Obesity Development in a Miniature Yucatan Pig Model: A Multi-compartmental Metabolomics Study on Cloned and Normal Pigs Fed Restricted or Ad Libitum High-Energy Diets.

Miniature-pig models for human metabolic disorders such as obesity and metabolic syndrome are gaining popularity. However, in-depth knowledge on the phenotypic and metabolic effects of metabolic dysregulation is lacking, and ad libitum feeding is not well-characterized in these pig breeds. Therefore, an investigation was performed into the metabolome of Yucatan minipigs fed ad libitum or restricted diets. Furthermore, we used cloned and conventional minipigs to assess if cloning reflects a presumably lowered variation between subjects. For 5 months, 17 female Yucatan minipigs were fed either ad libitum or restricted Western-style diets. Serum, urine, and liver tissues were collected and analyzed by non-targeted liquid chromatography-mass spectrometry metabolomics and by biochemical analyses. Several metabolic pathways were deregulated as a result of obesity and increased energy-dense feed intake, particularly the hepatic glutathione pathway and the pantothenic acid and tryptophan metabolic pathways in serum and urine. Although cloned minipigs were phenotypically similar to wild-type minipigs, the metabolomics analysis of serum and liver tissues showed several altered pathways, such as amino acid and purine metabolism. These changes, as an effect of cloning, could limit the use of cloned models in dietary intervention studies and provides no evidence of decreased variability between subjects.

Caseins from bovine colostrum and milk strongly bind piscidin-1, an antimicrobial peptide from fish.

A model system of bovine colostrum and piscidin, a fish-derived antimicrobial peptide, was developed to study potential interactions of antimicrobial peptides in colostrum. We did not detect any antimicrobial activity of colostrum using the radial plate diffusion assay; in fact colostrum completely abrogated activity of added piscidin. This could not be explained by degradation of piscidin by colostrum, which was less than ten percent. We found that colostrum even protected piscidin against degradation by added proteases. We further observed that colostrum and milk rapidly quenched the fluorescence of fluorescein-piscidin but not that of fluorescein. This effect was not seen with BSA and the specific quenching of fluorescein-piscidin by colostrum was saturably inhibited with unlabeled piscidin. Size exclusion chromatography indicated that fluorescein-piscidin bound to casein micelles with no apparent binding to IgG or whey proteins. Further, addition of pure caseins was able to quench fluorescence of fluorescein-piscidin and to inhibit the antimicrobial activity of piscidin. The interaction between caseins and piscidin could be dissociated by guanidine hydrochloride and recovered piscidin had antimicrobial activity against bacteria. Based on our results we propose that caseins could be carriers for antimicrobial peptides in colostrum and milk.

The effect of high-fat diet on the composition of the gut microbiota in cloned and non-cloned pigs of lean and obese phenotype.

The aim of this study was to investigate the effect of high-far-high-energy diet on cloned and non-cloned domestic pigs of both lean and obese phenotype and to evaluate if the lean cloned pigs had a lower inter-individual variation as compared with non-cloned pigs. The microbiota of colon and terminal ileum was investigated in cloned and non-cloned pigs that received a high-far-high-energy diet with either restricted or ad libitum access to feed, resulting in lean and obese phenotypes, respectively. The fecal microbiota of lean pigs was investigated by terminal restriction fragment length polymorphism (T-RFLP). The intestinal microbiota of lean and obese cloned and non-cloned pigs was analyzed by quantitative real time PCR and a novel high-throughput qPCR platform (Fluidigm). Principal component analysis (PCA) of the T-RFLP profiles revealed that lean cloned and non-cloned pigs had a different overall composition of their gut microbiota. The colon of lean cloned pigs contained relatively more bacteria belonging to the phylum Firmicutes and less from the phylum Bacteroidetes than obese cloned pigs as estimated by qPCR. Fluidigm qPCR results revealed differences in specific bacterial groups in the gut microbiota of both lean and obese pigs. Our results suggest that high-far-high-energy diet is associated with changes in the gut microbiota even in the absence of obesity. Overall, the cloned pigs had a different gut microbiota from that of non-cloned pigs. To our knowledge this is the first study to investigate the gut microbiota of cloned domestic pigs of lean and obese phenotype.

Cloning changes the response to obesity of innate immune factors in blood, liver, and adipose tissues in domestic pigs.

