Ginsenoside Rh2 Regulates the Calcium/ROS/CK1α/MLKL Pathway to Promote Premature Eryptosis and Hemolysis in Red Blood Cells.

Ginsenoside Rh2 (GRh2) exhibits significant potential as an anticancer agent; however, progress in developing chemotherapeutic drugs is impeded by their toxicity toward off-target tissues. Specifically, anemia caused by chemotherapy is a debilitating side effect and can be caused by red blood cell (RBC) hemolysis and eryptosis. Cells were exposed to GRh2 in the antitumor range and hemolytic and eryptotic markers were examined under different experimental conditions using photometric and cytofluorimetric methods. GRh2 caused Ca2+-independent, concentration-responsive hemolysis in addition to disrupted ion trafficking with K+ and Cl- leakage. Significant increases in cells positive for annexin-V-fluorescein isothiocyanate, Fluo4, and 2,7-dichlorofluorescein were noted upon GRh2 treatment coupled with a decrease in forward scatter and acetylcholinesterase activity. Importantly, the cytotoxic effects of GRh2 were mitigated by ascorbic acid and by blocking casein kinase 1α (CK1α) and mixed lineage kinase domain-like (MLKL) signaling. In contrast, Ca2+ omission, inhibition of KCl efflux, and isosmotic sucrose aggravated GRh2-induced RBC death. In whole blood, GRh2 selectively targeted reticulocytes and lymphocytes. Altogether, this study identified novel mechanisms underlying GRh2-induced RBC death involving Ca2+ buildup, loss of membrane phospholipid asymmetry and cellular volume, anticholinesterase activity, and oxidative stress. These findings shed light on the hematologic toxicity of GRh2 which is crucial for optimizing its utilization in cancer treatment.

Metabolic exhaustion and casein kinase 1α drive deguelin-induced premature red blood cell death.

1. Deguelin (DGN), a retinoid isolated from many plants, exhibits a potent anticancer activity against a wide spectrum of tumour cells. There is a dearth of evidence, however, regarding the toxicity of DGN to red blood cells (RBCs). This is relevant given the prevalent chemotherapy-associated anaemia observed in cancer patients.2. RBCs were exposed to 1-100 µM of DGN for 24 h at 37 °C. Haemolysis and related markers were photometrically measured while flow cytometry was employed to detect phosphatidylserine exposure through Annexin-V-FITC binding and light scatter properties. Additionally, cytosolic Ca2+ and reactive oxygen species were quantified using Fluo4/AM and H2DCFDA, respectively. DGN was also tested against specific signalling inhibitors in addition to vitamin C and ATP.3. DGN caused a significant increase in Annexin-V-positive cells which was accompanied by cell shrinkage without Ca2+ elevation or oxidative stress. DGN also elicited dose-responsive haemolysis which was ameliorated by preventing KCl efflux and in the presence of sucrose, D4476, and ATP. In whole blood, DGN significantly reduced the reticulocyte count and increased platelet distribution width and large cell count.4. DGN triggers premature RBC eryptosis and haemolysis through casein kinase 1α and ATP depletion, and exhibits a specific toxicity towards reticulocytes and platelets.

Stimulation of Hemolysis and Eryptosis by α-Mangostin through Rac1 GTPase and Oxidative Injury in Human Red Blood Cells.

BACKGROUND: Chemotherapy-related anemia is prevalent in up to 75% of patients, which may arise due to hemolysis and eryptosis. Alpha-mangostin (α-MG) is a polyphenolic xanthonoid found in the mangosteen tree (Garcinia mangostana) whose antitumor medicinal properties are well-established. Nevertheless, the potential toxic effects of α-MG on red blood cells (RBCs) have, as of yet, not been as well studied. METHODS: RBCs were exposed to 1-40 µM of α-MG for 24 h at 37 °C. Hemolysis and related markers were measured using colorimetric assays, eryptotic cells were identified through Annexin-V-FITC, Ca2+ was detected with Fluo4/AM, and oxidative stress was assessed through H2DCFDA using flow cytometry. The toxicity of α-MG was also examined in the presence of specific signal transduction inhibitors and in whole blood. RESULTS: α-MG at 10-40 µM caused dose-dependent hemolysis with concurrent significant elevation in K+, Mg2+, and LDH leakage, but at 2.5 µM it significantly increased the osmotic resistance of cells. A significant increase was also noted in Annexin-V-binding cells, along with intracellular Ca2+, oxidative stress, and cell shrinkage. Moreover, acetylcholinesterase activity was significantly inhibited by α-MG, whose hemolytic potential was significantly ameliorated by the presence of BAPTA-AM, vitamin C, NSC23766, and isosmotic sucrose but not urea. In whole blood, α-MG significantly depleted intracellular hemoglobin stores and was selectively toxic to platelets and monocytes. CONCLUSIONS: α-MG possesses hemolytic and eryptotic activities mediated through Ca2+ signaling, Rac1 GTPase activity, and oxidative injury. Also, α-MG leads to accelerated cellular aging and specifically targets platelet and monocyte populations in a whole blood milieu.

