Lipid Artifacts Research Articles

Improved signal-to-noise performance of MultiNet GRAPPA 1 H FID MRSI reconstruction with semi-synthetic calibration data.

To further develop MultiNet GRAPPA, a neural-network-based reconstruction, for lower SNR proton MRSI (1 H MRSI) data using adapted undersampling schemes and improved training sets. 1 H FID-MRSI data and an anatomical image for GRAPPA reconstruction were acquired in two slices in the human brain (n = 6) at 7T. MRSI data were retrospectively undersampled for a 4×, 6×, and 7× acceleration rate. Signal-to-noise, relative error (RE) between accelerated and fully sampled metabolic maps, RMS of the lipid artifacts, and fitting reliability were compared across acceleration rates, to the fully sampled data, and with different kinds and amounts of training images. Training with semi-synthetic images resulted in higher SNR and lower lipid RMS relative to training with acquired images from one or several subjects. SNR increased with the number of semi-synthetic training images and the 4× accelerated data retains ∼30% more SNR than other accelerated data. Spectra reconstructed with 20 semi-synthetic averages retained ∼100% more SNR and had ∼5% lower lipid RMS than those reconstructed with the center k-space points of one image as was originally proposed for very high SNR MRSI data and had higher fitting reliability. The metabolite RE was lowest when training with 20-semi-synthetic training images and highest when training with the center k-space points of one image. MultiNet GRAPPA is feasible with lower SNR 1 H MRSI data if 20-semi-synthetic training images are used at a 4× acceleration rate. This acceleration rate provided the best trade-off between scan time and spectral SNR.

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.

Reducing SAR in 7T brain fMRI by circumventing fat suppression while removing the lipid signal through a parallel acquisition approach

Ultra-high-field functional magnetic resonance imaging (fMRI) offers a way to new insights while increasing the spatial and temporal resolution. However, a crucial concern in 7T human MRI is the increase in power deposition, supervised through the specific absorption rate (SAR). The SAR limitation can restrict the brain coverage or the minimal repetition time of fMRI experiments. In the majority of today’s studies fMRI relies on the well-known gradient-echo echo-planar imaging (GRE-EPI) sequence, which offers ultrafast acquisition. Commonly, the GRE-EPI sequence comprises two pulses: fat suppression and excitation. This work provides the means for a significant reduction in the SAR by circumventing the fat-suppression pulse. Without this fat-suppression, however, lipid signal can result in artifacts due to the chemical shift between the lipid and water signals. Our approach exploits a reconstruction similar to the simultaneous-multi-slice method to separate the lipid and water images, thus avoiding undesired lipid artifacts in brain images. The lipid-water separation is based on the known spatial shift of the lipid signal, which can be detected by the multi-channel coils sensitivity profiles. Our study shows robust human imaging, offering greater flexibility to reduce the SAR, shorten the repetition time or increase the volume coverage with substantial benefit for brain functional studies.

Lipid-suppressed and tissue-fraction corrected metabolic distributions in human central brain structures using 2D 1 H magnetic resonance spectroscopic imaging at 7 T.

IntroductionMagnetic resonance spectroscopic imaging (MRSI) has the potential to add a layer of understanding of the neurobiological mechanisms underlying brain diseases, disease progression, and treatment efficacy. Limitations related to metabolite fitting of low signal‐to‐noise ratios data, signal variations due to partial‐volume effects, acquisition and extracranial lipid artifacts, along with clinically relevant aspects such as scan time constraints, are among the challenges associated with in vivo MRSI.MethodsThe aim of this work was to address some of these factors and to develop an acquisition, reconstruction, and postprocessing pipeline to derive lipid‐suppressed metabolite values of central brain structures based on free‐induction decay measurements made using a 7 T MR scanner. Anatomical images were used to perform high‐resolution (1 mm3) partial‐volume correction to account for gray matter, white matter (WM), and cerebral‐spinal fluid signal contributions. Implementation of automatic quality control thresholds and normalization of metabolic maps from 23 subjects to the Montreal Neurological Institute (MNI) standard atlas facilitated the creation of high‐resolution average metabolite maps of several clinically relevant metabolites in central brain regions, while accounting for macromolecular distributions.Partial‐volume correction improved the delineation of deep brain nuclei. We report average metabolite values including glutamate + glutamine (Glx), glycerophosphocholine, choline and phosphocholine (tCho), (phospo)creatine, myo‐inositol and glycine (mI‐Gly), glutathione, N‐acetyl‐aspartyl glutamate(and glutamine), and N‐acetyl‐aspartate in the basal ganglia, central WM (thalamic radiation, corpus callosum) as well as insular cortex and intracalcarine sulcus.ConclusionMNI‐registered average metabolite maps facilitate group‐based analysis, thus offering the possibility to mitigate uncertainty in variable MRSI data.

