Sex-Specific Differences in Lysine, 3-Hydroxybutyric Acid and Acetic Acid in Offspring Exposed to Maternal and Postnatal High Linoleic Acid Diet, Independent of Diet.

BACKGROUND: Linoleic acid (LA) is an essential polyunsaturated fatty acid (PUFA) that is required for foetal growth and development. Excess intake of LA can be detrimental for metabolic health due to its pro-inflammatory properties; however, the effect of a diet high in LA on offspring metabolites is unknown. In this study, we aimed to determine the role of maternal or postnatal high linoleic acid (HLA) diet on plasma metabolites in adult offspring. METHODS: Female Wistar Kyoto (WKY) rats were fed with either low LA (LLA) or HLA diet for 10 weeks prior to conception and during gestation/lactation. Offspring were weaned at postnatal day 25 (PN25), treated with either LLA or HLA diets and sacrificed at PN180. Metabolite analysis was performed in plasma samples using Nuclear Magnetic Resonance. RESULTS: Maternal and postnatal HLA diet did not alter plasma metabolites in male and female adult offspring. There was no specific clustering among different treatment groups as demonstrated by principal component analysis. Interestingly, there was clustering among male and female offspring independent of maternal and postnatal dietary intervention. Lysine was higher in female offspring, while 3-hydroxybutyric acid and acetic acid were significantly higher in male offspring. CONCLUSION: In summary, maternal or postnatal HLA diet did not alter the plasma metabolites in the adult rat offspring; however, differences in metabolites between male and female offspring occurred independently of dietary intervention.

Using metabolic profiling and gene expression analyses to explore molecular effects of replacing saturated fat with polyunsaturated fat-a randomized controlled dietary intervention study.

BACKGROUND: Replacing dietary saturated fatty acids (SFAs) with polyunsaturated fatty acids (PUFA) reduces the plasma low-density lipoprotein (LDL) cholesterol and subsequently the risk of cardiovascular disease. However, beyond changes in LDL cholesterol, we lack a complete understanding of the physiologic alterations that occur when improving dietary fat quality. OBJECTIVES: The aim of this study was to gain knowledge of metabolic alterations paralleling improvements in the fat quality of the diet. METHODS: We recently conducted an 8-wk, double-blind, randomized controlled trial replacing SFAs with PUFAs in healthy subjects with moderate hypercholesterolemia (n = 99). In the present substudy, we performed comprehensive metabolic profiling with multiple platforms (both nuclear magnetic resonance- and mass spectrometry-based technology) (n = 99), and analyzed peripheral blood mononuclear cell gene expression (n = 95) by quantitative real-time polymerase chain reaction. RESULTS: A large number of lipoprotein subclasses, myristoylcarnitine and palmitoylcarnitine, and kynurenine were reduced when SFAs were replaced with PUFAs. In contrast, bile acids, proprotein convertase subtilisin/kexin type 9, acetate, and acetoacetate were increased by the intervention. Some amino acids were also altered by the intervention. The mRNA levels of LXRA and LDLR were increased, in addition to several liver X receptor α target genes and genes involved in inflammation, whereas the mRNA levels of UCP2 and PPARD were decreased in peripheral blood mononuclear cells after replacing SFAs with PUFAs. Partial least squares-discriminant analysis showed that the 30 most important variables that contributed to class separation spanned all classes of biomarkers, and was in accordance with the univariate analysis. CONCLUSIONS: Applying metabolomics in randomized controlled dietary intervention trials has the potential to extend our knowledge of the biological and molecular effects of dietary fat quality. This study was registered at clinicaltrials.gov as NCT01679496.

Glutamate interactions with obesity, insulin resistance, cognition and gut microbiota composition.

