Mass conserved elementary kinetics is sufficient for the existence of a non-equilibrium steady state concentration.

Living systems are forced away from thermodynamic equilibrium by exchange of mass and energy with their environment. In order to model a biochemical reaction network in a non-equilibrium state one requires a mathematical formulation to mimic this forcing. We provide a general formulation to force an arbitrary large kinetic model in a manner that is still consistent with the existence of a non-equilibrium steady state. We can guarantee the existence of a non-equilibrium steady state assuming only two conditions; that every reaction is mass balanced and that continuous kinetic reaction rate laws never lead to a negative molecule concentration. These conditions can be verified in polynomial time and are flexible enough to permit one to force a system away from equilibrium. With expository biochemical examples we show how reversible, mass balanced perpetual reaction(s), with thermodynamically infeasible kinetic parameters, can be used to perpetually force various kinetic models in a manner consistent with the existence of a steady state. Easily testable existence conditions are foundational for efforts to reliably compute non-equilibrium steady states in genome-scale biochemical kinetic models.

Quantitative assignment of reaction directionality in a multicompartmental human metabolic reconstruction.

Reaction directionality is a key constraint in the modeling of genome-scale metabolic networks. We thermodynamically constrained reaction directionality in a multicompartmental genome-scale model of human metabolism, Recon 1, by calculating, in vivo, standard transformed reaction Gibbs energy as a function of compartment-specific pH, electrical potential, and ionic strength. We show that compartmental pH is an important determinant of thermodynamically determined reaction directionality. The effects of pH on transport reaction thermodynamics are only seen to their full extent when metabolites are represented as pseudoisomer groups of multiple protonated species. We accurately predict the irreversibility of 387 reactions, with detailed propagation of uncertainty in input data, and manually curate the literature to resolve conflicting directionality assignments. In at least half of all cases, a prediction of a reversible reaction directionality is due to the paucity of compartment-specific quantitative metabolomic data, with remaining cases due to uncertainty in estimation of standard reaction Gibbs energy. This study points to the pressing need for 1), quantitative metabolomic data, and 2), experimental measurement of thermochemical properties for human metabolites.

A variational principle for computing nonequilibrium fluxes and potentials in genome-scale biochemical networks.

We derive a convex optimization problem on a steady-state nonequilibrium network of biochemical reactions, with the property that energy conservation and the second law of thermodynamics both hold at the problem solution. This suggests a new variational principle for biochemical networks that can be implemented in a computationally tractable manner. We derive the Lagrange dual of the optimization problem and use strong duality to demonstrate that a biochemical analogue of Tellegen's theorem holds at optimality. Each optimal flux is dependent on a free parameter that we relate to an elementary kinetic parameter when mass action kinetics is assumed.

Integrated stoichiometric, thermodynamic and kinetic modelling of steady state metabolism.

The quantitative analysis of biochemical reactions and metabolites is at frontier of biological sciences. The recent availability of high-throughput technology data sets in biology has paved the way for new modelling approaches at various levels of complexity including the metabolome of a cell or an organism. Understanding the metabolism of a single cell and multi-cell organism will provide the knowledge for the rational design of growth conditions to produce commercially valuable reagents in biotechnology. Here, we demonstrate how equations representing steady state mass conservation, energy conservation, the second law of thermodynamics, and reversible enzyme kinetics can be formulated as a single system of linear equalities and inequalities, in addition to linear equalities on exponential variables. Even though the feasible set is non-convex, the reformulation is exact and amenable to large-scale numerical analysis, a prerequisite for computationally feasible genome scale modelling. Integrating flux, concentration and kinetic variables in a unified constraint-based formulation is aimed at increasing the quantitative predictive capacity of flux balance analysis. Incorporation of experimental and theoretical bounds on thermodynamic and kinetic variables ensures that the predicted steady state fluxes are both thermodynamically and biochemically feasible. The resulting in silico predictions are tested against fluxomic data for central metabolism in Escherichia coli and compare favourably with in silico prediction by flux balance analysis.

Reconstruction and Use of Microbial Metabolic Networks: the Core Escherichia coli Metabolic Model as an Educational Guide.

