Serum proteomic profiling in patients with drug-induced liver injury.

BACKGROUND: Idiosyncratic drug-induced liver injury (DILI) is a complex disorder that is difficult to predict, diagnose and treat. AIM: To describe the global serum proteome of patients with DILI and controls. METHODS: A label-free, mass spectrometry-based quantitative proteomic approach was used to explore protein expression in serum samples from 74 DILI patients (collected within 14 days of DILI onset) and 40 controls. A longitudinal analysis was conducted in a subset of 21 DILI patients with available 6-month follow-up serum samples. RESULTS: Comparison of DILI patients based on pattern, severity and causality assessment of liver injury revealed many differentially expressed priority 1 proteins among groups. Expression of fumarylacetoacetase was correlated with alanine aminotransferase (ALT; r = 0.237; P = 0.047), aspartate aminotransferase (AST; r = 0.389; P = 0.001) and alkaline phosphatase (r = -0.240; P = 0.043), and this was the only protein with significant differential expression when comparing patients with hepatocellular vs. cholestatic or mixed injury. In the longitudinal analysis, expression of 53 priority 1 proteins changed significantly from onset of DILI to 6-month follow-up, and nearly all proteins returned to expression levels comparable to control subjects. Ninety-two serum priority 1 proteins with significant differential expression were identified when comparing the DILI and control groups. Pattern analysis revealed proteins that are components of inflammation, immune system activation and several hepatotoxicity-specific pathways. Apolipoprotein E expression had the greatest power to differentiate DILI patients from controls (89% correct classification; AUROC = 0.97). CONCLUSION: This proteomic analysis identified differentially expressed proteins that are components of pathways previously implicated in the pathogenesis of idiosyncratic drug-induced liver injury.

Inactivation at various temperatures of bovine viral diarrhea virus in beef derived from persistently infected cattle.

Bovine viral diarrhea virus (BVDV) is a pestivirus that is enzootic in most cattle populations throughout the world. This virus is present throughout the body of persistently infected (PI) cattle. Previous research has not assessed the cooking temperature at which BVDV in meat from PI cattle can be inactivated. Therefore, muscle tissue from 6 PI cattle was harvested, refrigerated, frozen, and heated to various internal temperatures. The concentration of virus present was determined by virus isolation. Average cell culture infective doses (50% endpoint; CCID(50)) of BVDV per gram of frozen, uncooked meat from PI cattle were 10(5.85) CCID(50)/g of whole cuts and 10(6.02) CCID(50)/g of ground meat. The virus in whole and ground meat was consistently inactivated when cooked to temperatures greater than or equal to 75°C. A second objective of this research was to thoroughly reassess if Vero cells were permissive to BVDV infection in our laboratory to provide further indication of whether primates, including humans, might be susceptible to BVDV. Vero cells were not permissive to infection with any of 43 different strains of BVDV that readily replicated in Madin Darby bovine kidney cells. In conclusion, this bovine pathogen, which is not considered to be a human pathogen, can be inactivated by cooking ground or whole cuts of meat to 75°C or higher. Care should be taken to ensure that susceptible hosts such as pigs are not fed improperly cooked meat, meat by-products, or waste food originating from PI cattle.

Fat-induced membrane cholesterol accrual provokes cortical filamentous actin destabilisation and glucose transport dysfunction in skeletal muscle.