AIMS: To investigate the interactions among fecal and plasma glutamate levels, insulin resistance cognition and gut microbiota composition in obese and non-obese subjects. METHODS: Gut microbiota composition (shotgun) and plasma and fecal glutamate, glutamine and acetate (NMR) were analyzed in a pilot study of obese and non-obese subjects (n = 35). Neuropsychological tests [Trail making test A (TMT-A) and Trail making test B (TMT-B)] scores measured cognitive information about processing speed, mental flexibility and executive function. RESULTS: Trail-making test score was significantly altered in obese compared with non-obese subjects. Fecal glutamate and glutamate/glutamine ratio tended to be lower among obese subjects while fecal glutamate/acetate ratio was negatively associated with BMI and TMT-A scores. Plasma glutamate/acetate ratio was negatively associated with TMT-B. The relative abundance (RA) of some bacterial families influenced glutamate levels, given the positive association of fecal glutamate/glutamine ratio with Corynebacteriaceae, Coriobacteriaceae and Burkholderiaceae RA. In contrast, Streptococaceae RA, that was significantly higher in obese subjects, negatively correlated with fecal glutamate/glutamine ratio. To close the circle, Coriobacteriaceae/Streptococaceae ratio and Corynebacteriaceae/Streptococaceae ratio were associated both with TMT-A scores and fecal glutamate/glutamine ratio. CONCLUSIONS: Gut microbiota composition is associated with processing speed and mental flexibility in part through changes in fecal and plasma glutamate metabolism.

NMR metabolomic study of blood plasma in ischemic and ischemically preconditioned rats: an increased level of ketone bodies and decreased content of glycolytic products 24 h after global cerebral ischemia

Cardiac arrest is one of the leading causes of death among adults in older age. Understanding mechanisms how organism responds to ischemia at global level is essential for the prevention and ischemic patient’s treatment. In this study, we used a global cerebral ischemia induced by four-vessel occlusion as an established animal model for ischemic stroke to investigate metabolic changes after 24 h reperfusion, when transitions occur due to the onset of delayed neuronal death. We also focused on the endogenous phenomenon known as ischemic tolerance by the pre-ischemic treatment. The experiments were carried out on blood plasma samples as easily available and metabolically reflecting the overall changes in injured organism. Our results imply that disturbed glycolysis pathway, as a consequence of ischemic injury, leads to the increased level of ketone bodies (acetone, acetoacetate and β-hydroxybutyrate) along with increased utilization of triacylglycerols in plasma of ischemic and ischemically preconditioned rats. Complementary to, a decreased level of glycolytic intermediates (lactate, pyruvate, acetate) with increased level of glucose was found in ischemic and preconditioned animals. The protective effect of ischemic preconditioning on metabolome recovery was demonstrated by significantly increased level of creatine compared to ischemic, non-preconditioned rats. We also document that acetoacetate, pyruvate, lactate, and leucine have the best discriminatory power between ischemic and control plasma. Conclusively, our results provide evidence that NMR spectra analysis can identify specific group of metabolites present in plasma with the capability for discrimination between individual groups of animals. In addition, an excellent feasibility for the statistical discrimination among ischemic, preconditioned, and control rats can be applied regardless of native or deproteinated plasma and also regardless of noesy or cpmg NMR acquisition

NMR metabolomic study of blood plasma in ischemic and ischemically preconditioned rats: an increased level of ketone bodies and decreased content of glycolytic products 24 h after global cerebral ischemia.

Cardiac arrest is one of the leading causes of death among adults in older age. Understanding mechanisms how organism responds to ischemia at global level is essential for the prevention and ischemic patient's treatment. In this study, we used a global cerebral ischemia induced by four-vessel occlusion as an established animal model for ischemic stroke to investigate metabolic changes after 24 h reperfusion, when transitions occur due to the onset of delayed neuronal death. We also focused on the endogenous phenomenon known as ischemic tolerance by the pre-ischemic treatment. The experiments were carried out on blood plasma samples as easily available and metabolically reflecting the overall changes in injured organism. Our results imply that disturbed glycolysis pathway, as a consequence of ischemic injury, leads to the increased level of ketone bodies (acetone, acetoacetate and ß-hydroxybutyrate) along with increased utilization of triacylglycerols in plasma of ischemic and ischemically preconditioned rats. Complementary to, a decreased level of glycolytic intermediates (lactate, pyruvate, acetate) with increased level of glucose was found in ischemic and preconditioned animals. The protective effect of ischemic preconditioning on metabolome recovery was demonstrated by significantly increased level of creatine compared to ischemic, non-preconditioned rats. We also document that acetoacetate, pyruvate, lactate, and leucine have the best discriminatory power between ischemic and control plasma. Conclusively, our results provide evidence that NMR spectra analysis can identify specific group of metabolites present in plasma with the capability for discrimination between individual groups of animals. In addition, an excellent feasibility for the statistical discrimination among ischemic, preconditioned, and control rats can be applied regardless of native or deproteinated plasma and also regardless of noesy or cpmg NMR acquisition.

