Mouse genetics reveals Barttin as a genetic modifier of Joubert syndrome.

Genetic and phenotypic heterogeneity and the lack of sufficiently large patient cohorts pose a significant challenge to understanding genetic associations in rare disease. Here we identify Bsnd (alias Barttin) as a genetic modifier of cystic kidney disease in Joubert syndrome, using a Cep290-deficient mouse model to recapitulate the phenotypic variability observed in patients by mixing genetic backgrounds in a controlled manner and performing genome-wide analysis of these mice. Experimental down-regulation of Bsnd in the parental mouse strain phenocopied the severe cystic kidney phenotype. A common polymorphism within human BSND significantly associates with kidney disease severity in a patient cohort with CEP290 mutations. The striking phenotypic modifications we describe are a timely reminder of the value of mouse models and highlight the significant contribution of genetic background. Furthermore, if appropriately managed, this can be exploited as a powerful tool to elucidate mechanisms underlying human disease heterogeneity.

Reproducibility of 19 F-MR ventilation imaging in healthy volunteers.

PURPOSE: To assess the reproducibility of percentage ventilated lung volume (%VV) measurements in healthy volunteers acquired by fluorine (19 F)-MRI of inhaled perfluoropropane, implemented at two research sites. METHODS: In this prospective, ethically approved study, 40 healthy participants were recruited (May 2018-June 2019) to one of two research sites. Participants underwent a single MRI scan session on a 3T scanner, involving periodic inhalation of a 79% perfluoropropane/21% oxygen gas mixture. Each gas inhalation session lasted about 30 seconds, consisting of three deep breaths of gas followed by a breath-hold. Four 19 F-MR ventilation images were acquired per participant, each separated by approximately 6 minutes. The value of %VV was determined by registering separately acquired 1 H images to ventilation images before semi-automated image segmentation, performed independently by two observers. Reproducibility of %VV measurements was assessed by components of variance, intraclass correlation coefficients, coefficients of variation (CoV), and the Dice similarity coefficient. RESULTS: The MRI scans were well tolerated throughout, with no adverse events. There was a high degree of consistency in %VV measurements for each participant (CoVobserver1 = 0.43%; CoVobserver2 = 0.63%), with overall precision of %VV measurements determined to be within ± 1.7% (95% confidence interval). Interobserver agreement in %VV measurements revealed a high mean Dice similarity coefficient (SD) of 0.97 (0.02), with only minor discrepancies between observers. CONCLUSION: We demonstrate good reproducibility of %VV measurements in a group of healthy participants using 19 F-MRI of inhaled perfluoropropane. Our methods have been successfully implemented across two different study sites, supporting the feasibility of performing larger multicenter clinical studies.

Dynamic susceptibility contrast 19 F-MRI of inhaled perfluoropropane: a novel approach to combined pulmonary ventilation and perfusion imaging.

PURPOSE: To assess alveolar perfusion by applying dynamic susceptibility contrast MRI to 19 F-MRI of inhaled perfluoropropane (PFP). We hypothesized that passage of gadolinium-based contrast agent (GBCA) through the pulmonary microvasculature would reduce magnetic susceptibility differences between water and gas components of the lung, elevating the T2∗ of PFP. METHODS: Lung-representative phantoms were constructed of aqueous PFP-filled foams to characterize the impact of aqueous/gas phase magnetic susceptibility differences on PFP T2∗ . Aqueous phase magnetic susceptibility was modulated by addition of different concentrations of GBCA. In vivo studies were performed to measure the impact of intravenously administered GBCA on the T2∗ of inhaled PFP in mice (7.0 Tesla) and in healthy volunteers (3.0 Tesla). RESULTS: Perfluoropropane T2∗ was sensitive to modulation of magnetic susceptibility difference between gas and water components of the lung, both in phantom models and in vivo. Negation of aqueous/gas phase magnetic susceptibility difference was achieved in lung-representative phantoms and in mice, resulting in a ~2 to 3× elevation in PFP T2∗ (3.7 to 8.5 ms and 0.7 to 2.6 ms, respectively). Human studies demonstrated a transient elevation of inhaled PFP T2∗ (1.50 to 1.64 ms) during passage of GBCA bolus through the lung circulation, demonstrating sensitivity to lung perfusion. CONCLUSION: We demonstrate indirect detection of a GBCA in the pulmonary microvasculature via changes to the T2∗ of gas phase PFP within directly adjacent alveoli. This approach holds potential for assessing alveolar perfusion by dynamic susceptibility contrast 19 F-MRI of inhaled PFP, with concurrent assessment of lung ventilation properties, relevant to lung physiology and disease.

