Oxygen-derived free radicals producing activity and survival of activated polymorphonuclear leukocytes

Summary Activation of polymorphonuclear (PMN) leukocytes is known to generate oxygen free radicals (OFR). However the fate of activated PMN leukocytes is not known. We investigated the OFR producing (chemiluminescence) activity and the survival of the activated PMN leukocytes. The study was divided into two groups. Group I, In vivo study (n = 7): zymosan (8.4 mg/kg) was administered intravenously in the anesthetized dogs and the blood samples were collected before and after 5, 15, 30, 60 and 120 min of zymosan administration. This group represents the in vivo pre-stimulated PMN leukocytes; Group II, In vitro study (n = 7): the blood were collected from dogs and further divided into two groups. Group A (n = 7): non-stimulated, without any added zymosan and group B (n = 7): zymosan was added to stimulate PMN leukocytes. Blood samples from group A and B were also collected at various time intervals similar to in vivo studies. Oxygen free radical producing activity of PMN leukocytes was monitored by measuring luminoldependent chemiluminescence (CL). Opsonized zymosan was used to activate PMN leukocytes. The studies in which the PMN leukocytes were stimulated in in vivo, both oxygen derived free radicals and superoxide dismutase (SOD) inhibitable oxygen free radical CL decreased significantly for 60 min and tended to reach thereafter to the pre-stimulated values. The resting chemiluminescence (chemiluminescence without zymosan stimulation in the assay medium) increased significantly for 15 min reaching to pre-stimulated values at 30 min and thereafter. In in vitro studies, oxygen derived free radicals CL of pre-stimulated PMN leukocytes (Group B) was depressed for the whole duration of investigation while SOD inhibitable CL was depressed for only 60 min. There was approximately a two-fold increase in the resting CL within 5 min of PMN leukocyte activation and it remained high for the whole duration of study. The chemiluminescence of non-stimulated PMN leukocytes in vitro (group A) remained practically normal throughout the period of observation. In in vivo studies, total white blood cells (WBC) and PMN leukocyte counts decreased initially and tended to approach towards pre-stimulated values at the end of the protocol. There were no changes in these counts in in vitro studies. These results indicate that the capacity to generate OFR is decreased in the in vivo and in vitro pre-stimulated PMN leukocytes. However this activity recovers with time. This study also suggests that the activated PMN leukocytes are not destroyed.     

Chemiluminescence activity in whole blood phagocytes of dogs naturally infected with Leishmania infantum

Dogs are the domestic reservoir of Leishmania infantum, a vector-borne intracellular protozoan agent of human visceral leishmaniasis. The role of polymorphonuclear leukocytes (PMNs) in the immune defence against this parasite has been poorly studied. We have investigated the function of peripheral blood PMNs in naive beagle dogs that have been naturally exposed to phlebotomine vectors in an area highly endemic for canine leishmaniasis, and found infected by Leishmania at the end of the transmission season. Whole blood phagocyte oxidative metabolism was assessed by a rapid method that determines a luminol-amplified chemiluminescence (CL) emission. This was evaluated using either a soluble stimulant, phorbol mirystate acetate (PMA), or phagocytic stimuli, such as zymosan unopsonized (ZYM) or opsonized with autologous serum (OPZ). In blood samples taken 2 months after exposure to Leishmania transmission, data on CL emission revealed a significant decrease of reactive oxygen intermediates (ROI) production in the presence of both PMA and ZYM, compared with blood samples obtained from dogs before exposure. On the contrary, no variations in CL emission were detected in presence of OPZ. Our data indicate that immunological changes occur early in canine leishmaniasis and confirm that the role of PMNs and their products need to be clarified. Copyright © 2000 John Wiley & Sons, Ltd.     

