Impaired Cytoskeletal and Membrane Biophysical Properties of Acanthocytes in Hypobetalipoproteinemia – A Case Study

Abstract

Familial hypobetalipoproteinemia is a metabolic disorder mainly caused by mutations in the apolipoprotein B gene. In its homozygous form it can lead without treatment to severe ophthalmological and neurological manifestations. In contrast, the heterozygous form is generally asymptomatic but associated with a low risk of cardiovascular disease. Acanthocytes or thorny red blood cells (RBCs) are described for both forms of the disease. However, those morphological changes are poorly characterized and their potential consequences for RBC functionality are not understood. Thus, in the present study, we asked whether, to what extent and how acanthocytes from a patient with heterozygous familial hypobetalipoproteinemia could exhibit altered RBC functionality. Acanthocytes represented 50% of the total RBC population and contained mitoTracker-positive surface patches, indicating the presence of mitochondrial fragments. While RBC osmotic fragility, calcium content and ATP homeostasis were preserved, a slight decrease of RBC deformability combined with an increase of intracellular free reactive oxygen species were observed. The spectrin cytoskeleton was altered, showing a lower density and an enrichment in patches. At the membrane level, no obvious modification of the RBC membrane fatty acids nor of the cholesterol content were detected but the ceramide species were all increased. Membrane stiffness and curvature were also increased whereas transversal asymmetry was preserved. In contrast, lateral asymmetry was highly impaired showing: (i) increased abundance and decreased functionality of sphingomyelin-enriched domains; (ii) cholesterol enrichment in spicules; and (iii) ceramide enrichment in patches. We propose that oxidative stress induces cytoskeletal alterations, leading to increased membrane stiffness and curvature and impaired lipid lateral distribution in domains and spicules. In addition, ceramide- and spectrin-enriched patches could result from a RBC maturation defect. Altogether, the data indicate that acanthocytes are associated with cytoskeletal and membrane lipid lateral asymmetry alterations, while deformability is only mildly impaired. In addition, familial hypobetalipoproteinemia might also affect RBC precursors leading to disturbed RBC maturation. This study paves the way for the potential use of membrane biophysics and lipid vital imaging as new methods for diagnosis of RBC disorders.

Lauric Acid, a Dietary Saturated Medium-Chain Fatty Acid, Elicits Calcium-Dependent Eryptosis

“…This is in contrast to LA-induced acanthocytosis ( Figure 4 F,G), which was significantly abrogated upon the elimination of Ca 2+ ( Figure 4 F,H). Acanthocytes are susceptible to splenic digestion [ 73 ], again increasing the risk for hemolytic anemia [ 74 ]. Notably, membrane blebbing and surface granularity reflect increased stiffness which may be ascribed to cytoskeletal damage and oxidative injury, which, in turn, was also demonstrated to ensue following Ca 2+ influx ( Figure 5 and Figure 7 ).…”

A novel missense variant in ATP11C is associated with reduced red blood cell phosphatidylserine flippase activity and mild hereditary hemolytic anemia

“…RBC morphology was determined on peripheral blood smears and by scanning electron microscopy of glutaraldehyde‐fixed RBCs on filters. RBCs were prepared and analyzed as previously described 23,26 …”

“…Osmotic fragility through Hb release and RBC deformability using osmotic gradient ektacytometry with the Laser Optical Rotational Red Cell Analyzer (Lorrca; RR Mechatronics) were used according to the manufacturer's instructions 23,28 …”

“…RBCs were prepared and analyzed as previously described. 23,26 Osmotic fragility through Hb release and RBC deformability using osmotic gradient ektacytometry with the Laser Optical Rotational Red Cell Analyzer (Lorrca; RR Mechatronics) were used according to the manufacturer's instructions. 23,28 Dense RBCs (Hb concentration > 41 g/dL) were measured on the ADVIA hematology analyzer (Siemens).…”

“…23,26 Osmotic fragility through Hb release and RBC deformability using osmotic gradient ektacytometry with the Laser Optical Rotational Red Cell Analyzer (Lorrca; RR Mechatronics) were used according to the manufacturer's instructions. 23,28 Dense RBCs (Hb concentration > 41 g/dL) were measured on the ADVIA hematology analyzer (Siemens). RBC density separation was performed using the three Percoll gradients (59%, 70% and 78%) yielding four different fractions of RBCs according to density (i.e., cellular age) (from the top layer down to the bottom of the tube: fraction 1 containing the RBCs with the lowest density [i.e., youngest RBCs], up to fraction 4 containing the most dense RBCs [i.e., senescent RBCs]).…”