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Polyphenols, a complex group of secondary plant metabolites, including flavonoids and phenolic acids, have been studied in depth for their health-related benefits. The activity of polyphenols may, however, be hampered when consumed together with protein-rich food products, due to the interaction between polyphenols and proteins. To that end we have tested the bioavailability of representatives of a range of polyphenol classes when consumed for five days in different beverage matrices. In a placebo-controlled, randomized, cross-over study, 35 healthy males received either six placebo gelatine capsules consumed with 200 mL of water, six capsules with 800 mg polyphenols derived from red wine and grape extracts, or the same dose of polyphenols incorporated into 200 mL of either pasteurized dairy drink, soy drink (both containing 3.4% proteins) or fruit-flavoured protein-free drink . At the end of the intervention urine and blood was collected and analysed for a broad range of phenolic compounds using Gas Chromatography-Mass Spectrometry (GC-MS), Liquid Chromatography-Multiple Reaction Monitoring-Mass Spectrometry (LC-MRM-MS), and Nuclear Magnetic Resonance (NMR) spectroscopy techniques. The plasma and urine concentrations of the polyphenols identified increased with all formats, including the protein-rich beverages. Compared to capsule ingestion, consumption of polyphenol-rich beverages containing either dairy, soy or no proteins had minor to no effect on the bioavailability and excretion of phenolic compounds in plasma (118% ± 9%) and urine (98% ± 2%). We conclude that intake of polyphenols incorporated in protein-rich drinks does not have a major impact on the bioavailability of a range of different polyphenols and phenolic metabolites., Competing Interests: At time of design, execution, and data interpretation of this study all authors were full time employees of Unilever. Unilever markets polyphenol-rich products, mainly tea.

Scope: Consumption of a low-fat spread enriched with plant sterols (PS) and different low doses (<2 g/day) of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil reduces serum triglycerides (TGs) and low-density lipoprotein-cholesterol (LDL-Chol) and thus beneficially affects two blood lipid risk factors. Yet, their combined effects on TG and Chol in various lipoprotein subclasses have been investigated to a limited extent., Methods and Results: In a randomized, double-blind, placebo-controlled, parallel study, we determined TG and Chol in 13 LP subclasses in fasting serum of 282 hypercholesterolemic subjects, who consumed either a placebo spread or one of the four spreads containing PS (2.5 g/day) and EPA+DHA (0.0, 0.9, 1.3, and 1.8 g/day) for 4 weeks. After PS treatment, total LDL-Chol was reduced, which was not further changed by EPA+DHA. No shift in the LDL-Chol particle distribution was observed. The addition of EPA+DHA to PS dose-dependently reduced VLDL-Chol and VLDL-TG mainly in larger particles. Furthermore, the two highest doses of EPA+DHA increased Chol and TG in the larger HDL particles, while these concentrations were decreased in the smallest HDL particles., Conclusion: The consumption of a low-fat spread enriched with both PS and EPA+DHA induced shifts in the lipoprotein distribution that may provide additional cardiovascular benefits over PS consumption alone., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Gut microbial catabolites of black tea polyphenols (BTPs) have been proposed to exert beneficial cardiovascular bioactivity. This hypothesis is difficult to verify because the conjugation patterns and pharmacokinetics of these catabolites are largely unknown. The objective of our study was to identify, quantify, and assess the pharmacokinetics of conjugated BTP metabolites in plasma of healthy humans by means of an a priori untargeted LC-MS-based metabolomics approach. In a randomized, open, placebo-controlled, crossover study, 12 healthy men consumed a single bolus of black tea extract (BTE) or a placebo. The relative and, in several cases, absolute concentrations of a wide range of metabolites were determined using U(H)PLC-LTQ-Orbitrap-FTMS. Following BTE consumption, a kinetic response in plasma was observed for 59 BTP metabolites, 11 of these in a quantitative manner. Conjugated and unconjugated catechins appeared in plasma without delay, at 2-4 h, followed by a range of microbial catabolites. Interindividual variation in response was greater for gut microbial catabolites than for directly absorbed BTPs. The rapid and sustained circulation of conjugated catabolites suggests that these compounds may be particularly relevant to proposed health benefits of BTE. Their presence and effects may depend on individual variation in catabolic capacity of the gut microbiota.