Tumor size,leukocyte adherence inhibition and serum levels of tumor antigen in dogs with the canine transmissible venereal sarcoma

Summary Tumor antigen (TA) associated with the canine transmissible venereal sarcoma (CTVS) was detected in the sera of dogs bearing the tumor. Rabbit antisera specific for tumor antigen and 3 M KCl extracts of CTVS cells were used in both a competitive enzyme-linked immunosorbent assay (ELISA) and antigen-capture ELISA to quantify levels of circulating TA. In a study of 29 dogs bearing the transplanted CTVS, levels of circulating TA correlated positively with tumor volume. In a longitudinal study of four dogs receiving a transplant of 10⁸ viable CTVS cells, circulating CTVS antigen was detected transiently 2 days after transplantation, while persistent levels of TA associated with increasing tumor volume were demonstrable 19–34 days after transplantation. In three of four tumor-bearing dogs, levels of serum TA correlated inversely with values obtained with peripheral blood leukocytes in the leukocyte adherence inhibition (LAI) assay; elevated levels of circulating TA found in dogs with large (>7 cm³) tumors were associated with decreased LAI reactivity of peripheral blood leukocytes. TA could not be detected in sera 48–72 h after surgical removal of CTVS whereas LAI reactivity of peripheral blood leukocytes to CTVS antigen rebounded 1–3 weeks following tumor excision. Results of this study support the use of the competitive ELISA and LAI techniques in assessing levels of circulating tumor antigen, tumor burden and tumor-specific immunity.Supported by grants from the American Kennel Club, National Institutes of Health (CA23 469) and Funds for Research on Canine Diseases provided by the 1963 Connecticut Legislature     

Improved preparation of leukocytes for chemiluminescent study of human phagocytic leukocyte-generated reactive oxygen species

Leukocyte oxidative function was investigated in a more physiological milieu than currently used in the chemiluminescence (CL) technique. Heparinized blood was mixed with 6% dextran-T70 (9:1) and the leukocyte-rich plasma obtained without centrifugation was used for the CL experiments (phagocyte count was adjusted to 0.7 × 10⁶/mL with Hanks' buffer) (method A). In this medium, phagocytes responded to stimulation by opsonized zymosan, producing strong luminescence in the presence of 0.5 m? mol/L MCLA. CL was inhibited by superoxide dismutase, suggesting that the luminescence reaction was attributable to O. Granulocytes were also prepared by the usual method involving centrifugation and were then suspended in plasma (method B). Oxidative function of phagocytes prepared by the two methods was studied together with whole blood as aliquots diluted with Hanks' buffer up to a factor of 1000. Luminescence reached a peak value at a dilution factor of 16, but at very high dilutions luminescence decreased sharply. Significantly higher luminescence values were obtained with samples from method A. Luminescence of whole blood peaked at a dilution factor of 248 but it was less than the value obtained using samples prepared by method A or B. As samples prepared by method A contain all the leukocyte populations, platelets, residual red cells and plasma proteins, the assay of leukocyte-generated reactive oxygens using CL is attained in more physiological conditions than method B in which leukocytes may be damaged owing to repeated centrifugation and hypotonic shock.     

Three-peaked chemilluminescent response of human peripheral blood leukocytes following stimulation with phytohaemagglutinin (PHA)

Luminol-enhanced chemiluminescence (CL) was used to examine the response of various leukocyte populations following stimulation with a crude extract of Phaseolus vulgaris, namely phytohaemagglutinin (PHA-C). Populations stimulated included a human peripheral mixed leukocyte preparation (MLP), and purified preparations of lymphocytes, monocytes and polymorphonuclear leukocytes (PMNL). Mouse peritoneal exudate cells and the lymphocytic cells lines Molt #4 and Daudi were also stimulated. Following stimulation, a characteristic three-peaked chemiluminescent response was obtained from the MLP population. Little or no response was obtained from the purified lymphocytes. Monocytes produced a sharp peak corresponding to the second peak of the MLP response and PMNL produced a broad peak corresponding to the third peak of the MLP response. Mouse peritoneal exudate cells containing lymphocytes and monocytes/macrophages showed a two-peaked stimulation which corresponded to the first two peaks of the MLP response. Molt #4 and Daudi showed no chemiluminescence if stimulated individually, but if added to a MLP substantial enhancement of the first and second peaks was observed. These results indicate some form of lymphocyte/monocyte interaction leading to enhanced CL following PHA-C stimulation.     

