Emulsifying properties of legume proteins compared to ß-lactoglobulin and Tween 20 and the volatile release from oil-in-water emulsions.

The emulsifying properties of plant legume protein isolates (soy, pea, and lupin) were compared to a milk whey protein, ß-lactoglobulin (ß-lg), and a nonionic surfactant (Tween 20). The protein fractional composition was characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The following emulsion properties were measured: particle diameter, shear surface ζ-potential, interfacial tension (IT), and creaming velocity. The effect of protein preheat treatment (90 °C for 10 min) on the emulsifying behavior and the release of selected volatile organic compounds (VOCs) from emulsions under oral conditions was also investigated in real time using proton transfer reaction-mass spectrometry. The legume proteins showed comparable results to ß-lg and Tween 20, forming stable, negatively charged emulsions with particle diameter d3,2 < 0.4 µm, and maintained stability over 50 d. The relatively lower stability of lupin emulsions was significantly correlated with the low protein surface hydrophobicity and IT of the emulsion. After heating the proteins, the droplet size of pea and lupin emulsions decreased. The VOC release profile was similar between the protein-stabilized emulsions, and greater retention was observed for Tween 20-stabilized emulsions. This study demonstrates the potential application of legume proteins as alternative emulsifiers to milk proteins in emulsion products.

Volatile release and structural stability of ß-lactoglobulin primary and multilayer emulsions under simulated oral conditions.

The relationship between emulsion structure and the release of volatile organic compounds (VOCs) was investigated using a model mouth system under oral conditions (tongue mastication, artificial saliva, pH and salt). The VOCs were monitored on-line by proton transfer reaction mass spectrometry (PTR-MS). Two types of emulsion system were compared: primary and multilayer oil-in-water (P-O/W, M-O/W) emulsions consisting of soy oil coated by ß-lactoglobulin and pectin layers. The P-O/W emulsions showed intensive flocculation at pH 5 and above 200 mM NaCl where the electrostatic repulsive charge was at a minimum. Bridging and depletion flocculation were mostly observed for P-O/W emulsions containing artificial saliva with 1 wt% mucin. The VOC release was found to increase when the emulsion droplets flocculated, thus changing the oil volume phase distribution. The adsorbed pectin layer stabilised the emulsion structure under conditions of short-time oral processing, and hindered the release of hydrophobic VOCs.

Tongue pressure and oral conditions affect volatile release from liquid systems in a model mouth.

The release of volatile organic compounds (VOCs) into the mouth cavity is an integral part of the way flavor is perceived. An in vitro model mouth with an artificial tongue was developed to measure the dynamic release of VOCs from liquid model systems [e.g., aqueous solution, oil, and oil-in-water (O/W) emulsions] under oral conditions. The release of seven selected VOCs was affected by the different polarity and vapor pressure of the compounds and their affinity to the liquid system media. Different tongue pressure patterns were applied to the liquid systems, and the release of VOCs was monitored in real time using proton transfer reaction-mass spectrometry. The release was significantly more intense for longer tongue pressure duration and was influenced by the tongue altering the sample surface area and the distribution of the VOCs. The role of saliva (artificial versus human) and the sample temperature had a significant effect on VOC release. Saliva containing mucin and a higher sample temperature enhanced the release.

The effect of homogenization and milk fat fractions on the functionality of Mozzarella cheese.

Mozzarella cheese was manufactured from milk containing either a low (olein) or a high (stearin) melting point fraction of milk fat or anhydrous milk fat. The fat was dispersed into skim milk by homogenization at 2.6 MPa before being manufactured into cheese. The melting point of the milk fat did not affect the size or shape of the fat globules, nor was there any effect of homogenization on the polymorphic state of the milk fat. There were no changes in milk fat globule size and shape concomitant with the amount of free oil formed. The polymorphic state of the milk fat did affect the amount of free oil formed and the apparent viscosity of the cheese. The lower melting point fraction yielded a larger amount of free oil. The higher melting point fraction yielded a higher viscosity of melted cheese at 60 degrees C. Mozzarella cheese was also manufactured from homogenized milk, nonhomogenized milk, and a 1:1 ratio of the two, without altering the milk fat composition. Increasing the proportion of homogenized milk yielded a lower free oil content and higher viscosity of the cheese.