The objective of this study was to evaluate the usefulness of cloned pigs as porcine obesity models reflecting obesity-associated changes in innate immune factor gene expression profiles. Liver and adipose tissue expression of 43 innate immune genes as well as serum concentrations of six immune factors were analyzed in lean and diet-induced obese cloned domestic pigs and compared to normal domestic pigs (obese and lean). The number of genes affected by obesity was lower in cloned animals than in control animals. All genes affected by obesity in adipose tissues of clones were downregulated; both upregulation and downregulation were observed in the controls. Cloning resulted in a less differentiated adipose tissue expression pattern. Finally, the serum concentrations of two acute-phase proteins (APPs), haptoglobin (HP) and orosomucoid (ORM), were increased in obese clones as compared to obese controls as well as lean clones and controls. Generally, the variation in phenotype between individual pigs was not reduced in cloned siblings as compared to normal siblings. Therefore, we conclude that cloning limits both the number of genes responding to obesity as well as the degree of tissue-differentiated gene expression, concomitantly with an increase in APP serum concentrations only seen in cloned, obese pigs. This may suggest that the APP response seen in obese, cloned pigs is a consequence of the characteristic skewed gene response to obesity in cloned pigs, as described in this work. This should be taken into consideration when using cloned animals as models for innate responses to obesity.

Changes in the gut microbiota of cloned and non-cloned control pigs during development of obesity: gut microbiota during development of obesity in cloned pigs.

BACKGROUND: Obesity induced by a high-caloric diet has previously been associated with changes in the gut microbiota in mice and in humans. In this study, pigs were cloned to minimize genetic and biological variation among the animals with the aim of developing a controlled metabolomic model suitable for a diet-intervention study. Cloning of pigs may be an attractive way to reduce genetic influences when investigating the effect of diet and obesity on different physiological sites. The aim of this study was to assess and compare the changes in the composition of the gut microbiota of cloned vs. non-cloned pigs during development of obesity by a high-fat/high-caloric diet. Furthermore, we investigated the association between diet-induced obesity and the relative abundance of the phyla Firmicutes and Bacteroidetes in the fecal-microbiota. The fecal microbiota from obese cloned (n = 5) and non-cloned control pigs (n= 6) was investigated biweekly over a period of 136 days, by terminal restriction fragment length polymorphism (T-RFLP) and quantitative real time PCR (qPCR). RESULTS: A positive correlation was observed between body-weight at endpoint and percent body-fat in cloned (r=0.9, P<0.0001) and in non-cloned control pigs (r=0.9, P<0.0001). Shannon Weaver and principal component analysis (PCA) of the terminal restriction fragments (T-RFs) revealed no differences in the bacterial composition or variability of the fecal microbiota between the cloned pigs or between cloned and non-cloned control pigs. Body-weight correlated positively with the relative abundance of Firmicutes in both cloned (r=0.37; P<0.02) and non cloned-control pigs (r=0.45; P<0.006), and negatively with the abundance of Bacteroidetes in cloned pigs (r=-0.33, P<0.04), but not in the non-cloned control pigs. CONCLUSION: The cloned pigs did not have reduced inter-individual variation as compared to non-cloned pigs in regard to their gut microbiota in neither the obese nor the lean state. Diet-induced obesity was associated with an increase in the relative abundance of Firmicutes over time. Our results suggest that cloned pigs are not a more suitable animal model for gut microbiota-obesity related studies than non-cloned pigs. This study is the first to evaluate if cloned pigs provide a better animal model than conventional pigs in diet-intervention, obesity and gut microbiota research.

Orosomucoid expression profiles in liver, adipose tissues and serum of lean and obese domestic pigs, Göttingen minipigs and Ossabaw minipigs.

The acute phase protein orosomucoid (ORM) has anti-inflammatory and immunomodulatory effects, and may play an important role in the maintenance of metabolic homeostasis in obesity-induced low-grade inflammation. Even though the pig is a widely used model for obesity related metabolic symptoms, the expression of ORM has not yet been characterized in such pig models. The objective of this study was to investigate the expression of ORM1 mRNA in liver, visceral adipose tissue, subcutaneous adipose tissue (SAT) from the abdomen or retroperitoneal abdominal adipose tissue (RPAT) and SAT from the neck, as well as the serum concentration of ORM protein in three porcine obesity models; the domestic pig, Göttingen minipigs and Ossabaw minipigs. No changes in ORM1 mRNA expression were observed in obese pigs compared to lean pigs in the four types of tissues. However, obese Ossabaw minipigs, but none of the other breeds, showed significantly elevated ORM serum concentrations compared to their lean counterparts. Studies in humans have shown that the expression of ORM was unchanged in adipose tissue depots in obese humans with an increased serum concentration of ORM. Thus in this respect, obese Ossabaw minipigs behave more similarly to obese humans than the other two pig breeds investigated.