AIMS/HYPOTHESIS: Diminished cortical filamentous actin (F-actin) has been implicated in skeletal muscle insulin resistance, yet the mechanism(s) is unknown. Here we tested the hypothesis that changes in membrane cholesterol could be a causative factor, as organised F-actin structure emanates from cholesterol-enriched raft microdomains at the plasma membrane. METHODS: Skeletal muscle samples from high-fat-fed animals and insulin-sensitive and insulin-resistant human participants were evaluated. The study also used L6 myotubes to directly determine the impact of fatty acids (FAs) on membrane/cytoskeletal variables and insulin action. RESULTS: High-fat-fed insulin-resistant animals displayed elevated levels of membrane cholesterol and reduced F-actin structure compared with normal chow-fed animals. Moreover, human muscle biopsies revealed an inverse correlation between membrane cholesterol and whole-body glucose disposal. Palmitate-induced insulin-resistant myotubes displayed membrane cholesterol accrual and F-actin loss. Cholesterol lowering protected against the palmitate-induced defects, whereas characteristically measured defects in insulin signalling were not corrected. Conversely, cholesterol loading of L6 myotube membranes provoked a palmitate-like cytoskeletal/GLUT4 derangement. Mechanistically, we observed a palmitate-induced increase in O-linked glycosylation, an end-product of the hexosamine biosynthesis pathway (HBP). Consistent with HBP activity affecting the transcription of various genes, we observed an increase in Hmgcr, a gene that encodes 3-hydroxy-3-methyl-glutaryl coenzyme A reductase, the rate-limiting enzyme in cholesterol synthesis. In line with increased HBP activity transcriptionally provoking a membrane cholesterol-based insulin-resistant state, HBP inhibition attenuated Hmgcr expression and prevented membrane cholesterol accrual, F-actin loss and GLUT4/glucose transport dysfunction. CONCLUSIONS/INTERPRETATION: Our results suggest a novel cholesterolgenic-based mechanism of FA-induced membrane/cytoskeletal disorder and insulin resistance.

Kinetics of an acid-base catalyzed reaction (aspartame degradation) as affected by polyol-induced changes in buffer pH and pK values.

The kinetics of an acid-base catalyzed reaction, aspartame degradation, were examined as affected by the changes in pH and pK(a) values caused by adding polyols (sucrose, glycerol) to phosphate buffer. Sucrose-containing phosphate buffer solutions had a lower pH than that of phosphate buffer alone, which contributed, in part, to reduced aspartame reactivity. A kinetic model was introduced for aspartame degradation that encompassed pH and buffer salt concentrations, both of which change with a shift in the apparent pK(a) value. Aspartame degradation rate constants in sucrose-containing solutions were successfully predicted using this model when corrections (that is, lower pH, lower apparent pK(a) value, buffer dilution from the polyol) were applied. The change in buffer properties (pH, pK(a)) from adding sucrose to phosphate buffer does impact food chemical stability. These effects can be successfully incorporated into predictive kinetic models. Therefore, pH and pK(a) changes from adding polyols to buffer should be considered during food product development.

Physical properties and consumer liking of cookies prepared by replacing sucrose with tagatose.

The objective of this study was to investigate the suitability of tagatose, a minimally absorbed prebiotic monosaccharide, as a replacement for sucrose in cookies. A sucrose-containing cookie recipe was prepared as the control. Sucrose was replaced with tagatose at various levels ranging from 25% to 100%. Cookies containing fructose were also prepared for comparison due to the structural similarities between tagatose and fructose. The rheological properties of the dough were measured using texture profile analysis. The baked cookies were evaluated for spread, color, and hardness. For tagatose-containing cookies, the extent of likeness was evaluated by 53 untrained panelists using a 9-point hedonic scale. When sucrose was replaced by tagatose, doughs with similar rheological properties to the control resulted. The tagatose-containing cookies were harder and darker with a lower spread than the control. Sensory data indicated that panelists liked the brown color of the 100% tagatose cookies better than the control, but disliked their sweetness. Overall likeness scores of the control and cookies made by replacing half of the sucrose with tagatose were the same. Tagatose appears to be suitable as a partial replacer for sucrose in cookies based on similar dough properties, cookie properties, and likeness scores. Using tagatose to replace sucrose in foods would reduce the amount of metabolizeable sugars in the diet as well as provide the desirable prebiotic effect.

Caffeine content of prepackaged national-brand and private-label carbonated beverages.

