PUFA-Induced Metabolic Enteritis as a Fuel for Crohn's Disease.

BACKGROUND & AIMS: Crohn's disease (CD) globally emerges with Westernization of lifestyle and nutritional habits. However, a specific dietary constituent that comprehensively evokes gut inflammation in human inflammatory bowel diseases remains elusive. We aimed to delineate how increased intake of polyunsaturated fatty acids (PUFAs) in a Western diet, known to impart risk for developing CD, affects gut inflammation and disease course. We hypothesized that the unfolded protein response and antioxidative activity of glutathione peroxidase 4 (GPX4), which are compromised in human CD epithelium, compensates for metabolic perturbation evoked by dietary PUFAs. METHODS: We phenotyped and mechanistically dissected enteritis evoked by a PUFA-enriched Western diet in 2 mouse models exhibiting endoplasmic reticulum (ER) stress consequent to intestinal epithelial cell (IEC)-specific deletion of X-box binding protein 1 (Xbp1) or Gpx4. We translated the findings to human CD epithelial organoids and correlated PUFA intake, as estimated by a dietary questionnaire or stool metabolomics, with clinical disease course in 2 independent CD cohorts. RESULTS: PUFA excess in a Western diet potently induced ER stress, driving enteritis in Xbp1-/-IEC and Gpx4+/-IEC mice. ω-3 and ω-6 PUFAs activated the epithelial endoplasmic reticulum sensor inositol-requiring enzyme 1α (IRE1α) by toll-like receptor 2 (TLR2) sensing of oxidation-specific epitopes. TLR2-controlled IRE1α activity governed PUFA-induced chemokine production and enteritis. In active human CD, ω-3 and ω-6 PUFAs instigated epithelial chemokine expression, and patients displayed a compatible inflammatory stress signature in the serum. Estimated PUFA intake correlated with clinical and biochemical disease activity in a cohort of 160 CD patients, which was similarly demonstrable in an independent metabolomic stool analysis from 199 CD patients. CONCLUSIONS: We provide evidence for the concept of PUFA-induced metabolic gut inflammation which may worsen the course of human CD. Our findings provide a basis for targeted nutritional therapy.

Amp(1q) and tetraploidy are commonly acquired chromosomal abnormalities in relapsed multiple myeloma.

Long-term disease control in multiple myeloma (MM) is typically an unmet medical need, and most patients experience multiple relapses. Fluorescence in situ hybridization (FISH) is the standard technique to detect chromosomal abnormalities (CAs), which are important to estimate the prognosis of MM and the allocation of risk adapted therapies. In advanced stages, the importance of CAs needs further investigation. From 148 MM patients, two or more paired samples, at least one of which was collected at relapse, were analyzed by FISH. Using targeted next-generation sequencing, we molecularly investigated samples harboring relapse-associated CAs. Sixty-one percent of the patients showed a change in the cytogenetic profile during the disease course, including 10% who acquired high-risk cytogenetics. Amp(1q) (≥4 copies of 1q21), driven by an additional increase in copy number in patients who already had 3 copies of 1q21, was the most common acquired CA with 16% affected patients. Tetraploidy, found in 10% of the samples collected at the last time-point, was unstable over the course of the disease and was associated with TP53 lesions. Our results indicate that cytogenetic progression is common in relapsed patients. The relatively high frequency of amp(1q) suggests an active role for this CA in disease progression.

ost in promiscuity? An evolutionary and biochemical evaluation of HSD10 function in cardiolipin metabolism.