Effectiveness of fat suppression using a water-selective binomial-pulse excitation in chemical exchange saturation transfer (CEST) magnetic resonance imaging.

The purpose of this study was to characterize the individual contribution of multiple fat peaks to the measured chemical exchange saturation transfer (CEST) signal when using water-selective binomial-pulse excitation and to determine the effects of multiple fat peaks in the presence of B0 inhomogeneity. The excitation profiles of multiple binomial pulses were simulated. A CEST sequence with binomial-pulse excitation and modified point-resolved spectroscopy localization was then applied to the in vivo lumbar spinal vertebrae to determine the signal contributions of three distinct groups of lipid resonances. These confounding signal contributions were measured as a function of the irradiation frequency offset to determine the effect of the multi-peak nature of the fat signal on CEST imaging of exchange sites (at 1.0, 2.0 and 3.5ppm) and robustness in the presence of B0 inhomogeneity. Numerical simulations and in vivo experiments showed that water excitation (WE) using a 1-3-3-1 (WE-4) pulse provided the broadest signal suppression, which provided partial robustness against B0 inhomogeneity effects. Confounding fat signal contributions to the CEST contrasts at 1.0, 2.0 and 3.5ppm were unavoidable due to the multi-peak nature of the fat signal. However, these CEST sites only suffer from small lipid artifacts with ∆B0 spanning roughly from - 50 to 50Hz. Especially for the CEST site at 3.5ppm, the lipid artifacts are smaller than 1% with ∆B0 in this range. In WE-4-based CEST magnetic resonance imaging, B0 inhomogeneity is the limiting factor for fat suppression. The CEST sites at 1.0, 2.0ppm and 3.5ppm unavoidably suffer from lipid artifacts. However, when the ∆B0 is confined to a limited range, these CEST sites are only affected by small lipid artifacts, which may be ignorable in some cases of clinical applications.

Influence of fitting approaches in LCModel on MRS quantification focusing on age-specific macromolecules and the spline baseline.

Quantification of neurochemical concentrations from 1 H MR spectra is challenged by incomplete knowledge of contributing signals. Some experimental conditions hinder the acquisition of artifact-free spectra and impede the acquisition of condition-specific macromolecule (MM) spectra. This work studies differences caused by fitting solutions routinely employed to manage resonances from MM and lipids. High quality spectra (free of residual water and lipid artifacts and for which condition-specific MM spectra are available) are used to understand the influences of spline baseline flexibility and noncondition-specific MM on neurochemical quantification. Fitting with moderate spline flexibility or using noncondition-specific MM led to quantification that differed from when an appropriate, fully specified model was used. This occurred for all neurochemicals to an extent that varied in magnitude among and within approaches. The spline baseline was more tortuous when less constrained and when used in combination with noncondition-specific MM. Increasing baseline flexibility did not reproduce concentrations quantified under appropriate conditions when spectra were fitted using a MM spectrum measured from a mismatched cohort. Using the noncondition-specific MM spectrum led to quantification differences that were comparable in size with using a fitting model that had moderate freedom, and these influences were additive. Although goodness of fit was better with greater fitting flexibility, quantification differed from when fitting with a fully specified model that is appropriate for low noise data. Notable GABA and PE concentration differences occurred with lower estimates of measurement error when fitting with greater spline flexibility or noncondition-specific MM. These data support the need for improved metrics of goodness of fit. Attempting to correct for artifacts or absence of a condition-specific MM spectrum via increased spline flexibility and usage of noncondition-specific MM spectra cannot replace artifact-free data quantified with a condition-specific MM spectrum.