Rosmarinic Acid Elicits Calcium-Dependent and Sucrose-Sensitive Eryptosis and Hemolysis through p38 MAPK, CK1α, and PKC.

BACKGROUND: Rosmarinic acid (RA) possesses promising anticancer potential, but further development of chemotherapeutic agents is hindered by their toxicity to off-target tissue. In particular, chemotherapy-related anemia is a major obstacle in cancer therapy, which may be aggravated by hemolysis and eryptosis. This work presents a toxicity assessment of RA in human RBCs and explores associated molecular mechanisms. METHODS: RBCs isolated from healthy donors were treated with anticancer concentrations of RA (10-800 µM) for 24 h at 37 °C, and hemolysis and related markers were photometrically measured. Flow cytometry was used to detect canonical markers of eryptosis, including phosphatidylserine (PS) exposure by annexin-V-FITC, intracellular Ca2+ by Fluo4/AM, cell size by FSC, and oxidative stress by H2DCFDA. Ions and pH were assessed by an ion-selective electrode, while B12 was detected by chemiluminescence. RESULTS: RA elicited concentration-dependent hemolysis with AST and LDH release but rescued the cells from hypotonic lysis at sub-hemolytic concentrations. RA also significantly increased annexin-V-positive cells, which was ameliorated by extracellular Ca2+ removal and isosmotic sucrose. Furthermore, a significant increase in Fluo4-positive cells and B12 content and a decrease in FSC and extracellular pH with KCl efflux were noted upon RA treatment. Hemolysis was augmented by blocking KCl efflux and was blunted by ATP, SB203580, staurosporin, D4476, isosmotic urea, and PEG 8000. CONCLUSIONS: RA stimulates Ca2+-dependent and sucrose-sensitive hemolysis and eryptosis characterized by PS exposure, Ca2+ accumulation, loss of ionic regulation, and cell shrinkage. These toxic effects were mediated through energy deprivation, p38 MAPK, protein kinase C, and casein kinase 1α.

Geraniin inhibits whole blood IFN-γ and IL-6 and promotes IL-1ß and IL-8, and stimulates calcium-dependent and sucrose-sensitive erythrocyte death.

Correlations between circulating cytokine levels and disease states are well established, and pharmacological modulation of the immune response is thus an important aspect of the assessment of investigational new drugs. Moreover, chemotherapy-related anemia is a major obstacle in cancer treatment. Geraniin (GRN), a tannin extracted from Geranium and other plants, possesses promising antitumor potential. However, the effect of GRN on whole blood (WB) cytokine response and RBC physiology remains unexplored. Heparinized blood from consented, healthy adults was challenged with 100 ng/mL of lipopolysaccharide (LPS) with and without pretreatment with 10 µM of GRN for 24 h at 37 °C, and tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-1ß (IL-1ß), IL-6, IL-8, and IL-10 were assayed by ELISA. Moreover, single-cell RBC suspensions were treated with 5-100 µM of GRN for 24 or 48 h at 37 °C and cytotoxicity and canonical eryptotic markers were examined by flow cytometry. It was revealed that GRN significantly attenuated LPS-induced IFN-γ levels, increased IL-1ß, decreased IL-6 only in absence of LPS, and aggravated LPS-induced IL-8 while together with LPS significantly diminished IL-10. Furthermore, GRN induced dose-responsive, Ca2+-dependent, and sucrose-sensitive hemolysis, along with phosphatidylserine exposure and Ca2+ accumulation with no appreciable cell shrinkage or oxidative damage. GRN was also selectively toxic to platelets, significantly delayed reticulocyte maturation, and significantly disrupted leukocyte proportions. In conclusion, GRN regulates the WB cytokine response and promotes premature hemolysis and eryptosis. This study provides insights into the therapeutic utility of GRN in a highly relevant cellular model system.