Multifunctional proteins are challenging as it can be difficult to confirm pathomechanisms associated with disease-causing genetic variants. The human 17ß-hydroxysteroid dehydrogenase 10 (HSD10) is a moonlighting enzyme with at least two structurally and catalytically unrelated functions. HSD10 disease was originally described as a disorder of isoleucine metabolism, but the clinical manifestations were subsequently shown to be linked to impaired mtDNA transcript processing due to deficient function of HSD10 in the mtRNase P complex. A surprisingly large number of other, mostly enzymatic and potentially clinically relevant functions have been attributed to HSD10. Recently, HSD10 was reported to exhibit phospholipase C-like activity towards cardiolipins (CL), important mitochondrial phospholipids. To assess the physiological role of the proposed CL-cleaving function, we studied CL architectures in living cells and patient fibroblasts in different genetic backgrounds and lipid environments using our well-established LC-MS/MS cardiolipidomic pipeline. These experiments revealed no measurable effect on CLs, indicating that HSD10 does not have a physiologically relevant function towards CL metabolism. Evolutionary constraints could explain the broad range of reported substrates for HSD10 in vitro. The combination of an essential structural with a non-essential enzymatic function in the same protein could direct the evolutionary trajectory towards improvement of the former, thereby increasing the flexibility of the binding pocket, which is consistent with the results presented here.

The TMEM189 gene encodes plasmanylethanolamine desaturase which introduces the characteristic vinyl ether double bond into plasmalogens.

A significant fraction of the glycerophospholipids in the human body is composed of plasmalogens, particularly in the brain, cardiac, and immune cell membranes. A decline in these lipids has been observed in such diseases as Alzheimer's and chronic obstructive pulmonary disease. Plasmalogens contain a characteristic 1-O-alk-1'-enyl ether (vinyl ether) double bond that confers special biophysical, biochemical, and chemical properties to these lipids. However, the genetics of their biosynthesis is not fully understood, since no gene has been identified that encodes plasmanylethanolamine desaturase (E.C. 1.14.99.19), the enzyme introducing the crucial alk-1'-enyl ether double bond. The present work identifies this gene as transmembrane protein 189 (TMEM189). Inactivation of the TMEM189 gene in human HAP1 cells led to a total loss of plasmanylethanolamine desaturase activity, strongly decreased plasmalogen levels, and accumulation of plasmanylethanolamine substrates and resulted in an inability of these cells to form labeled plasmalogens from labeled alkylglycerols. Transient expression of TMEM189 protein, but not of other selected desaturases, recovered this deficit. TMEM189 proteins contain a conserved protein motif (pfam10520) with eight conserved histidines that is shared by an alternative type of plant desaturase but not by other mammalian proteins. Each of these histidines is essential for plasmanylethanolamine desaturase activity. Mice homozygous for an inactivated Tmem189 gene lacked plasmanylethanolamine desaturase activity and had dramatically lowered plasmalogen levels in their tissues. These results assign the TMEM189 gene to plasmanylethanolamine desaturase and suggest that the previously characterized phenotype of Tmem189-deficient mice may be caused by a lack of plasmalogens.

Adaptations of the 3T3-L1 adipocyte lipidome to defective ether lipid catabolism upon Agmo knockdown.

Little is known about the physiological role of alkylglycerol monooxygenase (AGMO), the only enzyme capable of cleaving the 1-O-alkyl ether bond of ether lipids. Expression and enzymatic activity of this enzyme can be detected in a variety of tissues including adipose tissue. This labile lipolytic membrane-bound protein uses tetrahydrobiopterin as a cofactor, and mice with reduced tetrahydrobiopterin levels have alterations in body fat distribution and blood lipid concentrations. In addition, manipulation of AGMO in macrophages led to significant changes in the cellular lipidome, and alkylglycerolipids, the preferred substrates of AGMO, were shown to accumulate in mature adipocytes. Here, we investigated the roles of AGMO in lipid metabolism by studying 3T3-L1 adipogenesis. AGMO activity was induced over 11 days using an adipocyte differentiation protocol. We show that RNA interference-mediated knockdown of AGMO did not interfere with adipocyte differentiation or affect lipid droplet formation. Furthermore, lipidomics revealed that plasmalogen phospholipids were preferentially accumulated upon Agmo knockdown, and a significant shift toward longer and more polyunsaturated acyl side chains of diacylglycerols and triacylglycerols could be detected by mass spectrometry. Our results indicate that alkylglycerol catabolism has an influence not only on ether-linked species but also on the degree of unsaturation in the massive amounts of triacylglycerols formed during in vitro 3T3-L1 adipocyte differentiation.