Effect of pulmonary blood flow on leukocyte uptake and release by dog lung

The effect of pulmonary blood flow on leukocyte uptake and release by the lung was examined in 10 anesthetized spontaneously breathing dogs. Pulmonary arterial and pulmonary venous blood was sampled with catheters placed into the right ventricle and aorta, respectively. Pulmonary blood flow was lowered by inflating a balloon catheter located in the inferior vena cava. In five experiments simultaneous blood samples were drawn from the right ventricle and aorta at 10-s intervals during a control period, a 2- to 3-min period of low flow, and a recovery period. In five additional experiments, less frequent samples were taken over periods of 15-60 min. Total leukocyte concentrations and differential counts were determined for each blood sample. The study shows that large numbers of leukocytes become sequestered within the lung when pulmonary blood flow is low and that an equivalent number of cells are released from the lung after deflation of the balloon catheter. Both the polymorphonuclear leukocytes and the lymphocytes were taken up by the lung when pulmonary blood flow was reduced. We conclude that pulmonary blood flow has a marked effect on the uptake and release of leukocytes by the dog lung.     

Inaccuracy of Venous Point-of-Care Glucose Measurements in Critically Ill Patients: A Cross-Sectional Study

Introduction

Current guidelines and consensus recommend arterial and venous samples as equally acceptable for blood glucose assessment in point-of-care devices, but there is limited evidence to support this recommendation. We evaluated the accuracy of two devices for bedside point-of-care blood glucose measurements using arterial, fingerstick and catheter venous blood samples in ICU patients, and assessed which factors could impair their accuracy.

Methods

145 patients from a 41-bed adult mixed-ICU, in a tertiary care hospital were prospectively enrolled. Fingerstick, central venous (catheter) and arterial blood (indwelling catheter) samples were simultaneously collected, once per patient. Arterial measurements obtained with Precision PCx, and arterial, fingerstick and venous measurements obtained with Accu-chek Advantage II were compared to arterial central lab measurements. Agreement between point-of-care and laboratory measurements were evaluated with Bland-Altman, and multiple linear regression models were used to investigate interference of associated factors.

Results

Mean difference between Accu-chek arterial samples versus central lab was 10.7 mg/dL (95% LA -21.3 to 42.7 mg/dL), and between Precision PCx versus central lab was 18.6 mg/dL (95% LA -12.6 to 49.5 mg/dL). Accu-chek fingerstick versus central lab arterial samples presented a similar bias (10.0 mg/dL) but a wider 95% LA (-31.8 to 51.8 mg/dL). Agreement between venous samples with arterial central lab was the poorest (mean bias 15.1 mg/dL; 95% LA -51.7 to 81.9). Hyperglycemia, low hematocrit, and acidosis were associated with larger differences between arterial and venous blood measurements with the two glucometers and central lab. Vasopressor administration was associated with increased error for fingerstick measurements.

Conclusions

Sampling from central venous catheters should not be used for glycemic control in ICU patients. In addition, reliability of the two evaluated glucometers was insufficient. Error with Accu-chek Advantage II increases mostly with central venous samples. Hyperglycemia, lower hematocrit, acidosis, and vasopressor administration increase measurement error.     

Production of leukotriene B4 and prostaglandin E2 by turbot (Scophthalmus maximus) leukocytes

Production of two eicosanoids derived from lipoxygenase and cyclooxygenase activities: leukotriene B₄ (LTB₄) and prostaglandin E₂ (PGE₂), respectively, have been simultaneously determined in turbot (Scophthalmus maximus) blood leucocyte and kidney macrophage supernatants by a reverse phase high performance liquid chromatography (HPLC) system coupled with a Diode–Array detector. Levels of LTB₄ after calcium ionophore challenge were 4.08 ng ml⁻¹ in blood leukocyte supernatants and 0.25 ng ml⁻¹ in kidney macrophage supernatants. The levels found for PGE₂ were 428.23 and 606.67 ng ml⁻¹ in blood leukocytes and kidney macrophage supernatants, respectively. When blood leukocytes were treated with the respective inhibitors for the enzymes implicated on the synthesis of both compounds an inhibition of 90.35% was observed for PGE₂ and 76.44% for LTB₄. The detection limit of the method was 0.15 ng ml⁻¹ for LTB₄ and 50 ng ml⁻¹ for PGE₂.     