Isolated and Combined Effect of Age and Gender on Neutrophil-Lymphocyte Ratio in the Hyperglycemic Saudi Population.

Inflammation is pivotal to the pathogenesis of diabetes mellitus (DM), but pathological alterations of the neutrophil−lymphocyte ratio (NLR), an emerging inflammatory index in DM management, remains understudied. The aim of this study is to examine the relationship between NLR and glycemic control in the Saudi population. Gender, age, WBC count, and fasting blood glucose (FBG) were obtained from Al-Borg Medical Laboratories for 14,205 subjects. Means, prevalence, risk measures, and the diagnostic accuracy of elevated NLR and hyperglycemia (HG) were evaluated. Subjects with elevated NLR (>3) had significantly higher FBG (105.10 ± 0.33 vs. 114.0 ± 2.81) and NLR was significantly elevated in impaired fasting glycemia (IFG; 1.21 ± 0.01 vs. 1.25 ± 0.01) and HG (1.21 ± 0.01 vs. 1.39 ± 0.02). Elevations of NLR in HG but not in IFG persisted across all age groups except young males and elderly females. The prevalence of elevated NLR in hyperglycemic subjects was 4.12% compared to 2.16% in subjects with normal FBG. HG was more prevalent in subjects with elevated NLR (17.33% vs. 12.46%) who had a relative risk (RR) of 1.68 (95% CI = 1.38−2.06, p < 0.0001) and an odds ratio (OR) of 1.94 (95% CI = 1.48−2.56, p < 0.0001) to be hyperglycemic. Nevertheless, NLR failed to discriminate individuals with normal FBG from those with HG based on ROC curve analysis. Pathological fluctuations in NLR may serve as supportive evidence in DM management.

Physcion Induces Hemolysis and Premature Phosphatidylserine Externalization in Human Erythrocytes.

The prevalence of cancer-associated anemia (CIA) is high, and the mechanisms governing its development remain poorly understood. Eryptosis, the suicidal cell death of red blood cells (RBCs), may account for CIA as it is triggered by clinically approved chemotherapeutics including cisplatin and paclitaxel. Physcion (PSN), an anthraquinone extracted from rhubarb and other plants, has shown great promise as an anticancer agent. However, the potential toxicity of PSN to RBCs remains elusive. RBCs were isolated from heparinized blood, and incubated with 10-100 µM of PSN for 24 h at 37 °C. Hemolysis was photometrically calculated from hemoglobin concentration in the medium at 405 nm, while flow cytometry was employed to investigate cardinal markers of eryptosis. Phosphatidylserine (PS) exposure was detected by Annexin-V-fluorescein isothiocyanate (FITC), intracellular calcium by Fluo4/AM, cellular volume from forward scatter (FSC), and oxidative stress by 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). PSN induced overt hemolysis at 50 and 100 µM which was not mediated through calcium influx, protein kinase C, casein kinase 1α, or receptor-interacting protein 1. Moreover, PSN caused significant increase in Annexin-V-FITC and Fluo4 fluorescence with no appreciable influence on FSC or DCF values. Accordingly, PSN stimulates premature eryptosis characterized by PS externalization and intracellular calcium overload without cell shrinkage or oxidative damage. In conclusion, this report shows, for the first time, that PSN is cytotoxic to RBCs by inducing hemolysis and programmed cell death which may limit its success as a chemotherapeutic agent.

Germinal center humoral autoimmunity independently mediates progression of allograft vasculopathy.