Comprehensive characterization of the prostate tumor microenvironment identifies CXCR4/CXCL12 crosstalk as a novel antiangiogenic therapeutic target in prostate cancer.

BACKGROUND: Crosstalk between neoplastic and stromal cells fosters prostate cancer (PCa) progression and dissemination. Insight in cell-to-cell communication networks provides new therapeutic avenues to mold processes that contribute to PCa tumor microenvironment (TME) alterations. Here we performed a detailed characterization of PCa tumor endothelial cells (TEC) to delineate intercellular crosstalk between TEC and the PCa TME. METHODS: TEC isolated from 67 fresh radical prostatectomy (RP) specimens underwent multi-omic ex vivo characterization as well as orthogonal validation of both TEC functions and key markers by immunohistochemistry (IHC) and immunofluorescence (IF). To identify cell-cell interaction targets in TEC, we performed single-cell RNA sequencing (scRNA-seq) in four PCa patients who underwent a RP to catalogue cellular TME composition. Targets were cross-validated using IHC, publicly available datasets, cell culture expriments as well as a PCa xenograft mouse model. RESULTS: Compared to adjacent normal endothelial cells (NEC) bulk RNA-seq analysis revealed upregulation of genes associated with tumor vasculature, collagen modification and extracellular matrix remodeling in TEC. PTGIR, PLAC9, CXCL12 and VDR were identified as TEC markers and confirmed by IF and IHC in an independent patient cohort. By scRNA-seq we identified 27 cell (sub)types, including endothelial cells (EC) with arterial, venous and immature signatures, as well as angiogenic tip EC. A focused molecular analysis revealed that arterial TEC displayed highest CXCL12 mRNA expression levels when compared to all other TME cell (sub)populations and showed a negative prognostic role. Receptor-ligand interaction analysis predicted interactions between arterial TEC derived CXCL12 and its cognate receptor CXCR4 on angiogenic tip EC. CXCL12 was in vitro and in vivo validated as actionable TEC target by highlighting the vessel number- and density- reducing activity of the CXCR4-inhibitor AMD3100 in murine PCa as well as by inhibition of TEC proliferation and migration in vitro. CONCLUSIONS: Overall, our comprehensive analysis identified novel PCa TEC targets and highlights CXCR4/CXCL12 interaction as a potential novel target to interfere with tumor angiogenesis in PCa.

The lipid environment modulates cardiolipin and phospholipid constitution in wild type and tafazzin-deficient cells.

Deficiency of the transacylase tafazzin due to loss of function variants in the X-chromosomal TAFAZZIN gene causes Barth syndrome (BTHS) with severe neonatal or infantile cardiomyopathy, neutropenia, myopathy, and short stature. The condition is characterized by drastic changes in the composition of cardiolipins, a mitochondria-specific class of phospholipids. Studies examining the impact of tafazzin deficiency on the metabolism of other phospholipids have so far generated inhomogeneous and partly conflicting results. Recent studies showed that the cardiolipin composition in cells and different murine tissues is highly dependent on the surrounding lipid environment. In order to study the relevance of different lipid states and tafazzin function for cardiolipin and phospholipid homeostasis we conducted systematic modulation experiments in a CRISPR/Cas9 knock-out model for BTHS. We found that-irrespective of tafazzin function-the composition of cardiolipins strongly depends on the nutritionally available lipid pool. Tafazzin deficiency causes a consistent shift towards cardiolipin species with more saturated and shorter acyl chains. Interestingly, the typical biochemical BTHS phenotype in phospholipid profiles of HEK 293T TAZ knock-out cells strongly depends on the cellular lipid context. In response to altered nutritional lipid compositions, we measured more pronounced changes on phospholipids that were largely masked under standard cell culturing conditions, therewith giving a possible explanation for the conflicting results reported so far on BTHS lipid phenotypes.

Novel ALDH3A2 mutations in structural and functional domains of FALDH causing diverse clinical phenotypes in Sjögren-Larsson syndrome patients.

