Fructose Reduces Mitochondrial Metabolism and Increases Extracellular BCAA during Insulin Resistance in C2C12 Myotubes.

Fructose is a commonly consumed monosaccharide implicated in developing several metabolic diseases. Previously, elevated branched-chain amino acids (BCAA) have been correlated with the severity of insulin resistance. Most recently, the effect of fructose consumption on the downregulation of BCAA catabolic enzymes was observed. Thus, this mechanistic study investigated the effects of physiologically attainable levels of fructose, both with and without concurrent insulin resistance, in a myotube model of skeletal muscle. METHODS: C2C12 mouse myoblasts were treated with fructose at a concentration of 100 µM (which approximates physiologically attainable concentrations in peripheral circulation) both with and without hyperinsulinemic-mediated insulin resistance. Gene expression was assessed by qRT-PCR, and protein expression was assessed by Western blot. Oxygen consumption rate and extracellular acidification rate were used to assess mitochondrial oxidative and glycolytic metabolism, respectively. Liquid chromatography-mass spectrometry was utilized to analyze leucine, isoleucine and valine concentration values. RESULTS: Fructose significantly reduced peak glycolytic and peak mitochondrial metabolism without altering related gene or protein expression. Similarly, no effect of fructose on BCAA catabolic enzymes was observed; however, fructose treatment resulted in elevated total extracellular BCAA in insulin-resistant cells. DISCUSSION: Collectively, these observations demonstrate that fructose at physiologically attainable levels does not appear to alter insulin sensitivity or BCAA catabolic potential in cultured myotubes. However, fructose may depress peak cell metabolism and BCAA utilization during insulin resistance.

The BCKDH kinase inhibitor BT2 promotes BCAA disposal and mitochondrial proton leak in both insulin-sensitive and insulin-resistant C2C12 myotubes.

Elevated circulating branched-chain amino acids (BCAAs) have been correlated with the severity of insulin resistance, leading to recent investigations that stimulate BCAA metabolism for the potential benefit of metabolic diseases. BT2 (3,6-dichlorobenzo[b]thiophene-2-carboxylic acid), an inhibitor of branched-chain ketoacid dehydrogenase kinase, promotes BCAA metabolism by enhancing BCKDH complex activity. The purpose of this report was to investigate the effects of BT2 on mitochondrial and glycolytic metabolism, insulin sensitivity, and de novo lipogenesis both with and without insulin resistance. C2C12 myotubes were treated with or without low or moderate levels of BT2 with or without insulin resistance. Western blot and quantitative real-time polymerase chain reaction were used to assess protein and gene expression, respectively. Mitochondrial, nuclei, and lipid content were measured using fluorescent staining and microscopy. Cell metabolism was assessed via oxygen consumption and extracellular acidification rate. Liquid chromatography-mass spectrometry was used to quantify BCAA media content. BT2 treatment consistently promoted mitochondrial uncoupling following 24-h treatment, which occurred largely independent of changes in expressional profiles associated with mitochondrial biogenesis, mitochondrial dynamics, BCAA catabolism, insulin sensitivity, or lipogenesis. Acute metabolic studies revealed a significant and dose-dependent effect of BT2 on mitochondrial proton leak, suggesting BT2 functions as a small-molecule uncoupler. Additionally, BT2 treatment consistently and dose-dependently reduced extracellular BCAA levels without altering expression of BCAA catabolic enzymes or pBCKDHa activation. BT2 appears to act as a small-molecule mitochondrial uncoupler that promotes BCAA utilization, though the interplay between these two observations requires further investigation.

5-Aza-2'-deoxycytidine-mediated DNA hypomethylation with and without concurrent insulin resistance suppresses myotube mitochondrial capacity.

