Composition Of Red Blood Cells Research Articles

Abstract 019: Radiomics analysis to predict etiology in large vessel occlusions.

Introduction Large vessel occlusions (LVO) are responsible of 40% of acute ischemic strokes (AIS). Nonetheless, nearly 30% of cases lack a clear source. Radiomics has emerged as a non‐invasive imaging tool that analyzes voxel‐by‐voxel signal intensity. We aim to correlate the histology of clots retrieved from LVO with radiomic features (RFs) to assess stroke etiology. Methods Patients diagnosed with AIS due to LVO between 2019 to 2024 were analyzed. Ten clots retrieved from mechanical thrombectomy were imaged using micro‐computed tomography (micro‐CT) and histologically analyzed. The three main clot components were red blood cells (RBCs), fibrin and calcium. 3D Slicer was used for 3D segmentations of each component. One hundred and thirty RFs were extracted using Py‐radiomics add‐in. NCCT and computed tomography angiography images were co‐registered with the corresponding slices obtained from histology. A cohort of 426 patient with NCCTs obtained at the time of presentation were then analyzed. Only clots with an identifiable hyperdense sign in NCCT were included. Stroke etiology was adjudicated based on TOAST, in cardioembolic, large artery atherosclerosis (LAA) and cryptogenic. Results The RFs total energy (TE), joint average (JA) and large dependence high gray level emphasis (LDHGLE) were specific for RBCs in micro‐CT (p<0.001, p 0.003, and p<0.002, respectively). Corresponding RFs of fibrin included minimum (MI) and 10 percentile (p .005, and p<0.001, respectively), while calcium RFs included difference variance (DV, p 0.05). TE, JA and LDHGLE were strongly correlated with clots that had at least 70% RBCs (Rho 0.654 and 0.652, respectively). MI was strongly correlated with clots that had at least >80% of fibrin (Rho 0.795). DV was not correlated to calcium composition in clots (Rho 0.400). The RFs of the NCCT segmentations of these clots had strong correlation with corresponding micro‐CT values of TE (Rho 0.687) JA (Rho 0.809) and LDHGLE (Rho 0.657). MI was negatively correlated (Rho ‐0.851). RFs analysis thresholds in the identification of clot components showed that TE (AUC 0.800, sensitivity 0.750, specificity 0.800, cutoff 38244.3089) and LDHGLE (AUC 0.750, sensitivity 0.750, specificity 0.800, cutoff: 52.64) had significant accuracy for determining clots with higher RBC composition in NCCT. MI (AUC: 0.350, sensitivity: 0.500, specificity: 0.400, cutoff: 32.3) did not have accuracy in the identification of fibrin. A total of 145 of 426 patients were included in the final analysis due to image quality purposes. Fifty patients have a stroke of cardioembolic origin, 45 due to LAA and 50 were cryptogenic. Applying TE and LDHGLE thresholds, thirty (60%) cardioembolic, 12 (27%) of LAA and 21 (42%) of cryptogenic strokes were mainly composed of RBCs (> 70% of the clot). Conclusion RFs are sensitive and specific to determine clots rich in RBC composition in NCCT. Cardioembolic clots have higher RBCs compared with LAA and cryptogenic. Radiomic analysis is a promising non‐invasive tool to determine stroke etiology.

Identifying ex vivo acute ischemic stroke thrombus composition using electrochemical impedance spectroscopy

Intra-procedural characterization of stroke thromboemboli might guide mechanical thrombectomy (MT) device choice to improve recanalization rates. Electrochemical impedance spectroscopy (EIS) has been used to characterize various biological tissues in real time but has not been used in thrombus. To perform a feasibility study of EIS analysis of thrombi retrieved by MT to evaluate: (1) the ability of EIS and machine learning to predict red blood cell (RBC) percentage content of thrombi and (2) to classify the thrombi as "RBC-rich" or "RBC-poor" based on a range of cutoff values of RBC. ClotbasePilot was a multicentric, international, prospective feasibility study. Retrieved thrombi underwent histological analysis to identify proportions of RBC and other components. EIS results were analyzed with machine learning. Linear regression was used to evaluate the correlation between the histology and EIS. Sensitivity and specificity of the model to classify the thrombus as RBC-rich or RBC-poor were also evaluated. Among 514 MT,179 thrombi were included for EIS and histological analysis. The mean composition in RBC of the thrombi was 36% ± 24. Good correlation between the impedance-based prediction and histology was achieved (slope of 0.9, R2 = 0.53, Pearson coefficient = 0.72). Depending on the chosen cutoff, ranging from 20 to 60% of RBC, the calculated sensitivity for classification of thrombi ranged from 77 to 85% and the specificity from 72 to 88%. Combination of EIS and machine learning can reliably predict the RBC composition of retrieved ex vivo AIS thrombi and then classify them into groups according to their RBC composition with good sensitivity and specificity.