Biochemical network reconstructions have become popular tools in systems biology. Metabolicnetwork reconstructions are biochemically, genetically, and genomically (BiGG) structured databases of biochemical reactions and metabolites. They contain information such as exact reaction stoichiometry, reaction reversibility, and the relationships between genes, proteins, and reactions. Network reconstructions have been used extensively to study the phenotypic behavior of wild-type and mutant stains under a variety of conditions, linking genotypes with phenotypes. Such phenotypic simulations have allowed for the prediction of growth after genetic manipulations, prediction of growth phenotypes after adaptive evolution, and prediction of essential genes. Additionally, because network reconstructions are organism specific, they can be used to understand differences between organisms of species in a functional context.There are different types of reconstructions representing various types of biological networks (metabolic, regulatory, transcription/translation). This chapter serves as an introduction to metabolic and regulatory network reconstructions and models and gives a complete description of the core Escherichia coli metabolic model. This model can be analyzed in any computational format (such as MATLAB or Mathematica) based on the information given in this chapter. The core E. coli model is a small-scale model that can be used for educational purposes. It is meant to be used by senior undergraduate and first-year graduate students learning about constraint-based modeling and systems biology. This model has enough reactions and pathways to enable interesting and insightful calculations, but it is also simple enough that the results of such calculations can be understoodeasily.

Quantitative assignment of reaction directionality in constraint-based models of metabolism: application to Escherichia coli.

Constraint-based modeling is an approach for quantitative prediction of net reaction flux in genome-scale biochemical networks. In vivo, the second law of thermodynamics requires that net macroscopic flux be forward, when the transformed reaction Gibbs energy is negative. We calculate the latter by using (i) group contribution estimates of metabolite species Gibbs energy, combined with (ii) experimentally measured equilibrium constants. In an application to a genome-scale stoichiometric model of Escherichia coli metabolism, iAF1260, we demonstrate that quantitative prediction of reaction directionality is increased in scope and accuracy by integration of both data sources, transformed appropriately to in vivo pH, temperature and ionic strength. Comparison of quantitative versus qualitative assignment of reaction directionality in iAF1260, assuming an accommodating reactant concentration range of 0.02-20mM, revealed that quantitative assignment leads to a low false positive, but high false negative, prediction of effectively irreversible reactions. The latter is partly due to the uncertainty associated with group contribution estimates. We also uncovered evidence that the high intracellular concentration of glutamate in E. coli may be essential to direct otherwise thermodynamically unfavorable essential reactions, such as the leucine transaminase reaction, in an anabolic direction.

The effect of ephedra and high fat dieting: a cause for concern! A case report.

The increased incidence of obesity in the world has resulted in more and more people attempting to lose weight through a variety of diets. Many of these diets employ caloric reduction through the elimination of certain food groups. These diets may initially be associated with weight loss (including water weight) but follow up reports of these diets show high drop out rates, proinflammatory changes which can precipitate heart disease and weight gain following cessation of these diets. Efforts to use prescription anorexic medications have been associated with valvular disease and other health concerns. Dissatisfaction with the medical community and a subsequent increase in the availability of information on the Internet, are only two of the reasons why people are looking at alternative medicine to assist with health care issues. This includes the use of herbal supplements for appetite suppression. A review of the literature reveals several problems with some of these supplements, including Ephedra. Potentially serious adverse effects include dysrhythmias, heart failure, myocardial infarction, changes in blood pressure, and death have occurred. Unfortunately, one half of all patients experiencing a myocardial infarction have total cholesterol levels below 150 mg/dL and/or no prior cardiac symptoms. This means that the development of inflammatory changes which can precipitate myocardial infarction may go unnoticed by conventional testing and unless markers of inflammation and coronary perfusion are looked for, changes which can precipitate myocardial infarction may go unnoticed until cardiac injury occurs. The following case presentation shows how an individual with exertional dyspnea and concerned about her weight was affected by both the ingestion of a low-carbohydrate diet and ephedra.

What effect, if any, does soy protein have on breast tissue?

Differences in breast tissue can be determined using breast-enhanced scintigraphy test (BEST) imaging. Minimal work in vivo has been done previously to determine the effects of soy protein on breast tissue. The authors' earlier work demonstrated reduction in inflammatory changes in breast tissue. This work was conducted to examine the effect of daily soy protein consumption on a larger group of women over the course of 1 year. Sixty-four premenopausal women were studied after initial BEST imaging evaluation revealed fibrocystic changes of the breast. Women were asked to consume a medical-grade soy protein on a daily basis, making no other dietary or lifestyle changes during that time. Each woman underwent BEST imaging 1 year later with the results compared to the initial findings. Women and their physicians reported a subjective reduction in both breast tenderness and fibrocystic disease (FCD). There was a nonstatistical reduction in both the average and maximal count breast activity following 1 year of daily soy consumption. There was a statistically significant reduction ( P < .01) in variability of tissue activity following 1 year of soy protein treatment. This is the first in vivo study looking at the effect of soy protein on breast tissue health. The findings are promising and showed both objective and subjective findings consistent with a reduction in fibrocystic disease of the breast. Further research is needed to confirm these findings in a greater number of women and to determine if soy protein has the same beneficial effect in atypia and breast cancer.

