scholarly journals Neutrophils Isolated from Septic Patients Exhibit Elevated Uptake of Vitamin C and Normal Intracellular Concentrations despite a Low Vitamin C Milieu

Antioxidants ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 1607
Author(s):  
Anitra C. Carr ◽  
Stephanie Bozonet ◽  
Juliet Pullar ◽  
Emma Spencer ◽  
Patrice Rosengrave ◽  
...  
Keyword(s):  
Vitamin C ◽  
Healthy Volunteers ◽  
Ex Vivo ◽  
Plasma Concentrations ◽  
Dehydroascorbic Acid ◽  
Neutrophil Function ◽  
Healthy Controls ◽  
Lower Plasma ◽  
Circulating Cells ◽  
Plasma Ascorbate

Vitamin C (ascorbate) plays an important role in neutrophil function and is accumulated by the cells either directly via vitamin C transporters (SVCT) or indirectly following oxidation to dehydroascorbic acid. Septic patients are known to have significantly depleted plasma ascorbate status, but little is known about the ascorbate content of their circulating cells. Therefore, we assessed the ascorbate concentrations of plasma, leukocytes and erythrocytes from septic patients and compared these to healthy controls. Non-fasting blood samples were collected from healthy volunteers (n = 20) and critically ill patients with sepsis (n = 18). The ascorbate content of the plasma and isolated neutrophils and erythrocytes was measured using HPLC and plasma myeloperoxidase concentrations were determined using ELISA. Ex vivo uptake of ascorbate and dehydroascorbic acid by neutrophils from septic patients was also assessed. Neutrophils isolated from septic patients had comparable intracellular ascorbate content to healthy volunteers (0.33 vs. 0.35 nmol/106 cells, p > 0.05), despite significantly lower plasma concentrations than the healthy controls (14 vs. 88 µmol/L, p < 0.001). In contrast, erythrocytes from septic patients had significantly lower intracellular ascorbate content than healthy controls (30 vs. 69 µmol/L, p = 0.002), although this was 2.2-fold higher than the matched plasma concentrations in the patients (p = 0.008). Higher concentrations of myeloperoxidase, a source of reactive oxygen species, were observed in the septic patients relative to healthy controls (194 vs. 14 mg/mL, p < 0.0001). In contrast to neutrophils from healthy volunteers, the neutrophils from septic patients demonstrated elevated uptake of extracellular ascorbate. Overall, neutrophils from septic patients exhibited comparable intracellular ascorbate content to those from healthy controls, despite the patients presenting with hypovitaminosis C. The mechanisms involved are currently uncertain, but could include increased generation of dehydroascorbic acid in septic patients, enhanced basal activation of their neutrophils or upregulation of their vitamin C transporters.

scholarly journals The Role of Physiological Vitamin C Concentrations on Key Functions of Neutrophils Isolated from Healthy Individuals

Nutrients ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 1363 ◽  
Author(s):  
Stephanie M. Bozonet ◽  
Anitra C. Carr
Keyword(s):  
Vitamin C ◽  
Immune Cell ◽  
Dehydroascorbic Acid ◽  
Optimal Level ◽  
Neutrophil Function ◽  
Neutrophil Extracellular Trap ◽  
Healthy Individuals ◽  
Healthy Humans ◽  
Ascorbate Uptake

