ß-Blockers and chronic obstructive pulmonary disease: inappropriate avoidance?

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the United States and is often accompanied by one or more comorbid conditions. While there are established morbidity and mortality benefits of ß-blocker (BB) use for certain cardiovascular conditions, data suggest that clinicians are often reluctant to prescribe them in the presence of COPD because of concerns for bronchoconstriction, despite evidence that they are typically well-tolerated among these patients. Treatment guidelines for COPD are consistent with those for cardiovascular disease management and support the role of BBs in management of particular cardiovascular conditions, even in the presence of severe COPD. Adherence to these guidelines could result in significant decreases in morbidity and mortality among patients with COPD. Additionally, current treatments for COPD are often linked to increased cardiovascular disease events. Further study is needed to clarify and guide therapeutic management in patients with COPD.

Cryoprotectant transport through articular cartilage for long-term storage: experimental and modeling studies.

OBJECTIVE: Long-term storage of articular cartilage (AC) remains challenging due to poor post-thaw viability. An initial step towards addressing this issue is characterizing cryoprotectant (CPA) transport, since ensuring adequate CPA equilibration throughout the tissue offers protection during cooling. This study takes a systematic approach in determining CPA transport rates through bovine AC and uses that information in mathematical models to determine CPA equilibration times. DESIGN: Diffusion of high concentration single (6.9 M dimethyl sulfoxide (DMSO)) and multi-component CPA solutions (VS55, 3.1 M DMSO+2.2 M 1,2-propanediol (PD)+3.1 M formamide (FM)) was measured through AC using (1)H nuclear magnetic resonance (NMR) imaging and localized spectroscopy, respectively. Using experimentally calculated effective diffusivities, diffusion models describing CPA transport through the tissue matrix and across chondrocyte membranes were combined to design a CPA addition and removal scheme for a cartilage plug of clinically relevant dimensions. RESULTS: (1)H NMR imaging and localized spectroscopy experiments suggested that the permeation of CPAs through AC (5 mm diameter, 5-10 mm in thickness) took on the order of 4 h for full equilibration at 22 degrees C. Imaging clearly showed the permeation of DMSO into cartilage over time and localized spectroscopy was able to distinguish the permeation rates of the individual VS55 components and water. Experimentally measured diffusivity values were used in CPA addition/removal simulations with a cartilage plug of clinically relevant dimensions (5 mm diameter, 2 mm in thickness). Results suggested a multi-step approach for adding and removing high concentration CPAs, with the addition and removal each taking approximately 2 h to complete. CONCLUSIONS: This study provides a foundation for designing CPA addition and removal protocols for effective long-term storage of cartilage tissue using a novel approach to measure CPA permeation.

Monitoring of dissolved oxygen and cellular bioenergetics within a pancreatic substitute.

This work investigated the use of nuclear magnetic resonance (NMR) spectroscopy in combination with a mathematical model of an encapsulated cell system as a method for rapidly assessing the status of a pancreatic substitute. To validate this method, an in vitro experiment was performed in which the encapsulated cells were perfused in an NMR-compatible system and the dissolved oxygen (DO) concentration of the perfusing medium was lowered from 0.20 to 0.05 mM, then returned to 0.20 mM in a stepwise fashion. The cellular metabolic activity and bioenergetics were evaluated by measuring the oxygen consumption rate (via DO sensors) and nucleotide triphosphate levels (via (31)P NMR). By incorporating a perfluorocarbon emulsion into the alginate beads, the cellular oxygenation state was monitored by measuring the average intrabead DO (AIDO) concentration by (19)F NMR. The in vitro measurements were then compared with model predictions based on the measured external DO concentration and time. Model-predicted cell growth and AIDO closely matched the experimentally acquired data. As the DO concentrations both external to and within the pancreatic substitute are needed to apply this methodology in vivo, the feasibility of measuring the DO concentration from two distinct bead populations implanted in the peritoneal cavity of mice was established. It is concluded that PFC incorporation and (19)F NMR measurements, in combination with a mechanistic model of the encapsulated system, allow the tracking of the state of a pancreatic substitute in vitro and potentially in vivo.

