Effect of extravascular hemolysis on the RES depression following thermal injury

This study was carried out to determine if the reticuloendothelial system (RES) uptake of damaged red blood cells (RBC) following thermal injury is sufficient to depress hepatic and splenic clearance function or to increase susceptibility to endotoxin shock. This was approached by determining the extent of the RES uptake of intact RBC (extravascular hemolysis) following thermal injury in rats, and by determining the effect on RES function of a similar degree of extravascular hemolysis induced in uninjured animals by the injection of heat damaged RBC. The RES uptake of three doses of heated RBC was characterized and the second largest dose caused a degree of extravascular hemolysis which was comparable to that associated with thermal injury. This latter dose of heated RBC depressed splenic clearance function but did not depress hapatic clearance function as determined using the smallest dose of heated RBC or formalinized sheep red blood cells as the test particles. Susceptibility to endotoxin shock was increased following the injection of the dose of heated RBC which stimulated the degree of extravascular hemolysis associated with thermal injury. Thus, the extravascular hemolysis associated with thermal injury is sufficient to depress splenic clearance function but not hepatic clearance function, and may contribute to the increased susceptibility to endotoxin shock following thermal injury. These findings further support the hypothesis that the hemolysis induced by thermal injury contributes to the depression of host defense associated with this form of injury.     

Depressing hepatic macrophage complement receptor function causes increased susceptibility to endotoxemia and infection.

Previous work has demonstrated that in vivo hepatic macrophage complement receptor clearance function is depressed after thermal injury. To determine whether impairment of complement receptor function is important in host defense, the present study evaluated the effect of the depression of complement receptor function in uninjured animals on susceptibility to endotoxin shock and bacterial infection. Hepatic complement receptor clearance function was evaluated by measuring the hepatic uptake of a test dose (2.9 X 10(8)/100 g) of rat erythrocytes coated with anti-erythrocyte immunoglobulin M (EIgM) or EIgG in rats. Depression of hepatic complement receptor function was induced by the injection of EIgG. The hepatic uptake of the test dose of EIgM or EIgG was depressed after the injection of 8.7 X 10(8) EIgG per 100 g and 17.4 X 10(8) EIgG per 100 g but not after the injection of 2.9 X 10(8) EIgG per 100 g. This effect was shown not to be due to a decrease in hepatic blood flow or a depletion of serum C3 and was, therefore, due to a depression in hepatic macrophage complement receptor clearance function. Susceptibility to endotoxin shock was increased with the dose of 8.7 X 10(8) EIgG per 100 g, and susceptibility to infection with Pseudomonas aeruginosa was increased with the dose of 17.4 X 10(8) EIgG per 100 g. Therefore, depression of hepatic macrophage complement receptor clearance function with EIgG is associated with depressed host defense.     

Activity of the Reticuloendothelial System and the Antibody Response: I. Effect of Stilboestrol on RES Activity and Localization of Sheep Erythrocytes in the Mouse

The stimulatory effect of stilboestrol on the RES of the mouse was assessed in terms of the blood clearance and organ uptake of colloidal carbon, sheep RBC and heat damaged mouse RBC, and comparison made between the effect on the RES of stilboestrol and endotoxin. Pretreatment with stilboestrol caused an increase of hepatic uptake of test materials and a reduction of carbon and SRBC localization in the spleen. Incorporation of [³H]thymidine and the proportion of labelled cells were increased in livers and spleens of stilboestrol treated animals.     

Humoral deficiency and reticuloendothelial depression after traumatic shock.

