13C-Glycogen deposition during pregnancy in the rat following routine meal feeding.

In vivo -NMR was employed to determine the hepatic fate of infused [1-]-d-glucose (200 mg/kg) following ad libitum or routine meal feeding (RMF) regimes imposed during pregnancy. Hepatic glycogen synthesis was measured immediately following the last meal in virgin, 10 and 20 day pregnant rats. No detectable incorporation of -glucose into glycogen was observed in 20 day pregnant and control fed virgin rats. In 20 day pregnant RMF rats, glycogen synthesis from -glucose occurred at a linear rate of 0.10/s (S.D. 0.018/s). By 50 min post-infusion, 13C-glycogen levels were 131% (p<0.01) higher than those seen for the 22 h starved and 2 h refed virgin group. Following 10 days of gestation, glucose incorporation into glycogen was maximal in both the ad libitum and RMF groups. Compared with the 20 day pregnant RMF group, the 10 day pregnant ad libitum and RMF rats produced 146% (p<0.001) and 315% (p<0.001) more incorporation of -glucose into the glycogen macromolecule, respectively. Hepatic glycogen values were similar for both 10 and 20 day pregnant ad libitum rats (65.7+/-4.7 and 58.8+/-4.5 mg/g weight) but lower in the RMF groups by 58% and 48%, respectively. In conclusion, meal feeding regimes in the pregnant rat alter carbohydrate control of the liver producing increased glycogen synthesis initially via direct incorporation of glucose into the macromolecule.

In vivo assessment of metabolic perturbations following alanine and glucagon administration using 31P-MRS in the rat.

This study set out to validate the use of 31P-NMR spectroscopy together with alanine +/- glucagon infusions to assess hepatic gluconeogenic flux in vivo. Bolus infusions of alanine (2.8 or 5.6 mmol/kg) +/- glucagon (250 microg/kg) were used. Maximal changes in the phosphomonoesters (PME), inorganic phosphate (Pi) and beta-NTP occurred 40 mins post infusion. PME increased 13.1% (p < 0.02) and 20.8% (P < 0.01) at 2.8 mmol/kg + glucagon and 5.6 mmol/kg +/- glucagon, respectively. Pi was unaltered at 2.8 mmol/kg but increased by 28.8% (P < 0.01) at 5.6 mmol/kg alanine + glucagon. beta-NTP decreased by 14.4% (P < 0.02) and 16.1% (P < 0.02) at 5.6 mmol/kg -/+ glucagon, respectively. This latter infusion showed slower recovery rates of NTP which remained 12.3% (P < 0.05) lower 70 min post infusion compared with pre-infusion values. 31 P-NMR analysis of liver extracts revealed that PME increases were partly due to 3-phosphoglycerate and corroborated reductions in beta-NTP and gamma-NTP: beta-NDP ratio upon infusion of 5.6 mmol/kg alanine +/- glucagon. Hepatic glucose output from perfused liver experiments showed no difference between alanine concentrations indicating maximal glucose output at the lower concentration. This study has shown that in vivo 31P-NMR in combination with alanine infusion, can be used to determine metabolic changes associated with gluconeogenesis.

Hepatic nucleotide triphosphate regeneration after hypothermic reperfusion in the pig model: an in vitro P-NMR study.

The aim of this study was to assess the possibility of regenerating nucleotide triphosphates (NTP) in the pig liver following its harvest and subsequent storage on ice. This study has used a pig model that allowed human donor liver retrieval techniques and methods of storage to be utilized. In vitro phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy was used to evaluate the changes associated with phosphorus containing metabolites such as NTP, phosphomonoesters (PME), phosphodiesters (PDE), and inorganic phosphate (Po). During 4 hr storage NTP levels were reduced to undetectable levels but its regeneration was possible over a period of 2 hr of oxygenated hypothermic reperfusion. Resynthesized NTP reached values that were only 30% reduced from pre-harvest values. There was a corresponding reduction in Pi over the same period. Glycolytic intermediates, 3-phosphoglycerate and 2,3 diphosphoglycerate, both increased significantly during the period of storage and subsequently declined following hypothermic reperfusion. Cellular damage, indicated by the concentrations of glycerophosphorylcholine (GPC) and glycerophosphorylethanolamine (GPE) was minimal during cold storage. However upon hypothermic reperfusion, concentrations of GPC and GPE reduced, indicating a degree of cellular damage caused by reperfusion. This study has shown for the first time that is possible to regenerate high energy phosphate nucleotides following a period of hypothermic reperfusion in a large, clinically related animal model. This technique warrants investigation clinically to improve the outcome of orthotopic liver transplantation. It also provides a method to study the effects of different preservation fluids and methods of storage and organ reperfusion.

