Red Blood Cell Fraction Research Articles

In-vivo platelet activation correlates with red cell anionic phospholipid exposure in patients with beta-thalassaemia major.

Several clinical and laboratory findings suggest the presence of a chronic hypercoagulable state in patients with beta-thalassaemia major (TM). We have previously shown that isolated TM red blood cells (RBC) strongly enhance prothrombin activation, suggesting an increased membrane exposure of procoagulant phospholipids (i.e. phosphatidylserine). In this study we quantitated the procoagulant activity of RBC in TM and thalassaemia intermedia (TI) patients. We also determined the fraction of activated platelets expressing p-selectin (CD62p) or CD63 in these subjects. Both assays were performed by dual-colour flow cytometry. A significantly (P < 0.01) higher fraction of FITC-annexin V-labelled RBC was found in TM and TI patients, compared to the controls. A highly significant correlation (P < 0.001) was found in TM patients between the number of RBC-bound annexin V molecules and the fraction of CD62p (p-selectin) or CD63-positive platelets. This association between annexin V binding to TM RBC and the expression of platelet activation markers was also found in individual TM patients over time. Thus, the procoagulant surface of TM RBC may accelerate thrombin generation in vivo which, in turn, triggers platelet activation.

Use of oxygen-15 to measure oxygen-carrying capacity of blood substitutes in vivo.

A method for determining oxygen-carrying capacity of blood substitutes has been developed using the short-lived cyclotron-produced positron-emitting isotope 15O. This method measures the oxygen-carrying capacity of the blood substitutes in vivo in the presence of red blood cells and allows determination of changes in the oxygen-carrying capacity over time after exchange transfusion. This method is applied to the blood substitutes of liposome-encapsulated hemoglobin (LEH) and cell-free hemoglobin (Hb). We have used 15O (half-life of 2 min) to quantitate the lung uptake and tissue delivery of [15O2]LEH. Lung uptake studies were performed in intubated, catheterized rats after a 40% exchange transfusion of bovine LEH (LEBH; 0.68 g Hb/kg body wt), human hemolysate LEH (LEHH; 1.0 g Hb/kg body wt), or free bovine hemoglobin (SFHS; 0.56 g Hb/kg body wt). A bolus inhalation of 15O2 (3-5 mCi) was given at 15 min, 3 h, and 24 h post-transfusion. Arterial blood samples were collected, spun, and separated into LEH, red blood cell, and plasma fractions. 15O activity and hemoglobin content were determined for each fraction. Oxygen-carrying capacity was calculated as a percentage of the original red blood cell fraction removed. For LEBH, the carrying capacity was 15% at 15 min, 13% at 3 h, and 1% at 24 h. For LEHH, the carrying capacity was 30% at 15 min, 26% at 3 h, and 19% at 24 h. The marked decrease in carrying capacity at 24 h for LEBH compared with LEHH was attributable to the increased formation of methemoglobin in the circulating LEBH rather than increased removal from circulation, because total hemoglobin concentrations measured for both LEH samples decreased at a similar rate during the 24 h. The presence of methemoglobin reductase and other naturally occurring antioxidants in the LEHH may be responsible for maintaining the higher levels of oxyhemoglobin. Oxygen-carrying capacity for SFHS also decreased over time but at a much sharper rate compared with both LEH formulations. The carrying capacity for SFHS of 8% measured at 15 min decreased to 0.3% at 3 h and undetectable levels at 24 h. This sharper decrease in carrying capacity for SFHS is attributable to the rapid removal of the hemoglobin from circulation.

Invasion and growth of Plasmodium falciparum is inhibited in fractionated thalassaemic erythrocytes