Circulating opsonin levels and reticuloendothelial (RE) phagocytic activity were investigated in anesthetized rats subjected to Noble-Collip drum (NCD) trauma. Reticuloendothelial function was assessed by colloid clearance kinetics and circulating opsonin levels by in vitro tissue slice bioassay. After sublethal shock, both hepatic RE phagocytosis and plasma opsonic activity were significantly (P less than 0.001) depressed in the 0.5- to 6-h posttrauma period. Pulmonary and bone marrow localization of the blood-borne test microparticles significantly (P less than 0.05) increased during hepatic RES depression. Hepatic RE cells from animals during the interval of posttraumatic in vivo phagocytic depression exhibited normal phagocytosis when incubated in normal pretrauma plasma and decreased phagocytic activity when incubated in posttrauma plasma. After sublethal shock, restoration or opsonin levels by 24 h after shock resulted in hepatic RE recovery. Plasma opsonin levels declined in direct relationship to the degree of trauma. Progressive hepatic RE failure was correlated with the progressive decline in circulating plasma opsonic activity. The findings indicate that opsonic depletion may be involved in the etiology of hepatic reticuloendothelial depression after traumatic shock.     

Effect of Kupffer cell phagocytosis of erythrocytes and erythrocyte ghosts on susceptibility to endotoxemia and bacteremia.

The phagocytosis of erythrocytes by macrophages has previously been shown to depress macrophage function. In this study we compared the effect of the phagocytosis of erythrocytes and erythrocyte ghosts by Kupffer cells on the duration of the depression of complement receptor clearance function and host defense against endotoxemia and bacteremia. Phagocytosis of erythrocytes and erythrocyte ghosts was induced in rats by the injection of rat erythrocytes or erythrocyte ghosts coated with anti-rat erythrocyte immunoglobulin G (EIgG and GIgG, respectively). The hepatic uptake of EIgG and GIgG (17.4 X 10(8)/100 g) occurred during the first 30 min after injection. The digestion of phagocytized EIgG and GIgG, as assessed by electron microscopy, was complete at 24 and 3 h after injection, respectively. The depression of Kupffer cell complement receptor clearance function caused by EIgG and GIgG returned to normal by 6 h after injection of EIgG and by 3 h after injection of GIgG. Phagocytosis of EIgG depressed the survival rate after endotoxemia and bacteremia when endotoxin or bacteria were injected at 30 min after EIgG. The survival rate returned to normal when the endotoxin and bacteria were injected at 12 and 6 h after the EIgG, respectively. Phagocytosis of GIgG did not depress the survival rate after endotoxemia and bacteremia. Thus, compared with erythrocytes, erythrocyte ghosts are more rapidly digested after phagocytosis, depress complement receptor function for a shorter period of time, and cause less depression of host defense. These findings indicate that the contents of erythrocytes play an important role in the impairment of host defense caused by the phagocytosis of erythrocytes by Kupffer cells.     

Image analysis of the effects of gonadectomy on colloidal carbon uptake in vivo by sinusoidal cells in liver and spleen of the mouse

PROBLEM: The presence of androgen and estrogen receptors in macrophages was recently described. This suggests that both sex hormones directly act on reticuloendothelial system (RES) activity. Studies of the effect of gonadectomy on uptake and clearance of foreign materials make clear a role of the testis and ovary in RES regulation. However, there is still some disagreement as to the gonadal influence on RES. METHOD OF STUDY: We applied a computed image analysis system to the investigation of the effects of gonadectomy on colloidal carbon uptake by the liver and spleen sinusoidal cells in mice. Mice were gonadectomized or sham-operated. After a few weeks, they received colloidal carbon with or without pretreatment with lipopolysaccharide (LPS). Thereafter, livers and spleens were taken for quantitative determination of the amounts of endocytosed carbon in the sinusoidal cells by means of digital image analysis. RESULTS: Ovariectomy did not significantly affect hepatic and splenic RES activities compared with sham-operated controls. Treatment of mice with LPS after ovariectomy also caused no significant changes of the RES activities compared with sham-operated and LPS-injected controls. In contrast, orchidectomy resulted in significant enhancement of splenic RES activity compared with sham-operated males, while hepatic RES activity did not significantly change with orchidectomy alone. However, LPS treatment to orchidectomized mice significantly augmented both hepatic and splenic RES activities compared with sham-operated and LPS-injected controls. CONCLUSION: The significant augmentation of RES activity by orchidectomy, but not by ovariectomy, indicates that male RES activity is physiologically downregulated in the presence of the testes. Furthermore, splenic RES is more sensitive to orchidectomy than hepatic RES.     