Are there differences in breast tissue as a result of hormone replacement therapy? Can BEST imaging distinguish these differences?

Although it has been speculated that estrogen therapy may promote changes in breast tissue that could lead to cancer, no information exists as to differences in breast tissue for women who do and do not take hormone replacement (HRT) therapy. This study seeks to determine if there are differences in the tissue of women taking HRT in contrast to those who do not and if these differences are apparent in cases of breast cancer, cellular atypia, fibrocystic (FCD) disease and normal breasts. A total of 327 non-pregnant, non-lactating, pre-menopausal women were enrolled in the study, including 139 women who were actively taking HRT and 188 women who never had taken HRT. Using breast enhanced scintigraphy test (BEST) imaging, differentiation of breast tissue was determined. The groups were then analyzed to determine the effect of hormone therapy within each category of breast tissue. Differentiation between normal, FCD, cellular atypia, and breast cancer represent statistically significant differences (p.001) in metabolic activity and vascularity as demonstrated by differences in both average count activity (ACA) and maximal count activity (MCA). The distinction between cellular atypia and infiltrating breast cancer was statistically (p.05) different when looking at the maximal activity. Normal breast tissue and breasts with FCD appear more homogenous with no statistical differences in variability in breast tissue. Tissue variability is statistically greater when localized processes, such as cellular atypia and breast cancer, are present. Differentiation of cellular metabolic activity in breast tissue can be statistically determined when looking at the average and maximal metabolic activity. The final distinction between cellular atypia and cancer occurs when a focal region of breast tissue becomes metabolically more active than the surrounding breast tissue as shown by statistical increases in MCA. These findings are confirmed by the increased metabolic variability seen in regions of cellular atypia and cancer compared with the homogenous metabolic activity present in normal and fibrocystic breasts.

Do women taking hormone replacement therapy (HRT) have a higher incidence of breast cancer than women who do not?

An estimated one third of all American and United Kingdom women take hormone therapy. In sharp contrast to these numbers, as many as one half of women diagnosed with breast cancer have taken hormones. Little additional information is available regarding the risk of breast cancer and even less is known about the association between hormone therapy and fibrocystic (FCD) disease or atypia of the breast. Three hundred women between 30 and 50 years of age were enrolled in this study, including 120 taking hormone replacement (HRT) therapy and 180 women who had never taken hormone therapy. These women were divided into four categories including those with normal breast tissue, those with FCD disease, those with cellular atypia, and those with breast cancer. Another group of women were also identified who had breast implants. Using breast enhanced scintigraphy (BEST) imaging, changes in breast tissue were determined and compared according to the use of HRT. Forty percent (122 of 300) had "normal" breasts, of whom 68.8% (84 of 122) did not take HRT. This accounted for 46.7% (84 of 180) of the women not taking hormone therapy, while only 31.7% (38 of 120) of the women taking HRT had normal breasts. This difference was statistically (p.001) significant. There was a greater incidence of breast abnormality in women taking HRT and a lower incidence in pathology among women not taking HRT when cumulatively analyzed for FCD, cellular atypia, and breast cancer. This difference was statistically significant (p.001) for women with breast cancer where 62.5% (10 of 16) were women taking HRT. Although the study was relatively small, it is the first such study to compare a continuum of changes in breast tissue according to the use of HRT. The study suggests that the initial empirical observations regarding higher incidence of HRT among women with breast cancer, may have a relationship to underlying changes in breast tissue that are associated with differences in mitochondrial content and activity. Further investigation is needed.

Sentinel node biopsy for orbital and ocular adnexal tumors.

PURPOSE: To describe a technique for sentinel node mapping and biopsy in patients with orbital or adnexal tumors. METHODS: Five patients with orbital and adnexal tumors were studied. Two patients had malignant eyelid melanomas (one of the skin and one of the conjunctiva), one with orbital invasion. Two patients had sebaceous gland carcinoma, and one patient had a mucoepidermoid carcinoma of the conjunctiva; 500 microCi of Technetium-99m sulfur nanocolloid (Nycomed Amersham, Princeton, NJ) diluted to 1.0 mL was injected intradermally at the lateral canthus. The patients were positioned as they would be during surgery. Lymphoscintigraphy was performed by means of anterior, lateral, and oblique views. The tracer was followed to the first lymphatic basin, and the sentinel node was identified. Cutaneous markers were placed to denote the site. During surgery, lymphoscintigraphy scans and a hand-held gamma probe were used to locate the sentinel node. Once excised, the sentinel node was sent for histopathology. Frozen sectioning confirmed the presence of lymphoid tissue. Permanent sections with immunohistochemical markers were performed to examine for metastatic disease. RESULTS: The sentinel node biopsy technique was applied to 5 patients with orbital and adnexal tumors. All lymph nodes were free of tumor on histopathologic examination. CONCLUSIONS: Sentinel node mapping and biopsy are possible for orbital and adnexal tumors. The morbidity of elective lymph node dissection and adjuvant radiotherapy can be avoided. Our results are preliminary, and further work must be done to identify the lymphatic basins of the orbit and ocular adnexa.