Red Blood Cells Protein Profile Is Modified in Breast Cancer Patients

Metastasis is the primary cause of death for most breast cancer (BC) patients who succumb to the disease. During the hematogenous dissemination, circulating tumor cells interact with different blood components. Thus, there are microenvironmental and systemic processes contributing to cancer regulation. We have recently published that red blood cells (RBCs) that accompany circulating tumor cells have prognostic value in metastatic BC patients. RBC alterations are related to several diseases. Although the principal known role is gas transport, it has been recently assigned additional functions as regulatory cells on circulation. Hence, to explore their potential contribution to tumor progression, we characterized the proteomic composition of RBCs from 53 BC patients from stages I to III and IV, compared with 33 cancer-free controls. In this work, we observed that RBCs from BC patients showed a different proteomic profile compared to cancer-free controls and between different tumor stages. The differential proteins were mainly related to extracellular components, proteasome, and metabolism. Embryonic hemoglobins, not expected in adults’ RBCs, were detected in BC patients. Besides, lysosome-associated membrane glycoprotein 2 emerge as a new RBCs marker with diagnostic and prognostic potential for metastatic BC patients. Seemingly, RBCs are acquiring modifications in their proteomic composition that probably represents the systemic cancer disease, conditioned by the tumor microenvironment.

Infant Red Blood Cell Arachidonic to Docosahexaenoic Acid Ratio Inversely Associates with Fat-Free Mass Independent of Breastfeeding Exclusivity.

The prevalence of childhood obesity has increased nearly ten times over the last 40 years, influenced by early life nutrients that have persistent effects on life-long metabolism. During the first six months, infants undergo accelerated adipose accumulation, but little is known regarding infant fatty acid status and its relationship to infant body composition. We tested the hypothesis that a low arachidonic to docosahexaenoic acid ratio (AA/DHA) in infant red blood cells (RBCs), a long-term indicator of fatty acid intake, would associate with more infant fat-free mass (FFM) and/or less adipose accumulation over the first 4 months of life. The fatty acid and composition of breastmilk and infant RBCs, as well as the phospholipid composition of infant RBCs, were quantified using targeted and unbiased lipid mass spectrometry from infants predominantly breastfed or predominantly formula-fed. Regardless of feeding type, FFM accumulation was inversely associated with the infant’s RBC AA/DHA ratio (p = 0.029, R2 = 0.216). Infants in the lowest AA/DHA ratio tertile had significantly greater FFM when controlling for infant sex, adiposity at 2 weeks, and feeding type (p < 0.0001). Infant RBC phospholipid analyses revealed greater peroxisome-derived ether lipids in the low AA/DHA group, primarily within the phosphatidylethanolamines. Our findings support a role for a low AA/DHA ratio in promoting FFM accrual and identify peroxisomal activity as a target of DHA in the growing infant. Both FFM abundance and peroxisomal activity may be important determinants of infant metabolism during development.