Vitamin C (ascorbate) is important for neutrophil function and immune health. Studies showing improved immune function have primarily used cells from scorbutic animals or from individuals with infectious conditions or immune cell disorders. Few studies have focused on the requirements of neutrophils from healthy adults. Therefore, we have investigated the role of vitamin C, at concentrations equivalent to those obtained in plasma from oral intakes (i.e., 50–200 µmol/L), on key functions of neutrophils isolated from healthy individuals. Cells were either pre-loaded with dehydroascorbic acid, which is rapidly reduced intracellularly to ascorbate, or the cells were activated in the presence of extracellular ascorbate. We measured the effects of enhanced ascorbate uptake on the essential functions of chemotaxis, oxidant production, programmed cell death and neutrophil extracellular trap (NET) formation. We found that neutrophils isolated from healthy individuals already had replete ascorbate status (0.35 nmol/106 cells), therefore they did not uptake additional ascorbate. However, they readily took up dehydroascorbic acid, thus significantly increasing their intracellular ascorbate concentrations, although this was found to have no additional effect on superoxide production or chemotaxis. Interestingly, extracellular ascorbate appeared to enhance directional mobilityin the presence of the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP). Stimulation of the cells in the presence of ascorbate significantly increased intracellular ascorbate concentrations and, although this exhibited a non-significant increase in phosphatidylserine exposure, NET formation was significantly attenuated. Our findings demonstrate the ability of neutrophils to regulate their uptake of ascorbate from the plasma of healthy humans to maintain an optimal level within the cell for proper functioning. Higher oral intakes, however, may help reduce tissue damage and inflammatory pathologies associated with NET formation.

scholarly journals Erythrocyte Cytochrome b Reductase 1 Protein Levels and Structure in Sickle Cell Patients

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3654-3654
Author(s):  
Richard Kulmacz ◽  
Hamidreza Farzaneh ◽  
Kathryn L McElhinney ◽  
Jose M. Rodriguez ◽  
Nicole C. Thomason ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Vitamin C ◽  
Sickle Cell ◽  
Dose Response ◽  
Healthy Controls ◽  
Rbc Membrane ◽  
Coomassie Blue ◽  
Plasma Ascorbate ◽  
Sds Page ◽  
And Control

Abstract In humans, vitamin C (ascorbic acid) is required as substrate or cofactor in a number of important cellular processes. Patients with sickle cell disease (SCD) often are deficient in vitamin C and thus potentially adversely affected. A major determinant of the cellular availability of ascorbate is the plasma level of the vitamin. This reflects the balance between dietary supply, uptake by tissues, excretion, oxidative reactions in the plasma, and RBC dependent recycling or irreversible decomposition of oxidized ascorbate. The mechanisms for depressed plasma ascorbate levels in SCD patients have not been identified, and the effectiveness and safety of dietary ascorbate supplements have not been established. As RBC dysfunction is central to SCD, we have focused on the possibility that SCD patients' RBC membranes have an altered level, structure or function of CYBRD1, a key enzyme in recycling oxidized plasma ascorbate (Fig. 1A). Materials and methods: Blood samples were collected in EDTA tubes from adult SCD patients at routine ambulatory clinic visits and from healthy African American adults. Protocols were approved by institutional review. RBC were isolated by centrifugation and lysed in hypotonic buffer; membranes were isolated by centrifugation and stored at -80 C. Electrophoretic and western blot analyses: RBC membrane proteins were separated by SDS-PAGE, stained with Coomassie Blue and analyzed by densitometry to quantitate total membrane protein (standard: BSA). Alternatively, separated proteins were transferred to nitrocellulose or PVDF and probed with antibodies against CYBRD1. Immunoreactive bands were visualized colorimetrically and quantitated by densitometry (standard: recombinant human CYBRD1 (rCYBRD1)). Mass spectroscopic analyses: CYBRD1 was extracted from RBC membranes with dodecyl maltoside, and further enriched by immunoprecipitation. The immunopurified mixture was separated by SDS-PAGE and stained with Coomassie Blue. Gel pieces containing the CYBRD1 monomers were subjected to in-gel digestion and MS analysis of the tryptic peptides. Results and conclusions: Detailed dose-response analyses found unsuspected positively cooperative behavior between 0 and ~2 ng CYBRD1 in both rCYBRD1 and RBC membrane samples. This invalidated calculations of RBC CYBRD1 content based on linear response to rCYBRD1. However, selected RBC membrane samples were routinely included as quality controls on many blots. Further, the dose-response curves for RBC membrane samples had a consistent shape that fit a simple saturable model with an x-axis offset parameter. It was thus possible to estimate the CYBRD1 content for 36 of the RBC samples using one of them (V7) as reference (Fig. 1B). This permitted our ongoing comparison of CYBRD1 content in patients and controls, and in SCD subtypes. RBC CYBRD1 content in 4 homozygous SCD subjects sampled at 5 regular clinic visits changed little over 7-9 months (averages: 0.32 ± 0.05; 0.25 ± 0.05; 0.41 ± 0.04; and 0.37 ± 0.04 ng CYBRD1/µg RBC membrane protein). This suggests that patients' RBC CYBRD1 contents are relatively stable despite the short RBC half-life, and that a single sampling is sufficient for cross sectional studies. There was no significant difference in CYBRD1 level among ambulatory HbSC (N=6) and HbSS (N=8) patientsand healthy controls (N=8)(Fig. 1C, top). However, there were notable differences in CYBRD1 protein modification, with significantly less modification in CYBRD1 from HbSS patients than either HbSC patients or healthy controls (Fig. 1C, bottom). Similar isoform banding patterns are seen for patient and control CYBRD1 with antibodies against either the N- and C-terminal peptides (Fig. 1D). Retention of epitopes at both ends of CYBRD1 in the isoforms argues against differential proteolysis being involved. The mass spectrometric analyses found extensive oxidative modification of multiple methionine residues, but similar patterns were seen in RBC CYBRD1 from an SCD patient and a healthy control. Phosphorylation was found at multiple sites in residues 200-285 of CYBRD1 from both patient and control, but the fraction phosphorylated appeared much too low to account for the large proportion of CYBRD1 in individual isoforms. Thus, the structural basis for the isoforms' differential gel mobility remains to be identified. Support: American Heart Association, 16GRNT29170013 (R. Kulmacz); NIH 5T35DK007676 (B. Kone) Disclosures No relevant conflicts of interest to declare.

