Towards a Field-Portable Real-Time Organic and Elemental Carbon Monitor.

Diesel particulate matter (DPM) has been classified as a carcinogen to humans by the International Agency for Research on Cancer. As a result of its potential carcinogenic nature, DPM exposure is regulated by the Mine Safety and Health Administration. Currently, diesel emissions in the workplace are monitored by collecting the aerosol onto filters, which are then sent to a laboratory for thermal-optical analysis using the NIOSH method 5040. This process can take days or even weeks, and workers can potentially be exposed to excessive levels of DPM before the problem is identified. Moreover, the delay involved in getting the loaded filter to the lab inevitably means the loss of some of the more volatile organic carbon. To remedy this delay, researchers from the National Institute for Occupational Safety and Health are seeking to develop a field-portable, real-time method for measuring elemental and organic carbons in DPM aerosols. In the current study, the use of mid-infrared spectrometry was investigated. It is believed that mid-infrared spectroscopy is more suitable for use in a real-time field-portable device than thermo-optical analysis methods. This article presents a method for measuring organic carbon (OC) and elemental carbon (EC) in DPM for a broad range of OC/EC ratios. The method has been successfully applied to laboratory-generated and mine samples.

Inhibition of xanthine oxidase by uric acid and its influence on superoxide radical production.

The inhibition of xanthine oxidase by its reaction product, uric acid, was studied by steady state kinetic analysis. Uric acid behaved as an uncompetitive inhibitor of xanthine oxidase with respect to the reducing substrate, xanthine. Under 50 microM xanthine and 210 microM oxygen, the apparent K(i) for uric acid was 70 microM. Uric acid-mediated xanthine oxidase inhibition also caused an increase in the percentage of univalent reoxidation of the enzyme (superoxide radical production). Steady-state rate equations derived by the King-Altman method support the formation of an abortive-inhibitory enzyme-uric acid complex (dead-end product inhibition). Alternatively, inhibition could also depend on the reversibility of the classical ping-pong mechanism present in xanthine oxidase-catalyzed reactions.

Physiology of the splanchnic circulation.

The splanchnic circulation is composed of gastric, small intestinal, colonic, pancreatic, hepatic, and splenic circulations, arranged in parallel with one another. The three major arteries that supply the splanchnic organs, cellac and superior and inferior mesenteric, give rise to smaller arteries that anastomose extensively. The circulation of some splanchnic organs is complicated by the existence of an intramural circulation. Redistribution of total blood flow between intramural vascular circuits may be as important as total blood flow. Numerous extrinsic and intrinsic factors influence the splanchnic circulation. Extrinsic factors include general hemodynamic conditions of the cardiovascular system, autonomic nervous system, and circulating neurohumoral agents. Intrinsic mechanisms include special properties of the vasculature, local metabolites, intrinsic nerves, paracrine substances, and local hormones. The existence of a multiplicity of regulatory mechanisms provides overlapping controls and restricts radical changes in tissue perfusion.

Increased injury following intermittent fetal hypoxia-reoxygenation is associated with increased free radical production in fetal rabbit brain.

Hypoxia associated with perinatal events can result in brain damage in the neonate. In labor and eclampsia, hypoxia can be intermittent, which may result in more severe damage than sustained hypoxia. The pathogenesis of brain injury in sustained ischemia involves free radical production; therefore, we investigated whether higher levels of free radicals contribute to the greater injury induced by repetitive ischemia. Brains were obtained from fetuses of near-term, pregnant rabbits subjected to repetitive ischemia-reperfusion (RIR), sustained uterine ischemia-reperfusion (IR), or a control protocol. Compared with controls, fetal brains from RIR or IR groups had more brain edema. Brains from RIR fetuses exhibited higher levels of lipid peroxidation, 3-nitrotyrosine, and nitrogen oxides, and lower total antioxidant capacity and cortical cellular viability than those of IR or control fetuses. Maternal administration of antioxidants following RIR and fetal bradycardia resulted in lower levels of fetal cortical and hippocampal cell death. Coadministration of Trolox and ascorbic acid resulted in less brain edema and liquefaction, and fewer hippocampal ischemic nuclei as compared with the saline control. Higher free radical production may be responsible for the greater fetal brain injury following repetitive hypoxia-reoxygenation. Maternal antioxidant treatment resulted in transplacental passage of antioxidants and amelioration of brain injury, and may be a viable clinical option following diagnosis of fetal distress.