Self‐adapting multi‐peak water‐fat reconstruction for the removal of lipid artifacts in chemical exchange saturation transfer (CEST) imaging

Artifacts caused by strong lipid signals pose challenges in body chemical exchange saturation transfer (CEST) imaging. This study aimed to develop an accurate water-fat reconstruction method based on the multi-echo Dixon technique to remove lipid artifacts in CEST imaging. It is well known that fat has multiple spectral peaks. Furthermore, RF pulses in CEST preparation saturate each fat peak at different levels, complicating fat modeling. Therefore, a self-adapting multi-peak model (SMPM) is proposed to update relative amplitudes of fat peaks using numerical calculation. With the SMPM-based updating, nonlinear least-squares fitting combined with IDEAL (Iterative Decomposition of water and fat with Echo Asymmetry and Least-squares estimation) algorithms was used for water-fat reconstruction and B0 mapping. The proposed method was compared with the reported 3-point Dixon method and the fixed multi-peak model in a phantom study using a fat-free Z-spectrum obtained from MR spectroscopy acquisition as the ground truth. This method was also validated by in vivo experiments on human breast. In the phantom experiments, the Z-spectrum from the SMPM-based method agreed well with the fat-free Z-spectrum from CEST-PRESS (point-resolved spectroscopy), validating the effective removal of lipid artifacts, while a decrease or a rise that appeared at -3.5 ppm was observed in the Z-spectrum from the 3-point method and the FMPM-based method, respectively. In the in vivo experiments, no lipid artifacts were observed in the Z-spectrum or the amide CEST map from the SMPM-based method in the fibro-glandular region of the breast with high fat fractions. The SMPM-based method successfully removes lipid artifacts and significantly improves the accuracy of CEST contrast.

Density-weighted concentric circle trajectories for high resolution brain magnetic resonance spectroscopic imaging at 7T.

PurposeFull‐slice magnetic resonance spectroscopic imaging at ≥7 T is especially vulnerable to lipid contaminations arising from regions close to the skull. This contamination can be mitigated by improving the point spread function via higher spatial resolution sampling and k‐space filtering, but this prolongs scan times and reduces the signal‐to‐noise ratio (SNR) efficiency. Currently applied parallel imaging methods accelerate magnetic resonance spectroscopic imaging scans at 7T, but increase lipid artifacts and lower SNR‐efficiency further. In this study, we propose an SNR‐efficient spatial‐spectral sampling scheme using concentric circle echo planar trajectories (CONCEPT), which was adapted to intrinsically acquire a Hamming‐weighted k‐space, thus termed density‐weighted‐CONCEPT. This minimizes voxel bleeding, while preserving an optimal SNR.Theory and MethodsTrajectories were theoretically derived and verified in phantoms as well as in the human brain via measurements of five volunteers (single‐slice, field‐of‐view 220 × 220 mm2, matrix 64 × 64, scan time 6 min) with free induction decay magnetic resonance spectroscopic imaging. Density‐weighted‐CONCEPT was compared to (a) the originally proposed CONCEPT with equidistant circles (here termed e‐CONCEPT), (b) elliptical phase‐encoding, and (c) 5‐fold Controlled Aliasing In Parallel Imaging Results IN Higher Acceleration accelerated elliptical phase‐encoding.ResultsBy intrinsically sampling a Hamming‐weighted k‐space, density‐weighted‐CONCEPT removed Gibbs‐ringing artifacts and had in vivo +9.5%, +24.4%, and +39.7% higher SNR than e‐CONCEPT, elliptical phase‐encoding, and the Controlled Aliasing In Parallel Imaging Results IN Higher Acceleration accelerated elliptical phase‐encoding (all P < 0.05), respectively, which lead to improved metabolic maps.ConclusionDensity‐weighted‐CONCEPT provides clinically attractive full‐slice high‐resolution magnetic resonance spectroscopic imaging with optimal SNR at 7T. Magn Reson Med 79:2874–2885, 2018. © 2017 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Z-spectrum appearance and interpretation in the presence of fat: Influence of acquisition parameters.