scholarly journals Reduced Plasma Ascorbate and Increased Proportion of Dehydroascorbic Acid Levels in Patients Undergoing Hemodialysis

Life ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1023
Author(s):  
Yuta Doshida ◽  
Mitsuyo Itabashi ◽  
Takashi Takei ◽  
Yuka Takino ◽  
Ayami Sato ◽  
...  
Keyword(s):  
Dehydroascorbic Acid ◽  
Hemodialysis Patients ◽  
Dialysis Patients ◽  
Lower Plasma ◽  
Established Method ◽  
Plasma Ascorbate ◽  
Study Results ◽  
Ckd Stage ◽  
Before And After ◽  
Low Levels

Ascorbate functions as an electron donor and scavenges free radicals. Dehydroascorbic acid (DHA), the oxidized form of ascorbate, is generated as a result of these reactions. While low plasma ascorbate levels have been reported in hemodialysis patients worldwide, no studies have measured DHA because it is not generalized. In this study, we aimed to clarify whether plasma ascorbate levels are low in dialysis patients and whether plasma ascorbate levels fluctuate before and after dialysis. Moreover, we applied our previously established method to measure the plasma ascorbate and DHA levels in chronic kidney disease (CKD) stage G3–G5 non-hemodialysis-dependent patients, and pre- and post-dialysis plasma ascorbate and DHA levels in CKD stage G5D hemodialysis patients. The sample size was calculated using G-power software. The pre-dialysis plasma total ascorbate levels, including DHA, were significantly (56%) lower in hemodialysis patients than in non-hemodialysis-dependent CKD patients. After dialysis, there was a 40% reduction in the plasma total ascorbate levels. Hemodialysis increased the post-dialysis plasma proportions of DHA from 37% to 55%. The study results demonstrated lower plasma total ascorbate levels in hemodialysis patients compared with in non-hemodialysis-dependent CKD patients; these low levels in hemodialysis patients were further reduced by hemodialysis and increased DHA proportion.

