Isolation rearing significantly perturbs brain metabolism in the thalamus and hippocampus.

Psychosocial neglect during childhood severely impairs both behavioral and physical health. The isolation rearing model in rodents has been employed by our group and others to study this clinical problem at a basic level. We previously showed that immediate early gene (IEG) expression in the hippocampus and medial prefrontal cortex (mPFC) is decreased in isolation-reared (IR) compared to group-reared (GR) rats. In the current study, we sought to evaluate: (1) whether these changes in IEG expression would be detected by the measurement of brain glucose metabolism using positron emission tomography (PET) with fluorodeoxyglucose (FDG) and (2) whether PET FDG could illuminate other brain regions with different glucose metabolism in IR compared to GR rats. We found that there were significant differences in FDG uptake in the hippocampus that were consistent with our findings for IEG expression (decreased mean FDG uptake in IR rats). In contrast, in the mPFC, the FDG uptake between IR and GR rats did not differ. Finally, we found decreased mean FDG uptake in the thalamus of the IR rats, a region we had not previously examined. The results suggest that PET FDG has the potential to be utilized as a biomarker of molecular changes in the hippocampus. Further, the differences found in thalamic brain FDG uptake suggest that further investigation of this region at the molecular and cellular levels may provide an important insight into the neurobiological basis of the adverse clinical outcomes found in children exposed to psychosocial deprivation.

Metabolic flux analysis of postburn hepatic hypermetabolism.

The hepatic response to severe injury is characterized by a marked upregulation of glucose, fatty acid, and amino acid turnover, which, if persistent, predisposes the patient to progressive organ dysfunction. To study the effect of injury on liver intermediary metabolism, metabolic flux analysis was applied to isolated perfused livers of burned and sham-burned rats. Intracellular fluxes were calculated using metabolite measurements and a stoichiometric balance model. Significant flux increases were found for multiple pathways, including mitochondrial electron transport, the TCA and urea cycles, gluconeogenesis, and pentose phosphate pathway (PPP). The burn-induced increase in gluconeogenesis did not significantly increase glucose output. Instead, glucose-6-phosphate was diverted into the PPP. These changes were paralleled by increases in glucose-6-phosphate dehydrogenase (G6PDH) and glutathione reductase (GR) activities. Given that G6PDH and GR are the most significant NADPH producers and consumers in the liver, respectively, and that GR is responsible for recycling the free radical scavenger glutathione, these data are consistent with the notion that hepatic metabolic changes are in part due to the induction of liver antioxidant defenses.

Cutaneous burn injury alters relative tricarboxylic acid cycle fluxes in rat liver.

Severe injury induces a hypermetabolic state in the liver; however, the pathways that are responsible for the increase in hepatic energy demand have not been identified. Relative fluxes in the tricarboxylic acid (TCA) cycle were determined in perfused livers from rats 4 days after administration of a cutaneous burn injury. The perfusate was supplemented with 5 mM uniformly labeled 13C-lactate to efficiently label intracellular metabolites. Flux ratios were calculated on the basis of (1) the 13C-labeling pattern of the glutamate and lactate isotopomers within the liver as determined by nuclear magnetic resonance spectroscopy and (2) an isotopomer mass balance model of the TCA cycle. Calculated flux ratios suggest that burn injury results in an increase in the contribution of pyruvate to the oxaloacetate pool at the expense of non-TCA cycle sources. Furthermore, a dramatic increase in 13C-labeling of glucose was observed in burned rat livers. These data taken together suggest that burn injury induces intrinsic changes in intrahepatic metabolism, including an alteration of the relative fluxes consistent with increased gluconeogenesis from lactate.

TCA cycle flux estimates from NMR- and GC-MS-determined [13C]glutamate isotopomers in liver.