Caffeine is a well-known stimulant that is added as an ingredient to various carbonated soft drinks. Due to its stimulatory and other physiological effects, individuals desire to know the exact amount of caffeine consumed from these beverages. This study analyzed the caffeine contents of 56 national-brand and 75 private-label store-brand carbonated beverages using high-performance liquid chromatography. Caffeine contents ranged from 4.9 mg/12 oz (IGA Cola) to 74 mg/12 oz (Vault Zero). Some of the more common national-brand carbonated beverages analyzed in this study with their caffeine contents were Coca-Cola (33.9 mg/12 oz), Diet Coke (46.3 mg/12 oz), Pepsi (38.9 mg/12 oz), Diet Pepsi (36.7 mg/12 oz), Dr Pepper (42.6 mg/12 oz), Diet Dr Pepper (44.1 mg/12 oz), Mountain Dew (54.8 mg/12 oz), and Diet Mountain Dew (55.2 mg/12 oz). The Wal-Mart store-brand beverages with their caffeine contents were Sam's Cola (12.7 mg/12 oz), Sam's Diet Cola (13.3 mg/12 oz), Dr Thunder (30.6 mg/12 oz), Diet Dr Thunder (29.9 mg/12 oz), and Mountain Lightning (46.5 mg/12 oz). Beverages from 14 other stores were also analyzed. Most store-brand carbonated beverages were found to contain less caffeine than their national-brand counterparts. The wide range of caffeine contents in carbonated beverages indicates that consumers would benefit from the placement of caffeine values on food labels.

Molecular dynamics of solid-state lysozyme as affected by glycerol and water: a neutron scattering study.

Glycerol has been shown to lower the heat denaturation temperature (T(m)) of dehydrated lysozyme while elevating the T(m) of hydrated lysozyme (. J. Pharm. Sci. 84:707-712). Here, we report an in situ elastic neutron scattering study of the effect of glycerol and hydration on the internal dynamics of lysozyme powder. Anharmonic motions associated with structural relaxation processes were not detected for dehydrated lysozyme in the temperature range of 40 to 450K. Dehydrated lysozyme was found to have the highest T(m) by. Upon the addition of glycerol or water, anharmonicity was recovered above a dynamic transition temperature (T(d)), which may contribute to the reduction of T(m) values for dehydrated lysozyme in the presence of glycerol. The greatest degree of anharmonicity, as well as the lowest T(d), was observed for lysozyme solvated with water. Hydrated lysozyme was also found to have the lowest T(m) by. In the regime above T(d), larger amounts of glycerol lead to a higher rate of change in anharmonic motions as a function of temperature, rendering the material more heat labile. Below T(d), where harmonic motions dominate, the addition of glycerol resulted in a lower amplitude of motions, correlating with a stabilizing effect of glycerol on the protein.

Invertase storage stability and sucrose hydrolysis in solids as affected by water activity and glass transition.

Research continues to differentiate the impact of water activity (a(W)) and the glass transition temperature (T(g)) on chemical reactions. Invertase with and without sucrose was incorporated into low and high molecular weight poly(vinylpyrrolidone) model systems (PVP-LMW and PVP-K30, respectively). Invertase activity and sucrose hydrolysis were monitored during storage at a(W) = 0.32-0.75 and 30 degrees C. Pseudo-first-order rate constants for activity loss in PVP-K30 were not different, regardless of the system being glassy or rubbery. In PVP-LMW, invertase stability decreased with increasing a(W). An a(W) > 0.62 was required for sucrose hydrolysis to occur in PVP-LMW. PVP molecular weight appeared to affect invertase stability and reactivity. No dramatic change around T(g) was found in either invertase stability or sucrose hydrolysis, suggesting that T(g)-dictated mobility has a minimal effect on these reactions in amorphous solids.

Thermally induced denaturation of lyophilized bovine somatotropin and lysozyme as impacted by moisture and excipients.

