Katja Dettmer

Lactic Acid and Acidification Inhibit TNF Secretion and Glycolysis of Human Monocytes

High concentrations of lactic acid (LA) are found under various pathophysiological conditions and are accompanied by an acidification of the environment. To study the impact of LA on TNF secretion, human LPS-stimulated monocytes were cultured with or without LA or the corresponding pH control. TNF secretion was significantly suppressed by low concentrations of LA (≤10 mM), whereas only strong acidification had a similar effect. This result was confirmed in a coculture model of human monocytes with multicellular tumor spheroids. Blocking synthesis of tumor-derived lactate by oxamic acid, an inhibitor of lactate dehydrogenase, reversed the suppression of TNF secretion in this coculture model. We then investigated possible mechanisms underlying the suppression. Uptake of [3-13C]lactate by monocytes was shown by hyphenated mass spectrometry. As lactate might interfere with glycolysis, the glycolytic flux of monocytes was determined. We added [1,2-13C2]glucose to the culture medium and measured glucose uptake and conversion into [2,3-13C2]lactate. Activation of monocytes increased the glycolytic flux and the secretion of lactate, whereas oxygen consumption was decreased. Addition of unlabeled LA resulted in a highly significant decrease in [2,3-13C2]lactate secretion, whereas a mere corresponding decrease in pH exerted a less pronounced effect. Both treatments increased intracellular [2,3-13C2]lactate levels. Blocking of glycolysis by 2-deoxyglucose strongly inhibited TNF secretion, whereas suppression of oxidative phosphorylation by rotenone had little effect. These results support the hypothesis that TNF secretion by human monocytes depends on glycolysis and suggest that LA and acidification may be involved in the suppression of TNF secretion in the tumor environment.

Advances in amino acid analysis

AbstractAmino acids are important targets for metabolic profiling. For decades, amino acid analysis has been accomplished by either cation-exchange or reversed-phase liquid chromatography coupled to UV absorbance or fluorescence detection of pre-column or post-column-derivatized amino acids. Recent years have seen great progress in the development of direct-infusion or hyphenated mass spectrometry in the analysis of free amino acids in physiological fluids, because mass spectrometry not only matches optical detection in sensitivity, but also offers superior selectivity. The advent of cryo-probes has also brought NMR spectroscopy within the detection limits required for the analysis of free amino acids. But there is still room for further improvement, including expansion of the analyte spectrum, reduction of sample preparation and analysis time, automation, and synthesis of affordable isotope standards. FigureFully automated gas chromatography-mass spectrometry analysis of amino acids.

Automated GC–MS analysis of free amino acids in biological fluids

A gas chromatography-mass spectrometry (GC-MS) method was developed for the quantitative analysis of free amino acids as their propyl chloroformate derivatives in biological fluids. Derivatization with propyl chloroformate is carried out directly in the biological samples without prior protein precipitation or solid-phase extraction of the amino acids, thereby allowing automation of the entire procedure, including addition of reagents, extraction and injection into the GC-MS. The total analysis time was 30 min and 30 amino acids could be reliably quantified using 19 stable isotope-labeled amino acids as internal standards. Limits of detection (LOD) and lower limits of quantification (LLOQ) were in the range of 0.03-12 microM and 0.3-30 microM, respectively. The method was validated using a certified amino acid standard and reference plasma, and its applicability to different biological fluids was shown. Intra-day precision for the analysis of human urine, blood plasma, and cell culture medium was 2.0-8.8%, 0.9-8.3%, and 2.0-14.3%, respectively, while the inter-day precision for human urine was 1.5-14.1%.

Lactate promotes glioma migration by TGF-β2–dependent regulation of matrix metalloproteinase-2

Lactate dehydrogenase type A (LDH-A) is a key metabolic enzyme catalyzing pyruvate into lactate and is excessively expressed by tumor cells. Transforming growth factor-beta2 (TGF-beta2) is a key regulator of invasion in high-grade gliomas, partially by inducing a mesenchymal phenotype and by remodeling the extracellular matrix. In this study, we tested the hypothesis that lactate metabolism regulates TGF-beta2-mediated migration of glioma cells. Small interfering RNA directed against LDH-A (siLDH-A) suppresses, and lactate induces, TGF-beta2 expression, suggesting that lactate metabolism is strongly associated with TGF-beta2 in glioma cells. Here we demonstrate that TGF-beta2 enhances expression, secretion, and activation of matrix metalloproteinase-2 (MMP-2) and induces the cell surface expression of integrin alpha(v)beta(3) receptors. In spheroid and Boyden chamber migration assays, inhibition of MMP-2 activity using a specific MMP-2 inhibitor and blocking of integrin alpha(v)beta(3) abrogated glioma cell migration stimulated by TGF-beta2. Furthermore, siLDH-A inhibited MMP2 activity, leading to inhibition of glioma migration. Taken together, we define an LDH-A-induced and TGF-beta2-coordinated regulatory cascade of transcriptional regulation of MMP-2 and integrin alpha(v)beta(3). This novel interaction between lactate metabolism and TGF-beta2 might constitute a crucial mechanism for glioma migration.

