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We have investigated the urinary and plasma metabolic phenotype of acute pancreatitis (AP) patients presenting to the emergency room at a single center London teaching hospital with acute abdominal pain using (1)H NMR spectroscopy and multivariate modeling. Patients were allocated to either the AP (n = 15) or non-AP patients group (all other causes of abdominal pain, n = 21) on the basis of the national guidelines. Patients were assessed for three clinical outcomes: (1) diagnosis of AP, (2) etiology of AP caused by alcohol consumption and cholelithiasis, and (3) AP severity based on the Glasgow score. Samples from AP patients were characterized by high levels of urinary ketone bodies, glucose, plasma choline and lipid, and relatively low levels of urinary hippurate, creatine and plasma-branched chain amino acids. AP could be reliably identified with a high degree of sensitivity and specificity (OPLS-DA model R(2) = 0.76 and Q(2)Y = 0.59) using panel of discriminatory biomarkers consisting of guanine, hippurate and creatine (urine), and valine, alanine and lipoproteins (plasma). Metabolic phenotyping was also able to distinguish between cholelithiasis and colonic inflammation among the heterogeneous non-AP group. This work has demonstrated that combinatorial biomarkers have a strong diagnostic and prognostic potential in AP with relevance to clinical decision making in the emergency unit.

Significant advances in understanding aging have been achieved through studying model organisms with extended healthy lifespans. Employing 1H NMR spectroscopy, we characterized the plasma metabolic phenotype (metabotype) of three long-lived murine models: 30% dietary restricted (DR), insulin receptor substrate 1 null (Irs1–/–), and Ames dwarf (Prop1df/df). A panel of metabolic differences were generated for each model relative to their controls, and subsequently, the three long-lived models were compared to one another. Concentrations of mobile very low density lipoproteins, trimethylamine, and choline were significantly decreased in the plasma of all three models. Metabolites including glucose, choline, glycerophosphocholine, and various lipids were significantly reduced, while acetoacetate, d-3-hydroxybutyrate and trimethylamine-N-oxide levels were increased in DR compared to ad libitum fed controls. Plasma lipids and glycerophosphocholine were also decreased in Irs1–/– mice compared to controls, as were methionine and citrate. In contrast, high density lipoproteins and glycerophosphocholine were increased in Ames dwarf mice, as were methionine and citrate. Pairwise comparisons indicated that differences existed between the metabotypes of the different long-lived mice models. Irs1–/– mice, for example, had elevated glucose, acetate, acetone, and creatine but lower methionine relative to DR mice and Ames dwarfs. Our study identified several potential candidate biomarkers directionally altered across all three models that may be predictive of longevity but also identified differences in the metabolic signatures. This comparative approach suggests that the metabolic networks underlying lifespan extension may not be exactly the same for each model of longevity and is consistent with multifactorial control of the aging process.

The biochemical effects of acute and chronic psychological stress have been investigated in male Sprague-Dawley rats using a combination of 1H NMR spectral analysis of plasma and conventional hematological analyses. Animals were subjected to 35 consecutive days of 6-h sessions of stress, and following a 9 day break, were stressed for a further 6-h period. Plasma samples were collected at 0, 1, 3, and 6 h on days 1, 9, 21, 35, and 44, measured using 600 MHz 1H NMR spectroscopy, and analyzed by Principal Components Analysis. Time-dependent biochemical effects of psychological stress on a range of endogenous metabolites were evident and were correlated with the intensity of the stress response as defined by corticosterone and hematological parameters. Following acute stress, increases in the levels of glucose and ketone bodies, and decreases in the levels of acetate, alanine, isoleucine, lactate, leucine, valine, and lipoproteins, were observed. Chronic stress-induced increases in plasma levels of alanine, lactate (day 9), and leucine, valine, and choline (day 44) and decreases in acetate (day 9) and lipoprotein concentrations were observed. Positive correlations between plasma corticosterone level and glucose and glycerol, and between plasma lipoprotein concentrations and hemoglobin levels, were established using Projection to Latent Structures (PLS) analysis. This study indicates the potential of using NMR-based metabonomic strategies for the characterization of endogenous metabolic perturbations induced by psychological stressors and lifestyle choices.