Numerical simulation of non-linear loading–unloading hysteresis behavior of blood clots

The stress–strain characteristics of a clot during loading/unloading mechanical cycles are significant features to assess the underlying mechanisms of thrombectomy, especially when multiple thrombectomy attempts are required. We investigated a damage model to predict loading/unloading response of clots. To study the validity of the model, we tested theoretical models to reproduce the experimentally obtained mechanical characteristics of clots under various conditions. Three types of clot analogs with different red blood cell (RBC) compositions were prepared. Cylindrical clot analogs were formed for the tensile and compression tests. Loading/unloading tests at 80% of strain were conducted, where the material parameters were determined by fitting the results to a theoretical curve combining the damage model and the elasto-plastic constitutive model. Through the computation for theoretical curves, unique characteristics of clots were revealed such that the hysteresis loss rate did not change by varying RBC contents, except for the clot created with 0% RBC composition, under compressive loading. In addition, the plastic strain decreased as the RBC content decreased under tensile loading, whereas it increased as the RBC content decreased under compressive loading. A three-dimensional finite element method (FEM) was employed with the determined parameters. The FEM could accurately reproduce the experimental stress–strain curves for all types of clot analogs and for both loading types up to a strain of 80%. The results indicate that the theoretical model which incorporates and combines the damage model and the elasto-plastic constitutive model is applicable to predict the non-linear stress–strain behavior of clots under loading and unloading.

Glucose-Dependent Insulinotropic Polypeptide Plasma Level Influences the Effect of n-3 PUFA Supplementation

Elevated glucose-dependent insulinotropic peptide (GIP) levels in obesity may predict the metabolic benefits of n-3 PUFA supplementation. This placebo-controlled trial aimed to analyze fasting and postprandial GIP response to 3-month n-3 PUFA supplementation (1.8 g/d; DHA:EPA, 5:1) along with caloric restriction (1200–1500 kcal/d) in obese subjects. Compliance was confirmed by the incorporation of DHA and EPA into red blood cells (RBCs). Blood analyses of glucose, insulin, non-esterified fatty acids (NEFAs), GIP and triglycerides were performed at fasting, and during an oral glucose tolerance test and a high fat mixed-meal tolerance test. Fatty acid composition of RBC was assessed by gas chromatography and total plasma fatty acid content and composition was measured by gas–liquid chromatography. The DHA and EPA content in RBCs significantly increased due to n-3 PUFA supplementation vs. placebo (77% vs. −3%, respectively). N-3 PUFA supplementation improved glucose tolerance and decreased circulating NEFA levels (0.750 vs. 0.615 mmol/L), as well as decreasing plasma saturated (1390 vs. 1001 µg/mL) and monounsaturated (1135 vs. 790 µg/mL) fatty acids in patients with relatively high GIP levels. The effects of n-3 PUFAs were associated with the normalization of fasting (47 vs. 36 pg/mL) and postprandial GIP levels. Obese patients with elevated endogenous GIP could be a target group for n-3 PUFA supplementation in order to achieve effects that obese patients without GIP disturbances can achieve with only caloric restriction.

Up-down biphasic volume response of human red blood cells to PIEZO1 activation during capillary transits.

In this paper we apply a novel JAVA version of a model on the homeostasis of human red blood cells (RBCs) to investigate the changes RBCs experience during single capillary transits. In the companion paper we apply a model extension to investigate the changes in RBC homeostasis over the approximately 200000 capillary transits during the ~120 days lifespan of the cells. These are topics inaccessible to direct experimentation but rendered mature for a computational modelling approach by the large body of recent and early experimental results which robustly constrain the range of parameter values and model outcomes, offering a unique opportunity for an in depth study of the mechanisms involved. Capillary transit times vary between 0.5 and 1.5s during which the red blood cells squeeze and deform in the capillary stream transiently opening stress-gated PIEZO1 channels allowing ion gradient dissipation and creating minuscule quantal changes in RBC ion contents and volume. Widely accepted views, based on the effects of experimental shear stress on human RBCs, suggested that quantal changes generated during capillary transits add up over time to develop the documented changes in RBC density and composition during their long circulatory lifespan, the quantal hypothesis. Applying the new red cell model (RCM) we investigated here the changes in homeostatic variables that may be expected during single capillary transits resulting from transient PIEZO1 channel activation. The predicted quantal volume changes were infinitesimal in magnitude, biphasic in nature, and essentially irreversible within inter-transit periods. A sub-second transient PIEZO1 activation triggered a sharp swelling peak followed by a much slower recovery period towards lower-than-baseline volumes. The peak response was caused by net CaCl2 and fluid gain via PIEZO1 channels driven by the steep electrochemical inward Ca2+ gradient. The ensuing dehydration followed a complex time-course with sequential, but partially overlapping contributions by KCl loss via Ca2+-activated Gardos channels, restorative Ca2+ extrusion by the plasma membrane calcium pump, and chloride efflux by the Jacobs-Steward mechanism. The change in relative cell volume predicted for single capillary transits was around 10-5, an infinitesimal volume change incompatible with a functional role in capillary flow. The biphasic response predicted by the RCM appears to conform to the quantal hypothesis, but whether its cumulative effects could account for the documented changes in density during RBC senescence required an investigation of the effects of myriad transits over the full four months circulatory lifespan of the cells, the subject of the next paper.

