Metabolomics reveals an entanglement of fasting leptin concentrations with fatty acid oxidation and gluconeogenesis in healthy children.

BACKGROUND: Leptin and adiponectin communicate with organ systems in order to regulate energetic and metabolic homeostasis. Their different points of action have been well characterized; however, no study has investigated their interrelationship with the metabolism at the molecular level in vivo. OBJECTIVE: To examine the associations of leptin and adiponectin with the metabolic profile reflecting the intercellular and interorgan communication as well as activated metabolic pathways. PATIENTS/METHODS: We measured plasma concentrations of leptin, adiponectin, and insulin along with concentrations of 196 metabolites in 400 healthy, fasting 8-years old German children who participated in the German Ulm Birth Cohort Study (UBCS). Using multiple linear mixed models, we evaluated the associations between hormones and metabolites. RESULTS: Leptin levels increased exponentially with increasing BMI. Leptin was furthermore strongly associated with alanine and aspartate (Bonferroni corrected P[PBF] = 5.7×10-8 and 1.7×10-6, respectively), and negatively associated to the sum of the non-esterified fatty acids (NEFA) and the sum of the long-chain acylcarnitines C12-C18 (PBF = 0.009 and 0.0001, respectively). Insulin showed a similar association pattern, although the associations were less strong than for leptin. Adiponectin was neither related to BMI nor to any metabolite. CONCLUSION: Although children were presumably metabolically similar, we found strong associations of insulin and leptin with the metabolite profile. High alanine concentrations and the lower concentrations of NEFA in children with high fasting leptin concentrations might arise from an increased gluconeogenesis and from the disinhibiting effect of leptin on the carnitine-palmitoyltransferase-1, respectively. As insulin had the same trend towards these associations, both hormones seem to be related to processes that provide the body with energy in fasting state.

Placental MFSD2a transporter is related to decreased DHA in cord blood of women with treated gestational diabetes.

BACKGROUND & AIMS: Maternal-fetal transfer of docosahexaenoic acid (DHA) is impaired by gestational diabetes mellitus (GDM), but the underlying mechanisms are still unknown. MFSD2a was recently recognized as a lyso-phospholipid (lyso-PL) transporter that facilitates DHA accretion in brain. The role of this transporter in placenta is uncertain. We evaluated effects of GDM and its treatment (diet or insulin) on phospholipid species, fatty acid profile in women, cord blood and placental fatty acid carriers. METHODS: Prospective observational study of pregnant women recruited in the third trimester (25 controls, 23 GDM-diet, 20 GDM-insulin). Fetal ultrasound was performed at gestational week 38. At delivery, maternal and neonatal anthropometry was performed, and fatty acids in total lipids and phospholipid species were analyzed in placenta, maternal and venous cord blood. Western-blot analyses were performed for placental fatty acid carriers. RESULTS: Fetal abdominal circumference z-score at 38 weeks tended to higher values in GDM (P = 0.071), pointing toward higher fetal fat accretion in these babies. DHA percentage in cord serum total lipids (P = 0.029) and lyso-PL (P = 0.169) were reduced in GDM. Placental MFSD2a was reduced in both GDM groups and was positively correlated to DHA values in cord serum total lipids (r = 0.388, P = 0.003). Among established placental lipid carriers, only FATP4 was correlated to DHA concentration in placental lyso-PL. In all compartments, DHA percentage was inversely correlated to fetal abdominal circumference. CONCLUSIONS: In offspring of women with GDM treated either with diet or insulin, higher fetal fat accretion and lower placental MFSD2a contribute to reduce DHA availability. Lyso-PL appear to contribute to materno-fetal DHA transport.

Tyrosine Is Associated with Insulin Resistance in Longitudinal Metabolomic Profiling of Obese Children.

In obese children, hyperinsulinaemia induces adverse metabolic consequences related to the risk of cardiovascular and other disorders. Branched-chain amino acids (BCAA) and acylcarnitines (Carn), involved in amino acid (AA) degradation, were linked to obesity-associated insulin resistance, but these associations yet have not been studied longitudinally in obese children. We studied 80 obese children before and after a one-year lifestyle intervention programme inducing substantial weight loss >0.5 BMI standard deviation scores in 40 children and no weight loss in another 40 children. At baseline and after the 1-year intervention, we assessed insulin resistance (HOMA index), fasting glucose, HbA1c, 2 h glucose in an oral glucose tolerance test, AA, and Carn. BMI adjusted metabolite levels were associated with clinical markers at baseline and after intervention, and changes with the intervention period were evaluated. Only tyrosine was significantly associated with HOMA (p < 0.05) at baseline and end and with change during the intervention (p < 0.05). In contrast, ratios depicting BCAA metabolism were negatively associated with HOMA at baseline (p < 0.05), but not in the longitudinal profiling. Stratified analysis revealed that the children with substantial weight loss drove this association. We conclude that tyrosine alterations in association with insulin resistance precede alteration in BCAA metabolism. This trial is registered with ClinicalTrials.gov Identifier NCT00435734.