Characterizing the relationships between genomic and phenotypic variation is essential to understanding disease etiology. Information-dense data sets derived from pathophysiological1, proteomic2,3 and transcriptomic4 profiling have been applied to map quantitative trait loci (QTLs). Metabolic traits, already used in QTL studies in plants5, are essential phenotypes in mammalian genetics to define disease biomarkers. Using a complex mammalian system, here we show chromosomal mapping of untargeted plasma metabolic fingerprints derived from NMR spectroscopic analysis6 in a cross between diabetic and control rats. We propose candidate metabolites for the most significant QTLs. Metabolite profiling in congenic strains provided evidence of QTL replication. Linkage to a gut microbial metabolite (benzoate) can be explained by deletion of a uridine diphosphate glucuronosyltransferase. Mapping metabotypic QTLs provides a practical approach to understanding genome-phenotype relationships in mammals and may uncover deeper biological complexity, as extended genome7 (microbiome) perturbations that affect disease processes through transgenomic effects8 may influence QTL detection.

Caloric restriction (CR) increases healthy life span in a range of organisms. The underlying mechanisms are not understood but appear to include changes in gene expression, protein function, and metabolism. Recent studies demonstrate that acute CR alters mortality rates within days in flies. Multitissue transcriptional changes and concomitant metabolic responses to acute CR have not been described. We generated whole genome RNA transcript profiles in liver, skeletal muscle, colon, and hypothalamus and simultaneously measured plasma metabolites using proton nuclear magnetic resonance in mice subjected to acute CR. Liver and muscle showed increased gene expressions associated with fatty acid metabolism and a reduction in those involved in hepatic lipid biosynthesis. Glucogenic amino acids increased in plasma, and gene expression for hepatic gluconeogenesis was enhanced. Increased expression of genes for hormone-mediated signaling and decreased expression of genes involved in protein binding and development occurred in hypothalamus. Cell proliferation genes were decreased and cellular transport genes increased in colon. Acute CR captured many, but not all, hepatic transcriptional changes of long-term CR. Our findings demonstrate a clear transcriptional response across multiple tissues during acute CR, with congruent plasma metabolite changes. Liver and muscle switched gene expression away from energetically expensive biosynthetic processes toward energy conservation and utilization processes, including fatty acid metabolism and gluconeogenesis. Both muscle and colon switched gene expression away from cellular proliferation. Mice undergoing acute CR rapidly adopt many transcriptional and metabolic changes of long-term CR, suggesting that the beneficial effects of CR may require only a short-term reduction in caloric intake.

Here, we study the intricate relationship between gut microbiota and host cometabolic phenotypes associated with dietary-induced impaired glucose homeostasis and nonalcoholic fatty liver disease (NAFLD) in a mouse strain (129S6) known to be susceptible to these disease traits, using plasma and urine metabotyping, achieved by 1 H NMR spectroscopy. Multivariate statistical modeling of the spectra shows that the genetic predisposition of the 129S6 mouse to impaired glucose homeostasis and NAFLD is associated with disruptions of choline metabolism, i.e., low circulating levels of plasma phosphatidylcholine and high urinary excretion of methylamines (dimethylamine, trimethylamine, and trimethylamine- N -oxide), coprocessed by symbiotic gut microbiota and mammalian enzyme systems. Conversion of choline into methylamines by microbiota in strain 129S6 on a high-fat diet reduces the bioavailability of choline and mimics the effect of choline-deficient diets, causing NAFLD. These data also indicate that gut microbiota may play an active role in the development of insulin resistance.

Partially purified lamb pregastric lipase, LPGL, isolated from the tongue and epiglottal region of lamb, was used to catalyze the hydrolysis of tributyrylglycerol, the dibutyryl esters of propanediol and the butyryl esters of propanol at pH 6.5, 35°C. The relative rates of hydrolysis for tributyrylglycerol: dibutyryl-1,2-propanediol: dibutyryl-1,3-propanediol: butyryl-1-propanol: butyryl-2-propanol were 100: 35.4: 12.5: 4.1: 1.1, respectively. Monohydrolysis stoichiometry was observed for tributyrylglycerol and the diesters with saturation kinetics. In contrast, hydrolysis of the monoesters was first-order in substrate. The catalyzed hydrolysis of tributyrylglycerol was ~20% inhibited in the presence of 100 mM EDTA, 10% and 60% in the presence of 1 M NaCl and LiCl, respectively, and ~30% in 10% v/v ethanol.