GC-MS Analysis of Short-Chain Fatty Acids in Feces, Cecum Content, and Blood Samples.

Short-chain fatty acids, the end products of fermentation of dietary fibers by the gut microbiota, have been shown to exert multiple effects on mammalian metabolism. For the analysis of short-chain fatty acids, gas chromatography-mass spectrometry is a very powerful and reliable method. Here, we describe a fast, reliable, and reproducible method for the separation and quantification of short-chain fatty acids in mouse feces, cecum content, and blood samples (i.e., plasma or serum) using gas chromatography-mass spectrometry. The short-chain fatty acids analyzed include acetic acid, propionic acid, butyric acid, valeric acid, hexanoic acid, and heptanoic acid.

Plasma metabolic biomarkers for discriminating individuals with alcohol use disorders from social drinkers and alcohol-naive subjects.

BACKGROUND: Alcohol use disorders (AUD) is a phase of alcohol misuse in which the drinker consumes excessive amount of alcohol and have a continuous urge to consume alcohol which may lead to various health complications. The current methods of alcohol use disorders diagnosis such as questionnaires and some biomarkers lack specificity and sensitivity. Metabolomics is a novel scientific field which may provide a novel method for the diagnosis of AUD by using a sensitive and specific technique such as nuclear magnetic resonance (NMR). METHODS: A cross sectional study was conducted on three groups: individuals with alcohol use disorders (n=30), social drinkers (n=54) and alcohol-naive controls (n=60). 1H NMR-based metabolomics was used to obtain the metabolic profiles of plasma samples. Data were processed by multivariate principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) followed by univariate and multivariate logistic regressions to produce the best fit-model for discrimination between groups. RESULTS: The OPLS-DA model was able to distinguish between the AUD group and the other groups with high sensitivity, specificity and accuracy of 64.29%, 98.17% and 91.24% respectively. The logistic regression model identified two biomarkers in plasma (propionic acid and acetic acid) as being significantly associated with alcohol use disorders. The reproducibility of all biomarkers was excellent (0.81-1.0). CONCLUSIONS: The applied plasma metabolomics technique was able to differentiate the metabolites between AUD and the other groups. These metabolites are potential novel biomarkers for diagnosis of alcohol use disorders.

NMR Metabolomics Investigates the Influence of Flavonoid-Enriched Rations on Chicken Plasma.

The use of flavonoids as dietary supplements is well established, mainly due to their intense antioxidant and anti-inflammatory properties. In the present study, hesperidin, naringin, and vitamin E were used as additives at different concentrations in poultry rations in order to achieve meat of improved quality. NMR metabolomics was applied to chicken blood serum samples to discern whether and how the enriched rations affected the animals' metabolic profile. Variations in the metabolic patterns according to sustenance consumption were traced by orthogonal projections to latent structures discriminant analysis (OPLS-DA) models and were attributed to specific metabolites by using S-line plots. In particular, serum samples from chickens fed with vitamin E displayed higher concentrations of glycine and succinic acid compared to control samples, which were mainly characterized by betaine, formic acid, and lipoproteins. Samples from chickens fed with hesperidin were characterized by increased levels of lactic acid, citric acid, creatine, carnosine, creatinine, phosphocreatine, anserine, glucose, and alanine compared to control samples. Lastly, naringin samples exhibited increased levels of citric and acetic acids. Results verify the scalability of NMR metabolomics to highlight metabolite variations among chicken serum samples in relation to food rations.

Serum analysis by 1H nuclear magnetic resonance spectroscopy: a new tool for distinguishing neuromyelitis optica from multiple sclerosis.

BACKGROUND: Neuromyelitis optica (NMO) and multiple sclerosis (MS), two inflammatory demyelinating diseases, are characterized by different therapeutic strategies. Currently, the only biological diagnostic tool available to distinguish NMO from MS is the specific serum autoantibody that targets aquaporin 4, but its sensitivity is low. OBJECTIVE: To assess the diagnostic accuracy of metabolomic biomarker profiles in these two neurological conditions, compared to control patients. METHODS: We acquired serum spectra (47 MS, 44 NMO and 42 controls) using proton nuclear magnetic resonance ((1)H-NMR) spectroscopy. We used multivariate pattern recognition analysis to identify disease-specific metabolic profiles. RESULTS: The (1)H-NMR spectroscopic analysis evidenced two metabolites, originating probably from astrocytes, scyllo-inositol and acetate, as promising serum biomarkers of MS and NMO, respectively. In 87.8% of MS patients, scyllo-inositol increased 0.15 to 3-fold, compared to controls and in 74.3% of NMO patients, acetate increased 0.4 to 7-fold, compared to controls. Using these two metabolites simultaneously, we can discriminate MS versus NMO patients (sensitivity, 94.3%; specificity, 90.2%). CONCLUSION: This study demonstrates the potential of (1)H-NMR spectroscopy of serum as a novel, promising analytical tool to discriminate populations of patients affected by NMO or MS.

