Biomedical subjects

E N Lightfoot

Publications and source records attributed to E N Lightfoot.

At least 19 recordsLinked to original sources

PubMed2004

Bioseparations.

Here we review key applications of separation technology in applied biology. We first sketch out the field as a whole, but then narrow our scope to the processing of fermentation products, particularly to high-value biologicals such as proteins and nucleotides. We go on to provide a qualitative overview describing the importance and general nature of this large field, major trends, and the strategies that have proven most fruitful in evolving effective separation and purification processes. We then give a detailed description of individual separations equipment and the principles governing their operation. We concentrate throughout on making the available literature accessible to the reader; we provide what is hoped to be a representative set of basic references. However, these references, in turn, include some that suggest promising new developments as well as a number of more specialized reviews. We hope that our overall result provides the reader with access to the most relevant literature.

Biopolymers↗

PubMed2003

Adsorptive membrane chromatography for purification of plasmid DNA.

Adsorptive membranes were investigated for the downstream processing of plasmid DNA by quantifying both separation efficiencies and adsorption uptake with the anion-exchange membranes. Separation efficiencies of the 10-ml Mustang-Q were measured using pulses of 6.1-kilo base pair plasmid DNA and lysozyme tracers, and comparing the responses for both conventional and reverse-flow operation. The plasmid exhibited nearly 200 plates/cm, almost as high efficiency as the protein despite the large difference in size. This behavior contrasts strongly with typical behavior for spherical porous particle packings, which predicted large decreases in efficiency with increases in tracer size. Batch adsorption isotherms for the 6.1-kilo base pair plasmid on small sheets of anion-exchange membranes at various ionic strengths showed high capacities for very large biomolecules. The maximum binding capacity for the membrane unit was calculated as 10 mg plasmid/ml, an order of magnitude greater than typical values reported for porous beads.

Adsorption↗

PubMed1998

Refining the scale-up of chromatographic separations.

The use of heavily loaded columns and complex processing conditions makes scale-up of chromatographic separations a non-trivial process. The wide ranges of process conditions that must be investigated demands that a large number of preliminary experiments must usually be made in small columns and laboratory-scale work stations. These preliminary data can be biased by improper column packing, poor distributors and dispersion in auxiliary apparatus, and it is important to understand these disturbing factors in detail. Moreover, it is precisely at this macroscopic level that our understanding of the chromatographic process is weakest, for large columns as well as small. This paper addresses three of these factors: Efficient elimination of peripheral effects and characterization of both header flow distribution and packing non-uniformity. This will be done using a variety of experimental and analytical approaches including nuclear magnetic resonance imaging, computational fluid dynamics and mass transfer, and careful experimentation.

Acetone↗

PubMed1997

Measuring column void volumes with NMR.

A novel method for measuring resin porosities and column void volumes with fluorine. NMR has not been developed. In situ measurements of the void volumes accessible to an array of fluorinated probe molecules are used to characterize the pore size distribution of the media. Application of this simple procedure is demonstrated for a commercially packed column and several bulk resins. The porosity distributions obtained by this technique are similar to those obtained by size exclusion chromatography. Unlike chromatographic tracer studies, however, this method does not require packed columns.

Magnetic Resonance Spectroscopy↗

PubMed1995

A model for multiple subcutaneous insulin injections developed from individual diabetic patient data.

Many diabetic patients taking multiple subcutaneous insulin injections cannot adjust their dosage appropriately to maintain blood glucose within a normal range. It is hard to predict how dosage changes and physiological fluctuations affect insulin levels and subsequently glucose control. To examine these issues, we have developed a model representing the link between dosage and blood insulin levels. Our model adequately predicts insulin concentrations for individual patients and could be incorporated into an overall glucose-insulin representation. More importantly, parameter and sensitivity analysis results highlight insulin kinetic features that are difficult to isolate in a clinical setting and that may significantly influence glucose dynamics. For example, large interpatient variation, measured quantitatively by model parameters, emphasizes the need for individualized design of insulin regimens. Intrapatient variations are also large in some patients. Improved control for these patients may only be possible through more frequent sampling and control action. The sensitivity coefficient for absorption suggests a significant overlapping injection effect that is not considered in present patient management strategies.

Diabetes Mellitus↗

PubMed1994

High-resolution chromatography of proteins in short columns and adsorptive membranes.

