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.

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.

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.

Magnetic susceptibility of molecular carbon: nanotubes and fullerite.

Elemental carbon can be synthesized in a variety of geometrical forms, from three-dimensional extended structures (diamond) to finite molecules (C(60) fullerite). Results are presented here on the magnetic susceptibility of the least well-understood members of this family, nanotubes and C(60) fullerite. (i) Nanotubes represent the cylindrical form of carbon, intermediate between graphite and fullerite. They are found to have significantly larger orientation-averaged susceptibility, on a per carbon basis, than any other form of elemental carbon. This susceptibility implies an average band structure among nanotubes similar to that of graphite. (ii) High-resolution magnetic susceptibility data on C(60) fullerite near the molecular orientational-ordering transition at 259 K show a sharp jump corresponding to 2.5 centimeter-gram-second parts per million per mole of C(60). This jump directly demonstrates the effect of an intermolecular cooperative transition on an intramolecular electronic property, where the susceptibility jump may be ascribed to a change in the shape of the molecule due to lattice forces.

Defects in carbon nanostructures.

Previous high-resolution electron microscopy (HREM) observations of the carbon nanotubes have led to a "Russian doll" structural model that is based on hollow concentric cylinders capped at both ends. The structures of the carbon nanotubes and particles were characterized here by bulk physical and chemical property measurements. The individual nanostructure is as compressible as graphite in the c axis, and such nanostructures can be intercalated with potassium and rubidium, leading to a saturation composition of "MC(8)." These results are counter to expectations that are based on a Russian doll structure. HREM after intercalation with potassium and deintercalation indicates that individual nanoparticles are a "paper-mache" of smaller graphite layers. Direct current magnetization and electron spin resonance measurements indicate that the electronic properties of the nanostructures are distinctly different from those of graphite. Although the nanostructures have distinct morphologies and electronic properties, they are highly defective and have a local structure similar to turbostratic graphite.

13C NMR Spectroscopy of KxC60: Phase Separation, Molecular Dynamics, and Metallic Properties.

The results of (13)C nuclear magnetic resonance (NMR) measurements on alkali fullerides KxC(60) are reported. The NMR spectra demonstrate that material with 0 < x < 3 is in fact a two-phase system at equilibrium, with x = 0 and x = 3. NMR lineshapes indicate that C(3-)(60) ions rotate rapidly in the K(3)C(60) phase at 300 K, while C(6)-(60) ions in the insulating K(6)C(60) phase are static on the time scale of the lineshape measurement. The temperature dependence of the (13)C spin-lattice relaxation rate in the normal state of K(3)C(60) is found to be characteristic of a metal, indicating the important role of the C(3-)(60) ions in the conductivity. From the relaxation measurements, an estimate of the density of electronic states at the Fermi level is derived.

Single-Crystal Epitaxial Thin Films of the Isotropic Metallic Oxides Sr1-xCaxRuO3 (0 le x le 1).

Single-crystal epitaxial thin films of the isotropic metallic oxides Sr1-xCaxRuO(3) (0

New phases of c60 synthesized at high pressure.

The fullerene C(60) can be converted into two different structures by high pressure and temperature. They are metastable and revert to pristine C(60) on reheating to 300 degrees C at ambient pressure. For synthesis temperatures between 300 degrees and 400 degrees C and pressures of 5 gigapascals, a nominal face-centered-cubic structure is produced with a lattice parameter a(o) = 13.6 angstroms. When treated at 500 degrees to 800 degrees C at the same pressure, C(60) transforms into a rhombohedral structure with hexagonal lattice parameters of a(o) = 9.22 angstroms and c(o) = 24.6 angstroms. The intermolecular distance is small enough that a chemical bond can form, in accord with the reduced solubility of the pressure-induced phases. Infrared, Raman, and nuclear magnetic resonance studies show a drastic reduction of icosahedral symmetry, as might occur if the C(60) molecules are linked.

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.

Effect of alpha2 macroglobulin on some kinetic parameters of trypsin.

1. Complex formation of trypsin with alpha2 macroglobulin results in marked changes of the Michaelis-Menten constant, pH optimum and sensitivity to ionic strength in a system using N-carbobenzoxy-glycylglycyl-L-arginine-2-naphthylamide as substrate. 2. In contrasts to the inhibition (50%) observed when alpha2 macroglobulin-bound trypsin is assayed under conditions optimal for the free enzyme, there is minimal reduction of activity when determinations are performed at a substrate concentration and pH optimal for the bound enzyme. 3. The changes in substrate concentration and ionic environment required for maximum activity of alpha2 macroglobulin-bound trypsin are similar to those observed with enzymes embedded in polyelectrolyte matrices and may reflect alterations in the microenvironment of the enzyme resulting from conformational changes of the macromolecule during interaction with trypsin. 4. Enzymatic activity of trypsin towards casein is greatly reduced by alpha2 macroglobulin, even under assay conditions optimal for the bound enzyme, confirming previous findings that access to the active center for high-molecular weight substrates is sterically hindered by alpha2 macroglobulin.

Patterns in visual interpretation of coronary arteriograms as detected by quantitative coronary arteriography.

In part 1 of a three-part study, 14 novice readers and 6 experienced cardiologists interpreted phantom images of known stenosis severity. No difference between the interpretations of experienced and novice readers was detectable. Visual estimates of "moderately" severe stenosis were 30% higher than actual percent diameter stenosis. In part 2 of the study, visual interpretation of percent diameter stenosis from 212 stenoses on 241 arteriograms was compared with quantitative coronary arteriographic assessment. The visual analysis overestimated disease severity in arteries with greater than or equal to 50% diameter stenosis (except for right coronary lesions) and underestimated severity in all arteries with less than 50% diameter stenosis. Of the 241 arteriograms, 40 had quantitative and visual analysis of all three coronary arteries for assessment of significant disease. In only 62% of the cases did visual and quantitative methods agree on the presence of severe disease; visual estimates diagnosed significantly (p less than 0.05) more three-vessel disease. In part 3 of the study, comparison of percent diameter stenosis by visual estimate with quantitative coronary arteriographic assessment before and after balloon angioplasty of 38 stenoses showed that visual interpretation significantly (p less than 0.001) overestimated initial lesion severity and underestimated stenosis severity after angioplasty.