Cell Wall Contents of Probiotics (Lactobacillus species) Protect Against Lipopolysaccharide (LPS)-Induced Murine Colitis by Limiting Immuno-inflammation and Oxidative Stress

Currently, there are no effective therapeutic agents to limit intestinal mucosal damage associated with inflammatory bowel disease (IBD). Based on several clinical studies, probiotics have emerged as a possible novel therapeutic strategy for IBD; however, their possible mechanisms are still poorly understood. Although probiotics in murine and human improve disease severity, very little is known about the specific contribution of cell wall contents of probiotics in IBD. Herein, we investigated the protective effects of cell wall contents of three Lactobacillus species in lipopolysaccharide (LPS)-induced colitis rats. LPS-sensitized rats were rendered colitic by colonic instillation of LPS (500 µg/rat) for 14 consecutive days. Concurrently, cell wall contents isolated from 10⁶ CFU of L. casei (LC), L. acidophilus (LA), and L. rhamnosus (LA) was given subcutaneously for 21 days, considering sulfasalazine (100 mg/kg, p.o.) as standard. The severity of colitis was assessed by body weight loss, food intake, stool consistency, rectal bleeding, colon weight/length, spleen weight, and histological analysis. Colonic inflammatory markers (myeloperoxidase activity, C-reactive protein, and pro-inflammatory cytokines) and oxidative stress markers (malondialdehyde, reduced glutathione, and nitric oxide) were also assayed. Cell wall contents of LC, LA, and LR significantly ameliorated the severity of colitis by reducing body weight loss and diarrhea and bleeding incidence, improving food intake, colon weight/length, spleen weight, and microscopic damage to the colonic mucosa. The treatment also reduced levels of inflammatory and oxidative stress markers and boosted anti-oxidant molecule. In conclusion, cell wall contents of LC, LA, and LR attenuate LPS-induced colitis by modulating immuno-inflammation and oxidative stress.

     

Determination of erythromycin in urine and plasma using microbore liquid chromatography with tris(2,2′-bipyridyl)ruthenium(II) electrogenerated chemiluminescence detection

Erythromycin is determined in both urine and plasma samples using microbore reversed-phase liquid chromatography with tris(2,2′-bipyridyl)ruthenium(II) [Ru(bpy)₃²⁺] electrogenerated chemiluminescence (ECL) detection. Ru(bpy)₃²⁺ is included in the mobile phase thus eliminating band broadening caused by post-column reagent addition. Extra column band broadening is an important concern in microbore liquid chromatography due to the small peak volumes. Erythromycin was studied in both water and biological samples. The detection limit for erythromycin in standards is 0.01 μM or 50 fmol injected with a S/N of 3 and a linear working range that extends four orders of magnitude. Human urine and blood plasma were also studied. Urine samples were diluted and filtered before injection. Ultrafiltration was used to remove protein from blood plasma samples prior to injection. Erythromycin was selectively detected in the body fluid samples without any further sample preparation. The detection limits obtained for erythromycin in urine and plasma are 0.05 and 0.1 μM, respectively, for 5 μl injected on a 150×1 mm I.D. C₁₈ column.     

Preparation of 111in leukocytes after hemolytic removal of erythrocytes

An optimized procedure is described for isolation and highefficiency radiolabeling of leukocytes using ¹¹¹In-oxine. The chief advantages over conventional methods include virtually no loss of leukocytes during washing and separation steps; a significant reduction in the time required to prepare leukocytes for radiolabeling compared to non-hemolytic preparations; a 28% increase in the average labeling efficiency obtained using ¹¹¹In-oxine; >95% cell viability as measured by the trypan blue exclusion test; elimination of contaminating red blood cells from the leukocyte pellet prior to labeling; and 80% survivability at 15 min post injection (measured as per cent of blood activity on leukocyte fraction).     

In vitro stimulation of human granulopoiesis by purified protein derivative (PPD)

Summary The effect of purified protein derivative (PPD) on human granulopoiesis was studied in an in vitro semisolid culture system of human bone marrow in which PPD was incorporated into the leukocyte feeder layers. We observed that preincubation of the feeder layers with PPD was necessary to induce a significant rise of agar culture colony-forming units (CFU-c) with a maximum of 3 days' preincubation and a dose of 200 g for 10 ⁶ leukocytes. A similar effect was obtained when a conditioned medium from PPD-stimulated leukocytes was used instead of feeder layers. We have found a significant correlation between the skin test response of the leukocyte donors to PPD and the colony-stimulating activity of their leukocytes exposed to PPD: these results suggest that PPD could stimulate human granulopoiesis by an indirect effect on CSF-producing mononuclear cells.     