Free oil and rheology of Cheddar cheese containing fat globules stabilized with different proteins.

Cheddar cheese was manufactured from recombined milk containing fat globules coated with alpha(s1)-CN (casein), alpha(s2)-CN, beta-CN, kappa-CN, alpha-lactalbumin, or beta-lactoglobulin. The effect of the coating on fat globule structure, free oil formation, and cheese rheology was investigated to determine if globule coating affected the physical structure of cheese. Fat globule size and shape were determined in cheese using confocal laser scanning microscopy, and the rheological properties measured by uniaxial compression after maturation for 35 and 70 d. Fat globules were elongated and clustered in the control cheese coated with native membrane material and in cheese where the globules were coated with alpha(s2)-CN, but were more circular and distinct than all others. Cheese containing globules coated with alpha(s2)-CN fractured at a lower strain and with a lower stress than other experimental cheeses. Free oil decreased in cheese as the stress at fracture of the cheese protein matrix increased. Strain at fracture increased as pH increased from 4.7 to 5.3. There was no correlation between free oil and fat globule circularity. Cheddar cheese aroma was not evident in experimental cheeses.

The effect of compression, stretching, and cooking temperature on free oil formation in mozzarella curd.

The effect of the extent and rate of compression and stretching on free oil formation in Mozzarella cheese curd was investigated at 55, 65, and 75 degrees C. Confocal laser scanning microscopy was used to determine the maximum cross-sectional diameter, cross-sectional area, elongation factor (maximum divided by minimum cross-sectional diameter), and circularity of fat globules in the cheese curd at the different temperatures, and after stretching or compression. Free oil was not significantly affected by the rate of biaxial compression from 50 to 2000 mm/min at 65 degrees C, the rate of tensile stretching from 1000 to 2500 mm/min at 60 degrees C, or the extent of biaxial compression from 40 to 80% of the original height at 1000 mm/min and 65 degrees C. Increasing the rate of stretching from 1000 to 2500 mm/min increased the elongation factor from 1.91 to 2.61. Cross-sectional area, maximum diameter, and circularity were not affected by the rate of biaxial compression. The extent of curd compression had no effect on the milk fat globule size and shape. Increasing the extent of stretching at 60 degrees C and at 1000 mm/min increased the free oil content (on a fat basis) from 23.8% (curd stretched by 1.4x) to 32.3% (stretched by 4.6x) and the elongation factor of the globules, but did not affect any of the other globule parameters. Increasing the temperature of the cooking-stretching water increased the cross-sectional area, diameter of the globules, and free oil content from 24.1% at 55 degrees C to 34.5% at 75 degrees C for curd compressed to 50% height at 1000 mm/min.

Dynamic rheology of renneted milk gels containing fat globules stabilized with different surfactants.

Anhydrous milk fat was emulsified with alpha s1-CN (casein), alpha s2-CN, beta-CN, kappa-CN, alpha-lactalbumin, beta-lactoglobulin, Tween 80, or phosphatidylcholine to produce a 30% fat cream in a 0.1 M imidazole pH 7 buffer. The creams were mixed with skim milk to yield a fat content of 3.4% and the viscoelastic properties of the recombined milks clotted with chymosin were measured. Recombined milk containing globules coated with the more amphipathic and phosphorylated alpha s2-CN and beta-CN clotted faster but gel firmness increased more slowly and weaker gels were formed. Gel firmness increased more rapidly for milks containing globules coated with of alpha s1-CN and kappa-CN that possess more uniformly distributed hydrophobic domains.

Percutaneous absorption of [3H]tretinoin and systemic exposure to mequinol after dermal application of 2% mequinol/0.01% [3H]tretinoin (Solagé) solution in healthy volunteers.