The development of humoral autoimmunity following organ transplantation is increasingly recognised, but of uncertain significance. We examine whether autoimmunity contributes independently to allograft rejection. In a MHC class II-mismatched murine model of chronic humoral rejection, we report that effector antinuclear autoantibody responses were initiated upon graft-versus-host allorecognition of recipient B cells by donor CD4 T-cells transferred within heart allografts. Consequently, grafts were rejected more rapidly, and with markedly augmented autoantibody responses, upon transplantation of hearts from donors previously primed against recipient. Nevertheless, rejection was dependent upon recipient T follicular helper (TFH) cell differentiation and provision of cognate (peptide-specific) help for maintenance as long-lived GC reactions, which diversified to encompass responses against vimentin autoantigen. Heart grafts transplanted into stable donor/recipient mixed haematopoietic chimeras, or from parental strain donors into F1 recipients (neither of which can trigger host adaptive alloimmune responses), nevertheless provoked GC autoimmunity and were rejected chronically, with rejection similarly dependent upon host TFH cell differentiation. Thus, autoantibody responses contribute independently of host adaptive alloimmunity to graft rejection, but require host TFH cell differentiation to maintain long-lived GC responses. The demonstration that one population of helper CD4 T-cells initiates humoral autoimmunity, but that a second population of TFH cells is required for its maintenance as a GC reaction, has important implications for how autoimmune-related phenomena manifest.

Are donor lymphocytes a barrier to transplantation tolerance?

PURPOSE OF REVIEW: Following solid organ transplantation (SOT), populations of donor lymphocytes are frequently found in the recipient circulation. Their impact on host alloimmunity has long been debated but remains unclear, and it has been suggested that transferred donor lymphocytes may either promote tolerance to the graft or hasten its rejection. We discuss possible mechanisms by which the interaction of donor passenger lymphocytes with recipient immune cells may either augment the host alloimmune response or inhibit it. RECENT FINDINGS: Recent work has highlighted that donor T lymphocytes are the most numerous of the donor leukocyte populations within a SOT and that these may be transferred to the recipient after transplantation. Surprisingly, graft-versus-host recognition of major histocompatibility complex class II on host B cells by transferred donor CD4 T cells can result in marked augmentation of host humoral alloimmunity and lead to early graft failure. Killing of donor CD4 T cells by host natural killer cells is critical in preventing this augmentation. SUMMARY: The ability of passenger donor CD4 T cells to effect long-term augmentation of the host humoral alloimmune response raises the possibility that ex-vivo treatment or modification of the donor organ prior to implantation may improve long-term transplant outcomes.

Bufalin reprograms erythrocyte lifespan through p38 MAPK and Rac1 GTPase.

Ample evidence indicates that bufalin (BFN), a cardiotonic steroid in Bufo toad toxin, possesses a potent anticancer activity mainly by stimulating apoptosis in cancer cells. Human red blood cells (RBCs) undergo eryptosis which contributes to a plethora of pathological conditions. No reports, however, have examined the potential toxicity of BFN to RBCs. This study aims to characterize the biochemical mechanisms governing the influence of BFN on the physiology and lifespan of RBCs. Isolated RBCs from healthy volunteers were exposed to anticancer concentrations of commercially available BFN from the skin of Bufo gargarizans (10-200 µM) for 24 h at 37 °C. Photometric assays were used to estimate hemolysis and hemolytic markers, and flow cytometry was used to detect eryptotic markers. Phosphatidylserine externalization was captured by fluorescein isothiocyante-labeled annexin V, cellular dimensions by light scatter patterns, and intracellular Ca2+ and reactive oxygen species (ROS) by fluorogenic dyes Fluo4/AM and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), respectively. BFN caused Ca2+-independent hemolysis and release of LDH, AST, CK, and K+, and increased annexin V-bound cells, cytosolic Ca2+, cell shrinkage, and ROS levels. BFN also disrupted Na+ and Mg2+ trafficking, and was sensitive to PEG 8000, sucrose, SB203580, and NSC 23766. In whole blood, BFN depleted hemoglobin stores, increased fragmented RBCs, and was selectively toxic to reticulocytes, lymphocytes, and platelets. In conclusion, BFN elicits premature RBC death, subject to regulation by p38 MAPK and Rac1 GTPase, and is detrimental to other peripheral blood cells. Altogether, these novel findings prompt cautious consideration of the toxin in anticancer therapy.

Stimulation of Calcium/NOS/CK1α Signaling by Cedrol Triggers Eryptosis and Hemolysis in Red Blood Cells.

