Calibration of citrus intake assessed by food frequency questionnaires using urinary proline betaine in an observational study setting.

BACKGROUND: Whether observational study can be employed to establish calibration equations for self-reported dietary intake using food biomarkers is unknown. OBJECTIVES: This study aims to demonstrate the feasibility of obtaining calibration equations based on food biomarkers and 7-d diet records (7DDRs) to correct measurement errors of food frequency questionnaires (FFQs) in an observational study setting. METHODS: The study population consisted of 669 males and 749 females from the Women's and Men's Lifestyle Validation Studies. In the training set, the biomarker-predicted intake derived by regressing 7DDR-assessed intake on urinary proline betaine concentration was regressed on the FFQ-assessed intake to obtain the calibration equations. The regression coefficients were applied to the test set to calculate the calibrated FFQ intake. We examined total citrus as well as individual citrus fruits/beverages. RESULTS: Urinary proline betaine was moderately correlated with orange juice intake (Pearson correlation [r] = 0.53 for 7DDR and 0.48 for FFQ) but only weakly correlated with intakes of orange (r = 0.12 for 7DDR and 0.15 for FFQ) and grapefruit (r = 0.14 for 7DDR and 0.09 for FFQ). The FFQ-assessed citrus intake was systematically higher than the 7DDR-assessed intake, and after calibrations, the mean calibrated FFQ measurements were almost identical to 7DDR assessments. In the test set, the mean intake levels from 7DDRs, FFQs, and calibrated FFQs were 62.5, 75.3, and 63.2 g/d for total citrus; 41.6, 42.5, and 41.9 g/d for orange juice; 11.8, 24.3, and 12.3 g/d for oranges; and 8.3, 9.3, and 8.6 g/d for grapefruit, respectively. We observed larger differences between calibrated FFQ and 7DDR assessments at the extreme ends of intake, although, on average, good agreements were observed for all citrus except grapefruit. CONCLUSIONS: Our 2-step calibration approach has the potential to be adapted to correct systematic measurement error for other foods/nutrients with established food biomarkers in a cost effective way.

Urine metabolic phenotyping in children with nocturnal enuresis and comorbid neurobehavioral disorders.

Nocturnal enuresis (NE) is a common problem among 10% school-aged children. The etiologies underlying childhood NE is complex and not fully understood nowadays. Nevertheless, increasing evidence suggests a potential link between neurobehavioral disorders and enuresis in children. In this study, we aimed to explore novel metabolomic insights into the pathophysiology of NE and also, its association with pediatric psychiatric problems. Urine collected from 41 bedwetting children and 27 healthy control children was analyzed by using 1H-nuclear magnetic resonance spectroscopy from August 2017 to December 2018. At regular follow-up, there were 14 children with refractory NE having a diagnosis of attention deficient hyperactivity disorder (ADHD) or anxiety. Eventually, we identified eight significantly differential urinary metabolites and particularly increased urinary excretion of betaine, creatine and guanidinoacetate linked to glycine, serine and threonine metabolism were associated with a comorbidity of neurobehavioral disorders in refractory bedwetting children. Notably, based on physiological functions of betaine acting as a renal osmolyte and methyl group donor, we speculated its potential role in modulation of renal and/or central circadian clock systems, becoming a useful urinary metabolic marker in diagnosis of treatment-resistant NE in children affected by these two disorders.

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.

Metabolomic analysis to detect urinary molecular changes associated with bipolar depression.

Bipolar disorder (BD) is a debilitating mental disorder with complex clinical manifestations and low diagnostic accuracy. Depressive episodes are most common in the course of BD with high comorbidity and suicide rates, which present greater clinical challenges than mania and hypomania episodes. However, there are no objective biomarkers for bipolar depression. The aim of this study was to detect urinary metabolite biomarkers that could be useful for the diagnosis of bipolar depression. Nuclear magnetic resonance spectroscopy was used to profile urine samples of patients with bipolar depression (n = 37) and healthy volunteers (n = 48). Data were analyzed using Orthogonal Partial Least Square Discriminant Analysis and t-test. Differential metabolites were identified (VIP > 1 and p < 0.05), and further analyzed using Metabo Analyst 3.0 to identify associated metabolic pathways. In total, we identified seven metabolites differentially expressed in patients with BD and healthy controls. Compared with healthy group, the levels of betaine, glycerol, hippuric acid, indole sulfate, trimethylamine oxide, and urea in urine samples of BD patients were significantly higher, while the level of inositol was significantly lower. Most of these small molecules are related to lipid metabolism and gut microbiota metabolism. These differential metabolites could provide critical insight into the pathological mechanisms of bipolar depression. The results of this study provide a meaningful reference for similar and further studies in the future.

