Complex I, V, and MDH2 deficient human skin fibroblasts reveal distinct metabolic signatures by 1 H HR-MAS NMR.

In this study, we investigated the metabolic signatures of different mitochondrial defects (two different complex I and complex V, and the one MDH2 defect) in human skin fibroblasts (HSF). We hypothesized that using a selective culture medium would cause defect specific adaptation of the metabolome and further our understanding of the biochemical implications for the studied defects. All cells were cultivated under galactose stress condition and compared to glucose-based cell culture condition. We investigated the bioenergetic profile using Seahorse XFe96 cell analyzer and assessed the extracellular metabolic footprints and the intracellular metabolic fingerprints using NMR. The galactose-based culture condition forced a bioenergetic switch from a glycolytic to an oxidative state in all cell lines which improved overall separation of controls from the different defect groups. The extracellular metabolome was discriminative for separating controls from defects but not the specific defects, whereas the intracellular metabolome suggests CI and CV changes and revealed clear MDH2 defect-specific changes in metabolites associated with the TCA cycle, malate aspartate shuttle, and the choline metabolism, which are pronounced under galactose condition.

Methods for Oxygen Determination in an NMR Bioreactor as a Surrogate Marker for Metabolomic Studies in Living Cell Cultures.

Nuclear magnetic resonance (NMR) approaches have been described as a powerful method for measuring oxygen in tissue cultures and body fluids by using relaxation time dependencies of substances on pO2. The present NMR study describes methods to longitudinally monitor global, in situ intracellular, and spatially resolved oxygen tension in culture media and 3D cell cultures using relaxation times of water without the need to use external sensors. 1H NMR measurements of water using a modified inversion recovery pulse scheme were employed for global, i.e., intra- and extracellular oxygen estimation in an NMR-bioreactor. The combination of 1H relaxation time T1 and diffusion measurements of water was employed for in situ cellular oxygen content determination. Spatially selective water relaxation time estimations were used for spatially resolved oxygen quantification along the NMR tube length. The inclusion in a study protocol of the presented techniques for oxygen quantification, as a surrogate marker of oxidative phosphorylation (OXPHOS), provides the possibility to measure mitochondrial respiration and metabolic changes simultaneously.

Targeting lactate dehydrogenase B-dependent mitochondrial metabolism affects tumor initiating cells and inhibits tumorigenesis of non-small cell lung cancer by inducing mtDNA damage.

Once considered a waste product of anaerobic cellular metabolism, lactate has been identified as a critical regulator of tumorigenesis, maintenance, and progression. The putative primary function of lactate dehydrogenase B (LDHB) is to catalyze the conversion of lactate to pyruvate; however, its role in regulating metabolism during tumorigenesis is largely unknown. To determine whether LDHB plays a pivotal role in tumorigenesis, we performed 2D and 3D in vitro experiments, utilized a conventional xenograft tumor model, and developed a novel genetically engineered mouse model (GEMM) of non-small cell lung cancer (NSCLC), in which we combined an LDHB deletion allele with an inducible model of lung adenocarcinoma driven by the concomitant loss of p53 (also known as Trp53) and expression of oncogenic KRAS (G12D) (KP). Here, we show that epithelial-like, tumor-initiating NSCLC cells feature oxidative phosphorylation (OXPHOS) phenotype that is regulated by LDHB-mediated lactate metabolism. We show that silencing of LDHB induces persistent mitochondrial DNA damage, decreases mitochondrial respiratory complex activity and OXPHOS, resulting in reduced levels of mitochondria-dependent metabolites, e.g., TCA intermediates, amino acids, and nucleotides. Inhibition of LDHB dramatically reduced the survival of tumor-initiating cells and sphere formation in vitro, which can be partially restored by nucleotide supplementation. In addition, LDHB silencing reduced tumor initiation and growth of xenograft tumors. Furthermore, we report for the first time that homozygous deletion of LDHB significantly reduced lung tumorigenesis upon the concomitant loss of Tp53 and expression of oncogenic KRAS without considerably affecting the animal's health status, thereby identifying LDHB as a potential target for NSCLC therapy. In conclusion, our study shows for the first time that LDHB is essential for the maintenance of mitochondrial metabolism, especially nucleotide metabolism, demonstrating that LDHB is crucial for the survival and proliferation of NSCLC tumor-initiating cells and tumorigenesis.