Distribution and localization of endotoxin in the reticulo-endothelial system (RES) and in the main vessels of the rat during shock

The fate of endotoxin was followed with immunohistochemistry and radio-labelled lipopolysaccharide (LPS) in organs of the reticulo-endothelial system (RES), in the great vessels and the thoracic duct of rats during a 14 day period after the injection of a shock-inducing amount of endotoxin. The immunohistochemical detectability of LPS in most tissues increased continuously during the first 48 hours, showing the strongest LPS staining in the liver and adrenal gland. Macrophages were found to be the most important cells of primary LPS uptake in all organs except the adrenal glands, where endotoxin was mainly present in phagocytic vacuoles of the cortical epithelium. The comparison of results obtained with the immunoperoxidase method and radioactivity measurements revealed that at a later stage of the experiment the persisting LPS in liver and spleen looses its antigenic activity. The correlation between the appearance of LPS positive macrophages and histological signs of tissue injury during endotoxin shock is striking.     

Protective effect of cysteine and methylprednisolone in lead acetate-endotoxin induced shock

To delineate possible mechanisms of lead acetate enhancement to endotoxin shock, liver parenchymal, and Kupffer cell function were evaluated in lead acetate-treated rats receiving low doses of S. enteritidis endotoxin. Additionally, the influence of methylprednisolone and cysteine, agents which have been demonstrated to prevent lead-induced endotoxin lethality was ascertained. Rats receiving lead acetate demonstrated profound hyperreactivity to endotoxin as denoted by increased mortality, impairment of bromsulphalein (BSP) removal, hypoglycemia, and increased blood lactate and pyruvate. Plasma activity of glutamic pyruvic transaminase (GPT), glutamic oxalacetic transaminase (GOT), and alkaline phosphatase was strikingly elevated. Serum activity of beta-glucuronidase and plasma acid phosphatase was also elevated. Methylprednisolone or cysteine treated rats were essentially refractory to the lethal effects of lead acetate and endotoxin. The enhanced survival of lead-endotoxin treated rats following administration of methylprednisolone or cysteine was associated with reduced impairment in BSP removal, a normal blood glucose, and significant reductions in blood lactate and pyruvate. Plasma activity of GPT, GOT, and alkaline phosphatase was also reduced. Slight reductions were observed in plasma activity of acid phosphatase although serum beta-glucuronidase activity was not significantly altered. Phagocytic activity, as measured by hepatic uptake of gelatinized RE test lipid emulsion, indicated a hypofunctional state of Kupffer cells in lead acetate-endotoxin treated rats. Since neither methylprednisolone, nor cysteine treatment modified the hypophagocytic state, Kupffer cell phagocytic dysfunction does not appear to play a major role in enhanced endotoxin susceptibility of lead acetate treated rats. The maintenance of parenchymal cell function in methylprednisolone or cysteine treated rats injected with lead and endotoxin supports the hypothesis of a contributory role for hepatic parenchymal cells in the pathophysiology of lead-endotoxin interaction.     

Opsonic fibronectin deficiency in the etiology of starvation-induced reticuloendothelial phagocytic dysfunction

The role of nutritional deficiencies in mediating host defense failure is increasingly recognized. This study evaluated the effect of starvation on serum opsonic fibronectin levels and reticuloendothelial function in the rat. Over a period of 5 days of starvation serum immunoreactive opsonic fibronectin had fallen to 62% of control levels (458 ± 20 μg/ml). This correlated closely (r = 0.85, P < 0.05) with a fall in bioassayable opsonic activity. Addition of purified opsonic fibronectin to plasma obtained from starved rats restored its ability to stimulate in vitro Kupffer cell phagocytic activity. Reticuloendothelial phagocytic function in vivo was significantly depressed after 5 days starvation as reflected by prolonged blood retention and decreased hepatic Kupffer cell uptake of a test colloid. Intravenous fibronectin therapy (2 mg/100 g), 1 hr prior to assessment of RE function, significantly (P < 0.05) improved but did not fully normalize blood clearance and hepatic uptake. These data suggest the RES phagocytic host defense failure in the nutritionally compromised patient may occur, in part, as a consequence of opsonic fibronectin deficiency.     