Fasting serum concentration of short-chain fatty acids in subjects with microscopic colitis and celiac disease: no difference compared with controls, but between genders.

BACKGROUND: Short-chain fatty acids (SCFAs), particularly propionic and butyric acids, have been shown to have many positive health effects. The amount and type of SCFAs formed from dietary fibre by the colonic microbiota depends on the substrate available and is reflected in blood. The total intake and type of dietary fibre in people with gastrointestinal diseases differs considerably from healthy subjects. OBJECTIVE: To compare fasting SCFA concentrations in subjects with microscopic colitis (MC), celiac disease and controls without these diseases. SCFAs were also analysed over 6.5 h in young healthy subjects, who had eaten a fibre-rich breakfast, to identify a possible peak concentration of SCFAs after a meal. METHODS: SCFAs in serum were pre-concentrated using hollow fibre-supported liquid membrane extraction and gas chromatography. RESULTS: The MC group had a higher concentration of valeric acid than the control group (p < 0.01). No significant differences in other SCFA concentrations were seen between groups, but the control group tended to have higher concentration of acetic acid (p = 0.1). Furthermore, males had higher concentrations of SCFAs (with the exception of valeric acid) than females (p < 0.05), which were independent of groups. The peaks for acetic, propionic and butyric acids came approximately 5 h, 6.5 h and 2-3 h, respectively, after breakfast. CONCLUSION: The fasting concentrations of SCFAs were quite similar, although the fibre intake had probably been quite different for a long time. The results might have been different if SCFAs had been recorded over a longer period.

Propionic and butyric acids, formed in the caecum of rats fed highly fermentable dietary fibre, are reflected in portal and aortic serum.

SCFA are important end products formed during colonic fermentation of dietary fibre (DF). It has been suggested that propionic and butyric acids affect metabolic parameters, low-grade systemic inflammation, insulin resistance and obesity. The aim of the present study was to investigate whether the various SCFA profiles observed after fermentation in the caecum of rats fed pectin, guar gum and fructo-oligosaccharides (FOS) were also represented in hepatic portal and aortic serum. The SCFA in serum were extracted using hollow fibre-supported liquid membrane extraction before GLC analysis. The concentrations of acetic, propionic and butyric acids in caecal content correlated well with those in portal serum (P< 0·001) for all the three diets. A weaker correlation was found for propionic and butyric acids between the caecal content and aortic serum (P< 0·05). Butyric acid concentration in caecal content was also reflected in the aortic serum (P= 0·019) of rats fed FOS. FOS gave rather low amounts of the SCFA, especially butyric acid, but caecal tissue weight was higher with FOS than with the other two diets. This may be explained by rapid fermentation and quick utilisation/absorption of the SCFA. The present study also showed that propionic acid was metabolised/utilised to a higher extent than butyric acid by colonocytes before reaching the liver. We conclude that the formation of propionic and butyric acids in the caecum is reflected by increased concentrations in the aortic blood. This approach may therefore simplify the evaluation and study of SCFA from DF in human subjects.

Selective cannabinoid-1 receptor blockade benefits fatty acid and triglyceride metabolism significantly in weight-stable nonhuman primates.