Type 2 diabetes is characterized by elevated blood glucose and reduced insulin sensitivity in target tissues. Moreover, reduced mitochondrial metabolism and expressional profile of genes governing mitochondrial metabolism (such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha [PGC-1α]) are also reduced during insulin resistance. Epigenetic regulation via DNA methylation of genes including PGC-1α may contribute to diminished mitochondrial capacity, while hypomethylation of PGC-1α (such as that invoked by exercise) has been associated with increased PGC-1α expression and favorable metabolic outcomes. The purpose of the present report is to characterize the effects of DNA hypomethylation on myotube metabolism and expression of several related metabolic targets. C2C12 myotubes were treated with 5-Aza-2'-deoxycytidine (5-Aza) for either 24 or 72 h both with and without hyperinsulinemic-induced insulin resistance. Mitochondrial and glycolytic metabolism were measured via oxygen consumption and extracellular acidification rate, respectively. Metabolic gene and protein expression were assessed via quantitative real time polymerase chain reaction and western blot analysis, respectively. Though expression of PGC-1α and other related targets remained unaltered, insulin resistance and 5-Aza treatment significantly reduced mitochondrial metabolism. Similarly, peak glycolytic metabolism was diminished by 5-Aza-treated cells, while basal glycolytic metabolism was unaltered. 5-Aza also reduced the expression of branched-chain amino acid (BCAA) catabolic components, however BCAA utilization was enhanced during insulin resistance with 5-Aza treatment. Together the present work provides proof-of-concept evidence of the potential role of DNA methylation in the regulation of mitochondrial metabolism and the potential interactions with insulin resistance in a model of skeletal muscle.

Social Network Changes and Disclosure Responses after Sexual Assault.

Social support after sexual assault is important for recovery, but violence and recovery may also challenge relationships. We examined functional and structural social support changes following sexual assault and their association with mental health. College women (N=544) with and without a sexual assault history completed a cross-sectional survey assessing current and past egocentric social networks. Functional support (perceived global support, assault disclosure, perceived helpfulness of responses) and structural support (network density, size, retention) were examined. Multilevel models revealed that, relative to non-survivors, survivors reported smaller, less dense past networks, but similarly sized current networks. Survivors retained less of their networks than non-survivors, and network members who provided unhelpful responses to disclosure were less likely to be retained. Structural equation modeling revealed that, among survivors, perceived unhelpful responses to disclosure and a greater loss of network members were associated with worse mental health. Findings suggest that survivors may experience a restructuring of social networks following sexual assault, especially when network members respond in unhelpful ways to disclosure. Although survivors appeared to build new relationships, this restructuring was associated with more mental health problems. It is possible that interventions to improve post-assault social network retention may facilitate recovery.

A Systematic Review of the Balloon Analogue Risk Task (BART) in Alcohol Research.

Risk-taking propensity has been crucial to the investigation of alcohol use and consequences. One measure, the balloon analogue risk task (BART), has been used consistently over the past two decades. However, it is unclear how this measure is related to alcohol outcomes. This paper systematically reviews the literature on the BART and alcohol outcomes. First, direct associations between the BART and alcohol use are reviewed including correlations, group comparisons, the BART's prediction of alcohol outcomes and BART performance after consuming alcohol. Then, potential moderators that explain when and for whom the BART is related to alcohol outcomes are reviewed. Finally, potential mechanisms that explain how the BART and alcohol outcomes are related are reviewed. This review reveals patterns in the BART suggesting risk-taking propensity may be related to changes in alcohol use over time; however, there is little evidence to suggest BART scores increase after consuming alcohol. Yet, additional research suggests adjusted average pump scores may be too simplistic for the amount of information the BART captures and understanding individual's patterns of responses on the BART is important for investigating its relation to alcohol outcomes. Finally, this review opens up several future directions for research to understand how risk-taking propensity is related to alcohol outcomes.

Workforce Planning Models for Oral Health Care: A Scoping Review.

BACKGROUND: For health care services to address the health care needs of populations and respond to changes in needs over time, workforces must be planned. This requires quantitative models to estimate future workforce requirements that take account of population size, oral health needs, evidence-based approaches to addressing needs, and methods of service provision that maximize productivity. The aim of this scoping review was to assess whether and how these 4 elements contribute to existing models of oral health workforce planning. METHODS: A scoping review was conducted. MEDLINE, Embase, HMIC, and EconLit were searched, all via OVID. Additionally, gray literature databases were searched and key bodies and policy makers contacted. Workforce planning models were included if they projected workforce numbers and were specific to oral health. No limits were placed on country. A single reviewer completed initial screening of abstracts; 2 independent reviewers completed secondary screening and data extraction. A narrative synthesis was conducted. RESULTS: A total of 4,009 records were screened, resulting in 42 included articles detailing 47 models. The workforce planning models varied significantly in their use of data on oral health needs, evidence-based services, and provider productivity, with most models relying on observed levels of service utilization and demand. CONCLUSIONS: This review has identified quantitative workforce planning models that aim to estimate future workforce requirements. Approaches to planning the oral health workforce are not always based on deriving workforce requirements from population oral health needs. In many cases, requirements are not linked to population needs, while in models where needs are included, they are constrained by the existence and availability of the required data. It is critical that information systems be developed to effectively capture data necessary to plan future oral health care workforces in ways that relate directly to the needs of the populations being served. KNOWLEDGE TRANSFER STATEMENT: Policy makers can use the results of this study when making decisions about the planning of oral health care workforces and about the data to routinely collect within health services. Collection of suitable data will allow for the continual improvement of workforce planning, leading to a responsive health service and likely future cost savings.

