Paul A.G. Butler

Assessment of erythrocyte phospholipid fatty acid composition as a biomarker for dietary MUFA, PUFA or saturated fatty acid intake in a controlled cross-over intervention trial

BackgroundDietary intervention trials rely on self-reported measures of intake for assessment of energy and macronutrient composition. Dietary fat intake is of particular interest due to strong associations with pathophysiology. In epidemiological trials phospholipid fatty acid composition may reflect composition of habitual diet, although strong correlations have been identified only for essential polyunsaturated fatty acids (PUFAs). Preliminary evidence shows that saturated fatty acids (SFA) C15:0 and C17:0 may be acceptable biomarkers. This study measured changes in erythrocyte membrane fatty acids during a period of strictly controlled fat feeding to investigate their use as a short-term marker of compliance, particularly for intake of SFAs.ResultsThis was a randomised cross-over trial in which diet was provided and strictly controlled. 20 healthy, male subjects were given a 40 energy % (en%) fat diet, high in saturated (high-SFA, 20 en%) or unsaturated (high-USFA, 24 en%) fatty acids for 2 periods of 3 weeks. Subjects were residential during intervention with all food and beverages provided. Dietary composition was verified by direct chemical analysis. Blood samples were collected on days 1,7,14, 21 and analysed for red blood cell (RBC) membrane fatty acid composition. Pearson correlation showed RBC fatty acid composition to mimic dietary composition by 3 weeks, but the relationships were weak. Of the SFAs only RBC C16:0 decreased in response to decreased dietary content on high-USFA treatment (ANOVA, diet, P < 0.05). Of the USFAs, higher levels of C18:1 MUFA, C20:4 and C22:6 long chain PUFA on high-USFA diet lead to higher C18:1, C20:4 and C22:6 within RBCs (ANOVA, time*diet, P < 0.05). Pearsons correlation was significant between dietary and RBC fatty acids during the 21d dietary manipulation for C18:1, and C20:5, C22:6 only (P < 0.05).ConclusionRBC membrane fatty acids cannot reliably be used as an independent measure of compliance for dietary SFA intake in short-term studies. The MUFA oleic acid and PUFAs EPA and DHA may be more useful as markers of compliance during short term intervention trials.

Influence of Grape-Harvesting Steps on Varietal Thiol Aromas in Sauvignon blanc Wines

The intense tropical fruit aroma of Sauvignon blanc wines has been associated with the varietal thiols 3-mercaptohexanol (3MH), derived from odorless precursors in the grape, and 3-mercaptohexyl acetate (3MHA), arising from 3MH during fermentation. Grapes and juice were sourced from five locations in Marlborough, New Zealand, taking hand-picked grapes and samples at four stages during the mechanical harvesting process and pressing, which were then fermented in replicated 750 mL bottles. With each set of juices, the highest concentrations of Cys-3MH and Glut-3MH were found in the juices pressed to 1 bar, but these juices produced wines with lower 3MH and 3MHA concentrations. With three of the juices, there was an increase in varietal thiol content for wines made from juices that had been machine harvested compared to the hand-picked samples, which matched earlier findings of lower 3MH and 3MHA levels in wines made from hand-picked grapes. Juices that were more oxidized, and which showed a higher absorbance at 420 nm, were found to produce wines with lower 3MH and 3MHA concentrations.

Ruminant pregastric lipases: experimental evidence of their potential as industrial catalysts in food technology☆

Abstract 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.

Detection of methamphetamine in indoor air using dynamic solid phase microextraction: a supplementary method to surface wipe sampling

Surface wipe sampling for methamphetamine is a standard protocol in many jurisdictions for sampling at suspected or known former clandestine methamphetamine laboratories, but this method relies on samples being taken from representatively contaminated surfaces. We have investigated whether a rapid sampling method for airborne methamphetamine can be used to supplement surface sampling. A dynamic solid phase microextraction (SPME) field sampler was constructed and tested in the field and in the laboratory. This device enabled large volumes of air to be passed over SPME fibres exposed during the comparatively short time (<2 h) that a testing company might be present at a former clandestine laboratory. The collected samples were then analyzed by gas chromatography-mass spectrometry. Airborne methamphetamine was detected with this method at former clandestine methamphetamine laboratory sites where surface wipe sampling showed surface methamphetamine concentrations greater than 40 μg/100 cm2.

Lamb pregastric lipase catalysed hydrolysis of 1,2,3-tri[(cis)-9-octadecenoyl]glycerol: temperature, pH and solvent isotope effects

Abstract 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.