The goal of this study was to determine whether administration of the CB1 cannabinoid receptor antagonist rimonabant would alter fatty acid flux in nonhuman primates. Five adult baboons (Papio Sp) aged 12.1 ± 4.7 yr (body weight: 31.9 ± 2.1 kg) underwent repeated metabolic tests to determine fatty acid and TG flux before and after 7 wk of treatment with rimonabant (15 mg/day). Animals were fed ad libitum diets, and stable isotopes were administered via diet (d31-tripalmitin) and intravenously (¹³C4-palmitate, ¹³C1-acetate). Plasma was collected in the fed and fasted states, and blood lipids were analyzed by GC-MS. DEXA was used to assess body composition and a hyperinsulinemic euglycemic clamp used to assess insulin-mediated glucose disposal. During the study, no changes were observed in food intake, body weight, plasma, and tissue endocannabinoid concentrations or the quantity of liver-TG fatty acids originating from de novo lipogenesis (19 ± 6 vs. 16 ± 5%, for pre- and posttreatment, respectively, P = 0.39). However, waist circumference was significantly reduced 4% in the treated animals (P < 0.04), glucose disposal increased 30% (P = 0.03), and FFA turnover increased 37% (P = 0.02). The faster FFA flux was consistent with a 43% reduction in these fatty acids used for TRL-TG synthesis (40 ± 3 vs. 23 ± 4%, P = 0.02) and a twofold increase in TRL-TG turnover (1.5 ± 0.9 vs. 3.1 ± 1.4 µmol·kg⁻¹·h⁻¹, P = 0.03). These data support the potential for a strong effect of CB1 receptor antagonism at the level of adipose tissue, resulting in improvements in fasting turnover of fatty acids at the whole body level, central adipose storage, and significant improvements in glucose homeostasis.

Dermal and pulmonary absorption of ethanol from alcohol-based hand rub.

BACKGROUND: Ethanol intoxication of healthcare workers (HCWs) using alcohol-based hand rubs (ABHRs) in the workplace is a potentially serious issue. This study quantified the level of ethanol absorption among HCWs after hygienic hand disinfection. METHODS: Eighty-six HCWs from Nancy University Hospital were tested before and after a 4-h shift. Participants used ABHR containing 70% ethanol. Levels of ethanol, acetaldehyde and acetate in blood and urine were determined using gas chromatography. A breathalyzer was used to measure the level of ethanol in expired air. RESULTS: Ethanol [mean concentration 0.076 (standard deviation 0.05) mg/L] was detected in the expired air of 28 HCWs 1-2 min post exposure. Ethanol, acetaldehyde and acetate were undetectable in blood after a 4-h shift, and urine tests were negative in all participants. CONCLUSION: Ethanol exposure from ABHR, particularly inhalation of vapours, resulted in positive breathalyzer readings 1-2 min after exposure. Dermal absorption of ethanol was not detected. Pulmonary absorption was detected but was below toxic levels.

NMR-based metabonomics reveals relationship between pre-slaughter exercise stress, the plasma metabolite profile at time of slaughter, and water-holding capacity in pigs.

Nuclear magnetic resonance (NMR)-based metabonomics was applied to investigate the effects of pre-slaughter exercise stress on the plasma metabolite profile at time of slaughter. The study included a total of 40 slaughter pigs, which were exposed to one of the following treatments: No pre-slaughter stress (control treatment), pre-slaughter exercise on a treadmill and subsequently 0, 1, or 3h rest prior to slaughter. NMR-based metabonomics revealed a clear difference in the plasma metabolite profile at time of slaughter between control pigs and pigs exercised without rest, which mainly could be ascribed to increased plasma lactate due to exercise. A resting period of 1 or 3h prior to slaughter reversed the stress-induced perturbations in the plasma metabolite profile. The plasma metabolite profile at time of slaughter was highly correlated with muscle temperature 1 min post-mortem, and a correlation to WHC was also demonstrated. Lactate was found to be the metabolite of importance for the association between the plasma metabolome and pH, temperature and WHC.

SIAM-Like phenomenon caused by low doses of alcohol.

BACKGROUND: Swift increase in alcohol metabolism (SIAM) is usually evoked by a large dose of ethanol, which is often demonstrated by an abrupt increase in oxygen uptake. SIAM was induced by low doses of ethanol and evaluated by pharmacokinetic analyses of ethanol and its metabolites. METHODS: Rabbits were initially administered 1.0 g/kg of ethanol solution and the same dose was given to the bolus group 6 hours after the first injection. The infusion group was administered 0.25 g/kg/h of ethanol 2 hours after the first injection. Blood concentrations of ethanol, acetaldehyde, and acetate were then determined and comparisons were made using pharmacokinetic parameters. RESULTS: A significantly higher ethanol elimination rate was observed after re-administration of ethanol to the bolus group. Other pharmacokinetic parameters were unaffected. The concentration at steady state (Css) for the infusion group was stable. A significantly higher level of mean residence time (MRT) in blood acetaldehyde was observed for the bolus group, whereas no MRT changes were observed for the infusion group. A significantly higher level of blood acetate Css was observed after re-administration of ethanol to the bolus group, following the changes in area under concentration and MRT. No Css changes were observed for the infusion group. The Css of acetate at stage 2 was significantly higher for the bolus group, compared to the infusion group. CONCLUSION: Low doses of ethanol enhanced alcohol metabolism in rabbits, according to a pharmacokinetic analysis of circulating ethanol concentrations. Simultaneous analyses of its metabolites followed the kinetic of ethanol.