Differences in MSU-induced Superoxide Responses by Neutrophils from Gout Subjects Compared to Healthy Controls and a Role for Environmental Inflammatory Cytokines and Hyperuricemia in Neutrophil Function and Survival

2010 ◽  
Vol 37 (6) ◽  
pp. 1228-1235 ◽  
Author(s):  
WILLIAM JOHN MARTIN ◽  
REBECCA GRAINGER ◽  
ANDREW HARRISON ◽  
JACQUIE L. HARPER
Keyword(s):  
Neutralizing Antibodies ◽  
Ex Vivo ◽  
Elevated Serum ◽  
Neutrophil Function ◽  
Superoxide Production ◽  
Healthy Controls ◽  
Serum Samples ◽  
Tnf Α ◽  
Neutrophil Superoxide ◽  
Factor Α

Objective.To determine whether monosodium urate (MSU) crystal-induced superoxide production is greater for neutrophils from patients with gout compared to asymptomatic hyperuricemic and healthy controls, and whether neutrophil functions are altered by an MSU crystal-induced inflammatory environment.Methods.Neutrophils were purified from the whole blood of study participants, restimulated with 500 mg MSU crystalsex vivo, and superoxide production measured using the colorimetric dye WST-1. Purified neutrophils were exposed to conditioned media from MSU crystal-activated blood monocyte cultures with and without neutralizing antibodies for interleukin 1ß (IL-1ß), IL-8 (CXCL8), IL-6, and tumor necrosis factor-α (TNF-α). Neutrophil superoxide production was measured and neutrophil apoptosis and IL-8 production were determined by flow cytometry. Serum samples were collected from participants and analyzed by Lincoplex bead array for IL-1ß, IL-8, IL-6, and TNF-α.Results.Neutrophils from gout and asymptomatic hyperuricemic subjects produced higher levels of MSU crystal-induced superoxide, and a weak trend toward elevated serum cytokines was observed compared to healthy controls. A correlation between serum uric acid and elevated neutrophil superoxide production was also observed. Neutrophils exposed to media from MSU crystal-activated monocytes exhibited enhanced superoxide production to MSU-crystal stimulation, increased IL-8 production, and extended cell survival that was predominantly dependent on IL-8, TNF-α and IL-6, respectively.Conclusion.Neutrophils from gout and asymptomatic hyperuricemic individuals are primed for enhanced MSU crystal-induced superoxide production that may be driven by a subclinical inflammatory cytokine environment combined with hyperuricemia. This inflammatory environment likely contributes to elevated neutrophil IL-8 production and survival in the absence of direct crystal stimulation. Asymptomatic hyperuricemia is not associated with suppressed neutrophil function.

Cellular and intracellular transport of vitamin C. The physiologic aspects

2013 ◽  
Vol 154 (42) ◽  
pp. 1651-1656 ◽  
Author(s):  
András Szarka ◽  
Tamás Lőrincz
Keyword(s):  
Ascorbic Acid ◽  
Vitamin C ◽  
Intracellular Transport ◽  
Initial Rate ◽  
Plasma Concentrations ◽  
Dehydroascorbic Acid ◽  
Reduced Form ◽  
Human Diet ◽  
Sodium Dependent ◽  
The One

Vitamin C requirement is satisfied by natural sources and vitamin C supplements in the ordinary human diet. The two major forms of vitamin C in the diet are L-ascorbic acid and L-dehydroascorbic acid. Both ascorbate and dehydroascorbate are absorbed along the entire length of the human intestine. The reduced form, L-ascorbic acid is imported by an active mechanism, requiring two sodium-dependent vitamin C transporters (SVCT1 and SVCT2). The transport of the oxidized form, dehydroascorbate is mediated by glucose transporters GLUT1, GLUT3 and possibly GLUT4. Initial rate of uptake of both ascorbate and dehydroascorbate is saturable with increasing external substrate concentration. Vitamin C plasma concentrations are tightly controlled when the vitamin is taken orally. It has two simple reasons, on the one hand, the capacity of the transporters is limited, on the other hand the two Na+-dependent transporters can be down-regulated by an elevated level of ascorbate. Orv. Hetil., 154 (42), 1651–1656.