Epidemiological and clinical studies have indicated that the thalassaemias may confer protection against malaria. The study reported here investigated this protective effect in vitro, using a new approach which controls for the potential effect of red cell size and age on the virulence of the parasite. A Percoll ™ density gradient method was used to separate α- and β-thalassaemic trait, haemoglobin H and normal red blood cells (RBC) into fractions of different density. Correlations between RBC density, age and size in fractions of all genotypes were established using red cell creatine as an index of cell age. The development of Plasmodium falciparum over 3 erythrocytic cycles (144 h) in whole blood as well as fractionated samples was monitored by slide microscopy and flow cytometry. A significantly reduced rate of parasite invasion and growth was demonstrated in RBC from all thalassaemic genotypes tested. Poor reinvasion rates were noted in the second and third cycles. Increased duration of culture and red cell age also had a greater negative impact on parasite growth in thalassaemic RBC. This poor growth rate was also associated with the arrest of parasite growth at the schizont stage (schizont maturation arrest) and the accumulation of abnormal, trophozoite/schizont stage parasites in the older thalassaemic RBC fractions. These findings suggest a defect in the number and viability of merozoites generated by parasites growing in thalassaemic RBC. Age related factors such as oxidant stress may play a key role in mediating this kind of protective mechanism and deserve further investigation.

Constitutional alterations in p16 in patients with uveal melanoma.

Chromosome 9p21 contains a susceptibility gene for cutaneous melanoma. Recent studies suggest that the gene responsible may be CDK41, since it encodes a putative cell cycle inhibitor, p16, and is frequently lost or rearranged in melanoma cell lines. In this study we examined whether germline alterations in CDK41 could be identified in patients with melanoma of the uveal tract. From an archive of bloods collected from patients with uveal melanoma, we identified 13 samples drawn from patients with a history in a family member of uveal (n = 6) or cutaneous (n = 7) melanoma. An additional 24 'control' bloods (without melanoma or any other primary malignancy in a family member), similar to the 'cases' in age and number of first-degree relatives, were also selected for study. For each sample, DNA was extracted from the red blood cell fraction. Using the polymerase chain reaction-single strand conformation polymorphism method, we screened for alterations in p16. Specific changes were characterized by DNA sequencing. Six nucleotide changes were detected in five (13.5%) of the 37 samples examined. An altered gene was found in one (7.7%) of the 13 patients with a family history (of intra-ocular melanoma) and four (16.7%) of the 24 patients with no family history (P = 0.64) of melanoma. In this series the group with a positive family history was predominantly female and most pedigrees involved matrilineal descent. In these data prevalence of germline alteration in p16 was similar in familial and sporadic cases. The results provide evidence against a significant role for p16 in familial clustering of intra-ocular and cutaneous melanomas.

K(86Rb) transport heterogeneity in the low-density fraction of sickle cell anemia red blood cells.

Previous studies have suggested ion transport heterogeneity among sickle cell anemia (SS) reticulocytes that could influence their dehydration susceptibility. We examined Ca2(+)-independent K transport in the lowest density (F1), reticulocyte-rich SS cells, measuring the effects of acidification, ouabain, and bumetanide on their unidirectional K(86Rb) fluxes. Unlike those of normal red blood cells and SS discocytes, the SS-F1 K(86Rb) fluxes were highly nonlinear, with large 5-min flux components (previously unobserved) and a more gradual decline over 60 min. Analysis revealed two distinct K pools: a rapid-turnover pool in a small fraction of cells, whose major ouabain-resistant K(86Rb) transport path showed distinctive properties including inhibition by high concentrations of bumetanide (> or = 1 mM) and stimulation at pH 7.0, and another heterogeneous, relatively slow-turnover pool, in most of the F1 cells, whose main ouabain-resistant K(86Rb) path was insensitive to bumetanide but was stimulated at pH 7.0, which is consistent with heterogeneous expression of the acid-sensitive K-Cl cotransport and with both rapid and slower generation of dehydrated SS cells.

A novel enzyme system for the reduction of 3-oxo bile acids in human red blood cells

7α,12α-Dihydroxy-3-oxo- and 3,7,12-trioxo-5β-cholanoic acids labeled with 18O atoms were incubated with human red blood cells, and the biotransformation products were separated and characterized by gas chromatography-mass spectrometry as the pentafluorobenzyl ester-trimethylsilyl and -dimethylethylsilyl ether derivatives with the negative ion chemical ionization mode. The reduced products, 3β,7α,12α-trihydroxy-5β-cholanoic acid for the former, and 3α-hydroxylated dioxo bile acid together with 3β-hydroxylated 7,12-dioxo-5β-cholanoic acid for the latter, were identified as metabolites. When 3-oxo bile acid was incubated with human blood denatured at 70°C for 2 min, no metabolites were formed. The enzymic reduction activity has been localized in the red blood cell fraction.