The effect of high-protein diets on coronary blood flow.

Recent research has demonstrated that successful simultaneous treatment of multiple risk factors including cholesterol, triglycerides, homocysteine, lipoprotein (a) [Lp(a)], fibrinogen, antioxidants, endothelial dysfunction, inflammation, infection, and dietary factors can lead to the regression of coronary artery disease and the recovery of viable myocardium. However, preliminary work revealed that a number of individuals enrolled in the original study went on popular high-protein diets in an effort to lose weight. Despite increasing numbers of individuals following high-protein diets, little or no information is currently available regarding the effect of these diets on coronary artery disease and coronary blood flow. Twenty-six people were studied for 1 year by using myocardial perfusion imaging (MPI), echocardiography (ECHO), and serial blood work to evaluate the extent of changes in regional coronary blood flow, regional wall motion abnormalities, and several independent variables known to be important in the development and progression of coronary artery disease. Treatment was based on homocysteine, Lp (a), C-reactive protein (C-RP), triglycerides, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, very low-density lipoprotein cholesterol, and fibrinogen levels. Each variable was independently treated as previously reported. MPI and ECHO were performed at the beginning and end of the study for each individual. The 16 people (treatment group/TG) studied modified their dietary intake as instructed. Ten additional individuals elected a different dietary regimen consisting of a "high-protein" (high protein group/HPG) diet, which they believed would "improve" their overall health. Patients in the TG demonstrated a reduction in each of the independent variables studied with regression in both the extent and severity of coronary artery disease (CAD) as quantitatively measured by MPI. Recovery of viable myocardium was seen in 43.75% of myocardial segments in these patients, documented with both MPI and ECHO evaluations. Individuals in the HPG showed worsening of their independent variables. Most notably, fibrinogen, Lp (a), and C-RP increased by an average of 14%, 106%, and 61% respectively. Progression of the extent and severity of CAD was documented in each of the vascular territories with an overall cumulative progression of 39.7%. The differences between progression and extension of disease in the HPG and the regression of disease in the TG were statistically (p<0.001) significant. Patients following recommended treatment for each of the independent variables were able to regress both the extent and severity of their coronary artery disease (CAD), as well as improve their myocardial wall motion (function) while following the prescribed medical and dietary guidelines. However, individuals receiving the same medical treatment but following a high-protein diet showed a worsening of independent risk factors, in addition to progression of CAD. These results would suggest that high-protein diets may precipitate progression of CAI) through increases in lipid deposition and inflammatory and coagulation pathways.

Reversing heart disease in the new millennium--the Fleming unified theory.

Nineteen people without prior history of documented heart disease were studied for 8 months to determine the effect of treatment based on an immunologic unified theory of vascular disease. Subjects underwent myocardial perfusion imaging to quantify the extent and severity of coronary artery disease, along with assessment of wall motion abnormalities and ejection fraction by both nuclear and echocardiographic methods. These tests were repeated at the end of the study. Treatment consisted of dietary changes, treatment of cholesterol, triglycerides, homocysteine, lipoprotein (a), fibrinogen, C-reactive protein, and infection. Patients who followed the dietary recommendations demonstrated statistically reduced disease in all three major coronary arteries, whereas those individuals who followed high-protein diets demonstrated statistically greater levels of disease.

A high-throughput search for electronic materials-thin-film dielectrics.

Parallel synthesis together with high-throughput screening was used to identify candidate materials for integrated circuit applications that demand a superior high permittivity dielectric thin film. Specifically, we developed a "continuous-composition spread" (CCS) technique to synthesize much of a pseudoternary oxide system in a single deposition and used this in conjunction with a high-throughput measurement protocol, thereby allowing each chemical system to be deposited and evaluated in about 24 h. This approach led to the identification of compositions in the Zr-Sn-Ti-O system with promising properties. The same technique was used to determine the optimum compositions as a function of processing parameters. Films with the composition Zr(.2)Sn(.2)Ti(.6)O(2) were then prepared using a conventional synthetic technique (on-axis sputtering) and were verified to have excellent properties. Thus, the CCS technique has demonstrated utility in rapidly identifying and developing a useful new material.