Sustained hypoxia-ischemia results in reactive nitrogen and oxygen species production and injury in the premature fetal rabbit brain.

Free radical-mediated injury is implicated in hypoxic-ischemic encephalopathy observed in neonates. We investigated in utero free radical production and injury following hypoxia-ischemia to premature fetal brain utilizing a rabbit model of acute placental insufficiency. Pregnant rabbits at 29 days gestation were randomized to uterine ischemia for 50 minutes (min) (hypoxia) or nonischemic controls. Fetal brains were obtained immediately after ischemia for oxidative and acute-injury markers or 24 hours (h) post-ischemia for histopathology. Nitrotyrosine formation, a marker of NO-derived species such as peroxynitrite, was observed only in hypoxic brains. Hypoxia resulted in a significant increase in nitrogen oxides, lipid peroxidation, and protein oxidation, with a concomitant decrease in total antioxidant capacity, compared with controls. Peroxynitrite addition to brain homogenate increased nitrogen oxides linearly (1:1), although protein carbonyls were unchanged. Concomitantly, in vitro cortical and hippocampal cell viability and ATP levels decreased, with an increase in brain edema in hypoxic brains. Fetuses delivered 24 h post-ischemia had increased hippocampal nuclear karyorrhexis on histology compared with controls. Antioxidant administration (ascorbic acid and Trolox) intraperitoneally ameliorated changes in cellular viability and brain edema. Acute fetal hypoxia-ischemia without reoxygenation results in increased nitrogen and oxygen free radical production that may cause brain injury. The merits of the described model are discussed.

Xanthine oxidase activity in the circulation of rats following hemorrhagic shock.

Reactive oxygen metabolites generated from xanthine oxidase play an important role in the pathogenesis of ischemia-induced tissue injury. In a hemorrhagic shock model of ischemia-reperfusion, the intracellular enzyme xanthine oxidase was released into the vasculature. This intravascular source of superoxide (O2.-) and hydrogen peroxide (H2O2) interacted reversibly with glycosaminoglycans of vascular endothelium and markedly concentrated xanthine oxidase at cell surfaces, enhancing its ability to produce extensive damage to remote tissues. Rats were made hypotensive by hemorrhage, maintained for 2h, and reinfused with shed blood. Blood samples were obtained prior to hemorrhage and 15, 30, 60, and 90 min after reperfusion for determination of xanthine oxidase (XO), lactate dehydrogenase (LDH), and alanine transaminase (AST). These enzymes were not significantly elevated in control animals. Reperfusion after hemorrhage-induced ischemia resulted in significantly elevated AST and LDH in both low heparin (100 U/h) and high heparin (1000 U/h) groups. Xanthine oxidase was detected in the circulation only after 90 min reperfusion in the low heparin group and was elevated during the entire reperfusion period in the high heparin group. Studies with cultured vascular endothelium showed significant heparin-reversible binding of XO to cellular glycosaminoglycans. These results suggest that XO can gain access to the circulation following ischemia, where it then binds to the vascular endothelial cells to produce site-specific oxidant injury to organs remote from the site of XO release.

A sensitive fluorometric assay for measuring xanthine dehydrogenase and oxidase in tissues.