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.

High-Throughput Quantitation of Proline Betaine in Foods and Suitability as a Valid Biomarker for Citrus Consumption.

Proline betaine has been proposed as a candidate dietary biomarker for citrus intake. To validate its suitability as a dietary biomarker and to gain insight into the range of this per-methylated amino acid in foods and beverages, a quick and accurate stable isotope dilution assay was developed for quantitative high-throughput HILIC-MS/MS screening of proline betaine in foods and urine after solvent-mediated matrix precipitation. Quantitative analysis of a variety of foods confirmed substantial amounts of proline betaine in citrus juices (140-1100 mg/L) and revealed high abundance in tubers of the vegetable Stachys affinis, also known as Chinese artichocke (∼700 mg/kg). Seafood including clams, shrimp, and lobster contained limited amounts (1-95 mg/kg), whereas only traces were detected in fish, cuttlefish, fresh meat, dairy products, fresh vegetable (<3 mg/kg), coffee, tea, beer, and wine (<7 mg/L). The human excretion profiles of proline betaine in urine were comparable when common portions of orange juice or fried Stachys tubers were consumed. Neither mussels nor beer provided enough proline betaine to detect significant differences between morning urine samples collected before and after consumption. As Stachys is a rather rare vegetable and not part of peoples' daily diet, the data reported here will help to monitor the subject's compliance in future nutritional human studies on citrus products or the exclusion of citrus products in the wash-out phase of an intervention study. Moreover, proline betaine measurement can contribute to the establishment of a toolbox of valid dietary biomarkers reflecting wider aspects of diet to assess metabolic profiles as measures of dietary exposure and indicators of dietary patterns, dietary changes, or effectiveness of dietary interventions.

Using NMR-Based Metabolomics to Evaluate Postprandial Urinary Responses Following Consumption of Minimally Processed Wheat Bran or Wheat Aleurone by Men and Women.

Wheat bran, and especially wheat aleurone fraction, are concentrated sources of a wide range of components which may contribute to the health benefits associated with higher consumption of whole-grain foods. This study used NMR metabolomics to evaluate urine samples from baseline at one and two hours postprandially, following the consumption of minimally processed bran, aleurone or control by 14 participants (7 Females; 7 Males) in a randomized crossover trial. The methodology discriminated between the urinary responses of control, and bran and aleurone, but not between the two fractions. Compared to control, consumption of aleurone or bran led to significantly and substantially higher urinary concentrations of lactate, alanine, N-acetylaspartate acid and N-acetylaspartylglutamate and significantly and substantially lower urinary betaine concentrations at one and two hours postprandially. There were sex related differences in urinary metabolite profiles with generally higher hippurate and citrate and lower betaine in females compared to males. Overall, this postprandial study suggests that acute consumption of bran or aleurone is associated with a number of physiological effects that may impact on energy metabolism and which are consistent with longer term human and animal metabolomic studies that used whole-grain wheat diets or wheat fractions.

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.

Validated method for the quantification of free and total carnitine, butyrobetaine, and acylcarnitines in biological samples.

A validated quantitative method for the determination of free and total carnitine, butyrobetaine, and acylcarnitines is presented. The versatile method has four components: (1) isolation using strong cation-exchange solid-phase extraction, (2) derivatization with pentafluorophenacyl trifluoromethanesulfonate, (3) sequential ion-exchange/reversed-phase (ultra) high-performance liquid chromatography [(U)HPLC] using a strong cation-exchange trap in series with a fused-core HPLC column, and (4) detection with electrospray ionization multiple reaction monitoring (MRM) mass spectrometry (MS). Standardized carnitine along with 65 synthesized, standardized acylcarnitines (including short-chain, medium-chain, long-chain, dicarboxylic, hydroxylated, and unsaturated acyl moieties) were used to construct multiple-point calibration curves, resulting in accurate and precise quantification. Separation of the 65 acylcarnitines was accomplished in a single chromatogram in as little as 14 min. Validation studies were performed showing a high level of accuracy, precision, and reproducibility. The method provides capabilities unavailable by tandem MS procedures, making it an ideal approach for confirmation of newborn screening results and for clinical and basic research projects, including treatment protocol studies, acylcarnitine biomarker studies, and metabolite studies using plasma, urine, tissue, or other sample matrixes.