Enamel Caries Detection and Diagnosis: An Analysis of Systematic Reviews.

Detection and diagnosis of caries-typically undertaken through a visual-tactile examination, often with supporting radiographic investigations-is commonly regarded as being broadly effective at detecting caries that has progressed into dentine and reached a threshold where restoration is necessary. With earlier detection comes an opportunity to stabilize disease or even remineralize the tooth surface, maximizing retention of tooth tissue and preventing a lifelong cycle of restoration. We undertook a formal comparative analysis of the diagnostic accuracy of different technologies to detect and inform the diagnosis of early caries using published Cochrane systematic reviews. Forming the basis of our comparative analysis were 5 Cochrane diagnostic test accuracy systematic reviews evaluating fluorescence, visual or visual-tactile classification systems, imaging, transillumination and optical coherence tomography, and electrical conductance or impedance technologies. Acceptable reference standards included histology, operative exploration, or enhanced visual assessment (with or without tooth separation) as appropriate. We conducted 2 analyses based on study design: a fully within-study, within-person analysis and a network meta-analysis based on direct and indirect comparisons. Nineteen studies provided data for the fully within-person analysis and 64 studies for the network meta-analysis. Of the 5 technologies evaluated, the greatest pairwise differences were observed in summary sensitivity points for imaging and all other technologies, but summary specificity points were broadly similar. For both analyses, the wide 95% prediction intervals indicated the uncertainty of future diagnostic accuracy across all technologies. The certainty of evidence was low, downgraded for study limitations, inconsistency, and indirectness. Summary estimates of diagnostic accuracy for most technologies indicate that the degree of certitude with which a decision is made regarding the presence or absence of disease may be enhanced with the use of such devices. However, given the broad prediction intervals, it is challenging to predict their accuracy in any future "real world" context.

Engineering plant membranes using droplet interface bilayers.

Droplet interface bilayers (DIBs) have become widely recognised as a robust platform for constructing model membranes and are emerging as a key technology for the bottom-up assembly of synthetic cell-like and tissue-like structures. DIBs are formed when lipid-monolayer coated water droplets are brought together inside a well of oil, which is excluded from the interface as the DIB forms. The unique features of the system, compared to traditional approaches (e.g., supported lipid bilayers, black lipid membranes, and liposomes), is the ability to engineer multi-layered bilayer networks by connecting multiple droplets together in 3D, and the capability to impart bilayer asymmetry freely within these droplet architectures by supplying droplets with different lipids. Yet despite these achievements, one potential limitation of the technology is that DIBs formed from biologically relevant components have not been well studied. This could limit the reach of the platform to biological systems where bilayer composition and asymmetry are understood to play a key role. Herein, we address this issue by reporting the assembly of asymmetric DIBs designed to replicate the plasma membrane compositions of three different plant species; Arabidopsis thaliana, tobacco, and oats, by engineering vesicles with different amounts of plant phospholipids, sterols and cerebrosides for the first time. We show that vesicles made from our plant lipid formulations are stable and can be used to assemble asymmetric plant DIBs. We verify this using a bilayer permeation assay, from which we extract values for absolute effective bilayer permeation and bilayer stability. Our results confirm that stable DIBs can be assembled from our plant membrane mimics and could lead to new approaches for assembling model systems to study membrane translocation and to screen new agrochemicals in plants.

Feasibility study: assessing the efficacy and social acceptability of using dental hygienist-therapists as front-line clinicians.

Background The oral health of the adult population has been improving in the United Kingdom decade upon decade. Over half of dental service activity in the National Health Service (NHS) is limited to a check-up without any further treatment. This raises a question as to whether check-ups could be provided by dental hygienist-therapists, rather than general dental practitioners. The aim of this study was to assess the feasibility of a definitive trial to evaluate the costs and effects of using dental hygienist-therapists to undertake the check-up.Methods/design Adult NHS patients were randomised into three arms in two dental practices: patients who only saw dental hygienist-therapists for a check-up, those that saw the general dental practitioner and dental hygienist-therapist alternatively and a control, where patients only saw the general dental practitioner for their check-up. The study ran for 15 months. The primary outcome measures of the study were patient recruitment, retention and fidelity. A parallel and embedded qualitative study was undertaken which recorded the views of participating patients to determine the social acceptability of the intervention.Results Sixty patients participated in the study. The initial recruitment rate for the study was 33.7%. This figure increased to over 82.1% when telephone calls or face-to-face recruitment was utilised. The retention rates were 60% for both the dental hygienist-therapist only group and the alternate general dental practitioner and dental hygienist-therapist group, compared to 70% for the general dental practitioner only group. Fifteen patients were interviewed in the qualitative study and supported a team approach to the provision of check-ups in the NHS. Conclusion This study demonstrates the feasibility of a definitive trial to evaluate the costs and effects of using dental-hygienist-therapists to undertake the check-up.