Optimized and accelerated 19 F-MRI of inhaled perfluoropropane to assess regional pulmonary ventilation.

PURPOSE: To accelerate 19 F-MR imaging of inhaled perfluoropropane using compressed sensing methods, and to optimize critical scan acquisition parameters for assessment of lung ventilation properties. METHODS: Simulations were performed to determine optimal acquisition parameters for maximal perfluoropropane signal-to-noise ratio (SNR) in human lungs for a spoiled gradient echo sequence. Optimized parameters were subsequently employed for 19 F-MRI of inhaled perfluoropropane in a cohort of 11 healthy participants using a 3.0 T scanner. The impact of 1.8×, 2.4×, and 3.0× undersampling ratios on 19 F-MRI acquisitions was evaluated, using both retrospective and prospective compressed sensing methods. RESULTS: 3D spoiled gradient echo 19 F-MR ventilation images were acquired at 1-cm isotropic resolution within a single breath hold. Mean SNR was 11.7 ± 4.1 for scans acquired within a single breath hold (duration = 18 s). Acquisition of 19 F-MRI scans at shorter scan durations (4.5 s) was also demonstrated as feasible. Application of both retrospective (n = 8) and prospective (n = 3) compressed sensing methods demonstrated that 1.8× acceleration had negligible impact on qualitative image appearance, with no statistically significant change in measured lung ventilated volume. Acceleration factors of 2.4× and 3.0× resulted in increasing differences between fully sampled and undersampled datasets. CONCLUSION: This study demonstrates methods for determining optimal acquisition parameters for 19 F-MRI of inhaled perfluoropropane and shows significant reduction in scan acquisition times (and thus participant breath hold duration) by use of compressed sensing.

Murine Joubert syndrome reveals Hedgehog signaling defects as a potential therapeutic target for nephronophthisis.

Nephronophthisis (NPHP) is the major cause of pediatric renal failure, yet the disease remains poorly understood, partly due to the lack of appropriate animal models. Joubert syndrome (JBTS) is an inherited ciliopathy giving rise to NPHP with cerebellar vermis aplasia and retinal degeneration. Among patients with JBTS and a cerebello-oculo-renal phenotype, mutations in CEP290 (NPHP6) are the most common genetic lesion. We present a Cep290 gene trap mouse model of JBTS that displays the kidney, eye, and brain abnormalities that define the syndrome. Mutant mice present with cystic kidney disease as neonates. Newborn kidneys contain normal amounts of lymphoid enhancer-binding factor 1 (Lef1) and transcription factor 1 (Tcf1) protein, indicating normal function of the Wnt signaling pathway; however, an increase in the protein Gli3 repressor reveals abnormal Hedgehog (Hh) signaling evident in newborn kidneys. Collecting duct cells from mutant mice have abnormal primary cilia and are unable to form spheroid structures in vitro. Treatment of mutant cells with the Hh agonist purmorphamine restored normal spheroid formation. Renal epithelial cells from a JBTS patient with CEP290 mutations showed similar impairments to spheroid formation that could also be partially rescued by exogenous stimulation of Hh signaling. These data implicate abnormal Hh signaling as the cause of NPHP and suggest that Hh agonists may be exploited therapeutically.

Noninvasive in vivo magnetic resonance measures of glutathione synthesis in human and rat liver as an oxidative stress biomarker.