The historical importance of pregastric enzymes in cheese-making is reviewed and the potential for extending their use is discussed in terms of requiring an understanding of their physicochemical parameters. Commericial extracts from the tongues and epiglotti of suckling lambs and calves and adult goats have been processed to yield partially purified samples of the primary pregastric lipase (PGL). The N-terminal sequence and molecular weight of lamb PGL have been determined. The activity of lamb and goat PGLs against tributyrin has been determined over a range of pH and temperature values. Optimum conditions were pH 6.4, 43°C, and pH 6.0, 52°C, for lamb and goat PGL respectively. The possible influence of the development of a ruminant multi-chambered stomach on the difference in optimal temperature is discussed. A lengthening of the carboxylic acid chain of homoacid triglycerides causes a decrease in hydrolytic activity of lamb PGL but in all cases only a single free fatty acid was released. Against a series of 4-nitrophenylalkanoate esters, maximum activity was observed against the decanoate ester but, in contrast to hydrolysis of the acetate ester which exhibited full Michaelis-Menten kinetics with increasing substrate concentration, activity against the decanoate ester was restricted to the monomeric substrate. Taurocholate inhibits the activity of lamb PGL at concentrations >8 mM. Values of pK2 equal to 6.69 and 7.92 respectively have been determined for lamb PGL. Attempts to interesterify coconut oil and cocoa butter, and tributyrin and tricaprylin, catalysed by calf PGL were unsuccessful, although positive results obtained using Candida cylindracea encourage further investigation of alternative methods for immobilizing the PGL. Finally, anhydrous milk fat has been hydrolysed by calf, lamb and goat PGLs and the differences in relative amounts of released free fatty acids have been used to explain the differences in taste which arise when Parmesan cheese is produced using different sources of PGL.

Three lamb pregastric enzymes, isolated from the commercial extract from the tongue and epiglottal region of lamb, have been used to catalyze the hydrolysis of a series of 4-nitrophenylalkanoate esters (C2–C12) at 37°C, pH 7.2 and maximum activity was obtained against the decanoate ester in all cases. Burst kinetics were observed for activity of the principal lipase component against the decanoate ester. This enzyme was also used as a catalyst for the hydrolysis of monoacid triglycerides (C4:0 to C10:0) at 35°C, pH 6.5 and maximum activity was obtained against tributyrin (C4:0). A suggestion is made for orientation of ester substrates within the active site of the enzymes.

A partially purified pregastric lipase from lambs was used as a catalyst for the hydrolysis of tributyroylglycerol. The reaction was followed by pH-stat autotitration of the released butyric acid. Integral values for pH and temperature were selected for determination of full Michaelis-Menten plots, and a kinetic surface was derived to characterize the activity of the enzyme preparation over a wide combination of these conditions. This surface enclosed a region of maximum activity centered on optimal pH (pH 6.4) and temperature (43°C), although optima were not sharply delineated. The activity in the surrounding region was generally high, and the value of the Michaelis-Menten constant was < 0.2 mM, indicating that tributyroylglycerol was generally a highly favored substrate.

Lamb pregastric lipase, extracted from the tongue and epiglottal region of lamb, has been partially purified and used to catalyse the hydrolysis of (9–10 3 H) (1,2,3-tris-[( cis )-9-octadecenoyl]glycerol) over the pH range 5.50–7.50 and temperature range 20.0–40.0°C. Michaelis-Menten plots were constructed for each reaction condition, and allowed evaluation of K m and k cat . The values of k cat have been fitted to a three-dimensional activity profile. The optimum pH for reactivity was 6.3 ± 0.3 and the optimum temperature 32 ± 3°C. Under optimum conditions the values of k cat and K m were 0.073 μmol · min −1 · mg −1 and 9 mM, respectively. The reaction has also been studied in D 2 O as solvent at pD = 6.50 and T = 30.0°C. The kinetic isotope effects were 1.46 and 1.31 for the rate determining acylation and deacylation steps, respectively. The activity of the enzyme was inhibited by the presence of the bile salt, sodium taurocholate.

The widely-used blood anticoagulants citrate and EDTA give rise to prominent peaks in (1)H NMR spectra of plasma samples collected in epidemiological and clinical studies, and these cause varying levels of interference in recovering biochemical information on endogenous metabolites. To investigate both the potential metabolic information loss caused by these substances and any possible inter-molecular interactions between the anticoagulants and endogenous components, the (1)H NMR spectra of 40 split human plasma samples collected from 20 individuals into either citrate or EDTA have been analysed. Endogenous metabolite peaks were selectively obscured by large citrate peaks or those from free EDTA and its calcium and magnesium complexes. It is shown that the endogenous metabolites that give rise to peaks obscured by those from EDTA or citrate almost invariably also have other resonances that allow their identification and potential quantitation. Also, metabolic information recovery could be maximised by use of spectral editing techniques such as spin-echo, diffusion-editing and J-resolved experiments. The NMR spectral effects of any interactions between the added citrate or EDTA and endogenous components were found to be negligible. Finally, identification of split samples was feasible using simple multivariate statistical approaches such as principal components analysis. Thus even when legacy epidemiological plasma samples have been collected using the NMR-inappropriate citrate or EDTA anticoagulants, useful biochemical information can still be recovered effectively.