Short chromatographic columns prepared from stacks of microporous adsorptive membranes are promising for preparative-scale fractionation of even rather closely related proteins, but careful selection of operating conditions is needed for success. It has been shown that existing devices exhibit very low internal diffusional resistance, and that resolution is almost totally independent of percolation velocity. Total column length is short, however, and the number of plates exhibited under isocratic low-loading conditions is small, on the order of 100. Simulations using a large survey of protein thermodynamic data show that one can frequently obtain excellent protein separations in these short columns by using the sensitivity of protein adsorption equilibria to eluting solvent composition. In fact, some proteins can be separated in a single stage utilizing this 'on-off' behavior and properly selected solvent gradients. Solely on-off, or differential elution, behavior cannot often be depended upon under the mild conditions need for preparative operations. Carefully programmed gradient elution can frequently produce acceptable purification by maximizing both differential elution and differential migration, resulting in protein separations in columns of less than fifty plates. Means for doing this are described for some simple situations, and criteria are provided for selecting modulator gradient schedules.

Animals↗

PubMed1993

Comparing steady counterflow separation with differential chromatography.

Separation of closely related solutes by steady solid-fluid counterflow is compared with differential separation in a fixed chromatographic bed. Analogous expressions for exit concentration and mean residence time in the two systems are presented. A counterpart to chromatographic resolution is derived for binary steady counterflow separations. Estimated counterflow savings in product-concentration dilution, solvent volume requirement and solid-phase volume requirement obtained with these expressions relative to comparable chromatographic operations are compared with experimental results from adsorptive, simulated moving beds. Analysis of a size-exclusion protein separation suggests counterflow substantially decreases solvent and resin usage relative to conventional, batch operation.

Chromatography, Liquid↗

PubMed1993

Biopulping process design and kinetics.

Biopulping is the solid-state fermentation of wood chips as a pretreatment for mechanical pulping processes. The two organisms that are currently of the greatest interest for biopulping are the white-rot fungi, Phanerochaete chrysosporium and Ceriporiopsis subvermispora. P. chrysosporium has been shown to successfully biopulp wood (33% energy savings; 39% improvement in tear index) without the need for sterilization of the wood or nutrient supplementation. Demonstrating the practical and economical feasibility of the biopulping process requires process modeling based on accurate kinetic data. Techniques to monitor dry weight loss and growth rate as functions of time using carbon dioxide production data have been developed. Growth was shown to be linear with time on unsupplemented chips and exponential with time on supplemented chips.

Journal Article↗

PubMed1987

Extending the quasi-steady state concept to analysis of metabolic networks.

A means is proposed for evaluating enzyme effectiveness in vivo via a simplified dynamic description of the metabolic reaction network within which the enzyme operates. The basis of the method is application of sensitivity analysis to a quasi-steady approximation of a complete dynamic model, and its implementation centers on interpreting the transient relations of selected intermediates following a perturbation to the system of interest: for many important situations such relations can be simply interpreted to give a useful global measure of enzyme effectiveness. This method is found to be successful for estimating phosphofructokinase and pyruvate kinase activity in the human red cell, and it appears promising as a basis for developing a means for detecting enzyme abnormalities caused by environmental or genetic factors. This method may also prove useful for comparative studies of glycolysis in different types of cells. The analysis presented is based on available models of red cell glycolysis, but the results are not highly sensitive to ambiguities in the system model. The approach suggested appears to provide an effective means for describing system dynamics and determining the behavior of an individual enzyme in an intact system by making a first-order allowance for interaction with the system as a whole. Requirements for success of this approach remain to be identified in detail, but effective time-scale separation is probably the key.

Erythrocytes↗

PubMed1986

Hydrogen washout in bone cortex and periosteum.

Residence time distributions of hydrogen in bone of anesthetized dogs and rabbits were used to estimate local blood perfusion rates and to characterize the important transport processes taking place. The hydrogen was administered by inhalation, and the concentrations in the bone were measured by embedded platinum microelectrodes. Mean residence times varied significantly both with position and time, and it was found preferable to calculate residence time from moments of the residence time distribution rather than the downslope method. Moreover, the downslope on a semilogarithmic scale continued to decrease with the increase in observation time. For the tissue investigated, simple compartmental models are inadequate even for the small regions characterized by the electrodes. This means that a large number of Haldanian compartments are needed even to characterize local washout behavior. The significance of this finding for the selection of decompression schedules is briefly discussed.

Animals↗

PubMed1985

Mathematical modelling of dynamics and control in metabolic networks. III. Linear reaction sequences.

Kinetics of linear sequences of enzymatic reactions converting a single substrate into a single product are examined with emphasis on obtaining the relationship between the individual kinetic parameters and overall dynamic behavior. Chains of reactions exhibiting irreversible Michaelis-Menten kinetics are examined via scaling, linearization and modal analysis. The modal analysis gives the conditions under which the quasi-steady state assumption is applicable for one reaction relative to another in such a reaction sequence. The linearized description permits characterization of the transient response in terms of temporal moments. The moments provide useful physical insight and also provide a basis for systematic model reduction.