Improved preservation solutions for organ storage: a dynamic study of hepatic metabolism.

BACKGROUND: Organ cold storage times may be extended by modifications to organ preservation solutions. METHODS: Three preservation solutions were investigated for their ability to maintain viable hepatic bioenergetics in stored pig livers: modified University of Wisconsin (mUW); mUW+adenosine (1.34 g/L), and mUW+ iloprost (10(-8)mol/L), a prostacyclin analogue. Using human liver retrieval and storage techniques, pig livers were stored on ice for either 2 or 16 hr, after which phosphorus-31 spectra were collected every 2 min during the period of cold ischemia and hypothermic reperfusion (HtR). During HtR, metabolite concentration changes associated with phosphomonoesters, inorganic phosphate, gamma-nucleotide triphosphate (NTP), and beta-NTP were measured for all solutions. RESULTS: After a 2-hr storage, beta-NTP regeneration in mUW+iloprost produced +57.7% (P<0.01) more beta-NTP, at a faster initial rate of +66.3% (P<0.001), compared with mUW, and mUW+adenosine regenerated +35.6% (P<0.05) more beta-NTP, compared with mUW. Storage for 16 hr did not slow the rates of regeneration, and the total NTP produced during the course of the experiment remained unchanged for the respective preservation solutions. Cessation of HtR invoked a net accumulation of nucleotide diphosphate, indicating differential kinetics of adenine nucleotide hydrolysis. CONCLUSION: This large animal model study suggests significant improvements to human organ preservation solutions using prostacyclin analogues and adenosine with respect to hepatic bioenergetics.

Conflicting MRI signals from gliosis and neuronal vacuolation in prion diseases.

Magnetic resonance imaging (MRI) has given inconsistent results when used as a non-invasive diagnostic tool for Creutzfeldt-Jakob disease (CJD). In order to understand this finding, we studied a hamster model of scrapie by in vivo MRI and histopathology. Vacuolation of neurones/neuropil and gliosis were found to correlate with hypo-intense and hyper-intense changes in the conventional T2-weighted MR images, respectively. These opposing effects were shown to give rise to normal images of a scrapie-affected brain undergoing severe neurodegeneration, and may underlie the variability of previous CJD MRI data.

Progressive suppression of muscle glucose utilization during pregnancy.

Glucose utilization indices (GUIs) were measured in heart and a range of skeletal muscles in conscious, unrestrained, virgin or pregnant rats in the absorptive and post-absorptive phases. A clear effect of pregnancy to diminish muscle GUIs was identified, the magnitude of which was greatest in late gestation in the absorptive phase. Differences in the time courses and magnitudes of the response to pregnancy were observed between individual muscles. The effects of pregnancy are discussed in relation to an increased availability of lipid fuels and to decreased insulin and glucose concentrations.

The role of magnetic resonance spectroscopy in the assessment of kidney viability.

Renal transplant programmes are seriously limited by the continuing shortage of donor organs. Kidneys from marginal and non-heart-beating donors are increasingly being used, but their viability may be compromised. There is currently no rapid yet accurate method for assessing donor organ viability which can be applied within the window of opportunity between harvesting and implantation. Magnetic resonance spectroscopy (MRS) is a non-invasive technique which is being increasingly applied to delineate biochemical changes in vivo. Studies in animal models and humans now suggest that phosphorus-31 MRS may be useful in the non-invasive assessment of isolated donor kidney viability.

Liver transplantation: current and potential applications of magnetic resonance spectroscopy.