Isolation and Characterization of Purified Bile Salt Stimulated Human Milk Lipase

A method has been developed for the isolation and purification of bile-salt-stimulated human milk lipase. This method yields up to six times more enzyme than other reported methods and the specific activity is compara ble. The concentration of BSSL recovered from the whole milk was 0.65 percent of the original protein content. The molecular weight of the isolated protein was 120 kDa. During the course of the purification, both protein content and specific activity were monitored and the esterase and lipase activities of the isolated product were characterized in the presence of sodium taurocholate. Five separate isolations were carried out with the introduction of minor varia tions in the procedure, but the catalytic properties of the product remain unchanged.

Bile-Salt-Stimulated Human Milk Lipase: Interaction with Proteins:

The initial rates of hydrolysis of triolein, catalyzed by bile-salt-stimulated hu man milk lipase, BSSL, were measured at pH 7.5 and 37 ° C, in the presence of selected proteins, namely immunoglobulin A, α-lactalbumin, lactoferrin, hen egg white lysozyme, pancreatic lipase, myoglobin and the very surface active protein melittin. The esterase activity of the enzyme against 4-nitro- phenylacetate was also measured in the presence of a number of different samples of lactoferrin. Under the conditions used, α-lactalbumin and hen egg white lysozyme had almost no effect on the lipase activity. Immunoglobulin A was slightly inhibitory; lactoferrin, pancreatic lipase and myoglobin were all partially inhibitory; and melittin was capable of almost completely inac tivating the lipase.

Dynamic solid phase microextraction analysis for airborne methamphetamine: quantitation using isotopically substituted methamphetamine

A vapour dosing system was developed that gave constant concentrations of methamphetamine in the range 1 to 10 μg m−3. This was used to calibrate the response of solid phase microextraction (SPME) fibres in passive and dynamic sampling modes. Exposure of 100 μm polydimethylsiloxane SPME fibres to a constant concentration of 1–5 μg m−3 methamphetamine over 1–70 min followed by GC-MS analysis produced a curvilinear pre-steady state sorption curve sufficiently reproducible to enable calibration of the SPME absorption. There was no evidence for loss of methamphetamine from the polydimethylsiloxane fibre under static ambient conditions for 5 h or during exposure to 1 L min−1 airflow in a dynamic SPME sampler for 90 min at room temperature. Sequential exposure of SPME fibres to methamphetamine and d9-methamphetamine showed that both analytes were retained. These results demonstrate that solid phase microextraction can be used with pre-loaded isotopically substituted methamphetamine as an internal standard for accurate quantitation of airborne methamphetamine. The isotopic labeling experiments also showed that the dosing system had a small but significant reservoir of methamphetamine, even though it was constructed from mainly inert materials and much of it was at elevated temperature. We therefore recommend that separate vapour dosing units be used for labeled and unlabeled methamphetamine.

Bile-salt-stimulated human milk lipase catalysed hydrolysis of 1,2,3-tri [(cis)-9-octadecenoyl] glycerol: Solvent isotope effect

Abstract Bile salt stimulated lipase from human milk has been used to catalyse the hydrolysis of (9–10 3 H) (1,2,3-tri[( cis )-9-octadecenoyl]glycerol) in emulsion media containing buffer dissolvedin H 2 O and D 2 O. The reactions were carried out in the presence of 20 mM taurocholate at pH = pD = 7.5 and 37.5°C. Analysis of the data gave values of V max equal to 194 and 61.8 Bq·s −1 , and of K m equal to 3.48 and 2.91 mM, for reactions in H 2 O and D 2 O, respectively. The solvent isotope effects for acylation and deacylation were equal to 2.63 and 3.14, respectively. A mechanism has been proposed which explains the observed effects and takes into account the known amino acid sequence of the active site and the residues involved in catalysis.

Effect of temperature and pH on the esterase activity of bile-salt-stimulated human milk lipase

The initial rate constants of hydrolysis of 4-nitrophenyl acetate, catalyzed by bile-salt-stimulated human milk lipase, have been measured over the tempera ture range 20 ° C to 37 ° C, in 0.1 M buffer solutions in the pH range 6-10, and in the presence of 2 mM sodium taurocholate. From these data, the values of the energy of activation have been calculated and found to be independent of pH. Analysis of the data has also led to determination of the values of the disso ciation constants, pK1 (8.20) and pK2 (8.70) (at 25°C), and heats of formation, ΔH° (40.2 and 59.4 kJ mol -1, respectively), of the essential ionizable residues of the enzyme.