Comparative accuracy of different members of the dental team in detecting malignant and non-malignant oral lesions.

OBJECTIVES: Role substitution between primary care dentists (PCDs) and dental hygienists and therapists is increasingly being used in a number of different countries. Opponents to this development argue that it is unsafe and frequently cite the potential for missing oral malignancy as an inherent danger. The aim of the present study was to determine the comparative diagnostic test accuracy of different members of the dental team when differentiating between standardised photographs of mouth cancer, potentially malignant disorders and benign oral lesions. METHODS: A total of 192 dental professionals, comprising 96 PCDs, 63 DH-Ts, nine hospital-based dental staff and 24 other dental professionals were sampled purposively. Following orientation, participants were asked to score 90 clinical photographs that depicted cases of oral squamous cell carcinoma, potentially malignant disorders and non-malignant lesions of the oral mucosa. For each photograph participants were asked to determine whether they felt the lesion was representative of carcinoma, a potentially malignant disorder (test positive), or whether the lesion was benign (test negative). They were also asked to record their confidence in their decision on a 0-10 scale. Judgement decisions were compared against the known histopathological diagnosis of each lesion. Sensitivity and specificity were calculated for each participant and clinical group. RESULTS: The diagnostic test accuracy of PCDs and DH-Ts was similar. There was a median sensitivity of 81% Interquartile range (IQR) 19%) for PCDs and 77% (IQR 19%) for DH-T, with specificity of 73% (IQR 16%) and 69% (IQR 17%) respectively. DH-Ts missed fewer frank malignant lesions compared to PCDs. CONCLUSION: The performance of PCDs and DH-Ts when differentiating between mouth cancer, potentially malignant disorders and benign lesions is comparable. DH-Ts should be regarded as being as competent as PCDs as front-line healthcare workers with regard to detection of mouth cancer. However, considerable heterogeneity in detection was found within both clinical groups, suggesting that training remains paramount.

The efficacy of screening for common dental diseases by hygiene-therapists: a diagnostic test accuracy study.

Regularly attending adult patients are increasingly asymptomatic and not in need of treatment when attending for their routine dental examinations. As oral health improves further, using the general dental practitioner to undertake the "checkup" on regular "low-risk" patients represents a substantial and potentially unnecessary cost for state-funded systems. Given recent regulatory changes in the United Kingdom, it is now theoretically possible to delegate a range of tasks to hygiene-therapists. This has the potential to release the general dental practitioner's time and increase the capacity to care. The aim of this study is to compare the diagnostic test accuracy of hygiene-therapists when screening for dental caries and periodontal disease in regularly attending asymptomatic adults who attend for their checkup. A visual screen by hygiene-therapists acted as the index test, and the general dental practitioner acted as the reference standard. Consenting asymptomatic adult patients, who were regularly attending patients at 10 practices across the Northwest of England, entered the study. Both sets of clinicians made an assessment of dental caries and periodontal disease. The primary outcomes measured were the sensitivity and specificity values for dental caries and periodontal disease. In total, 1899 patients were screened. The summary point for sensitivity of dental care professionals when screening for caries and periodontal disease was 0.81 (95% CI, 0.74 to 0.87) and 0.89 (0.86 to 0.92), respectively. The summary point for specificity of dental care professionals when screening for caries and periodontal disease was 0.87 (0.78 to 0.92) and 0.75 (0.66 to 0.82), respectively. The results suggest that hygiene-therapists could be used to screen for dental caries and periodontal disease. This has important ramifications for service design in public-funded health systems.

Osmotic equilibrium and elastic properties of eel intestinal vesicles.