Determination of Intra- and Extracellular Metabolic Adaptations of 3D Cell Cultures upon Challenges in Real-Time by NMR.

NMR flow devices provide longitudinal real-time quantitative metabolome characterisation of living cells. However, discrimination of intra- and extracellular contributions to the spectra represents a major challenge in metabolomic NMR studies. The present NMR study demonstrates the possibility to quantitatively measure both metabolic intracellular fingerprints and extracellular footprints on human control fibroblasts by using a commercially available flow tube system with a standard 5 mm NMR probe. We performed a comprehensive 3D cell culture system characterisation. Diffusion NMR was employed for intra- and extracellular metabolites separation. In addition, complementary extracellular footprints were determined. The implemented perfused NMR bioreactor system allowed the determination of 35 metabolites and intra- and extracellular separation of 19 metabolites based on diffusion rate differences. We show the reliability and sensitivity of NMR diffusion measurements to detect metabolite concentration changes in both intra- and extracellular compartments during perfusion with different selective culture media, and upon complex I inhibition with rotenone. We also demonstrate the sensitivity of extracellular footprints to determine metabolic variations at different flow rates. The current method is of potential use for the metabolomic characterisation of defect fibroblasts and for improving physiological comprehension.

Determination of bile acids from human gallbladder by 1 H-MRS-Protocol optimization and estimation of reproducibility.

Bile exerts multiple functions in the liver and gut and is involved in multiple disease processes. It is secreted continuously from the liver and stored in the gallbladder until needed, and closely reflects the available bile acid pool. The study objective was therefore to develop a reliable MRS protocol and to assess variability of bile acid determination in human gallbladder. MRS measurements were performed on a 3 T MR scanner with 20 subjects to optimize protocols (26 measurements) and conduct a prospective reproducibility study (18 measurements). Measurements were carried out with subjects lying in either supine (23 scans) or prone positions (21 scans) to compare results from the two positions. For reproducibility determination, six of the 20 volunteers (three males, three females, age = 34.9 ± 10.9 years, BMI = 23.4 ± 2.1 kg/m2 ) were measured three times: back to back to assess technical variability and once again after three weeks to assess total variability, including additional physiological variability. A single voxel was measured in the gallbladder with respiratory triggering. For quantification, apparent T2 times were determined and a non-water-suppressed spectrum was acquired. Total bile acids, glycine and taurine conjugated bile acids, and lipids including choline-containing phospholipids were determined. Higher quality and reliability of gallbladder spectra were obtained with subjects measured in prone compared with supine position. All measurements of the reproducibility sub-study were of sufficient quality to be included in the analysis. Average coefficients of variation within subjects for the main compounds were 37% for total variation (including physiological and technical variation) and 24% for technical variation alone. These values were much smaller than those between subjects, which were >54% for both back-to-back and three weeks separated measurements. These results suggest diagnostic applicability of the method, especially for longitudinal studies aiming at non-invasive characterization of bile composition in humans with various diseases and/or interventional maneuvers.

Live monitoring of cellular metabolism and mitochondrial respiration in 3D cell culture system using NMR spectroscopy.

