In Vivo 2-Hydroxyglutarate Monitoring With Edited MR Spectroscopy for the Follow-up of IDH-Mutant Diffuse Gliomas: The IDASPE Prospective Study.

BACKGROUND AND OBJECTIVES: D-2-hydroxyglutarate (2HG) characterizes IDH-mutant gliomas and can be detected and quantified with edited MRS (MEGA-PRESS). In this study, we investigated the clinical, radiologic, and molecular parameters affecting 2HG levels. METHODS: MEGA-PRESS data were acquired in 71 patients with glioma (24 untreated, 47 treated) on a 3 T system. Eighteen patients were followed during cytotoxic (n = 12) or targeted (n = 6) therapy. 2HG was measured in tumor samples using gas chromatography coupled to mass spectrometry (GCMS). RESULTS: MEGA-PRESS detected 2HG with a sensitivity of 95% in untreated patients and 62% in treated patients. Sensitivity depended on tumor volume (>27 cm3; p = 0.02), voxel coverage (>75%; p = 0.002), and expansive presentation (defined by equal size of T1 and FLAIR abnormalities, p = 0.04). 2HG levels were positively correlated with IDH-mutant allelic fraction (p = 0.03) and total choline levels (p < 0.001) and were higher in IDH2-mutant compared with IDH1 R132H-mutant and non-R132H IDH1-mutant patients (p = 0.002). In patients receiving IDH inhibitors, 2HG levels decreased within a few days, demonstrating the on-target effect of the drug, but 2HG level decrease did not predict tumor response. Patients receiving cytotoxic treatments showed a slower decrease in 2HG levels, consistent with tumor response and occurring before any tumor volume change on conventional MRI. At progression, 1p/19q codeleted gliomas, but not the non-codeleted, showed detectable in vivo 2HG levels, pointing out to different modes of progression characterizing these 2 entities. DISCUSSION: MEGA-PRESS edited MRS allows in vivo monitoring of 2-hydroxyglutarate, confirming efficacy of IDH inhibition and suggests different patterns of tumor progression in astrocytomas compared with oligodendrogliomas.

Diagnostic performance of edited 2HG MR spectroscopy of central glioma in the clinical environment.

OBJECTIVE: Oncometabolite D-2-hydroxyglutarate (2HG) is pooled in isocitrate dehydrogenase (IDH)-mutant glioma cells. Detecting 2HG by MR spectroscopy (MRS) has been proven viable in the last decade but has not entirely found its way into the clinical routine. This study aimed to explore the adoption of 2HG MRS while acknowledging factors that influence its performance in the clinical environment. METHODS: Thirty-nine MR spectra were acquired and reported prospectively in patients with suspected glioma using a 3 T system with Mescher-Garwood point-resolved spectroscopy (MEGA-PRESS) sequence utilizing averaged free induction decay (FID) signals. Postprocessing and evaluation of spectra were performed with jMRUI and LCModel. 2HG concentration estimates, 2HG/Cr ratio, together with quality measures, including Cramér-Rao lower bounds (CRLBs), full-width at half-maximum (FWHM) values, and signal-to-noise ratio (SNR) were calculated using LCModel. Immunohistochemistry and genomic analysis results used as a ground truth were available for 15 patients. RESULTS: The threshold for test positivity was set according to the ROC curve at 1 mM. Calculated sensitivity was 57.14% (95% CI 0.20-0.88), specificity 87.5% (95% CI 0.46-0.99), positive predictive value 80%, and negative predictive value 70%. Overall diagnostic accuracy was 73.33% (95% CI 0.45-0.92). The 2HG/Cr ratio with the cutoff value 0.085 significantly improved sensitivity and overall diagnostic accuracy [85.71%, 95% CI 0.42-1.00 and 86.67%, (95% CI 0.60-0.98), respectively]. CONCLUSION: Multiple factors compromising spectral quality in the clinical adoption of edited 2HG MRS resulted in diminished sensitivity but clinically acceptable specificity. Furthermore, the 2HG/Cr ratio performs better than the sole 2HG concentration estimate in the pre-operative setting.

Improving D-2-hydroxyglutarate MR spectroscopic imaging in mutant isocitrate dehydrogenase glioma patients with multiplexed RF-receive/B0 -shim array coils at 3 T.