The conversion of xanthine dehydrogenase to a free radical producing oxidase is an important component of oxygen-mediated tissue injury. Current assays for these enzymes are of limited sensitivity, making it difficult to analyze activities in organ biopsies or cultured cells. The xanthine oxidase-catalyzed conversion of pterin (2-amino-4-hydroxypteridine) to isoxanthopterin provides the basis for a fluorometric assay which is 100-500 times more sensitive than the traditional spectrophotometric assay of urate formation from xanthine. Enzyme activity as low as 0.1 pmol min-1 ml-1 can be measured with the fluorometric pterin assay. Xanthine oxidase is assayed in the presence of pterin only, while combined xanthine dehydrogenase plus oxidase activity is determined with methylene blue which replaces NAD+ as an electron acceptor. The relative proportions and specific activities of xanthine oxidase and dehydrogenase determined by the fluorometric pterin assay are comparable with the spectrophotometric measurement of activities present in rat liver, intestine, kidney, and plasma. The assay has been successfully applied to brain, human kidney, and cultured mammalian cells, where xanthine dehydrogenase and oxidase activities are too low to detect spectrophotometrically.

Endothelial injury from a circulating mediator following rat liver ischemia.

Ischemia-reperfusion to the liver results in increased microvascular permeability in a nonischemic lung. We hypothesized that a circulatory mediator released from ischemic liver contributed to endothelial cell (EC) damage. Isolated rat livers, made ischemic for 2 h, were reperfused for 10 min. Bovine ECs were incubated for 5 h with pooled liver effluent collected before ischemia (Baseline) or after 10 min of reperfusion (Reperfusion). In the Reperfusion group, there was increased endothelial cell injury, as determined by release of 8-[14C]adenine, (39 +/- 2%) compared to the Baseline group (22 +/- 2%). Permeability of ECs to rhodamine B-labeled dextran (70,000 Mr) was also increased in the Reperfusion group by 54 +/- 9%. There was no significant attenuation in EC injury following incubation with reperfusion effluent stored for 24 h, supplementation with antioxidants (superoxide dismutase + catalase), or inhibition of xanthine oxidase with allopurinol or tungstate. We conclude that the reperfused liver releases a long-lived circulatory mediator of EC injury, which may produce the clinical microvascular injury observed following hepatic ischemia. The mechanism of injury in our model is independent of oxidants or oxidants generated from the circulating xanthine oxidase released from reperfused ischemic liver.

Antioxidant mechanisms of isoflavones in lipid systems: paradoxical effects of peroxyl radical scavenging.

Oxidation of lipids has been implicated in the pathophysiology of atherosclerosis. It has been suggested that scavenging of lipid peroxyl radicals contribute to the antiatherosclerotic effects of naturally occurring compounds such as the isoflavones. This group of polyphenolics includes genistein and is present in relatively high concentrations in food products containing soy. Soy isoflavones are capable of inhibiting lipoprotein oxidation in vitro and suppressing formation of plasma lipid oxidation products in vivo. However, key aspects of the antioxidant mechanisms remain unknown. In this study the antioxidant effects of genistein and other soy isoflavones on lipid peroxidation initiated by mechanistically diverse oxidants was investigated. Although isoflavones inhibited lipid peroxidation stimulated by both metal-dependent and independent processes, the concentration required for these effects were relatively high compared to those found in vivo. Interestingly, however, isoflavones were not consumed and remained in the native state over the time during which inhibition of lipid peroxidation was observed. This was also the case under conditions where synergistic inhibition of LDL oxidation was observed with ascorbate. Furthermore, in an oxidation system driven solely by peroxyl radicals, isoflavones were found to be relatively poor peroxyl radical scavengers. Consistent with the apparent lack of reactivity with lipid-derived oxidants, isoflavones were also relatively resistant to oxidation mediated by the potent oxidant peroxynitrite. The potential antioxidant mechanisms of isoflavones are discussed in the context of possible reactivities of isoflavone-derived phenoxyl radicals.

Xanthine oxidoreductase release after descending thoracic aorta occlusion and reperfusion in rabbits.

