RBC Fractions Research Articles

An in vitro evaluation of intravenous lipid emulsion on three common canine toxicants.

To determine whether intravenous lipid emulsion (ILE) therapy significantly reduces the concentration of baclofen, ibuprofen, and/or bromethalin in canine whole blood over time. Seven 500 mL bags of canine DEA 1.1 negative blood were divided into aliquots of 125 mL and randomly assigned to one of three treatment groups (baclofen, ibuprofen, bromethalin) or four control groups (a positive control for each treatment group and a negative control group). Injectable ibuprofen (200 mg/kg), baclofen (8 mg/kg), or bromethalin (3 mg/kg) was apportioned into 125 mL aliquots of canine whole blood and incubated for 30 min at 38.5°C. ILE (12.4 mL, Intralipid® ) was added to each sample and the solution vortexed [215 rpm for 15 min at 37°C (98.6°F)]. Samples were obtained at designated time points (0, 15, 30, 60, 180, 360 min), centrifuged, and separated into serum and RBC fractions. Serum samples were ultracentrifuged (22,000 g for 10 min at 37°C) to separate lipid rich and poor fractions. Samples were stored at -80°C prior to analysis. A significant decrease in total drug concentration was established for bromethalin and its metabolite desmethylbromethalin compared to positive controls. ILE significantly reduced desmethylbromethalin at the 30-and 360-min time points. The remainder of the desmethylbromethalin time points did not reach significance. Bromethalin concentration was significantly reduced at all time points compared to positive controls. Neither baclofen nor ibuprofen had significant changes in concentration. ILE therapy was effective at reducing the total drug concentration of bromethalin and its metabolite desmethylbromethalin supporting the lipid sink theory. As a single compartment in vitro study, this study does not evaluate other proposed mechanisms of action of ILE therapy. ILE therapy may have other means of significantly decreasing lipophilic drug concentration in cases of toxicosis.