Urinary amino acid analysis: A comparison of iTRAQ®–LC–MS/MS, GC–MS, and amino acid analyzer

Urinary amino acid analysis is typically done by cation-exchange chromatography followed by post-column derivatization with ninhydrin and UV detection. This method lacks throughput and specificity. Two recently introduced stable isotope ratio mass spectrometric methods promise to overcome those shortcomings. Using two blinded sets of urine replicates and a certified amino acid standard, we compared the precision and accuracy of gas chromatography/mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) of propyl chloroformate and iTRAQ derivatized amino acids, respectively, to conventional amino acid analysis. The GC-MS method builds on the direct derivatization of amino acids in diluted urine with propyl chloroformate, GC separation and mass spectrometric quantification of derivatives using stable isotope labeled standards. The LC-MS/MS method requires prior urinary protein precipitation followed by labeling of urinary and standard amino acids with iTRAQ tags containing different cleavable reporter ions distinguishable by MS/MS fragmentation. Means and standard deviations of percent technical error (%TE) computed for 20 amino acids determined by amino acid analyzer, GC-MS, and iTRAQ-LC-MS/MS analyses of 33 duplicate and triplicate urine specimens were 7.27+/-5.22, 21.18+/-10.94, and 18.34+/-14.67, respectively. Corresponding values for 13 amino acids determined in a second batch of 144 urine specimens measured in duplicate or triplicate were 8.39+/-5.35, 6.23+/-3.84, and 35.37+/-29.42. Both GC-MS and iTRAQ-LC-MS/MS are suited for high-throughput amino acid analysis, with the former offering at present higher reproducibility and completely automated sample pretreatment, while the latter covers more amino acids and related amines.

Metabolite extraction from adherently growing mammalian cells for metabolomics studies: optimization of harvesting and extraction protocols.

Trypsin/ethylenediaminetetraacetic acid (EDTA) treatment and cell scraping in a buffer solution were compared for harvesting adherently growing mammalian SW480 cells for metabolomics studies. In addition, direct scraping with a solvent was tested. Trypsinated and scraped cell pellets were extracted using seven different extraction protocols including pure methanol, methanol/water, pure acetone, acetone/water, methanol/chloroform/water, methanol/isopropanol/water, and acid–base methanol. The extracts were analyzed by GC-MS after methoximation/silylation and derivatization with propyl chloroformate, respectively. The metabolic fingerprints were compared and 25 selected metabolites including amino acids and intermediates of energy metabolism were quantitatively determined. Moreover, the influence of freeze/thaw cycles, ultrasonication and homogenization using ceramic beads on extraction yield was tested. Pure acetone yielded the lowest extraction efficiency while methanol, methanol/water, methanol/isopropanol/water, and acid–base methanol recovered similar metabolite amounts with good reproducibility. Based on overall performance, methanol/water was chosen as a suitable extraction solvent. Repeated freeze/thaw cycles, ultrasonication and homogenization did not improve overall metabolite yield of the methanol/water extraction. Trypsin/EDTA treatment caused substantial metabolite leakage proving it inadequate for metabolomics studies. Gentle scraping of the cells in a buffer solution and subsequent extraction with methanol/water resulted on average in a sevenfold lower recovery of quantified metabolites compared with direct scraping using methanol/water, making the latter one the method of choice to harvest and extract metabolites from adherently growing mammalian SW480 cells.

Urinary Metabolite Quantification Employing 2D NMR Spectroscopy

Two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy is a fairly novel method for the quantification of metabolites in biological fluids and tissue extracts. We show in this contribution that, compared to 1D 1H spectra, superior quantification of human urinary metabolites is obtained from 2D 1H-13C heteronuclear single-quantum correlation (HSQC) spectra measured at natural abundance. This was accomplished by the generation of separate calibration curves for the different 2D HSQC signals of each metabolite. Lower limits of detection were in the low to mid micromolar range and were generally the lower the greater the number of methyl groups contained in an analyte. The quantitative 2D NMR data obtained for a selected set of 17 urinary metabolites were compared to those obtained independently by means of gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry of amino acids and hippurate as well as enzymatic and colorimetric measurements of creatinine. As a typical application, 2D-NMR was used for the investigation of urine from patients with inborn errors of metabolism.