Magnetic resonance spectroscopy (MRS) allows the noninvasive measurement of whole organ metabolism due to the presence of the MR-sensitive nucleus phosphorus 31 in adenosine triphosphate (ATP), its precursors, and break-down products. In small animal liver transplant studies it has been used to analyze the metabolic effects of cold and warm ischemia, hypothermic reperfusion, and the relative efficacy of different organ preservation solutions. In recent large animal studies MRS has been developed to provide continuous dynamic information on ATP metabolism during graft reperfusion and the bioenergetic consequences of altering preservation solutions. These basic experimental data need to be critically evaluated in human liver transplantation. Encouraging preliminary data on many possible clinical applications have already been obtained, such as the assessment of human donor liver viability and posttransplant graft function. At present, the cost and technically demanding nature of MRS may restrict its application to research units.

Bioenergetic targeting during organ preservation: (31)P magnetic resonance spectroscopy investigations into the use of fructose to sustain hepatic ATP turnover during cold hypoxia in porcine livers.

During liver preservation, ATP supplies become depleted, leading to loss of cellular homeostatic controls and a cascade of ensuing harmful changes. Anaerobic glycolysis is unable to prolong ATP production for a significant period because of metabolic blockade. Our aim was to promote glycolysis during liver cold hypoxia by supplying fructose as an additional substrate, compared to supplementation with an equivalent concentration of glucose. Porcine livers (two groups; n = 5 in each) were retrieved by clinical harvesting techniques and subjected to two cycles of cold hypoxia and oxygenated hypothermic reperfusion. In the second cycle of reperfusion, the perfusate was supplemented with either 10 mmol/L glucose (Group 1) or 10 mmol/L fructose (Group 2). During reperfusion in both groups, similar levels of ATP were detected by phosphorus magnetic resonance spectroscopy ((31)P MRS). However, during subsequent hypoxia, ATP was detected for much longer periods in the fructose-perfused group. The rate of ATP loss was sevenfold slower during hypoxia in the presence of fructose than in the presence of glucose (ATP consumption of -7.2 x 10(-3)% total (31)P for Group 1 versus -1.0 x 10(-3)% total (31)P for Group 2; P < 0. 001). The changes in ATP were mirrored by differences in other MRS-detectable intermediates; e.g., inorganic phosphate was significantly higher during subsequent hypoxia in Group 1 (45.7 +/- 2.7% total (31)P) than in Group 2 (33.7 +/- 1.1% total (31)P; P < 0. 01). High-resolution MRS of liver tissue extracts demonstrated that fructose was metabolized mainly via fructose 1-phosphate. We conclude that fructose supplied by brief hypothermic perfusion may improve the bioenergetic status of cold hypoxic livers by sustaining anaerobic glycolysis via a point of entry into the pathway that is different from that for glucose.

A longitudinal magnetic resonance imaging (MRI) study of differences in abdominal fat distribution between normal mice, and lean overexpressers of mitochondrial uncoupling protein-3 (UCP-3).

AIM: To characterize evolution and distribution of abdominal adipose fat between 6 and 18 weeks of age in an animal model of energy consumption based on mice overexpressing the mitochondrial uncoupler protein 3 (UCP-3). METHODS: T2-weighted multislice MRI was performed six times during the 12 week study; visceral, subcutaneous and intermuscular fat depots were quantified. RESULTS: The overexpressor (UCP-3tg) mice consistently have less subcutaneous, visceral, interskeletal muscle and total fat throughout the experiment. Mean (standard error) volumes (ml) of the three distinct depots change between week 6 and week 18 as follows: wild type: subcutaneous 1.93 (0.28) to 6.18 (0.47), visceral 2.15 (0.34) to 6.37 (0.64), intermuscular 0.23 (0.04) to 0.53 (0.03); UCP-3tg: subcutaneous 1.47 (0.17) to 4.07 (0.57), visceral 1.18 (0.04) to 3.69 (0.59), intermuscular 0.23 (0.01) to 0.32 (0.04). Although they eat more (4.3 g compared with 3.4 g per day) the UCP-3tg's always weigh less than controls. In wild-type control animals, increases of all fat pools between week 6 and week 18 is highly significant, as it is for subcutaneous, visceral and total pools in the UCP-3tg animals. The UCP-3tg mice, however, show no significant absolute or relative increase in intermuscular fat; UCP-3 is predominantly overexpressed in skeletal muscle. CONCLUSION: MRI provides an excellent approach to comparative studies of fat distribution in animal models of energy expenditure such as the UCP-3tg mouse.