Enzymes↗

PubMed1985

Mathematical modelling of dynamics and control in metabolic networks. IV. Local stability analysis of single biochemical control loops.

The objective of the study presented herein is to describe the dynamic behavior of a single biochemical control loop, a simple system but an important element of metabolic networks. This loop is a self-regulated sequence of reactions that converts an initial substrate (S) into a final product (P). It consists of three basic elements: (1) a regulated reaction, where the concentration of P controls the flux (I) into the system. This element serves as the control element in the feedback circuit. (2) a sequence of unregulated reactions that leads to the formation of P. This process is to be regulated so that the production rate of P meets a desired target. (3) a process (R) that removes P from the loop to another part of the metabolic network. A mathematical description is formulated that consists of two differential equations and two unspecified functions that represent the reaction rates of I and R. This description is scaled to clarify functional dependence and to attempt a separation of genetic and process determined parameters. The global dynamic behavior of the model is assessed qualitatively by examining the occurrence of static and dynamic bifurcations, multiple steady states or sustained oscillations respectively, via local stability analysis. General criteria for both types of bifurcations are developed without specifying the functional form of I and R, but explicitly accounting for the kinetic properties of the reaction chain. A particularly simple criterion is found for static bifurcations which can appear only for loops with positive feedback, i.e. when the regulated reaction is activated by P. This criterion only contains the properties of I and R. The criteria for dynamic bifurcations, which occur when the feedback interaction is inhibitory, are more complex. These depend strongly on the properties of the reaction chain, and oscillations are favored if the dynamic operator describing the reaction sequence is of high order or if it contains time delays.

Feedback↗

PubMed1985

Mathematical modelling of dynamics and control in metabolic networks. V. Static bifurcations in single biochemical control loops.

Here we expand an earlier study of feedback activation in simple linear reaction sequences by searching the parameter space of biologically realistic rate laws for multiple stable steady states. The impetus for this work is to seek the origin of decision making strategies at the metabolic level, with particular emphasis on the switching between the operating conditions needed to meet changing substrate availability and organism requirements. The control loop considered herein is a linear reaction chain in which the end product of the reaction sequence feedback activates the first reaction in the sequence to produce feedback control. It has been found that the criteria for the existence of multiple steady state solutions in such loops involve only the kinetics of the regulatory enzyme controlling the first reaction and that of end product removal. The effects of these kinetics are examined here using two representative models for the regulatory enzyme: the lumped controller, based on Hill-type kinetics, and the symmetry model. The behavior of these two models is qualitatively similar, and both show the characteristics needed for switching between low and high substrate utilization. The removal rate is assumed to be of the Michaelis-Menten type. Judicious scaling of the governing equations permits separation of genetically determined kinetic parameters from concentration dependent ones. This allows us to conclude that, for a fixed set of kinetic parameters, the steady state flux through the loop can be switched between stable steady states by merely varying metabolite or enzyme concentrations. In particular, when the initial substrate exceeds a certain critical level, the loop can be "switched on" (by a discontinuous increase in the flux through the chain), and similarly, when it falls below a critical level, the pathway is shut down. Similar effects can be realized by varying the ratios of enzyme concentrations. It is proposed that by identifying these critical points one can gain significant insight into the objectives of decision making at the metabolic level.

Enzymes↗

PubMed1985

Physicochemical characterization of cryogenically ground, size separated, fibrogenic particles.

A method for cryogenically grinding and separating (by size) fibrogenic minerals in the 1-micron size range is described and verified for chrysotile asbestos, quartz, forsterite (an olivine), and tantalum with a battery of analytical tests. Through use of energy dispersive X-ray spectroscopy, neutron activation analysis, X-ray photoelectron spectroscopy, and X-ray diffraction analysis it is shown that the grinding and separation procedure described does not alter the mineral composition, preserves the trace element composition, maintains the surface composition, and preserves the crystalline structure. Further, investigation of electrokinetic properties of these dusts by electrophoretic quasielastic light scattering is described. The small size dispersity of these samples facilitates use of this technique for the determination of the apparent electrokinetic charge and estimations of surface charge density at ionic strengths below physiological. It is suggested that analyses of the type described here be an integral part of studies of the fibrogenic, immunologic, or toxicologic properties of such minerals. This work has been performed in conjunction with the authors' studies of the effects of these particulates on macrophage ultrastructure and immunologic function in vitro.

Chemical Phenomena↗

PubMed1984

Mathematical modelling of dynamics and control in metabolic networks. I. On Michaelis-Menten kinetics.

