-N-oxide, carnitine, and acetylcarnitine were measured by liquid chromatography with mass spectrometric detection., Results: Fenofibrate increased the median betaine excretion from 7.5 to 25.8 mmol/mole creatinine (median increase 3-fold), P < .001. The median increase in N,N-dimethylglycine excretion was 2-fold (P < .001). Median choline excretion increased 12% (significant, P = .029). Participants with higher initial excretions tended to have larger increases (P < .001 in all 3 cases). Fenofibrate did not significantly change the median excretions of myo-inositol, taurine, trimethylamine-N-oxide, and carnitine. The excretion of acetylcarnitine decreased 4-fold on treatment, with no correlation between the baseline and after-treatment excretions. Changes in all urine components tested, except trimethylamine-N-oxide, positively correlated with changes in betaine excretion even when the median excretions before and after were not significantly different., Conclusions: Fibrates increase betaine, and to a lesser extent N,N-dimethylglycine and choline, excretion. Other osmolytes are not elevated. Because the increase in betaine excretion depends on the baseline excretion, large increases in excretion in the metabolic syndrome and diabetes (where baseline excretions are high) could be expected. Replacement with betaine supplements may be considered., (Copyright © 2014 National Lipid Association. Published by Elsevier Inc. All rights reserved.)

We have conducted for the first time an experimental study of pharmacokinetics of dicholine succinate (DCS) for different ways of its administration in rats The quantitative evaluation of DCS and its metabolites was performed by the radioactive isotope technique. Various parameters of DCS pharmacokinetics were estimated, including the dose dependence of drug content in the blood plasma, total bioavailability, distribution kinetics, and the main ways of DCS excretion.

Inflammation is closely associated with pathogenesis of various metabolic disorders, cardiovascular diseases, and cancers. To understand the systems responses to localized inflammation, we analyzed the dynamic metabolic changes in rat plasma and urine associated with the carrageenan-induced self-limiting pleurisy using NMR spectroscopy in conjunction with multivariate data analysis. Fatty acids in plasma were also analyzed using GC-FID/MS with the data from clinical chemistry and histopathology as complementary information. We found that in the acute phase of inflammation rats with pleurisy had significantly lower levels in serum albumin, fatty acids, and lipoproteins but higher globulin level and larger quantity of pleural exudate than controls. The carrageenan-induced inflammation was accompanied by significant metabolic alterations involving TCA cycle, glycolysis, biosyntheses of acute phase proteins, and metabolisms of amino acids, fatty acids, ketone bodies, and choline in acute phase. The resolution process of pleurisy was heterogeneous, and two subgroups were observed for the inflammatory rats at day-6 post treatment with different metabolic features together with the quantity of pleural exudate and weights of thymus and spleen. The metabolic differences between these subgroups were reflected in the levels of albumin and acute-phase proteins, the degree of returning to normality for multiple metabolic pathways including glycolysis, TCA cycle, gut microbiota functions, and metabolisms of lipids, choline and vitamin B3. These findings provided some essential details for the dynamic metabolic changes associated with the carrageenan-induced self-limiting inflammation and demonstrated the combined NMR and GC-FID/MS analysis as a powerful approach for understanding biochemical aspects of inflammation.

In this work, untargeted NMR metabonomics was employed to evaluate the effects of pregnancy on the metabolite composition of maternal urine, thus establishing a control excretory trajectory for healthy pregnancies. Urine was collected for independent groups of healthy nonpregnant and pregnant women (in first, second, third trimesters) and multivariate analysis performed on the corresponding NMR spectra. Models were validated through Monte Carlo Cross Validation and permutation tests and metabolite correlations measured through Statistical Total Correlation Spectroscopy. The levels of 21 metabolites were found to change significantly throughout pregnancy, with variations observed for the first time to our knowledge for choline, creatinine, 4-deoxyerythronic acid, 4-deoxythreonic acid, furoylglycine, guanidoacetate, 3-hydroxybutyrate, and lactate. Results confirmed increased aminoaciduria across pregnancy and suggested (a) a particular involvement of isoleucine and threonine in lipid oxidation/ketone body synthesis, (b) a relation of excreted choline, taurine, and guanidoacetate to methionine metabolism and urea cycle regulation, and (c) a possible relationship of furoylglycine and creatinine to pregnancy, based on a tandem study of nonfasting confounding effects. Results demonstrate the usefulness of untargeted metabonomics in finding biomarker metabolic signatures for healthy pregnancies, against which disease-related deviations may be confronted in future studies, as a base for improved diagnostics and prediction.