Cerebral proton and phosphorus-31 magnetic resonance spectroscopy in patients with subclinical hepatic encephalopathy.

BACKGROUND/AIMS: In vivo magnetic resonance spectroscopy can be used to study cerebral metabolism non-invasively. We aimed to correlate 1H and 31P magnetic resonance spectral abnormalities in the brains of patients with subclinical hepatic encephalopathy. METHODS: Eighteen patients were studied at 1.5T, with combined 1H and 31P magnetic resonance spectra obtained from multiple voxels in the cerebral cortex and basal ganglia. Peak area ratios of choline, glutamine/glutamate, relative to creatine in the 1H spectra and percentage phosphomonoesters, phosphodiesters and betaNTP signals relative to total 31P signals in the 31P spectra were measured. RESULTS: Six patients did not complete the full examination - 31P results are available from 12 patients only. Relative to creatine, there were reductions in choline and elevations in glutamine/glutamate, varying across the brain with choline significantly reduced in occipital cortex (p<0.05) and glutamine/glutamate most significantly elevated in temporo-parietal cortex (p<0.0001). Percentage phosphomonoester (p<0.05), phosphodiester (p<0.05) and betaNTP (p<0.005) signals were significantly decreased in basal ganglia spectra. No correlation was found between the magnitude of 1H and 31P MRS changes, except between percentage phosphodiester decrease and glutamine/glutamate to creatine increase in occipital cortex. CONCLUSION: The results of this study point to a multifactorial aetiology for this condition.

Rapid reversal of hepatic steatosis, and reduction of muscle triglyceride, by rosiglitazone: MRI/S studies in Zucker fatty rats.

AIM: This study aimed to chart the time course and durability of the effects of rosiglitazone, a potent thiazolidinedione-based peroxisome proliferator-activated receptor gamma agonist, on hepatic steatosis and intramyocellular lipid in an animal model of obesity, the Zucker Fatty (ZF) rat. METHODS AND RESULTS: Rosiglitazone (3 mg/kg/day p.o.) significantly reduced both liver fat content (by 59%; p < 0.05) and size (11.5%; p < 0.05) in male ZF rats that received between 3 days and 1 week of treatment, and these reductions were maintained for at least 12 weeks. Liver fat content measured by magnetic resonance spectroscopy (MRS) correlated closely and positively with plasma insulin levels (reduced by 89% within a week, r = 0.8) and with postmortem histological fat fractional volume (r = 0.89). Similarly, liver volume measured by magnetic resonance imaging (MRI) correlated closely with postmortem wet weight (r = 0.99). MRS also showed, and numbers of lipid vacuoles counted in transmission electron micrographs confirmed, that rosiglitazone significantly reduced the elevated intramyocellular lipid seen in ZF rat skeletal muscle by at least 40% (p < 0.05). CONCLUSIONS: Localized MRS and MRI showed that rosiglitazone reversed the hepatic steatosis, hepatomegaly and intramyocellular lipid, characteristic of the ZF rat, an animal model of obesity.

Evidence for altered hepatic gluconeogenesis in patients with cirrhosis using in vivo 31-phosphorus magnetic resonance spectroscopy.

BACKGROUND AND AIMS: Alterations in gluconeogenesis in the diseased liver can be assessed non-invasively using magnetic resonance spectroscopy by measuring changes in phosphomonoester resonance which contains information regarding several metabolites, including the phosphorylated intermediates of the gluconeogenic pathway. METHODS: 31P magnetic resonance spectroscopy was used to determine changes in phosphomonoesters following bolus infusions of 2.8 mmol/kg L-alanine in five patients with functionally compensated cirrhosis and in five patients with functionally decompensated cirrhosis. RESULTS: Compared with six healthy volunteers, baseline phosphomonoester values were elevated by 35% (p<0.05) in the compensated cirrhosis group and by 57% (p<0.01) in the decompensated cirrhosis group. Following alanine infusion, phosphomonoesters in healthy volunteers increased by 46% from baseline values (p<0.01), in patients with compensated cirrhosis by 27% (p<0.02) but those with decompensated cirrhosis showed no increase from baseline. There was a reduction in the percentage of inorganic phosphate signal in all subjects. CONCLUSIONS: By analysing changes in phosphomonoester and inorganic phosphate resonances it is possible to discern clear metabolic differences between healthy volunteers and patients with cirrhosis of varying severity using magnetic resonance spectroscopy. Those patients with functionally decompensated cirrhosis have higher percentage baseline phosphomonoester values but the absence of phosphomonoester elevation following L-alanine infusion suggests that they are unable to mount a significant metabolic response with a progluconeogenic stimulus.