Ulk1 Kinase Mediates Clearance of Free α-Globin in β-Thalassemia

Normal erythroid maturation is highly regulated to maximize the production of HbA heterotetramer (α2β2) and minimize the accumulation of potentially toxic free α- or β-globin subunits. In β-thalassemia, β-globin gene (HBB) mutations cause buildup of free α-globin, which forms intracellular precipitates that impair erythroid cell maturation and viability. Endogenous mechanisms exist to detoxify free α-globin, as evidenced by clinical observations that most individuals with HBB haploinsufficiency (β-thalassemia trait) are asymptomatic. Previously, we showed that free α-globin is eliminated in erythroid precursors by the ubiquitin-proteasome (UPS) system and by a lysosomal-dependent process presumed to be autophagy (Khandros et al., Blood 2012;119:5265). Our current study investigates the latter.Atg5 is required for basal autophagy and autophagosome formation via conjugation with Atg12 in most cell types. We investigated whether Atg5 played a selective role in eliminating free α-globin during unbalanced globin chain synthesis. β-thalassemic mice (HbbTh3/+) carrying a conditional Atg5 allele (Atg5fl) in which exon 3 is flanked by loxP sites were interbred with mice expressing erythroid-specific Cre recombinase (EpoR-Cre). Loss of Atg5 in β-thalassemic erythroid precursors inhibited autophagy, as evidenced by the accumulation of the markers p62/SQSTM1 and LC3. However, there was minimal effect on the β-thalassemic phenotype as measured by RBC count, Hb, hematocrit, reticulocyte count, and spleen weight. Moreover, loss of Atg5 did not alter the level of insoluble α-globin in β-thalassemic RBC fractions, as assessed by triton-acetic acid-urea (TAU) gel electrophoresis to resolve the α- and β-globin chains. Thus, Atg5, a key component of canonical autophagy, is not required for the elimination of free α-globin in β-thalassemia.The Ulk1 (unc-51−like kinase 1) protein kinase participates in canonical and alternative autophagy pathways and mediates mitophagy during erythropoiesis. To determine the role of Ulk1 in a-globin degradation, we introduced a null allele into HbbTh3/+ mice. Ulk1−/− HbbTh3/+ mice were born at normal Mendelian ratio, but exhibited a high rate of perinatal death. Only 20% of double-mutant mice survived for 30 days (n=5), compared to 70% of Ulk1+/+ HbbTh3/+ mice (n=13) (P <0.05). To examine the consequences of Ulk1 loss in β-thalassemic erythroblasts, we transplanted whole bone marrow cells from double mutants and controls (CD45.2) into lethally irradiated wild-type hosts (CD45.1; >87% engraftment after 30 days). Loss of Ulk1 exacerbated the β-thalassemic phenotype, as evidence by reduced Hb (10.85 ± 0.18 vs 10.21 ± 0.18 g/dL, n=14 vs 8; P <0.01), reduced RBC number (7.37 ± 0.13 vs 6.35 ± 0.09 × 106/mL; P <0.0001), increased reticulocyte count (17.99 ± 2.76 vs 39.41 ± 2.14%; P <0.0001), and increased spleen weight (0.24 ± 0.01 vs 0.34 ± 0.03 g; P <0.01). Moreover, insoluble α-globin was increased by approximately 2-fold in Ulk1−/− HbbTh3/+ reticulocytes, ascompared to Ulk1+/+ HbbTh3/+ cells (Figure). To assess the turnover of newly synthesized free α-globin, we pulse-labeled reticulocytes with 35S amino acids and chased with or without a proteasome inhibitor (MG132) or a lysosome inhibitor (chloroquine or bafilomycin A1). Analysis of control (Ulk1+/+ HbbTh3/+) reticulocytesshowed that newly synthesized insoluble free α-globin was cleared by separate proteasomal and lysosomal pathways. In marked contrast, the lysosomal-dependent α-globin degradation pathway was completely eliminated in Ulk1−/− HbbTh3/+ reticulocytes. Thus, we conclude that Ulk1 mediates the degradation of free α-globin in β-thalassemia.Our findings illustrate a new mechanism through which protein quality-control pathways can modulate the severity of β-thalassemia by eliminating unstable free α-globin, and they open up possibilities for new therapeutic approaches, as Ulk1 activity can be modulated pharmacologically. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Endocannabinoid hydrolases are differentially distributed in human blood fractions and differentially influenced by thrombin

Plasma levels of the endocannabinoids anandamide and 2‐arachidonoylglycerol have been reported in a number of studies using both humans and laboratory animals, with typically much higher levels of 2‐arachidonoylglycerol than anandamide. In this investigation, we sought to characterize activities of the two primary endocannabinoid hydrolases, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) responsible for anandamide and 2‐arachidonoylglycerol hydrolysis, respectively, in different fractions from human blood.Following approval from the School of Life Sciences Ethics Committee, University of Nottingham, overnight fasting blood samples were taken in the morning from otherwise healthy volunteers. Blood fractions were isolated, including platelet‐rich plasma, packed erythrocytes (RBC), washed platelets, plasma and serum. Thrombin was employed to trigger clot formation, particularly with RBC and platelet‐rich plasma. Following the generation of a clot, the supernatant layer from a low speed centrifugation, equivalent to serum, was harvested and assayed for endocannabinoid hydrolase activity. The activities of FAAH and MAGL were quantified using both radiometric and ABPP assays.Of the blood fractions examined, MAGL activity was highest in platelets (92 % of the MAGL activity in whole blood), while FAAH activity was highest in RBC. Intriguingly, expressing the FAAH activity of various fractions relative to whole blood summated to 854 %, with RBC constituting 824 % of the whole blood activity. There were no differences between male and female volunteers in the levels and pattern of enzyme activities in the different blood fractions. Assessing enzyme activities from individual donors in these fractions on three visits separated by two week intervals showed no significant differences. The level of FAAH and MAGL activities was relatively stable in washed platelet and RBC samples after being stored in liquid nitrogen, while more than 50 % of their activities were lost after 3 days of storage at −80 °C.Interestingly, FAAH activity was low but detectable, while MAGL was essentially undetectable in serum. Thrombin addition to a washed platelet or RBC fraction caused clot formation within 5 min. FAAH activity in the supernatant fraction following thrombin exposure was much higher from RBC relative to platelets, in accordance with the activities in those fractions. In contrast, the activity of MAGL was significantly reduced in RBC and platelet fractions induced by thrombin compared to nonactivated erythrocytes and platelets, with negligible activities present in the releasate.In summary, we have identified a differential distribution of endocannabinoid hydrolases across different fractions of human blood, and identified the potential for FAAH, but not MAGL, release from RBC and platelets during thrombus formation.Support or Funding InformationFunded by the Saudi Cultural Bureau