Dietary protein intake affects amino acid and acylcarnitine metabolism in infants aged 6 months.

CONTEXT: The protective effect of breast-feeding against later obesity may be explained by the lower protein content compared with formula milk. However, the metabolic mechanisms remain unknown. OBJECTIVE: We studied the metabolic response to a higher or lower protein supply in infancy. DESIGN AND SETTING: The Childhood Obesity Project study is a double-blind, randomized, multicenter intervention trial. Infants were randomized to receive a higher (HP) or lower protein (LP) content infant formula or were breast-fed. PATIENTS AND INTERVENTIONS: Plasma samples of 691 infants who received formula milk with different protein content (HP, 2.05 g per 100 mL; LP, 1.25 g per 100 mL) or were breast-fed were collected. MAIN OUTCOME MEASURES: Changes in plasma amino acid and acylcarnitine concentrations of 6-month-old infants according to different dietary protein supply were determined by liquid chromatography coupled to tandem mass spectrometry. RESULTS: Twenty-nine metabolites differed significantly between the formula groups. Branched-chain amino acids (BCAAs) were the most discriminant metabolites. Their degradation products, the short-chain acylcarnitines C3, C4, and C5, were also significantly elevated in the HP group. A breakpoint analysis confirmed that with increasing BCAAs, the ratio between acylcarnitines and BCAAs decreases. Long-chain acylcarnitines were decreased in HP infants. CONCLUSIONS: BCAAs seem to play a pivotal role in the effect of a high-protein diet on ß-oxidation and fat storage. We provide new evidence for a possible saturation of the BCAA degradation pathway that may represent the mechanism by which high-protein intake affects the metabolic regulation. Moreover, it appears to inhibit the initial step of the ß-oxidation, thus leading to high early weight gain and body fat deposition.

Changes in the serum metabolite profile in obese children with weight loss.

PURPOSE: Childhood obesity is an increasing problem and is accompanied by metabolic disturbances. Recently, we have identified 14 serum metabolites by a metabolomics approach (FIA-MS/MS), which showed altered concentrations in obese children as compared to normal-weight children. Obese children demonstrated higher concentrations of two acylcarnitines and lower levels of three amino acids, six acyl-alkyl phosphatidylcholines, and three lysophosphatidylcholines. The aim of this study was to analyze whether these alterations normalize in weight loss. METHODS: We analyzed the changes of these 14 metabolites by the same metabolic kit as in our previous study in serum samples of 80 obese children with substantial weight loss (BMI-SDS reduction >0.5) and in 80 obese children with stable weight status all participating in a 1-year lifestyle intervention. RESULTS: In the children without weight change, no significant changes of metabolite concentrations could be observed. In children with substantial weight loss, glutamine, methionine, the lysophosphatidylcholines LPCaC18:1, LPCaC18:2, and LPCa20:4, as well as the acyl-alkyl phosphatidylcholine PCaeC36:2 increased significantly, while the acylcarnitines C12:1 and C16:1, proline, PCaeC34:1, PCaeC34:2, PCaeC34:3, PCaeC36:3, and PCaeC38:2 did not change significantly. CONCLUSIONS: The changes of glutamine, methionine, LPCaC18:1, LPCaC18:2, LPCa20:4, and PCaeC36:2 seem to be related to the changes of dieting or exercise habits in lifestyle intervention or to be a consequence of overweight since they normalized in weight loss. Further studies should substantiate our findings.

Rapid growth and childhood obesity are strongly associated with lysoPC(14:0).