MR spectroscopic imaging (MRSI) noninvasively maps the metabolism of human brains. In particular, the imaging of D-2-hydroxyglutarate (2HG) produced by glioma isocitrate dehydrogenase (IDH) mutations has become a key application in neuro-oncology. However, the performance of full field-of-view MRSI is limited by B0 spatial nonuniformity and lipid artifacts from tissues surrounding the brain. Array coils that multiplex RF-receive and B0 -shim electrical currents (AC/DC mixing) over the same conductive loops provide many degrees of freedom to improve B0 uniformity and reduce lipid artifacts. AC/DC coils are highly efficient due to compact design, requiring low shim currents (<2 A) that can be switched fast (0.5 ms) with high interscan reproducibility (10% coefficient of variation for repeat measurements). We measured four tumor patients and five volunteers at 3 T and show that using AC/DC coils in addition to the vendor-provided second-order spherical harmonics shim provides 19% narrower spectral linewidth, 6% higher SNR, and 23% less lipid content for unrestricted field-of-view MRSI, compared with the vendor-provided shim alone. We demonstrate that improvement in MRSI data quality led to 2HG maps with higher contrast-to-noise ratio for tumors that coincide better with the FLAIR-enhancing lesions in mutant IDH glioma patients. Smaller Cramér-Rao lower bounds for 2HG quantification are obtained in tumors by AC/DC shim, corroborating with simulations that predicted improved accuracy and precision for narrower linewidths. AC/DC coils can be used synergistically with optimized acquisition schemes to improve metabolic imaging for precision oncology of glioma patients. Furthermore, this methodology has broad applicability to other neurological disorders and neuroscience.

Synthesis of [1-13 C-5-12 C]-alpha-ketoglutarate enables noninvasive detection of 2-hydroxyglutarate.

Isocitrate dehydrogenase 1 (IDH1) mutations that generate the oncometabolite 2-hydroxyglutarate (2-HG) from α-ketoglutarate (α-KG) have been identified in many types of tumors and are an important prognostic factor in gliomas. 2-HG production can be determined by hyperpolarized carbon-13 magnetic resonance spectroscopy (HP-13 C-MRS) using [1-13 C]-α-KG as a probe, but peak contamination from naturally occurring [5-13 C]-α-KG overlaps with the [1-13 C]-2-HG peak. Via a newly developed oxidative-Stetter reaction, [1-13 C-5-12 C]-α-KG was synthesized. α-KG metabolism was measured via HP-13 C-MRS using [1-13 C-5-12 C]-α-KG as a probe. [1-13 C-5-12 C]-α-KG was synthesized in high yields, and successfully eliminated the signal from C5 of α-KG in the HP-13 C-MRS spectra. In HCT116 IDH1 R132H cells, [1-13 C-5-12 C]-α-KG allowed for unimpeded detection of [1-13 C]-2-HG. 12 C-enrichment represents a novel method to circumvent spectral overlap, and [1-13 C-5-12 C]-α-KG shows promise as a probe to study IDH1 mutant tumors and α-KG metabolism.

Adaptive laboratory evolution accelerated glutarate production by Corynebacterium glutamicum.

BACKGROUND: The demand for biobased polymers is increasing steadily worldwide. Microbial hosts for production of their monomeric precursors such as glutarate are developed. To meet the market demand, production hosts have to be improved constantly with respect to product titers and yields, but also shortening bioprocess duration is important. RESULTS: In this study, adaptive laboratory evolution was used to improve a C. glutamicum strain engineered for production of the C5-dicarboxylic acid glutarate by flux enforcement. Deletion of the L-glutamic acid dehydrogenase gene gdh coupled growth to glutarate production since two transaminases in the glutarate pathway are crucial for nitrogen assimilation. The hypothesis that strains selected for faster glutarate-coupled growth by adaptive laboratory evolution show improved glutarate production was tested. A serial dilution growth experiment allowed isolating faster growing mutants with growth rates increasing from 0.10 h-1 by the parental strain to 0.17 h-1 by the fastest mutant. Indeed, the fastest growing mutant produced glutarate with a twofold higher volumetric productivity of 0.18 g L-1 h-1 than the parental strain. Genome sequencing of the evolved strain revealed candidate mutations for improved production. Reverse genetic engineering revealed that an amino acid exchange in the large subunit of L-glutamic acid-2-oxoglutarate aminotransferase was causal for accelerated glutarate production and its beneficial effect was dependent on flux enforcement due to deletion of gdh. Performance of the evolved mutant was stable at the 2 L bioreactor-scale operated in batch and fed-batch mode in a mineral salts medium and reached a titer of 22.7 g L-1, a yield of 0.23 g g-1 and a volumetric productivity of 0.35 g L-1 h-1. Reactive extraction of glutarate directly from the fermentation broth was optimized leading to yields of 58% and 99% in the reactive extraction and reactive re-extraction step, respectively. The fermentation medium was adapted according to the downstream processing results. CONCLUSION: Flux enforcement to couple growth to operation of a product biosynthesis pathway provides a basis to select strains growing and producing faster by adaptive laboratory evolution. After identifying candidate mutations by genome sequencing causal mutations can be identified by reverse genetics. As exemplified here for glutarate production by C. glutamicum, this approach allowed deducing rational metabolic engineering strategies.