A systematic approach is described for building validated PLS models that predict cholesterol and triglyceride concentrations in lipoprotein subclasses in fasting serum from a normolipidemic, healthy population. The PLS models were built on diffusion-edited (1)H NMR spectra and calibrated on HPLC-derived lipoprotein subclasses. The PLS models were validated using an independent test set. In addition to total VLDL, LDL, and HDL lipoproteins, statistically significant PLS models were obtained for 13 subclasses, including 5 VLDLs (particle size 64-31.3 nm), 4 LDLs (particle size 28.6-20.7 nm) and 4 HDLs (particle size 13.5-9.8 nm). The best models were obtained for triglycerides in VLDL (0.82 < Q(2) <0.92) and HDL (0.69 < Q(2) <0.79) subclasses and for cholesterol in HDL subclasses (0.68 < Q(2) <0.96). Larger variations in the model performance were observed for triglycerides in LDL subclasses and cholesterol in VLDL and LDL subclasses. The potential of the NMR-PLS model was assessed by comparing the LPD of 52 subjects before and after a 4-week treatment with dietary supplements that were hypothesized to change blood lipids. The supplements induced significant (p < 0.001) changes on multiple subclasses, all of which clearly exceeded the prediction errors.

Background: Resilience or the ability of our body to cope with daily-life challenges has been proposed as a new definition of health, with restoration of homeostasis as target resultant of various physiological stress responses. Challenge models may thus be a sensitive measure to study the body's health. The objective of this study was to select a dietary challenge model for the assessment of inflammatory resilience. Meals are a challenge to metabolic homeostasis and are suggested to affect inflammatory pathways, yet data in literature are limited and inconsistent., Method: The kinetic responses of three different dietary challenges and a water control challenge were assessed on various metabolic and inflammatory markers in 14 healthy males and females using a full cross-over study design. The dietary challenges included glucose (75 g glucose in 300 ml water), lipids (200 ml whipping cream) and a mix of glucose and lipids (same amounts as above), respectively. Blood samples were collected at baseline and at 0.5, 1, 2, 4, 6, 8 and 10 h after consumption of the treatment products. Inflammation (IFNγ, IL-1β, IL-6, IL-8, IL-10, IL-12p70, TNF-α CRP, ICAM-1, VCAM-1, SAA, E-selectin, P-selectin, thrombomodulin, leukocytes, neutrophils, lymphocytes) and clinical (e.g. glucose, insulin, triglycerides) markers as well as gene expression in blood cells and plasma oxylipin profiles were measured., Results: All three dietary challenges induced changes related to metabolic control such as increases in glucose and insulin after the glucose challenge and increases in triglycerides after the lipid challenge. In addition, differences between the challenges were observed for precursor oxylipins and some downstream metabolites including DiHETrE's and HODE's. However, none of the dietary challenges induced an acute inflammatory response, except for a modest increase in circulating leukocyte numbers after the glucose and mix challenges. Furthermore, subtle, yet statistically significant increases in vascular inflammatory markers (sICAM-1 and sVCAM-1) were found after the mix challenge, when compared to the water control challenge., Conclusions: This study shows that dietary glucose and lipid challenges did not induce a strong acute inflammatory response in healthy subjects, as quantified by an accurate and broad panel of parameters.

The colonic microbial degradation of a polyphenol-rich black tea extract (BTE) and red wine/grape juice extract (RWGE) was compared in a five-stage in vitro gastrointestinal model (TWINSHIME). Microbial metabolism of BTE and RWGE polyphenols in the TWINSHIME was studied subsequently in single- and continuous-dose experiments. A combination of liquid or gas chromatography with mass spectrometry (LC-MS or GC-MS) and NMR-based metabolic profiling was used to measure selected parent polyphenols, their microbial degradation into phenolic acids, and the production of short-chain fatty acids (SCFAs) in different colon compartments. Acetate production was increased by continuous feeding of BTE but not RWGE. During RWGE feeding, gallic acid and 4-hydroxyphenylpropionic acid remained elevated throughout the colon, while during BTE feeding, they were consumed in the distal colon, while 3-phenylpropionic acid was strongly produced. Gut microbial production of phenolics and SCFAs is dependent on colon location and polyphenol source, which may influence potential health benefits.