Distribution of 203Pb during lead-potentiated endotoxic shock

Lead salts markedly sensitize rats to lethal endotoxemia. Since this lethal synergism may reflect altered clearance and toxicity of lead, tissue distribution of ²⁰³Pb was evaluated with or without a concurrent dose of Salmonella enteritidis endotoxin. Intravenous injection of 0.5 μCi of ²⁰³Pb with 2 mg/100 g body wt. of lead acetate carrier was assessed at 1 and 5 hr. Hepatic uptake of the heavy metal comprised 60–70% of the total injected dose with lesser amounts being localized in order of decreasing concentration in kidney, spleen, lung, heart, and brain. With the exception of decreased renal distribution (32%), endotoxin did not significantly alter tissue distribution of the isotope. Electron-dense deposits were observed in hepatic Kupffer cells and as extracellular deposits in hepatic sinusoids. Cysteine, methylprednisolone, or methyl palmitate prevented lead-potentiated endotoxic shock and associated hepatic dysfunction. However, only cysteine caused a marked tissue redistribution of lead as evidenced by an 87% reduction in hepatic uptake of lead and a concomitant 11-fold increase in renal uptake. This increased renal localization was confirmed by the presence of crystalline deposits of electron-dense material in intracytoplasmic vacuoles or vesicles in renal proximal tubules. The hepatic sequestration of lead provides a basis for altered hepatic function and consequent sensitization to endotoxin. Cysteine may prevent this lethal synergism by suppressing hepatic localization of the heavy metal.     

Does red blood cell size correlate with red blood cell age in mouse?

Mouse red blood cells (RBC) can be fractionated according to their size by counterflow centrifugation. The mean corpuscular hemoglobin content and the enzyme activities (ASAT, LDH, PK and acetylcholinesterase) increase when the mean corpuscular volume (MCV) rises. However, the in-vivo survival of size-separated RBC is similar whatever their MCV is; thus, counterflow centrifugation is not a suitable procedure to achieve an age fractionation of mouse RBC. Moreover, RBC subpopulations collected by counterflow centrifugation are different from those obtained when RBC are fractionated according to their density.     

Human red blood cell choline uptake with age and Alzheimer's disease

Since previous studies suggested that blood choline homeostasis is altered in aging and in Alzheimer's disease, choline uptake was examined in human red blood cells (RBC) from young adults, intellectually-intact elderly controls and outpatients with Alzheimer's disease. Eadie-Hofstee analysis of uptake by RBC from young controls indicated two components; thus, group comparisons were done with 1 and 50 microM choline in the media. Temperature-dependent choline uptake at low and high choline concentrations increased in RBC from elderly controls (62-66%) or Alzheimer patients (52-54%) compared to young controls. These changes in transport were not directly related to altered RBC choline content, since RBC choline concentrations did not vary significantly between groups. However, plasma choline content was significantly elevated in elderly controls and Alzheimer patients compared to young control values. The RBC to plasma ratio of choline was reduced in elderly compared to young controls, whereas the ratio in Alzheimer patients was between the two other groups. Thus, abnormalities in RBC choline uptake and plasma choline content were not exacerbated in Alzheimer patients, and these results do not support suggestions that Alzheimer's disease is a form of generalized accelerated aging. The striking changes in RBC choline uptake and plasma choline content in elderly subjects do indicate age-related changes in systemic choline homeostasis and these abnormalities may contribute to the predisposition of the elderly to neurological diseases.     