Assessment of Blood Clot Composition by Spectral Optical Coherence Tomography: An In Vitro Study.

PurposeOptical coherence tomography (OCT) has the potential for in vivo clot composition characterization in difficult mechanical embolectomy cases. We performed an in vitro study to determine the OCT characteristics of red blood cells (RBCs) and fibrin rich clots.Materials and MethodsAnalogues of 5 compositions of clots (5% to 95% RBCs from Group A to E) were created from human blood. The blood mixture was injected into the bifurcation of a 3D printed bifurcated silicone tube. The OPTISTM Integrated System (St. Jude Medical Inc.) was used to identify the magnitude of OCT signals from different compositions of clots. Martius Scarlett Blue trichrome (MSB) staining was performed to confirm the composition of RBCs and fibrin in each clot.ResultsGroup A and B showed less signal attenuation (less than 30%) from its surface to the inside, which indicated high penetration (low-back scattering). Group C indicated intermediate signal attenuation (60%) from its surface to inside the clots, in which signals were found even at the periphery of the clot. Group D and E were superficially signal rich with more signal attenuation (more than 80%) from its surface to the inside indicating low penetration (high-back scattering). Signal-free shadowing was shown in 3 clots in Group E. MSB staining indicated color change (from red in fibrin-rich clots to yellow in RBC-rich clots).ConclusionDifferent compositions of clots can be assessed using OCT. Fibrin-rich clots have homogeneous signals with high penetration, while RBC-rich clots can be recognized as superficially signal rich with low penetration.

Platelet-rich emboli are associated with von Willebrand factor levels and have poorer revascularization outcomes

Background and aimsPlatelets and von Willebrand factor (vWF) are key factors in thrombosis and thus are likely key components of acute ischemic stroke (AIS) emboli. We aimed to characterize platelet...

High red blood cell composition in clots is associated with successful recanalization during intra-arterial thrombectomy.

We evaluated the composition of individual clots retrieved during intra-arterial thrombectomy in relation to recanalization success, stroke subtype, and the presence of clot signs on initial brain images. We analyzed clot and interventional data from 145 retrieval trials performed for 37 patients (69.5±14.0 years, 20 men, large artery atherosclerosis, n = 7; cardioembolism, n = 22; undetermined etiology, n = 8) who had undergone intra-arterial thrombectomy. Rates of clot retrieval and successful recanalization (Arterial Occlusive Lesion score of 2–3) for separate retrieval trials were evaluated. The area occupied by red blood cell (RBC), fibrin/platelets, and white blood cell (WBC) was measured from digitized images of hematoxylin-eosin stained clots. Compositional differences were compared according to recanalization success, stroke subtype, and the presence of hyperdense clot sign on initial computed tomography and/or blooming artifact on magnetic resonance image. Of the 145 total retrieval trials (3.4±2.4 times per patient), clot was retrieved in 93 trials (64%), while recanalization was successful in 73 (50%). Fibrin/platelets (63%) occupied the greatest area in retrieved clots, followed by RBCs (33%) and WBCs (4%). Clots retrieved from successful recanalization exhibited higher RBC composition (37%) than those retrieved from non-recanalization trials (20%, p = 0.001). RBC composition was higher in cardioembolic stroke (38%) rather than large artery atherosclerosis (23%) and undetermined etiology (26%, p = 0.01). Clots exhibiting clot signs (40%) had higher RBC composition than those without clot signs (19%, p = 0.001). RBC-rich clots were associated with successful recanalization of intra-arterial thrombectomy, cardioembolic stroke, and the presence of clot-signs on initial brain images.