Solagé is a combination product composed of 2% mequinol (4-hydroxyanisole) and 0.01% tretinoin (all-trans-retinoic acid) in an ethanolic solution, which is being studied for its safety and efficacy as a topical treatment for disorders of skin hyperpigmentation. The purpose of this study was to evaluate the extent of percutaneous absorption of [3H]tretinoin and to estimate the systemic exposure to mequinol from this combination product when topically applied to the backs of healthy subjects. Eight subjects received bid topical applications of nonradiolabelled 2% mequinol/0.01% tretinoin solution on a 400 cm2 area of the back for 14 days. The subjects then received a single topical application of 2% mequinol/0.01% [3H]tretinoin solution. After 12 h, the radiolabelled dose was removed and bid treatment with nonradiolabelled 2% mequinol/0.01% tretinoin solution was continued for 7 days. Plasma, urine and faecal samples were analysed for total radioactivity and plasma was analysed for both mequinol and tretinoin by GC/MS procedure. Mean percutaneous absorption of [3H]tretinoin based on the cumulative recoveries of radioactivity in the urine and faeces was about 4.5% (median 2.18%). Tretinoin concentrations in plasma did not increase above endogenous levels. This was consistent with the concentrations of radioactivity in plasma, which showed an average Cmax of 91 pg-eq/mL (median 26 ng/mL). Average Cmax and AUC(0-12 h) values for mequinol were 10 ng/mL and 33 ng h/mL, respectively. Based on the results of this study, systemic toxicity from topical application of tretinoin in this formulation is unlikely, because percutaneous absorption of tretinoin is minimal and because endogenous levels of tretinoin are not increased following bid dosing with this combination formulation. The safety of mequinol in this combination formulation is supported by the low systemic exposures of the subjects in this study compared with the systemic exposures at the highest doses in the dermal toxicity studies in mice (16.6-fold) and rats (34.6-fold).

Oral bioavailability and disposition characteristics of irbesartan, an angiotensin antagonist, in healthy volunteers.

Absolute oral bioavailability and disposition characteristics of irbesartan, an angiotensin II receptor antagonist, were investigated in 18 healthy young male volunteers. Subjects received [14C] irbesartan as a 30-minute intravenous infusion (50 mg), [14C] irbesartan orally as a solution (50 mg or 150 mg), or irbesartan capsule (50 mg). Irbesartan was rapidly and almost completely absorbed after oral administration, and exhibited a mean absolute oral bioavailability of 60% to 80%. Mean total body clearance was approximately 157 mL/min, and renal clearance was 3.0 mL/min. Volume of distribution at steady state was 53 L to 93 L, and terminal elimination half-life was approximately 13 to 16 hours. Hepatic extraction ratio was low (0.2). There were no major circulating metabolites, and approximately 80% of total plasma radioactivity was attributable to unchanged irbesartan. Regardless of route of administration, approximately 20% of dose was recovered in urine and the remainder in feces.

Biotransformation of irbesartan in man.

The metabolism of irbesartan, a highly selective and potent nonpeptide angiotensin II receptor antagonist, has been investigated in humans. An aliquot of pooled urine from healthy subjects given a 50-mg oral dose of [14C]irbesartan was added as a tracer to urine from healthy subjects that received multiple, 900-mg nonradiolabeled doses of irbesartan. Urinary metabolites were isolated, and structures were elucidated by mass spectroscopy, proton NMR, and high-performance liquid chromatography (HPLC) retention times. Irbesartan and the following eight metabolites were identified in human urine: (1) a tetrazole N2-beta-glucuronide conjugate of irbesartan, (2) a monohydroxylated metabolite resulting from omega-1 oxidation of the butyl side chain, (3, 4) two different monohydroxylated metabolites resulting from oxidation of the spirocyclopentane ring, (5) a diol resulting from omega-1 oxidation of the butyl side chain and oxidation of the spirocyclopentane ring, (6) a keto metabolite resulting from further oxidation of the omega-1 monohydroxy metabolite, (7) a keto-alcohol resulting from further oxidation of the omega-1 hydroxyl of the diol, and (8) a carboxylic acid metabolite resulting from oxidation of the terminal methyl group of the butyl side chain. Biotransformation profiles of pooled urine, feces, and plasma samples from healthy male volunteers given doses of [14C]irbesartan were determined by HPLC. The predominant drug-related component in plasma was irbesartan (76-88% of the plasma radioactivity). None of the metabolites exceeded 9% of the plasma radioactivity. Radioactivity in urine accounted for about 20% of the radiolabeled dose. In urine, irbesartan and its glucuronide each accounted for about 5 to 10% of the urinary radioactivity. The predominant metabolite in urine was the omega-1 hydroxylated metabolite, which constituted about 25% of the urinary radioactivity. In feces, irbesartan was the predominant drug-related component (about 30% of the radioactivity), and the primary metabolites were monohydroxylated metabolites and the carboxylic acid metabolite. Irbesartan and these identified metabolites constituted 90% of the recovered urinary and fecal radioactivity from human subjects given oral doses of [14C]irbesartan.