Mutations in ALDH3A2 cause Sjögren-Larsson syndrome (SLS), a neuro-ichthyotic condition due to the deficiency of fatty aldehyde dehydrogenase (FALDH). We screened for novel mutations causing SLS among Indian ethnicity, characterized the identified mutations in silico and in vitro, and retrospectively evaluated their role in phenotypic heterogeneity. Interestingly, asymmetric distribution of nonclassical traits was observed in our cases. Nerve conduction studies suggested intrinsic-minus-claw hands in two siblings, a novel neurological phenotype to SLS. Genetic testing revealed five novel homozygous ALDH3A2 mutations in six cases: Case-1-NM_000382.2:c.50C>A, NP_000373.1:p.(Ser17Ter); Case-2-NM_000382.2:c.199G>T, NP_000373.1:p.(Glu67Ter); Case-3-NM_000382.2:c.1208G>A, NP_000373.1:p.(Gly403Asp); Case-4-NM_000382.2:c.1325C>T, NP_000373.1:p.(Pro442Leu); Case-5 and -6 NM_000382.2:c.1349G>A, NP_000373.1:p.(Trp450Ter). The mutations identified were predicted to be pathogenic and disrupt the functional domains of the FALDH. p.(Pro442Leu) at the C-terminal α-helix, might impair the substrate gating process. Mammalian expression studies with exon-9 mutants confirmed the profound reduction in the enzyme activity. Diminished aldehyde-oxidizing activity was observed with cases-2 and 3. Cases-2 and 3 showed epidermal hyperplasia with mild intracellular edema, spongiosis, hypergranulosis, and perivascular-interstitial lymphocytic infiltrate and a leaky eosinophilic epidermis. The presence of keratin-containing milia-like lipid vacuoles implies defective lamellar secretion with p.(Gly403Asp). This study improves our understanding of the clinical and mutational diversity in SLS, which might help to fast-track diagnostic and therapeutic interventions of this debilitating disorder.

Fatty acyl availability modulates cardiolipin composition and alters mitochondrial function in HeLa cells.

The molecular assembly of cells depends not only on the balance between anabolism and catabolism but to a large degree on the building blocks available in the environment. For cultured mammalian cells, this is largely determined by the composition of the applied growth medium. Here, we study the impact of lipids in the medium on mitochondrial membrane architecture and function by combining LC-MS/MS lipidomics and functional tests with lipid supplementation experiments in an otherwise serum-free and lipid-free cell culture model. We demonstrate that the composition of mitochondrial cardiolipins strongly depends on the lipid environment in cultured cells and favors the incorporation of essential linoleic acid over other fatty acids. Simultaneously, the mitochondrial respiratory complex I activity was altered, whereas the matrix-localized enzyme citrate synthase was unaffected. This raises the question on a link between membrane composition and respiratory control. In summary, we found a strong dependency of central mitochondrial features on the type of lipids contained in the growth medium. This underlines the importance of considering these factors when using and establishing cell culture models in biomedical research. In summary, we found a strong dependency of central mitochondrial features on the type of lipids contained in the growth medium.

Molecular structural diversity of mitochondrial cardiolipins.

Current strategies used to quantitatively describe the biological diversity of lipids by mass spectrometry are often limited in assessing the exact structural variability of individual molecular species in detail. A major challenge is represented by the extensive isobaric overlap present among lipids, hampering their accurate identification. This is especially true for cardiolipins, a mitochondria-specific class of phospholipids, which are functionally involved in many cellular functions, including energy metabolism, cristae structure, and apoptosis. Substituted with four fatty acyl side chains, cardiolipins offer a particularly high potential to achieve complex mixtures of molecular species. Here, we demonstrate how systematically generated high-performance liquid chromatography-mass spectral data can be utilized in a mathematical structural modeling approach, to comprehensively analyze and characterize the molecular diversity of mitochondrial cardiolipin compositions in cell culture and disease models, cardiolipin modulation experiments, and a broad variety of frequently studied model organisms.