In vivo compartmental kinetics of Plasmodium falciparum histidine-rich protein II in the blood of humans and in BALB/c mice infected with a transgenic Plasmodium berghei parasite expressing histidine-rich protein II

BackgroundThe Plasmodium falciparum histidine-rich protein II (PfHRP2) is a common biomarker used in malaria rapid diagnostic tests (RDTs), but can persist in the blood for up to 40 days following curative treatment. The persistence of PfHRP2 presents a false positive limitation to diagnostic interpretation. However, the in vivo dynamics and compartmentalization underlying PfHRP2 persistence have not been fully characterized in the plasma and erythrocyte (RBC) fraction of the whole blood.MethodsThe kinetics and persistence of PfHRP2 in the plasma and RBC fractions of the whole blood were investigated post-treatment in human clinical samples and samples isolated from BALB/c mice infected with a novel transgenic Plasmodium berghei parasite engineered to express PfHRP2 (PbPfHRP2).ResultsPfHRP2 levels in human RBCs were consistently 20–40 times greater than plasma levels, even post-parasite clearance. PfHRP2 positive, DNA negative, once-infected RBCs were identified in patients that comprised 0.1–1% of total RBCs for 6 and 12 days post-treatment, even post-atovaquone–proguanil regimens. Transgenic PbPfHRP2 parasites in BALB/c mice produced and exported tgPfHRP2 to the RBC cytosol similar to P. falciparum. As in humans, tgPfHRP2 levels were found to be approximately 20-fold higher within the RBC fraction than the plasma post-treatment. RBC localized tgPfHRP2 persisted longer than tgPfHRP2 in the plasma after curative treatment. tgPfHRP2 positive, but DNA negative once-infected RBCs were also detected in mouse peripheral blood for 7–9 days after curative treatment.ConclusionsThe data suggest that persistence of PfHRP2 is due to slower clearance of protein from the RBC fraction of the whole blood. This appears to be a result of the presence PfHRP2 in previously infected, pitted cells, as opposed to PfHRP2 binding naïve RBCs in circulation post-treatment. The results thus confirm that the extended duration of RDT positivity after parasite clearance is likely due to pitted, once-infected RBCs that remain positive for PfHRP2.

Desaturation Is Related to the Presence of Dense Red Blood Cells in Sickle Cell Patients and Is Reversed By Hydroxyurea