BACKGROUND: Despite the growing interest in the early-origins-of-later-disease hypothesis, little is known about the metabolic underpinnings linking infant weight gain and childhood obesity. OBJECTIVE: To discover biomarkers reflective of weight change in the first 6 months and overweight/obesity at age 6 years via a targeted metabolomics approach. DESIGN: This analysis comprised 726 infants from a European multicenter randomized trial (Childhood Obesity Programme, CHOP) for whom plasma blood samples at age 6 months and anthropometric data up to the age of 6 years were available. 'Rapid growth' was defined as a positive difference in weight within the first 6 months of life standardized to WHO growth standards. Weight change was regressed on each of 168 metabolites (acylcarnitines, lysophosphatidylcholines, sphingomyelins, and amino acids). Metabolites significant after Bonferroni's correction were tested as predictors of later overweight/obesity. RESULTS: Among the overall 19 significant metabolites, 4 were associated with rapid growth and 15 were associated with a less-than-ideal weight change. After adjusting for feeding group, only the lysophosphatidylcholine LPCaC14:0 remained significantly associated with rapid weight gain (ß = 0.18). Only LPCaC14:0 at age 6 months was predictive of overweight/obesity at age 6 years (OR 1.33; 95% CI 1.04-1.69). CONCLUSION: LPCa14:0 is strongly related to rapid growth in infancy and childhood overweight/obesity. This suggests that LPCaC14:0 levels may represent a metabolically programmed effect of infant weight gain on the later obesity risk. However, these results require confirmation by independent cohorts.

Three-month B vitamin supplementation in pre-school children affects folate status and homocysteine, but not cognitive performance.

BACKGROUND: Suboptimal vitamin B status might affect cognitive performance in early childhood. We tested the hypothesis that short-term supplementation with folic acid and selected B vitamins improves cognitive function in healthy children in a population with relatively low folate status. METHODS: We screened 1,002 kindergarten children for suboptimal folate status by assessing the total urinary para-aminobenzoylglutamate excretion. Two hundred and fifty low ranking subjects were recruited into a double blind, randomized, controlled trial to receive daily a sachet containing 220 µg folic acid, 1.1 mg vitamin B2, 0.73 mg B6, 1.2 µg B12 and 130 mg calcium, or calcium only for 3 months. Primary outcomes were changes in verbal IQ, short-term memory and processing speed between baseline and study end. Secondary outcomes were urinary markers of folate and vitamin B12 status, acetyl-para-aminobenzoylglutamate and methylmalonic acid, respectively, and, in a subgroup of 120 participants, blood folate and plasma homocysteine. RESULTS: Pre- and post-intervention cognitive measurements were completed by 115 children in the intervention and 122 in the control group. Compared to control, median blood folate increased by about 50% (P for difference, P < 0.0001). Homocysteine decreased by 1.1 µmol/L compared to baseline, no change was seen in the control group (P for difference P < 0.0001) and acetyl-para-aminobenzoylglutamate was 4 nmol/mmol higher compared to control at the end of the intervention (P < 0.0001). We found no relevant differences between the groups for the cognitive measures. CONCLUSION: Short-term improvement of folate and homocysteine status in healthy children does not appear to affect cognitive performance.

Association between plasma nonesterified fatty acids species and adipose tissue fatty acid composition.

Fatty acid composition of adipose tissue (AT) is an established long-term biomarker for fatty acid (FA) intake and status, but AT samples are not easily available. Nonesterified FA composition in plasma (pNEFA) may be a good indicator of AT FA composition, because pNEFA are mainly generated by AT lipolysis. We investigated the correlation of 42 pNEFA and subcutaneous as well as visceral AT FA in 27 non-diabetic women with a median BMI of 36 kg/m(2) (Q0.25: 25 kg/m(2); Q0.75: 49 kg/m(2)). Close correlations of pNEFA and AT FA were found for odd-chain FA (15∶0 r = 0.838 and 0.862 for subcutaneous and visceral AT, respectively) and omega-3 FA (22∶6 r = 0.719/0.535), while no significant or low correlations were found for other FA including 18∶1 (r = 0.384/0.325) and 20∶4 (r = 0.386/0.266). Close correlations of pNEFA and AT FA were found for essential fatty acids, like 18∶2 (r = 0.541/0.610) and 20∶5 (r = 0.561/0.543). The lower correlation for some pNEFA species with AT FA indicates that the variation of most pNEFA is significantly affected by other FA sources and flux of FA to tissue, in addition to release from AT. A relevant influence of BMI on the level of correlation was shown for saturated FA. NEFA analysis in fasted plasma can serve as a virtual AT biopsy for some FA, and as a biomarker for intake of dairy products and sea fish.

Quantification of urinary folate catabolites using liquid chromatography-tandem mass spectrometry.