Viremia induced by influenza virus

A mouse model of influenza A/PR/8 virus infection was adopted to investigate the blood and various tissues of intranasally infected mice for the presence of viral RNA by using the nested polymerase chain reaction. The nucleoprotein gene was detected in the red blood cell fraction from 1 to 5 days post-inoculation, while it was found in the lung and brain up to 14 days and in the liver, spleen, kidney, heart, and skeletal muscle up to 7 days. The virus-specific messenger RNA was transiently found in these organs. When mice received the uv-inactivated virus, viremia did not occur. The prior transfer of the hyperimmune serum prevented pneumonia but not bronchitis, and viremia was totally abolished. These results suggest: (1) viremia occurs during the acute phase of infection, (2) the virus is present in various organs and there the virus gene is transiently expressed, and (3) the virus enters the blood stream possibly through capillaries of the infected alveolar wall. Viremia may influence the pathogenesis of influenza.

IN VITRO INVESTIGATION OF THE FACTORS CONTRIBUTING TO THE UNIQUE CO2 TRANSPORT PROPERTIES OF BLOOD IN THE SEA LAMPREY (PETROMYZON MARINUS)

In vitro experiments were conducted to determine the factors contributing to the unusual distribution of CO2 in the blood of the sea lamprey. When rainbow trout red blood cells (RBCs) were equilibrated with a 3 % CO2:nitrogen mixture in either normal saline or sodium-free saline, the extracellular total carbon dioxide content (CCO2ext) was highly dependent upon the fraction of RBCs in the suspension. In contrast, when lamprey RBCs were equilibrated in normal saline, the CCO2ext decreased with increasing hematocrit. In the absence of extracellular sodium, however, the CCO2ext in the lamprey RBC suspension also became positively correlated with hematocrit. These results suggest that the membrane of sea lamprey RBCs may be somewhat permeable to bicarbonate, but that transmembrane bicarbonate movements may only be detectable in vitro when Na+/H+ exchange is inactivated. Also in contrast to the results for rainbow trout, the changes in CCO2 that occurred in lamprey RBC suspensions following a step increase in PCO2 were not associated with any change in RBC chloride concentration and were not markedly affected by the chloride/bicarbonate exchange inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). When lamprey RBCs in sodium-free saline were treated with an ionophore for anions, tributyl tin chloride (TBTC), however, the distribution of anions across the RBC membrane came to resemble that of the trout. Furthermore, the relationship between CCO2ext and hematocrit in suspensions of TBTC-treated lamprey RBCs also resembled that of trout in normal saline. Thus, these results demonstrate that both the presence of Na+/H+ exchange and the absence of significant anion exchange contribute to the unique CO2 transport properties of sea lamprey blood.

Identification and characterization of dehydrocholic acid reductase system in the cytosol of human red blood cells.

We conducted in vivo and in vitro studies of the reductive metabolism of the cholagogue, dehydrocholic acid (DHCA). Immediately after the intravenous administration of 1 g of DHCA in normal subjects (n = 6), the concentration of the reductive metabolite, 3 alpha-hydroxy-7,12-dioxo-cholanoic acid (unconjugated form), increased sharply in the systemic circulation, rising to 95.8 microM 10 min after administration. The results of in vitro experiments with DHCA and whole blood showed that 3 alpha-hydroxy-7,12-dioxocholanoic acid were produced from DHCA. In vitro experiments using DHCA and the red blood cell fraction, and DHCA and the red blood cell cytoplasmic fraction gave similar results to those described above with whole blood. However, a reductive metabolite was not formed by the incubation of DHCA and the red blood cell membrane fraction. These findings indicated that, contrary to the conventional theory that intravenously administered DHCA is subjected to reductive metabolism only in the liver, reduction also occurs in the systemic circulation, and the mechanism for this reductive metabolism is present in the cytoplasmic fraction of red blood cells. Further investigation to characterize this reductive metabolic system revealed an optimum temperature of 37 degrees C, an optimum pH of 7.4, a Km value of 2.0 x 10(-3) M, and inactivation by heat treatment (70 degrees C for 2 min).