The endothermic thermal transitions (i.e., denaturation) of lyophilized recombinant bovine somatotropin (rbSt) and lysozyme as seen via differential scanning calorimetry were evaluated with respect to moisture and excipients. The denaturation temperature, Tm, of rbSt and lysozyme decreased with increasing moisture irrespective of the excipient. However, the magnitude of the decrease elicited by moisture was dependent on the type of excipient. Furthermore, the effect of the excipient was dependent on the moisture content; excipients decreased Tm in low moisture solids (i.e., < 5% moisture) and increased it in hydrated solids (i.e., > 15% moisture). In the dry state (< 1% moisture), the addition of 50% sucrose, sorbitol, or glycerol lowered the Tm of rbSt from 161 degrees C to 136, 120, and 83 degrees C, respectively, indicating a destabilizing mechanism. Likewise, the Tm of lysozyme decreased from 156 degrees C to 142, 128, and 97 degrees C due to the addition of sucrose, sorbitol, and glycerol, respectively. At higher moisture contents, the excipients promoted a higher transition temperature at a given moisture content than the pure protein systems, indicating a stabilizing mechanism. An increase in the enthalpy of unfolding for dehydrated lysozyme was noted with increasing levels of moisture and/or excipient, despite the observed decrease in Tm. The thermal stability, or Tm, of the dehydrated proteins appeared to be correlated to the glass transition temperature (Tg) of the excipient, which in turn should be related to the Tg of the system. The lower the Tg of the excipient, the greater was the degree of destabilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of surfactants on the physical stability of recombinant human growth hormone.

The physical stability of a human growth hormone (hGH) formulation upon exposure to air/water interfaces (with vortex mixing) and to nonisothermal stress [determined by differential scanning calorimetry (DSC)] was investigated. The effect of these stresses on the formation of soluble and insoluble aggregates was studied. The aggregates were characterized and quantified by size exclusion-HPLC and UV spectrophotometry. Vortex mixing of hGH solutions (0.5 mg/mL) in phosphate buffer, pH 7.4, for just 1 min caused 67% of the drug to precipitate as insoluble aggregates. These aggregates were noncovalent in nature. Non-ionic surfactants prevented the interfacially induced aggregation at their critical micelle concentration (cmc) for Pluronic F-68 (polyoxyethylene polyoxypropylene block polymer) and Brij 35 (polyoxyethylene alkyl ether) and above the cmc for Tween 80 (polyoxyethylene sorbitan monooleate). However, the same surfactants failed to stabilize hGH against thermal stress in DSC studies. Higher concentrations of surfactants actually destabilized hGH as evidenced by the decrease in the onset temperature for the denaturation endotherm.

Impact of moisture on thermally induced denaturation and decomposition of lyophilized bovine somatotropin.

The nonisothermal transitions of lyophilized recombinant bovine somatotropin (rbSt) as seen via differential scanning calorimetry were evaluated with respect to moisture content. The transition peak temperature of rbSt decreased with increasing moisture from 161 degrees C in the dry state to a plateau of 65 degrees C at 28% moisture, which is similar to that of rbSt in solution. Using high performance liquid chromatography, this irreversible endothermic transition consisted primarily of unfolding, hydrophobic aggregation, and some covalent modifications. In the dry state, covalent modifications, including polymerization into compounds of higher molecular weight, were more prominent, while in the presence of moisture, hydrophobic aggregation was most prominent. The irreversibility and scan rate dependence of the endothermic phenomena supports the kinetic nature of the transition rather than a simple equilibrium between globular and unfolded states. The apparent activation energy for the net transition (i.e., unfolding, hydrophobic aggregation, and covalent modifications) was 57 kcal/mol for rbSt at 9.9% moisture. The observed enthalpy of the transition increased, decreased, then approximately leveled off as a function of increasing moisture content. This can be explained by the increasingly significant contribution of the exothermic aggregation at higher moisture contents.

Aspartame stability in commercially sterilized flavored dairy beverages.

The objective of this research was to evaluate the stability of aspartame in commercially sterilized skim milk beverages that contained different buffer salts, buffer concentrations, and flavor. The effects of pH and temperature on aspartame stability in these dairy beverages were also studied. The pH and storage temperature appeared to be the two most important factors for a successful dairy beverage sweetened with aspartame. The half-lives were 1 to 4 d at 30 degrees C and 24 to 58 d at 4 degrees C. Decreasing the pH from 6.7 to 6.4 doubled the stability of aspartame. The type and concentration of buffer had only a minor influence on the aspartame stability. The addition of vanilla did not enhance the degradation of aspartame in dairy beverages.

Aspartame degradation as a function of "water activity".