Quantitative Analysis of Human Red Blood Cell Proteome.

Red blood cells (RBCs) are the most abundant cell type in the human body. RBCs and, in particular, their plasma membrane composition have been extensively studied for many years. During the past decade proteomics studies have extended our knowledge on RBC composition; however, these studies did not provide quantitative insights. Here we report a large-scale proteomics investigation of RBCs and their "white ghost" membrane fraction. Samples were processed using the multienzyme digestion filter-aided sample preparation (MED-FASP) and analyzed using Q-Exactive mass spectrometer. Protein abundances were computed using the total protein approach (TPA). The validation of the data with stable isotope-labeled peptide-based protein quantification followed. Our in-depth analysis resulted in the identification of 2650 proteins, of which 1890 occurred at more than 100 copies per cell. We quantified 41 membrane transporter proteins spanning an abundance range of five orders of magnitude. Some of these, including the drug transporter ABCA7 and choline transporters SLC44A1 and SLC44A2, have not previously been identified in RBC membranes. Comparison of protein copy numbers assessed by proteomics showed a good correlation with literature data; however, abundances of several proteins were not consistent with the classical references. Because we validated our findings by a targeted analysis using labeled standards, our data suggest that some older reference data from a variety of biochemical approaches are inaccurate. Our study provides the first "in-depth" quantitative analysis of the RBC proteome and will promote future studies of erythrocyte structure, functions, and disease.

Early infant adipose deposition is positively associated with the n-6 to n-3 fatty acid ratio in human milk independent of maternal BMI.

Background/ObjectivesExcessive infant weight gain in the first 6-months of life is a powerful predictor of childhood obesity and related health risks. In mice, omega-6 fatty acids (FA) serve as potent ligands driving adipogenesis during early development. The ratio of omega-6 relative to omega-3 (n-6/n-3) FA in human milk (HM) has increased 3-fold over the last 30 years, but the impact of this shift on infant adipose development remains undetermined. This study investigated how maternal obesity and maternal dietary FA (as reflected in maternal red blood cells (RBC) composition) influenced HM n-6 and n-3 FAs, and whether the HM n-6/n-3 ratio was associated with changes in infant adipose deposition between 2-weeks and 4-months postpartum.Subjects/MethodsForty-eight infants from normal-weight (NW), overweight (OW) and obese (OB) mothers were exclusively or predominantly breastfed over the first 4 months of lactation. Mid-feed HM and maternal RBC were collected at either transitional (2-weeks) or established (4-months) lactation, along with infant body composition assessed using air-displacement plethysmography. The FA composition of HM and maternal RBC was measured quantitatively by lipid mass spectrometry.ResultsIn transitional and established HM, DHA was lower (P=0.008; 0.005) and the AA/DHA+EPA ratio was higher (P=0.05; 0.02) in the OB relative to the NW group. Maternal prepregnancy BMI and AA/ DHA+EPA ratios in transitional and established HM were moderately correlated (P=0.018; 0.001). Total infant fat mass was increased in the upper AA/DHA+EPA tertile of established HM relative to the lower tertile (P=0.019). The amount of changes in infant fat mass and % body fat were predicted by AA/EPA+DHA ratios in established HM (P=0.038; 0.010).ConclusionsPerinatal infant exposures to a high AA/EPA+DHA ratio during the first 4-months of life, which is primarily reflective of maternal dietary FA, may significantly contribute to the way infants accumulate adipose.

Novel methodology to replicate clot analogs with diverse composition in acute ischemic stroke.

Translational research on clot composition may be advanced by the use of clot analogs for the preclinical evaluation of mechanical thrombectomy devices. This work describes a novel set of clot analogs to represent a diverse range of fibrin and red blood cell (RBC) compositions for use in acute ischemic stroke (AIS) occlusion models. Fresh whole blood obtained from ovine species was used to create seven different clot analog types. Five replicates were formed for each clot type. Varying amounts of whole blood constituents were mixed with thrombotic factors to create clots of varying compositions. Following histological processing, five sections from each clot were stained with H&E and Martius Scarlet Blue. Fibrin, RBC and white blood cell compositions were quantified. Histological examination demonstrated that the clot types had a distinct RBC and fibrin composition. No significant difference in composition was shown between replicates (p>0.05), indicating that the method of clot formation was reproducible. Percentage fibrin composition of the clot types was 1%, 8%, 31%, 38%, 64%, 79%, and 100%. A significant difference in fibrin and RBC composition between clot types was observed (p<0.05). Seven different clot types were developed to replicate common AIS thrombi. These clot analogs may be beneficial for the preclinical evaluation of endovascular therapies, and may be applied to interventional technique training.