Fetal hemoglobin and potassium in isolated transferrin receptor- positive dense sickle reticulocytes

A subset of sickle cells have an increased density at the reticulocyte stage of development, indicating that they are either abnormally dense upon release from the bone marrow or become dense quickly in the circulation. These cells are of interest because they most likely have severely disrupted cation regulation and a short lifespan. Based on the distribution of fetal hemoglobin (HbF) in the density fractions of sickle red blood cells (RBCs) and in vitro studies of cellular K+ loss, it seems likely that HbF content is an important in vivo determinant of dense cell formation. In this study, we tested the hypothesis that young, dense cells have low HbF content. Sickle RBCs were first separated into light and dense fractions. Reticulocytes were isolated from unfractionated cells and from each density fraction with an immunomagnetic technique directed against transferrin receptors (TfR) and assayed for the percentage of HbF and K+/Hb ratio. TfR+ reticulocytes isolated from unfractionated cells had a much lower HbF content when compared with all the unfractionated RBCs. This is most likely caused by enrichment of F cells because of a longer circulation life span. Heavy TfR+ reticulocytes had a K+/Hb ratio similar to that measured in the entire dense population and contained very low levels of HbF, averaging 2.5% of the level in all RBCs, 11.7% of the level in all TfR+ reticulocytes, and 4.0% of the level in all dense RBCs. These findings suggest that TfR+ dense cells derive predominantly from non-F cells. Furthermore, the amount of HbF in the circulating dense cells suggests that many of these cells do not derive from the TfR+ dense cells.

Fetal Hemoglobin and Potassium in Isolated Transferrin Receptor-Positive Dense Sickle Reticulocytes

A subset of sickle cells have an increased density at the reticulocyte stage of development, indicating that they are either abnormally dense upon release from the bone marrow or become dense quickly in the circulation. These cells are of interest because they most likely have severely disrupted cation regulation and a short lifespan. Based on the distribution of fetal hemoglobin (HbF) in the density fractions of sickle red blood cells (RBCs) and in vitro studies of cellular K+ loss, it seems likely that HbF content is an important in vivo determinant of dense cell formation. In this study, we tested the hypothesis that young, dense cells have low HbF content. Sickle RBCs were first separated into light and dense fractions. Reticulocytes were isolated from unfractionated cells and from each density fraction with an immunomagnetic technique directed against transferrin receptors (TfR) and assayed for the percentage of HbF and K+/Hb ratio. TfR+ reticulocytes isolated from unfractionated cells had a much lower HbF content when compared with all the unfractionated RBCs. This is most likely caused by enrichment of F cells because of a longer circulation life span. Heavy TfR+ reticulocytes had a K+/Hb ratio similar to that measured in the entire dense population and contained very low levels of HbF, averaging 2.5% of the level in all RBCs, 11.7% of the level in all TfR+ reticulocytes, and 4.0% of the level in all dense RBCs. These findings suggest that TfR+ dense cells derive predominantly from non-F cells. Furthermore, the amount of HbF in the circulating dense cells suggests that many of these cells do not derive from the TfR+ dense cells.

Oxygen Transport Properties in Malaria-Infected Rodents — A Comparison Between Infected and Noninfected Erythrocytes