Noninvasive measurement of viable cell number in tissue-engineered constructs in vitro, using 1H nuclear magnetic resonance spectroscopy.

Noninvasive monitoring of tissue-engineered constructs is of critical importance for accurate characterization of constructs and their remodeling in vitro and in vivo. This study investigated the utility of (1)H NMR spectroscopy to noninvasively quantify viable cell number in tissue-engineered substitutes in vitro. Agarose disk-shaped constructs containing betaTC3 cells were employed as the model tissue-engineered system. Two construct prototypes containing different initial cell numbers were monitored by localized, water-suppressed 1H NMR spectroscopy over the course of 13 days. (1)H NMR measurements of the total choline resonance at 3.2 ppm were compared with results from the traditional cell viability assay MTT and with insulin secretion rates. Results show a strong linear correlation between total choline and MTT (R (2) = 0.86), and between total choline and insulin secretion rate (R (2) = 0.90). Overall, this study found noninvasive measurement of total choline to be an accurate and nondestructive assay for monitoring viable betaTC3 cell numbers in tissue-engineered constructs. The applicability of this method to in vivo monitoring is also discussed.

In vivo noninvasive monitoring of a tissue engineered construct using 1H NMR spectroscopy.

Direct, noninvasive monitoring of tissue engineered substitutes containing live, functional cells would provide valuable information on dynamic changes that occur postimplantation. Such changes include remodeling both within the construct and at the interface of the implant with the surrounding host tissue, and may result in changes in the number of viable cells in the construct. This study investigated the use of 1H NMR spectroscopy in noninvasively monitoring the viable cell number within a tissue engineered construct in vivo. The construct consisted of mouse betaTC3 insulinomas in a disk-shaped agarose gel, surrounded by a cell-free agarose gel layer. Localized 1H NMR spectra were acquired from within implanted constructs, and the total choline resonance was measured. Critical issues that had to be addressed in accurately quantifying total choline from the implanted cells included avoiding signal from host tissue and correcting for interfering signal from diffusing solutes. In vivo NMR measurements were correlated with MTT assays and NMR measurements performed in vitro on explanted constructs. Total choline measurements accurately and noninvasively quantified viable betaTC3 cell numbers in vivo, in the range of 1 x 10(6) to more than 14 x 10(6) cells, and monitored changes in viable cell number that occurred in the same construct over time. This is the first study using NMR techniques to monitor viable cell numbers in an implanted tissue substitute. It established architectural characteristics that a construct should have to be amenable to NMR monitoring, and it set the foundation for future in vivo investigations with other tissue engineered implants.

Effects of short-term hypoxia on a transformed cell-based bioartificial pancreatic construct.

Hypoxia is an adverse condition that can jeopardize the function of a bioartificial pancreatic construct. In this study we have investigated the effects of short-term hypoxic exposure (up to 24 h) on the bioenergetic status, metabolism, and insulin secretion of perfused pancreatic constructs composed of alginate/poly-L-lysine/alginate (APA) encapsulated mouse insulinoma betaTC3 cells. The bioenergetic status of the encapsulated cells was monitored noninvasively with the aid of 31P NMR spectroscopy, while glucose, lactate, and insulin concentrations were measured with off-line assays from media samples removed from the perfusion loop. Our results demonstrate that in freshly prepared constructs insulin secretion was not affected by the hypoxic conditions, although intracellular ATP concentration decreased and glucose consumption increased. Alternatively, in constructs that were maintained in our perfusion system for at least 10 days, identical hypoxic conditions resulted in a decreased insulin secretion concomitant to a decreased intracellular ATP concentration and increased glucose consumption. These results suggest that the effects of hypoxia on a transformed cell-based pancreatic construct are not constant throughout the duration of an in vitro culture. The observed differences are attributed to the significant cell growth and rearrangement that occurs with time during an in vitro culture of the constructs.