Production of abnormal hemoglobin (HbS) in sickle-cell disease (SCD) results in its polymerization in deoxygenated conditions and in sickled-RBC formation. Dense RBCs (DRBCs), defined as density >1.11 and characterized by increased rigidity, viscosity and HbS concentration (main polymerization factor), are absent in normal AA subjects, but present at percentages that vary from 1 SCD patient to another but remain stable throughout adulthood for each patient. Polymerized, but not nonpolymerized, HbS has reduced affinity for oxygen, demonstrated by the rightward shift of the oxygen-dissociation curve, leading to disturbances in oxygen transport. We recently described a correlation between %DRBCs and some clinical SCD manifestations. Notably, some SCD patients have unexplained, very low oxygen saturation (SpO2), without heart or lung dysfunctions. %DRBC variability within SCD patients could be the main pathophysiological explanation of those manifestations. This study was undertaken to determine whether a link exists between the %DRBCs and Hb affinity for oxygen, and to look for a potential clinical implication for SCD patients.92 patients (44.6 ± 7.7 years; 51 women and 41 men) were included in the study. Blood samples were obtained at steady state to measure hemorheological and hematological parameters. Using a Percoll-gradient fractionation method, total RBCs were separated into non-DRBCs (NDRBC) (d<1.11) and DRBCs (d>1.11) fractions. The %DRBCs was determined using the phthalate-gradient method. P50 in venous blood gases was measured with a radiometry analyzer. Oxygen-affinity curves of Hb dissociation and association in RBC fractions were obtained with dual wavelength spectrophotometry. All patients had a 6-minute walking test (6MWT) and 10 of them (38.1 ± 6.1 years; 6 men and 4 women) had done so before and after >6 months (>6M) on hydroxyurea (HU). Times <90% and <88% transcutaneous SpO2 of Hb and a SpO2 decrease ≥4% during the test (delta SpO2 >4%) were evaluated to investigate the physiological impact on patients during exertion. Patients, divided into quartiles according to their values (Q1: 0–25th centile, Q2–Q3: 25th–75th centiles, and Q4: >75th centile), were analyzed for the times <88% SpO2, <90% SpO2 and with delta SpO2 >4%, for the distance walked.DRBCs had increased MCHC and decreased %HbF and 2,3 DPG, leading to more polymerization and modulation of Hb affinity for oxygen, compared to NDRBCs. Moreover, dissociation and association curves of SS RBC fractions differed (compared to AA RBCs), with rightward shifts of NDRBCs and, more importantly, DRBC-association curves, thereby confirming the role of HbS polymerization in the loss of affinity (Fig 1). Bivariate analysis showed that the P50 was positively correlated with the %DRBCs (P<0.0001, r²=0.34), reflecting a link between the total Hb–oxygen affinity and %DRBCs in SCD patients. Conversely, P50 and %HbF were negatively correlated (P<0.0001, r²=0.25). The clinical impact of %DRBCs was studied with the 6MWT. Q4 patients for the times <90% SpO2 and <88% SpO2 had higher %DRBCs than Q1 patients (P=0.03 and P=0.04, respectively). No between-group differences were observed for the times <90% SpO2 and <88% SpO2 for Hb or %HbF. Finally, in agreement with our previous demonstration that HU strongly decreased the %DRBCs >M6 of therapy and that the %DRBCs impacted the time at low SpO2, the 10 SCD patients’ 6MWT results before and >M6 of HU therapy showed significantly decreased times <90% SpO2 (P=0.002) and <88% SpO2 (P=0.01) (Fig 2), and with delta SPO2 <4% (P=0.02). In conclusion, according to our results, the %DRBCs directly affects SCD patients’ SpO2 during exercise; HU improves oxygen affinity in correlation with the %DRBC decline. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Determination of miosis threshold from whole-body vapor exposure to sarin in African green monkeys

We determined the threshold concentration of sarin vapor exposure producing miosis in African green monkeys ( Chlorocebus aethiops). Monkeys ( n = 8) were exposed to a single concentration of sarin (0.069–0.701 mg/m 3) for 10 min. Changes in pupil size were measured from photographs taken before and after the exposure. Sarin EC 50 values for miosis were determined to be 0.166 mg/m 3 when miosis was defined as a 50% reduction in pupil area and 0.469 mg/m 3 when miosis was defined as a 50% reduction in pupil diameter. Monkeys were also evaluated for behavioral changes from sarin exposure using a serial probe recognition test and performance remained essentially unchanged for all monkeys. None of the concentrations of sarin produced specific clinical signs of toxicity other than miosis. Sarin was regenerated from blood sampled following exposure in a concentration-dependent fashion. Consistent with a predominant inhibition of acetylcholinesterase (AChE), more sarin was consistently found in RBC fractions than in plasma fractions. Further, elimination of regenerated sarin from RBC fractions was slower than from plasma fractions. Blood samples following exposure also showed concentration-dependent inhibition of AChE activity and, to a lesser extent, butyrylcholinesterase activity. At the largest exposure concentration, AChE inhibition was substantial, reducing activity to approximately 40% of baseline. The results characterize sarin exposure concentrations that produce miosis in a large primate species in the absence of other overt signs of toxicity. Further, these results extend previous studies indicating that miosis is a valid early indicator for the detection of sarin vapor exposure.