High-risk human papillomavirus testing of women with cytological low-grade squamous intraepithelial lesions.

BACKGROUND: Testing for carcinogenic human papillomavirus (HPV) types has been suggested to evaluate patients with atypical squamous cells of unknown significance. We wanted to know whether this test was clinically useful in patients with low-grade squamous intraepithelial lesions (SIL) detected by Papanicoloau (Pap) smear. METHODS AND MATERIALS: We tested 159 women with low-grade SIL Pap smears for high-risk HPV types, using the Hybrid Capture HPV DNA Assay (Digene Corp., Silver Springs, MD) during evaluation by colposcopy and cervical biopsies. We constructed a receiver-operating characteristic curve and calculated sensitivity, specificity, and predictive values for the presence of high-risk virus and cervical intraepithelial neoplasia (CIN). RESULTS: The receiver-operating characteristic curve suggested that 3.0 relative light units was the best cutoff for a positive test, rather than the manufacturer-recommended 1.0 relative light units. The sensitivity for detecting CIN was 0.858 [95% confidence interval (Cl); 0.792-0.925] and specificity was 0.623 (95% Cl, 0.492-0.753). The pretest probability of CIN was 0.667 in this group of patients, and the positive predictive value was 0.820. CONCLUSION: The Hybrid Capture HPV DNA Assay is of limited use in predicting those low-grade SIL Pap smears will have histologically confirmed CIN.

Biotransformation of pravastatin sodium in humans.

Pravastatin sodium (PV) is a potent cholesterol-lowering agent that acts by inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Biotransformation profiles of PV in pooled human urine, plasma, and feces from healthy male volunteers given single 19.2-mg oral or 9.9-mg iv doses of [14C]PV were determined by HPLC. The predominant drug-related component in urine, plasma, and feces corresponded to intact PV; in the pooled urine samples, PV constituted 29 and 69% of the radioactivity after the po and iv doses, respectively. The delta 4.5-3 alpha-hydroxy isomer of PV constituted 10% (po) and 2% (iv), and 6-epi-PV constituted 3% (po) and 1% (iv) of the urinary radioactivity. Negligible amounts of the lactones of PV or its isomers were detected in urine, plasma, or feces. At least 15 other metabolites were also present; none of these accounted for more than 6% of the total urinary radioactivity. For metabolite isolation, an aliquot of pooled urine samples, obtained after administration of the radioactive dose, was added as a tracer to urine samples obtained from healthy subjects after administration of single nonradiolabeled 40-mg oral doses of PV. Urinary metabolites were concentrated on an XAD-2 column, extracted with ethyl acetate, and purified by extensive preparative HPLC. In addition to isolation and identification of unchanged drug and the two isomeric metabolites described above, eight other metabolites were isolated and structural assignments were made based on HPLC, UV spectra, mass spectral analysis, and proton NMR.(ABSTRACT TRUNCATED AT 250 WORDS)

High-performance liquid chromatographic method for the radiometric determination of [14C] bucromarone in human plasma utilizing non-radiolabeled bucromarone as an internal standard.