Mechanism of transfer of immune complexes from red blood cell CR1 to monocytes.

Complement receptor 1 (CR1) on primate red blood cells (RBC) binds most complement-fixing immune complexes in the circulation. It has been postulated that by binding them, RBC keep immune complexes in the intravascular space and deliver them to the tissue macrophages of the mononuclear phagocyte system. We have developed an in vitro model to study the transfer of RBC-bound immune complexes (heat-aggregated IgG and DNA-anti-DNA) to phagocytic cells (human monocytes). Transfer of immune complexes from RBC to monocytes occurred significantly more rapidly than monocyte uptake of the same immune complexes from solution. In the transfer process, complex-bearing RBC were not bound or sequestered by the monocytes. To define the monocyte receptors involved in binding immune complexes from the RBC surface, monocyte receptors were blocked with MoAbs (anti-CR1, anti-FcRII) or EDTA (to block CR3). Monocyte binding of immune complexes primarily used CR1 with a small contribution from FcRII, and with little or no contribution from CR3 and FcRI. Uptake of immune complexes from solution employed the same monocyte receptors as binding of complexes from the RBC surface. Immune complexes in solution bound to RBC and to monocytes with equally high avidity (approximately 1 x 10(11) l/M), but monocytes expressed a 15-20-fold greater number of immune complex binding sites. We propose that immune complexes distribute between RBC and monocytes according to the binding capacity of these cells, such that at equal or high RBC/monocyte ratios as would be seen in the circulation immune complexes bind to RBC, but at low RBC/monocyte ratios (as would be seen in the sinusoidal circulation of the liver and spleen), most immune complexes bind to monocytes. To define the pathway by which immune complexes move from RBC to monocytes, their release from RBC CR1 was examined. Under various conditions, the dissociation rate was extremely slow, and did not increase with the addition of monocyte supernatants. To examine whether factor I-mediated processing of immune complexes enhances binding of immune complexes to monocytes, RBC-bound complexes were released with factor I, and binding of these 'processed' immune complexes to monocytes was examined. Monocyte binding of these processed immune complexes was slower than of control ones; furthermore, performance of transfer experiments at 4 degrees C, which significantly shows enzymatic processes, did not decrease the rate of immune complex transfer from RBC to monocytes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Elevation of creatine in red blood cells in vegetarians and nonvegetarians after creatine supplementation.

The purpose of this study was to examine the effect of a 5-day creatine (CR) supplementation period on red blood cell (RBC) CR uptake in vegetarian and nonvegetarian young women. Blood samples were collected from lacto-ovo vegetarians (VG, n = 6, age 21.8 +/- 1.9 yrs) and nonvegetarians (NV, n = 6, age 21.7 +/- 1.9 yrs) before and after a 5-day CR loading period (0. 3g CR/kg lean body mass/day), and from a control group of nonvegetarians (NV, n = 5, age 22.0 +/- 0.7 yrs) who did not supplement with creatine. RBC and plasma samples were analyzed for the presence of creatine. Significant increases (p < .05) in RBC and plasma CR levels were found for vegetarians and nonvegetarians following supplementation. The initial RBC CR content was significantly lower (p < .05) in the vegetarian group. There was no significant difference between vegetarians and nonvegetarians in final RBC CR content, suggesting that a ceiling had been reached. As the uptake into both muscle and RBC is moderated by creatine transporter proteins, analysis of the uptake of CR into RBC may reflect the uptake of CR into muscle, offering an alternative to biopsies.     

Effect of splenic sequestration of erythrocytes on splenic clearance function and susceptibility to septic peritonitis.