BACKGROUND: Because of the interplay between mitochondrial respiration and cellular metabolism, the simultaneous monitoring of both cellular processes provides important insights for the understanding of biological processes. NMR flow systems provide a unique window into the metabolome of cultured cells. Simplified bioreactor construction based on commercially available flow systems increase the practicability and reproducibility of bioreactor studies using standard NMR spectrometers. We therefore aim at establishing a reproducible NMR bioreactor system for metabolic 1H-NMR investigations of small molecules and concurrent oxygenation determination by 19F-NMR, with in depth description and validation by accompanying measures. METHODS: We demonstrate a detailed and standardized workflow for the preparation and transfer of collagen based 3D cell culture of high cell density for perfused investigation in a 5 mm NMR tube. Self-constructed gas mixing station enables 5% CO2 atmosphere for physiological pH in carbon based medium and is perfused by HPLC pump. RESULTS & DISCUSSION: Implemented perfused bioreactor allows detection of perfusion rate dependent metabolite content. We show interleaved dynamic profiling of 26 metabolites and mitochondrial respiration. During constant perfusion, sequential injection of rotenone/oligomycin and 2-deoxy-glucose indicated immediate activation and deactivation of glycolytic rate and full inhibition of oxygen consumption. We show sensitivity to detect substrate degradation rates of major mitochondrial fuel pathways and were able to simultaneously measure cellular oxygen consumption.

1H HR-MAS NMR Based Metabolic Profiling of Lung Cancer Cells with Induced and De-Induced Cisplatin Resistance to Reveal Metabolic Resistance Adaptations.

Cisplatin (cisPt) is an important drug that is used against various cancers, including advanced lung cancer. However, drug resistance is still a major ongoing problem and its investigation is of paramount interest. Here, a high-resolution magic angle spinning (HR-MAS) NMR study is presented deciphering the metabolic profile of non-small cell lung cancer (NSCLC) cells and metabolic adaptations at different levels of induced cisPt-resistance, as well as in their de-induced counterparts (cells cultivated in absence of cisPt). In total, fifty-three metabolites were identified and quantified in the 1H-HR-MAS NMR cell spectra. Metabolic adaptations to cisPt-resistance were detected, which correlated with the degree of resistance. Importantly, de-induced cell lines demonstrated similar metabolic adaptations as the corresponding cisPt-resistant cell lines. Metabolites predominantly changed in cisPt resistant cells and their de-induced counterparts include glutathione and taurine. Characteristic metabolic patterns for cisPt resistance may become relevant as biomarkers in cancer medicine.

Carbon source regulates polysaccharide capsule biosynthesis in Streptococcus pneumoniae.

The exopolysaccharide capsule of Streptococcus pneumoniae is an important virulence factor, but the mechanisms that regulate capsule thickness are not fully understood. Here, we investigated the effects of various exogenously supplied carbohydrates on capsule production and gene expression in several pneumococcal serotypes. Microscopy analyses indicated a near absence of the capsular polysaccharide (CPS) when S. pneumoniae was grown on fructose. Moreover, serotype 7F pneumococci produced much less CPS than strains of other serotypes (6B, 6C, 9V, 15, and 23F) when grown on glucose or sucrose. RNA-sequencing revealed carbon source-dependent regulation of distinct genes of WT strains and capsule-switch mutants of serotypes 6B and 7F, but could not explain the mechanism of capsule thickness regulation. In contrast, 31P NMR of whole-cell extract from capsule-knockout strains (Δcps) clearly revealed the accumulation or absence of capsule precursor metabolites when cells were grown on glucose or fructose, respectively. This finding suggests that fructose uptake mainly results in intracellular fructose 1-phosphate, which is not converted to CPS precursors. In addition, serotype 7F strains accumulated more precursors than did 6B strains, indicating less efficient conversion of precursor metabolites into the CPS in 7F, in line with its thinner capsule. Finally, isotopologue sucrose labeling and NMR analyses revealed that the uptake of the labeled fructose subunit into the capsule is <10% that of glucose. Our findings on the effects of carbon sources on CPS production in different S. pneumoniae serotypes may contribute to a better understanding of pneumococcal diseases and could inform future therapeutic approaches.

Consensus-based technical recommendations for clinical translation of renal BOLD MRI.