Changes in volume of intestinal brush border membrane vesicles of the European eel Anguilla anguilla were measured as vesicles were exposed to media with different osmotic pressures. Preparing the vesicles in media of low osmotic pressure allowed the effects of a small hydrostatic pressure to become a significant factor in the osmotic equilibration. By applying LaPlace's law to relate pressure and volume and assuming a linear relation between membrane tension and area expansion, we estimate an initial membrane tension at 4.02 x 10(-5) N cm(-1) and an area compressibility elastic modulus at 0. 87 x 10(-3) N cm(-1). The elastic modulus estimate falls in the low range of values reported for membranes from other tissues in other species. This lower modulus quantitatively accounts for why eel intestinal vesicles show measurable changes in volume in hypotonic media while rabbit kidney vesicles do not.

Kinetics of water transport in eel intestinal vesicles.

Brush border membrane vesicles, BBMV, from eel intestinal cells or kidney proximal tubule cells were prepared in a low osmolarity cellobiose buffer. The osmotic water permeability coefficient P(f) for eel vesicles was not affected by pCMBS and was measured at 1.6 x 10(-3) cm sec(-1) at 23 degrees C, a value lower than 3.6 x 10(-3) cm sec(-1) exhibited by the kidney vesicles and similar to published values for lipid bilayers. An activation energy E(a) of 14.7 Kcal mol(-1) for water transport was obtained for eel intestine, contrasting with 4.8 Kcal mol(-1) determined for rabbit kidney proximal tubule vesicles using the same method of analysis. The high value of E(a), as well as the low P(f) for the eel intestine is compatible with the absence of water channels in these membrane vesicles and is consistent with the view that water permeates by dissolution and diffusion in the membrane. Further, the initial transient observed in the osmotic response of kidney vesicles, which is presumed to reflect the inhibition of water channels by membrane stress, could not be observed in the eel intestinal vesicles. The P(f) dependence on the tonicity of the osmotic shock, described for kidney vesicles and related to the dissipation of pressure and stress at low tonicity shocks, was not seen with eel vesicles. These results indicate that the membranes from two volume transporter epithelia have different mechanisms of water permeation. Presumably the functional water channels observed in kidney vesicles are not present in eel intestine vesicles. The elastic modulus of the membrane was estimated by analysis of swelling kinetics of eel vesicles following hypotonic shock. The value obtained, 0.79 x 10(-3) N cm(-1), compares favorably with the corresponding value, 0. 87 x 10(-3) N cm(-1), estimated from measurements at osmotic equilibrium.

Mechanical properties of brush border membrane vesicles from kidney proximal tubule.

The mechanical properties of brush border membrane vesicles, BBMV, from rabbit kidney proximal tubule cells, were studied by measuring the initial and final equilibrium volumes of vesicles subjected to different osmotic shocks, using cellobiose as the impermeant solute in the preparation buffer. An elevated intracellular hydrostatic pressure was inferred from osmotic balance requirements in dilute solutions. For vesicles prepared in 18 and 85 mosM solutions, these pressures are close to 17 mosM (290 mm Hg). The corresponding membrane surface tension is 6.0 x 10(-5) N cm-1 while the membrane surface area is expanded by at least 2.2%. When these vesicles are exposed to very dilute solutions the internal hydrostatic pressure rises to an estimated 84 mosM (1444 mm Hg) just prior to lysis. The corresponding maximal surface tension (pre-lysis) is 18.7 x 10(-5) N cm-1, and the maximal expansion of membrane area is 6.8%. The calculated area compressibility elastic modulus was 2.8 x 10(-3) N cm-1.

Water permeability of brush border membrane vesicles from kidney proximal tubule.

Brush border membrane vesicles (BBMV) maintain an initial hydrostatic pressure difference between the intra- and extravesicular medium, which causes membrane strain and surface area expansion (Soveral, Macey & Moura, 1997). This has not been taken into account in prior osmotic water permeability Pf evaluations. In this paper, we find further evidence for the pressure in the variation of stopped-flow light scattering traces with different vesicle preparations. Response to osmotic shock is used to estimate water permeability in BBMV prepared with buffers of different osmolarities (18 and 85 mosM). Data analysis includes the dissipation of both osmotic and hydrostatic pressure gradients. Pf values were of the order of 4 x 10(-3) cm sec-1 independent of the osmolarity of the preparation buffer. Arrhenius plots of Pf vs. 1/T were linear, showing a single activation energy of 4.6 kcal mol-1. The initial osmotic response which is significantly retarded is correlated with the period of elevated hydrostatic pressure. We interpret this as an inhibition of Pf caused by membrane strain and suggest how this inhibition may play a role in cell volume regulation in the proximal tubule.