Bladder cancer is one of the leading lethal cancers worldwide. With the high risk of recurrence for bladder cancer following the initial diagnoses, lifelong monitoring of patients is necessary. The lack of adequate sensitivity and specificity of current noninvasive monitoring approaches including urine cytology, other urine tests, and imaging, underlines the importance of studies that focus on the detection of more reliable biomarkers for this cancer. The emerging area of metabolomics, which deals with the analysis of a large number of small molecules in a single step, promises immense potential for discovering metabolite markers for screening and monitoring treatment response and recurrence in patients with bladder cancer. Since naturally-occurring canine transitional cell carcinoma of the urinary bladder is very similar to human invasive bladder cancer, spontaneous canine transitional cell carcinoma has been applied as a relevant animal model of human invasive transitional cell carcinoma. In this study, we have focused on profiling the metabolites in urine from dogs with transitional cell carcinoma and healthy control dogs combining nuclear magnetic resonance spectroscopy and statistical analysis methods. (1)H NMR-based metabolite profiling analysis was shown to be an effective approach for differentiating samples from dogs with transitional cell carcinoma and healthy controls based on a partial least square-discriminant analysis of the NMR spectra. In addition, there were significant differences in the levels of six individual metabolites between samples from dogs with transitional cell carcinoma and the control group based on the Student's t-test. These metabolites were selected to build a separate partial least square-discriminant analysis model that was then used to test the classification accuracy. The result showed good classification between transitional cell carcinoma and control groups with the area under the receiver operating characteristic curve of 0.85. The sensitivity and specificity of the model were 86% and 78%, respectively. These results suggest that urine metabolic profiling may have potential for early detection of bladder cancer and of bladder cancer recurrence following treatment, and may enhance our understanding of the mechanisms involved., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: In 1998 choline Adequate Intakes of 425 and 450 mg/d were established for nonpregnant and pregnant women, respectively. However, to our knowledge, no dose-response studies have been conducted to evaluate the effects of pregnancy or maternal choline intake on biomarkers of choline metabolism., Objective: We sought to quantify the effects of pregnancy and maternal choline intake on maternal and fetal indicators of choline metabolism., Design: Healthy pregnant (n = 26; 27 wk gestation) and nonpregnant (n = 21) women were randomly assigned to receive 480 or 930 mg choline/d for 12 wk. Fasting blood samples and placental tissue and umbilical cord venous blood were collected and analyzed for choline and its metabolites., Results: Regardless of the choline intake, pregnant women had higher circulating concentrations of choline (30%; P < 0.001) but lower concentrations of betaine, dimethylglycine, sarcosine, and methionine (13-55%; P < 0.001). Obligatory losses of urinary choline and betaine in pregnant women were ∼2-4 times as high (P ≤ 0.02) as those in nonpregnant women. A higher choline intake yielded higher concentrations of choline, betaine, dimethylglycine, and sarcosine (12-46%; P ≤ 0.08) in both pregnant and nonpregnant women without affecting urinary choline excretion. The higher maternal choline intake also led to a doubling of dimethylglycine in cord plasma (P = 0.002)., Conclusion: These data suggest that an increment of 25 mg choline/d to meet the demands of pregnancy is insufficient and show that a higher maternal choline intake increases the use of choline as a methyl donor in both maternal and fetal compartments. This trial was registered at clinicaltrials.gov as NCT01127022.