Delaying aging and the aging-associated decline in protein homeostasis by inhibition of tryptophan degradation

Toxicity of aggregation-prone proteins is thought to play an important role in aging and age-related neurological diseases like Parkinson and Alzheimer’s diseases. Here, we identify tryptophan 2,3-dioxygenase (tdo-2), the first enzyme in the kynurenine pathway of tryptophan degradation, as a metabolic regulator of age-related α-synuclein toxicity in a Caenorhabditis elegans model. Depletion of tdo-2 also suppresses toxicity of other heterologous aggregation-prone proteins, including amyloid-β and polyglutamine proteins, and endogenous metastable proteins that are sensors of normal protein homeostasis. This finding suggests that tdo-2 functions as a general regulator of protein homeostasis. Analysis of metabolite levels in C. elegans strains with mutations in enzymes that act downstream of tdo-2 indicates that this suppression of toxicity is independent of downstream metabolites in the kynurenine pathway. Depletion of tdo-2 increases tryptophan levels, and feeding worms with extra l-tryptophan also suppresses toxicity, suggesting that tdo-2 regulates proteotoxicity through tryptophan. Depletion of tdo-2 extends lifespan in these worms. Together, these results implicate tdo-2 as a metabolic switch of age-related protein homeostasis and lifespan. With TDO and Indoleamine 2,3-dioxygenase as evolutionarily conserved human orthologs of TDO-2, intervening with tryptophan metabolism may offer avenues to reducing proteotoxicity in aging and age-related diseases.

Nuclear magnetic resonance and mass spectrometry-based milk metabolomics in dairy cows during early and late lactation

Milk production in dairy cows has dramatically increased over the past few decades. The selection for higher milk yield affects the partitioning of available nutrients, with more energy being allocated to milk synthesis and less to physiological processes essential to fertility and fitness. In this study, the abundance of numerous milk metabolites in early and late lactation was systematically investigated, with an emphasis on metabolites related to energy metabolism. The aim of the study was the identification and correlation of milk constituents to the metabolic status of the cows. To investigate the influence of lactation stage on physiological and metabolic variables, 2 breeds of different productivity were selected for investigation by high-resolution nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. We could reliably quantify 44 different milk metabolites. The results show that biomarkers such as acetone and beta-hydroxybutyrate are clearly correlated to the metabolic status of the individual cows during early lactation. Based on these data, the selection of cows that cope well with the metabolic stress of early lactation should become an option.

Low urinary indoxyl sulfate levels early after transplantation reflect a disrupted microbiome and are associated with poor outcome

Indole, which is produced from l-tryptophan by commensal bacteria expressing tryptophanase, not only is an important intercellular signal in microbial communities, but also modulates mucosal barrier function and expression of pro- and anti-inflammatory genes by intestinal epithelial cells. Here, we hypothesized that decreased urinary excretion of 3-indoxyl sulfate (3-IS), the major conjugate of indole found in humans, may be a marker of gut microbiota disruption and increased risk of developing gastrointestinal (GI) graft-versus-host-disease. Using liquid chromatography/tandem mass spectrometry, 3-IS was determined in urine specimens collected weekly within the first 28 days after allogeneic stem cell transplantation (ASCT) in 131 patients. Low 3-IS levels within the first 10 days after ASCT were associated with significantly higher transplant-related mortality (P = .017) and worse overall survival (P = .05) 1 year after ASCT. Least absolute shrinkage and selection operator regression models trained on log-normalized counts of 763 operational taxonomic units derived from next-generation sequencing of the hypervariable V3 region of the 16S ribosomal RNA gene showed members of the families of Lachnospiraceae and Ruminococcaceae of the class of Clostridia to be associated with high urinary 3-IS levels, whereas members of the class of Bacilli were associated with low 3-IS levels. Risk factors of early suppression of 3-IS levels were the type of GI decontamination (P = .01), early onset of antibiotic treatment (P = .001), and recipient NOD2/CARD15 genotype (P = .04). In conclusion, our findings underscore the relevance of microbiota-derived indole and metabolites thereof in mucosal integrity and protection from inflammation.