trans-3-Methylglutaconyl CoA isomerization-dependent protein acylation.

3-methylglutaconic (3MGC) aciduria is associated with a growing number of discrete inborn errors of metabolism. Herein, an antibody-based approach to detection/quantitation of 3MGC acid has been pursued. When trans-3MGC acid conjugated keyhole limpet hemocyanin (KLH) was inoculated into rabbits a strong immune response was elicited. Western blot analysis provided evidence that immune serum, but not pre-immune serum, recognized 3MGC-conjugated bovine serum albumin (BSA). In competition ELISAs using isolated immune IgG, the limit of detection for free trans-3MGC acid was compared to that for cis-3MGC acid and four structurally related short-chain dicarboxylic acids. Surprisingly, cis-3MGC acid yielded a much lower limit of detection (∼0.1 mg/ml) than trans-3MGC acid (∼1.0 mg/ml) while all other dicarboxylic acids tested were poor competitors. The data suggest trans-3MGC- isomerized during, or after, conjugation to KLH such that the immunogen was actually comprised of KLH harboring a mixture of cis- and trans-3MGC haptens. To investigate this unexpected isomerization reaction, trans-3MGC CoA was prepared and incubated at 37 °C in the presence of BSA. Evidence was obtained that non-enzymatic isomerization of trans-3MGC CoA to cis-3MGC CoA precedes intramolecular catalysis to form cis-3MGC anhydride plus CoASH. Anhydride-dependent acylation of BSA generated 3MGCylated BSA, as detected by anti-3MGC immunoblot. The results presented provide an explanation for the unanticipated detection of 3MGCylated proteins in a murine model of primary 3MGC aciduria. Furthermore, non-enzymatic hydrolysis of cis-3MGC anhydride represents a potential source of cis-3MGC acid found in urine of subjects with 3MGC aciduria.

UPLC-QTof-MSE Metabolomics Reveals Changes in Leaf Primary and Secondary Metabolism of Hop (Humulus lupulus L.) Plants under Drought Stress.

The hop (Humulus lupulus L.) is an important specialty crop used in beer production. Untargeted UPLC-QTof-MSE metabolomics was used to determine metabolite changes in the leaves of hop plants under varying degrees of drought stress. Principal component analysis revealed that drought treatments produced qualitatively distinct changes in the overall chemical composition of three out of four genotypes tested (i.e., Cascade, Sultana, and a wild var. neomexicanus accession but not Aurora), although differences among treatments were smaller than differences among genotypes. A total of 14 compounds consistently increased or decreased in response to drought stress, and this effect was generally progressive as the severity of drought increased. A total of 10 of these marker compounds were tentatively identified as follows: five glycerolipids, glutaric acid, pheophorbide A, abscisic acid, roseoside, and dihydromyricetin. Some of the observed metabolite changes likely occur across all plants under drought conditions, while others may be specific to hops or to the type of drought treatments performed.

Quantitative analysis of 2-hydroxyglutarate as a controversial oncometabolite in malignant gliomas.