UNLABELLED: Oxidative stress (OS) plays a central role in the progression of liver disease and in damage to liver by toxic xenobiotics. We have developed methods for noninvasive assessment of hepatic OS defenses by measuring flux through the glutathione (GSH) synthesis pathway. (13) C-labeled GSH is endogenously produced and detected by in vivo magnetic resonance after administration of [2-(13) C]-glycine. We report on a successful first-ever human demonstration of this approach as well as preclinical studies demonstrating perturbed GSH metabolism in models of acute and chronic OS. Human studies employed oral administration of [2-(13) C]-glycine and (13) C spectroscopy on a 3T clinical magnetic resonance (MR) imaging scanner and demonstrated detection and quantification of endogenously produced (13) C-GSH after labeled glycine ingestion. Plasma analysis demonstrated that glycine (13) C fractional enrichment achieved steady state during the 6-hour ingestion period. Mean rate of synthesis of hepatic (13) C-labeled GSH was 0.32 ± 0.18 mmole/kg/hour. Preclinical models of acute OS and nonalcoholic steatohepatitis (NASH) comprised CCl4 -treated and high-fat, high-carbohydrate diet-fed Sprague-Dawley rats, respectively, using intravenous administration of [2-(13) C]-glycine and observation of (13) C-label metabolism on a 7T preclinical MR system. Preclinical studies demonstrated a 54% elevation of GSH content and a 31% increase in flux through the GSH synthesis pathway at 12 hours after acute insult caused by CCl4 administration, as well as a 23% decrease in GSH content and evidence of early steatohepatitis in the model of NASH. CONCLUSION: Our data demonstrate in vivo (13) C-labeling and detection of GSH as a biomarker of tissue OS defenses, detecting chronic and acute OS insults. The methods are applicable to clinical research studies of hepatic OS in disease states over time as well as monitoring effects of therapeutic interventions.

Diurnal variation in skeletal muscle and liver glycogen in humans with normal health and Type 2 diabetes.

In health, food carbohydrate is stored as glycogen in muscle and liver, preventing a deleterious rise in osmotically active plasma glucose after eating. Glycogen concentrations increase sequentially after each meal to peak in the evening, and fall to fasting levels thereafter. Skeletal muscle accounts for the larger part of this diurnal buffering capacity with liver also contributing. The effectiveness of this diurnal mechanism has not been previously studied in Type 2 diabetes. We have quantified the changes in muscle and liver glycogen concentration with 13C magnetic resonance spectroscopy at 3.0 T before and after three meals consumed at 4 h intervals. We studied 40 (25 males; 15 females) well-controlled Type 2 diabetes subjects on metformin only (HbA1c (glycated haemoglobin) 6.4±0.07% or 47±0.8 mmol/mol) and 14 (8 males; 6 females) glucose-tolerant controls matched for age, weight and body mass index (BMI). Muscle glycogen concentration increased by 17% after day-long eating in the control group (68.1±4.8 to 79.7±4.2 mmol/l; P=0.006), and this change inversely correlated with homoeostatic model assessment of insulin resistance [HOMA-IR] (r=-0.56; P=0.02). There was no change in muscle glycogen in the Type 2 diabetes group after day-long eating (68.3±2.6 to 67.1±2.0 mmol/mol; P=0.62). Liver glycogen rose similarly in normal control (325.9±25.0 to 388.1±30.3 mmol/l; P=0.005) and Type 2 diabetes groups (296.1±16.0 to 350.5±6.7 mmol/l; P<0.0001). In early Type 2 diabetes, the major physiological mechanism for skeletal muscle postprandial glycogen storage is completely inactive. This is directly related to insulin resistance, although liver glycogen storage is normal.

Human brain 7Li-MRI following low-dose lithium dietary supplementation in healthy participants.

BACKGROUND: Lithium is an effective mood stabiliser, but its mechanism of action is incompletely defined. Even at very low doses, lithium may have neuroprotective effects, but it is not clear if these relate to brain lithium concentration in vivo. We have developed magnetic resonance imaging (7Li-MRI) methods to detect lithium in the brain following supplementation at a very low dose. METHODS: Lithium orotate supplements were taken by nine healthy adult male subjects (5 mg daily) for up to 28 days, providing 2-7 % of the lithium content of a typical therapeutic lithium carbonate dose. One-dimensional 7Li-images were acquired on a 3.0 T MRI scanner. All subjects were scanned on day 14 or 28; seven were scanned on both, one at baseline and one after 7-days washout. RESULTS: 7Li-MR signal amplitude was broadly stable between days 14 and 28. Two subjects had notably higher 7Li-signal intensities (approximately 2-4×) compared to other study participants. LIMITATIONS: Lithium adherence was self-reported by all participants without formal validation. The coarse spatial resolution necessary for detection of low concentrations of 7Li exhibits imperfect spatial separation of signal from adjacent pixels. CONCLUSIONS: 7Li-MRI performed using a clinical 3T scanner demonstrated detection of lithium in the brain at very low concentration, in the range of approximately 10-60 mM. The methods are suited to studies assessing low dose lithium administration in psychiatric and neurodegenerative disorders, and permit the comparison of different lithium salt preparations at a time of emerging interest in the field.

Hepatic cholesteryl ester accumulation in lysosomal acid lipase deficiency: non-invasive identification and treatment monitoring by magnetic resonance.