Background: Cedrol (CRL) is a sesquiterpene alcohol present in the essential oils of coniferous trees including Cupressus and Juniperus genera. CRL has shown potent anticancer activity by virtue of apoptosis. Red blood cells (RBCs), although devoid of mitochondria and nucleus, can undergo hemolysis and eryptosis which contribute to chemotherapy-induced anemia (CIA). In this work, we explored the hemolytic and eryptotic potential of CRL in human RBCs as a safety assessment of the sesquiterpene as an anticancer agent. Methods: RBCs from healthy donors were treated with anticancer concentrations of CRL for 24 h at 37°C with varying experimental manipulations. Hemolysis was photometrically assessed by measuring hemoglobin release whereas flow cytometry was employed to detect phosphatidylserine (PS) exposure by annexin-V-FITC, intracellular Ca2+ by Fluo4/AM, cell volume by forward scatter (FSC), and oxidative stress by 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). Results: Significant, concentration-responsive hemolysis was noted upon CRL exposure with concomitant K+, LDH, and AST leakage. CRL also significantly increased annexin-V-positive cells and Fluo4 fluorescence and reduced FSC. Moreover, the cytotoxicity of CRL was significantly ameliorated in the presence of L-NAME, D4476, and PEG 8,000 but was aggravated by urea and sucrose. Conclusion: CRL stimulates hemolysis and eryptosis characterized by PS exposure, Ca2+ overload, and cell shrinkage. The hemolytic activity of CRL was mediated through nitric oxide synthase and casein kinase 1α. Blocking either enzyme may attenuate the toxicity of CRL to RBCs and prevent undesirable side effects associated with its anticancer applications.

Tamoxifen induces eryptosis through calcium accumulation and oxidative stress.

Chemotherapy-related anemia is a major obstacle in anticancer therapy. Tamoxifen (TAM) is an antiestrogen prescribed for breast cancer patients with hemolytic potential and apoptotic properties in nucleated cells. However, the eryptotic activity of TAM has hitherto escaped the efforts of investigators. RBCs from apparently healthy volunteers were treated with 1-50 µM of TAM for 24 h at 37 °C. Hemoglobin leakage and LDH, AST, and AChE activities were photometrically determined while K+, Na+, and Mg2+ were detected by ion-selective electrode. Flow cytometry was used to identify eryptotic cells by annexin-V-FITC, intracellular Ca2+ by Fluo4/AM, sell size and morphology by FSC and SSC signals, respectively, and oxidative stress by H2DCFDA. Whole blood was also exposed to 30 µM of TAM for 24 h at 37 °C to examine the toxicity of TAM to WBCs and platelets. TAM caused Ca2+-independent, dose-responsive hemolysis accompanied by K+, LDH, and AST leakage without improving the mechanical stability of RBCs in hypotonic environments. TAM treatment also increased the proportion of cells positive for annexin-V-FITC, Fluo4, and DCF, along with diminished FSC and SSC signals and AChE activity. Notably, TAM toxicity was aggravated by sucrose but abrogated by vitamin C, PEG 8000, and urea. Moreover, TAM exhibited distinct cytotoxic profiles against leukocytes and platelets. TAM-induced eryptosis is characterized by breakdown of membrane asymmetry, inhibition of AChE activity, Ca2+ accumulation, cell shrinkage, and oxidative stress. Vitamin C, PEG 8000, and urea may hold promise to subvert the undesirable toxic effects of TAM on RBCs.

Stimulation of Hemolysis and Eryptosis by ß-Caryophyllene Oxide.

BACKGROUND: Eryptosis stimulated by anticancer drugs can lead to anemia in patients. ß-caryophyllene oxide (CPO) is an anticancer sesquiterpene present in various plants; however, its effect on the structure and function of human red blood cells (RBCs) remains unexplored. The aim of this study was to investigate the hemolytic and eryptotic activities and underlying molecular mechanisms of CPO in human RBCs. METHODS: Cells were treated with 10-100 µM of CPO for 24 h at 37 °C, and hemolysis, LDH, AST, and AChE activities were photometrically assayed. Flow cytometry was employed to determine changes in cell volume from FSC, phosphatidylserine (PS) externalization by annexin-V-FITC, intracellular calcium by Fluo4/AM, and oxidative stress by 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). Cells were also cotreated with CPO and specific signaling inhibitors and antihemolytic agents. Furthermore, whole blood was exposed to CPO to assess its toxicity to other peripheral blood cells. RESULTS: CPO induced concentration-responsive hemolysis with LDH and AST leakage, in addition to PS exposure, cell shrinkage, Ca2+ accumulation, oxidative stress, and reduced AChE activity. The toxicity of CPO was ameliorated by D4476, staurosporin, and necrosulfonamide. ATP and PEG 8000 protected the cells from hemolysis, while urea and isotonic sucrose had opposite effects. CONCLUSIONS: CPO stimulates hemolysis and eryptosis through energy depletion, Ca2+ buildup, oxidative stress, and the signaling mediators casein kinase 1α, protein kinase C, and mixed lineage kinase domain-like pseudokinase. Development of CPO as an anticancer therapeutic must be approached with prudence to mitigate adverse effects on RBCs using eryptosis inhibitors, Ca2+ channel blockers, and antioxidants.