There is a great effort to connect the accumulation of 2-hydroxyglutarate (2-HG) oncometabolite with cellular onco-epigenetic status and subsequently predict the prognoses of glioma patients. In this observational study, the concentrations of D- and L- 2-HG were determined in 57 tumor tissue samples of glioma patients (n=57) WHO grade I through IV (astrocytoma, oligodendroglioma, secondary glioblastoma, and glioblastoma multiforme) in vitro. Also, genetic mutation status on isocitrate dehydrogenase 1 and 2 (IDH 1/2) was determined from these samples. The objective of this study was to confirm or to reject the hypothesis of the direct correlation of 2-HG concentration in tumor tissue and the results from IDH 1/IDH 2 point mutation analyses. The concentrations of 2-HG were quantified using high sensitive HPLC and Q-TOF HRMS spectrometer setup. Concurrently, the genetic mutation analyses of both IDH 1 (cytosolic) and IDH 2 (mitochondrial) were performed by the isolation of tumor tissue DNA, PCR amplification, and subsequent Sanger forward sequencing. Our results indicate that there is no definite correlation between the two as we identified cases of glioma tumors with significantly increased concentration of one or both L- and D- 2-HG but no IDH 1/2 mutations (44% 2-HG positive cases).

Robust detection of oncometabolic aberrations by 1H-13C heteronuclear single quantum correlation in intact biological specimens.

Magnetic resonance (MR) spectroscopy has potential to non-invasively detect metabolites of diagnostic significance for precision oncology. Yet, many metabolites have similar chemical shifts, yielding highly convoluted 1H spectra of intact biological material and limiting diagnostic utility. Here, we show that hydrogen-carbon heteronuclear single quantum correlation (1H-13C HSQC) offers dramatic improvements in sensitivity compared to one-dimensional (1D) 13C NMR and significant signal deconvolution compared to 1D 1H spectra in intact biological settings. Using a standard NMR spectroscope with a cryoprobe but without specialized signal enhancing features such as magic angle spinning, metabolite extractions or 13C-isotopic enrichment, we obtain well-resolved 2D 1H-13C HSQC spectra in live cancer cells, in ex vivo freshly dissected xenografted tumors and resected primary tumors. This method can identify tumors with specific oncometabolite alterations such as IDH mutations by 2-hydroxyglutarate and PGD-deleted tumors by gluconate. Results suggest potential of 1H-13C HSQC as a non-invasive diagnostic in precision oncology.

Comparative Value of 2-Hydroxyglutarate-to-Lipid and Lactate Ratio versus 2-Hydroxyglutarate Concentration on MR Spectroscopic Images for Predicting Isocitrate Dehydrogenase Mutation Status in Gliomas.

Purpose: To compare the ability of 2-hydroxyglutarate (2HG)-to-lipid and lactate (2HG/[lipid + lactate]) ratio with the ability of 2HG concentration alone to predict the isocitrate dehydrogenase (IDH) mutation status in patients with glioma. Materials and Methods: In this retrospective study, consecutive patients with histopathologically proven glioma were enrolled between July 2016 and February 2019. A total of 79 patients were enrolled (mean age, 44 years; 49 men). The 2HG concentration and other MR spectroscopic parameters were measured by single-voxel point-resolved spectroscopy before surgery. The diagnostic performance of the 2HG concentration and 2HG/(lipid + lactate) ratio were calculated. Internal validation was assessed by the bootstrap approach with 1000 bootstrap resamples. Differences in the predictive accuracy of 2HG/(lipid + lactate) ratio and 2HG concentration were determined by calculating the integrated discrimination improvement. The diagnostic accuracy (sensitivity, specificity, and area under the receiver operating characteristic curve [AUC]) of these measures was also compared separately in patients with glioblastomas and patients with lower-grade gliomas. Results: Of the 79 enrolled patients, 28 had IDH mutations and 51 had wild-type IDH. The sensitivity, specificity, and AUC of 2HG concentration for predicting IDH-mutant gliomas were 89% (25 of 28), 67% (34 of 51), and 0.80 (95% confidence interval [CI]: 0.70, 0.88; C statistic, 0.80), respectively. The sensitivity, specificity, and AUC of the 2HG/(lipid + lactate) ratio for predicting IDH-mutant gliomas were 79% (22 of 28), 92% (47 of 51), and 0.90 (95% CI: 0.81, 0.96; C statistics, 0.90), respectively. The optimal cutoff value for the 2HG/(lipid + lactate) ratio was 0.63. The 2HG/(lipid + lactate) ratio was significantly better for predicting IDH mutation status than the 2HG concentration alone (P < .01). In glioblastoma, the 2HG/(lipid + lactate) ratio was also better for predicting IDH mutations than the 2HG concentration alone, with borderline significance (P = .052). In lower-grade glioma, the 2HG/(lipid + lactate) ratio and the 2HG concentration showed comparable diagnostic performance (P = .72). Conclusion: The 2HG/(lipid + lactate) ratio is more accurate for predicting IDH mutation status in patients with glioma than the 2HG concentration alone.Keywords: Brain/Brain Stem, CNS, MR-Imaging, MR-Spectroscopy, Neoplasms-Primary, Neuro-Oncology© RSNA, 2020.