Harmonization of acquisition and analysis protocols is an important step in the validation of BOLD MRI as a renal biomarker. This harmonization initiative provides technical recommendations based on a consensus report with the aim to move towards standardized protocols that facilitate clinical translation and comparison of data across sites. We used a recently published systematic review paper, which included a detailed summary of renal BOLD MRI technical parameters and areas of investigation in its supplementary material, as the starting point in developing the survey questionnaires for seeking consensus. Survey data were collected via the Delphi consensus process from 24 researchers on renal BOLD MRI exam preparation, data acquisition, data analysis, and interpretation. Consensus was defined as ≥ 75% unanimity in response. Among 31 survey questions, 14 achieved consensus resolution, 12 showed clear respondent preference (65-74% agreement), and 5 showed equal (50/50%) split in opinion among respondents. Recommendations for subject preparation, data acquisition, processing and reporting are given based on the survey results and review of the literature. These technical recommendations are aimed towards increased inter-site harmonization, a first step towards standardization of renal BOLD MRI protocols across sites. We expect this to be an iterative process updated dynamically based on progress in the field.

Selective galactose culture condition reveals distinct metabolic signatures in pyruvate dehydrogenase and complex I deficient human skin fibroblasts.

INTRODUCTION: A decline in mitochondrial function represents a key factor of a large number of inborn errors of metabolism, which lead to an extremely heterogeneous group of disorders. OBJECTIVES: To gain insight into the biochemical consequences of mitochondrial dysfunction, we performed a metabolic profiling study in human skin fibroblasts using galactose stress medium, which forces cells to rely on mitochondrial metabolism. METHODS: Fibroblasts from controls, complex I and pyruvate dehydrogenase (PDH) deficient patients were grown under glucose or galactose culture condition. We investigated extracellular flux using Seahorse XF24 cell analyzer and assessed metabolome fingerprints using NMR spectroscopy. RESULTS: Incubation of fibroblasts in galactose leads to an increase in oxygen consumption and decrease in extracellular acidification rate, confirming adaptation to a more aerobic metabolism. NMR allowed rapid profiling of 41 intracellular metabolites and revealed clear separation of mitochondrial defects from controls under galactose using partial least squares discriminant analysis. We found changes in classical markers of mitochondrial metabolic dysfunction, as well as unexpected markers of amino acid and choline metabolism. PDH deficient cell lines showed distinct upregulation of glutaminolytic metabolism and accumulation of branched-chain amino acids, while complex I deficient cell lines were characterized by increased levels in choline metabolites under galactose. CONCLUSION: Our results show the relevance of selective culture methods in discriminating normal from metabolic deficient cells. The study indicates that untargeted fingerprinting NMR profiles provide physiological insight on metabolic adaptations and can be used to distinguish cellular metabolic adaptations in PDH and complex I deficient fibroblasts.

Correction to: Selective galactose culture condition reveals distinct metabolic signatures in pyruvate dehydrogenase and complex I deficient human skin fibroblasts.

The original version of this article contained an error in Table 2. The text in the second header line should read "GAL supernatant" and "GAL Medium" instead of "GLC supernatant" and "GLC Medium". The corrected Table 2 is given below. The original article has been corrected.

Argininosuccinate neurotoxicity and prevention by creatine in argininosuccinate lyase deficiency: An in vitro study in rat three-dimensional organotypic brain cell cultures.

The urea cycle disorder (UCD) argininosuccinate lyase (ASL) deficiency, caused by a defective ASL enzyme, exhibits a wide range of phenotypes, from life-threatening neonatal hyperammonemia to asymptomatic patients, with only the biochemical marker argininosuccinic acid (ASA) elevated in body fluids. Remarkably, even without ever suffering from hyperammonemia, patients often develop severe cognitive impairment and seizures. The goal of this study was to understand the effect on the known toxic metabolite ASA and the assumed toxic metabolite guanidinosuccinic acid (GSA) on developing brain cells, and to evaluate the potential role of creatine (Cr) supplementation, as it was described protective for brain cells exposed to ammonia. We used an in vitro model, in which we exposed three-dimensional (3D) organotypic rat brain cell cultures in aggregates to different combinations of the metabolites of interest at two time points (representing two different developmental stages). After harvest and cryopreservation of the cell cultures, the samples were analyzed mainly by metabolite analysis, immunohistochemistry, and western blotting. ASA and GSA were found toxic for astrocytes and neurons. This toxicity could be reverted in vitro by Cr. As well, an antiapoptotic effect of ASA was revealed, which could contribute to the neurotoxicity in ASL deficiency. Further studies in human ASL deficiency will be required to understand the biochemical situation in the brain of affected patients, and to investigate the impact of high or low arginine doses on brain Cr availability. In addition, clinical trials to evaluate the beneficial effect of Cr supplementation in ASL deficiency would be valuable.