Cellular chemiluminescence of activated phagocytes of whole blood

Both, the phagocytic process and the activation of phagocytes with soluble stimuli are accompanied by increased production of reactive oxygen species (ROS). Chemiluminescence (CL) measurement is a simple and sensitive method for the detection of ROS generation. Phagocytes (mainly polymorphonuclear leukocytes, PMNL) were stimulated with soluble stimulus or via phagocytosis in diluted whole blood, and the generation of Luminol-enhanced CL was registered. The time dependence of CL, determined in whole blood, corresponds to the CL from isolated leukocytes. A relationship between peak CL and the number of leukocytes as well as of PMNL was observed. The specific CL, i.e. the CL response related to a defined PMNL number, increases with the age of investigated healthy individuals. No correlations were found between CL and the capacity of PMNL to ingest zymosan particles. Relations between CL and spontaneous platelet aggregation suggest, that reactivity of blood platelets may be a contributing factor to the kinetics of the CL signal in our test system. The inhibition of CL by the sulphydryl reagents diamide and fever few extract indicate the role of cellular sulphydryl groups for phagocyte function. Measurement of CL in whole blood is proved to be a simple assay for assessment of PMNL function and allows measurements in very small blood samples (greater than or equal to 10 ul).     

Effects of fish oil on lymphocyte proliferation,cytokine production and intracellular signalling in weanling pigs

It has been widely documented that fish oil attenuates inflammatory responses partially via down-regulation of T-lymphocyte function. To determine the anti-inflammatory role of fish oil in weanling pigs, we investigated the effects of fish oil and its functional constituents on peripheral blood lymphocyte proliferation, cytokine production and subsequent intracellular signalling in inflammatory-challenged weanling pigs and in in vitro cultured lymphocytes. Fish oil (7%) or corn oil (7%) was supplemented to 72 crossbred pigs (7.6 ± 0.3 kg BW and 28 ± 3 days of age) in a 2 × 2 factorial experiment that included an Escherichia coli lipopolysaccharide (LPS) challenge (challenged or not challenged). On day 14 and 28 of the experiment, 200 μg/kg BW of LPS or an equivalent amount of sterile saline was administered to the pigs by intraperitoneal injection. Blood samples were collected on days 15 and 29 to determine peripheral blood lymphocyte proliferation, interleukin-1β (IL-1β) and interleukin-2 (IL-2) production. The results showed that inflammatory challenge decreased average daily gain (P < 0.05) and average daily feed intake (P < 0.05) during days 15 - 28. Fish oil supplementation had no effect on growth performance. Inflammatory challenge increased lymphocyte proliferative response to concanavalin A (Con A) (P < 0.05) following each challenge. Fish oil tended to suppress (P < 0.1) the proliferation following the first challenge. Similarly, fish oil tended to reduce IL-1β production (P < 0.1) following the second challenge and IL-2 (P < 0.1) production following the first challenge in both challenged and unchallenged pigs compared with corn oil. In parallel in vitro experiments, peripheral blood lymphocytes of weanling pigs were incubated with various concentrations of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or linoleic acid (LA) (0, 20, 40, 60, 80, 100 μg/ml). EPA, DHA and high levels of LA predominantly suppressed IL-1β (P < 0.05), IL-2 (P < 0.05) production and subsequent lymphocyte proliferation (P < 0.05). Low levels of LA increased (P < 0.05) IL-2 production. Compared with LA, EPA resulted in a stronger inhibition of lymphocyte proliferation (P < 0.05) and IL-2 (P < 0.01), and DHA resulted in a stronger inhibition of IL-1β (P < 0.05) and IL-2 (P < 0.01). To elucidate the mechanism(s) by which fish oil and its functional constituents suppressed lymphocyte function, the kinetics of intracellular [Ca^{2 +}]_i and protein kinase C activity were determined in in vitro experiments. EPA, DHA and LA exerted very similar dose-dependent stimulatory effects on intracellular Ca^{2 +}. EPA and DHA inhibited protein kinase C activity (P < 0.05), while LA had no significant effect (P > 0.05). These results suggest that fish oil and its functional constituents (EPA and DHA) exerted an anti-inflammatory effect by down-regulation of lymphocyte activation in weanling pigs, possibly by manipulation of intracellular signalling.     