Abstract This study was performed to investigate oxygen transport properties in whole blood (WB) of malaria-infected rats as well as in infected erythrocytes (IE) and noninfected erythrocytes (NIE) separated by density centrifugation. One week after inoculation with Plasmodium berghei, mean parasitemia was 26.5% and high correlations were found between parasitemia and hemoglobin concentration ([Hb]; r = -.902), mean cellular Hb concentration (MCHC; r = -.712), MetHb (r = .923), and base excess (r = -.922). Compared with control animals (C), the oxygen affinity was lower in WB under standard (pH 7.40) and simulated “in vivo” (pH 7.00) conditions (difference in P50, 5.7 and 5.1 mm Hg, respectively; 2P &lt; .01, 2P &lt; .05). In IE Hb and 2,3-biphosphoglycerate (2,3-BPG) concentrations were decreased (MCHC: IE 14.6 +/- 1.0, NIE 33.1 +/- 1.7 g/100 mL; [2,3-BPG]: IE 2.0 +/- 0.6, NIE 7.6 +/- 1.8 mmol/L), whereas [MetHb] and [ATP] were increased ([MetHb]: IE 19.0 +/- 3.7, NIE 0.7% +/- 0.8%; [ATP]: IE 33.5 +/- 2.4, NIE 6.2 +/- 1.0 mumol/g Hb). At pH 7.40, half-saturation oxygen tension (P50) was reduced in IE (29.6 +/- 2.6, NIE 39.2 +/- 5.4 mm Hg, 2P &lt; .001), which correlates with lower [2,3-BPG], increased MetHb content, and higher intrinsic Hb- O2 affinity. However, at pH 7.00, the oxygen affinity was lower in IE when compared with NIE, which was most likely due to high [ATP] in IE. The resulting Bohr coefficients (BC) calculated for CO2 and lactic acid were extremely high in IE and low in NIE (at 50% O2-saturation BCCO2: IE -1.04 +/- 0.06, NIE -0.26 +/- 0.10, 2P &lt; .001; BCLac: IE -0.82 +/- 0.16, NIE -0.47 +/- 0.07, 2P &lt; .001), which was caused by different [2,3-BPG] and [ATP] as well as probably by structural changes of the Hb molecule. The O2 capacity was 14.1 mL per 100 mL erythrocytes in IE compared with 44.4 mL/100 mL in NIE. On the basis of the calculated arterio-venous O2 difference under “in vivo” conditions, the infected red blood cell fraction transports 30% of the O2 amount delivered to the tissues by the noninfected cells (IE 8.0, NIE 26.9 mL/100 mL red blood cells). We conclude that the O2 transport in malaria infected blood is not only affected by the degree of anemia but also by the percentage of infected erythrocytes.

Oxygen transport properties in malaria-infected rodents--a comparison between infected and noninfected erythrocytes

This study was performed to investigate oxygen transport properties in whole blood (WB) of malaria-infected rats as well as in infected erythrocytes (IE) and noninfected erythrocytes (NIE) separated by density centrifugation. One week after inoculation with Plasmodium berghei, mean parasitemia was 26.5% and high correlations were found between parasitemia and hemoglobin concentration ([Hb]; r = -.902), mean cellular Hb concentration (MCHC; r = -.712), MetHb (r = .923), and base excess (r = -.922). Compared with control animals (C), the oxygen affinity was lower in WB under standard (pH 7.40) and simulated "in vivo" (pH 7.00) conditions (difference in P50, 5.7 and 5.1 mm Hg, respectively; 2P < .01, 2P < .05). In IE Hb and 2,3-biphosphoglycerate (2,3-BPG) concentrations were decreased (MCHC: IE 14.6 +/- 1.0, NIE 33.1 +/- 1.7 g/100 mL; [2,3-BPG]: IE 2.0 +/- 0.6, NIE 7.6 +/- 1.8 mmol/L), whereas [MetHb] and [ATP] were increased ([MetHb]: IE 19.0 +/- 3.7, NIE 0.7% +/- 0.8%; [ATP]: IE 33.5 +/- 2.4, NIE 6.2 +/- 1.0 mumol/g Hb). At pH 7.40, half-saturation oxygen tension (P50) was reduced in IE (29.6 +/- 2.6, NIE 39.2 +/- 5.4 mm Hg, 2P < .001), which correlates with lower [2,3-BPG], increased MetHb content, and higher intrinsic Hb-O2 affinity. However, at pH 7.00, the oxygen affinity was lower in IE when compared with NIE, which was most likely due to high [ATP] in IE. The resulting Bohr coefficients (BC) calculated for CO2 and lactic acid were extremely high in IE and low in NIE (at 50% O2-saturation BCCO2: IE -1.04 +/- 0.06, NIE -0.26 +/- 0.10, 2P < .001; BCLac: IE -0.82 +/- 0.16, NIE -0.47 +/- 0.07, 2P < .001), which was caused by different [2,3-BPG] and [ATP] as well as probably by structural changes of the Hb molecule. The O2 capacity was 14.1 mL per 100 mL erythrocytes in IE compared with 44.4 mL/100 mL in NIE. On the basis of the calculated arterio-venous O2 difference under "in vivo" conditions, the infected red blood cell fraction transports 30% of the O2 amount delivered to the tissues by the noninfected cells (IE 8.0, NIE 26.9 mL/100 mL red blood cells). We conclude that the O2 transport in malaria infected blood is not only affected by the degree of anemia but also by the percentage of infected erythrocytes.