Hepatitis C virus infection triggers a tumor-like glutamine metabolism.

Chronic infection with hepatitis C virus (HCV) is one of the main causes of hepatocellular carcinoma. However, the molecular mechanisms linking the infection to cancer development remain poorly understood. Here we used HCV-infected cells and liver biopsies to study how HCV modulates the glutaminolysis pathway, which is known to play an important role in cellular energetics, stress defense, and neoplastic transformation. Transcript levels of glutaminolytic factors were quantified in Huh7.5 cells or primary human hepatocytes infected with the Japanese fulminant hepatitis 1 HCV strain as well as in biopsies of chronic HCV patients. Nutrient deprivation, biochemical analysis, and metabolite quantification were performed with HCV-infected Huh7.5 cells. Furthermore, short hairpin RNA vectors and small molecule inhibitors were used to investigate the dependence of HCV replication on metabolic changes. We show that HCV modulates the transcript levels of key enzymes of glutamine metabolism in vitro and in liver biopsies of chronic HCV patients. Consistently, HCV infection increases glutamine use and dependence. We finally show that inhibiting glutamine metabolism attenuates HCV infection and the oxidative stress associated with HCV infection. CONCLUSION: Our data suggest that HCV establishes glutamine dependence, which is required for viral replication, and, importantly, that glutamine addiction is a hallmark of tumor cells. While HCV induces glutaminolysis to create an environment favorable for viral replication, it predisposes the cell to transformation. Glutaminolytic enzymes may be interesting therapeutic targets for prevention of hepatocarcinogenesis in chronic hepatitis C. (Hepatology 2017;65:789-803).

Metabolic profiling of listeria rhombencephalitis in small ruminants by 1 H high-resolution magic angle spinning NMR spectroscopy.

Listeria rhombencephalitis is caused by infection with Listeria monocytogenes and is associated with a high mortality rate in humans and ruminants. Little is known about the metabolic changes associated with neurolisteriosis in particular and infectious central nervous system (CNS) diseases in general. The purpose of our study was to investigate the metabolic changes associated with listeria rhombencephalitis in small ruminants (goats and sheep) as a model for inflammatory CNS disease by 1 H high-resolution magic angle spinning nuclear magnetic resonance (1 H HR-MAS NMR) spectroscopy of brain biopsies obtained from the brainstem and thalamus. Statistical analysis revealed distinct differences in the metabolic profile of brainstem biopsies, the primary location of listeria rhombencephalitis with moderate or severe inflammatory changes. N-Acetylaspartate (NAA), N-acetylaspartylglutamate, choline, myo-inositol and scyllo-inositol were decreased, and glycine, phosphocholine, taurine and lactate were increased, in the diseased group (n = 13) in comparison with the control group (n = 12). In the thalamus, which showed no or only mild inflammatory changes in the majority of animals, no statistically significant metabolic changes were observed. However, trends for metabolic alterations were partly the same as those found in the brainstem, including NAA, choline and lactate. This may be an indicator of metabolic changes occurring in the early stages of the disease. Therefore, further research with a larger number of animals is needed to evaluate the presence of subtle metabolic changes associated with mild inflammatory changes in the thalamus. In conclusion, 1 H HR-MAS NMR investigation of listeria rhombencephalitis identified brain metabolite changes, offering new insights into the disease pathophysiology.

Longitudinal investigation of the metabolome of 3D aggregating brain cell cultures at different maturation stages by 1H HR-MAS NMR.