Amino acid-derived betaines dominate as urinary markers for rye bran intake in mice fed high-fat diet--A nontargeted metabolomics study.

SCOPE: Bioprocessing of whole grain cereals may affect the bioavailability of phytochemicals associated with grain fiber and ultimately lead to different health outcomes. Here, we studied the impact of long-term feeding with intact and bioprocessed rye bran on the urinary phytochemical profile of mice. METHODS AND RESULTS: Nontargeted hydrophilic interaction chromatography-ESI-qTOF-MS metabolite profiling approach was applied on urine samples collected from three groups of diet-induced obese mice fed for 8 weeks with one of the three diets: high-fat (HF) control diet, HF diet enriched with intact rye bran, or HF diet enriched with bioprocessed rye bran. The most striking finding was the increased urinary excretion of several amino-acid derived betaines after both rye diets. These included proline betaine, alanine betaine, valine betaine, phenylalanine betaine, pipecolic acid betaine, and trigonelline, but not glycine betaine. Furthermore, bioprocessing may have improved the bioavailability of rye-derived phytochemicals, as higher increase in, e.g. ferulic acid and benzoxazinoid metabolites were observed in urine of mice fed with bioprocessed than intact rye bran. CONCLUSION: Urinary excretion of various betaines was greatly increased in mice fed rye brans. Furthermore, bioprocessing of rye bran appears to serve as a beneficial way to improve the bioavailability of various phytochemicals.

Development and validation of a simple UHPLC-MS/MS method for the simultaneous determination of trimethylamine N-oxide, choline, and betaine in human plasma and urine.

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.

Variation of betaine, N,N-dimethylglycine, choline, glycerophosphorylcholine, taurine and trimethylamine-N-oxide in the plasma and urine of overweight people with type 2 diabetes over a two-year period.

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.

Fenofibrate causes elevation of betaine excretion but not excretion of other osmolytes by healthy adults.

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.

Extreme urinary betaine losses in type 2 diabetes combined with bezafibrate treatment are associated with losses of dimethylglycine and choline but not with increased losses of other osmolytes.

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.

Assessment of urinary betaine as a marker of diabetes mellitus in cardiovascular patients.

Abnormal urinary excretion of betaine has been demonstrated in patients with diabetes or metabolic syndrome. We aimed to identify the main predictors of excretion in cardiovascular patients and to make initial assessment of its feasibility as a risk marker of future diabetes development. We used data from 2396 patients participating in the Western Norway B-vitamin Intervention Trial, who delivered urine and blood samples at baseline, and in the majority at two visits during follow-up of median 39 months. Betaine in urine and plasma were measured by liquid-chromatography-tandem mass spectrometry. The strongest determinants of urinary betaine excretion by multiple regression were diabetes mellitus, age and estimated glomerular filtration rate; all p<0.001. Patients with diabetes mellitus (n = 264) had a median excretion more than three times higher than those without. We found a distinct non-linear association between urinary betaine excretion and glycated hemoglobin, with a break-point at 6.5%, and glycated hemoglobin was the strongest determinant of betaine excretion in patients with diabetes mellitus. The discriminatory power for diabetes mellitus corresponded to an area under the curve by receiver-operating characteristics of 0.82, and betaine excretion had a coefficient of reliability of 0.73. We also found a significant, independent log-linear relation between baseline betaine excretion and the risk of developing new diabetes during follow-up. The good discriminatory power for diabetes, high test-retest stability and independent association with future risk of new diabetes should motivate further investigation on the role of betaine excretion in risk assessment and long-term follow-up of diabetes mellitus.

Urinary metabolomics in newborns infected by human cytomegalovirus: a preliminary investigation.