Filterability of density-separated equine erythrocytes, neutrophils and mononuclear cells

The filterability of density-separated fractions of red blood cells (RBC), neutrophils and mononuclear cells was evaluated to determine their relative contribution to resistance to blood flow. Increasing RBC density was associated with smaller cell size, greater haemoglobin concentration, and improved filterability. Filtration of RBC, after adrenaline-induced splenic contraction, failed to identify a population of non-deformable RBC. Leucocytes filtered less readily compared to RBC. Neutrophils and mononuclear cells produced similar initial pressure increments but mononuclear cells had a greater potential to plug pores in the filters. Our results indicate both RBC and leucocytes may contribute to resistance to blood flow. A subpopulation of non-deformable RBC could not be identified in circulating blood or in blood after adrenaline-induced splenic contraction.

Decreased concentration of hemoglobin, accumulation of lipid oxidation products and unchanged skeletal muscle in Atlantic salmon (Salmo salar) fed low dietary vitamin E

Atlantic salmon (Salmo salar) fry, initial weight 0.16 g, were fed a semipurified diet with 0, 15, 30, 60 or 120 mg dl-α-tocopheryl acetate/kg. After 24 weeks, the first two of these groups were extinct, and the fish receiving 30 mg/kg were clearly vitamin E deficient. Vitamin E deficient fish had low hemoglobin levels, characterized by a combination of reduced cellular hemoglobin concentration, red cell volume and red cell number, and an increased number and fraction of immature red blood cells. The hemoglobin concentration decreased over the decreasing range of experimental dl-ga-tocopheryl acetate levels. Therefore, even if 60 mg dl-α-tocopheryl acetate/kg gave good survival, this level was clearly physiologically suboptimal. Ceroid accumulated in the liver of fish fed 30 mg vitamin E/kg, and autofluorescent inclusions were found in the red blood cells of fish fed 30 and 60 mg vitamin E/kg. Degeneration of skeletal muscle was not observed in the present study.

Determinants of red blood cell deformability in relation to cell age

Red blood cell (RBC) deformability was determined with an ektacytometer in fractions separated on the basis of differences in cell volume or density. Deformability was measured with ektacytometry (rpm-scan and osmo-scan). We studied three groups of RBC fractions:1. By counterflow centrifugation we obtained fractions of different cell age which showed a slight decrease in mean corpuscular haemoglobin concentration (MCHC) and an increase in surface-to-volume (S/V) ratio in fractions with older cells. 2. By Percoll fractionation fractions were obtained which showed a pronounced increase in (MCHC) but no change in S/V ratio. 3. By a combination of both fractionation techniques, fractions were obtained which showed an increased MCHC and an increase in S/V ratio. Deformability in group 1,2 and 3 showed respectively no change, a moderate decrease and a pronounced decrease in fractions of older cells. A decline in deformability occurs during the aging process of the red blood cell. This decline in deformability in old red cells is greater than originally thought. This decline is the result of an increase in haemoglobin concentration and a second factor, probably a decrease in membrane elasticity.

Human red blood cell aging at 5,050-m altitude: a role during adaptation to hypoxia.

To test the hypothesis that the human red blood cell aging process participates actively in the adaptation to hypoxia, we studied some physical and biochemical hematologic variables in 10 volunteers at sea level (SL) and after 1 (1WK) or 5 wk (5WK) of exposure to 5,050-m altitude. The 2,3-diphosphoglycerate-to-hemoglobin ratio (2,3-DPG/Hb) was 0.88 +/- 0.03 (mol/mol) at SL and increased to 1.08 +/- 0.03 (P = 0.002) and 1.28 +/- 0.05 (P < 0.0001) at 1WK and 5WK, respectively. The average red blood cell density (D50), which is inversely proportional to the fraction of young red blood cells and is therefore an index of the red blood cell aging process, was 1.1053 +/- 0.0007 g/ml at SL and decreased to 1.1046 +/- 0.0008 g/ml (NS) and 1.1018 +/- 0.0008 g/ml (P < 0.001) at 1WK and 5WK, respectively. D50 was correlated with 2,3-DPG/Hb at SL (P = 0.004), only weakly at 5WK (P = 0.1), but not at all at 1WK. The arterial O2 saturation was correlated with the change of 2,3-DPG/Hb in 1WK (P = 0.02) and that of D50 in 5WK (P = 0.04). It is concluded that short-term (1WK) increase of 2,3-DPG/Hb is not associated with the erythropoietic response but is presumably due to respiratory alkalosis. By contrast, after prolonged hypoxia (5WK), erythropoiesis may provide an efficient way for increasing blood 2,3-DPG through an augmented proportion of young red blood cells.