A double surrogate approach for the quantitation of 2-Hydroxyglutarate - An oncometabolite in human brain tumors via LC-MS/MS.

We are reporting a validated LC-MS/MS assay for the quantitation of 2-Hydroxyglutarate (2-HG), an endogenous oncometabolite, in human brain tumors. To address the challenges of endogenous levels of 2-HG in biological matrix and the limited availability of human brain tumors, a surrogate analyte in conjunction with a surrogate matrix approach was used (stable isotope labeled 2-HG in monkey brain homogenate). Human brain tumor homogenate samples were spiked with stable isotope labeled internal standard, processed by protein precipitation, and analyzed using LC-MS/MS. The mass spectrometer was optimized to ensure equal molar response between 2-HG - the authentic analyte and 13C5 2-HG - the surrogate analyte. Parallelism was successfully demonstrated between human brain tumor homogenate (the authentic matrix) and monkey brain homogenate (the surrogate matrix). The linear analytical range of the assay was set at 0.15-150 µM. This validated LC-MS/MS method demonstrated excellent accuracy and precision. For 13C5 2-HG in monkey brain homogenate and human brain tumor homogenate, the overall accuracy was between 98.4 % and 102 %, and the intra- and inter-assay precision (%CV) were less than 5.7 %. 2-HG was found to be stable in human brain tumor homogenate for at least 26 h at room temperature, 454 days when stored at 70 °C and after five freeze/thaw cycles. The assay has been successfully applied to human brain tumor samples to support clinical studies. Novel Aspect: A surrogate matrix and surrogate analyte approach to quantify endogenous 2-HG concentrations in human brain tumors.

In vivo 1 H MRS detection of cystathionine in human brain tumors.

PURPOSE: To report the technical aspects of noninvasive detection of cystathionine in human brain glioma with edited MRS, and to investigate possible further acquisition improvements for robust quantification of this metabolite. METHODS: In vivo 1 H MR spectra were acquired at 3 T in 15 participants with an isocitrate dehydrogenase-mutated glioma using a MEGA-PRESS (MEscher GArwood point resolved spectroscopy) sequence previously employed for 2-hydroxyglutarate detection (TR = 2 s, TE = 68 ms). The editing pulse was applied at 1.9 ppm for the edit-on condition and at 7.5 ppm for the edit-off condition. To evaluate the editing efficiency, spectra were acquired in 1 participant by placing the editing pulse for the edit-on condition at 1.9, 2.03, and 2.16 ppm. Cystathionine concentration was quantified using LCModel and a simulated basis set. To confirm chemical shifts and J-coupling values of cystathionine, the 1 H NMR cystathionine spectrum was measured using a high-resolution 500 MHz spectrometer. RESULTS: In 12 gliomas, cystathionine was observed in the in vivo edited MR spectra at 2.72 and 3.85 ppm and quantified. The signal intensity of the cystathionine resonance at 2.72 ppm increased 1.7 and 2.13 times when the editing pulse was moved to 2.03 and 2.16 ppm, respectively. Cystathionine was not detectable in normal brain tissue. CONCLUSION: Cystathionine can be detected in vivo by edited MRS using the same protocol as for 2-hydroxyglutarate detection. This finding may enable a more accurate, noninvasive investigation of cellular metabolism in glioma.

In vivo Metabolic Profiles as Determined by 31P and short TE 1H MR-Spectroscopy : No Difference Between Patients with IDH Wildtype and IDH Mutant Gliomas.