Expression of innate immune response genes in liver and three types of adipose tissue in cloned pigs.

The pig has been proposed as a relevant model for human obesity-induced inflammation, and cloning may improve the applicability of this model. We tested the assumptions that cloning would reduce interindividual variation in gene expression of innate immune factors and that their expression would remain unaffected by the cloning process. We investigated the expression of 40 innate immune factors by high-throughput quantitative real-time PCR in samples from liver, abdominal subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT), and neck SAT in cloned pigs compared to normal outbred pigs. The variation in gene expression was found to be similar for the two groups, and the expression of a small number of genes was significantly affected by cloning. In the VAT and abdominal SAT, six out of seven significantly differentially expressed genes were downregulated in the clones. In contrast, most differently expressed genes in both liver and neck SAT were upregulated (seven out of eight). Remarkably, acute phase proteins (APPs) dominated the upregulated genes in the liver, whereas APP expression was either unchanged or downregulated in abdominal SAT and VAT. The general conclusion from this work is that cloning leads to subtle changes in specific subsets of innate immune genes. Such changes, even if minor, may have phenotypic effects over time, e.g., in models of long-term inflammation related to obesity.

Liquid chromatography-mass spectrometry based metabolomics study of cloned versus normal pigs fed either restricted or ad libitum high-energy diets.

Genetically identical cloned pigs should in principle eliminate biological variation and provide more pronounced effects when subjected to, e.g., dietary interventions, but little is known about how phenotype and phenotypic variation is affected by cloning. Therefore, an investigation of the metabolome of cloned pigs compared to normal control pigs was performed to elucidate the variation and possible differences in the metabolic phenotypes during a dietary intervention. A total of 19 control pigs and 17 cloned pigs were given the same high-energy dense diet either ad libitum or in a restricted manner (60% of ad libitum) for ∼6 months, and plasma was subjected to liquid chromatography-mass spectrometry nontargeted metabolomics and biochemical analyses. Low systemic levels of IGF-1 could indicate altered growth conditions and energy metabolism in cloned pigs. In response to ad libitum feeding, clones had a decreased energy intake and lower weight gain compared to controls, and plasma lipid profiles were changed accordingly. Elevated lactate and decreased creatine levels implied an increased anaerobic metabolism in ad libitum fed clones. Less interindividual variation between cloned pigs was however not established, suggesting a strong role for epigenetics and/or the gut microbiota to develop variation.

Metabolomic phenotyping of a cloned pig model.

BACKGROUND: Pigs are widely used as models for human physiological changes in intervention studies, because of the close resemblance between human and porcine physiology and the high degree of experimental control when using an animal model. Cloned animals have, in principle, identical genotypes and possibly also phenotypes and this offer an extra level of experimental control which could possibly make them a desirable tool for intervention studies. Therefore, in the present study, we address how phenotype and phenotypic variation is affected by cloning, through comparison of cloned pigs and normal outbred pigs. RESULTS: The metabolic phenotype of cloned pigs (n = 5) was for the first time elucidated by nuclear magnetic resonance (NMR)-based metabolomic analysis of multiple bio-fluids including plasma, bile and urine. The metabolic phenotype of the cloned pigs was compared with normal outbred pigs (n = 6) by multivariate data analysis, which revealed differences in the metabolic phenotypes. Plasma lactate was higher for cloned vs control pigs, while multiple metabolites were altered in the bile. However a lower inter-individual variability for cloned pigs compared with control pigs could not be established. CONCLUSIONS: From the present study we conclude that cloned and normal outbred pigs are phenotypically different. However, it cannot be concluded that the use of cloned animals will reduce the inter-individual variation in intervention studies, though this is based on a limited number of animals.

Mass spectrometric-based protein chips for detection of food-derived bioactive components.