Background Metabolic profiling of biofluid specimens is an established method for investigating disease states in clinical studies but is only recently being applied to large-scale human population studies. As part of protocol development for the UK Biobank study, a (1)H nuclear magnetic resonance (NMR)-based metabonomic analysis of specimen storage effects and analytical reproducibility was carried out using urine and serum specimens from 40 volunteers. Methods Aliquots of each specimen were stored for t = 0 and t = 24 h at 4 degrees C prior to freezing, and in the case of serum samples for a further 12 h (t = 36), to determine whether the storage times affected specimen composition and quality. A blinded split-specimen matching exercise was implemented to assign candidate spectral pairs stored for different times using multivariate statistical analysis of the NMR data. Results Using a chemometric strategy, split specimens at time t = 0 and t = 24 or 36 h after storage at 4 degrees C were easily paired and the split-specimen matching task was reduced to a workable size. (1)H NMR profiling established that the t = 24 h urine and serum groups showed no systematic metabolite changes, indicating biochemical stability. Some small differences in serum specimens stored for t = 36 h at 4 degrees C were detectable only by multivariate analysis, and were attributed to generalized alterations in proteins and protein fragments, and possibly trimethylamine-N-oxide. No other specific metabolite was implicated. Conclusions For the purposes of NMR-based analysis, storage of urine and serum for up to t = 24 h at 4 degrees C does not detectably affect the metabolic profile and the methodology is robust. Future application of multivariate methods to data-rich studies should substantially enhance information recovery from epidemiological studies.

The objective was to determine whether the ini- tial pregastric lipase catalyzed hydrolysis of a triacylglycerol to 1,2(2,3)-diacylglycerol was a consequence of sn-specific hy- drolysis. The identity of the reaction products for the enzyme- assisted hydrolysis and uncatalyzed acyl-transfer reaction se- quence of tributyrylglycerol was assigned by 13 C nuclear mag- netic resonance. The optical activity of the product 1,2-dibutyryl-sn-glycerol (yield >50%, pH 6.5, 35°C, 13 min) was solvent dependent, being −2.92° (c ~1.3, CHCl 3 ) and +3.32° (c ~1.2, pyridine), and confirmation of sn-3 specificity by pregastric lipase was obtained. JAOCS 75, 1061-1062 (1998).

13C nuclear magnetic resonance (n.m.r.) spectra were obtained at 50 and 100 MHz for mixtures of propanediol, propane-1,2-diol dibutyrate, propane-1,2-diol 1-butyrate and propane-1,2-diol 2-butyrate in CDCl3. Distortionless enhancement by polarization transfer (DEPT) and two-dimensional 13C–1H correlation spectroscopy were used to confirm shift assignments. Spectra for the 1- and 2-monoesters showed strongly different inductive effects due to the position of the butyryl chain. These experiments demonstrate the desirability of using 13C n.m.r. rather than 1H n.m.r. spectroscopy in quantitative speciation of acylated diol complex hydrolysis mixtures.

The speciation of mixed butyrylglycerols (glycerol butyrates) and propanediol butyrate esters in the product mix from lamb pregastric lipase-catalysed hydrolysis of tributyrylglycerol and propane-1,2-diol dibutyrate has been examined by 13C n.m.r. spectroscopy. Samples from the quenched reaction mixture were extracted and allowed to stand in emulsion systems made up in bis tris propane buffer or water, pH 7·0, and in the absence of enzyme. There is clear evidence of uncatalysed conversion of rac-1,2-dibutyrylglycerol into the 1,3-isomer to form an equilibrium mixture containing c. 60–67% 1,3-isomer, and of conversion of propane-1,2-diol 1-butyrate into propane-1,2-diol 2-butyrate to form an equilibrium mixture containing c. 67% 2-monoester. Conversion kinetics to reach equilibrium are first order. Rate constants for acyl transfer of the diacylglycerol are 0·48 h-1 (in water) and 0·68 h-1 (in buffer) at 50°C, while those for acyl transfer of the 1-monoester are 0·72 h-1 (50°C) and 0·35 h-1 (35°C).