PURPOSE: Previous ex vivo spectroscopic data from tissue samples revealed differences in phospholipid metabolites between isocitrate dehydrogenase mutated (IDHmut) and IDH wildtype (IDHwt) gliomas. We investigated whether these changes can be found in vivo using 1H-decoupled 31P magnetic resonance spectroscopic imaging (MRSI) with 3D chemical shift imaging (CSI) at 3 T in patients with low and high-grade gliomas. METHODS: The study included 33 prospectively enrolled, mostly untreated patients who met spectral quality criteria according to the World Health Organization (WHO II n = 7, WHO III n = 17, WHO IV n = 9; 25 patients IDHmut, 8 patients IDHwt). The MRSI protocol included 1H decoupled 31P MRSI with 3D CSI (3D 31P CSI), 2D 1H CSI and a 1H single voxel spectroscopy sequence (TE 30 ms) from the tumor area. For 1H MRS, absolute metabolite concentration values were calculated (phantom replacement method). For 31P MRS, metabolite intensity ratios were calculated for the choline (C) and ethanolamine (E)-containing metabolites. RESULTS: In our patient cohort we did not find significant differences for the ratio of phosphocholine (PC) and phosphoethanolamine (PE), PC/PE, (p = 0.24) for IDHmut compared to IDHwt gliomas. Furthermore, we found no elevated ratios of glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE), GPC/GPE, (p = 0.68) or GPC/PE (p = 0.12) for IDHmut gliomas. Even the ratio (PC+GPC)/(PE+GPE) showed no significant differences with respect to mutation status (p = 0.16). Nonetheless, changes related to tumor grade regarding intracellular pH (pHi) and phospholipid metabolism as well as absolute metabolite concentrations of co-registered 2D 1H CSI data for tumor and control tissue showed the anticipated results. CONCLUSION: Using 3D-CSI data acquisition, in vivo 31P MR spectroscopic measurement of phospholipid metabolites could not distinguish between IDHmut and IDHwt.

3D high-resolution imaging of 2-hydroxyglutarate in glioma patients using DRAG-EPSI at 3T in vivo.

PURPOSE: To develop 3D high-resolution imaging of 2-hydroxyglutarate (2HG) at 3T in vivo. METHODS: Echo-planar spectroscopic imaging with dual-readout alternated-gradients (DRAG-EPSI), which was recently reported for 2D imaging of 2HG at 7T, was tested for 3D imaging of 2HG at 3T. The frequency drifts and acoustic noise induced by DRAG-EPSI were investigated in comparison with conventional EPSI. Four patients with IDH-mutant gliomas were enrolled for 3D imaging of 2HG and other metabolites. A previously reported 2HG-tailored TE 97-ms PRESS sequence preceded the DRAG-EPSI readout gradients. Unsuppressed water, acquired with EPSI, was used as reference for multi-channel combination, eddy-current compensation, and metabolite quantification. Spectral fitting was conducted with the LCModel using in-house basis sets. RESULTS: With gradient strength of 4 mT/m and slew rate of 20 mT/m/ms, DRAG-EPSI produced frequency drifts smaller by 5.5-fold and acoustic noise lower by 25 dB compared to conventional EPSI. In a 19-min scan, 3D DRAG-EPSI provided images of 2HG with precision (CRLB <10%) at a resolution of 10 × 10 × 10 mm3 for a field of view of 240 × 180 × 80 mm3 . 2HG was estimated to be 5 mM in a pre-treatment patient. In 3 post-surgery patients, 2HG estimates were 3-6 mM, and the 2HG distribution was different from the water-T2 image pattern or highly concentrated in the post-contrast enhancing region. CONCLUSION: Together with 2HG-optimized PRESS, DRAG-EPSI provides an effective tool for reliable 3D high-resolution imaging of 2HG at 3T in vivo.

Fitting algorithms and baseline correction influence the results of non-invasive in vivo quantitation of 2-hydroxyglutarate with 1 H-MRS.