Chemical exchange saturation transfer (CEST) MRI is increasingly evolving from brain to body applications. One of the known problems in the body imaging is the presence of strong lipid signals. Although their influence on the CEST effect is acknowledged, there was no study that focuses on the interplay among echo time, fat fraction, and Z-spectrum. This study strives to address these points, with the emphasis on the application in the breast. Z-spectra were simulated in phase and out of phase of the main fat peak at -3.4 ppm, with the fat fraction varying from 0 to 100%. The magnetization transfer ratio asymmetry in two ranges, centering at the exchanging pool and at 3.5 ppm approximately opposite the nonexchanging fat pool, were calculated and were plotted against fat fraction. The results were verified in phantoms and in vivo. The results demonstrate the combined influence of fat fraction and echo time on the Z-spectrum for gradient echo based CEST acquisitions. The influence is straightforward in the in-phase images, but it is more complicated in the out-of-phase images, potentially leading to erroneous CEST contrast. This study provides a basis for understanding the origin and appearance of lipid artifacts in CEST imaging, and lays the foundation for their efficient removal. Magn Reson Med 79:2731-2737, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Improved volume selective (1) H MR spectroscopic imaging of the prostate with gradient offset independent adiabaticity pulses at 3 tesla.

Volume selection in (1) H MR spectroscopic imaging (MRSI) of the prostate is commonly performed with low-bandwidth refocusing pulses. However, their large chemical shift displacement error (CSDE) causes lipid signal contamination in the spectral range of interest. Application of high-bandwidth adiabatic pulses is limited by radiofrequency (RF) power deposition. In this study, we aimed to provide an MRSI sequence that overcomes these limitations. Measurements were performed at 3 T with an endorectal receive coil. A semi-LASER sequence was equipped with low RF power demanding gradient-modulated offset-independent adiabaticity (GOIA) refocusing pulses with WURST(16,4) modulation, with a 10 kHz bandwidth. Simulations and phantom studies verified that the GOIA pulses select slices with a flat top and sharp edges and minimal CSDE. The sequence timing was tuned to an optimal citrate signal shape at an echo time of 88 ms. Patient studies (n = 10) demonstrated that high quality spectra with reduced lipid artifacts can be obtained from the whole prostate. Compared with PRESS acquisition at 145 ms the signal-to-noise ratio (SNR) of citrate is increased up to 2.6 and choline up to 1.3. An MRSI sequence of the prostate is presented with minimized spectral lipid contamination and improved SNR, to facilitate routine clinical acquisition of metabolic data.

Minimizing lipid signal bleed in brain (1) H chemical shift imaging by post-acquisition grid shifting.

Low spatial resolution in conventional 1H brain chemical shifting imaging (CSI) studies causes partial volume error (PVE) or signal "bleed" that is especially deleterious to voxels near the scalp. The standard spatial apodization approach adversely affects spatial resolution. Here, a novel automated post-processing strategy of partial volume correction employing grid shifting ("PANGS") is presented, which minimizes residual PVE without compromising spatial resolution. PANGS shifts the locations of the reconstruction coordinates in a designated region of image space-the scalp, to match the tissue "centers-of-mass" instead of the geometric centers of each voxel, by iteratively minimizing the PVE from the scalp into outside voxels. PANGS' performance was evaluated by numerical simulation, and in 3 Tesla 1H CSI human studies employing outer volume suppression and long echo times. PANGS reduced lipid contamination of cortical spectra by up to 86% (54% on average). Metabolite maps exhibited significantly less lipid artifact than conventional and spatially-filtered CSI. All methods generated quantitatively identical spectral peak areas from central brain locations, but spatial filtering increased spectral linewidths and reduced spatial resolution. PANGS significantly reduces lipid artifacts in 1H brain CSI spectra and metabolite maps, and improves metabolite detection in cortical regions without compromising resolution.