Effects of Whole-Body VX Vapor Exposure on Lethality in Rats

Male and female rats were whole-body exposed to VX vapor in a 1000-L single-pass exposure chamber. Estimated exposure dosages producing lethal (LCT50) effects in 50% of exposed male and female rats were established for 10, 60, and 240 min exposure durations. A potency comparison with GB and GF shows that VX becomes increasingly more potent than these G agents with increasing exposure duration. VX is approximately 4–30 times more potent than GB and 5–15 times more potent than GF. Gender differences in the estimated median dosages were not significant at the 10, 60, and 240 min exposure durations. An empirical toxic load model was developed and the toxic load exponent for lethality (n) in the equation Cn× T = k was determined to be n = 0.92. The VX–G regeneration assay was successfully used as a biomarker for the presence of VX in the blood plasma and RBC fractions of the blood 24 h postexposure.

The Oxidative Status of Valinomycin-Resistant Normal and Thalassemic Red Cells (RBCs).

Normal high-K+, low-Na+ RBCs, suspended in low-K+ media and permeabilized to K+ with valinomycin, become dehydrated from net loss of KCl and water. A very small fraction of light, normal RBC and larger fractions of light, sickle cell anemia (SCA) and beta-thalassemia RBC were found to be “valinomycin-resistant” (val-res) due to their Na+/K+ gradient dissipation (PNAS 2000; 97: 8050; BLOOD 2000; 96:24b). In thalassemia and SCA, although the primary lesions involve the globin genes, the major damage to the RBC membranes is mediated by oxidative stress. We previously showed (Cytometry 2004; 60:73) that thalassemic RBC have higher reactive oxygen species (ROS) and lower reduced glutathione (GSH) levels than normal RBC before or after in vitro oxidant stress (treatment with hydrogen peroxide). Here, we examined the oxidative status of val-res RBC from normal and beta-thalassemia major blood. RBC suspended in a plasma-like buffer containing 15 mM KCl and 10 mM valinomycin for 45 min were then layered on arabinogalactone (Larex) with density δ=1.091, and spun at 15,000 g for 30 min. Val-res cells were identified as the low density (δ<1.091 g/ml) RBCs recovered from the interphase layer. The percent val-res RBC in beta-thalassemic samples (n = 10), was 84-fold higher (4.2 ± 0.4% (mean ± SD), range 2.5 to 6.0%) than in normal samples (0.05 ± 0.06%, range 0.02 to 0.1%) (n =10). To determine the oxidative status of the RBC, the cells were washed with PBS and stained for intracellular contents of ROS and GSH, using 2′-7′-dichlorofluoresein and Mercury Orange, respectively. RBC were analyzed by flow cytometry, using gating based on size and granularity. The Mean Fluorescence Channel (MFC) for each fluorochrome was computed. The results showed that valinomycin treatment, per se, did not affect ROS and GSH contents: MFC of the stained un-fractionated RBC was similar before and after treatment with valinomycin, indicating that large changes in MCHC had little or no effect on these measurements. In addition, the unfractionated RBC had ROS and GSH values comparable to those of the high density (val-sensitive) RBC which were recovered from the pellet of valinomycin-treated RBC following Larex fractionation. Measurements on six normal and six beta-thalassemic blood samples indicated that in each case val-res RBC had higher ROS (3.5-10 fold) and lower GSH (2.5-8 fold) levels than the unfractionated RBC or the val-sensitive RBC of the same sample. Compared with val-res cells from normal blood, thalassemic val-res RBC had higher capacity to produce ROS (1.7-fold) and had a lower GSH level (1.5-fold) compared with normal val-res RBC. These results confirm that, as with SCA, beta-thalassemia blood contains a higher percent of val-res RBC than normal blood. They show, further, that (i) both normal and thalassemic val-res RBC have higher oxidative status than other cells (val-sensitive) in the same sample; and that (ii) thalassemic val-res RBC have higher oxidative status than val-res RBC in normal blood. The present results are consistent with the possibility that oxidative stress may contribute to the generation of val-res RBCs, but do not establish a cause-effect relationship. Further studies will be needed to elucidate the origin and significance of these cells.

Blood distribution of rapamycin.