Cardiopulmonary and other organ dysfunction often occurs after operation on the descending thoracic aorta. Though there are multiple causes of organ dysfunction in this setting, free radical injury may play a prominent role. Xanthine oxidoreductase, an enzyme that generates oxidants after exposure to ischemia, could be released from ischemic liver and intestine during reperfusion. To test this hypothesis, we created aortic occlusion in eight rabbits for 40 minutes by inflation of a 4F Fogarty balloon catheter in the descending thoracic aorta. Eight sham-operated rabbits served as a control group. Two hours of reperfusion followed removal of the balloon catheter. Hemodynamic and acid-base status were maintained near baseline values during reperfusion. Plasma samples were obtained for determination of the activity of the hepatocellular enzymes xanthine oxidoreductase, aspartate aminotransferase, alanine transferase, and lactate dehydrogenase. Plasma xanthine oxidoreductase activity increased significantly (p < 0.001) during reperfusion (729 +/- 140 microU/ml, mean +/- standard error of the mean) compared with baseline (132 +/- 18 microM/mL). The other enzymes followed a similar pattern of release. We report the release of xanthine oxidoreductase in an animal model that simulates the situation of human thoracic aorta operations. The oxidants produced by the circulating xanthine oxidoreductase observed during reperfusion would likely be toxic to vascular endothelium, potentially contributing to multiple organ dysfunction.

Xanthine oxidase inactivation attenuates postocclusion shock after descending thoracic aorta occlusion and reperfusion in rabbits.

"Declamping shock" is observed after aortic crossclamping, with hypovolemia, hypotension, and metabolic acidemia invariably present. We hypothesized that oxidants derived from xanthine oxidase influence the resuscitative interventions required to maintain baseline hemodynamic and acid-base status after aortic occlusion and reperfusion in rabbits. We also hypothesized that inactivation of xanthine oxidase with sodium tungstate could reduce systemic injury as assessed by the release of lactate dehydrogenase and alkaline phosphatase. To test these hypotheses, we established aortic occlusion in rabbits (n = 10, standard diet; n = 8, tungstate diet) for 40 minutes by inflation of a 4F Fogarty catheter in the descending thoracic aorta followed by 2 hours of reperfusion. Sham-operated rabbits (n = 10, standard diet; n = 9, tungstate diet) served as controls. Tungstate-pretreated rabbits required significantly less Ringer's solution (28%), phenylephrine (68%), and sodium bicarbonate (30%) during reperfusion (p < 0.005). Lactate dehydrogenase and alkaline phosphatase release during reperfusion was significantly attenuated by tungstate pretreatment (p < 0.05). Tungstate pretreatment resulted in plasma xanthine oxidase activities significantly lower than those in the sham group administered a standard diet (p = 0.007). Resuscitation requirements and systemic injury were reduced by inactivation of xanthine oxidase in a rabbit model that simulates the situation of human thoracic aorta operations.

alpha 1-tubulin expression in proximally axotomized mouse cortical neurons.

This paper further characterizes the response to axotomy of mouse transcallosal cortical neurons, a population of neurons that seems to be particularly refractory to regeneration. Mouse transcallosal cortical neurons did not upregulate mRNA for the growth-associated protein alpha 1-tubulin following axotomy, even when the axonal distance from injury to cell body was only 100-300 microns. Previous experiments had found no upregulation of another growth-associated protein, GAP-43, by transcallosal neurons following axotomy 1-2 mm from the cell body. These latest results establish that this population of neurons fails to respond to axotomy even when it is extremely proximal and that this failure is not a peculiarity specific to one growth-associated protein but is indicative of a generally poor regenerative response.

Comparison of partial and complete arterial occlusion models for studying intestinal ischemia.