Lipid suppression in CSI with spatial priors and highly undersampled peripheral k‐space

Mapping 1H brain metabolites using chemical shift imaging is hampered by the presence of subcutaneous lipid signals, which contaminate the metabolites by ringing due to limited spatial resolution. Even though chemical shift imaging at spatial resolution high enough to mitigate the lipid artifacts is infeasible due to signal-to-noise constraints on the metabolites, the lipid signals have orders of magnitude of higher concentration, which enables the collection of high-resolution lipid maps with adequate signal-to-noise. The previously proposed dual-density approach exploits this high signal-to-noise property of the lipid layer to suppress truncation artifacts using high-resolution lipid maps. Another recent approach for lipid suppression makes use of the fact that metabolite and lipid spectra are approximately orthogonal, and seeks sparse metabolite spectra when projected onto lipid-basis functions. This work combines and extends the dual-density approach and the lipid-basis penalty, while estimating the high-resolution lipid image from 2-average k-space data to incur minimal increase on the scan time. Further, we exploit the spectral-spatial sparsity of the lipid ring and propose to estimate it from substantially undersampled (acceleration R=10 in the peripheral k-space) 2-average in vivo data using compressed sensing and still obtain improved lipid suppression relative to using dual-density or lipid-basis penalty alone.

SPECIAL semi‐LASER with lipid artifact compensation for 1H MRS at 7 T

The measurement of full metabolic profiles at ultrahigh fields including low concentrated or fast-relaxing metabolites is usually achieved by applying short echo time sequences. One sequence beside stimulated echo acquisition mode that was proposed in this regard is spin echo full intensity-acquired localized spectroscopy. Typical problems that are still persistent for spin echo full intensity-acquired localized spectroscopy are B(1) inhomogeneities especially for signal acquisition with surface coils and chemical shift displacement artifacts due to limited B(1) amplitudes when using volume coils. In addition, strong lipid contaminations in the final spectrum can occur when only a limited number of outer volume suppression pulses is used. Therefore, an adiabatic short echo time (= 19 ms) spin echo full intensity-acquired localized spectroscopy semilocalization by adiabatic selective refocusing sequence is presented that is less sensitive to strong B(1) variations and that offers increased excitation and refocusing pulse bandwidths than regular spin echo full intensity acquired localized spectroscopy. Furthermore, the existence of the systematic lipid artifact is identified and linked to unfavorable effects due to the preinversion localization pulse. A method to control this artifact is presented and validated in both phantom and in vivo measurements. The viability of the proposed sequence was further assessed for in vivo measurements by scanning 17 volunteers using a surface coil and moreover acquiring additional volume coil measurements. The results show well-suppressed lipid artifacts, good signal-to-noise ratio, and reproducible fitting results in accordance with other published studies.

Simple baseline correction for 1H MRSI data of the prostate

Proton magnetic resonance spectroscopic imaging of the prostate can detect and localise tumour tissue in patients with prostate cancer. Pattern recognition methods have the potential to discriminate tumour from normal tissue using the spectral patterns of magnetic resonance spectroscopic imaging data thus providing a powerful tool for diagnosis and monitoring treatment of prostate cancer. Prostate magnetic resonance spectroscopic imaging data acquisition has been optimized for a flat baseline, including a long echo time and the use of water and fat suppression pulses. Despite this acquisition method, lipid resonances may still occur particularly at the margins of the prostate. Correcting the baseline by removing lipid artifacts is, therefore, a necessary processing step. We propose a simple baseline correction method for prostate magnetic resonance spectroscopic imaging through subtraction of a single simulated-lorentzian resonance. This method is shown to restore flat baselines to a test set of spectra and compares favorably with a state-space modeling approach.