Bioactive components in the diet influence our health and well-being beyond that of simple supply of energy and raw materials for biochemical reactions. However, the complex chemistry and composition of our food does not make the identification of potential bioactive components a straightforward task. Bioassays for a multitude of functionalities have to be performed for thousands of different food-derived molecules in order to identify important interactions. Our approach is to directly identify those food molecules that interact with cellular targets based on mass spectrometric (MS) techniques through immobilization of cellular target molecules on glass chips and incubation with various foods. Food-derived molecules that bind with high affinity can then be directly analyzed by MS. We have chosen bovine colostrum, a potent bioactive food, and cellular receptors of the innate immune response as our model for proof of concept.

Colostrum and bioactive, colostral peptides differentially modulate the innate immune response of intestinal epithelial cells.

Characterization and identification of peptides with bioactivity from food have received considerable interest recently since such bioactive components must be adequately documented if they are part of functional food claims. We have characterized peptides from colostrum or those generated by a simulated gastrointestinal digest (GI) and tested them for bioactivity using murine intestinal (mIC(c12)) cells and compared with bioactivity of intact colostrum. The peptides were recovered in the permeate after dialysis. The presence of peptides in the permeate was confirmed by C(18) RP-HPLC, determination of free amino termini and MALDI MS. The bioactivity of the intact colostrum and colostral peptides in the permeate was tested using mIC(c12) cells stimulated in the absence or presence of different bacterial ligands that mediate cellular activation through stimulation of Toll-like receptors (TLR). Whereas intact colostrum generally reduced TLR-mediated signaling, the isolated peptides seemed to either stimulate or reduce the immune response depending on the bacterial ligand used for stimulation. Interestingly, the most potent bioactive peptides originated from nondigested colostrum, which had only been subject to endogenous protease activity. Identified peptides in the nondigested colostrum originated exclusively from the casein fraction of colostrum as shown by MALDI MS/MS identification. Thus, multiple components with different bioactivities towards the innate immune response appear in bovine colostrum.

Characterization of peroxides formed by riboflavin and light exposure of milk. Detection of urate hydroperoxide as a novel oxidation product.

Characterization of peroxides by size exclusion chromatography (SEC) of milk following exposure to riboflavin and light showed that hydrogen peroxide was the most abundant peroxide formed since it could be removed by catalase. Formation of peroxides after separation by SEC showed that hydrogen peroxide formation was primarily increased in the presence of caseins and ascorbate, although whey proteins also were found to contribute. Caseins and beta-lactoglobulin also formed catalase-resistant peroxides, presumably protein hydroperoxides. A catalase-resistant and unstable peroxide was observed in fractions containing urate. Experiments performed with pure urate suggested that urate radicals reacted further with superoxide leading to a urate hydroperoxide. Electron paramagnetic resonance spectroscopy using spin-traps showed that the presence of oxygen was required for urate radical formation, which could be assigned as nitrogen-centered radicals. These results suggest a new route during light-induced oxidation sensitized by flavins, in effect making urate pro-oxidative.

Characterization of major radical scavenger species in bovine milk through size exclusion chromatography and functional assays.

Radical scavenging activities of bovine milk components were quantified following size exclusion chromatography (SEC) with postcolumn characterization of fractions using the scavenging of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radicals (ABTS*(+)) in the Trolox equivalent antioxidant capacity (TEAC) assay and peroxyl radicals formed from cleavage of 2,2'-azobis(2-amidinopropane) (AAPH) in the oxygen radical absorbance capacity (ORAC) fluorometric assay. Caseins were quantitatively the major radical scavenger species in both assays, whereas beta-lactoglobulin (beta-lg) and alpha-lactalbumin (alpha-la) were much less active and only in the peroxyl radical assay. The radical scavenging activity of the caseins could be quantitatively accounted for by their constituent amino acids, as there were no effects of denaturing agents or complete digestion with proteases. In contrast, the activities of the whey proteins were dependent on denaturation or partial hydrolysis and dominated by the free thiol in beta-lg. A component in milk serum with a molecular mass of approximately 100 kDa contributed significantly to both ABTS*(+) and peroxyl radical scavenging but was absent in whey. This radical scavenger was identified as beta-casein. The only significant low molecular weight radical scavenger species were identified as ascorbate and urate in both assays.

A novel, simple and sensitive ligand affinity capture method for detecting molecular interactions by MALDI mass spectrometry.