Eriocitrin Disrupts Erythrocyte Membrane Asymmetry through Oxidative Stress and Calcium Signaling and the Activation of Casein Kinase 1α and Rac1 GTPase.

BACKGROUND: Hemolysis and eryptosis result in the premature elimination of circulating erythrocytes and thus contribute to chemotherapy-related anemia, which is extremely prevalent in cancer patients. Eriocitrin (ERN), a flavanone glycoside in citrus fruits, has shown great promise as an anticancer agent, but the potential toxicity of ERN to human erythrocytes remains unstudied. METHODS: Erythrocytes were exposed to anticancer concentrations of ERN (10-100 µM) for 24 h at 37 °C, and hemolysis and associated markers were quantified using colorimetric assays. Eryptosis was assessed by flow cytometric analysis to detect phosphatidylserine (PS) exposure by annexin-V-FITC, intracellular Ca2+ using Fluo4/AM, and oxidative stress with 2-,7-dichlorodihydrofluorescin diacetate (H2DCFDA). ERN was also tested against specific signaling inhibitors and anti-hemolytic agents. RESULTS: ERN caused significant, concentration-dependent hemolysis at 20-100 µM. ERN also significantly increased the percentage of eryptotic cells characterized by Ca2+ elevation and oxidative stress. Furthermore, the hemolytic activity of ERN was significantly ameliorated in the presence of D4476, NSC23766, isosmotic urea and sucrose, and polyethylene glycol 8000 (PEG). In whole blood, ERN significantly elevated MCV and ESR, with no appreciable effects on other peripheral blood cells. CONCLUSIONS: ERN promotes premature erythrocyte death through hemolysis and eryptosis characterized by PS externalization, Ca2+ accumulation, membrane blebbing, loss of cellular volume, and oxidative stress. These toxic effects, mediated through casein kinase 1α and Rac1 GTPase, can be ameliorated by urea, sucrose, and PEG. Altogether, these novel findings are relevant to the further development of ERN as an anticancer therapeutic.

Association of Platelet-Monocyte Ratio with Dyslipidemia in Saudi Arabia: A Large, Population-Based Study.

BACKGROUND: Abnormal lipid metabolism predisposes to cardiovascular disease. However, dyslipidemia is often asymptomatic leading to its underdiagnosis. Therefore, it is of utmost importance to identify biomarkers that reflect an abnormal lipid profile and trigger the specific investigation of lipid metabolism. The platelet-monocyte ratio (PMR) is a severely understudied index whose association with disturbed lipid markers remains unknown. METHODS: A cross-sectional study of the association between PMR and comprehensive lipid profile including total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglycerides (TG), TC/HDL, LDL/HDL, and TG/HDL in 14,269 Saudi subjects was designed. Prevalence, risk measures, association, and the diagnostic performance (i.e., area under the curve (AUC)) were evaluated. RESULTS: Median PMR was significantly elevated in subjects with high TC (p < 0.01), TG, TC/HDL, LDL/HDL, TG/HDL, and LDL and reduced in those with low HDL (all p < 0.0001) compared to normal subjects. The increase in PMR was abolished when only males with high TC were considered. Except for TC and LDL, all other abnormal markers were significantly more prevalent when PMR was lower (higher for HDL) than a certain cutoff specific for each parameter. Moreover, the odds of having PMR readings above or below the selected cutoffs are significantly higher with all lipid abnormalities. PMR was also weakly but significantly and differentially correlated with all forms of dyslipidemia (p < 0.0001). Notably, the highest diagnostic accuracy of PMR was observed for reduced HDL (AUC = 0.608, p < 0.0001) and elevated TG/HDL (AUC = 0.596, p < 0.0001). CONCLUSIONS: PMR is a novel, inexpensive, and readily available index that is associated with all forms of dyslipidemia, suggesting its potential use in related disorders.