Red Blood Cell Fatty Acid Levels in Patients with Autosomal Dominant Retinitis Pigmentosa

Fatty acid profiles were determined in red blood cell (RBC), plasma lipids and lipid fractions from 50 patients with autosomal dominant retinitis pigmentosa (ADRP) and 20 normal-sighted controls. In plasma of affected patients, significant increases in ω6 long-chain derivatives compared to controls resulted in elevated fatty acid unsaturation whereas, in RBCs, significant decreases in the levels of ω6 and ω3 long-chain polyunsaturates and a reduced unsaturation index were demonstrated (P < 0·01). The disproportionate levels of long-chain fatty acids in plasma vs. RBCs were consistent with abnormal function of acyl group transfer from plasma lipids to target tissues such as RBCs or, possibly, retina. Metabolic lipid anomalies were evaluated in two sub-groups of patients with ADRP; ten with the highest and ten with the lowest RBC lipid levels of the ω3 fatty acid, docosahexaenoic acid (DHA). Fatty acid product/precursor relationships in the sub-groups revealed reduced chain elongation and ω3 fatty acid desaturation associated with reduced ratios of rod-to-cone electroretinographic amplitudes. Based on these results, we suggest that the heterogenic ADRP population includes a sub-group of patients characterized by abnormal fatty acid metabolism that may effect membrane structure and visual function.

Enhanced botulinal toxin development in beef sausages containing decolourized red blood cell fractions

Toxin production by Clostridium botulinum was studied in a model cured beef sausage containing decolourized dried bovine red blood cells (RBC), including intact RBC, acetone-treated RBC, enzyme-treated RBC, peroxide-treated RCB or plasma. Samples were formulated with beef shoulder, curing agents and spores of proteolytic strains of Clostridium botulinum. Vacuum packaged samples were heated to 72°C, stored at 28°C, and tested weekly. Sausages contained iron levels proportional to the iron in the blood fraction. Residual nitrite levels varied between < 10–40 μg g-1. Toxin was detected earlier in samples containing higher levels of iron except for acetone-treated RBC. Higher pH values were associated with shorter times to toxin detection. We conclude that the RBC decolourization method can significantly modulate Cl. botulinum growth and toxigenesis.

A comparison of tumor and normal tissue microvascular hematocrits and red cell fluxes in a rat window chamber model

This laboratory has previously used a window chamber model to measure red blood cell velocity in mammary tumors and normal granulation tissues of the F-344 rat. Because red cell flux and hematocrit more accurately reflect the oxygen carrying potential of blood, we used this model to measure these parameters. Red blood cells were labeled with fluorescein isothiocyanate, and 0.2 ml. packed cells were injected intravenously into rats bearing an 8 to 10 day old R-3230 mammary carcinoma. β-phycoerythrin (0.15 mg.) was also injected and served as a plasma dye to outline the blood vessels. A sample of peripheral blood was then taken and analyzed by flow cytometry to determine the labeled fraction of red blood cells. Flowing tumor and normal tissue vessels were recorded onto a VCR, and these video images were used to determine vascular length and diameter, RBC flux and velocity, and hematocrit. Median vessel diameter and log e (red blood cell flux) were significantly greater in tumors than in normal tissues ( p = 0.007 and p < 0.025, respectively). After controlling for these variables, the median tumor hematocrit of 19% was not significantly greater than the median normal tissue hematocrit of 15%. This technique provides a nontoxic and reproducible method that is now being used to assist in the in vivo definition of tumor oxygenation.