Non-invasive assessment of ATP regeneration potential of the preserved donor liver. A 31P MRS study in pig liver.

We have developed a quick, non-invasive method for measuring the ability of an isolated preserved liver to regenerate high energy phosphate nucleotides without the need for biopsy. Using 31P MRS we have monitored the hepatic energetics of intact cold preserved pig liver using standard clinical harvesting and storage techniques. Following cold storage for 2 h the livers were hypothermically reperfused with oxygenated modified University of Wisconsin preservation fluid. Prior to reperfusion MRS detectable adenosine diphosphate plus adenosine triphosphate was negligible; however, the spectrum showed intense resonances from phosphomonoesters and inorganic phosphate, as a consequence of adenosine triphosphate hydrolysis during cold preservation. Following a 10-min period of hypothermic reperfusion, regeneration of adenosine triphosphate occurred with a concurrent decline in inorganic phosphate and phosphomonoester, both of which are associated with adenosine triphosphate synthesis. The capacity of the liver to regenerate adenosine triphosphate following a 24-h period of cold storage was reduced by approximately 40% (p < 0.01) of the total amount achieved following the shorter cold storage time. Adenosine triphosphate regeneration rates were biphasic and were decreased upon prolonged storage, with the initial rate being reduced from 40.6 x 10(-2).min-1 (standard deviation (sd) 2.70 x 10(-2).min-1) to 14.8 x 10(-2).min-1 (sd; 2.4 x 10(-2).min-1) and the secondary rate from 1.77 x 10(-2).min-1 (sd; 0.18 x 10(-2).min-1) to 0.84 x 10(-2).min-1 (sd; 0.45 x 10(-2).min-1). MR images of the liver during the period of hypothermic reperfusion were also performed providing an assessment for the degree of hepatic vascular perfusion. This non-invasive, 31P MRS assessment of hepatic energetics in a clinically relevant animal model has great potential for the understanding of graft preservation injury.

Assessment of quantitative artificial neural network analysis in a metabolically dynamic ex vivo 31P NMR pig liver study.

Quantitative artificial neural network analysis for 1550 ex vivo 31P nuclear magnetic resonance spectra from hypothermically reperfused pig livers was assessed. These spectra show wide ranges of metabolite concentrations and have been analyzed using metabolite prior knowledge based lineshape fitting analysis which had proved robust in its biochemical interpretation. This finding provided a good opportunity to assess the performance of artificial neural network analysis in a biochemically complex situation. The results showed high correlations (0.865 < or = R < or = 0.992) between the lineshape fitting and artificial neural network analysis for the metabolite values, and the artificial neural network analysis was able to fully represent the trends in the metabolic fluctuations during the experiments.

Incorporation of metabolite prior knowledge for data analysis: biochemical implications of dynamic 31P NMR ex vivo pig liver studies.

A semi-automated, metabolite prior-knowledge-based, lineshape fitting analysis has been developed to assess the dynamic biochemical changes found in ex vivo 31P NMR pig liver preservation studies. Due to the inherent experimental limitations of the ex vivo study and the complexity of the composite phosphorus resonances, metabolite information obtained in vitro was incorporated into the ex vivo analysis. This approach has allowed complete metabolite analysis (phosphomonoesters, inorganic phosphate, phosphodiesters and nucleotide triphosphates) in over 2000 spectra in a fraction of the time compared with more conventional analysis methods. The developed analysis will enable complete and rapid assessment of the biochemical changes in ongoing cold preservation studies of the pig liver which will result in thousands of ex vivo 31P NMR spectra. It is also envisaged that comparative studies on human donor livers will be carried out, in which this type of analysis would be the method of choice. Moreover, this kind of analysis approach could be advantageous in many complex in vivo NMR spectroscopy applications.

In vivo and in vitro hepatic 31P magnetic resonance spectroscopy and electron microscopy of the cirrhotic liver.