As a starting point for modeling of metabolic networks this paper considers the simple Michaelis-Menten reaction mechanism. After the elimination of diffusional effects a mathematically intractable mass action kinetic model is obtained. The properties of this model are explored via scaling and linearization. The scaling is carried out such that kinetic properties, concentration parameters and external influences are clearly separated. We then try to obtain reasonable estimates for values of the dimensionless groups and examine the dynamic properties of the model over this part of the parameter space. Linear analysis is found to give excellent insight into reaction dynamics and it also gives a forum for understanding and justifying the two commonly used quasi-stationary and quasi-equilibrium analyses. The first finding is that there are two separate time scales inherent in the model existing over most of the parameter space, and in particular over the regions of importance here. Full modal analysis gives a new interpretation of quasi-stationary analysis, and its extension via singular perturbation theory, and a rationalization of the quasi-equilibrium approximation. The new interpretation of the quasi-steady state assumption is that the applicability is intimately related to dynamic interactions between the concentration variables rather than the traditional notion that a quasi-stationary state is reached, after a short transient period, where the rates of formation and decomposition of the enzyme intermediate are approximately equal. The modal analysis reveals that the generally used criterion for the applicability of quasi-stationary analysis that total enzyme concentration must be much less than total substrate concentration, et much less than St, is incomplete and that the criterion et much less than Km much less than St (Km is the well known Michaelis constant) is the appropriate one. The first inequality (et much less than Km) guarantees agreement over the longer time scale leading to quasi-stationary behavior or the applicability of the zeroth order outer singular perturbation solution but the second half of the criterion (Km much less than St) justifies zeroth order inner singular perturbation solution where the substrate concentration is assumed to be invariant. Furthermore linear analysis shows that when a fast mode representing the binding of substrate to the enzyme is fast it can be relaxed leading to the quasi-equilibrium assumption. The influence of the dimensionless groups is ascertained by integrating the equations numerically, and the predictions made by the linear analysis are found to be accurate.(ABSTRACT TRUNCATED AT 400 WORDS)

Diffusion↗

PubMed1984

Mathematical modelling of dynamics and control in metabolic networks. II. Simple dimeric enzymes.

The dynamics of enzyme cooperativity are examined by studying a homotropic dimeric enzyme with identical reaction sites, both of which follow irreversible Michaelis-Menten kinetics. The problem is approached via scaling and linearization of the governing mass action kinetic equations. Homotropic interaction between the two sites are found to depend on three dimensionless groups, two for the substrate binding step and one for the chemical transformation. The interaction between the two reaction sites is shown capable of producing dynamic behavior qualitatively different from that of a simple Michaelis-Menten system; when the two sites interact to increase enzymatic activity over that of two independent monomeric enzymes (positive cooperativity) damped oscillatory behavior is possible, and for negative cooperativity in the chemical transformation step a multiplicity of steady states can occur, with one state unstable and leading to runaway behavior. Linear analysis gives significant insight into system dynamics, and their parametric sensitivity, and a way to identify regions of the parameter space where the approximate quasi-stationary and quasi-equilibrium analyses are appropriate.

Allosteric Regulation↗

PubMed1984

The effects of axial diffusion and permeability barriers on the transient response of tissue cylinders. II. Solution in time domain.

A mathematical description of transient mass transfer in a Krogh tissue cylinder, for which a solution in transform space was presented previously, is solved in the time domain. The solution is found in the form of an expansion in terms of the eigenfunctions of a non-self-adjoint differential operator, with the eigenvalues being found by way of a computational scheme which makes use of the known characteristics of the constitutive compartments of the system. The solution is compared with previous solutions of both a complete and an approximate nature, and two modifications of the single-phase axial dispersion model are found to be especially useful: the previously-used flow-limited approximation is satisfactory for highly permeable solutes, while the apparently novel barrier-limited approximation is accurate for poorly permeable solutes at the early times of most experimental interest. Although the neglect of axial diffusion does not affect the qualitative nature of the solution, e.g. in predicting a bimodal response curve, significant discrepancies shed doubt on this practice when truly impulsive inputs are used. The results obtained raise several questions regarding existing approaches to interpretation of indicator dilution experiments. These include the use of extraction ratios and of exponential extrapolation of the tails of response curves.

Capillary Permeability↗

PubMed1984

Microcirculatory mass transfer.

The dynamics of Krogh tissue cylinders and related structures are critically reviewed to determine the roles of underlying transport and reaction processes, and the interactions between them. Emphasis is put on the gaining of insight through efficient scaling procedures, and discussion is organized about the time constants characteristic of these structures and the processes occurring in them. The basis of discussion is a new analytic solution technique which provides a formal description of indefinitely large arrays of parallel interacting elements and which includes both axial diffusion and uniform convection as well as first or zero order reaction within each element. The solution has the form of an expansion in the eigenfunctions of a non-self-adjoint differential operator. Comparison of model predictions with previously available results identifies the useful parameter ranges of analytic approximations and determines the accuracy of existing numerical procedures.

Biological Transport, Active↗