Folate catabolites p-aminobenzoylglutamate (pABG) and p-acetamidobenzoylglutamate (apABG) in human urine result from break-down of endogenous folate pools and are potential biomarkers of folate status. There is growing interest in analysis of these non-invasive indicators of folate status, since widespread diseases such as cancer, arteriosclerosis and dementia may be linked to disturbed availability of folates. Determination of pABG and apABG in human urine is challenging due to their low urinary concentrations and due to interferences with other urinary compounds. To address these analytical difficulties, we developed an improved LC-MS/MS method with chemical derivatization for fast, selective and sensitive quantification of pABG and apABG in human urine. Forming butyl esters of urinary folate catabolites pABG and apABG improves ionization efficiency as well as enables selective chromatographic separation on standard C18 reversed-phase column material. In contrast to some previously proposed methods for folate catabolites, the new method allows precise differentiation of apABG from pABG. Partial degradation of apABG during derivatization is exactly accounted for using a second differentially labeled stable isotope internal standard. This method is highly sensitive and covers the full range of physiologically occurring concentrations (from 2 to 1000nmol/L), with volume requirements of only 80µL urine. Method performance has been validated according to widely accepted standard recommendations. Use of two stable isotope-labeled internal standards and qualifier ion monitoring for both analytes ensure correct identification and unbiased quantification. With run times of less than 2.5min per sample and cost-efficient sample preparation, this method allows exact quantitation of urinary folate catabolites pABG and apABG for large-scale non-invasive screening of folate status in clinical and epidemiological trials.

Folate catabolites in spot urine as non-invasive biomarkers of folate status during habitual intake and folic acid supplementation.

BACKGROUND: Folate status, as reflected by red blood cell (RCF) and plasma folates (PF), is related to health and disease risk. Folate degradation products para-aminobenzoylglutamate (pABG) and para-acetamidobenzoylglutamate (apABG) in 24 hour urine have recently been shown to correlate with blood folate. AIM: Since blood sampling and collection of 24 hour urine are cumbersome, we investigated whether the determination of urinary folate catabolites in fasted spot urine is a suitable non-invasive biomarker for folate status in subjects before and during folic acid supplementation. STUDY DESIGN AND METHODS: Immediate effects of oral folic acid bolus intake on urinary folate catabolites were assessed in a short-term pre-study. In the main study we included 53 healthy men. Of these, 29 were selected for a 12 week folic acid supplementation (400 µg). Blood, 24 hour and spot urine were collected at baseline and after 6 and 12 weeks and PF, RCF, urinary apABG and pABG were determined. RESULTS: Intake of a 400 µg folic acid bolus resulted in immediate increase of urinary catabolites. In the main study pABG and apABG concentrations in spot urine correlated well with their excretion in 24 hour urine. In healthy men consuming habitual diet, pABG showed closer correlation with PF (rs = 0.676) and RCF (rs = 0.649) than apABG (rs = 0.264, ns and 0.543). Supplementation led to significantly increased folate in plasma and red cells as well as elevated urinary folate catabolites, while only pABG correlated significantly with PF (rs = 0.574) after 12 weeks. CONCLUSION: Quantification of folate catabolites in fasted spot urine seems suitable as a non-invasive alternative to blood or 24 hour urine analysis for evaluation of folate status in populations consuming habitual diet. In non-steady-state conditions (folic acid supplementation) correlations between folate marker (RCF, PF, urinary catabolites) decrease due to differing kinetics.

Determination of creatinine in human urine with flow injection tandem mass spectrometry.

BACKGROUND/AIMS: Excretion of urinary compounds in spot urine is often estimated relative to creatinine. For the growing number of liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays of urine-excreted molecules, a fast and accurate method for determination of creatinine is needed. METHODS: A high-throughput flow injection tandem mass spectrometry method for exact quantitation of creatinine in urine has been developed and validated. Sample preparation used only two-step dilution for protein precipitation and matrix dilution. Flow injection analysis without chromatographic separation allowed for total run times of 1 min per sample. Creatinine concentrations were quantitated using stable isotope dilution tandem mass spectrometry. Selectivity and coelution-free quantitation were assured by qualifier ion monitoring. RESULTS: Method validation revealed excellent injection repeatability of 1.0% coefficient of variation (CV), intraday precision of 1.2% CV and interday precision of 2.4% CV. Accuracy determined from standard addition experiments was 106.1 ± 3.8%. The linear calibration range was adapted to physiological creatinine concentrations. Comparison of quantitation results with a routinely used method (Jaffé colorimetric assay) proved high agreement (R(2) = 0.9102). CONCLUSIONS: The new method is a valuable addition to the toolbox of LC-MS/MS laboratories where excretion of urinary compounds is studied. The 'dilute and shoot' approach to isotope dilution tandem mass spectrometry makes the new method highly accurate as well as cost- and time-efficient.