BACKGROUND & AIMS: Lysosomal Acid Lipase (LAL) deficiency is a rare metabolic storage disease, caused by a marked reduction in activity of LAL, which leads to accumulation of cholesteryl esters (CE) and triglycerides (TG) in lysosomes in many tissues. We used (1)H magnetic resonance (MR) spectroscopy to characterize the abnormalities in hepatic lipid content and composition in patients with LAL deficiency, and in ex vivo liver tissue from a LAL deficiency rat model. Secondly, we used MR spectroscopy to monitor the effects of an enzyme replacement therapy (ERT), sebelipase alfa (a recombinant human lysosomal acid lipase), on hepatic TG and CE content in the preclinical model. METHODS: Human studies employed cohorts of LAL-deficient patients and NAFLD subjects. Rat experimental groups comprised ex vivo liver samples of wild type, NAFLD, LAL-deficient, and LAL-deficient rats receiving 4weeks of sebelipase alfa treatment. Hepatic (1)H MR spectroscopy was performed using 3T (human) and 7T (preclinical) MRI scanners to quantify hepatic cholesterol and triglyceride content. RESULTS: CE accumulation was identified in LAL deficiency in both human and preclinical studies. A significant decrease in hepatic CE was observed in LAL-deficient rats following treatment with sebelipase alfa. CONCLUSIONS: We demonstrate an entirely non-invasive method to identify and quantify the hepatic lipid signature associated with a rare genetic cause of fatty liver. The approach provides a more favorable alternative to repeated biopsy sampling for diagnosis and disease progression / treatment monitoring of patients with LAL deficiency and other disorders characterised by increased free cholesterol and/or cholesteryl esters.

Liver and muscle glycogen repletion using 13C magnetic resonance spectroscopy following ingestion of maltodextrin, galactose, protein and amino acids.

The present study evaluated whether the inclusion of protein (PRO) and amino acids (AA) within a maltodextrin (MD) and galactose (GAL) recovery drink enhanced post-exercise liver and muscle glycogen repletion. A total of seven trained male cyclists completed two trials, separated by 7 d. Each trial involved 2 h of standardised intermittent cycling, followed by 4 h recovery. During recovery, one of two isoenergetic formulations, MD-GAL (0.9 g MD/kg body mass (BM) per h and 0.3 g GAL/kg BM per h) or MD-GAL-PRO+AA (0.5 g MD/kg BM per h, 0.3 g GAL/kg BM per h, 0.4 g whey PRO hydrolysate plus l-leucine and l-phenylalanine/kg BM per h) was ingested at every 30 min. Liver and muscle glycogen were measured after depletion exercise and at the end of recovery using 1H-13C-magnetic resonance spectroscopy. Despite higher postprandial insulin concentations for MD-GAL-PRO+AA compared with MD-GAL (61.3 (se 6.2) v. 29.6 (se 3.0) mU/l, (425.8 (se 43.1) v. 205.6 (se 20.8) pmol/l) P= 0.03), there were no significant differences in post-recovery liver (195.3 (se 2.6) v. 213.8 (se 18.0) mmol/l) or muscle glycogen concentrations (49.7 (se 4.0) v. 51.1 (se 7.9) mmol/l). The rate of muscle glycogen repletion was significantly higher for MD-GAL compared with MD-GAL-PRO+AA (5.8 (se 0.7) v. 3.7 (se 0.6) mmol/l per h, P= 0.04), while there were no significant differences in the rate of liver glycogen repletion (15.0 (se 2.5) v. 13.0 (se 2.7) mmol/l per h). PRO and AA within a MD-GAL recovery drink, compared with an isoenergetic mix of MD-GAL, did not enhance but matched liver and muscle glycogen recovery. This suggests that the increased postprandial insulinaemia only compensated for the lower MD content in the MD-GAL-PRO+AA treatment.

In vivo MR studies of glycine and glutathione metabolism in a rat mammary tumor.

