Masking foods for food challenge: practical aspects of masking foods for a double-blind, placebo-controlled food challenge.

In diagnosing a food allergy or food intolerance, a double-blind, placebo-controlled food challenge (DBPCFC) with the suspected food or food substance is the only method available for objective confirmation of an assumed relationship between a suspected agent and a complaint. When the use of capsules as a vehicle for DBPCFC with the suspected agent is not feasible, recipes have to be developed for masking the suspected food with another food. We describe demands and other aspects of the development of recipes for DBPCFC purposes. The taste, smell, color, and texture of the suspected agent have to be hidden in such a way that the patient cannot tell which of the two foods (the active food or the placebo food) contains the suspected agent. Once developed, the recipes have to be judged carefully to ensure that the foods do not contain ingredients other than the suspected agent that can possibly provoke complaints in the patient. Besides ordinary equipment such as cutlery, glasses, and dishes, the minimal equipment needed for preparing the recipes is an accurate balance. The more laborious a recipe is to make, the more equipment is needed. The development of recipes and the preparation of masked foods are time-consuming, which makes DBPCFC with masked foods difficult to perform in daily clinical practice. Performing a DBPCFC with masked foods in daily clinical practice can be a great challenge to the imagination and creativity of a dietitian.

Vitamin C status in elderly women: a comparison between women living in a nursing home and women living independently.

The vitamin C status in blood fractions in 135 elderly women aged 65 years and older was studied within the framework of the Dutch Nutrition Surveillance System. Mean (+/- standard deviation) vitamin C intake (mg/day) was lower among women living in a nursing home (54 +/- 27 mg/day) than among women living in service flats (97 +/- 55 mg/day) and women living independently (132 +/- 44 mg/day). (Service flats are apartments in which the rent includes housekeeping and, when ordered, meal service.) Marginal vitamin C values (< 23 mumol/L) in blood fractions and even levels as low as those found in clinical scurvy (< 11 mumol/L) were frequently observed. In the nursing home, 35% of the women had plasma vitamin C values below 11 mumol/L, and 23% had values between 11 and 23 mumol/L. Blood levels were not significantly affected by age, smoking status, or use of particular drugs but were strongly (r = .47 or, after logarithmic transformation, r = .64) associated with daily intake of vitamin C. Low intake of vitamin C resulted from an overall low food consumption and selective restriction of food products rich in vitamin C. Vitamin C losses caused by food preparation practices and distribution in the nursing home's catering system reduced actual vitamin C intake levels but these losses were not substantially greater than those that are assumed to occur as a result of preparation practices by women living independently.

Stabilization of the nuclear matrix by disulfide bridges: identification of matrix polypeptides that form disulfides.

The molecular structure of the nuclear matrix is still poorly understood. We have tried to assess which proteins are important structural elements by examining the process of stabilization of the nuclear matrix by sodium tetrathionate. Sodium tetrathionate stabilizes the nuclear matrix by oxidizing sulfhydryl groups to disulfides. We show that tetrathionate-stabilized matrices are disassembled in buffers containing SDS, indicating that the stabilized nuclear matrix is not a continuous network of cross-linked proteins. Using monobromobimane, a thiol-specific fluorescent reagent, we show that many protein thiols in the stabilized matrix are oxidized. By chromatography on activated thiol-Sepharose we estimated that about 50% of the matrix proteins had oxidized sulfhydryl groups. The protein composition of the material bound to activated thiol-Sepharose was similar to that of the not-bound material. A few proteins are highly enriched in the fraction that was bound to the column. This indicates that many matrix protein species are partially oxidized and that some proteins are completely oxidized. The oxidized protein thiols are found in relatively large complexes as determined by SDS gel-electrophoresis under nonreducing conditions. These results are interpreted in terms of protein-protein interactions in the matrix. The possible role of thiols and disulfides in the in vivo organization of the nucleus is discussed.

Transport-associated phosphorylation of 2-deoxyglucose in rat adipocytes.

The relationship between ATP levels and 2-deoxyglucose uptake was investigated. When the concentration in the medium lies between 1 and 10 mM 2-deoxyglucose uptake causes a marked decrease in ATP level. This could partly be explained by an inhibiting effect of 2-deoxyglucose and 2-deoxyglucose 6-phosphate on ATP synthesis in the mitochondria. A good correlation between the various ATP levels induced by 2,4-dinitrophenol and the rate of uptake of 5 microM and 0.5 mM (but not 5 mM) 2-deoxyglucose was observed. The addition of glucose and 2-deoxyglucose to cells incubated in the presence of trace amounts of 2-deoxy-[1-14C]glucose induced marked changes in the uptake of the tracer that were associated with a rapid decline in ATP level. It appeared that the phosphorylation of 2-deoxyglucose is an important step in the uptake of the sugar. It is hypothesized that the processes of transport and phosphorylation of 2-deoxyglucose are coupled in rat adipocytes.

Differential effects of triphenyltin and 8-azido-ATP on the ATP synthesis, ATP-Pi exchange, and ATP hydrolysis in liposomes containing ATP synthase and bacteriorhodopsin.