In vitro monitoring of total choline levels in a bioartificial pancreas: (1)H NMR spectroscopic studies of the effects of oxygen level.

This investigation implements specifically designed solvent-suppressed adiabatic pulses whose properties make possible the long-term monitoring of (1)H NMR detectable metabolites from alginate/poly-l-lysine/alginate (APA)-encapsulated betaTC3 cells. Our encapsulated preparations were maintained in a perfusion bioreactor for periods exceeding 30 days. During this prolonged cultivation period, the cells were exposed to repetitive hypoxic episodes of 4 and 24 h. The ratio of the total choline signal (3.20 ppm) to the reference signal (observed at 0.94 ppm assigned to isoleucine, leucine, and valine) decreased by 8-10% for the 4-h and by 20-32% for the 24-h episodes and returned to its prehypoxic level upon reoxygenation. The decrease in the mean value of total choline to reference signal ratio for three 4-h and two 24-h episodes in two different cultures was highly significant (P<0.01). The rate of recovery by this ratio was slower than the rates of recovery by oxygen consumption, lactate production, or glucose consumption. A step-up in oxygen level led to a new, higher value for the total choline to reference ratio. From spectra of extracts at 400 MHz, it was determined that 63.6% of the total choline signal is due to intracellular phosphorylcholine. Therefore, it is inferred that the observed changes in total choline signal are linked to an oxygen level dependence of the intracellular phosphorylcholine. Several possible mechanisms in which oxygen may influence phosphorylcholine metabolism are suggested. In addition, the implications of these findings to the development of a noninvasive monitoring method for tissue-engineered constructs composed of encapsulated cells are discussed.

Development of a bioartificial pancreas: I. long-term propagation and basal and induced secretion from entrapped betaTC3 cell cultures.

Bioartificial pancreatic constructs based on immunoisolated, insulin-secreting cells have the potential for providing effective, long-term treatment of type I (insulin-dependent) diabetes. Use of insulinoma cells, which can be amplified in culture, relaxes the tissue availability limitation that exists with normal pancreatic islet transplantations. We have adopted mouse insulinoma betaTC3 cells entrapped in calcium alginate/poly-L-lysine/alginate (APA) beads as our model system for a bioartificial pancreas, and we have characterized the effects of long-term propagation and of glucose concentration step changes on the bioenergetic status and on the metabolic and secretory activities of the entrapped cells. Cell bioenergetics were evaluated nonivasively by phosphorus-31 nuclear magnetic resonance ((31)P NMR) spectroscopy, and metabolic and secretory parameters by assaying cell culture medium. Data indicate that net cell growth occurred between days 3 and 10 of the experiment, resulting in an approximate doubling of the overall metabolic and secretory rates and of the intracellular metabolite levels. Concurrently, a reorganization of cell distribution within the beads was observed. Following this growth period, the measured metabolic and secretory parameters remained constant with time. During glucose step changes in the perfusion medium from a high concentration of 12 to 15 mM to 0 mM for 4.5 h to the same high glucose concentration, the oxygen consumption rate was not affected, whereas insulin secretion was always glucose-responsive. Intracellular nucleotide triphosphates did not change during 0 mM glucose episodes performed early in culture history, but they declined by 20% during episodes performed later in the experiment. It is concluded that the system of APA-entrapped betaTC3 cells exhibits several of the desirable characteristics of a bioartificial pancreas device, and that a correlation between ATP and the rate of insulin secretion from betaTC3 cells exists for only a domain of culture conditions. These findings have significant implications in tissue engineering a long-term functional bioartificial endocrine pancreas, in developing noninvasive methods for assessing construct function postimplantation, and in the biochemical processes associated with insulin secretion.

Development of a bioartificial pancreas: II. Effects of oxygen on long-term entrapped betaTC3 cell cultures.