Inverse association between docosahexaenoic acid and mortality in patients on hemodialysis during over 10 years

We have previously conducted a cohort study to investigate n-3 polyunsaturated fatty acids (PUFAs) in red blood cells (RBCs) and risk of all-cause mortality in hemodialysis (HD) patients over 5 years and found that n-3 PUFAs, especially docosahexaenoic acid (DHA), might be an independent predictor of all-cause mortality. In the present study, we extended the study for another 5 years to determine whether DHA levels in RBCs still predict the mortality of HD patients during a 10-year study period. The study cohort consisted of 176 patients (64.1 ± 12.0 [mean ± standard deviation] years of age, 96 men and 80 women) under HD treatment. The fatty acid composition of patients' RBCs was analyzed by gas chromatography. During the study period of 10 years, 97 deaths occurred. After adjustment for 10 confounding factors, the hazard ratio of all-cause mortality of the HD patients in the highest DHA tertile (>8.1%) was 0.52 (95% confidence interval 0.30-0.91) compared with those in the lowest DHA tertile (<7.2%). However, other n-3 PUFAs such as eicosapentaenoic acid and docosapentaenoic acid (n-3) did not reveal any significant correlations. The level of DHA in RBCs could be an independent predictor of all-cause mortality in HD patients even during a long period of follow-up.

Improved Extraction of Saturated Fatty Acids but not Omega‐3 Fatty Acids from Sheep Red Blood Cells Using a One‐Step Extraction Procedure

Several methods are available to extract total lipid and methylate fatty acids from a range of samples including red blood cells (RBC). Fatty acid analysis of human RBC can be undertaken using a two-step extraction and methylation or a combined one-step extraction and methylation procedure. The lipid composition of sheep RBC differs significantly from that of humans and may affect their extraction. The purpose of the current study was to examine the efficiency of extraction of lipid and detection of fatty acids from sheep RBC using a one-step procedure. Fatty acids were analysed using a one-step extraction and methylation procedure using methanol:toluene and acetyl chloride in comparison with a two-step procedure involving extraction of lipid using chloroform:methanol and separate methylation. Concentrations of saturated fatty acids including C16:0 and C18:0 were significantly higher (42.6 and 33.9% respectively) following extraction using the one-step procedure compared with the two-step procedure. However, concentrations of some polyunsaturated fatty acids, including C20:5n-3 and C22:6n-3 were not significantly different between either procedure. The improved detection of fatty acids may be related to the ability of different solvents to extract different lipid fractions. The differential extraction of lipids and detection of fatty acids from sheep RBC may have important implications in studies examining the effect of dietary treatment on the possible health benefits of fatty acids.

Human and great ape red blood cells differ in plasmalogen levels and composition

BackgroundPlasmalogens are ether phospholipids required for normal mammalian developmental, physiological, and cognitive functions. They have been proposed to act as membrane antioxidants and reservoirs of polyunsaturated fatty acids as well as influence intracellular signaling and membrane dynamics. Plasmalogens are particularly enriched in cells and tissues of the human nervous, immune, and cardiovascular systems. Humans with severely reduced plasmalogen levels have reduced life spans, abnormal neurological development, skeletal dysplasia, impaired respiration, and cataracts. Plasmalogen deficiency is also found in the brain tissue of individuals with Alzheimer disease.ResultsIn a human and great ape cohort, we measured the red blood cell (RBC) levels of the most abundant types of plasmalogens. Total RBC plasmalogen levels were lower in humans than bonobos, chimpanzees, and gorillas, but higher than orangutans. There were especially pronounced cross-species differences in the levels of plasmalogens with a C16:0 moiety at the sn-1 position. Humans on Western or vegan diets had comparable total RBC plasmalogen levels, but the latter group showed moderately higher levels of plasmalogens with a C18:1 moiety at the sn-1 position. We did not find robust sex-specific differences in human or chimpanzee RBC plasmalogen levels or composition. Furthermore, human and great ape skin fibroblasts showed only modest differences in peroxisomal plasmalogen biosynthetic activity. Human and chimpanzee microarray data indicated that genes involved in plasmalogen biosynthesis show cross-species differential expression in multiple tissues.ConclusionWe propose that the observed differences in human and great ape RBC plasmalogens are primarily caused by their rates of biosynthesis and/or turnover. Gene expression data raise the possibility that other human and great ape cells and tissues differ in plasmalogen levels. Based on the phenotypes of humans and rodents with plasmalogen disorders, we propose that cross-species differences in tissue plasmalogen levels could influence organ functions and processes ranging from cognition to reproduction to aging.