Blood indices of omega-3 and omega-6 polyunsaturated fatty acids are altered in hyperglycemia.

Polyunsaturated fatty acids (PUFAs) may favorably influence the risk and clinical course of diabetes mellitus (DM). In particular, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and arachidonic acid (AA) alleviate oxidative injury and insulin resistance characteristic of DM. Uncertainty still remains, however, as to the composition and proportions of blood PUFAs in relation to fasting blood glucose levels. This study, thus, aims to examine the patterns of blood PUFA indices in normoglycemic (NG) and hyperglycemic (HG) Saudi subjects. Age, gender, FA profiles, and laboratory records of 143 subjects collected from September 2014 to March 2018 were retrospectively analyzed. Means, prevalence rates, associations, risk measures, and the diagnostic accuracy of PUFAs were determined. HG subjects had significantly lower AA (0.70%, 95% CI: 0.59-0.80% vs 0.46%, 95% CI: 0.38-0.53%) and higher EPA/AA ratio (0.36, 95% CI: 0.30-0.42 vs 0.69, 95% CI: 0.61-0.77). Gender-wise comparisons revealed that ώ-6/ώ-3 ratio was the only PUFA index significantly elevated in HG males (0.36, 95% CI: 0.26-0.45 vs 5.68, 95% CI: 4.98-6.38) while both DHA (2.91%, 95% CI: 2.54-3.29% vs 3.37%, 95% CI: 3.13-3.60%) and ώ-3 index (3.1%, 95% CI: 2.70-3.49% vs 3.63%, 95% CI: 3.38-3.88%) were significantly elevated in HG females. Furthermore, reduced AA and elevated EPA/AA ratio were more prevalent in HG subjects (26.53 vs 28.72 and 30.61 vs 38.29, respectively) and exhibited the highest diagnostic accuracy for HG among all PUFA indices. Altogether, our study revealed that distinct, gender-specific blood PUFA indices are differentially regulated in HG subjects which may be valuable for DM management.

Induction of hemolysis and eryptosis by occupational pollutant nickel chloride is mediated through calcium influx and p38 MAP kinase signaling.

OBJECTIVES: Nickel (Ni) is an abundant environmental hazard and an occupational pollutant. Exposure to Ni compounds is prevalent in electroplating workers and in the printing industry, among others. The toxicity of Ni manifests as dermatological, gastrointestinal, respiratory, allergic, and cardiovascular symptoms. In particular, hyperbilirubinemia and reticulocytosis have been detected in intoxicated subjects; an observation possibly implicating selective red blood cell (RBC) toxicity. Herein, the interaction of nickel chloride (NiCl2) with human RBCs and associated molecular mechanisms are described. MATERIAL AND METHODS: Cells from healthy donors were incubated for 24 h at 37°C in the presence or absence of 0.5‒10 mM of NiCl2, and cytotoxicity was determined through hemoglobin leakage by colorimetry under different experimental conditions. Eryptotic markers were also identified by flow cytofluorometry using Annexin-V-FITC tagging for phosphatidylserine (PS) exposure, light scatter properties for cellular dimensions, Fluo4/AM labeling for intracellular calcium, and H2DCFDA staining for reactive oxygen species (ROS). Additionally, small molecule inhibitors were used to probe the signaling pathways involved. RESULTS: It was found that NiCl2 at 10 mM caused profound intracellular calcium overload and significant calcium-dependent hemolysis. Also, NiCl2 reduced forward scatter and increased side scatter, Annexin- positive cells, and ROS levels. Importantly, NiCl2-induced hemolysis was significantly attenuated by the exclusion of extracellular calcium, and in the presence of p38 MAP kinase (MAPK) inhibitor SB203580. CONCLUSIONS: It is concluded that NiCl2 induces p38 MAPK-dependent hemolysis, and stimulates the canonical features of premature eryptosis. This report presents the first description of the molecular mechanisms underlying the hemolytic and eryptotic potential of NiCl2 and, thus, may explain changes in hematological parameters observed in poisoning victims. Int J Occup Med Environ Health. 2022;35(1):1-11.

Patterns of 25-Hydroxyvitamin D3, Calcium Status, and Anemia in the Saudi Population: A Cross-Sectional Study.