Tissue-engineered pancreatic constructs based on immunoisolated, insulin-secreting cells are promising in providing an effective, relatively inexpensive, long-term treatment for type I (insulin-dependent) diabetes. An in vitro characterization of construct function under conditions mimicking the in vivo environment is essential prior to any extensive animal experimentation. Encapsulated cells may experience hypoxic conditions postimplantation as a result of one or more of the following: the design of the construct; the environment at the implantation site; or the development of fibrosis around the construct. In this work, we studied the effects of 3- and 4-day-long hypoxic episodes on the metabolic and secretory activities and on the levels of intracellular metabolites detectable by phosphorus-31 nuclear magnetic resonance ((31)P NMR) of alginate/poly-L-lysine/alginate entrapped betaTC3 mouse insulinomas continuously perfused with culture medium. Results show that, upon decreasing the oxygen concentration in the surrounding medium, the encapsulated cell system reached a new, lower metabolic and secretory state. Hypoxia drove the cells to a more anaerobic glycolytic metabolism, increased the rates of glucose consumption (GCR) and lactate production (LPR), and reduced the rates of oxygen consumption (OCR) and insulin secretion (ISR). Furthermore, hypoxia reduced the levels of intracellular nucleotide triphosphates (NTP) and phosphorylcholine (PC) and caused a rapid transient increase in inorganic phosphate (P(i)). Upon restoration of the oxygen concentration in the perfusion medium, all parameters returned to their prehypoxic levels within 2 to 3 days following either gradual unidirectional changes (ISR, NTP, PC) or more complicated dynamic patterns (OCR, GCR, LPR). A further increase in oxygen concentration in the perfusion medium drove OCR, ISR, NTP, PC, and P(i) to new, higher levels. It is concluded that (31)P NMR spectroscopy can be used for the prolonged noninvasive monitoring of the bioenergetic changes of encapsulated betaTC3 cells occurring with changes in oxygen tension. The data also indicate that the oxygen-dependent states might be related to the total number of viable, metabolically active cells supported by the particular oxygen level to which the system is exposed. These findings have significant implications in developing and non-invasively monitoring a tissue-engineered bioartificial pancreas based on transformed beta cells, as well as in understanding the biochemical events pertaining to insulin secretion from betaTC3 insulinomas.

Effects of alginate composition on the metabolic, secretory, and growth characteristics of entrapped beta TC3 mouse insulinoma cells.

The effects of alginate composition on cell growth as well as the metabolic and secretory profile of transformed beta-cells entrapped in alginate/poly-L-lysine/alginate (APA) solid beads were investigated following entrapment of beta TC3 mouse insulinoma cells in alginate composed of either high mannuronic acid or high guluronic acid residues. Entrapped cultures were maintained in spinner flasks for 40-60 days. The pattern of cell growth and the overall rates of glucose consumption and insulin secretion were investigated. Cultures of beta TC3 cells entrapped in alginate composed predominantly of mannuronic acid units (77%) displayed a linear increase in the rates of glucose consumption and insulin secretion concomitant with an increase in cell population in the periphery of the beads. Conversely, cultures of beta TC3 cells entrapped in alginate composed predominantly of high guluronic acid units (69%) displayed a decrease in the rates of glucose consumption and insulin secretion during the first three weeks of culture, followed by a rapid recovery that surpassed the initial rates by day 40. This biphasic pattern was concomitant to a decrease in viable cells during the first three weeks as ascertained by histology, followed by an increase in cell proliferation. Cell growth in high guluronic acid alginate took place at random locations throughout the solid bead and not in the periphery, as was the case in high mannuronic acid alginate preparations. Possible reasons for these differences and the significance of these findings in the context of a bioartificial pancreas composed of APA entrapped transformed cells are discussed.

Role of ATP and Pi in the mechanism of insulin secretion in the mouse insulinoma betaTC3 cell line.