Unequivocal Mapping of Molecular Ether Lipid Species by LC-MS/MS in Plasmalogen-Deficient Mice.

Deficient ether lipid biosynthesis in rhizomelic chondrodysplasia punctata and other disorders is associated with a wide range of severe symptoms including small stature with proximal shortening of the limbs, contractures, facial dysmorphism, congenital cataracts, ichthyosis, spasticity, microcephaly, and mental disability. Mouse models are available but show less severe symptoms. In both humans and mice, it has remained elusive which of the symptoms can be attributed to lack of plasmanyl or plasmenyl ether lipids. The latter compounds, better known as plasmalogens, harbor a vinyl ether double bond conferring special chemical and physical properties. Discrimination between plasmanyl and plasmenyl ether lipids is a major analytical challenge, especially in complex lipid extracts with many isobaric species. Consequently, these lipids are often neglected also in recent lipidomic studies. Here, we present a comprehensive LC-MS/MS based approach that allows unequivocal distinction of these two lipid subclasses based on their chromatographic properties. The method was validated using a novel plasmalogen-deficient mouse model, which lacks plasmanylethanolamine desaturase and therefore cannot form plasmenyl ether lipids. We demonstrate that plasmanylethanolamine desaturase deficiency causes an accumulation of plasmanyl species, a too little studied but biologically important substance class.

The prognostic value of additional copies of 1q21 in multiple myeloma depends on the primary genetic event.

Hyperdiploidy (HRD) and specific immunoglobulin heavy locus (IGH) translocations are primary chromosomal abnormalities (CA) in multiple myeloma (MM). In this retrospective study of 794 MM patients we aimed to investigate clinical features and common CA including gain(1q) in separate subgroups defined by primary CA. In the entire group, we confirmed that gain(1q) was associated with short time to next treatment and adverse overall survival (OS). The impact was worse for four or more copies of 1q21 as compared to three copies. However, in a subgroup of patients with clonal gain(11q) and without known primary IGH translocations (CG11q), already three copies of 1q21 were associated with a poor outcome; in the absence of gain(1q), patients in this subgroup had a remarkably long median OS of more than nine years. These cases were associated with HRD, coexpression of CD56 and CD117, male gender, and IgG subtype. In non-CG11q patients, four or more copies of 1q21 (but not three copies) had a significant adverse impact on outcome. Several associations with CA and clinical findings were observed for the defined subgroups. As an example, we found a predominance of early tetraploidy, plasma cell leukemia, and female gender in the t(14;16) subgroup. Our results underscore the importance of subgrouping in MM.

Genotype and phenotype variability in Sjögren-Larsson syndrome.

The Sjögren-Larsson syndrome (SLS) is a rare autosomal recessive disorder caused by pathogenic variants in the ALDH3A2 gene, which codes for fatty aldehyde dehydrogenase (FALDH). FALDH prevents the accumulation of toxic fatty aldehydes by converting them into fatty acids. Pathogenic ALDH3A2 variants cause symptoms such as ichthyosis, spasticity, intellectual disability, and a wide range of less common clinical features. Interpreting patient-to-patient variability is often complicated by inconsistent reporting and negatively impacts on establishing robust criteria to measure the success of SLS treatments. Thus, with this study, patient-centered literature data was merged into a concise genotype-based, open-access database (www.LOVD.nl/ALDH3A2). One hundred and seventy eight individuals with 90 unique SLS-causing variants were included with phenotypic data being available for more than 90%. While the three lead symptoms did occur in almost all cases, more heterogeneity was observed for other frequent clinical manifestations of SLS. However, a stringent genotype-phenotype correlation analysis was hampered by the considerable variability in reporting phenotypic features. Consequently, we compiled a set of recommendations of how to generate comprehensive SLS patient descriptions in the future. This will be of benefit on multiple levels, for example, in clinical diagnosis, basic research, and the development of novel treatment options for SLS.

A novel assay for the introduction of the vinyl ether double bond into plasmalogens using pyrene-labeled substrates.