The ATP hydrolysis activity of purified ATP synthase reconstituted in liposomes was inhibited by triphenyltin in a manner different from that of other thiol-specific reagents. In liposomes containing ATP synthase and bacteriorhodopsin, ATP hydrolysis and ATP-Pi exchange were inhibited by triphenyltin to a greater extent than the ATP synthesis, in contrast to what was found with an F1-specific inhibitor, 8-azido-ATP. The possibility is discussed that ATP hydrolysis and ATP synthesis are differently coupled to proton conduction through F0.

The phosphate potential maintained by mitochondria in State 4 is proportional to the proton-motive force.

Evidence is presented for a proportional relationship between the extramitochondrial phosphate potential (delta Gexp) and the proton-motive force (delta mu H+) across the mitochondrial membrane in rat-liver mitochondria oxidising succinate in State 4, when delta mu H+ is varied by addition of uncouplers or malonate. This relationship was found when precautions were taken to minimise interference with the determination of delta Gexp and delta mu H+ by intramitochondrial nucleotides, adenylate kinase activity, the quenching method, and delta mu H+-dependent changes in matrix volume. A non-proportional delta Gexp/delta mu H+ relationship was obtained when these precautions were omitted. Our results do not support mosaic protonic coupling, but are not necessarily in conflict with other localised coupling schemes.

Effect of cryosolvent on transient kinetics of the glutamate dehydrogenase reaction.

The transient and steady-state kinetics of the oxidative deamination of L-glutamate by glutamate dehydrogenase and NADP in both aqueous solution and 30% methanol are compared. Methanol causes an approximately 5-fold tightening of the enzyme--L-glutamate binary complex and an approximately 2-fold reduction of the interaction parameter for the ternary enzyme--NADP--L-glutamate complex. The most dramatic effect of methanol on the time course of the reaction is what appears to be a conversion of the enzyme at substoichiometric initial levels of reactant NADP to a form from which product alpha-ketoglutarate does not readily dissociate. This conversion appears only at NADP concentrations over one-third of the enzyme active site concentration.

Temperature-dependent delta C0p generated by a shift in equilibrium between macrostates of an enzyme.

Substantial negative heat capacity changes (delta C0p' s) have frequently been observed to accompany the formation of protein-ligand complexes. Glutamate dehydrogenase and horse liver alcohol dehydrogenase, however, have been reported to form binary complexes with coenzyme with negligible delta H0p' and only small delta C0p' s. Although many intriguing mechanisms have been proposed to account for the observed phenomena, there is little direct experimental evidence available which might provide a basis for evaluating the contributions of delta C0p' s of complex formation from the various mechanistic sources or even for distinguishing between them. However, if, as Eftink and Biltonen have suggested, a shift in equilibrium between macrostates contributes significantly to an observed delta C0p' s for a given reaction, it should be possible to characterize such a system by measuring the temperature dependence of the delta C0p'. Despite this, few studies have determined delta H0' values at more than two temperatures. We have now measured the temperature dependence of the delta H0' (and, thereby, that of the delta C0p') of the formation of an enzyme-reduced coenzyme complex in an attempt to provide such a basis and have found that the entire delta C0p' of complex formation is accounted for by a temperature-induced shift of an equilibrium between the different forms of the free enzyme.

pH-dependent thermodynamic parameters of the glutamate dehydrogenase-alpha-ketoglutarate-NADPH complex.

The enthalpy of formation of the reactive bovine glutamate dehydrogenase-alpha-ketoglutarate-NADPH complex has been measured as a function of pH, at two temperatures and in two buffer systems having different enthalpies of ionization. The results demonstrate the existence of an extensive two-way traffic of protons between the buffer and the complex itself. While the pattern in a single buffer is too complex to resolve within the experimental limitations imposed by the system, we develop a theory which shows that the difference in delta H0H+ between buffers of different enthalpies of ionization has a simpler, more easily resolvable form. From such a relationship we show that in the process of binding of a molecule of NADPH to the enzyme-alpha-ketoglutarate complex, a proton has been shifted from the buffer to the complex and that this is due to the shift of the pK of an ionized group on the enzyme to a much higher value when the complex is formed. The delta Cp of formation of the complex is -450 cal . deg.-1 . M-1, and is pH independent. Implications of these results for the interpretation of enthalpies of enzyme-complex formation obtained at a single pH, and at one temperature, with one buffer system are mentioned. The complexity of the pattern of the dependence of delta H0, taken together with the magnitudes of the individual features of these dependencies, suggest that a number of large pK shifts occur when the enzyme-NADPH-alpha-ketoglutarate complex is formed. It is quite probable that these shifts represent a sizeable fraction of the machinery of the catalytic mechanism.

The thermodynamics of a negatively interacting allosteric effector system. The glutamate dehydrogenase . NADPH . ADP complexes.