The aim of the present study was to establish the developmental profile of metabolic changes of 3D aggregating brain cell cultures by 1H high-resolution magic angle spinning (HR-MAS) NMR spectroscopy. The histotypic 3D brain aggregate, containing all brain cell types, is an excellent model for mechanistic studies including OMICS analysis; however, their metabolic profile has not been yet fully investigated. Chemometric analysis revealed a clear separation of samples from the different maturation time points. Metabolite concentration evolutions could be followed and revealed strong and various metabolic alterations. The strong metabolite evolution emphasizes the brain modeling complexity during maturation, possibly reflecting physiological processes of brain tissue development. The small observed intra- and inter-experimental variabilities show the robustness of the combination of 1H-HR-MAS NMR and 3D brain aggregates, making it useful to investigate mechanisms of toxicity that will ultimately contribute to improve predictive neurotoxicology. Graphical Abstract ᅟ.

Differentiation of prostate cancer lesions with high and with low Gleason score by diffusion-weighted MRI.

OBJECTIVES: To differentiate prostate cancer lesions with high and with low Gleason score by diffusion-weighted-MRI (DW-MRI). METHODS: This prospective study was approved by the responsible ethics committee. DW-MRI of 84 consenting prostate and/or bladder cancer patients scheduled for radical prostatectomy were acquired and used to compute apparent diffusion coefficient (ADC), intravoxel incoherent motion (IVIM: the pure diffusion coefficient D t, the pseudo-diffusion fraction F p and the pseudo-diffusion coefficient D p), and high b value (as acquired and Hessian filtered) parameters within the index lesion. These parameters (separately and combined in a logistic regression model) were used to differentiate lesions depending on whether whole-prostate histopathological analysis after prostatectomy determined a high (≥7) or low (6) Gleason score. RESULTS: Mean ADC and D t differed significantly (p of independent two-sample t test < 0.01) between high- and low-grade lesions. The highest classification accuracy was achieved by the mean ADC (AUC 0.74) and D t (AUC 0.70). A logistic regression model based on mean ADC, mean F p and mean high b value image led to an AUC of 0.74 following leave-one-out cross-validation. CONCLUSIONS: Classification by IVIM parameters was not superior to classification by ADC. DW-MRI parameters correlated with Gleason score but did not provide sufficient information to classify individual patients. KEY POINTS: • Mean ADC and diffusion coefficient differ between high- and low-grade prostatic lesions. • Accuracy of trivariate logistic regression is not superior to using ADC alone. • DW-MRI is not a valid substitute for biopsies in clinical routine yet.

Metabolic Profiling of Cells in Response to Drug Treatment using 1H High-resolution Magic Angle Spinning (HR-MAS) NMR Spectroscopy.

High-resolution magic angle spinning (HR-MAS) is an NMR technique that provides access to well resolved liquid-like 1H NMR spectra of semi-solid samples. Therefore, 1H HR-MAS NMR spectroscopy has become an important tool for the direct analysis of biological samples such as tissues and cells in a mostly non-destructive way. Here, we focus on the application of HR-MAS NMR combined with multivariate statistical methods used for metabolic profiling of cells and in particular for the study of cellular metabolic responses to drug exposure. The principles of HR-MAS and the metabolomic approach are briefly described. As an example, a study on the metabolic response of different cell types towards treatment with a highly cytotoxic hexacationic ruthenium metallaprism as potential anti-cancer drug is presented. Specific metabolites and metabolic pathways are suggested to be associated with the cellular response. The study demonstrates the potential of HR-MAS metabolomics applied to cells for addressing the intracellular processes involved in the treatment with organometallic drugs.

Renal Blood Oxygenation Level-dependent Imaging in Longitudinal Follow-up of Donated and Remaining Kidneys.