Infusion of 13C-labeled lactate into rabbits and the subsequent measurement of glutamate isotopomers by 13C nuclear magnetic resonance (NMR) spectroscopy enables one to calculate relative flow rates associated with the tricarboxylic acid (TCA) cycle, albeit with a lower precision than one would obtain using a perfused organ. Two factors contribute to the lower precision in the determination of relative flow rates for the in vivo system: 1) a poorly defined pyruvate input and 2) low levels of 13C-enriched oxaloacetate and acetyl-CoA isotopomers, which give rise to weaker glutamate isotopomer NMR signals. To help overcome these limitations, we introduce a procedure to 1) include experimental data from gas chromatography-mass spectrometry (GC-MS) and 2) account for the uncertainty in the labeling of the input to pyruvate by creating the labeling as a measurement that is subject to measurement error. The effects of the uncertainties in the input labeling, NMR data, and MS data are evaluated via a Monte Carlo method. The change in the precision of the relative fluxes for the cases of high/low NMR and high/low MS precision is given. An uncertainty in the lactate measurements of up to 10% does not add significantly to the imprecision of the relative flow rates.

Effect of burn injury on glucose and nitrogen metabolism in the liver: preliminary studies in a perfused liver system.

BACKGROUND: The direct impact of burn injury on liver metabolism was studied in a rat liver perfusion system to remove the influence of systemic factors that modulate liver metabolism. METHODS: Seven animals received a burn injury covering 20% of the total body surface area, and seven were sham burned. The in situ liver perfusion studies were carried out in these animals after 3 days of isonitrogenous-isocaloric enteral feeding. In each study oxygen consumption and the rates of uptake and release of glucose, urea, and various amino acids were measured. RESULTS: Burn injury significantly increased urea production (18.5 +/- 0.4 versus 12.2 +/- 0.6 micromol/gm liver/hr and oxygen consumption (3.23 +/- 0.17 versus 1.21 +/- 0.03 micromol/gm liver/min) in the liver but did not alter the rate of gluconeogenesis. The change in amino acid concentrations in the perfusion medium implies an increased net protein breakdown. CONCLUSIONS: Our study indicates that (1) burn injury induces a hypermetabolic state in the liver, (2) the observed enhancement of gluconeogenesis in vivo after burn in probably regulated by factors outside the liver, and (3) the liver itself plays an active role in up-regulating urea production in burn injury. Identifying intrahepatic factors that up-regulate urea production may provide an "intrahepatic approach" to ameliorate the severe nitrogen loss after burn injury.

An analysis of transport resistances in the operation of BIAcore; implications for kinetic studies of biospecific interactions.

The mass-transfer processes that affect kinetic measurements of biospecific interactions between one species in a flowing solution and another species immobilized in a thin hydrogel instrument were analysed by means of convection-diffusion reaction models. The specific purpose was to identify experimental design considerations for kinetic measurements using the BIAcore instrument. Numerical solutions identified three different regimes of operation. A kinetic regime exists at low values of Damkohler number (Da), when the intrinsic kinetics are slow and the diffusion is relatively fast. This allows for the accurate determination of kinetic constants. A limiting value of Da, above which mass-transfer limitations appear, is presented as a function of Peclet number, Pe. At higher Da values, the reaction occurs in the mass-transfer-controlled regime where the reaction-rate is independent of the intrinsic kinetics. It was observed that, frequently, the reaction occured in an intermediate regime where, although the mass-transfer rate was not strictly limiting, substantial concentration gradients were present. Analysing the data in this regime by direct application of kinetic equations underestimates the association rate constant. Even when the reaction was not limited by mass-transfer in the flow channel, it may have been affected by steric hindrance to transport in the hydrogel, if a large concentration of capturing antibody or ligand was immobilized. The primary effect of the hindrance was to lower the soluble-species (analyte) concentration in the hydrogel when compared to the bulk solution. Non-uniformity of conditions within the hydrogel in the presence of steric hindrance had a significant effect on the observed reaction. The effect was most prominent at higher analyte concentration, when the rate constant showed an apparent reduction as the reaction progressed.

Coupling of antibody-binding fragments to solid-phase supports: site-directed binding of F(ab')2 fragments.