Disorders in choline metabolism are related to disease conditions. We developed a stable-isotope dilution ultra performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method for the simultaneous quantification of acetylcholine (ACh), betaine, choline, and dimethylglycine (DMG). We used this method to measure concentrations of the analytes in plasma and urine in addition to other biological fluids after a protein precipitation by acetonitrile. The detection limits were between 0.35 nmol/L (for ACh in urine) and 0.34 μmol/L (for betaine in urine). ACh concentrations were not detectable in plasma. Intraassay and interassay coefficient of variation (CVs) were all <10.0% in biological fluids, except for DMG in cerebrospinal fluid (CV=12.44%). Mean recoveries in urine pool samples were between 99.2% and 103.9%. The urinary excretion of betaine, choline, and DMG was low, with approximately 50.0% higher excretion of choline in females compared to males. Median urinary excretion of ACh were 3.44 and 3.92 μmol/mol creatinine in males and females, respectively (p=0.689). Plasma betaine concentrations correlated significantly with urinary excretions of betaine (r=0.495, p=0.027) and choline (r=0.502, p=0.024) in females. Plasma choline concentrations correlated significantly with urinary excretion of ACh in males (r=0.419, p=0.041) and females (r=0.621, p=0.003). The new method for the simultaneous determination of ACh, betaine, choline, and DMG is sensitive, precise, and fast enough to be used in clinical investigations related to the methylation pathway., (Copyright © 2010 Elsevier B.V. All rights reserved.)

In order to determine trace choline in human urine, a flow injection analysis (FIA) system has been developed by coupling an enzyme reactor with an electrochemiluminescence (ECL) detector. The enzyme reactor is prepared by covalently immobilizing choline oxidase (ChOx) onto the aminopropyl-controlled pore glass beads, which are then carefully packed into a micro column. The enzyme reactor catalyzes the production of hydrogen peroxide that is in direct proportion to the concentration of choline. In this study, the enzymatically produced hydrogen peroxide was detected by an ECL detector positioned at the down stream of an enzyme reactor based on the luminol/H(2)O(2) ECL system. Under the optimized condition, the enzymatic FIA/ECL provided high sensitivity for the determination of choline with the detection limit as low as 0.05 microM (absolute detection limit was at sub pmol level). The method was successfully applied in the determination of choline in the samples of human urine, and the analytical results were in good agreement with those obtained by using the microbore HPLC with an immobilized enzyme reactor-electrochemical detection system., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

Background: An adequate intake of 550 mg choline/d was established for the prevention of liver dysfunction in men, as assessed by measuring serum alanine aminotransferase concentrations., Objective: This controlled feeding study investigated the influence of choline intakes ranging from 300 to 2200 mg/d on biomarkers of choline status. The effect of the methylenetetrahydrofolate reductase (MTHFR) C677T genotype on choline status was also examined., Design: Mexican American men (n = 60) with different MTHFR C677T genotypes (29 677TT, 31 677CC) consumed a diet providing 300 mg choline/d plus supplemental choline intakes of 0, 250, 800, or 1900 mg/d for total choline intakes of 300, 550, 1100, or 2200 mg/d, respectively, for 12 wk; 400 mug/d as dietary folate equivalents and 173 mg betaine/d were consumed throughout the study., Results: Choline intake affected the response of plasma free choline and betaine (time x choline, P < 0.001); the highest concentrations were observed in the 2200 mg/d group. Phosphatidylcholine (P = 0.026) and total cholesterol (P = 0.002) were also influenced by choline intake; diminished concentrations were observed in the 300 mg/d group. Phosphatidylcholine was modified by MTHFR genotype (P = 0.035; 677TT < 677CC). After a methionine load (100 mg/kg body wt), choline intakes of 1100 and 2200 mg/d attenuated (P = 0.016) the rise in plasma homocysteine, as did the MTHFR 677TT genotype (P < 0.001). Serum alanine aminotransferase was not influenced by the choline intakes administered in this study., Conclusions: These data suggest that 550 mg choline/d is sufficient for preventing elevations in serum markers of liver dysfunction in this population under the conditions of this study; higher intakes may be needed to optimize other endpoints.