Purpose To determine renal oxygenation changes associated with uninephrectomy and transplantation in both native donor kidneys and transplanted kidneys by using blood oxygenation level-dependent (BOLD) MR imaging. Materials and Methods The study protocol was approved by the local ethics committee. Thirteen healthy kidney donors and their corresponding recipients underwent kidney BOLD MR imaging with a 3-T imager. Written informed consent was obtained from each subject. BOLD MR imaging was performed in donors before uninephrectomy and in donors and recipients 8 days, 3 months, and 12 months after transplantation. R2* values, which are inversely related to tissue partial pressure of oxygen, were determined in the cortex and medulla. Longitudinal R2* changes were statistically analyzed by using repeated measures one-way analysis of variance with post hoc pair-wise comparisons. Results R2* values in the remaining kidneys significantly decreased early after uninephrectomy in both the medulla and cortex (P < .003), from 28.9 sec(-1) ± 2.3 to 26.4 sec(-1) ± 2.5 in the medulla and from 18.3 sec(-1) ± 1.5 to 16.3 sec(-1) ± 1.0 in the cortex, indicating increased oxygen content. In donors, R2* remained significantly decreased in both the medulla and cortex at 3 (P < .01) and 12 (P < .01) months. In transplanted kidneys, R2* remained stable during the first year after transplantation, with no significant change. Among donors, cortical R2* was found to be negatively correlated with estimated glomerular filtration rate (R = -0.47, P < .001). Conclusion The results suggest that BOLD MR imaging may potentially be used to monitor renal functional changes in both remaining and corresponding transplanted kidneys. (©) RSNA, 2016.

Diffusion tensor imaging of the human kidney: Does image registration permit scanning without respiratory triggering?

PURPOSE: To investigate if image registration of diffusion tensor imaging (DTI) allows omitting respiratory triggering for both transplanted and native kidneys MATERIALS AND METHODS: Nine kidney transplant recipients and eight healthy volunteers underwent renal DTI on a 3T scanner with and without respiratory triggering. DTI images were registered using a multimodal nonrigid registration algorithm. Apparent diffusion coefficient (ADC), the contribution of perfusion (FP ), and the fractional anisotropy (FA) were determined. Relative root mean square errors (RMSE) of the fitting and the standard deviations of the derived parameters within the regions of interest (SDROI ) were evaluated as quality criteria. RESULTS: Registration significantly reduced RMSE in all DTI-derived parameters of triggered and nontriggered measurements in cortex and medulla of both transplanted and native kidneys (P < 0.05 for all). In addition, SDROI values were lower with registration for all 16 parameters in transplanted kidneys (14 of 16 SDROI values were significantly reduced, P < 0.04) and for 15 of 16 parameters in native kidneys (9 of 16 SDROI values were significantly reduced, P < 0.05). Comparing triggered versus nontriggered DTI in transplanted kidneys revealed no significant difference for RMSE (P > 0.14) and for SDROI (P > 0.13) of all parameters. In contrast, in native kidneys relative RMSE from triggered scans were significantly lower than those from nontriggered scans (P < 0.02), while SDROI was slightly higher in triggered compared to nontriggered measurements in 15 out of 16 comparisons (significantly for two, P < 0.05). CONCLUSION: Registration improves the quality of DTI in native and transplanted kidneys. Diffusion parameters in renal allografts can be measured without respiratory triggering. In native kidneys, respiratory triggering appears advantageous. J. Magn. Reson. Imaging 2016;44:327-334.

Direct determination of phosphate sugars in biological material by (1)H high-resolution magic-angle-spinning NMR spectroscopy.

The study aim was to unambiguously assign nucleotide sugars, mainly UDP-X that are known to be important in glycosylation processes as sugar donors, and glucose-phosphates that are important intermediate metabolites for storage and transfer of energy directly in spectra of intact cells, as well as in skeletal muscle biopsies by (1)H high-resolution magic-angle-spinning (HR-MAS) NMR. The results demonstrate that sugar phosphates can be determined quickly and non-destructively in cells and biopsies by HR-MAS, which may prove valuable considering the importance of phosphate sugars in cell metabolism for nucleic acid synthesis. As proof of principle, an example of phosphate-sugar reaction and degradation kinetics after unfreezing the sample is shown for a cardiac muscle, suggesting the possibility to follow by HR-MAS NMR some metabolic pathways. Graphical abstract Glucose-phosphate sugars (Glc-1P and Glc-6P) detected in muscle by 1H HR-MAS NMR.