Plasmanylethanolamine desaturase (PEDS) (EC 1.14.99.19) introduces the 1-prime double bond into plasmalogens, one of the most abundant phospholipids in the human body. This labile membrane enzyme has not been purified and its coding sequence is unknown. Previous assays for this enzyme used radiolabeled substrates followed by multistep processing. We describe here a straight-forward method for the quantification of PEDS in enzyme incubation mixtures using pyrene-labeled substrates and reversed-phase HPLC with fluorescence detection. After stopping the reaction with hydrochloric acid in acetonitrile, the mixture was directly injected into the HPLC system without the need of lipid extraction. The substrate, 1-O-pyrenedecyl-2-acyl-sn-glycero-3-phosphoethanolamine, and the lyso-substrate, 1-O-pyrenedecyl-sn-glycero-3-phosphoethanolamine, were prepared from RAW-12 cells deficient in PEDS activity and were compared for their performance in the assay. Plasmalogen levels in mouse tissues and in cultured cells did not correlate with PEDS levels, indicating that the desaturase might not be the rate limiting step for plasmalogen biosynthesis. Among selected mouse organs, the highest activities were found in kidney and in spleen. Incubation of intact cultivated mammalian cells with 1-O-pyrenedecyl-sn-glycerol, extraction of lipids, and treatment with hydrochloric or acetic acid in acetonitrile allowed sensitive monitoring of PEDS activity in intact cells.

Regulation of ribosomal DNA amplification by the TOR pathway.

Repeated regions are widespread in eukaryotic genomes, and key functional elements such as the ribosomal DNA tend to be formed of high copy repeated sequences organized in tandem arrays. In general, high copy repeats are remarkably stable, but a number of organisms display rapid ribosomal DNA amplification at specific times or under specific conditions. Here we demonstrate that target of rapamycin (TOR) signaling stimulates ribosomal DNA amplification in budding yeast, linking external nutrient availability to ribosomal DNA copy number. We show that ribosomal DNA amplification is regulated by three histone deacetylases: Sir2, Hst3, and Hst4. These enzymes control homologous recombination-dependent and nonhomologous recombination-dependent amplification pathways that act in concert to mediate rapid, directional ribosomal DNA copy number change. Amplification is completely repressed by rapamycin, an inhibitor of the nutrient-responsive TOR pathway; this effect is separable from growth rate and is mediated directly through Sir2, Hst3, and Hst4. Caloric restriction is known to up-regulate expression of nicotinamidase Pnc1, an enzyme that enhances Sir2, Hst3, and Hst4 activity. In contrast, normal glucose concentrations stretch the ribosome synthesis capacity of cells with low ribosomal DNA copy number, and we find that these cells show a previously unrecognized transcriptional response to caloric excess by reducing PNC1 expression. PNC1 down-regulation forms a key element in the control of ribosomal DNA amplification as overexpression of PNC1 substantially reduces ribosomal DNA amplification rate. Our results reveal how a signaling pathway can orchestrate specific genome changes and demonstrate that the copy number of repetitive DNA can be altered to suit environmental conditions.

Tetrahydrobiopterin and alkylglycerol monooxygenase substantially alter the murine macrophage lipidome.

Tetrahydrobiopterin is a cofactor synthesized from GTP with well-known roles in enzymatic nitric oxide synthesis and aromatic amino acid hydroxylation. It is used to treat mild forms of phenylketonuria. Less is known about the role of tetrahydrobiopterin in lipid metabolism, although it is essential for irreversible ether lipid cleavage by alkylglycerol monooxygenase. Here we found intracellular alkylglycerol monooxygenase activity to be an important regulator of alkylglycerol metabolism in intact murine RAW264.7 macrophage-like cells. Alkylglycerol monooxygenase was expressed and active also in primary mouse bone marrow-derived monocytes and "alternatively activated" M2 macrophages obtained by interleukin 4 treatment, but almost missing in M1 macrophages obtained by IFN-γ and lipopolysaccharide treatment. The cellular lipidome of RAW264.7 was markedly changed in a parallel way by modulation of alkylglycerol monooxygenase expression and of tetrahydrobiopterin biosynthesis affecting not only various ether lipid species upstream of alkylglycerol monooxygenase but also other more complex lipids including glycosylated ceramides and cardiolipins, which have no direct connection to ether lipid pathways. Alkylglycerol monooxygenase activity manipulation modulated the IFN-γ/lipopolysaccharide-induced expression of inducible nitric oxide synthase, interleukin-1ß, and interleukin 1 receptor antagonist but not transforming growth factor ß1, suggesting that alkylglycerol monooxygenase activity affects IFN-γ/lipopolysaccharide signaling. Our results demonstrate a central role of tetrahydrobiopterin and alkylglycerol monooxygenase in ether lipid metabolism of murine macrophages and reveal that alteration of alkylglycerol monooxygenase activity has a profound impact on the lipidome also beyond the class of ether lipids.