Plasma D Dimer: A Useful Marker of Fibrin Breakdown in Renal Failure

1989 ◽  
Vol 61 (03) ◽  
pp. 522-525 ◽  
Author(s):  
M P Gordge ◽  
R W Faint ◽  
P B Rylance ◽  
H Ireland ◽  
D A Lane ◽  
...  
Keyword(s):  
Renal Failure ◽  
Renal Function ◽  
Acute Renal Failure ◽  
Monoclonal Antibodies ◽  
Diabetic Nephropathy ◽  
Renal Disease ◽  
Plasma Concentrations ◽  
Significant Negative Correlation ◽  
Healthy Controls ◽  
D Dimer

SummaryD dimer and other large fragments produced during the breakdown of crosslinked fibrin may be measured by enzyme immunoassay using monoclonal antibodies. In 91 patients with renal disease and varying degrees of renal dysfunction, plasma D dimer showed no correlation with renal function, whereas FgE antigen, a fibrinogen derivative which is known to be cleared in part by the kidney, showed a significant negative correlation with creatinine clearance. Plasma concentrations of D dimer were, however, increased in patients with chronic renal failure (244 ± 3l ng/ml) (mean ± SEM) and diabetic nephropathy (308 ± 74 ng/ml), when compared with healthy controls (96 ± 13 ng/ml), and grossly elevated in patients with acute renal failure (2,451 ± 1,007 ng/ml). The results indicate an increase in fibrin formation and lysis, and not simply reduced elimination of D dimer by the kidneys, and are further evidence of activated coagulation in renal disease. D dimer appears to be a useful marker of fibrin breakdown in renal failure.

Pharmacokinetics and Effects on Fibrinolytic and Coagulation Parameters of Two Doses of Recombinant Tissue-Type Plasminogen Activator in Healthy Volunteers

1986 ◽  
Vol 56 (01) ◽  
pp. 001-005 ◽  
Author(s):  
M Verstraete ◽  
C A P F Su ◽  
P Tanswell ◽  
W Feuerer ◽  
D Collen
Keyword(s):  
Healthy Volunteers ◽  
Plasminogen Activator ◽  
Degradation Products ◽  
Plasma Concentrations ◽  
State Level ◽  
Compartment Model ◽  
Tissue Type ◽  
Maximum Plasma ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

SummaryPharmacokinetics and pharmacological effects of two intravenous doses of recombinant tissue-type plasminogen activator (rt-PA) (40 and 60 mg over 90 min) were determined in healthy volunteers. Mean maximum plasma concentrations were 1080 and 1560 ng/ml respectively. The steady state level during subsequent maintenance infusion of 30 mg over 6 h was 250 ng/ml. The pharmacokinetics of rt-PA showed a bi-exponential disappearance from plasma consistent with a 2-compartment model of t½α = 5.7 min, a t½β = 1.3 h and a total clearance of 380 ml/min.Mean fibrinogen levels at the end of the infusions of 40 mg or 60 mg rt-PA over 90 min, measured in thawed plasma samples collected on citrate/aprotinin, decreased to 74% and 57% of the preinfusion values respectively. Plasminogen fell to 55% and 48%, and α2-antiplasmin to 28% and 18% of initial values. No further decrease of these parameters was observed during the infusion of 30 mg rt-PA over 6 h. Only 2% of the preinfusion fibrinogen levels could be recovered as fibrinogen-fibrin degradation products. This moderate extent of systemic fibrinogenolysis is much less than that reported for therapeutic i.v. infusions of streptokinase.