Plant sterols (PS) are well known to reduce serum levels of total cholesterol and LDL-cholesterol. Lipidomics potentially provides detailed information on a wide range of individual serum lipid metabolites, which may further add to our understanding of the biological effects of PS. In this study, lipidomics analysis was applied to serum samples from a placebo-controlled, parallel human intervention study (n = 97) of 4-week consumption of two PS-enriched, yoghurt drinks differing in fat content (based on 0.1% vs. 1.5% dairy fat). A comprehensive data analysis strategy was developed and implemented to assess and compare effects of two different PS-treatments and placebo treatment. The combination of univariate and multivariate data analysis approaches allowed to show significant effects of PS intake on the serum lipidome, and helped to distinguish them from fat content and non-specific effects. The PS-enriched 0.1% dairy fat yoghurt drink had a stronger impact on the lipidome than the 1.5% dairy fat yoghurt drink, despite similar LDL-cholesterol lowering effects. The PS-enriched 0.1% dairy fat yoghurt drink reduced levels of several sphingomyelins which correlated well with the reduction in LDL-cholesterol and can be explained by co-localization of sphingomyelins and cholesterol on the surface of LDL lipoprotein. Statistically significant reductions in serum levels of two lysophosphatidylcholines (LPC(16:1), LPC(20:1)) and cholesteryl arachidonate may suggest reduced inflammation and atherogenic potential. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0384-2) contains supplementary material, which is available to authorized users.

Red wine and grape polyphenols are considered to promote cardiovascular health and are involved in multiple biological functions. Their overall impact on the human metabolome is not known. Therefore, exogenous and endogenous metabolic effects were determined in fasting plasma and 24 h urine from healthy male adults consuming a mix of red wine and grape juice extracts (WGM) for 4 days in a placebo-controlled, crossover study. Syringic acid, 3-hydroxyhippuric acid, pyrogallol, 3-hydroxyphenylacetic acid, and 3-hydroxyphenylpropionic acid were confirmed as the strongest urinary markers of WGM intake. Overall, WGM had a mild impact on the endogenous metabolism. Most noticeable were changes in several amino acids deriving from tyrosine and tryptophan. Reductions in the microbial metabolites p-cresol sulfate and 3-indoxylsulfuric acid and increases in indole-3-lactic acid and nicotinic acid were observed in urine. In plasma, tyrosine was reduced. The results suggest that short-term intake of WGM altered microbial protein fermentation and/or amino acid metabolism.

The metabolic impact of polyphenol-rich red wine and grape juice consumption in humans was studied using a metabolomics approach. Fifty-eight men and women participated in a placebo-controlled, double-crossover study in which they consumed during a period of 4 wk, either a polyphenol-rich 2:1 dry mix of red wine and red grape juice extracts (MIX) or only a grape juice extract (GJX). Twenty-four-hour urine samples were collected after each intervention. (1)H NMR spectroscopy was applied for global metabolite profiling, while GC-MS was used for focused profiling of urinary phenolic acids. Urine metabolic profiles after intake of both polyphenol-rich extracts were significantly differentiated from placebo using multilevel partial least squares discriminant analysis. A significant 35% increase in hippuric acid excretion (p<0.001) in urine was measured after the MIX consumption as) or only a red grape juice dry extract (GJX). 24-h urine samples were collected after each intervention. 1H-NMR spectroscopy was applied for global metabolite profiling, while gas chromatography-mass spectrometry (GC-MS) was used for focused profiling of urinary phenolic acids. Urine metabolic profiles after intake of both polyphenol-rich extracts were significantly differentiated from placebo using multilevel partial least squares discriminant analysis (ML-PLS-DA). A significant 35% increase in hippuric acid excretion (p<0.001) in urine was measured after the MIX consumption compared with placebo, whereas no change was found after GJX consumption. GC-MS-based metabolomics of urine allowed identification of 18 different phenolic acids, which were significantly elevated following intake of either extract. Syringic acid, 3- and 4-hydroxyhippuric acid and 4-hydroxymandelic acid were the strongest urinary markers for both extracts. MIX and GJX consumption had a slightly different effect on the excreted phenolic acid profile and on endogenous metabolite excretion, possibly reflecting their different polyphenol composition.

An integration of metabolomics and pharmacokinetics (or nutrikinetics) is introduced as a concept to describe a human study population with different metabolic phenotypes following a nutritional intervention. The approach facilitates an unbiased analysis of the time-response of body fluid metabolites from crossover designed intervention trials without prior knowledge of the underlying metabolic pathways. The method is explained for the case of a human intervention study in which the nutrikinetic analysis of polyphenol-rich black tea consumption was performed in urine over a period of 48 h. First, multilevel PLS-DA analysis was applied to the urinary 1H NMR profiles to select the most differentiating biomarkers between the verum and placebo samples. Then, a one-compartment nutrikinetic model with first-order excretion, a lag time, and a baseline function was fitted to the time courses of these selected biomarkers. The nutrikinetic model used here fully exploits the crossover structure in the data by fitting the data from both the treatment period and the placebo period simultaneously. To demonstrate the procedure, a selected set of urinary biomarkers was used in the model fitting. These metabolites include hippuric acid, 4-hydroxyhippuric acid and 1,3-dihydroxyphenyl-2-O-sulfate and derived from microbial fermentation of polyphenols in the gut. Variations in urinary excretion between- and within the subjects were observed, and used to provide a phenotypic description of the test population.