Short chain fatty acids exchange: Is the cirrhotic, dysfunctional liver still able to clear them?

BACKGROUND & AIMS: Prebiotics are increasingly used to improve gut integrity. A presumed mechanism of their beneficial action is the synthesis of short chain fatty acids (SCFA: acetate, propionate and butyrate). High systemic concentrations of propionate and butyrate are toxic and can adversely affect the patient. In physiological situations the liver uses propionate and butyrate for energy metabolism. The aim of the present study was to investigate to which extent patients with liver cirrhosis are still able to metabolize portal derived SCFA in the liver. METHODS: Twelve patients with liver cirrhosis and an intrahepatic portosystemic shunt (TIPSS) were studied. Blood was sampled from the femoral artery, portal and hepatic vein. Organ plasma flow was measured. Net release or uptake was calculated by multiplying the arteriovenous differences by plasma flow. SCFA plasma concentrations were measured using LC-MS. RESULTS: Arterial concentrations were 124+/-12, 8+/-1 and 10+/-1micromol/l for acetate, propionate and butyrate, respectively. The gut produced 32.5+/-13.0, 4.8+/-1.3 and 6.2+/-2.1micromolkgbw(-1)h(-1) of acetate, propionate and butyrate, respectively. Assuming 70% portosystemic shunting, hepatic uptake of propionate and butyrate was 3.1+/-0.9 and 5.2+/-1.4micromolkgbw(-1)h(-1). Hepatic uptake of acetate was non significant (12.1+/-12.3micromolkgbw(-1)min(-1)). As a consequence of shunting, part of total acetate escaped from the splanchnic bed, which equalled 34.9+/-14.7micromolkgbw(-1)h(-1). CONCLUSION: The liver of patients with stable cirrhosis is able to use butyrate and propionate, most likely preventing increased systemic concentrations. This suggests that prebiotics can be administered safely, but monitoring butyrate levels may be advisable in patients with diminished liver function.

Continuous lactation in dairy cows: effect on milk production and mammary nutrient supply and extraction.

Reports over the past decade have indicated that normal lactational performance can be achieved in genetically superior and high-producing dairy cows, even when the dry period between 2 lactations is omitted. The hypothesis tested in this experiment was that normal lactogenesis I and metabolic function may be achievable in continuously milked high-yielding dairy cows as a result of the genetic selection for lactation performance and hence longevity of mammary epithelial cells. The milk production and mammary nutrient uptake in response to omission of the dry period for cows with an expected peak milk yield higher than 45 kg/d were studied in 28 Holstein dairy cows managed without bovine somatotropin. Performance and metabolic parameters were followed in late gestation and in the following early lactation. Fourteen cows were milked continuously throughout late gestation, and another 14 dairy cows experienced a 7-wk prepartum dry period. Continuous milking during the prepartum period reduced milk production in the following early lactation period by >20%. The reduced milk production could not be readily ascribed to inefficiency of the mechanisms responsible for nutrient uptake by the lactating mammary epithelial cells, nor to systemic endocrine changes. This suggests that lowered mammary nutrient uptake must have been associated with reduced mammary blood flow, metabolic activity, or both, most likely as a result of disturbed lactogenesis I prepartum or lactogenesis II postpartum triggered by as yet unknown local mechanisms. Milk protein content was elevated by 0.4 percentage units in the continuously milked cows. The underlying reason is unknown, but given the current pricing system for milk, it deserves to be further investigated.

[Metabonomic study of syndrome of liver qi stagnation and spleen deficiency in rats].