Nonesterified fatty acid determination for functional lipidomics: comprehensive ultrahigh performance liquid chromatography-tandem mass spectrometry quantitation, qualification, and parameter prediction.

Despite their central importance for lipid metabolism, straightforward quantitative methods for determination of nonesterified fatty acid (NEFA) species are still missing. The protocol presented here provides unbiased quantitation of plasma NEFA species by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Simple deproteination of plasma in organic solvent solution yields high accuracy, including both the unbound and initially protein-bound fractions, while avoiding interferences from hydrolysis of esterified fatty acids from other lipid classes. Sample preparation is fast and nonexpensive, hence well suited for automation and high-throughput applications. Separation of isotopologic NEFA is achieved using ultrahigh-performance liquid chromatography (UPLC) coupled to triple quadrupole LC-MS/MS detection. In combination with automated liquid handling, total assay time per sample is less than 15 min. The analytical spectrum extends beyond readily available NEFA standard compounds by a regression model predicting all the relevant analytical parameters (retention time, ion path settings, and response factor) of NEFA species based on chain length and number of double bonds. Detection of 50 NEFA species and accurate quantification of 36 NEFA species in human plasma is described, the highest numbers ever reported for a LC-MS application. Accuracy and precision are within widely accepted limits. The use of qualifier ions supports unequivocal analyte verification.

Quantification of 22 plasma amino acids combining derivatization and ion-pair LC-MS/MS.

Time efficient and comprehensive quantification of amino acids continues to be a challenge. We developed a sensitive and precise method for quantitative analysis of amino acids from very small plasma and serum volumes. Ion-pair chromatography of amino acid butyl esters proved to provide an optimal combination of selectivity, sensitivity and robustness. 10 µL of plasma or serum are added to precipitation reagent containing stable isotope standards. After protein precipitation, the supernatants is dried and incubated with 3N butanolic HCl for improving chromatographic separation and ionization efficiency. Amino acid butyl esters are separated using ion-pair (heptafluorobutyric acid) reversed-phase chromatography coupled to triple quadrupole mass spectrometry. The established method enables quantitative analysis of 22 amino acids, all 20 proteinogenic amino acids, ornithine and citrulline. Cysteine is measured as cystine. The combination of precipitation, derivatization and chromatographic separation effectively avoids ion suppression and coelution. Simultaneous with quantification, analyte identity is verified in each sample using qualifier ions. The micro-method is very sensitive and accurate. The intra-assay precision for the analysis of plasma was 2.6-10.1%. Absolute accuracy as determined by comparison of external reference samples was 82-117.7%. Excellent linearity of detection response was demonstrated for all compounds in the range representative for clinical samples from infants and adults. Lower limits of quantification were in the range of 1 µmol/L for all analytes. In conclusion, the method is ideally suited for cost-effective high-throughput analysis of large numbers of samples in clinical studies and metabolomics research.

Aqueous normal phase chromatography improves quantification and qualification of homocysteine, cysteine and methionine by liquid chromatography-tandem mass spectrometry.

Elevation of plasma homocysteine concentration is recognized as an independent predictor of cardiovascular disease risk. Therefore, quantification of homocysteine and related sulphur amino acids cysteine and methionine from plasma samples is routinely performed in clinical laboratories. Due to the highly hydrophilic character of these amino acids, previously reported LC-MS methods often suffered from very short chromatographic retention resulting in inadequate separation from matrix background and possible co-eluents. In the present method, aqueous normal phase (ANP) chromatography was introduced to improve chromatographic separation for liquid chromatography-electrospray ionization tandem mass spectrometry. Selective qualification of analytes and internal standards was achieved by qualifier ion monitoring. Using this enhanced selectivity, spurious co-eluents were identified and separated from the analyte signal by optimization of chromatographic conditions. Method validation proved high precision and accuracy (intra-assay reproducibility 1.2-4.3% CV, inter-assay reproducibility 3.4-6.1% CV, accuracy 91.3-105.9%). Total cycle time of 7 min and low costs per sample allow high-throughput application in clinical diagnostics and research trials.