Membrane stress causes inhibition of water channels in brush border membrane vesicles from kidney proximal tubule.

Brush border membrane vesicles (BBMV) from rabbit kidney proximal tubule cells, prepared with different internal solute concentrations (cellobiose buffer 13, 18 or 85 mosM) developed an hydrostatic pressure difference across the membrane of 18.7 mosM, that causes a membrane tension close to 5 x 10(-5) N cm-1. When subjected to several hypertonic osmotic shocks an initial delay of osmotic shrinkage (a lag time), corresponding to a very small change in initial volume was apparent. This initial osmotic response, which is significantly retarded, was correlated with the initial period of elevated membrane tension, suggesting that the water permeability coefficient is inhibited by membrane stress. We speculate that this inhibition may serve to regulate cell volume in the proximal tubule.

Independence of water and solute pathways in human RBCs.

We have investigated the permeability of the human red blood cell to four di-hydroxy alcohols, 1,2PD (1,2 propanediol), 1,3PD (1.3 propanediol), 1,4BD (1,4 butanediol), and 2,3BD (2,3 butanediol), and to water by using a recently developed ESR stopped-flow method which is free from artifacts found in light scattering methods. Numerical solutions of the Kedem-Katchalsky equations fit to experimental data yielded the following permeability coefficients: P1,2PD = 3.17 x 10(-5) cm sec-1, P1,3PD = 1.75 x 10(-5) cm sec-1, P1,4BD = 2.05 x 10(-5) cm sec-1, P2,3BD = 7.32 x 10(-5) cm sec-1. Reflection coefficients (sigma) were evaluated by comparing data fit with assumed values of sigma = 0.6, 0.8 and 1.0. In all four cases the best fit was obtained with sigma = 1.0. Treatment of cells with PCMBS (para-chloro mercuri-benzene-sulfonate) was followed by a large (> 10-fold) decrease in water permeability with virtually no change in alcohol permeability. We conclude that these alcohols do not permeate the water channels to any significant extent, and discuss some of the problems in light scattering measurements of reflection coefficients that could lead to erroneous values for sigma.

Estimate of the number of urea transport sites in erythrocyte ghosts using a hydrophobic mercurial.

In this paper a variety of mercurials, including a pCMB-nitroxide analogue, were used to study urea transport in human red cell ghosts. It was determined that the rate of inhibition for pCMBS, pCMB, pCMB-nitroxide, and chlormerodrin extended over four orders of magnitude consistent with their measured oil/water partition coefficients. From these results, we concluded that a significant hydrophobic barrier limits access to the urea inhibition site, suggesting that the urea site is buried in the bilayer or in a hydrophobic region of the transporter. In contrast, the rate of water inhibition by the mercurials ranged by only a factor of four and did not correlate with their hydrophobicities. Thus, the water inhibition site may be more directly accessible via the aqueous phase. Under conditions that leave water transport unaffected, we determined that < or = 32,000 labeled sites per cell corresponded to complete inhibition of urea transport. This rules out major transmembrane proteins such as band 3, the glucose carrier, and CHIP28 as candidates for the urea transporter. In contrast, this result is consistent with the Kidd (Jk) antigen being the urea transporter with an estimated 14,000 copies per cell. From the experimental number of urea sites, a turnover number between 2-6 x 10(6) sec-1 at 22 degrees C is calculated suggesting a channel mechanism.

Hemoglobin polymerization in sickle cells studied by circular polarized light scattering.

We have studied intracellular polymerization of hemoglobin S in suspensions of small populations of sickle cells using circular polarized light scattering. We argue that the preferential scattering of right circular polarized light (as expressed by measurements of the S14 Mueller scattering matrix element) directly reflects the amount of polymer inside cells. This technique has made it possible to investigate the effect of oxygen tension, cell density and osmotic stress on intracellular hemoglobin polymerization. Using S14 to determine hemoglobin polymer, we show that the polymer increases with deoxyhemoglobin concentration, that cells containing higher hemoglobin concentrations show significantly more polymer than cells containing less hemoglobin, and that polymerization occurs in sickle-trait cells in hypertonic solutions as the oxygen tension in the suspension is reduced. We also present kinetic measurements of polymerization, including that induced by osmotic shock. Finally, we demonstrate that the total light scattered (S11 Mueller scattering matrix element) that is routinely measured simultaneously with S14 can be used to estimate the percent of reduced (deoxy) Hb in the sample. These experiments demonstrate the potential of this technique to monitor hemoglobin polymerization simultaneously with oxygen dissociation under a wide variety of physiological conditions.