A new method is introduced for the analysis of 'omics' data derived from crossover designed drug or nutritional intervention studies. The method aims at finding systematic variations in metabolic profiles after a drug or nutritional challenge and takes advantage of the crossover design in the data. The method, which can be considered as a multivariate extension of a paired t test, generates different multivariate submodels for the between- and the within-subject variation in the data. A major advantage of this variation splitting is that each submodel can be analyzed separately without being confounded with the other variation sources. The power of the multilevel approach is demonstrated in a human nutritional intervention study which used NMR-based metabolomics to assess the metabolic impact of grape/wine extract consumption. The variations in the urine metabolic profiles are studied between and within the human subjects using the multilevel analysis. After variation splitting, multilevel PCA is used to investigate the experimental and biological differences between the subjects, whereas a multilevel PLS-DA model is used to reveal the net treatment effect within the subjects. The observed treatment effect is validated with cross model validation and permutations. It is shown that the statistical significance of the multilevel classification model ( p << 0.0002) is a major improvement compared to a ordinary PLS-DA model ( p = 0.058) without variation splitting. Finally, rank products are used to determine which NMR signals are most important in the multilevel classification model.

Flavonoids, a subclass of polyphenols, are major constituents of many plant-based foods and beverages, including tea, wine and chocolate. Epidemiological studies have shown that a flavonoid-rich diet is associated with reduced risk of cardiovascular diseases. The majority of the flavonoids survive intact until they reach the colon where they are then extensively metabolized into smaller fragments. Here, we describe the development of GC-MS-based methods for the profiling of phenolic microbial fermentation products in urine, plasma, and fecal water. Furthermore, the methods are applicable for profiling products obtained from in vitro batch culture fermentation models. The methods incorporate enzymatic deconjugation, liquid-liquid extraction, derivatization, and subsequent analysis by GC-MS. At the level of individual compounds, the methods gave recoveries better than 80% with inter-day precision being better than 20%, depending on the matrix. Limits of detection were below 0.1 microg/ml for most phenolic acids. The newly developed methods were successfully applied to samples from human and in-vitro intervention trials, studying the metabolic impact of flavonoid intake. In conclusion, the methods presented are robust and generally applicable to diverse biological fluids. Its profiling character is useful to investigate on a large scale the gut microbiome-mediated bioavailability of flavonoids.

Intermittent claudication has proved to be a good in vivo model for ischaemia-reperfusion. For assessment of ischaemia-reperfusion damage, the known biochemical markers all have disadvantages with respect to sensitivity and interference with other physiological events. In this work, we studied the metabolic effects of ischaemia-reperfusion in patients with intermittent claudication, and the effects of vitamin C and E intervention, using both traditional biochemical measurements and 1H-NMR-based metabonomics on urine and plasma. The 1H-NMR spectra were subjected to multivariate modelling using principal components discriminant analysis, and the observed clusters were validated using joint deployment of univariate analysis of variance and Tukey-Kramer honestly significant difference (HSD) testing. The study involved 14 patients with intermittent claudication and three healthy volunteers, who were monitored during a walking test, before and after a vitamin C/E intervention, and after a washout period. The effect of exercise was only observable for a limited number of biochemical markers, whereas 1H NMR revealed an effect in line with anaerobic ATP production via glycolysis in exercising (ischaemic) muscle of the claudicants. Thus, the beneficial effect of vitamins C and E in claudicants was more pronounced when observed by metabonomics than by traditional biochemical markers. The main effect was more rapid recovery from exercise to resting state metabolism. Furthermore, after intervention, claudicants tended to have lower concentrations of lactate and glucose and several other citric acid cycle metabolites, whereas acetoacetate was increased. The observed metabolic changes in the plasma suggest that intake of vitamin C/E leads to increased muscle oxidative metabolism., (Copyright (c) 2008 John Wiley & Sons, Ltd.)