The incorporation of aspartame into an increasing number of foods necessitates evaluation of its degradation kinetics as a function of "water activity" (aw). The kinetics of degradation were followed in model systems as a function of initial pH, temperature, and aw. An increase in aw, for each 0.1 units in the 0.3 to 0.7 range, resulted in about a 30-80% increase in degradation rate, which then decreased only slowly up to dilute solution. The presence of oil increased the degradation rate at high aw, but glucose had no effect on the rate of aspartame loss. The activation energies for loss ranged from 25 to 20 kcal/mole, decreasing as aw increased, as expected. The rates as a function of pH showed that the actual pH of the water in the condensed phase, based on the Bronsted relationship, may be very different than the initial pH. This caused a shift in the pH at which the fastest rate of degradation occurred, as aw increased.

Inhibitors of dihydropteroate synthase: substituent effects in the side-chain aromatic ring of 6-[[3-(aryloxy)propyl]amino]-5-nitrosoisocytosines and synthesis and inhibitory potency of bridged 5-nitrosoisocytosine-p-aminobenzoic acid analogues.

We previously reported that 6-(methylamino)-5-nitrosoisocytosine (5) is a potent inhibitor (I50 = 1.6 microM) of Escherichia coli dihydropteroate synthase. It was noted that 6-amino substituents larger than methyl were detrimental to binding, although the adverse steric effect could be overcome by a positive ancillary binding contribution of a phenyl ring attached at the terminus of certain 6-alkylamino substituents. We selected the 6-[[3-(aryloxy)propyl]amino]-5-nitrosoisocytosine structure as a parent system and explored the effects of aromatic substituents on synthase inhibition. The nature of the aryl substitution influences binding, as shown by a 30-fold range of inhibitory potencies observed for the 15 aryl analogues (I50 values = 0.6-18 microM), although there is no apparent correlation between synthase inhibition and the electronic or hydrophobic characteristics of the aryl substituents. To explore the possibility that the aryl ring of these inhibitors might interact with the synthase binding site for the substrate p-aminobenzoic acid (PABA), three compounds were synthesized in which a PABA analogue is bridged to the nitrosoisocytosine moiety by linkage to an amino group at C-6 of the isocytosine. The bridged analogues significantly inhibited the synthase (I50 values = 2.5-8.9 microM) but were of unexceptional potency compared with other members of the (aryloxy)propyl series. Structure-activity considerations and inhibition kinetics did not support the PABA binding site as the synthase region that interacts with the aryl ring of these inhibitors. Despite the potent synthase inhibition exhibited by many of the nitrosoisocytosines studied, none of the 18 new analogues showed significant antibacterial activity.

Monocyclic pteridine analogues. Inhibition of Escherichia coli dihydropteroate synthase by 6-amino-5-nitrosoisocytosines.

A variety of 5,6-disubstituted isocytosine derivatives were evaluated in vitro as inhibitors of dihydropteroate synthase from Escherichia coli. A number of 6-(alkylamino)-5-nitrosoisocytosines have in vitro potency equivalent with or superior to that of therapeutically effective sulfonamide inhibitors of the synthase. The sulfonamide drugs are known to compete for the p-aminobenzoic acid binding site of the synthase, and kinetic analysis of inhibition of the synthase by 6-(methylamino)-5-nitrosoisocytosine (16; I50 = 1.6 microM) and by the 6-(3-phenoxypropyl) amino analogue (33; I50 = 3.7 microM) indicated that the nitrosoisocytosine inhibitors compete with the pteridine substrate for the enzyme. Structure-activity studies demonstrated that the enzyme surface has a low tolerance for steric bulk in the region surrounding the isocytosine 6-amino function. However, this steric intolerance may be counterbalanced to a significant degree by positive allosteric interactions achieved by certain analogues that have a 6-(omega-phenylalkyl)amino substituent. For example, 6-[(7-phenylheptyl)amino]-5-nitrosoisocytosine (28) is as effective an inhibitor (I50 = 1.4 microM) as the 6-methylamino compound 16. Although several members of the 5-nitroso series were potent synthase inhibitors, none of the nitrosoisocytosines exhibited significant antibacterial activity. This observation may reflect poor transport of these compounds through the bacterial cell wall or, alternatively, may result from a rapid metabolic inactivation process.