The metabolism of glycine into glutathione was monitored noninvasively in vivo in intact rat mammary adenocarcinomas (R3230Ac) by MRI and MRS. Metabolism was tracked by following the isotope label from intravenously infused [2-(13)C]-glycine into the glycinyl residue of glutathione. Signals from [2-(13)C]-glycine and γ-glutamylcysteinyl-[2-(13)C]-glycine ((13)C-glutathione) were detected by nonlocalized (13)C spectroscopy, as these resonances are distinct from background signals. In addition, using spectroscopic imaging methods, heterogeneity in the in vivo tumor distribution of glutathione was observed. In vivo spectroscopy also detected isotope incorporation from [2-(13)C]-glycine into both the 2- and 3-carbons of serine. Analyses of tumor tissue extracts showed single- and multiple-label incorporation from [2-(13)C]-glycine into serine from metabolism through the serine hydroxymethyltransferase and glycine cleavage system pathways. Mass spectrometric analysis of extracts also showed that isotope-labeled serine is further metabolized via the trans-sulfuration pathway, as (13)C isotope labels appear in both the glycinyl and cysteinyl residues of glutathione. Our studies demonstrate the use of MRI and MRS for the monitoring of tumor metabolic processes central to oxidative stress defense.

Effects of raising muscle glycogen synthesis rate on skeletal muscle ATP turnover rate in type 2 diabetes.

Mitochondrial dysfunction has been implicated in the pathogenesis of type 2 diabetes. We hypothesized that any impairment in insulin-stimulated muscle ATP production could merely reflect the lower rates of muscle glucose uptake and glycogen synthesis, rather than cause it. If this is correct, muscle ATP turnover rates in type 2 diabetes could be increased if glycogen synthesis rates were normalized by the mass-action effect of hyperglycemia. Isoglycemic- and hyperglycemic-hyperinsulinemic clamps were performed on type 2 diabetic subjects and matched controls, with muscle ATP turnover and glycogen synthesis rates measured using (31)P- and (13)C-magnetic resonance spectroscopy, respectively. In diabetic subjects, hyperglycemia increased muscle glycogen synthesis rates to the level observed in controls at isoglycemia [from 19 ± 9 to 41 ± 12 µmol·l(-1)·min(-1) (P = 0.012) vs. 40 ± 7 µmol·l(-1)·min(-1) in controls]. This was accompanied by a modest increase in muscle ATP turnover rates (7.1 ± 0.5 vs. 8.6 ± 0.7 µmol·l(-1)·min(-1), P = 0.04). In controls, hyperglycemia brought about a 2.5-fold increase in glycogen synthesis rates (100 ± 24 vs. 40 ± 7 µmol·l(-1)·min(-1), P = 0.028) and a 23% increase in ATP turnover rates (8.1 ± 0.9 vs. 10.0 ± 0.9 µmol·l(-1)·min(-1), P = 0.025) from basal state. Muscle ATP turnover rates correlated positively with glycogen synthesis rates (r(s) = 0.46, P = 0.005). Changing the rate of muscle glucose metabolism in type 2 diabetic subjects alters demand for ATP synthesis at rest. In type 2 diabetes, skeletal muscle ATP turnover rates reflect the rate of glucose uptake and glycogen synthesis, rather than any primary mitochondrial defect.

Quantitative lithium magnetic resonance spectroscopy in the normal human brain on a 3 T clinical scanner.

Lithium (Li) is a core for many neuropsychiatric conditions. The safe serum range of Li treatment is narrow, and regular monitoring by blood test is required, although serum levels are thought to be a poor indicator of Li concentration in the brain itself. Brain Li concentration can be measured by magnetic resonance spectroscopy. However, little data exist in the healthy human brain, and there are no studies of the relaxation properties of brain (7)Li at 3 T. Here, 11 healthy male subjects were prescribed Li over a period of 11 days. In seven subjects, the in vivo T(1) of (7)Li was measured to be 2.1 ± 0.7 s. In the remaining subjects, spectroscopic imaging (1D) yielded a mean brain (7)Li concentration of 0.71 ± 0.1 mM, with no significant difference between gray and white matter. Mean serum concentration was 0.9 ± 0.16 mM, giving a mean brain/serum ratio of 0.78 ± 0.26.

Inhibition of lipolysis in Type 2 diabetes normalizes glucose disposal without change in muscle glycogen synthesis rates.