Understanding the biochemical events associated with glucose-stimulated insulin secretion by pancreatic beta cells is of importance in gaining insight into both the pathophysiology of diabetes and the development of tissue-engineered bioartificial pancreatic substitutes. We have investigated the effects of glucose concentration on the bioenergetic status and on the metabolic and secretory functions exhibited by mouse insulinoma betaTC3 cells entrapped in calcium alginate/poly-L-lysine/alginate (APA) beads. Cells entrapped in APA beads constitute a possible implantable bioartificial pancreas for the long-term treatment of insulin-dependent diabetes mellitus. Our results show that, in entrapped betaTC3 cells, the oxygen consumption rate and the intracellular nucleotide triphosphate levels are unaffected by a step change in glucose concentration from 16 mM to 0 mM for 4.5 h and then back to 16 mM. The intracellular Pi level and the ammonia production rate were doubled, while insulin secretion was decreased 10-fold, upon switching from 16 mM to 0 mM glucose. The implications of these findings in the context of pancreatic beta cell biochemistry and the mechanism of the 'Fuel Hypothesis' are discussed.

Effects of oxygen on metabolic and secretory activities of beta TC3 cells.

We have investigated the rates of glucose consumption, lactate production and insulin secretion by mouse insulinoma beta TC3 cells exposed to high glucose and oxygen concentrations in the range of 132 mmHg (normoxia) to 0 mmHg (anoxia). The rates of glucose consumption and lactate production, and the yield of lactate on glucose were 6.4 +/- 0.2 nmol/h - 10(5) cells, 7.7 +/- 0.5 nmol/h - 10(5) cells, and 1.2 +/- 0.1 respectively, at oxygen concentrations between 132-25 mmHg. These values increased gradually as the oxygen concentration was reduced below 25 mmHg, reaching a maximum value of 12.8 +/- 0.4, 23.8 +/- 1.1, 1.9 +/- 0.1 respectively, at complete anoxia. Insulin secretion remained constant at 360 +/- 24 pmol/h - 10(8) cells at oxygen concentrations between 132-7 mmHg, but was inhibited at lower oxygen concentrations, dropping to 96 +/- 24 pmol/h - 10(8) cells at 0 mmHg. The rate of insulin secretion in the presence of high glucose under anoxia was significantly higher than the rate of basal secretion (28.2 +/- 3.0 pmol/h - 10(8) cells) at normoxia. The secretory properties of beta TC3 cells at low oxygen concentrations may have implications in the development of a diffusion-based bioartificial tissue constructs for the long-term treatment of Insulin Dependent Diabetes Mellitus.

Influence of acid loading, extracellular pH and uremia on intracellular pH in muscle.

UNLABELLED: Our previous work has shown that chronic metabolic acidosis can induce changes in protein and amino acid metabolism in muscle. The relationship of these metabolic responses to changes in muscle pH is unknown. To examine the role that acute acidosis might have on intracellular pH in rats, we evaluated the influence an acute infusion of HCl had on intracellular pH using 31P-NMR. Male rats fed 22% protein and weighing between 150 and 170 g were infused with 150 mM HCl (4 mmol/kg) over a 2- or 4-hour period. Baseline blood pH was 7.36 and dropped to 7.04 at the end of the infusion. Despite this, no changes in intracellular pH were seen. In contrast, we found that chronic acid loading (4 mmol NH4Cl/100 g/day for 5 days) produces a small change in muscle pH (0.05 pH units, p < 0.05). Chronic uremia did not change resting muscle pH despite a decrease in extracellular pH to 7.23 even though metabolic changes are well documented. CONCLUSIONS: (1) An acute acid load does not alter intracellular pH while chronic metabolic acidosis does reduce intracellular pH. (2) The lack of an acute change in intracellular pH suggests that intracellular buffering capacity changes over time.

Evaluation of 1H magnetic resonance spectroscopic imaging as a diagnostic tool for the lateralization of epileptogenic seizure foci.