The effect of splenic sequestration of erythrocytes (RBC) on splenic clearance function and susceptibility to septic peritonitis was studied. Homologous RBC were treated with 20 mM phenylhydrazine hydrochloride in vitro and injected into rats at doses of 5, 15, 75, and 150 mg of hemoglobin per 100 g. Splenic 93, 23, and 13% of the injected dose, respectively, whereas less than 2.1% of the same doses of washed RBC were localized in the spleen. The total quantity of phenylhydrazine-treated RBC sequestered by the spleen was similar for the three larger doses (40 to 45 mg of hemoglobin per g of tissue), indicating that this is the capacity of the spleen to take up this type of RBC in 234 h. The phenylhydrazine-treated RBC in doses of 15, 75, and 150 mg of hemoglobin per 100 g depressed the clearance rate and splenic localization of a test dose of phenylhydrazine-treated RBC, whereas splenic clearance function was unchanged after a dose of 5 mg of hemoglobin per 100 g. Splenectomy or injection of doses of phenylhydrazine-treated RBC which depressed splenic clearance function increased the susceptibility to septic peritonitis induced by cecal ligation and puncture. After thermal injury, splenic clearance function was found to be depressed. Also, splenic localization of RBC 24 h after thermal injury was found to be approximately equal to that after injection of the doses of phenylhydrazine-treated RBC which depressed splenic clearance function. It is concluded that splenic sequestration of RBC can depress splenic clearance function and increase susceptibility to peritonitis, and that this may be one mechanism for the increased susceptibility to infection seen after thermal injury.     

Role of zinc in the abatement of hepatocellular damage and mortality incidence in endotoxemic rats.

Intraperitoneal administration of zinc (ZnIP) as zinc chloride prior to or simultaneously with a lethal quantity of intraperitoneally administered Salmonella typhimurium endotoxin significantly protected rats against toxin-induced mortality and hepatocellular damage. Pretreatment with amounts of zinc chloride ranging from 0.4 to 2.0 mg/100 g of body weight resulted in 80 to 100% survival compared with 10% survival in untreated control rats at 24 h after endotoxin treatment. Zinc chloride treatment in excess of 2.0 mg/100 g of body weight appeared to be toxic and provided diminished protection. In contrast with the protection obtained with ZnIP, intravenously administered zinc did not provide protection. The effectiveness of ZnIP to enhance survival if it was given after endotoxin was greatly diminished as a function of time after endotoxin. The extent of hepatocellular damage was assessed at various times after endotoxin administration in ZnIP-treated and untreated rats by measurement of plasma ornithine carbamoyltransferase activity and histological examination of liver sections. Endotoxin absorption from the peritoneal cavity and hepatic uptake were studied by using 51Cr-labeled endotoxin. ZnIP pretreatment significantly reduced 51Cr-labeled endotoxin content of blood and liver when compared to untreated controls, and effectively prevented endotoxin-induced elevations in plasma ornithine carbamoyltransferase activity and hepatic tissue necrosis. These data indicate that protection afforded by ZnIP treatment results as a consequence of the ability of zinc to diminish absorption of the toxin from the peritoneal cavity and subsequent hepatic uptake.     

Investigation of effect of cortisone and endotoxin on mesangial function

The effect of endotoxin and cortisone treatment on the mesangial handling of ferritin in rats was examined. Experimental conditions were chosen where the RES was saturated, this enabled us to test for a direct effect on the mesangium. We were unable to detect any influence of either endotoxin or cortisone on the degree of uptake, or the rate of elimination of ferritin from the mesangium. Endotoxin did appear to affect the intra-glomerular distribution of ferritin, which was seen mainly as larger nodules. The iron core of ferritin was found to persist for longer than the antigenic (protein) portion, indicating that some degree of breakdown of ferritin molecules occured within the mesangium.     