Selecting drug candidates based on toxicity is an important step in early drug development. In this case study, it is shown how metabonomics is applied to a ranking study, in which drug candidates with equal pharmacological activities are selected based on least toxic side effects. The metabonomic analyses were carried out on an animal study that followed an established protocol for pilot toxicology/ranking studies in rats, however, not specifically modified for a metabonomic assessment. It is shown how conditions not specificially adopted for metabonomics investigations can significantly influence the metabolic profiles recorded by NMR. Furthermore, it is shown how the multivariate analysis of the NMR spectra identified an extreme excretion of an endogenous metabolite into urine induced by two out of the five drug candidates. The subsequent structure elucidation by two-dimensional NMR experiments and a subsequent validation by spiking experiments identified the metabolite as choline. The discussion of the mechanistic background for the excretion of choline, which is usually well-conserved in the body, results in two hypotheses of either a massive degradation of cell membranes or an inhibition of the choline oxidation. Although the validation of these hypotheses needs a follow-up study, the finding of a increased excretion of the important metabolite choline warrants exclusion of these two compounds as viable drug candidates from a metabonomics point of view.

Objective: To assess for novel markers of muscle damage using urinary muscle metabolites by 1H magnetic resonance spectroscopy in patients with juvenile idiopathic inflammatory myopathy (IIM)., Methods: Creatine (Cr), choline (Cho), betaine (Bet), glycine (Gly), trimethylamine oxide (TMAO), and several other metabolites were measured in first morning void urine samples from 45 patients with juvenile IIM and from 35 healthy age-matched controls, and correlated with measures of myositis disease activity and damage. Urinary metabolite to age-adjusted creatinine (Cn) ratios were examined., Results: Age-adjusted initial Cr:Cn, Cho:Cn, Bet:Cn, Gly:Cn, and TMAO:Cn ratios were higher in patients with juvenile IIM than controls (P < 0.01). Cr:Cn ratios showed significant correlations with physician-assessed global disease damage (Spearman rs = 0.37; P = 0.01), Steinbrocker functional class (rs = 0.35; P = 0.02), serum Cr (rs = 0.72; P = 0.001), and lactate dehydrogenase (rs = 0.34; P = 0.03) levels. Cho:Cn (rs = 0.3; P = 0.05), Gly:Cn (rs = 0.33; P = 0.03), and TMAO:Cn (rs = 0.36; P = 0.02) ratios showed a significant correlation with serum aldolase levels. Cho:Cn ratios also showed a significant correlation with aspartate aminotransferase levels (rs = 0.35; P = 0.02). A linear regression model was used to evaluate the factors influencing urinary Cr:Cn ratios in the 43 patients with data sets available at the initial visit. The regression model explained 73% of the variation in Cr:Cn ratios. The most significant factor was the physician-assessed global disease damage (R2 = 0.50, P = 0.015)., Conclusion: Urinary Cr:Cn, Cho:Cn, Bet:Cn, Gly:Cn, and TMAO:Cn ratios are elevated in juvenile IIM and Cr:Cn correlates strongly with global disease damage. The Cr:Cn ratio may have potential utility as a marker of myositis disease damage.

Background: A simple and reliable method is needed to assess disease activity and monitor the efficacy of therapy in polymyositis (PM) and dermatomyositis (DM). This study used in vitro proton ((1)H) magnetic resonance spectroscopy (MRS) to explore whether excretion of urinary metabolites can be used as a reliable marker of disease in PM and DM patients., Methods: Urine samples were obtained from PM/DM patients (n=34), healthy controls (50) and subjects with known muscle-wasting conditions including adult-onset muscular dystrophy (8), stroke patients (10), rheumatoid arthritis (RA) patients on steroids (13) and not on steroids (16) and patients with alcoholic myopathy (12). Levels of urinary metabolites were then correlated with creatine kinase (CK) activities and quadriceps muscle strength., Results: Creatine was detected in the urine in 26 of 35 patients with PM/DM, four of 60 cases with other medical disorders (including one with adult-onset dystrophy, one with a stroke and two with RA who were not on steroids) and 10 of 50 healthy controls. The urinary creatine/creatinine ratio exceeded 0.4 in 20 patients with PM/DM but no patients with other medical disorders and no healthy controls. These differences were highly significant (P<0.001) by Kruskal-Wallis test (comparing all groups) and by Mann-Whitney U-tests (comparing individual groups with PM/DM cases). Citrate, glycine, choline-containing compounds and taurine levels were significantly increased in PM/DM when compared with controls. There were positive correlations between CK activities and choline-containing compounds (r=0.78, P=0.0006) and also between CK activities and betaine (r=0.57, P=0.026)., Conclusions: This study shows significant differences in the urinary levels of creatine, choline-containing metabolites, betaine and citrate in PM/DM subjects compared with controls, although further work is required to elucidate the underlying metabolic processes.