Suppression of lipolysis by acipimox is known to improve insulin-stimulated glucose disposal, and this is an important phenomenon. The mechanism has been assumed to be an enhancement of glucose storage as glycogen, but no direct measurement has tested this concept or its possible relationship to the reported impairment in insulin-stimulated muscle ATP production. Isoglycaemic-hyperinsulinaemic clamps with [13C]glucose infusion were performed on Type 2 diabetic subjects and matched controls with measurement of glycogen synthesis by 13C MRS (magnetic resonance spectroscopy) of muscle. 31P saturation transfer MRS was used to quantify muscle ATP turnover rates. Glucose disposal rates were restored to near normal in diabetic subjects after acipimox (6.2 ± 0.8 compared with 4.8 ± 0.6 mg·kgffm⁻¹·min⁻¹; P<0.01; control 6.6 ± 0.5 mg·kgffm⁻¹·min⁻¹; where ffm, is fat-free mass). The increment in muscle glycogen concentration was 2-fold higher in controls compared with the diabetic group, and acipimox administration to the diabetic group did not increase this (2.0 ± 0.8 compared with 1.9 ± 1.1 mmol/l; P<0.05; control, 4.0 ± 0.8 mmol/l). ATP turnover rates did not increase during insulin stimulation in any group, but a modest decrease in the diabetes group was prevented by lowering plasma NEFAs (non-esterified fatty acids; 8.4 ± 0.7 compared with 7.1 ± 0.5 µmol·g⁻¹·min⁻¹; P<0.05; controls 8.6 ± 0.8 µmol·g⁻¹·min⁻¹). Suppression of lipolysis increases whole-body glucose uptake with no increase in the rate of glucose storage as glycogen but with increase in whole-body glucose oxidation rate. ATP turnover rate in muscle exhibits no relationship to the acute metabolic effect of insulin.

Non-invasive monitoring of L-2-oxothiazolidine-4-carboxylate metabolism in the rat brain by in vivo 13C magnetic resonance spectroscopy.

The cysteine precursor L-2-oxothiazolidine-4-carboxylate (OTZ, procysteine) can raise cysteine concentration, and thus glutathione levels, in some tissues. OTZ has therefore been proposed as a prodrug for combating oxidative stress. We have synthesized stable isotope labeled OTZ (i.e. L-2-oxo-[5-(13)C]-thiazolidine-4-carboxylate, (13)C-OTZ) and tracked its uptake and metabolism in vivo in rat brain by (13)C magnetic resonance spectroscopy. Although uptake and clearance of (13)C-OTZ was detectable in rat brain following a bolus dose by in vivo spectroscopy, no incorporation of isotope label into brain glutathione was detectable. Continuous infusion of (13)C-OTZ over 20 h, however, resulted in (13)C-label incorporation into glutathione, taurine, hypotaurine and lactate at levels sufficient for detection by in vivo magnetic resonance spectroscopy. Examination of brain tissue extracts by mass spectrometry confirmed only low levels of isotope incorporation into glutathione in rats treated with a bolus dose and much higher levels after 20 h of continuous infusion. In contrast to some previous studies, bolus administration of OTZ did not alter brain glutathione levels. Even a continuous infusion of OTZ over 20 h failed to raise brain glutathione levels. These studies demonstrate the utility of in vivo magnetic resonance for non-invasive monitoring of antioxidant uptake and metabolism in intact brain. These types of experiments can be used to evaluate the efficacy of various interventions for maintenance of brain glutathione.

Measuring the acute effect of insulin infusion on ATP turnover rate in human skeletal muscle using phosphorus-31 magnetic resonance saturation transfer spectroscopy.

Mitochondrial dysfunction has been proposed to underlie the insulin resistance of type 2 diabetes. However, the relative time course of insulin action in stimulating ATP turnover rate and glucose uptake in skeletal muscle has not been examined. These two parameters were measured in young healthy subjects using the (31)P MRS saturation transfer method in conjunction with the euglycaemic hyperinsulinaemic clamp technique respectively. Glucose infusion rate rose rapidly from 0 to 2.90 ± 0.11 mg/kg(ffm)/min during the first 10 min of insulin infusion and further to 6.17 ± 0.57 mg/kg(ffm)/min between 15 and 45 min. In contrast, baseline ATP turnover rate was 9.0 ± 0.4 µmol/g/min of muscle and did not change during the first 45 min of insulin infusion. Between 50 and 80 minutes ATP turnover rate increased by 8% and remained steady to 150 minutes (9.7 ± 0.5 µmol/g/min of muscle, p = 0.03 vs baseline). The in vivo time course of insulin stimulation of skeletal muscle ATP turnover rate is not consistent with a rate limiting effect upon the initiation of insulin-stimulated glycogen synthesis.

Postmortem interval alters the water relaxation and diffusion properties of rat nervous tissue--implications for MRI studies of human autopsy samples.