OBJECTIVE: To determine the changes of the plasma metabolic phenotype in rats with chronic restraint stress (rats with syndrome of liver qi stagnation and spleen deficiency), so as to reveal the biological features of the syndrome of liver qi stagnation and spleen deficiency, and to examine potential application of metabonomic analysis in studies of syndromes in traditional Chinese medicine. METHODS: Twenty-four male Sprague-Dawley rats were randomly divided into four groups: group A, 7 d normal control group; group B, 21 d normal control group; group C, 7 d stress group; and group D, 21 d stress group, with 6 rats in each group. Chronic restraint was used to induce stress in rats. Blood was collected from the cardio-ventricle under anesthesia on the 8th (groups A and C) or 22nd day (groups B and D) and detected by using the Fourier variable superconducting nuclear magnetic resonance (NMR) spectrometer (Varian UnityInova 600 M). Free induction decay signals were transferred into one-dimensional NMR spectrogram via 32 k Fourier transformation. Segmental integral calculus (0.04 ppm per segment) was performed from 4.5-0.5 ppm (Carr-Purcell-Meiboom-Gill, CPMG) or 6.0-0 ppm (longitudinal eddy-delay, LED) as defaulted 1H spectra values by using the VNMR software. Data were saved as text or excel files after normalization and then used for pattern recognition analyses. All the data were analyzed by principal component analysis (PCA) using the SIMCA-P 10.0 software (Umetrics AB, Umea, Sweden). RESULTS: The PCA analysis of rat plasma (1)H NMR spectra revealed different metabolic spectra between stress and control groups, which were consistent with alterations of in vivo metabolisms in rats under stress stimuli. Compared with the normal control group, rats with repeated stress displayed significant changes in spectral peak shapes of acetate, lactate, tyrosine, low-density lipoprotein, and unknown compounds (3.44 ppm). These altered metabolites can be used as biomarkers of syndrome of liver qi stagnation and spleen deficiency for further studies. CONCLUSION: The (1)H NMR spectra of metabolites in the rat blood are differentially changed after chronic stress. Specific, characteristic metabolic products can be identified by analyses of metabonomics, which lead to interpretation of biological feature of Chinese medicine syndromes. Therefore, metabonomic analysis is an approach with good development prospects to studies of TCM syndromes.

Effect of beer ingestion on the plasma concentrations and urinary excretion of purine bases: one-month study.

To investigate the effect of long-term beer ingestion on the plasma concentrations and urinary excretion of purine bases, 5 healthy males participated in the present study, during which they ingested beer every evening for 30 days. Blood and 24-hour urine samples were collected in the morning one day before and 14 and 30 days after the initiation of the beer ingestion. During the beer ingestion period, the plasma concentration and the urinary excretion of uric acid were increased significantly, while uric acid clearance was not decreased. Further, purine ingestion was not significantly different throughout the study. These results suggest that production of uric acid by ethanol ingestion was the main contributor to the increased plasma uric acid. Therefore, patients with gout should be encouraged to avoid drinking large amounts of beer on a daily basis.

D-lactate production and excretion in diarrheic calves.

The origin of D-lactate, the most important acid contributing to metabolic acidosis in the diarrheic calf, is unknown. We hypothesized that because D-lactate is produced only by microbes, gastrointestinal fermentation is the source. The objective of this study was to determine whether D-lactate production occurs in the rumen, colon, or both, and to measure D- and L-lactate concentrations in urine. Fecal, rumen, blood, and urine samples were obtained from 16 diarrheic and 11 healthy calves. Serum electrolyte concentrations were measured in both groups, and blood gas analyses were performed for diarrheic calves. All samples were analyzed for D- and L-lactate by high performance liquid chromatography (HPLC). Diarrheic calves were generally hyperkalemic with high serum anion gap, depressed serum bicarbonate, and low blood pH. L-lactate was markedly higher in rumen contents (22.7 mmol/ L [median]) and feces (8.6 mmol/L) of diarrheic calves than healthy calves (0.5 mmol/L and 5.1 mmol/L, respectively), but not different in serum or urine. Rumen, fecal, serum, and urine D-lactate concentrations were all significantly higher (P < .05) in diarrheic calves (17.0, 25.4, 13.9, and 19.2 mmol/L, respectively) than in healthy calves (0.5, 9.1, 1.4, and 0.5 mmol/L, respectively). Higher D-lactate concentrations in the rumen and feces of diarrheic calves suggests these sites as the source of D-lactate in blood and urine.