The purpose of this study was to assess whether a visual examination of 1H spectroscopic images could correctly lateralize patients with intractable temporal lobe epilepsy. 20 patients with intractable temporal lobe epilepsy and 10 volunteers were included in this study. Spectroscopic images were analysed using a protocol based on visual inspection. Images of the metabolites N-acetyl aspartate (NAA), choline (Cho), creatine (Cr) and lactate were obtained from a transverse plane oriented along the sylvian fissure. Images from each individual were evaluated independently by six reviewers. Results of the lateralization procedure obtained from the visual examinations were compared with those obtained from quantitative analysis of the spectra and with those obtained by magnetic resonance imaging (MRI), positron emission tomography (PET), neuropsychological examinations, and electroencephalographic (EEG) recordings. NAA images were found to be the most effective, amongst metabolite images, in lateralizing the epileptogenic lobe. Using the site selected for resection as the definition of the correct lateralization, 70% of the patients who underwent temporal lobectomy were correctly lateralized by the majority of the examiners using the visual inspection protocol. Based on the results of this study it is concluded that visual examination of 1H spectroscopic images is potentially valid in lateralizing patients with intractable temporal lobe seizures. Confidence in the visual interpretation increased as the difference in NAA signal intensity between the temporal lobes increased. The threshold above which the majority of the examiners correctly lateralized the patients was approximately 15% in NAA signal loss in the ipsilateral lobe.

Experimental acidemia and muscle cell pH in chronic acidosis and renal failure.

To test whether muscle intracellular pH (pHi) decreases when extracellular pH falls, 31P-nuclear magnetic resonance was utilized in rats made acidotic by infusion of HCl, gavage-feeding NH4Cl, or induction of chronic renal failure (CRF). A 2- or 4-h HCl infusion did not lower muscle pHi, even though serum bicarbonate fell to 5 mM. With chronic acidemia, blood pH was 7.15 +/- 0.01 vs. 7.38 +/- 0.02 in pair-fed controls, and muscle pHi was 7.09 +/- 0.01 and 7.14 +/- 0.02, respectively (P < 0.01). pHi in muscle of CRF rats (7.16 +/- 0.01) did not differ from sham-operated, pair-fed controls (7.19 +/- 0.01) despite a blood pH of 7.23 +/- 0.05 in CRF vs. 7.39 +/- 0.01 in controls. Because ion transport is abnormal in CRF, we examined whether recovery of pHi is impaired when muscles of six CRF and control rats were exercised to tetany by stimulation of the sciatic nerve. Neither pHi nor the recovery of pHi differed between CRF and control rats. We conclude that pHi is maintained in muscle in uremia and that signals other than changing pHi must be necessary to disrupt metabolism.

Towards the development of a bioartificial pancreas: immunoisolation and NMR monitoring of mouse insulinomas.

A promising method for diabetes treatment is the implantation of immunoisolated cells secreting insulin in response to glucose. Cell availability limits the application of this approach at a medically-relevant scale. We explore the use of transformed cells that can be grown to large homogeneous populations in developing artificial pancreatic tissues. We also investigate the use of NMR in evaluating, non-invasively, cellular bioenergetics in the tissue environment. The system employed in this study consisted of mouse insulinoma beta TC3 cells entrapped in calcium alginate/poly-L-lysine (PPL)/alginate beads. The PPL layer imposed a molecular weight cutoff of approximately 60 kDa, allowing nutrients and insulin to diffuse through but excluding high molecular weight antibodies and cytotoxic cells of the host. We fabricated a radiofrequency coil that can be double-tuned to 1H and 31P, and an NMR-compatible perfusion bioreactor and support circuit that can maintain cells viable during prolonged studies. The bioreactor operated differentially, was macroscopically homogeneous and allowed the acquisitions of 1H images and 31P NMR spectra in reasonable time intervals. Results indicated that entrapment had little effect on cell viability; that insulin secretion from beads was responsive to glucose; and that the bioenergetics of perfused, entrapped cells were not grossly different from those of cells never subjected to the immobilization procedure. These findings offer promise for developing an artificial pancreatic tissue for diabetes treatment based on continuous cell lines.