Rapamycin (RAPA) is a potent new immunosuppressive drug. Although blood concentration monitoring of RAPA is being performed in preclinical and clinical trials, little is known regarding the blood distribution of the drug. Such information would have an impact on the medium used for analysis of the drug. The distribution of RAPA was investigated by spiking human whole blood having an initial temperature of either 4 degrees C or 22 degrees C with a constant amount of 3H-RAPA and increasing amounts of RAPA to a final concentration of 5-100 micrograms/L. The drug concentration spans the range seen when immunosuppressive doses of the drug are administered. This was followed by incubation of the blood at 37 degrees C for 0 to 60 min before separation of cells. The dpm in the resulting plasma and RBC fractions was determined by scintillation counting. The plasma to formed blood elements and plasma to whole blood ratios were 0.05 +/- 0.051 and 0.09 +/- 0.016, respectively (mean +/- SD, n = 50). The distribution did not exhibit any temperature or concentration dependence. The proportion of the drug among cellular components was as follows (mean % distribution +/- SD); RBC 94.5 +/- 4.9%; plasma 3.1 +/- 2.5%; lymphocytes 1.01 +/- 1.02%; and granulocytes 1.0 +/- 0.88%. The free or unbound fraction of RAPA over the plasma concentration range of 5-100 micrograms/L as determined by ultracentrifugation was 2.5 +/- 0.2%. The drug was found to be associated primarily with nonlipoprotein fractions in plasma. The results suggest from an analytical perspective that whole blood as compared with plasma would be the most suitable medium for analysis due to the higher concentrations found in the former.

Magnesium, zinc and copper in plasma and blood cellular components in children with IDDM

The levels of magnesium (Mg), zinc (Zn) and copper (Cu) in the plasma, erythrocytes (RBC) and polymorphonuclear (PMN) and lymphocyte (L), leukocytes (WBC) of 45 diabetic children were compared to those of 12 normal children and were related to the diabetic control via HbA 1 and fasting blood sugar (FBS) assessments. Mineral levels were determined via Zeeman-effect atomic absorption spectrophotometry following separation of plasma, RBC, and WBC fractions (PMN vs. M). ANOVA (four-way, blood components, by two-way, diabetic vs. normal children) was significant for Mg only ( F = 4.60, P < 0.004). Plasma Mg and MWBC Mg were significantly lower in children with diabetes (780 ± 16 vs. 860 ± 29 μmol/l and 519 ± 33 vs. 866 ± 86 μg/10 10 cells, respectively). RBC Zn was significantly lower in diabetic youngsters by t-test (0.48 ± 0.012 vs. 0.57 ± 0.046 per μmol/g Hgb, t = 2.79, P < 0.004), but the ANOVA for Zn was not significant. Cu level differences were not significant. HbA 1 was predicted only by PMN-WBC Mg ( F = 8.78, P < 0.04) and FBS by none. In conclusion, the mineral status of these diabetic children was altered in regard to Mg, but was mainly independent of diabetic control.

Studies on the pathogenesis of the erythrocyte destruction associated with the anemia of inflammatory disease.

Density-separated feline RBC were analyzed for surface immunoglobulin and in vitro susceptibility to macrophage phagocytosis before and after introduction of a sterile abscess. Prior to abscess induction, least dense cells did not have detectable surface immunoglobulin G (IgG) as a direct antiglobulin reaction was negative. Two of four intermediate and three of four most dense cell fractions had detectable surface IgG as indicated by a positive direct antiglobulin reaction. On Days 3 and 5 after induction of sterile abscesses all three RBC fractions were positive and the size of the RBC clumps tended to increase. In vitro macrophage phagocytosis of all three RBC fractions increased significantly after abscess induction. The phagocytosis could be partially blocked by preincubation of macrophages with feline IgG.

Sialic acid analysis and tritium-labelling of sialoglycoproteins of mouse erythrocytes infected with Plasmodium berghei.