A method to covalently bind antibody fragments, via their carboxyl termini to solid supports, is presented. The strategy involves: (1) reversibly blocking all the accessible carboxyl groups on the antibody molecule with phenylhydrazine, (2) exposing the carboxyl termini of the fragment by enzymatic digestion with pepsin and (3) subsequently coupling the fragment to an appropriate support. Experiments with an anti-bovine serum albumin monoclonal antibody and C-14 phenylhydrazine revealed that the blocking step was nearly completely reversible with a dilute solution of FeCl3. Radioiodinated blocked F(ab')2 fragments were then coupled to an amino-functionalized Sepharose 4B column, and characterized as to their coupling capacity (mass of protein coupled/ml of bead), and antigen-binding activity. The coupling capacity of the blocked fragments was found to be 12%, half the coupling efficiency of unmodified radioiodinated F(ab')2. The antigen-binding capacity (mol antigen bound per mol antibody coupled) for the blocked F(ab')2, on the other hand, was found to be 1.9, which was approx. 3.5-times greater than for the unmodified F(ab')2. Comparisons with other conventional coupling techniques were also made. These preliminary studies suggest that this technique can provide one with the means to obtain more uniform and active populations of immobilized antibody fragments.

Immunoadsorption: strategies for antigen elution and production of reusable adsorbents.

Immunoadsorption is a powerful and generalizable method for protein purification that exploits the fine specificity of antigen-antibody interactions. In spite of its potential utility, the more widespread process scale use of immunoadsorption has been limited by the high cost of the antibody and the lack of gentle elution schemes that completely preserve the activity of both the immunoadsorbent and the eluted product. In this report, we review common chemical elution strategies such as pH, ionic strength, chaotropic salts, denaturants, and organic solvents as well as physical techniques such as pressure, electrokinetics, and temperature. In general, selection of elution strategies has largely been an empirical art, balancing stability of the immunoadsorbent and the eluted product and efficiency. The limitations of the available choices demonstrate that more attention must be placed on the antibody. Techniques which assist in the identification or creation of new antibodies with improved binding properties and resistance to degradation, e.g., screening and/or rational protein engineering, are also discussed.

Immunoaffinity purification: basic principles and operational considerations.

Immunoaffinity purification has become an important technique in biotechnology. In this review the basic principles of immunoaffinity separations are described with respect to the stages of operation and potential application. The most commonly used support materials, activation procedures, and coupling chemistries are compared to one another for suitability in various applications. Individual operational steps for fixed bed immunoadsorbents including loading, washing, elution and regeneration are described in terms of both theory and practice. Factors influencing adsorbent stability are identified, and alternative operation and configuration strategies are discussed in light of their application to immunoaffinity systems.

Role of association on protein adsorption isotherms. Beta-lactoglobulin A adsorbed on a weakly hydrophobic surface.

This paper explores the role of association on the adsorption isotherms of beta-lactoglobulin A on a weakly hydrophobic stationary phase at 4 degrees C and mobile phases of 0.85 M and 1 M ammonium sulfate, pH 4.5. The isotherms, obtained by frontal analysis, show an S-shape and the corresponding Scatchard plots indicate positive cooperativity. The slopes and intercepts of the Scatchard plots at low solute concentration are analyzed in terms of two species--a promoter and a higher order stronger adsorbing species. An explicit equation of the isotherm is developed based on this model, and this expression is shown to reproduce the isotherm shape using the appropriate derived parameters. It is further shown from this equation that a Langmuir-shaped adsorption isotherm can be obtained if the higher order associate or aggregate binds weaker to the support than the promoter. These results indicate that protein-protein interactions and the formation of associates can play a significant role on the shape of the isotherm and ultimately on the behavior of the species in preparative scale chromatography.

Protein aggregation in high-performance liquid chromatography: hydrophobic interaction chromatography of beta-lactoglobulin A.

Aggregation of beta-lactoglobulin A under acidic buffer conditions was studied in hydrophobic interaction chromatography. At high ammonium sulfate concentrations, pH 4.5 and 4 degrees C, UV chromatograms revealed a maximum of three peaks for beta-lactoglobulin A concentrations greater than 5 mg/mL, suggesting three distinct aggregate species. The size of the smallest aggregate (tetramer) and its stoichiometric relationship to the other two aggregates (octamer and dodecamer) were determined from the chromatographic data and a simple mass balance model. These stoichiometries agreed with those determined in a separate study by on-line low-angle laser light scattering. In addition, the association constants describing the formation of octamer from two tetramer molecules and the formation of dodecamer from the octameric and tetrameric species were found to be (2.4 +/- 0.5) X 10(4) M-1 and (3.3 +/- 0.8) X 10(3) M-1, respectively. Analysis of the beta-lactoglobulin A system is based on a model in which aggregates form in solution upon injection before adsorbing to the column matrix. The column retains those species formed in solution and induces little change in the relative amounts of each species. These results illustrate another example by which multiple peaks can arise in high-performance liquid chromatography, beyond the previously described studies of protein conformational changes during chromatography.