Myohemerythrin from the sipunculid, Phascolopsis gouldii: purification, properties and amino acid sequence.

Two previously unknown isoforms, labelled iso I and iso II, of the oxygen-carrying protein, myohemerythrin, have been isolated from carcasses of the sipunculid worm, Phascolopsis gouldii. The two isoforms have non-identical N-terminal amino acid sequences and slightly different absorption spectra in the met form. Far-ultraviolet circular dichroism shows that iso I contains approximately 69% alpha-helix. The complete amino acid sequence for iso I was obtained. The molecular weight calculated from this amino acid sequence and including the active site Fe-O-Fe unit, is 13,829. All of the physical and chemical properties of iso I noted above, including the amino acid sequence, are very similar to those of T. zostericola myohemerythrin. Except for the amino acid sequence, these properties are also very similar to that of a subunit in hemerythrin, the octameric analog found in hemerythrocytes. Only 58 of the 113 residues in P. gouldii hemerythrin are conserved in iso I. Sequence comparisons were used to help identify residues responsible for maintaining the common tertiary and diiron site structures in hemerythrin and myohemerythrin. The seven iron ligand residues previously identified in crystal structures of hemerythrin and myohemerythrin are conserved in iso I. However, none of the ten residue pairs previously identified as engaging in direct salt-bridge or hydrogen bond interactions between subunits in the hemerythrin octamer are conserved in iso I.

An in vivo examination of rat brain during sepsis with 31P-NMR spectroscopy.

Neurological symptoms including lethargy, obtundation, and confusion are early and common findings in patients with sepsis. The etiology of the mental status changes that occur during severe infection is not known. We investigated the effects of sepsis on the levels of high-energy phosphates to determine whether decreased energy metabolism was a factor in the depressed neurological state. The time course of changes in brain pH and brain high-energy phosphate metabolites during an Escherichia coli infusion was determined from sequential phosphorus-31 nuclear magnetic resonance (31P-NMR) spectra of ketamine-xylazine-anesthetized rats. A second group of rats received 0.9% saline infusion and served as a control group. Despite severe obtundation and near loss of righting reflex, the rats in the septic group had no significant differences in the brain pH, the ratio of phosphocreatine (PCr) to beta-adenosine 5'-triphosphate (beta-ATP), or in the ratio of PCr to Pi. The only significant decrease in brain high-energy phosphates or pH occurred terminally in the septic rat group and corresponded with a rapidly falling arterial blood pressure. We conclude that the severe neurological depression that is characteristic of sepsis is not due to decreased levels of brain high-energy phosphates or brain acidosis.

Purification and properties of a membrane-bound NADH-cytochrome-b5 reductase from erythrocytes of the sipunculid worm, Phascolopsis gouldii.

The purification to homogeneity of the membrane-bound NADH-cytochrome-b5 reductase from erythrocytes of the sipunculid, Phascolopsis gouldii is reported. This highly purified reductase has allowed more detailed characterizations of its molecular and kinetic properties than was possible in a previous study (Utecht, R.E. and Kurtz, D.M., Jr. (1988) Biochim. Biophys. Acta 953, 164-178). The reductase has a molecular weight of 34,000 and contains FAD as the prosthetic group. In aqueous solution containing 0.5 vol% Triton X-100, the reductase forms an aggregate of Mr approximately 220,000. A higher purity preparation of P. gouldii erythrocyte b5 was also obtained. The combination of purified, solubilized reductase and cytochrome b5 was shown to catalyze the quantitative two-electron reduction of [Fe(III),Fe(III)]methemerythrin to [Fe(II),Fe(II)]deoxyhemerythrin by NADH. The P. gouldii NADH-cytochrome b5 reductase is the first from hemerythrin-containing erythrocytes to be purified and characterized. This methemerythrin reduction system appears to be analogous to methemoglobin reductases from vertebrate erythrocytes.