Oxygen free radicals in volume overload heart failure

It has been suggested that oxygen free radicals (OFR) depress the excitation-contraction coupling in cardiac muscle. It is possible that a decrease in the cardiac contractility in the failing heart may be due to an increased OFR producing activity of polymorphonuclear (PMN) leukocytes. We studied the OFR producing activity (chemiluminescence) of PMN leukocytes from blood in dogs with heart failure due to chronic volume overload. The animals were divided into two groups: I) normal, (n = 10): II) dogs with mitral insufficiency (MI) of 6 to 9 months duration, (n = 10). Hemodynamic studies were done to establish the presence of heart failure. Blood samples were collected to measure PMN leukocyte chemiluminescence. There was a decrease in the cardiac index and index of myocardial contractility (dp/dt/IIP) and an increase in the left ventricular end-diastolic pressure in dogs with MI indicating left ventricular failure. The peak chemiluminescent activity of the PMN leukocytes in blood of dogs with failure was about four folds greater than that in the blood from normal dogs. These results suggest that there may be an increased OFR generation in dogs with volume overload heart failure. The decrease in the myocardial contractility in the failing heart might be due to an increase in the OFR produced by the PMN leukocytes.     

Flow cytometric analysis of glyoxalase-1 expression in human leukocytes

Altered glyoxalase‐1 (GLO‐1) activity and expression is associated with the development of late diabetic complications, malignancy and oxidative stress‐ and aging‐related diseases. In the present study, we developed a flow cytometry method for GLO‐1 detection in human leukocytes isolated from peripheral blood samples to investigate GLO‐1 expression in leukocyte subsets from type 1 and 2 diabetes mellitus patients (n = 11) and healthy subjects (n = 8). The flow cytometry analysis of GLO‐1 in leukocytes showed that expression index of GLO‐1‐positive cells was slightly increased in mononuclear leukocytes from diabetic patients. This result correlated with the increase in GLO‐1 activity in the whole blood samples of type 2 diabetes patients. In conclusion, the present study demonstrates that flow cytometry is suitable for the detection of the GLO‐1 enzyme in human leukocytes and that this method could be used to investigate the fast adaptation of the glyoxalase system related to the pathogenesis of late complications of diabetes mellitus and other glycation stress‐related disorders. Copyright © 2011 John Wiley & Sons, Ltd.     

Chemiluminescence of bronchoalveolar lavage cells as an indicator of leukocyte activation in rats with a hyperreactive bronchus

Activation and generation of inflammatory mediators by different leukocytes may be important in the pathogenesis of airway hyperreactivity. We studied the effect of active sensitization with ovalbumin as antigen and i.v. treatment with Sephadex particles on bronchial reactivity (BR) in rats and its possible relation to leukocyte infiltration (LI) and activation (LA) in bronchoalveolar lavage (BAL). A marked BR to aerosols of serotonin (5-HT) and ovalbumin was found in Sephadex treated animals but not in control animals. In parallel to this a marked increase in BAL cell count from Sephadex-treated animals compared to controls was seen. This increase in BAL cell count corresponded with a clear augmentation of spontaneous, buffer-induced and C₃Z-induced, luminol-amplified CL. We deduce that detection of CL of BAL cells from rats might be used for studying inflammatory mechanisms which lead to a hyperreactive bronchus.     

Acid-base disequilibrium in the venous blood of rainbow trout (Oncorhynchus mykiss)