Mucosal albumin clearance was measured in jejunal segments of dogs under control conditions and following complete or partial arterial occlusion of varying durations (1, 2, 3, or 4 hours). The rate of albumin clearance was estimated from the luminal perfusion rate and the activity of protein bound 125I in the perfusate and plasma. Partial and total arterial occlusions of 60 minutes to 4 hours' duration produced significant increases in mucosal albumin clearance. The magnitude of the rise in albumin clearance was directly related to the duration of ischemia in both total and partial arterial occlusion models. However, the magnitude of the increase in albumin clearance was significantly greater with total arterial occlusion for any given duration of ischemia. The albumin clearance results obtained in the present study compare favorably with previously reported morphologic changes in the intestinal mucosa produced by both total and partial occlusion of the superior mesenteric artery. The agreement between morphologic and physiologic measurements indicates that mucosal albumin clearance may be a useful tool for studying the pathophysiology of intestinal ischemia.

Role of oxygen-derived free radicals in digestive tract diseases.

Evidence is presented that supports a role of oxygen free radicals in the pathogenesis of various disorders of the digestive system. In the intestine, there is evidence that oxygen radicals play an important role in the endothelial and epithelial damage associated with certain models of ischemia. The mechanism for superoxide production in this condition differs from that described for other pathologic states (i.e., oxygen toxicity and neutrophil-mediated inflammation). This mechanism involves the reaction of xanthine oxidase, hypoxanthine, and molecular oxygen to produce a burst of oxygen radicals with reperfusion of the ischemic bowel. Evidence implicating oxygen radicals in inflammatory disorders of the digestive tract (i.e., pancreatitis), radiation injury, and hepatic cirrhosis is also presented. The available data suggest that oxygen radicals appear to be a fundamental mechanism of tissue injury in the pathogenesis of various disorders of the digestive system.

Role of oxygen free radicals in shock, ischemia, and organ preservation.

Oxygen radicals appear to be involved in the microvascular and parenchymal cell injury associated with various pathologic disorders. Studies indicate that oxygen radicals increase microvascular permeability by creating large leakage sites predominantly in the small venules. The highly reactive hydroxyl radical appears to be responsible for the microvascular alterations associated with oxygen radical production. There is considerable indirect evidence implicating oxygen radicals in the pathogenesis of circulatory shock. The oxygen radicals are probably formed by the enzyme xanthine oxidase when intravascular volume is restored. Similar biochemical processes appear to be involved in reperfusion injury to the kidney and skin. Evidence is also presented that implicates oxygen radicals in the reperfusion injury associated with organ preservation and transplantation.

Death of transcallosal neurons after close axotomy.

The extent of cell death after axotomy may limit potential recovery after brain injury. We wished to determine the effect of axotomizing lesions on survival of transcallosally projecting cortical neurons. Transcallosal neurons were prelabeled by retrograde transport of the fluorescent dyes Fluoro-Gold and True Blue. A transcortical stab wound divided the field of labeled cortical cells into axotomized and unaxotomized groups. Little difference in labeled cell density was seen over the first few days after injury. Animals surviving at least 2 weeks after injury had clear loss of axotomized neurons. By 1 month after injury, the vast majority of axotomized labeled cells appeared to have died. Quantitative evaluation of labeled cells showed that the region of cortex within 1 mm of the axotomizing injury had less than 10% of the expected neuronal density in animals surviving at least 4 weeks after injury. Close axotomy appears to cause dramatic loss of transcallosal neurons even in adult animals.

Effect of proximal axotomy on GAP-43 expression in cortical neurons in the mouse.