Quasi-elastic light scattering of antigen-antibody complexes.

Many biological properties of immune complexes (IC) depend upon their size. Quasi-elastic light scattering (QLS) was used to measure a mean equivalent hydrodynamic radius (Rh) and variance of the distribution of model IC composed of bovine serum albumin (BSA) as antigen (Ag) and combinations of two or three well-characterized monoclonal antibodies (MAb) which bound noncompetitively to unique epitopes on BSA. With the molar ratio (X) of each MAb to Ag fixed, Rh increased with concn. Rh was maximal at equivalence (X = 0.5) with two MAb and at slight MAb excess (X = 0.67) with three MAb. The largest Rh with two MAb was about 200 A, and Rh was uniformly different amongst the three combinations of two MAb IC. The largest hydrodynamic radius of individual complexes which formed with two MAb was estimated to be about 400 A; even larger individual complexes were formed with three MAb. Size changes following alteration of solution concns were also followed with QLS. Kinetics of two MAb IC association were too fast to observe; dissociation following large dilution (40-fold) required 5-10 min to attain a new steady state, much less at small dilution. With three MAb, Rh dropped sharply in 5 min and became steady after 1-2 hr. These results suggest that conventional chromatographic and ultracentrifugation techniques for studying IC size, involving large dilution and long measurement time, provide misleading results. Association of three MAb produced a rapid initial increase of Rh in several min, followed by diverse behavior which depended upon concn. From high to low concn, these included (1) exponential growth of Rh with time and appearance of visible macroscopic particles; (2) metastable states for several hr followed by slow growth to large size over several days, leading to formation of particles; and (3) rapid growth to steady state conditions with no visible particles. This heretofore unobserved equilibrium and kinetic behavior of model IC in solution may be reflected in the behavior of more complex, naturally occurring IC.

Monoclonal antibodies to bovine serum albumin: affinity and specificity determinations.

A panel of 12 monoclonal antibodies (MAb) to bovine serum albumin (BSA) was developed and characterized as to their physiochemical and immunological properties. Affinity constants of the MAb varied over a wide range from 10(5) to 10(8) M-1. MAb were assembled into several groups of non- or minimally interacting antibodies by analysis of competitive binding experiments, and BSA domain and subdomain specificities of the MAb were assigned by analysis of results of MAb binding to purified BSA fragments. Further fine specificity delineation was accomplished by examination of cross-reactivity patterns to several mammalian albumins. The data suggest that some of the low affinity MAb recognize sites on different portions of the BSA molecule, indicating that similar epitopes exist on different domains of the BSA molecule.

A new technique for mapping epitope specificities of monoclonal antibodies using quasi-elastic light scattering spectroscopy.

Quasi-elastic light scattering (QLS) was used to determine relative epitope specificities of a group of anti-bovine serum albumin (BSA) monoclonal antibodies (MAb). QLS is a non-invasive technique which can determine the mean size and size distributions of macromolecular scatterers by analysis of the fluctuations in the intensity of laser light scattering. When two MAbs are mixed together with antigen, QLS detects the complex formation which results from the Ag-Ab reaction, and can easily distinguish between the large complexes formed by interaction of non-competitive MAbs and the smaller complexes formed by competitive MAbs. In this report, the competitive or non-competitive behavior of six anti-BSA MAbs were assessed by radioimmunoassay (RIA) and QLS analysis. The results obtained by QLS analysis confirmed the RIA findings indicating that the six MAbs examined can be categorized into three distinct, non-interacting groups. QLS analysis represents a simple, and extremely rapid technique for epitope mapping studies.