Current research increasingly recognizes the human gut microbiome as a metabolically versatile biological 'digester' that plays an essential role in regulating the host metabolome. Gut microbiota recover energy and biologically active molecules from food that would otherwise be washed out of the intestinal tract without benefit. In this study, a protocol for NMR-based metabolite profiling has been developed to access the activity of the microbiome. The physicochemical properties of fecal metabolites have been found to strongly affect the reproducibility and coverage of the profiles obtained. Metabolite profiles generated by water and methanol extraction of lyophilized feces are reproducible and comprise a variety of different compounds including, among others, short-chain fatty acids (e.g. acetate, propionate, butyrate, isobutyrate, isovalerate, malate), organic acids (e.g. succinate, pyruvate, fumarate, lactate), amino acids, uracil, trimethylamine, ethanol, glycerol, glucose, phenolic acids, cholate, and lipid components. The NMR profiling approach was validated on fecal samples from a double-blinded, placebo-controlled, randomized cross-over study, in which healthy human subjects consumed a placebo and either a grape juice extract or a mix of grape juice and wine extract over a period of 4 weeks, each. The considerable inter- and intra-individual variability observed originates in the first instance from variable metabolite concentrations rather than from variable metabolite compositions, suggesting that different colonic flora share general biochemical characteristics metabolizing different substrates to specific metabolic patterns. Whereas the grape juice extract did not induce changes in the metabolite profiles as compared with the placebo, the mixture of grape juice and wine extract induced a reduction in isobutyrate, which may indicate that polyphenols are able to modulate the microbial ecology of the gut.

The purpose of this study was to compare the effects of black and green tea consumption on human metabolism. Seventeen healthy male volunteers consumed black tea, green tea, or caffeine in a randomized crossover study. Twenty-four-hour urine and blood plasma samples were analyzed by NMR-based metabonomics, that is, high-resolution 1H NMR metabolic profiling combined with multivariate statistics. Green and black tea consumption resulted in similar increases in urinary excretion of hippuric acid and 1,3-dihydroxyphenyl-2-O-sulfate, both of which are end products of tea flavonoid degradation by colonic bacteria. Several unidentified aromatic metabolites were detected in urine specifically after green tea intake. Interestingly, green and black tea intake also had a different impact on endogenous metabolites in urine and plasma. Green tea intake caused a stronger increase in urinary excretion of several citric acid cycle intermediates, which suggests an effect of green tea flavanols on human oxidative energy metabolism and/or biosynthetic pathways.

Rationale and Objectives: We sought to explore the feasibility of magnetic resonance imaging (MRI) of the prostate at 3T, with the knowledge of potential drawbacks of MRI at high field strengths., Material and Method: MRI, dynamic MRI, and 1H-MR spectroscopic imaging were performed in 10 patients with prostate cancer on 1.5T and 3T whole-body scanners. Comparable scan protocols were used, and additional high-resolution measurements at 3T were acquired. For both field strengths the signal-to-noise ratio was calculated and image quality was assessed., Result: At 3T the signal-to-noise ratio improved. This resulted in increased spatial MRI resolution, which significantly improved anatomic detail. The increased spectral resolution improved the separation of individual resonances in MRSI. Contrast-enhanced time-concentration curves could be obtained with a doubled temporal resolution., Conclusions: Initial results of endorectal 3T 1H-MR spectroscopic imaging in prostate cancer patients showed potential advantages: the increase in spatial, temporal, and spectral resolution at higher field strength may result in an improved accuracy in delineating and staging prostate cancer.

Purpose: To differentiate prostate carcinoma from healthy peripheral zone and central gland using quantitative dynamic contrast-enhanced (DCE) magnetic resonance (MR) imaging and two-dimensional (1)H MR spectroscopic imaging (MRSI) combined into one clinical protocol., Materials and Methods: Twenty-three prostate cancer patients were studied with a combined DCE-MRI and MRSI protocol. Cancer regions were localized by histopathology of whole mount sections after radical prostatectomy. Pharmacokinetic modeling parameters, K(trans) and k(ep), as well as the relative levels of the prostate metabolites citrate, choline, and creatine, were determined in cancer, healthy peripheral zone (PZ), and in central gland (CG)., Results: K(trans) and k(ep) were higher (P < 0.05) in cancer and in CG than in normal PZ. The (choline + creatine)/citrate ratio was elevated in cancer compared to the PZ and CG (P < 0.05). While a (choline + creatine)/citrate ratio above 0.68 was found to be a reliable indicator of cancer, elevated K(trans) was only a reliable cancer indicator in the diagnosis of individual patients. K(trans) and (choline + creatine)/citrate ratios in cancer were poorly correlated (Pearson r(2) = 0.07), and thus microvascular and metabolic abnormalities may have complementary value in cancer diagnosis., Conclusion: The combination of high-resolution spatio-vascular information from dynamic MRI and metabolic information from MRSI has excellent potential for improved localization and characterization of prostate cancer in a clinical setting. J. Magn. Reson. Imaging 2004;20:279-287., (Copyright 2004 Wiley-Liss, Inc.)