A novel radiometric high-performance liquid chromatographic (HPLC) method was developed for the determination of [14C]bucromarone in human plasma. The procedure involved the addition of non-radiolabeled bucromarone hydrochloride to each plasma sample as an internal standard; the plasma sample was then extracted, and the bucromarone was separated from its metabolites and endogenous compounds by reversed phase HPLC. The concentration of [14C]bucromarone in each plasma sample was calculated from the ratio of the amount of radioactivity in the eluate fraction corresponding to bucromarone and the peak height of the ultraviolet absorbance (210 nm) of the non-radiolabeled bucromarone used as an internal standard. The lower limit of quantitation for bucromarone free base in this assay was 8 ng/ml when [14C]bucromarone succinate had a specific activity of 0.5 microCi/mg. The coefficients of variation for the experimentally determined concentrations of bucromarone in spiked plasma samples were 6.8 and 14.3% at concentrations of 80 and 20 ng/ml, respectively. This method was used to determine concentrations of bucromarone in the plasma of healthy volunteers who were given intravenous infusions of [14C]bucromarone succinate. In general, the methodology should be applicable to any radiolabeled compound that possesses appreciable ultraviolet absorbance.

Disposition of fosinopril sodium in healthy subjects.

1 Fosinopril sodium is the first phosphorus-containing angiotensin-converting enzyme (ACE) inhibitor to be studied clinically as an antihypertensive agent. It is an ester prodrug that is hydrolysed in vivo to the active diacid ACE inhibitor, SQ 27, 519. 2 In a three-way crossover study, nine healthy male subjects (age range 20-34 years) each received an intravenous 7.5 mg dose of SQ 27, 519-[14C] and two oral 10 mg doses of [14C]-fosinopril sodium, administered as a capsule and in solution. 3 After the intravenous dose of SQ 27, 519, the 0 to 96 h recovery of radioactivity averaged 44 and 46% of the dose in urine and faeces, respectively, indicating substantial biliary secretion. Only intact SQ 27, 519 was detected in the plasma, urine, and faeces following the intravenous dose of SQ 27, 519. 4 After oral doses of fosinopril sodium, about 75% of the radioactivity in plasma and urine was present as SQ 27, 519; the remainder corresponded mainly to a beta-glucuronide conjugate of SQ 27, 519 (15-20%), and a monohydroxylated analogue of SQ 27, 519 (about 5%). Negligible amounts of fosinopril sodium were present, indicating complete hydrolysis of the prodrug. 5 For the solution and capsule doses, respectively, the oral absorption of fosinopril sodium averaged 32% and 36% and the oral bioavailability of SQ 27, 519 averaged 25% and 29%. 6 The average values for clearance (39 ml min-1), renal clearance (17 ml min-1), Vss (10 1), and plasma protein binding (approximately 95%), indicated that SQ 27, 519 was slowly cleared from the body and not distributed extensively into extravascular sites.

Relationship between metabolism and pharmacologic activity of SQ 27,786, an angiotensin converting enzyme inhibitor with potent diuretic activity.

SQ 27,786 is a sulfhydryl-containing angiotensin converting enzyme (ACE) inhibitor, which also possesses potent diuretic activity in dogs after intravenous administration. The absorption, distribution, metabolism and elimination of 35S-labeled SQ 27,786 was studied in dogs to determine if the observed pharmacologic activities were intrinsic to this compound or the result of metabolism to separate ACE-inhibitory and diuretic moieties. The poor pharmacologic activity observed after oral administration was found to be due to poor absorption of the ACE-inhibitory-diuretic compound. The results of this study indicated that SQ 27,786 was excreted largely intact, either as the parent compound, the symmetrical disulfide of the parent compound, or as mixed disulfides of the parent compound with endogenous sulfhydryl compounds (e.g., SQ 27,786-L-cysteine) in a manner similar to captopril. It was concluded that the observed diuretic and ACE inhibitory activities were the result of intact SQ 27,786 and not of metabolites resulting from cleavage of the molecule to separate diuretic and ACE inhibitory moieties.

Reactions of nucleosides with glyoxal and acrolein.