Background: Previous investigations have shown that plasma free choline decreases during long distance running., Objective: This study was undertaken to determine if body choline status changes during a marathon run and whether performance is thereby adversely affected., Design: Twenty-three accomplished marathon runners 25 to 49 years of age were studied before and after the 1997 Houston-Methodist Marathon. Fasting blood and five-hour urine samples were obtained in the morning, 14 days prior to the race, immediately after the race and approximately 48 hours after completion of the race. Runners were asked to predict their finish times two weeks prior to the race. Performance was indicated by the ratio of predicted to actual time., Results: Both plasma free and phospholipid-bound choline concentrations as well as urinary free choline concentration decreased immediately following the race (19.2+/-4.5 to 14.6+/-4.2 nmol/mL, p=0.005, and 2565.2+/-516.4 to 2403.4+/-643.0 nmol/mL, p=0.068, respectively) and, except for the phospholipid-bound choline, rebounded towards baseline after 48 hours (15.6+/-3.2 and 2299.9+/-426.7 nmol/mL), although plasma concentrations remained significantly below baseline. Plasma free and phospholipid-bound choline concentrations were significantly correlated (r=0.46, p=0.0001), although urinary free choline concentration was not correlated with either. There was no correlation between plasma free, phospholipid-bound or urinary free choline concentration and actual finish time or the ratio of predicted to actual finish time. However, the percent decrease in urinary free choline concentration was significantly correlated with the ratio of predicted to actual time (r=0.47, p=0.036). No relationship was seen between this ratio and the percent decrease in either plasma free or phospholipid-bound choline concentrations immediately after the race., Conclusion: Our finding of both decreased free and phospholipid-bound choline suggests the decrease in choline status is related to accelerated choline metabolism or enhanced choline uptake by tissues rather than decreased hepatic choline release. The role of choline supplementation during endurance running requires further investigation.

Thioether methyltransferase (S-adenosyl-L-methionine: thioether S-methyltransferase; EC 2.1.1.96) catalyzes the methylation of X in compounds of the type R-X-R'(X = S, Se, Te), yielding a methyl onium ion. Previous results using mice have demonstrated a role for thioether methyltransferase in the conversion and clearance of thioethers by methylation to more water-soluble methyl sulfonium ions suitable for excretion in the urine. A potential major physiological source of thiethers is reactions catalyzed by microsomal thiol methyltransferase (S-adenosyl-L-methionine: thiol S-methyltransferase; EC 2.1.1.9), which has been shown to methylate a diverse range of aliphatic sulfhydryl compounds. This study provides evidence for the sequential methylation of the aliphatic thiol, 2-mercaptoethanol, first to the methyl thioether, 2-(methylthio)ethanol, by thiol methyltransferase followed by methylation of this methyl thioeter to the dimethyl sulfonium ion, 2-(-dimethylthio)ethanol, by thioether methyltransferase. This sequence of reactions was demonstrated in vivo by injecting mice i.p. with radioactive 2-mercaptoethanol and analyzing the labeled methylated products, 2-(methylthio)ethanol and 2(dimethylthio)ethanol, in the urine by HPLC. In addition, the system converting 2-mercaptoethanol to 2-(dimethylthio)ethanol was reconstituted in vitro using solubilized mouse liver microsomes as a source of thiol methyltransferase and purified thioether methyltransferase from mouse lung. The results of these in vivo and in vitro studies established the sequential methylation of 2-mercaptoethanol by these two enzymes.