The potential of multiparametric MRI parameters for differentiating between reversibly and irreversibly damaged brain tissue was investigated in an experimental model of focal brain ischemia in the rat. The middle cerebral artery (MCA) was occluded by intraluminal suture insertion for 60 or 90 min, followed by 4.5 h of reperfusion. The apparent diffusion coefficient (ADC) of brain water, T(1) and T(2) relaxation times, and CBF(i), an MR-derived index of cerebral perfusion, were repeatedly measured and correlated with the outcome from the ischemic impact. A novel user-independent approach for segmentation of ADC maps into classes of increasing injury was introduced to define regions of interest (ROIs) in which these parameters were evaluated. MCA occlusion led to a graded decline of ADC, which corresponded with both the severity of flow reduction and an increase in T(1) and T(2) relaxation times. Removal of the suture led to a triphasic restitution of blood flow consisting of a fast initial rise, a secondary decline, and final normalization. Postischemic reperfusion led to a rise of ADC irrespective of the duration of ischemia. However, the quality of recovery declined with increasing severity of the ischemic impact. Throughout the observation time, T(1) and T(2) showed a continuous increase, the intensity of which correlated with the severity of ADC decline during ischemia. Particularly with longer ischemia time, elevated T(2) in combination with reduced ADC yielded a lower probability of recovery during recirculation, while intraischemic perfusion information contributed less to the prediction of outcome. In conclusion, the combination of MR parameters at the end of ischemia correlated with the probability of tissue recovery but did not permit reliable differentiation between reversibly and irreversibly damaged tissue., (Copyright 2002 Wiley-Liss, Inc.)

Perfusion-weighted imaging (PWI), using the method of arterial spin tagging, is strongly T(1)-dependent. This translates into a high field dependency of the perfusion signal intensity. In order to determine the expected signal improvement at higher magnetic fields we compared perfusion-weighted images in rat brain at 4.7 T and 7 T. Application of PWI to focal ischemia and functional activation of the brain and the use of two different anesthetics allowed the observation of a wide range of flow values. For all these (patho-)physiological conditions switching from 4.7 T to 7 T resulted in a significant increase of mean perfusion signal intensity by a factor of 2.96. The ratio of signal intensities of homotopic regions in the ipsi- and contralateral hemisphere was field-independent. The relative contribution of a) T(1) relaxation time, b) net magnetization, c) the Q-value of the receiver coils and d) the degree of adiabatic inversion to the signal improvement at higher field strength were discussed. It was shown that the main parameters contributing to the higher signal intensity are the lengthening of T(1) and the higher magnetization at the higher magnetic field.

Temporary focal ischaemia was induced in wild-type C57Black/6 mice by thread occlusion of the middle cerebral artery (MCA). Recirculation was started after 60 min and maintained for 24 h, after which the mouse brain was frozen in situ. Development of the cerebral infarct was monitored by diffusion-, perfusion- and T(2)-weighted magnetic resonance imaging (MRI) during ischaemia, during the early reperfusion period of 90 min, and at 24 h after reperfusion. Ischaemia caused a marked reduction of the perfusion signal intensity and of the apparent diffusion coefficient (ADC) of tissue water in the ipsilateral MCA territory. In sham-operated control animals ADC remained unchanged. Hemispheric lesion volume after 1 h MCA occlusion was 53 +/- 6% (n = 6), as defined by an ADC decrease of more than 20%. Recirculation reduced hemispheric lesion volume to only 27 +/- 13%, while there was a trend towards secondary lesion growth at 24 h. Post-ischaemic recovery of perfusion was slow, heterogeneous and incomplete. A region-of-interest analysis showed only partial and transient recovery of the ADC, particularly in the dorsolateral cortex and lateral caudate putamen, which may be explained by inadequate reperfusion in these regions. Detailed MRI studies of cerebral ischaemia and reperfusion may now also be performed in the transgenic mice., (Copyright 1999 John Wiley & Sons, Ltd.)

Quantitative imaging contrast is evaluated which allows the selective measurement of the blood oxygenation state during cerebral ischemia within a multiparametric imaging study on rats. In a first step, the ambiguities arising in T*(2)-weighted images due to T*(2) heterogeneity are eliminated by calculating T*(2) maps. Then, 1/T'(2) maps are calculated according to 1/T'(2) = 1/T*(2) - 1/T(2) to eliminate nonsusceptibility-induced changes of 1/T*(2) after the induction of stroke. This is of particular importance in the presence of vasogenic edema. The changes Delta(1/T'(2)) after the onset of ischemia selectively quantify the variations of the deoxyhemoglobin content during the development of the infarct. The presented results are not available from conventionally recorded parameters of a stroke study and, together with perfusion-weighted images, form a powerful combination to analyze the oxygen consumption and the metabolism of the tissue. Magn Reson Med 42:1027-1032, 1999., (Copyright 1999 Wiley-Liss, Inc.)