Hyperpolarized [U-(2) H, U-(13) C]Glucose reports on glycolytic and pentose phosphate pathway activity in EL4 tumors and glycolytic activity in yeast cells.

PURPOSE: A resonance at ∼181 ppm in the (13) C spectra of tumors injected with hyperpolarized [U-(2) H, U-(13) C]glucose was assigned to 6-phosphogluconate (6PG), as in previous studies in yeast, whereas in breast cancer cells in vitro this resonance was assigned to 3-phosphoglycerate (3PG). These peak assignments were investigated here using measurements of 6PG and 3PG (13) C-labeling using liquid chromatography tandem mass spectrometry (LC-MS/MS) METHODS: Tumor-bearing mice were injected with (13) C6 glucose and the (13) C-labeled and total 6PG and 3PG concentrations measured. (13) C MR spectra of glucose-6-phosphate dehydrogenase deficient (zwf1Δ) and wild-type yeast were acquired following addition of hyperpolarized [U-(2) H, U-(13) C]glucose and again (13) C-labeled and total 6PG and 3PG were measured by LC-MS/MS RESULTS: Tumor (13) C-6PG was more abundant than (13) C-2PG/3PG and the resonance at ∼181 ppm matched more closely that of 6PG. (13) C MR spectra of wild-type and zwf1Δ yeast cells showed a resonance at ∼181 ppm after labeling with hyperpolarized [U-(2) H, U-(13) C]glucose, however, there was no 6PG in zwf1Δ cells. In the wild-type cells 3PG was approximately four-fold more abundant than 6PG CONCLUSION: The resonance at ∼181 ppm in (13) C MR spectra following injection of hyperpolarized [U-(2) H, U-(13) C]glucose originates predominantly from 6PG in EL4 tumors and 3PG in yeast cells.

Non-enzymatic glycolysis and pentose phosphate pathway-like reactions in a plausible Archean ocean.

The reaction sequences of central metabolism, glycolysis and the pentose phosphate pathway provide essential precursors for nucleic acids, amino acids and lipids. However, their evolutionary origins are not yet understood. Here, we provide evidence that their structure could have been fundamentally shaped by the general chemical environments in earth's earliest oceans. We reconstructed potential scenarios for oceans of the prebiotic Archean based on the composition of early sediments. We report that the resultant reaction milieu catalyses the interconversion of metabolites that in modern organisms constitute glycolysis and the pentose phosphate pathway. The 29 observed reactions include the formation and/or interconversion of glucose, pyruvate, the nucleic acid precursor ribose-5-phosphate and the amino acid precursor erythrose-4-phosphate, antedating reactions sequences similar to that used by the metabolic pathways. Moreover, the Archean ocean mimetic increased the stability of the phosphorylated intermediates and accelerated the rate of intermediate reactions and pyruvate production. The catalytic capacity of the reconstructed ocean milieu was attributable to its metal content. The reactions were particularly sensitive to ferrous iron Fe(II), which is understood to have had high concentrations in the Archean oceans. These observations reveal that reaction sequences that constitute central carbon metabolism could have been constrained by the iron-rich oceanic environment of the early Archean. The origin of metabolism could thus date back to the prebiotic world.