Suppression of lipid artifacts in amide proton transfer imaging

Amide proton transfer (APT) imaging is a type of chemical exchange saturation transfer imaging in which the amide protons of cellular proteins and peptides are saturated and detected via the water resonance. To study this effect, conventional magnetization transfer and direct saturation effects in the frequency-dependent water saturation spectrum (z-spectrum) need to be removed by asymmetry analysis with respect to the water frequency offset. When using echo planar imaging, it was found that unequal pericranial fat saturation at equidistant higher and lower frequencies with respect to water leads to a lipid artifact in APT asymmetry images. It is demonstrated that a chemical-shift-selective refocusing pulse in combination with crusher gradients can suppress this artifact and provide high-quality images.

Removal of lipid artifacts in 1H spectroscopic imaging by data extrapolation

Proton MR spectroscopic imaging (MRSI) of human cerebral cortex is complicated by the presence of an intense signal from subcutaneous lipids, which, if not suppressed before Fourier reconstruction, causes ringing and signal contamination throughout the metabolite images as a result of limited k-space sampling. In this article, an improved reconstruction of the lipid region is obtained using the Papoulis-Gerchberg algorithm. This procedure makes use of the narrow-band-limited nature of the subcutaneous lipid signal to extrapolate to higher k-space values without alteration of the metabolite signal region. Using computer simulations and in vivo experimental studies, the implementation and performance of this algorithm were examined. This method was found to permit MRSI brain spectra to be obtained without applying any lipid suppression during data acquisition, at echo times of 50 ms and longer. When applied together with optimized acquisition methods, this provides an effective procedure for imaging metabolite distributions in cerebral cortical surface regions.

Reduced lipid contamination in in vivo 1H MRSI using time-domain fitting and neural network classification

It is a well-known problem that metabolite maps, reconstructed from in vivo 1H MRSI data sets, may suffer from contamination caused by the presence of strong lipid signals. In the present investigation, the lipid problem was addressed by applying specific signal processing and data-analysis techniques, combined with pattern recognition based on the concept of the artificial neural network. In order to arrive at images, cleaned from lipid artifacts, we have applied our previously introduced interative and noniterative time-domain fitting procedures. Furthermore, reduction in computational time of the image reconstructions could be realized by using information provided by a neural network classification of the spectra, calculated from the MRSI data sets.

A novel core lipid isolated from the aceticlastic methanogen, Methanothrix concilii GP6

The membrane lipids of Methanothrix concilii are based on two types of core lipid structures, namely, the ubiquitous 2,3-di- O-phytanyl- sn-glycerol and a second previously unidentified diether core lipid, each accounting for 70 and 30% (wt. %), respectively, of the total polar lipids. Preparation of the novel diether core lipid from either purified glycolipid-1 (GL-1) or total polar lipid in 2.5% methanolic HCl resulted in the formation of several lipid artifacts, thereby necessitating the utilization of new hydrolytic conditions. We found that mild acid hydrolysis in 0.18% methanolic HCl produced only one core lipid product from GL-1 or two core lipids from the total polar lipid fraction. This new core lipid material differed from the authentic C 20,20 diether in that it contained a hydroxyl group on C-3 of the phytanyl chain at the sn-3 position of glycerol. The full chemical designation for this novel diether is: 2- O-[3,7,11,15-tetramethyl]hexadecy l-3- O-[3′ -hydroxy-3′,7′,11′,15′-tetramethyl]hexadecyl- sn-glycerol. 1H-NMR of the total polar lipid extract could detect the presence of this new core lipid by exploiting the appearance of the methyl (C-17′) singlet at 1.24 ppm. The presence of this methyl singlet in the 1H-NMR spectrum would subsequently determine whether a strong or a mild acid hydrolysis should be used for the preparation of core lipids in methanogens.