1 H-MRS enables non-invasive detection of 2-hydroxyglutarate (2-HG), an oncometabolite accumulating in gliomas carrying mutations in the isocitrate dehydrogenase (IDH) genes. Reliable 2-HG quantitation requires reproducible post-processing, deployment of fitting algorithms and quantitation methods. We prospectively enrolled 38 patients with suspected or recently diagnosed gliomas (IDH mutated n = 26). The MRI protocol included a 1 H single voxel PRESS sequence with volumes of usually 8 mL or more (20 × 20 × 20 mm3 ) at TE  = 97 ms and 180° pulse spacing. Our aim was to evaluate the reliability of 2-HG quantitation comparing two frequently used software tools and their respective options of baseline correction (jMRUI with the time domain methods AQSES and QUEST, and LCModel, which analyzes the frequency domain data). For AQSES, degrees of freedom for baseline correction constrains were varied. For LCModel, baseline correction was obtained with and without correction of the unknown background term (predefined macromolecules, lipids). Tissue concentrations were calculated based on the phantom replacement method. Quantitation of 2-HG levels showed similar mean 2-HG tissue concentrations for IDH mutated tumors (2.65mM, range 3.06-2.20) for all methods. Bland-Altman plots (difference plots) did not reveal a systematic bias (fixed bias) for any of the algorithms tested, and we were able to show a significant correlation regarding 2-HG concentration at the same echo time with few statistical outliers (parametric correlation). However, evaluation of outliers suggested that in vivo quantitation of 2-HG is affected not only by the fitting domain (time or frequency), but also by the baseline correction, which is a major contributing factor to the result of 2-HG fitting. Clinical application of 2-HG quantitation as a prognostic or predictive biomarker, particularly in multicenter trials, requires standardized use of fitting methods and baseline correction procedures.

Point-of-Care Tissue Analysis Using Miniature Mass Spectrometer.

The combination of direct sampling ionization and miniature mass spectrometer presents a promising technical pathway of point-of-care analysis in clinical applications. In this work, a miniature mass spectrometry system was used for analysis of tissue samples. Direct tissue sampling coupled with extraction spray ionization was used with a home-built miniature mass spectrometer, Mini 12. Lipid species in tissue samples were well profiled in rat brain, kidney, and liver in a couple of minutes. By incorporating a photochemical (Paternò-Büchi) reaction, fast identification of lipid C═C location was realized. Relative quantitation of the lipid C═C isomer was performed by calculating the intensity ratio C═C diagnostic product ions, by which FA 18:1 (Δ9)/FA 18:1 (Δ11) was found to change significantly in mouse cancerous breast tissue samples. Accumulation of 2-hydroxylglutarate in human glioma samples, not in normal brains, can also be easily identified for rapid diagnosis.

Rapid diagnosis of IDH1-mutated gliomas by 2-HG detection with gas chromatography mass spectrometry.

The metabolic genes encoding isocitrate dehydrogenase (IDH1, 2) are frequently mutated in gliomas. Mutation of IDH defines a distinct subtype of glioma and predicts therapeutic response. IDH mutation has a remarkable neomorphic activity of converting α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG), which is now commonly referred to as an oncometabolite and biomarker for gliomas. PCR-sequencing (n = 220), immunohistochemistry staining (IHC, n = 220), and gas chromatography mass spectrometry (GC-MS, n = 87) were applied to identify IDH mutation in gliomas, and the sensitivity and specificity of these strategies were compared. PCR-sequencing and IHC staining are reliable for retrospective assessment of IDH1 mutation in gliomas, but both methods usually take 1-2 days, which hinders their application for rapid diagnosis. GC-MS-based methods can detect 2-HG qualitatively and quantitatively, offering information on the IDH1 mutation status in gliomas with the sensitivity and specificity being 100%. Further optimization of the GC-MS based methodology (so called as the mini-column method) enabled us to determine 2-HG within 40 min in glioma samples without complex or time-consuming preparation. Most importantly, the ratio of 2-HG/glutamic acid was shown to be a reliable parameter for determination of mutation status. The mini-column method enables rapid identification of 2-HG, providing a promising strategy for intraoperative diagnosis of IDH1-mutated gliomas in the future.

900MHz 1H-/13C-NMR analysis of 2-hydroxyglutarate and other brain metabolites in human brain tumor tissue extracts.