1. [3H]-triethylcholine (TEC) was actively transported by the renal tubule of the chicken at a rate 85% that of simultaneously administered p-aminohippuric acid (PAH). 2. TEC was demonstrated to be transported by the organic cation transport system in the kidney through inhibition with quinine and the bio-cation choline. 3. When the infusion of TEC was increased to 2 times 10(-6) mol kg(-1) min(-1) reaching the infused kidney, the transport of [3H]-TEC was inhibited, suggesting that an excretory transport maximum for TEC in the renal tubules had been reached. 4. The excretion of both choline and acetylcholine was enhanced by TEC loads as low as 1 times 10(-18) mol kg(-1) min(-1). Enhancement continued as TEC infusion was increased up to approximately 1 times 10(-7) mol kg(-1) min(-1) at which point this enhancement was converted to inhibition. 5. Possible mechanisms for the biphasic effect of TEC on organic cation transport are discussed.

Microelectrodes with 2-micronm-tip diameters arranged to record differentially between this ascending limbs (ALH) and ascending vasa recta (AVR), gave values of 1.95 +/- 0.17 mV, ALH positive, in hydropenia with mineral oil bathing the kidney. Average values remained in the range of 1-2 mV when the ALH and AVR values were obtained sequentially, when the kidney was bathed in Ringer solution, when 5-8 Momega Ling-Gerard microelectrodes were used in the ALH, or when the hamsters were in saline diuresis. These results contradict reports of earlier studies with high impedance Ling-Gerard electrodes that the PD was -9 mV, ALH negative. Perfusion of ALH in saline diuresis with solutions of various compositions provided estimates of ionic transport numbers: tNa+ = 0.33 +/- 0.01, tK+ = 0.00 +/- 0.02, tC1- = 0.67 +/- 0.02. When the perfusion solution was designed to have the same Na+, K+, and C1- concentrations as AVR plasma, the PD was 1.36 +/- .20 mV; when ouabain or furosemide were included (10(-5) M), the PD declined 1.35 +/- 0.21 mV and 1.41 +/- 0.28 mV, respectively. The results suggest that active C1- transport is mainly responsible for the PD, but that diffusion potentials can contribute.

Exogenous choline was administered into the wing vein of chickens until steady-state plasma choline levels were achieved. Both plasma and urine were analyzed for free choline by a choline kinase, radiochemical microassay that did not require prior extraction of choline from the biological fluids. Choline was infused at rates from 0.5 to 20.0 mumol/kg-min. Total and urinary clearance were assessed at the steady state reached in each experiment. At the endogenous level of plasma choline of 0.019 mM, total clearance of choline from the plasma was about 50 ml/kg-min and urinary clearance was 0.06 ml/kg-min. There was no significant increase in urinary clearance of choline produced by infusion loads from 0.5 to 1.0 mumol/kg-min. However, as the infusion of choline was increased further, extraurinary clearance decreased while the contribution of the kidneys to total clearance of choline from the plasma increased. At the choline infusion rate of 12.5 mumol/kg-min, plasma choline was 0.5 mM and urinary choline clearance had reached a maximum value of 18.5 ml/kg-min for two kidneys, removing more than 70% of the infused choline.

Study on 24-hour, 30- and 60-day urine collected from 1500 newborn infants pointed out in some cases an abnormal excretion of some of the 40 urinary metabolites investigated. The analysis of urinary metabolites allows to detect a relation between abnormal excretion of urine metabolites and various types of "inborn errors of metabolism".

The bodily response to injury is an integrated and protective mechanism involving all systems, and hence it should be considered as a whole. The nervous system, especially the psychic center of the cerebral cortex, seems to initiate these responses. In the present study the neurohumoral response to trauma was assessed in experimental animals and clinical subjects. Immediately after trauma acetylcholine levels increased, suggesting enhanced activity of the central nervous system. This was followed by predominant activity of the sympathetic nervous system, as evidenced by increased catecholamine levels. During the later postoperative period, histamine levels also increased. There appears to be a good correlation between increased histamine content and fibroblastic proliferation in the traumatized area. These observations suggest that levels of the neurohumors increase in a definite sequence after trauma, facilitating the process of repair, regeneration, and normal convalescence. Therefore, trauma should be minimized during repair and the patient should receive proper sedation and rest during the pre- and postoperative periods, since each quantum of response depends upon the amount of sensory input that occurs at the time of trauma and in the post-traumatic period.