Hyperintense lesions on T2-weighted MR images of the brain, predominantly located in the basal ganglia, the brainstem and cerebellum, are a frequent finding in patients with neurofibromatosis type 1. Nature and significance of these lesions are still unknown so that the term 'unidentified bright objects' (UBOs) has been introduced to allow an unbiased description. We analyzed brain MRI scans of 31 children with definite diagnosis of neurofibromatosis type 1 according to the NIH criteria. High-intensity lesions on T2-weighted images were present in 86% of the patients. They did not correlate to other MRI findings such as optic pathway gliomas and were not indicative of intellectual impairment. Additionally, brain MR imaging of Nf1 knockout mice was performed to find out if similar abnormalities are present in this animal model. A total of 9 Nf1 knockout mice was examined on a dedicated animal MRI scanner at 4.7 Tesla but no evidence of high-signal intensity lesions on T2-weighted images was found. Therefore, the Nf1 mouse model seems to be unhelpful in providing further insights into the histological basis of hyperintense MRI abnormalities in NF1 patients.

Hearts of wild-type and cytosolic muscle creatine kinase (M-CK)-knockout mice were perfused with Krebs-Henseleit buffer containing 10 mM glucose and 5 mM pyruvate and studied during pacing at 400 and 600 beats/min and during K+ arrest. Phosphocreatine (PCr) and ATP concentrations in M-CK-deficient hearts were not significantly different from those in wild-type hearts. With the use of 31P NMR saturation transfer, the flux mediated predominantly by mitochondrial creatine kinase (Mi-CK) was clearly detected in M-CK-deficient hearts. Mi-CK flux was 4.8 +/- 0.6 and 4.5 +/- 0.6 mM/s during pacing at 400 and 600 beats/min, respectively, and was 3. 5 +/- 0.4 mM/s during cardiac arrest. In control hearts total CK flux was 7.8 +/- 1.1 and 6.6 +/- 1.3 mM/s during pacing at 400 and 600 beats/min, respectively, and decreased to 3.8 +/- 0.5 mM/s during arrest. It is suggested that the relative contribution of Mi-CK to the total NMR-measured CK flux in the wild-type heart is higher than that of the homodimeric M-CK isoform (MM-CK).

A mathematical model of the compartmentalized energy transfer in cardiac cells is described and used for interpretation of novel experimental data obtained by using phosphorus NMR for determination of the energy fluxes in the isolated hearts of transgenic mice with knocked out creatine kinase isoenzymes. These experiments were designed to study the meaning and importance of compartmentation of creatine kinase isoenzymes in the cells in vivo. The model was constructed to describe quantitatively the processes of energy production, transfer, utilization, and feedback between these processes. It describes the production of ATP in mitochondrial matrix space by ATP synthase, use of this ATP for phosphocreatine production in the mitochondrial creatine kinase reaction coupled to the adenine nucleotide translocation, diffusional exchange of metabolites in the cytoplasmic space, and use of phosphocreatine for resynthesis of ATP in the myoplasmic creatine kinase reaction. It accounts also for the recently discovered phenomenon of restricted diffusion of adenine nucleotides through mitochondrial outer membrane porin pores (VDAC). Practically all parameters of the model were determined experimentally. The analysis of energy fluxes between different cellular compartments shows that in all cellular compartments of working heart cells the creatine kinase reaction is far from equilibrium in the systolic phase of the contraction cycle and approaches equilibrium only in cytoplasm and only in the end-diastolic phase of the contraction cycle. Experimental determination of the relationship between energy fluxes by a 31P-NMR saturation transfer method and workload in isolated and perfused heart of transgenic mice deficient in MM isoenzyme of the creatine kinase, MM-/-showed that in the hearts from wild mice, containing all creatine kinase isoenzymes, the energy fluxes determined increased 3-4 times with elevation of the workload. By contrast, in the hearts in which only the mitochondrial creatine kinase was active, the energy fluxes became practically independent of the workload in spite of the preservation of 26% of normal creatine kinase activity. These results cannot be explained on the basis of the conventional near-equilibrium theory of creatine kinase in the cells, which excludes any difference between creatine kinase isoenzymes. However, these apparently paradoxical experimental results are quantitatively described by a mathematical model of the compartmentalized energy transfer based on the steady state kinetics of coupled creatine kinase reactions, compartmentation of creatine kinase isoenzymes in the cells, and the kinetics of ATP production and utilization reactions. The use of this model shows that: (1) in the wild type heart cells a major part of energy is transported out of mitochondria via phosphocreatine, which is used for complete regeneration of ATP locally in the myofibrils--this is the quantitative estimate for PCr pathway; (2) however, in the absence of MM-creatine kinase in the myofibrils in transgenic mice the contraction results in a very rapid rise of ADP in cytoplasmic space, that reverses the mitochondrial creatine kinase reaction in the direction of ATP production. In this way, because of increasing concentrations of cytoplasmic ADP, mitochondrial creatine kinase is switched off functionally due to the absence of its counterpart in PCr pathway, MM-creatine kinase. This may explain why the creatine kinase flux becomes practically independent from the workload in the hearts of transgenic mouse without MM-CK. Thus, the analysis of the results of studies of hearts of creatine kinase-deficient transgenic mice, based on the use of a mathematical model of compartmentalized energy transfer, show that in the PCr pathway of intracellular energy transport two isoenzymes of creatine kinase always function in a coordinated manner out of equilibrium, in the steady state, and disturbances in functioning of one of them inevitably result