PURPOSE: To perform in vitro high-resolution 900 MHz magnetic resonance spectroscopy (NMR) analysis of human brain tumor tissue extracts and analyze for the oncometabolite 2-hydroxyglutarate (2HG) and other brain metabolites, not only for 1H but also for 13C with indirect detection by heteronuclear single quantum correlation (HSQC). MATERIAL AND METHODS: Four surgically removed human brain tumor tissue samples were used for extraction and preparation of NMR samples. These tissue samples were extracted with 4% perchloric acid and chloroform, freeze-dried, then dissolved into 0.28 mL of deuterium oxide (D2O, 99.9 atom % deuterium) containing 0.025 wt % sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TSP). All samples were adjusted to pH range of 6.9-7.1 before finally transferred to 5 mm Shigemi™ NMR microtube. NMR experiments were performed on Bruker DRX 900 MHz spectrometer with 1H/13C/15N Cryo-probe™ with Z-gradient, without further temperature control for the samples. All chemical shift values were presented relative to TSP at 0.00 ppm for both 1H and 13C. 1H 1D, 1H-13C HSQC, 1H-1H correlation spectroscopy (COSY) and 1H-13C heteronuclear multiple bond correlation (HMBC) spectra were acquired and analyzed. RESULTS: 2-hydroxyglutarate, an oncometabolite associated with gliomas with IDH mutations, was successfully detected and assigned by both 1H-13C HSQC and 1H-1H COSY experiments as well as 1H 1D experiments in two of the tissue samples. In particular, to our knowledge this work shows the first example of detecting 900 MHz 13C-NMR spectral lines of 2-hydroxyglutarate in human brain tumor tissue samples. In addition to the oncometabolite 2-hydroxyglutarate, at least 42 more metabolites were identified from our series of NMR experiment. CONCLUSION: The detection of 2-hydroxyglutarate and other metabolites can be facilitated by homonuclear and heteronuclear two-dimensional 900 MHz NMR spectroscopy even in case of real tumor tissue sample extracts without physical separation of metabolites.

One-pot two-strain system based on glucaric acid biosensor for rapid screening of myo-inositol oxygenase mutations and glucaric acid production in recombinant cells.

The development of D-glucaric acid (GA) production in recombinant cells has leapt forward in recent years, and higher throughput screening and selection of better-performing recombinant cells or biocatalysts is in current demand. A biosensor system which converts GA concentration into fluorescence signal in Escherichia coli was developed in 2016, but its application has rarely been reported. Herein, an effective high-throughput screening approach independent of special-purpose devices such as microfluidic platforms was established and tentatively applied. In this one-pot two-strain system, GA producers-bacterial or yeast cells containing the GA biosynthetic pathway-were sorted with the help of another E. coli strain acting as a GA biosensor. The identification of highly active mutants of myo-inositol oxygenase through this system validates its effectiveness in sorting E. coli cells. Subsequently, accurate ranking of the GA synthesis capacity of a small library of Saccharomyces cerevisiae strains containing distinct GA synthesis pathways demonstrated that this optimized one-pot two-strain system may also be used for eukaryotic producer strains. These results will assist in research into metabolic engineering for GA production and development of biosensor applications.

Metabolite-cycled density-weighted concentric rings k-space trajectory (DW-CRT) enables high-resolution 1 H magnetic resonance spectroscopic imaging at 3-Tesla.

Magnetic resonance spectroscopic imaging (MRSI) is a promising technique in both experimental and clinical settings. However, to date, MRSI has been hampered by prohibitively long acquisition times and artifacts caused by subject motion and hardware-related frequency drift. In the present study, we demonstrate that density weighted concentric ring trajectory (DW-CRT) k-space sampling in combination with semi-LASER excitation and metabolite-cycling enables high-resolution MRSI data to be rapidly acquired at 3 Tesla. Single-slice full-intensity MRSI data (short echo time (TE) semi-LASER TE = 32 ms) were acquired from 6 healthy volunteers with an in-plane resolution of 5 × 5 mm in 13 min 30 sec using this approach. Using LCModel analysis, we found that the acquired spectra allowed for the mapping of total N-acetylaspartate (median Cramer-Rao Lower Bound [CRLB] = 3%), glutamate+glutamine (8%), and glutathione (13%). In addition, we demonstrate potential clinical utility of this technique by optimizing the TE to detect 2-hydroxyglutarate (long TE semi-LASER, TE = 110 ms), to produce relevant high-resolution metabolite maps of grade III IDH-mutant oligodendroglioma in a single patient. This study demonstrates the potential utility of MRSI in the clinical setting at 3 Tesla.