In vivo 31P magnetic resonance spectroscopy (MRS) provides direct biochemical information on hepatic metabolic processes. To assess in vivo changes in hepatic 31P MRS in liver transplant candidates, we studied 31 patients with cirrhosis of varying aetiology; 14 with compensated cirrhosis (Pugh's score < or = 7) and 17 with decompensated cirrhosis (Pugh's score > or = 8). Underlying cellular abnormalities were characterised using in vitro 31P MRS and electron microscopy. In vitro spectra were obtained from liver extracts, freeze-clamped at recipient hepatectomy, from all subjects. Electron microscopy of liver tissue was also performed in 17 cases. Relative to nucleotide triphosphates, elevations in phosphomonoesters and reductions in phosphodiesters were observed in vivo with worsening liver function. In vitro spectra showed elevated phosphoethanolamine and phosphocholine, and reduced glycerophosphorylethanolamine and glycerophosphorylcholine, mirroring the in vivo changes, but no distinction was noted between compensated and decompensated cirrhosis. With electron microscopy, functional decompensation was associated with reduced endoplasmic reticulum in parenchymal liver disease, but elevated levels in biliary cirrhosis. We conclude that in vivo spectral abnormalities in cirrhosis are consistent with alterations in phospholipid metabolism and quantity of endoplasmic reticulum. However, in individual patients the biopsy results do not always mirror in vivo findings.

In vivo and in vitro hepatic phosphorus-31 magnetic resonance spectroscopy and electron microscopy in chronic ductopenic rejection of human liver allografts.

BACKGROUND: In vivo hepatic phosphorus-31 magnetic resonance spectroscopy (MRS) provides non-invasive information about phospholipid metabolism. AIMS: To delineate MRS abnormalities in patients with chronic ductopenic rejection (CDR) and to characterise spectral changes by in vitro MRS and electron microscopy. PATIENTS AND METHODS: Sixteen liver transplant recipients (four with CDR; 12 with good graft function) and 29 controls (23 healthy volunteers; six patients with biliary duct strictures) were studied with in vivo 31P MRS. Peak area ratios of phosphomonoesters (PME) and phosphodiesters (PDE), relative to nucleotide triphosphates (NTP) were measured. In vitro MRS and electron microscopy were performed on biopsy specimens from five patients with CDR, freeze clamped at retransplantation. Phosphoethanolamine (PE), phosphocholine (PC), glycerophosphorylethanolamine (GPE), and glycerophosphorylcholine (GPC) concentrations were measured. RESULTS: The 12 patients with good graft function displayed no spectral abnormalities in vivo; the four patients with CDR showed significantly elevated PME:NTP (p < 0.01) and PDE:NTP ratios (p < 0.005). Patients with biliary strictures had significant differences in PME:NTP (p < 0.01) from patients with CDR, but not in mean PDE:NTP. In vitro spectra from CDR patients showed elevated PE and PC, mirroring the in vivo changes in PME, but reduced GPE and GPC concentrations were observed, at variance with the in vivo PDE findings. On electron microscopy, there was no proliferation in hepatocyte endoplasmic reticulum. CONCLUSIONS: The increase in PME:NTP reflects altered phospholipid metabolism in patients with CDR, while the increase in PDE:NTP may represent a significant contribution from bile phospholipid.

Mice overexpressing human uncoupling protein-3 in skeletal muscle are hyperphagic and lean.

Uncoupling protein-3 (UCP-3) is a recently identified member of the mitochondrial transporter superfamily that is expressed predominantly in skeletal muscle. However, its close relative UCP-1 is expressed exclusively in brown adipose tissue, a tissue whose main function is fat combustion and thermogenesis. Studies on the expression of UCP-3 in animals and humans in different physiological situations support a role for UCP-3 in energy balance and lipid metabolism. However, direct evidence for these roles is lacking. Here we describe the creation of transgenic mice that overexpress human UCP-3 in skeletal muscle. These mice are hyperphagic but weigh less than their wild-type littermates. Magnetic resonance imaging shows a striking reduction in adipose tissue mass. The mice also exhibit lower fasting plasma glucose and insulin levels and an increased glucose clearance rate. This provides evidence that skeletal muscle UCP-3 has the potential to influence metabolic rate and glucose homeostasis in the whole animal.