The reaction catalyzed by bovine L-glutamate dehydrogenase is subjected to allosteric activation by ADP. We have measured the thermodynamic parameters (delta G0, delta H0, delta S0, and delta Cp) of the formation of the various possible binary and ternary complexes formed between the enzyme, NADPH, and either ADP or its analogs, adenosine, AMP, and ATP. delta H0 and delta Cp have been measured by flow calorimetry; delta G0 values obtained by calorimetry itself, differences spectroscopy, or gel filtration have been selected on the basis of accuracy under the conditions required for the formation of each complex. The data are interpreted in terms of "interaction parameters," the differences between the thermodynamic parameters of the formation of a ternary complex and the sum of those of the two related binary complexes. Both adnosine and ATP appear to loosen the binding of NADPH by simply preventing a subsite interaction of NADPH. AMP appears to have only minor and probably secondary effects. The negative effect of the binding of ADP on that of NADPH, however, involves the formation of a new interaction, which is exothermic, entropy compensated, has a moderately large negative delta Cp, and does not occur in either binary complex.

Effect of ammonia on the glutamate dehydrogenase catalyzed oxidative deamination of L-glutamate. The steady state.

Ammonia is known to inhibit the steady-state rate of oxidation of L-glutamate catalyzed by glutamate dehydrogenase. We reported previously [Brown, A., Colen, A. H., & Fisher, H. F. (1978) Biochemistry 17, 2031] kinetic evidence supporting the formation in the initial rapid phase of a complex which is composed of enzyme, reduced coenzyme, alpha-ketoglutarate, and ammonia. We show here that the effects of ammonia on the steady-state reaction can be correlated with transient-state kinetic effects related to the concentration of that ammonia-containing complex. These results indicate the existence of alternate reaction pathways which become important at high ammonia concentrations. These new pathways provide an additional route for the release of NADPH from the enzyme surface. The expanded mechanism shows that the noncompetitive product inhibition by ammonia can occur without the simultaneous presence of ammonia and L-glutamate on the enzyme. This mechanism also accommodates the observed substrate inhibition by L-glutamate.

The effects of methanol on the glutamate dehydrogenase reaction at 0 degrees C.

The effects of 0-30% methanol (vol/vol) on the Km an Vm values for both the forward and reverse directions of the L-glutamate dehydrogenase reaction were determined at 0 degrees C. The decrease in temperature alone had very little effect on these parameters. However, in the forward reaction, 30% methanol resulted in a 14-fold decrease in the Km value for glutamate, a slight decrease in the Km value for NADP, and a thirty-fold decrease in Vm. Substrate inhibition by glutamate was observed at concentrations greater than 4 mM. In the reverse reaction, 30% methanol caused a decrease in the Km values for alpha-ketoglutarate and ammonia and a 10-fold decrease in Vm. Substrate inhibition by both alpha-ketoglutarate and NADPH was observed at concentrations of either substrate above 0.03 mM. The dependence of Km for glutamate and Vm values for the forward reaction on methanol concentration suggests that they are similarly affected by methanol, in direct contrast to results obtained for NADP. Methanol appeared to cause a general tightening of complexes, which may arise from an effect on the "activities" of species in solution. The use of methanol not only allows for the study of reaction intermediates by slowing the reaction with the cryogenic method, but may also serve as a mechanistic probe by affecting several polarity as well as Km, Vm, and K1 values.

Effect of ammonia on the glutamate dehydrogenase catalyzed oxidative deamination of L-glutamate: production of an ammonia-containing intermediate in the "burst" phase.

We have studied the effects of ammonium acetate on the transient "burst" phase of the oxidation of L-glutamate by glutamate dehydrogenase. Two measurable changes are observed in the "burst" phase as ammonium acetate concentration is increased: (i) an increase in the apparent first-order rate constant, kapp, and (ii) a decrease in the amplitude of the absorbance change measured at 320 nm. The increase in kapp shows a hyperbolic dependence on ammonium acetate concentration and is independent of glutamate concentration. The results demonstrate the existence of an intermediate immediately following hydrogen transfer. The intermediate contains enzyme, reduced coenzyme, ammonia, and alpha-ketoglutarate moieties and is in equilibrium with the known complex consisting of enzyme, reduced coenzyme, and alpha-ketoglutarate. At high concentrations of ammonium acetate, the equilibrium favors the ammonia containing complex.

Transient-state kinetics of L-glutamate dehydrogenase: mechanism of alpha-ketoglutarate inhibition in the burst phase.

Stopped-flow studies of the initial burst of NADPH production accompanying the oxidative deamination of L-glutamate by L-glutamate dehydrogenase and NADP+ were performed in the presence of alpha-ketoglutarate, a product of the reaction. Both binary enzyme-alpha-ketoglutarate and ternary enzyme--NADP+-alpha-ketoglutarate complexes are inhibitory in the burst presence of the enzyme-catalyzed reaction. Order-of-addition experiments show the binary complex to form rapidly, in the 3 ms dead time of the stopped-flow instrument. There is a distinct lag, however, in the achievement of the full ternary complex inhibitory effect unless the enzyme is preincubated with both NADP+ and alpha-ketoglutarate prior to initiation of the catalytic reaction with L-glutamate. The formation of an inhibitory enzyme--NADP+-alpha-ketoglutarate complex appears to be sufficiently slow to give a delayed kinetic response when alpha-ketoglutarate is added to the reaction system.