静脉输入休克大鼠肠淋巴液对正常大鼠红细胞的损伤作用

目的: 观察静脉输入休克大鼠肠淋巴液对正常大鼠红细胞参数、代谢以及血液黏度的影响,探讨休克肠淋巴液对红细胞的损伤作用。方法: 将引流休克1~3 h的大鼠肠淋巴液离心去细胞后,以等量生理盐水稀释,经股静脉输入正常大鼠(2 mL/kg),时间为30 min;另一组大鼠输入等量生理盐水作为对照组。输液结束后2.5 h,经腹主动脉取血,检测红细胞常规参数、三磷酸腺苷(ATP)、乳酸(LA)、2,3-二磷酸甘油酸(2,3-DPG)含量、内外液离子浓度以及血液黏度等指标。结果: 静脉输入休克肠淋巴液降低了正常大鼠红细胞数目、血红蛋白浓度、血细胞比容和ATP含量,使红细胞平均体积、2,3-DPG和LA含量及全血还原黏度显著增高,但对红细胞内外液离子浓度、全血黏度和血浆黏度无明显影响。结论: 静脉输入休克肠淋巴液引起正常大鼠红细胞能量代谢障碍,从而导致红细胞体积与全血还原黏度增加,即休克肠淋巴液可损伤红细胞。     

Changes in the susceptibility of red blood cells to oxidative and osmotic stress following submaximal exercise

Red blood cell (RBC) susceptibility to oxidative and osmotic stress in vitro was investigated in cells from trained and untrained men before and after submaximal exercise. Whilst no significant change in peroxidative haemolysis occurred immediately after 1 h of cycling at 60% of maximal aerobic capacity ( _max), a 20% increase was found 6 h later in both groups (P<0.05). The RBC osmotic fragility decreased by 15% immediately after exercise (P<0.001) and this was maintained for 6 h (Ps<0.001). There was an associated decrease in mean cell volume (P<0.05). Training decreased RBC susceptibility to peroxidative haemolysis (P<0.025) but it did not influence any other parameter. These exercise-induced changes were smaller in magnitude but qualitatively similar to those found in haemopathological states involving haem-iron incorporation into membrane lipids and the short-circuiting of antioxidant protection. To explore this similarity, a more strenuous and mechanically stressful exercise test was used. Running at 75% _max for 45 min reduced the induction time of O₂ uptake (peroxidation), consistent with reduced antioxidation capacity, and increased the maximal rate of O2 uptake in RBC challenged with cumene hydroperoxide (P<0.001). The proportion of high-density RBC increased by 10% immediately after running (P<0.001) but no change in membrane-incorporated haem-iron occurred. In contrast, treatment of RBC with oxidants (20–50 mol·l^–1 in vitro increased cell density and membrane incorporation of haem-iron substantially. These results showed that single episodes of submaximal exercise caused significant changes in RBC susceptibility to oxidative and osmotic stress. Such responses may account for the increase in RBC turnover found in athletes undertaking strenuous endurance training.     

Direct detection of red blood cell fragments: a new flow cytometric method to evaluate hemolysis in blood pumps

Pump induced hemolysis is presently evaluated by measuring plasma free hemoglobin (fHb). However, this method has disadvantages because quantification of fHb depends on hematocrit (HCT) and hemoglobin (Hb) levels. The aim of this work was to devise a hemoglobin independent method, capable of quantifying cell trauma directly by measuring the number of red blood cell (RBC) fragments. Whole blood flow cytometry was used to quantify circulating RBC fragments derived from a roller pump (Sarns, Inc. Model 2 M 6,002) and a centrifugal pump (Gyro C1E3, Kyocera Corp.). The pumps were tested in a mock circuit for 2 hr (5 L/min flow against 100 mm Hg pressure head). Red blood cell fragments were quantified by a phycoerythrin (PE) labeled glycophorin A antibody specific for erythrocytes. Red blood cell fragments were smaller than the intact RBC population and overlapped in size with the platelet population (based on forward- and side-light scattering measurements). For the roller pump, the values for RBC fragments increased from 1,090 +/- 260/microl at 0 min to 14,880 +/- 5,900/microl after 120 min. In contrast, using the centrifugal pump, there was little increase in RBC fragments (from 730 +/- 270/microl at 0 min to 1,400 +/- 840/microl after 120 min). Flow cytometry can be used for the rapid, sensitive, hemoglobin independent evaluation of pump induced RBC trauma.