The sensitive, specific growth response to choline activity of the thermophilic enteric yeast Torulopsis pintolopessi enables estimation of free and bound choline activity in rat and human fluids and tissues- as little as 10 ng/ml of choline is measurable. Unlike other microbial assays, estimation of unbound (free) choline activity is not influenced by methionine or phospholipids. The method also distinguishes differences in choline activity of fluids and tissues from choline-deficient and choline-replete rats. Free and bound choline activity in blood, red blood cells, plasma, and liver from choline-deficient rats were almost 2-fold lower than from choline-supplemented animals. Free and bound choline activity in whole brain from choline-deficient rats were signifigantly higher (more than 2-fold). The application of the T. pintolopessi method in studying choline status in man and reasons for high choline activity in brain of choline-deficient rats are suggested.

1) Rats and mice were given large oral or subcutaneous doses of (-)-L-, (+)-D- and DL-carnitine (5 mg/g body weight). The carnitine metabolites, beta-methylcholine and acetonyltrimethylammonium, were isolated from the urine by special methods, and determined as their characteristic derivatives (2,4-dinitrophenylhydrazone and butyric ester) by thin-layer chromatography or photometry. 2) beta-Methylcholine, the product of carnitine decarboxylase, was not excreted, even when animals were heavily dosed with both carnitine isomers, with or without starvation. 3) After the administration of (+)-D- and DL-carnitine, both species excreted acetonyltrimethylammonium, which is already known as the spontaneous decarboxylation product of dehydrocarnitine (product of carnitine dehydrogenase) in bacteria. Injection of 0.71 mmol (+)-D-carnitine resulted in the excretion of 5.0 mumol (average) acetonyltrimethylammonium per mouse during the 48 h post injection. Under the same conditions, rats produced up to 40 mumol acetonyltrimethylammonium. The ratio of excreted acetonyltrimethylammonium to injected (+)-D-carnitine depended on the method of administration and the dose. 4) Production of the pharmacologically active (+)-acetyl-L-beta-methylcholine is not to be expected, following high exogenous doses of (-)-L-carnitine or (-)-acetyl-L-carnitine. The chief metabolites are trimethylamine, trimethylamine oxide and gamma-butyrobetaine (this journal 361, 1059), and both the (-)-L-carnitine pool and exogenous (-)-L-carnitine are dehydrogenated or decarboxylated only to a very small extent, if at all. When DL-carnitine is used therapeutically, the formation of acetonyltrimethylammonium must be taken into account.

Methylamines and polyols are known to behave as organic osmolytes in the adaptation of many cells to hyperosmolar conditions. Using 1H nuclear magnetic resonance spectroscopy to analyze perchloric acid extracts we have examined several tissues in the rat for the presence of these compounds. Methylamines such as glycerophosphorylcholine, choline and betaine were observed in the renal inner medulla, urinary bladder, urine, liver, brain, and plasma. Myoinositol was relatively abundant in the renal inner medulla and brain whereas sorbitol was detected only in the inner medulla. A variety of unidentified compounds was also detected in each tissue. Although these methylamines and polyols are known to respond to osmotic changes in the renal inner medulla, their responses in other tissues remain to be investigated.

Studies are reported on the specific enzymic microdetermination of free choline in human semen. The concentration of choline in normal semen was found to be 0.9 - 1.4 mg/ml. The minimum detectable concentration of choline using the method described was 1.5 micrograms. Normal levels of choline in vaginal fluid, saliva, serum and urine could not be detected by this procedure.

Growth of Escherichia coli was inhibited in a defined minimal medium by high concentrations of electrolytes and sugars in direct relation to their osmotic strength. Choline, betaine, proline, and human urine increased resistance to these substances. In contrast, the toxic effect of urea was not altered directly by betaine or urine, but was reduced in the presence of other osmolytes. The osmolyte protective effect was augmented by betaine. The osmoprotective effect of betaine and urine was confirmed with 40 strains of enteric bacteria. Urine from 19 healthy subjects contained osmoprotective activity greater than that observed with betaine. A methanol extract of urine was found to be highly protective. Although betaine was present in the extract, it could not account for all the protective activity. Urine contains additional low-molecular-weight osmoprotective agents.