Schizont-infected red blood cells (SI-RBC) from Plasmodium berghei-infected mice contain between 2 and 10 times as much sialic acid as uninfected RBC from the same blood (99-550 micrograms/10(10) RBC versus 33-65 micrograms/10(10) RBC). Total RBC samples from infected animals containing up to 63% ring- and trophozoite-infected cells had identical sialic acid contents to purified RBC samples (of less than 3% parasitaemia) from the same blood (52-64 micrograms/10(10) RBC). We conclude that RBC containing immature parasites have the same sialic acid content as uninfected RBC from infected blood and that total cellular sialic acid increases during maturation to the schizont stage. Uninfected RBC from infected blood had 25-50% as much sialic acid as normal mouse RBC (33-65 micrograms/10(10) RBC versus 126 micrograms/10(10) RBC). There were no qualitative changes in RBC sialic acids, all RBC samples having 60-70% N-acetylneuraminic acid, 30-40% N-acetyl-9-O-acetylneuraminic acid and 5-10% N-glycolylneuraminic acid. The quantitative changes we observed during infection must reflect changes in murine sialoglycoconjugates, as we have shown elsewhere that Plasmodia do not synthesize or contain sialic acids. Since the sialic acid composition of mouse serum glycoconjugates is quite different to that of the RBC fractions studied here, the quantitative data suggest that part of the sialic acids of the uninfected RBC has been transferred to SI-RBC. With higher molar ratios of periodate to substrate than generally used, we were able to radio-isotopically label normal murine sialoglycoproteins on SI-RBC and purified uninfected RBC from infected blood by the periodate/NaB3H4 method. Several new proteins were then tritiated with SI-RBC but these proteins may be intracellular and could even lack sialic acid.

Red blood cell tocopherol and liver tocopherol in hyperlipemic rats as compared with plasma tocopherol.

In rats with hyperlipemia induced by Triton WR-1339, changes in tocopherol concentrations in plasma and RBC were compared with those in the liver and its subcellular fractions, microsomes and mitochondria. After daily injection with Triton, plasma total lipids at 3 days and 7 days, respectively, showed elevations 6.5 times and 15 times as high as those in the control rats, and triglycerides showed the most predominant elevation. With the hyperlipemia, the concentrations of tocopherol in RBC and the subcellular fractions decreased, as plasma lipids and plasma tocopherol increased, while no change occurred in tocopherol concentrations in liver homogenates. The changes in the ratio of tocopherol to total lipids in plasma coincided with changes in tocopherol concentrations in the RBC and subcellular fractions.

Differential binding of IgG anti-D and IgG autoantibodies to reticulocytes and red blood cells.

125I IgG anti-D binding to reticulocytes obtained by density fractionation is reduced relative to that bound to all other red cell (RBC) fractions. Maximum D antigen reactivity occurs following reticulocyte maturation with no detectable change in D reactivity of mature RBC throughout their life span. Reticulocytes have in the range of about 60% of the content of mature RBC. Previously reported increased anti-D agglutinability and binding to old RBC it is not due to an intrinsic increase in D antigen with age, but results from an 'apparent' decrease in anti-D binding to young RBC fractions due to reticulocyte enrichment. IgG RBC autoantibodies obtained by elution from the RBC of eight Coombs-positive blood donors, probably associated with alpha-methyldopa (alpha-MD) administration, showed decreased binding to reticulocytes as determined by 125I protein A (PA). Reticulocytes bound about 70% of the IgG bound to mature RBC, indicating that the membrane antigenic determinant defined by these autoantibodies was incompletely expressed in the reticulocyte. This difference in IgG autoantibody binding between reticulocytes and mature RBC is similar to the decreased D antigen content of reticulocytes and consistent with an autoantibody determinant associated with the Rh complex. Direct testing of density fractionated Coombs-positive RBC in four out of five patients with autoimmune haemolytic anaemia (AIHA) showed reduced quantities of IgG on reticulocytes. The distribution of IgG between reticulocytes and mature RBC may be useful in serologically characterizing patients with AIHA and in identifying subpopulations of patients with this disorder.