A simple and sensitive ligand affinity capture method (LAC) was developed to detect biotinylated biomolecules bound to a biotin-avidin base by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI ToF MS). Glass slides covered with a metal film for MALDI MS applications were treated with amino-silane and derivatized with biotin followed by binding of avidin. Washing buffers with high ionic strength increased the specificity of the subsequent binding of biotinylated biomolecules to the avidin layer. A combined thin layer-dried droplet method using alpha-cyano-4-hydroxycinnamic acid (CHCA) in acetone or ethyl acetate resulted in the most intense ions of biotinylated polymyxin B, whereas the matrix conditions did not influence the detection of angiotensin II. Addition of biotinylated biomolecules in the low femtomole to low picomole range resulted in sufficient ion intensity for detection by the LAC method. The LAC concept was extended by binding of biotinylated lipopolysaccharide to the biotin-avidin base followed by preferential capture and specific detection of the binding antagonist polymyxin B.

Inhibition of lactoperoxidase-catalyzed 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and tyrosine oxidation by tyrosine-containing random amino acid copolymers.

Oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) by lactoperoxidase was found to be inhibited by tyrosine-containing random amino acid copolymers but not by tyrosine. Both electrostatic effects and polymer size were found to be important by comparison of negatively and positively charged copolymers of varying lengths, with poly(Glu, Tyr)4:1 ([E 4Y 1] approximately 40) as the strongest competitive inhibitor (EC 50 approximately 20 nM). This polymer did not form dityrosine in the presence of lactoperoxidase (LPO) and peroxide. Furthermore, incubation with tert-butyl hydroperoxide, as opposed to hydrogen peroxide, resulted in a peculiar long lag phase of the reaction between the redox intermediate compound II and [E 4Y 1] approximately 40, indicating a very tight association between enzyme and inhibitor. We propose that interactions between multiple positively charged areas on the surface of LPO and the polymer are required for optimal inhibition.

Short-term effects of selenium supplementation of cows' feed on the content and distribution of selenium, copper and zinc in bovine milk, whey and blood plasma.

The effect of selenium supplementation of feed on the Se content in bovine milk, whey and plasma, and on the distribution of Se, Zn and Cu in whey and plasma was investigated. In a cross-over study two groups of cows were given a basal feed with 0.16 ppm selenite (approx. 3 mg Se/d) with or without 25 mg yeast Se/d for 2 weeks. In the supplemented group the Se content increased 10-fold in milk, 10-fold in whey and 2-fold in plasma, and after the cessation of the supplementation, selenium in milk decreased with a calculated half-life of 3.5 d. In another experiment, two groups of cows were given either 100 mg yeast Se/d for 1 week or only the basal feed. The increase in Se content in both whole and defatted milk was 40-50-fold, and in whey it was approx. 20-fold. Size-exclusion chromatography of whey using inductively coupled plasma mass spectrometry for detection showed that supplementation increased the proportion of Se in the beta-lactoglobulin-alpha-lactalbumin fraction. Distribution of Cu and Zn was essentially unaffected. In plasma, supplementation increased the Se content in all major Se fractions like selenoprotein P, albumin and low-molecular-weight compounds, but the distribution profiles of Zn and Cu underwent no major changes. The study showed for the first time the rapid kinetics of the Se increase and decrease in milk after the initiation and cessation of supplementation, respectively, and the preferential appearance of Se in the beta-lactoglobulin-alpha-lactalbumin fraction of whey. Milk highly enriched in selenium will be a useful tool for different research purposes.

A short-term intervention trial with selenate, selenium-enriched yeast and selenium-enriched milk: effects on oxidative defence regulation.

Increased Se intakes have been associated with decreased risk of cancer and CVD. Several mechanisms have been proposed, including antioxidant effects through selenoproteins, induction of carcinogen metabolism and effects on the blood lipid profile. In a 4 x 1 week randomised, double-blind cross-over study, healthy young men supplemented their usual diet with selenate, Se-enriched yeast, Se-enriched milk or placebo (Se dose was 300 microg/d for selenate and Se-enriched yeast, and about 480 microg/d for Se-enriched milk) followed by 8-week washout periods. All Se sources increased serum Se levels after supplementation for 1 week. The effect of the organic forms did not differ significantly and both increased serum Se more than selenate. Conversely, thrombocyte glutathione peroxidase (GPX) was increased in the periods where subjects were supplemented with selenate but not in those where they were given Se-enriched yeast or Se-enriched milk. We found no effect on plasma lipid resistance to oxidation, total cholesterol, TAG, HDL- and LDL-cholesterol, GPX, glutathione reductase (GR) and glutathione S-transferase (GST) activities measured in erythrocytes, GPX and GR activities determined in plasma, or GR and GST activities in thrombocytes. Leucocyte expression of genes encoding selenoproteins (GPX1, TrR1 and SelP), and of electrophile response element-regulated genes (GCLC, Fra1 and NQO1) were likewise unaffected at all time points following intervention. We conclude that thrombocyte GPX is specifically increased by short-term selenate supplementation, but not by short-term supplementation with organic Se. Short-term Se supplementation does not seem to affect blood lipid markers or expression and activity of selected enzymes and a transcription factor involved in glutathione-mediated detoxification and antioxidation.