CT and MRI Early Vessel Signs Reflect Clot Composition in Acute Stroke

The purpose of this study was to provide the first correlative study of the hyperdense middle cerebral artery sign (HMCAS) and gradient-echo MRI blooming artifact (BA) with pathology of retrieved thrombi in acute ischemic stroke. Noncontrast CT and gradient-echo MRI studies before mechanical thrombectomy in 50 consecutive cases of acute middle cerebral artery ischemic stroke were reviewed blinded to clinical and pathology data. Occlusions retrieved by thrombectomy underwent histopathologic analysis, including automated quantitative and qualitative rating of proportion composed of red blood cells (RBCs), white blood cells, and fibrin on microscopy of sectioned thrombi. Among 50 patients, mean age was 66 years and 48% were female. Mean (SD) proportion was 61% (±21) fibrin, 34% (±21) RBCs, and 4% (±2) white blood cells. Of retrieved clots, 22 (44%) were fibrin-dominant, 13 (26%) RBC-dominant, and 15 (30%) mixed. HMCAS was identified in 10 of 20 middle cerebral artery stroke cases with CT with mean Hounsfield Unit density of 61 (±8 SD). BA occurred in 17 of 32 with gradient-echo MRI. HMCAS was more commonly seen with RBC-dominant and mixed than fibrin-dominant clots (100% versus 67% versus 20%, P=0.016). Mean percent RBC composition was higher in clots associated with HMCAS (47% versus 22%, P=0.016). BA was more common in RBC-dominant and mixed clots compared with fibrin-dominant clots (100% versus 63% versus 25%, P=0.002). Mean percent RBC was greater with BA (42% versus 23%, P=0.011). CT HMCAS and gradient-echo MRI BA reflect pathology of occlusive thrombus. RBC content determines appearance of HMCAS and BA, whereas absence of HMCAS or BA may indicate fibrin-predominant occlusive thrombi.

Positive Association Between DNA Strand Breaks in Peripheral Blood Mononuclear Cells and Polyunsaturated Fatty Acids in Red Blood Cells From Women

Lipid peroxidation of polyunsaturated fatty acids (PUFA) generates reactive products that may cause DNA damage. To examine the possible relationship between DNA damage in peripheral blood mononuclear cells (PBMC) and the concentration of PUFA in red blood cells (RBC), endogenous DNA strand breaks, formamidopyrimidine DNA glycosylase (FPG) sites, and hydrogen peroxide (H2O2) sensitive sites were evaluated by the comet assay in blood samples from 98 Icelandic women. Fatty acid composition of RBC was analyzed by gas chromatography. Endogenous DNA strand breaks in PBMC correlated positively with the concentration of total PUFA, total n-3 PUFA, docosahexaenoic acid, linoleic acid, oleic acid, and palmitic acid in RBC. However, there was no association between FPG sites or H2O2 sensitive sites in DNA in PBMC and the concentration of total PUFA or total saturated fatty acid in RBC. As there was no association between oxidative DNA damage or sensitivity of DNA to oxidative stress and the concentration of PUFA in RBC, the positive association between endogenous DNA strand breaks in PBMC and the concentration of total PUFA in RBC is probably not related to oxidative stress.

Lysophospholipid Acylation in RBC.