BACKGROUND: Emerging evidence suggests an intricate relationship between vitamin D, Ca2+, and inflammation-driven anemia. We, thus, investigated the patterns of serum 25(OH)D3, Ca2+, ferritin, and iron in healthy and anemic members of the Saudi population. METHODS: A population-based, retrospective, cross-sectional study was designed to analyze data for 14,229 subjects, aged 3-110 years, obtained from Al-Borg Medical Laboratories, over a six-year period (2014-2020). Gender and age differences were analyzed for 25(OH)D3, Ca2+, hemoglobin, ferritin, and iron. RESULTS: Vitamin D deficiency was extremely prevalent (98.47%) irrespective of age or gender, despite an increasing trend with age, in clear contrast to serum Ca2+. Ferritin was significantly lower in young adult and adult females, compared to elderly females, whereas iron was significantly reduced in females; in particular, adult females compared to young adults or elderly adults. Only anemic adult males had significantly lower 25(OH)D3, while Ca2+ was consistently significantly diminished in anemics of all age groups, independent of gender. Notably, hypocalcemic subjects were 2.36 times more likely to be anemic. Moreover, ferritin, but not iron, was significantly diminished in anemics, which was only evident in young adults and adults. However, both ferritin and iron showed positive correlation with hematocrit, hemoglobin, MCH, MCHC, and MCV. CONCLUSIONS: Despite being significantly lower in anemics, 25(OH)D3 is not particularly associated with anemia, while hypocalcemia is associated with an increased risk for anemia. Assessment of vitamin D and Ca2+ status may be valuable in the clinical management of anemia in the Saudi population.

Demography and blood donation trends in Saudi Arabia: A nationwide retrospective, cross-sectional study.

Background: Blood product supply and utilization are understudied in Saudi Arabia. This study evaluates the trends in Saudi blood banks readiness, donors' demography, and blood product utilization and wastage. Study design and methods: A retrospective, cross-sectional study of records obtained from the Ministry of Health (MOH) was initiated to report trends and statistics on annual whole blood donors and blood product utility from 2010 to 2020. Data collected in 2020 was further characterized for donors' demographics, laboratory readiness, and staffing. Results: The average number of annual blood donors over the last decade (2010-2020) was 325,847.3 ± 43,160. The forecasted blood donation and dispatch trends suggest a significant increase in blood demand (R2 = 0.7582) over annual donation rates (R2 = 0.2356). In 2020, 342,460 nationwide blood donations were registered in governmental donation centers and females constituted a mere 2.5 %. Approximately 60 % of whole blood donation was voluntary, 36% was compensatory, and 4% was part of driving license renewal. The highest blood donation rate per 1,000 inhabitants was observed in Taif (69.8) and Alqonfoda (45.0). Eastern directory and Madinah had the most successful donation campaigns attracting 53% and 50% of total annual donations, respectively. Notably, Tabouk, Hai'l, and Albaha had the highest blood product wastage medians. Conclusion: Blood donation rates and impetus, staffing ratios, and laboratory readiness and wastage varied among the various directories. Laboratory managers and medical directors need to increase efforts to refine current guidelines in order to comply with the transformation plan of the health sector.

Calcium-oxidative stress signaling axis and casein kinase 1α mediate eryptosis and hemolysis elicited by novel p53 agonist inauhzin.

Inauhzin (INZ) is a novel p53 agonist with antitumor activity. Anemia is a common side effect of chemotherapy and may arise from red blood cell (RBC) hemolysis or eryptosis. In this study, we investigate the mechanisms of INZ toxicity in human RBCs. RBCs were isolated from healthy donors and treated with antitumor concentrations of INZ (5-500 µM) for 24 h at 37 °C. Hemoglobin was photometrically measured, and cells were stained with Annexin-V-FITC for phosphatidylserine (PS), Fluo4/AM for calcium, and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) for oxidative stress. INZ caused significant dose-responsive, calcium-dependent hemolysis starting at 40 µM. Furthermore, INZ significantly increased Annexin-positive cells and Fluo4 and DCF fluorescence. The cytotoxicity of INZ was also significantly mitigated in presence of D4476. INZ possesses hemolytic and eryptotic potential characterized by cell membrane scrambling, intracellular calcium overload, cell shrinkage, and oxidative stress secondary to calcium influx from the extracellular space.