The structural determination of the products formed by the reaction of derivatives of glyoxal with guanine are reviewed, as are the applications of these reactions. Conditions have now been defined for the use of glyoxal as a probe for microdetermination of the structure of unidentified nucleic acid components. Unexpectedly, 3-methylguanine shows a positive reaction with glyoxal. The adduct produced has been isolated and characterized. A new ring is formed by substitution at the 1- and N2-positions of 3-methylguanine in this product. Acrolein reacts smoothly with 1-methylcytosine, cytidine and deoxycytidine at pH 4. The structures of the adducts have been determined. The amino group of cytosine adds to the double bond of acrolein, while the aldehyde binds to the 3-position, forming a new ring. Adenosine, deoxyadenosine and 9-methyladenine react in an analogous manner with acrolein, with ring formation involving the amino group and the N-1 of adenine. The orientation of addition is similar to that in the cytosine series. Guanosine reacts with excess acrolein at pH 4 to form a bis-adduct. Two new rings are fused to the guanine structure, one at N-1 and the amino group, and the other involving N-7 and C-8. A number of derivatives of this product have been obtained by glycosyl cleavage and imidazole-ring-opening reactions. However, the assignment of the orientation of addition for both acroleins remains tentative in this series.

Disposition of [14C]aztreonam in rats, dogs, and monkeys.

[14C]aztreonam was administered as single 25-mg/kg doses to dogs (intravenously and subcutaneously) and monkeys (intramuscularly and intravenously) and as single 50-mg/kg doses (intramuscularly and intravenously) to rats. In rats and dogs, radioactive moieties were excreted primarily in urine; in monkeys, they were excreted about equally in urine and feces. Unchanged aztreonam accounted for 77 to 86% of the radioactivity excreted in the urine of rats, dogs, and monkeys; SQ 26,992, the metabolite resulting from hydrolysis of the monobactam ring, accounted for 10 to 15%; and minor, unidentified metabolites accounted for the remainder. In rats with cannulated bile ducts, about 15% of an intramuscular dose was excreted in bile in 24 h; the bile contained a greater percentage of metabolites than that found in urine. In dogs, the apparent elimination half-life of aztreonam in serum was 0.7 h after intravenous administration. Aztreonam and SQ 26,992 accounted for most of the radioactivity in the sera of dogs and monkeys. Serum protein binding of aztreonam and its metabolites ranged from 28 to 35% in dogs and from 49 to 59% in monkeys. In the three species studied, aztreonam was most extensively metabolized in monkeys; SQ 26,992 and other minor metabolites from monkey urine were tested and found to be devoid of any significant antimicrobial activity.

Metabolism of o-[methyl-14C]toluidine in the F344 rat.

1. Following a single dose (400 mg/kg s.c.) of o-[methyl-14C]toluidine to male F344 rats, 56% of the 14C was recovered in the 24 h urine, 2.3% in the faeces and 1% as exhaled 14CO2. After 48 h, 83.9% of the 14C appeared in the urine, 3.3% in the faeces and 1.4% was exhaled. 2. Ether-extractable urinary metabolites were separated by h.p.l.c. and identified as: o-toluidine (5.1% dose); azoxytoluene (0.2%); o-nitrosotoluene (less than or equal to 0.1%); N-acetyl-o-toluidine (0.2%); N-acetyl-o-aminobenzyl alcohol (0.3%); 4-amino-m-cresol (0.6%); N-acetyl-4-amino-m-cresol (0.3%); anthranilic acid (0.3%) and N-acetylanthranilic acid (0.3%). 3. Acid-conjugated urinary metabolites (51% of dose), separated by paper electrophoresis and by Sephadex LH-20 chromatography, were identified as sulphates of 4-amino-m-cresol (27.8% dose), N-acetyl-4-amino-m-cresol (8.5%), and 2-amino-m-cresol (2.1%), and glucuronides of 4-amino-m-cresol (2.6%), N-acetyl-4-amino-m-cresol (2.8%) and N-acetyl-o-aminobenzyl alcohol. Evidence for a double acid conjugate of 4-amino-m-cresol was also found. 4. These results show that N-acetylation and hydroxylation at the 4 position of o-toluidine are major metabolic pathways in the rat. Minor pathways include hydroxylation at the 6 position, oxidation of the methyl group and oxidation of the amino group. Sulphate conjugates predominate over glucuronides by a ratio of 6:1.