As an approach to understanding why central neurons fail to regenerate, we have studied the response to proximal axotomy of transcallosal neurons of the cerebral cortex of the mouse. Anatomical studies have indicated only very slight regenerative responses by this population of cortical neurons. To further examine the regenerative response of these cells, we have looked by in situ hybridization at the expression of GAP-43 mRNA following axotomy caused by a stab wound delivered within about 200 microm to 1.25 mm of the cell body. Axotomized transcallosal neurons were compared with near-by unaxotomized transcallosal neurons, as well as with distant unaxotomized cortical neurons in the contralateral hemisphere. All three populations of neurons had been pre-labeled with Fluoro-Gold to allow identification. No up-regulation of GAP-43 mRNA above background levels was detected for axotomized cortical neurons at 1, 3 or 7 days after injury. In contrast, increases in mean silver grain density of up to 8-fold were measured in axotomized spinal cord motor neurons used as positive controls. Thus, as a population, the transcallosal cortical pyramidal neurons did not show a significant regenerative response, as monitored by GAP-43 upregulation, even with very close axotomy. These results identify this population of neurons as among the least regenerative studied, and suggest that, on a molecular level, inherent neuronal properties play a role in the limited regenerative response to brain injury.

Glutamate-pyruvate transaminase protects against glutamate toxicity in hippocampal slices.

Elimination of glutamate through enzymatic degradation is an alternative to glutamate receptor blockade in preventing excitotoxic neuronal injury. Glutamate pyruvate transaminase (GPT) is a highly active glutamate degrading enzyme that requires pyruvate as a co-substrate. This study examined the ability of GPT to protect neurons of the hippocampal slice preparation against glutamate toxicity. Two methods were used to elevate the concentration of glutamate in the peri-neuronal space. In an endogenous release paradigm, slices were incubated with 100-500 microM L-trans-pyrrolidine-2,4-dicarboxylate (PDC), an inhibitor of glutamate re-uptake. One hour of exposure to PDC in normal, pyruvate-free slice maintenance medium caused a dose dependent increase in neuronal death assessed 24 h later by propidium iodide uptake in dead cell nuclei. GPT (10 U/ml) decreased neuronal death caused by exposure to PDC at all PDC concentrations tested. Neuroprotection in this model was not dependent on added or non-physiologic levels of pyruvate. In a different paradigm, glutamate was added directly to the normal, pyruvate-free slice maintenance medium and not rinsed away, exposing the slices to a range of 1-5 mM glutamate for an extended period. Twenty-four hours later, neuronal death was again assessed by propidium iodide uptake. GPT was again neuroprotective, decreasing neuronal death in the range from 3 to 5 mM glutamate. In the setting of incubation with this large load of glutamate, neuroprotection by GPT was enhanced by adding pyruvate to the medium. GPT is an effective neuroprotectant against glutamate excitotoxicity. When exposure is limited to endogenously released glutamate, neuroprotection by GPT is not dependent on added pyruvate.

Tetanus toxin fragment C as a vector to enhance delivery of proteins to the CNS.

The non-toxic neuronal binding domain of tetanus toxin (tetanus toxin fragment C, TTC) has been used as a vector to enhance delivery of potentially therapeutic proteins to motor neurons from the periphery following an intramuscular injection. The unique binding and transport properties of this 50-kDa polypeptide suggest that it might also enhance delivery of proteins to neurons after direct injection into the CNS. Using quantitative fluorimetry, we found that labeled TTC showed vastly superior retention within brain tissue after intracerebral injection compared to a control protein (bovine serum album). Fluorescence microscopy revealed that injected TTC was not retained solely in a restricted deposit along the needle track, but was distributed through gray matter in a pattern not previously described. The distribution of injected protein within the extracellular space of the gray matter and neuropil was also seen after injection of a recombinant fusion protein comprised of TTC linked to the enzyme superoxide dismutase (TTC-SOD-1). Injections of native SOD-1 in contrast showed only minimal retention of protein along the injection track. Immunohistochemistry demonstrated that both TTC and TTC-SOD-1 were distributed in a punctate perineuronal and intraneuronal pattern similar to that seen after their retrograde transport, suggesting localization primarily in synaptic boutons. This synaptic distribution was confirmed using HRP-labeled TTC with electron microscopy along with localization within neuronal endosomes. We conclude that TTC may be a useful vector to enhance neuronal delivery of potentially therapeutic enzymes or trophic factors following direct injection into the brain.