High-resolution imaging of human autopsy tissues may improve our understanding of in vivo MRI findings, but interpretation is complicated because samples are obtained by immersion fixation following a postmortem interval (PMI). This study tested the hypotheses that immersion fixation and PMI's from 0-24 h would alter the water relaxation and diffusion properties in rat cortical slice and spinal cord models of human nervous tissue. Diffusion data collected from rat cortical slices at multiple diffusion times (10-60 ms) and b-values (7-15,000 s/mm(2)) were analyzed using a two-compartment model with exchange. Rat spinal cords were characterized with standard diffusion tensor imaging (21 directions, b=1250 s/mm(2)). Switching from perfusion- to immersion-fixation at 0 h PMI altered most MRI properties of rat cortical slices and spinal cords, including a 22% decrease in fractional anisotropy (P<0.001). After 4 h PMI, cortical slice T(1) and T(2) increased 22% and 65% respectively (P<0.001), transmembrane water exchange decreased 23% (P<0.001) and intracellular proton fraction increased 25% (P=0.002). After 6 h PMI, spinal cord white matter fractional anisotropy had decreased 38% (P<0.001). MRI property changes were observed for PMIs up to 24 h. The MRI changes correlated with protease activity and histopathological signs of autolysis. Thus, immersion fixation and/or even short PMIs (4-6 h) altered the MRI properties of rat nervous tissue. This suggests comparisons between in vivo clinical MRI and MRI data from human autopsy tissues should be interpreted with caution.

Aldehyde fixative solutions alter the water relaxation and diffusion properties of nervous tissue.

Chemically-fixed nervous tissues are well-suited for high-resolution, time-intensive MRI acquisitions without motion artifacts, such as those required for brain atlas projects, but the aldehyde fixatives used may significantly alter tissue MRI properties. To test this hypothesis, this study characterized the impact of common aldehyde fixatives on the MRI properties of a rat brain slice model. Rat cortical slices immersion-fixed in 4% formaldehyde demonstrated 21% and 81% reductions in tissue T(1) and T(2), respectively (P < 0.001). The T(2) reduction was reversed by washing slices with phosphate-buffered saline (PBS) for 12 h to remove free formaldehyde solution. Diffusion MRI of cortical slices analyzed with a two-compartment analytical model of water diffusion demonstrated 88% and 30% increases in extracellular apparent diffusion coefficient (ADC(EX)) and apparent restriction size, respectively, when slices were chemically-fixed with 4% formaldehyde (P

Noninvasive in vivo detection of glutathione metabolism in tumors.

Magnetic resonance spectroscopic imaging has been used to follow glutathione metabolism and evaluate glutathione heterogeneity in intact tumor tissue. Stable isotope-labeled glutathione was detected in s.c. implanted fibrosarcoma tumors in anesthetized rats following infusion of [2-13C]glycine. Using 1H-decoupled 13C magnetic resonance spectroscopy, the appearance of [2-13C]glycine at 42.4 ppm and the subsequent incorporation of this isotope label into the glycyl residue of glutathione at 44.2 ppm can be detected. The identity and relative concentrations of labeled metabolites observed in the in vivo spectrum were confirmed in studies of tissue extracts. The high level of isotopic enrichment and the concentration of glutathione in tumor tissue allow for collection of spatially localized spectra using 13C chemical shift imaging methods. These data provide the first direct images of glutathione in intact tumor tissue and show metabolic heterogeneity. This method may lead to the ability to monitor changes in tumor tissue redox state that may ultimately affect diagnosis, monitoring, and treatment.

Observation of anomalous diffusion in excised tissue by characterizing the diffusion-time dependence of the MR signal.

This report introduces a novel method to characterize the diffusion-time dependence of the diffusion-weighted magnetic resonance (MR) signal in biological tissues. The approach utilizes the theory of diffusion in disordered media where two parameters, the random walk dimension and the spectral dimension, describe the evolution of the average propagators obtained from q-space MR experiments. These parameters were estimated, using several schemes, on diffusion MR spectroscopy data obtained from human red blood cell ghosts and nervous tissue autopsy samples. The experiments demonstrated that water diffusion in human tissue is anomalous, where the mean-square displacements vary slower than linearly with diffusion time. These observations are consistent with a fractal microstructure for human tissues. Differences observed between healthy human nervous tissue and glioblastoma samples suggest that the proposed methodology may provide a novel, clinically useful form of diffusion MR contrast.