Assessing the independent effect of dietary counseling and hypolipidemic medications on serum lipids.

Determination of changes in total cholesterol (TC) and triglyceride (TG) levels has focused primarily on hypolipidemic drug effects. Changes resulting from dietary effect alone versus diet and drug effect have not yet been fully established. Seventy subjects were enrolled into four treatment groups to determine the impact of diet and drug effect upon TC and TG. Group 1 (n = 28) served as the control group and received no dietary counseling or drug therapy. Group 2 (n = 22) received dietary counseling. Group 3 (n = 7) underwent dietary counseling for six months and drug therapy for eighteen months. Subjects in groups 1-3 were monitored for eighteen months. Patients in group 4 (n = 13) were followed up for thirty-six months. No intervention occurred during the first eighteen months, and hypolipidemic medications were used during the second eighteen-month period. Subjects in groups 1 and 4 received no specific dietary counseling and demonstrated no significant improvement over the course of the study. Patients in groups 2 and 3 showed significant reductions in both TC and TG. The improvement in TC seen for patients in group 3 was reduced after dietary counseling ceased. Dietary intervention is necessary if patients are to statistically significantly reduce TC and TG levels. Drug therapy demonstrated the expected reductions in both TC and TG but did not statistically significantly lower lipid levels without concomitant dietary counseling. When dietary counseling and hypolipidemic medications are used together, reductions in TC and TG values are even greater than those seen with dietary effect alone. Diet control alone appears to significantly reduce TC and TG levels, resulting in reduced need for antianginal medications.

Training physicians and health care providers to accurately read coronary arteriograms. A training program.

UNLABELLED: Patterns in visual interpretation of coronary arteriograms (CAs) frequently cause incorrect assessment of percent diameter stenosis (%DS). These errors result in overestimating the results of angioplasty as well as of the number of arteries significantly affected by coronary artery (CAD) disease. METHODS: Forty-one physicians, nurses, and students participated in the standardization of 45 Kodachromes (39 arteries, 6 phantoms) and 5 photographic reproductions. Eleven of the 41 participated in a three-part training program designed to eliminate errors and improve accuracy of interpreting %DS from CAs. RESULTS: Improvement in reading %DS was seen in 69% of CAs with statistical (P < or = 0.05) improvement in one third of these cases, whose narrowings ranged from 4% to 84%DS. Variability of reporting was reduced in 26% of the cases. Skewing, representing an overestimation of "severe" disease and underestimation of "less severe" disease, was reduced with statistical improvement (P < or = 0.05) in reported %DS noted after training. Similar improvement was seen with phantoms but not in photographic images where the arterial edges were outlined. CONCLUSION: The outcomes of clinical management, invasive and interventional (mechanical, thrombolytic) procedures, as well as research studies depend in part upon the accuracy of reading %DS from CAs. Most studies to date have completed using extremely unreliable estimates of %DS with resultant problems in data interpretation. The use of this standardized training program has led to significant improvement in accurately assessing CAD.

Treating hyperlipidemia in the elderly.

BACKGROUND: Determination of the effects of dietary modification and hyperlipidemic medications in the elderly (> sixty-five years of age) patient has not been significantly investigated to date despite knowledge that elevated cholesterol (TC) and triglyceride (TG) levels increase the risk of coronary artery disease (CAD). METHODS: Twenty-seven individuals were placed into one of three treatment groups and longitudinally followed up to examine the effects of diet and hyperlipidemic medications on TC and TG levels. Group 1 (n = 14) received neither dietary nor drug therapy. Group 2 (n = 9) received dietary counseling without concomitant hyperlipidemic medications. Subjects in group 3 (n = 4) underwent dietary instruction for six months and hyperlipidemic medication(s) for eighteen months. RESULTS: Subjects in group 1 demonstrated a statistical increase in TC (P < or = 0.001) during the study. Patients in groups 2 (P < or = 0.001) and 3 (P < or = 0.05) demonstrated statistical improvement in TC reduction during dietary counseling. The effect on TC was blunted in group 3 after dietary counseling was discontinued. Reductions in TG levels were significant (P < or = 0.001) only for patients in group 2. CONCLUSION: Elderly individuals were able to significantly reduce both TC and TG levels by dietary modification alone. Minimal improvement was seen with the addition of hyperlipidemic medications.