Human cytomegalovirus (HCMV) represents one of the most significant viral risks of birth defects and long-term sequelae. The severity of the infection depends on the form of the disease, which can be symptomatic or asymptomatic with or without sequelae. The aim of this study was to investigate in a population of newborns the impact of HCMV infection on the urine metabolome by using (1)H-nuclear magnetic resonance (NMR) spectroscopy combined with multivariate statistical analysis. Twenty-three children born from women with a primary HCMV infection during pregnancy were recruited. Twelve were HCMV infected at birth whereas eleven were not infected (control). The (1)H-NMR spectra were analyzed using a PLS-DA mathematical model in order to identify the discriminant metabolites between the asymptomatic and the control group. The most important metabolites characterizing the clustering of the samples were: myoinositol, glycine, 3-hydroxybutyrate, 3-aminoisobutyrate, creatine, taurine and betaine. These findings suggest the use of metabolomics as a useful new tool in the investigation of HCMV congenital infection.

[Urine metabonomic study of intervention effects of Morinda officinalis how. on 'kidney-yang deficiency syndrome'].

To investigate the intervention effects of Morinda officinalis How. on 'Kidney-yang deficiency syndrome' induced by hydrocortisone in rats, the metabolic profiles of rat urine were characterized using proton nuclear magnetic resonance and principal component analysis (PCA) was applied to study the trajectory of urinary metabolic phenotype of rats with 'Kidney-yang deficiency syndrome' under administration of M. officinalis at different time points. Meanwhile, the intervention effects of M. officinalis on urinary metabolic potential biomarkers associated with 'Kidney-yang deficiency syndrome' were also discussed. The experimental results showed that in accordance to the increased time of administration, an obvious tendency was observed that clustering of the treatment group moved gradually closed to that of the control group. Eight potential biomarkers including citrate, succinate, alpha-ketoglutarate, lactate, betaine, sarcosine, alanine and taurine were definitely up- or down-regulated. In conclusion, the effectiveness of M. oficinalis on 'Kidney-yang deficiency syndrome' is proved using the established metabonomic method and the regulated metabolic pathways involve energy metabolism, transmethylation and transportation of amine. Meanwhile, the administration of M. officinalis can alleviate the kidney impairment induced by 'Kidney-yang deficiency syndrome'.

Metabolic profiling in Maturity-onset diabetes of the young (MODY) and young onset type 2 diabetes fails to detect robust urinary biomarkers.

It is important to identify patients with Maturity-onset diabetes of the young (MODY) as a molecular diagnosis determines both treatment and prognosis. Genetic testing is currently expensive and many patients are therefore not assessed and are misclassified as having either type 1 or type 2 diabetes. Biomarkers could facilitate the prioritisation of patients for genetic testing. We hypothesised that patients with different underlying genetic aetiologies for their diabetes could have distinct metabolic profiles which may uncover novel biomarkers. The aim of this study was to perform metabolic profiling in urine from patients with MODY due to mutations in the genes encoding glucokinase (GCK) or hepatocyte nuclear factor 1 alpha (HNF1A), type 2 diabetes (T2D) and normoglycaemic control subjects. Urinary metabolic profiling by Nuclear Magnetic Resonance (NMR) and ultra performance liquid chromatography hyphenated to Q-TOF mass spectrometry (UPLC-MS) was performed in a Discovery set of subjects with HNF1A-MODY (n = 14), GCK-MODY (n = 17), T2D (n = 14) and normoglycaemic controls (n = 34). Data were used to build a valid partial least squares discriminate analysis (PLS-DA) model where HNF1A-MODY subjects could be separated from the other diabetes subtypes. No single metabolite contributed significantly to the separation of the patient groups. However, betaine, valine, glycine and glucose were elevated in the urine of HNF1A-MODY subjects compared to the other subgroups. Direct measurements of urinary amino acids and betaine in an extended dataset did not support differences between patients groups. Elevated urinary glucose in HNF1A-MODY is consistent with the previously reported low renal threshold for glucose in this genetic subtype. In conclusion, we report the first metabolic profiling study in monogenic diabetes and show that, despite the distinct biochemical pathways affected, there are unlikely to be robust urinary biomarkers which distinguish monogenic subtypes from T2D. Our results have implications for studies investigating metabolic profiles in complex traits including T2D.