The formation, distribution and utilization of acyl-CoA plays a crucial role in plasma membrane phospholipid turnover in red blood cells (RBC). Upon de-acylation of glycero-phospholipids (PL) via the action of phospholipase, re-acylation of the lysophospholipids (LPL) requires activity of two enzymes of the Lands pathway. Long-chain acyl-CoA synthetases (ACSL) activate fatty acids to acyl-CoA which are subsequently ligated to LPL by LysoPhosphoLipid Acyl Transferase (LPLAT) a family of enzymes with exclusive specificity for the polar group of LPL (phosphatidic acid, choline, serine and ethanolamine). We recently identified ACSL6 as the enzyme responsible for the activation of fatty acid in RBC. None of the family members of LPLAT have been identified in RBC to date. LPC, either generated in the RBC or taken up from plasma, is rapidly acylated by RBC suggesting an important role for Lysohosphatidylcholine-acyl transferase (LPCAT) in RBC. We report the identification and characterization of LPCAT, the enzyme that generates PC from LPC and acylCoA. We identified the RNA expression of LPCAT, an approximately 60kD protein, in reticulocytes, confirming proteomic studies suggesting the presence of this protein in adult RBC membranes.). It is a modular protein containing an acyltransferase domain at the amino-terminus, three predicted membrane spanning domains, and a putative calcium binding site at the C-terminus, distinguishing it from the lysophosphatidic acid acyltransferease (LPAAT). The putative LPCAT was expressed in E. coli. It was found in the E. coli membrane fraction, and was able to use oleoyl-CoA and LPC as substrates to generate PC. Lysophosphatidic acid (LPA) was not acylated by this protein. In contrast the previously identified LPAAT (1) expressed in E. coli, utilized LPA but not LPC, indicating LPL specificity of these enzymes. Radioactive fatty acid added to RBC is also incorporated in phosphatidyl ethanolamine (PE) and phosphatidyl serine (PS). Sequence analysis suggests that two other proteins present in the genome of mammals are homologues of LPCAT. We hypothesize that these putative acyltransferases are responsible for the acylation of lysophosphatidyl ethanolamine (LPE) and lysophosphatidyl serine (LPS). These proteins are essential to maintain a proper glycerophospholipid composition of the RBC membrane and thereby viability of the cells. A dysfunction of this system may underlie the observed differences in phospholipid molecular species composition in subpopulations of sickle cells contributing to sickle cell pathology. A complete description of these proteins involved in the maintenance of glycerophospholipid composition of RBC will aid to better understand the maintenance of plasma membrane lipid composition of all mammalian cells.

Dietary Essential Fatty Acid Deficiency Differentially Affects Tissues of Rats

Quantitative variations of polyunsaturated fatty acids (PUFA) were studied in various tissues: red blood cells (RBC), hepatic microsomes, kidney, skeletal muscle and heart of young rats fed either a control diet (n = 7) or an essential fatty acid (EFA)-deficient diet (n = 7). After 4 wk, the EFA-[deficient rats had significantly lower proportions of (n-6) and (n-3) fatty acids in RBC, hepatic microsomes and kidney than the control group. Paradoxically, normal proportions of arachidonic acid [20:4(n-6)] and 5,8,11,14,17-eicosapentaenoic acid [20:5(n-3)] were retained in heart and skeletal muscle despite generally lower proportions of the precursors, 18:2(n-6) and 18:3(n-3). Morever, absolute levels of 20:4(n-6) and 20:5(n-3) in skeletal muscle of the EFA deficient group were significantly higher than in controls and 22:5(n-3) and 22:6(n-3) levels were comparable. This suggests that fatty acid proportions alone, without any consideration of long-chain polyunsaturated fatty acid quantities, may not reflect the (n-6) and (n-3) PUFA status of individual tissues. This study indicates that diet-[induced changes in the PUFA composition of RBC, which are often used in clinical investigations, do not fully reflect the changes in the fatty acid composition of organs, and that individual tissues respond differently to EFA deficiency. The conservation of proportional and absolute levels of 20:5(n-3) and 20:4(n-6), and the decrease in the more unsaturated homologues in the heart, suggest that this organ may avidly retain 20:5(n-3) and 20:4(n-6) in order to maintain eicosanoid production.