Acid-base equilibria/disequilibria were evaluated in vivo in post-branchial arterial blood and pre-branchial venous blood of freshwater rainbow trout (Oncorhynchus mykiss). This was accomplished using arterial and venous extracorporeal circuits in conjunction with a stopped-flow apparatus. After the abrupt stoppage of circulating post-branchial blood within the stopped-flow apparatus, pH increased slowly ([Delta]pH = +0.032 ± 0.004 pH units; n = 15), thus confirming the existence of an acid-base disequilibrium state in the arterial blood of rainbow trout. The slow downstream pH changes were unaffected by prior treatment of fish with the carbonic anhydrase inhibitor benzolamide (1.2 mg kg^-1; [Delta]pH = +0.032 ± 0.01 pH units; n = 5) but were eliminated after intra-vascular injection of 10 mg kg^-1 bovine carbonic anhydrase ([Delta]pH = -0.011 ± 0.003 pH units; n = 8). These results demonstrate that the acid-base disequilibrium in the arterial blood reflects a total absence of extracellular carbonic anhydrase activity. Similar stopped-flow experiments revealed the existence of a reduced, yet significant, acid-base disequilibrium in the venous blood circulating within the caudal vein ([Delta]pH = +0.004 ± 0.003 pH units; n = 15). Selective inhibition of extracellular carbonic anhydrase using benzolamide did not significantly influence the magnitude of the venous pH disequilibrium ([Delta]pH = +0.007 ± 0.007 pH units; n = 8) whereas intra-vascular injection of carbonic anhydrase eliminated the pH disequilibrium. These results demonstrate that extracellular carbonic anhydrase, although reported to be present within the skeletal muscle of rainbow trout, does not accelerate post-capillary pH changes in the venous circulation.     

Particle-induced myeloperoxidase release in serially diluted whole blood quantifies the number and the phagocytic activity of blood neutrophils and opsonization capacity of plasma.

Luminol-amplified chemiluminescence (CL) from phagocytes has previously been shown to be almost completely dependent on the release of myeloperoxidase (MPO) from azurophilic granules. We measured the luminol-amplified chemiluminescence response (WBCL) by using serially diluted whole blood. In these experiments, non-opsonized and serum-opsonized zymosan (NWBCL and OWBCL, respectively) were used concurrently as phagocytosable particles. We found two whole-blood dilution ranges with clinical significance: first, <0.04% of whole blood in the reaction volume, where MPO released by the zymosan-activated leukocyte population came almost totally from neutrophils and the OWBCL response could be exploited as a measure of a neutrophil count in a given blood specimen, despite the pathophysiological state of the host. In contrast, the NWBCL response was two-fold in blood samples from bacterial infection patients compared to those of controls and patients with viral infection, suggesting the use of NWBCL for the differential diagnosis of bacterial infections from viral infections; second, 0.16-1.2% of whole blood in the reaction volume, where the opsonization capacity of plasma (OC(50)) can be determined. We also found that at whole blood content >0.04%, erythrocytes quickly start to absorb chemiluminescence light, and that at whole blood content >1.2%, plasma proteins, most probably albumin and fibrinogen, start to inhibit MPO release.     

Generation of Nitric Oxide by Peripheral Blood Leukocytes and Platelets in Healthy Humans and Patients with Thermal Trauma

The generation of nitric oxide (NO) by human peripheral blood leukocytes and platelets has been studied in healthy subjects and patients with burns (with the affected area varying from 10 to 45% of the body surface). Differential centrifugation was used to isolate leukocytes and platelets from the blood. The leukocyte suspension was diluted with a complete medium to a concentration of 1 × 10⁷ cells/ml, and the platelet suspension, to 1 × 10⁸ cells/ml; the suspensions were then cultured for 15 h (37°C). The concentration of nitrite, an NO metabolite, was determined using the Griss reaction. The relative production of NO by leukocytes of healthy subjects and patients was 0.75 ± 0.06 and 2.93 ± 0.16 mol/l, respectively (p < 0.001), and its relative production by platelets of healthy subjects and patients was 2.15 ± 0.14 and 3.62 ± 0.13 mol/l, respectively (p < 0.01). The absolute generation of NO by leukocytes of healthy subjects and patients is 0.47 ± 0.05 and 3.02 ± 0.28 mol/l, respectively (p < 0.001), and its absolute generation by platelets of healthy subjects and patients was 7.70 ± 0.55 and 14.68 ± 0.84 mol/l, respectively (p < 0.001). Thus, the absolute production of NO by platelets is 16 times higher than the absolute production of NO by leukocytes of healthy subjects. Stress increases the generation of NO by both leukocytes and platelets. The absolute generation of NO by platelets in thermal trauma is positively correlated with the plasma content of fibrinogen in the patients.