Comparison of technetium-99m teboroxime tomography with automated quantitative coronary arteriography and thallium-201 tomographic imaging.

Technetium-99m (Tc-99m) teboroxime is a new perfusion tracer that is highly extracted and rapidly cleared by the myocardium. To determine the feasibility of Tc-99m teboroxime imaging in the diagnosis of patients with suspected coronary artery disease, 30 patients underwent single photon emission computed tomography imaging with Tc-99m teboroxime (25.2 +/- 1 mCi) at peak exercise and again 60 min later at rest. All patients underwent either a thallium stress test (n = 26) or automated quantitative coronary arteriography (n = 25), or both, without intervening revascularization or infarction. Images were reviewed by two investigators who had no knowledge of clinical data. Coronary lesions with greater than or equal to 50% diameter narrowing by quantitative coronary arteriography were considered significant. Both thallium and Tc-99m teboroxime detected disease in all patients with two or three vessel disease. One vessel disease was detected with Tc-99m teboroxime in 9 of 10 patients and with thallium in 8 of 10 (p = NS). In patients without angiographically significant disease. Tc-99m teboroxime demonstrated normal perfusion in six of eight patients and thallium in three of five (p = NS). Overall, when presence or absence of disease detected by Tc-99m teboroxime or thallium was compared with quantitative coronary arteriography, there was no difference between Tc-99m teboroxime and thallium. These results suggest that Tc-99m teboroxime is comparable to thallium as an imaging agent. The rapid biologic half-life, 5.3 min, allows studies to be completed in 60 to 90 min.

Nuclear imaging analysis of human low-density lipoprotein biodistribution in rabbits and monkeys.

We have evaluated the biodistribution of human low-density lipoprotein (LDL) radiolabeled with 99mTc or with 123I-tyramine cellobiose in rabbits and in rhesus monkeys. Biodistribution was assessed after intravenous injection of radiolabeled LDL by quantitative analysis of scintigrams, counting of excreta, and counting of tissues at necropsy. Both rabbits and monkeys showed lower renal uptake (123I:99mTc approximately 1:3, as regional percent injected activity corrected for physical decay) and excretion (1:2 to 1:4), but higher hepatic (1.5:1 to 2:1) and cardiac (1.7:1 to 4:1) uptake of 123I than of 99mTc. Adrenals were visualized in normolipemic animals with 123I-tyramine cellobiose-LDL but not with 99mTc-LDL. Hyperlipemic animals showed increased cardiac (up to six-fold) and decreased hepatic activity (by 50%-60%) of both radionuclides. We conclude that 123I-tyramine cellobiose-LDL is better suited than 99mTc-LDL for dynamic studies of LDL metabolism in vivo.

Accumulation of indium-111-labeled human low density lipoprotein in the rabbit aorta: Implications for nuclear imaging of vascular lesions.

Nuclear imaging of atheromata must distinguish lesions from both blood pool and normal arterial tissue. We have examined spatial and temporal variations of indium-111-labeled human low density lipoprotein (LDL) accumulation in rabbit aortas. LDL-derived In-111 activity was time-independent in lesion-resistant regions of aortas from normal and hypercholesterolemic animals (mean 2.9 × 10(-6) percent injected activity per milligram tissue [%IA/mg]) and in lesion-prone regions of normal aortas (mean 7.1 × 10(-6) %IA/mg). In contrast, activity in sudanophilic lesions of hypercholesterolemic rabbit aortas reached a peak of 31 × 10(-6) %IA/mg at 92 hours postinjection. The mean ratio between activity in lesions versus lesion-resistant regions described a broad convex curve with minima of 4:1 at 14 hours and 136 hours and a peak of 14:1 measured at 72 hours postinjection. The mean ratio between In-111 in lesions and blood followed a sigmoid curve, rising exponentially from 1:25 at 14 hours to 1:3 by 72 hours postinjection. We conclude that optimal signal-to-noise ratios for monitoring atheroma-associated LDL-derived radioactivity occur late, not before about 3 days postinjection. Therefore, LDL labeled with In-111 or even longer-lived radionuclides holds the greatest promise for effective clinical nuclear imaging of atherosclerosis.

A new, highly sensitive and specific assay for chymotrypsin.

1. A simple, highly sensitive, specific fluorometric method for the determination of chymotrypsin is described. 2. The new substrate utilized in this assay, N-glutaryl-glycyl-glycyl-l-phenylalanine beta-naphthylamide (GGPNA), is readily soluble in water, stable and highly specific for chymotrypsin. It is not degraded by a large excess of carboxypeptidase B, elastase, thrombin or plasmin and is virtually resistant to trypsin. 3. GGPNA is extremely sensitive to the action of chymotrypsin and permits detection of enzyme concentrations as low as 1 ng/ml. Linearity between enzyme concentration and fluorescence produced is maintained up to at least 3000 ng/ml. 4. alpha2-Macroglobulin-bound chymotrypsin hydrolyzes GGPNA at a rate about 2/3 of that exhibited by the free enzyme. 5. Bile pigments in amounts normally found in duodenal juice or traces of blood do not interfere with the assay. 6. GG PNA which releases beta-naphthylamine upon hydrolysis is suitable also for colorimetric and histological determination of chymotrypsin.

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.

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.