P-31 nuclear magnetic resonance (NMR) is uniquely suited to measure the kinetics of the phosphoryl-exchange reaction catalyzed by creatine kinase in intact mammalian tissue, especially striated muscle. Recently developed transgenic mouse models of the creatine kinase iso-enzyme system open novel opportunities to assess the functional importance of the individual iso-enzymes and their relative contribution to the total in situ flux through the CK reaction. This chapter reviews the most recent findings from NMR flux measurements on such genetic models of CK function. Findings in intact mouse skeletal and cardiac muscle in vivo are compared to data from purified mitochondrial and cytosolic creatine kinase in vitro. The relevance of findings in transgenic animals for the function of CK in wild-type tissue is described and the perspectives of transgenic techniques in future quantitative studies on the creatine kinase iso-enzyme system are indicated.

The kinetic properties of the cytoplasmic and the mitochondrial iso-enzymes of creatine kinase from striated muscle were studied in vitro and in vivo. The creatine kinase (CK) iso-enzyme family has a multi-faceted role in cellular energy metabolism and is characterized by a complex pattern of tissue-specific expression and subcellular distribution. In mammalian tissues, there is always co-expression of at least two different CK isoforms. As a result, previous studies into the role of CK in energy metabolism have not been able to directly differentiate between the individual CK species. Here, we describe experiments which were directed at achieving this goal. First, we studied the kinetic properties of the muscle-specific cytoplasmic and mitochondrial CK isoforms in purified form under in vitro conditions, using a combination of P-31 NMR and spectrophotometry. Secondly, P-31 NMR measurements of the flux through the CK reaction were carried out on intact skeletal and heart muscle from wild-type mice and from transgenic mice, homozygous for a complete deficiency of the muscle-type cytoplasmic CK isoform. Skeletal muscle and heart were compared because they differ strongly in the relative abundance of the CK isoforms. The present data indicate that the kinetic properties of cytoplasmic and mitochondrial CK are substantially different, both in vitro and in vivo. This finding particularly has implications for the interpretation of in vivo studies with P-31 NMR.

Michaelis- and dissociation constants of sarcomeric mitochondrial creatine kinase (Mi(b)-CK) in solution were determined by enzyme assay and compared to those of cytosolic MM-CK under identical conditions at pH 7.4 and 25 degrees C. Saturation transfer 31P-NMR was used to determine the steady state fluxes mediated by Mi-CK and MM-CK in solution. The NMR detected fluxes of both Mi-CK and MM-CK exhibited, as expected, a linear dependence on Vmax (Vmax range 0-9 mM.s-1). Interestingly, the oligomeric state of Mi-CK, with the Mi-CK octamer/dimer ratio ranging from 2 to 9, did not have a significant effect on the flux/Vmax ratio. Furthermore, the flux/Vmax ratio of Mi-CK was twice as high as that of MM-CK under similar conditions (flux/Vmax for Mi-CK was 0.31 and for MM-CK was 0.15). This difference was primarily due to a 4-fold higher apparent affinity for MgADP of Mi-CK compared to MM-CK (K(m)(MgADP) = 22 +/- 9 microM and 80 +/- 17 microM, resp.). The NMR observed fluxes were in agreement with the fluxes as calculated from the rate equation, using the appropriate metabolite concentrations and the kinetic constants from the spectrophotometric assays. Thus we conclude, that Mi-CK and MM-CK, when in solution, catalyse an exchange-reaction, the flux of which is fully observable by saturation transfer 31P-NMR.