Recovery of erythrocyte Li +/Na + countertransport and choline transport from lithium therapy

Li +/Na + countertransport, choline transport, and intracellular Li + and choline were measured in the RBC of a manic-depressive subject as a function of time after termination of Li + therapy. Countertransport recovered from its inhibition by Li + in 10 days. This recovery involved a decrease in the Michaelis-Menten parameter K m, without change in V max. RBC choline decreased, and choline influx rose, much more slowly back towards pre-Li + values over the course of two months. Thus, choline transport was still inhibited long after Li + levels in RBC and plasma had become undetectable. Measurements on age-separated RBC fractions showed that recovery of choline transport was mainly in the young-cell fraction. Hence inhibition of choline transport by Li + may be irreversible at the level of the individual RBC.

Binding of immunoglobulin classes to subpopulations of human red blood cells separated by density-gradient centrifugation

Human erythrocytes (RBC) from whole blood were separated according to their specific densities by centrifugation on a polyvinyl-pyrrolidine- coated colloidal silica matrix (Percoll) into four major subpopulations. By indirect immunofluorescence assay, the most dense RBC subpopulation, with specific density greater than 1.110 g/ml (3%-5% of total RBC), was positive for membrane-bound immunoglobulin; the remaining, less dense subpopulations were negative. IgG was present on 85%-95%, IgM on 28%-32%, and IgA on 15%-20% of the RBC in the most dense population. When these immunoglobulins were eluted, radiolabeled, and used in binding studies with autologous RBC fractions subjected to thermal and/or enzymatic treatment, they reacted specifically with the less dense RBC subpopulations. These results suggest that previously cryptic antigens were revealed by the activity of neuraminidase on the plasma membranes of the treated RBC.

Binding of immunoglobulin classes to subpopulations of human red blood cells separated by density-gradient centrifugation

Human erythrocytes (RBC) from whole blood were separated according to their specific densities by centrifugation on a polyvinyl-pyrrolidine-coated colloidal silica matrix (Percoll) into four major subpopulations. By indirect immunofluorescence assay, the most dense RBC subpopulation, with specific density greater than 1.110 g/ml (3%-5% of total RBC), was positive for membrane-bound immunoglobulin; the remaining, less dense subpopulations were negative. IgG was present on 85%-95%, IgM on 28%-32%, and IgA on 15%-20% of the RBC in the most dense population. When these immunoglobulins were eluted, radiolabeled, and used in binding studies with autologous RBC fractions subjected to thermal and/or enzymatic treatment, they reacted specifically with the less dense RBC subpopulations. These results suggest that previously cryptic antigens were revealed by the activity of neuraminidase on the plasma membranes of the treated RBC.

Indoleamine compartmentation in human blood

Improved methods are described for the fluorimetric determination of 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and tryptophan (TRP) in plasma, platelet and RBC blood compartments. The methods for 5-HT and 5-HIAA utilize ion exchange chromatography and allow simultaneous determinations to be made on the same blood sample. The tryptophan assay is a modification of an existing extraction method. All of the 5-HT in whole blood was found to be associated with the platelet fraction (122.6 ng/ ml), whereas all 5-HIAA was distributed between the plasma (78.3 ng/ml) and RBC (124.0 ng/ml) compartments. Tryptophan was found in all three compartments although the majority of this amino acid was in the plasma (4.3 μg/ml) and RBC (1.7 μg/ml) fractions.

Cellular variation in the uptake and metabolism of cortisol by canine erythrocytes.

ABSTRACT Samples of blood taken from 30 dogs were incubated with 1 μg cortisol/ml blood for 15 minutes. The RBC rapidly took up cortisol in varying amounts with the mean uptake being slightly greater than could be accounted for by equal distribution between plasma and RBC. The actual steroidal concentration associated with the RBC varied from 37.7 to 79.1 % in these 30 animals. It is suggested that some of the discrepancies in the literature relative to the quantitative association of cortisol with mammalian RBC may be related to the different kinds of RBC used to study uptake, as well as individual variation in cellular uptake. Also the finding of substantial quantities of cortisol associated with the RBC fractions of dog's blood suggests that no reliable estimates of blood steroidal concentration can be achieved without a knowledge of the quantity associated with the RBC. Cortisol reductase enzyme systems could not be detected in these cells. It is concluded that canine anucleated RBC do not possess the capacity to catabolize cortisol.