n-6 and n-3 fatty acids ratio and vitamin E in porcine maternal diet influence the antioxidant status and immune cell eicosanoid response in the progeny.

Five groups of lactating sows were fed diets containing 8% of either added rapeseed oil, fish oil or sunflower oil and 60 mg vitamin E/kg feed, or the diets with sunflower oil and fish oil, respectively, supplemented with 500 mg vitamin E/kg. Supplementation of vitamin E to the sows increased the concentration of alpha-tocopherol of the muscle, and addition of sunflower oil decreased the activity of glutathione peroxidase in liver cytosol compared to fish oil and rapeseed oil. The composition of fatty acids of alveolar macrophages (AM) of piglets was influenced by the dietary fat sources provided the sows, i.e., the ratio of n-6:n-3 fatty acids was highest in AM of piglets suckling sows of the sunflower oil treatments, and lowest in AM of piglets suckling sows fed fish oil with the rapeseed oil treatment in between. The ex vivo synthesis of prostaglandin E(2) and thromboxane B(2) in AM of piglets suckling sows fed sunflower oil was elevated compared to piglets suckling sows fed fish oil. Vitamin E supplementation to sows enhanced the synthesis of these eicosanoids, and also the concentration of alpha-tocopherol in the AM of the piglets.

Significance of vitamin E supplementation, dietary content of polyunsaturated fatty acids, and preslaughter stress on oxidative status in pig as reflected in cell integrity and antioxidative enzyme activities in porcine muscle.

The present study investigates the combined effects of feed-induced increase in polyunsaturated fatty acids (PUFA) content and/or alpha-tocopherol content in pig muscles and preslaughter stress on cell integrity. Cell integrity was determined by plasma lactate dehydrogenase (LDH) activity, and antioxidative status of muscle was measured by activities of the antioxidative enzymes catalase, superoxide dismutase, and glutathione peroxidase. Preslaughter stress increased LDH activity, reflecting loss in cell membrane integrity independent of increased content of PUFA and/or alpha-tocopherol. However, feed-induced increase of PUFA decreased the LDH activity in plasma immediately after slaughter. Catalase activity in the muscle tissue increased as a consequence of the high-PUFA diet, which may indicate an increased demand caused by introduction of oxidative labile PUFA.

Elucidation of membrane destabilization in post-mortem muscles using an extracellular paramagnetic agent (Gd-DTPA): an NMR study.

The effect of Gd-DTPA on the development in NMR relaxation of skeletal rabbit muscles post-mortem was investigated by dynamic low-field (0.47 T) relaxation measurements from 4 min post-mortem and until 23 h post-mortem. Twelve rabbits were included in the study, and half of the animals were administered 0.2 mmol of Gd-DTPA iv 15 min before sacrifice, while the other half was administered an isotonic salt solution. A significant effect of Gd-DTPA treatment corresponding to a 25% reduction in the T(1) relaxation time was observed. T(2) relaxation was decomposed into two components reflecting intra- and extracellular components (T(2)()alpha and T(2)()beta, respectively), and Gd-DTPA treatment was found to affect both components. However, around 150 min post-mortem a dramatic increase in the difference between control and Gd-DTPA-treated rabbits was observed in the relaxation time of the intracellular water population (T(2)()alpha). Electrical stimulation of the muscles resulted in a significantly earlier onset of the increased effect of Gd-DTPA on the T(2)()alpha population. The increased effect of Gd-DTPA treatment on the T(2)()alpha component is believed to reflect leakage of water from the muscle cells due to membrane destabilization, known to be promoted by electrical stimulation. Accordingly, the present study demonstrates how Gd-DTPA can be used for probing membrane integrity in post-mortem muscles known to be of importance for subsequent water distribution and final water-holding capacity.