Pharmacokinetics and Safety Assessment of Anti-HIV Dapivirine Vaginal Microbicide Rings with Multiple Dosing

2020 ◽  
Author(s):  
Lungwani Muungo
Keyword(s):  
Ex Vivo ◽  
Oral Treatment ◽  
Plasma Concentrations ◽  
Vaginal Ring ◽  
Vaginal Fluid ◽  
Cervical Tissue ◽  
Double Blind ◽  
Group A ◽  
Group B ◽  
Anti Hiv

Objectives: Self-administered vaginal rings are a promising method for delivery of topical anti-HIV microbicidesand might offer an adherence advantage over daily or coitally-dependent dosage forms such as gels. This trialassessed the safety and pharmacokinetic aspects of the Dapivirine Vaginal Ring-004 when worn as multiple rings oversequential periods of ring use by healthy, sexually-active, HIV-negative women.Methods: This double-blind trial was conducted among 48 women (18-40 years). Participants were randomlyassigned to two groups (A or B) and received (3:1) either the dapivirine or a placebo vaginal ring. Group A used tworings over a 56-day period and Group B used three rings over a 57-day period. Safety evaluations were conductedthroughout the trial. Dapivirine concentrations were measured in plasma, vaginal fluid and cervical tissue samplescollected during and after the 56 days (Group A) or 57 days (Group B) of vaginal ring use.Results: Ring-004 was safe and well tolerated in all participants. The pharmacokinetic profile demonstrated arapid increase in plasma and vaginal fluid concentrations and achieved concentrations in vaginal fluids and cervicaltissue well above the in vitro IC99 in cervical tissue (3.3 ng/mL) that were sustained for a 28 to 35-day ring use period(approximately 3000 times higher in vaginal fluids and 14 -1000 times higher in cervical tissue). Drug levels wereassociated with significant inhibitory activity of genital secretions against HIV ex vivo, a biomarker of pharmacodynamics.Individual plasma dapivirine concentrations did not exceed 553 pg/mL and were well below plasma concentrations atthe maximum tolerated dose for oral treatment (mean Cmax 2286 ng/mL).Conclusions: The consecutive use of several rings over a period of up to 57 days was safe and well tolerated, andPK data indicate that a single Ring-004 is likely to be protective for at least 35 days.

scholarly journals Viscoelastometry for detecting oral anticoagulants

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Philipp Groene ◽  
Daniela Wagner ◽  
Tobias Kammerer ◽  
Lars Kellert ◽  
Andreas Giebl ◽  
...  
Keyword(s):  
Healthy Volunteers ◽  
Prospective Observational Study ◽  
Oral Anticoagulants ◽  
Plasma Concentrations ◽  
Factor Xa ◽  
Clotting Time ◽  
Emergency Situations ◽  
Tissue Plasminogen ◽  
The Impact

Abstract Background Determination of anticoagulant therapy is of pronounced interest in emergency situations. However, routine tests do not provide sufficient insight. This study was performed to investigate the impact of anticoagulants on the results of viscoelastometric assays using the ClotPro device. Methods This prospective, observational study was conducted in patients receiving dabigatran, factor Xa (FXa)-inhibitors, phenprocoumon, low molecular weight heparin (LMWH) or unfractionated heparin (UFH) (local ethics committee approval number: 17–525-4). Healthy volunteers served as controls. Viscoelastometric assays were performed, including the extrinsic test (EX-test), intrinsic test (IN-test) Russel’s viper venom test (RVV-test), ecarin test (ECA-test), and the tissue plasminogen activator test (TPA-test). Results 70 patients and 10 healthy volunteers were recruited. Clotting time in the EX-test (CTEX-test) was significantly prolonged versus controls by dabigatran, FXa inhibitors and phenprocoumon. CTIN-test was prolonged by dabigatran, FXa inhibitors and UFH. Dabigatran, FXa inhibitors and UFH significantly prolonged CTRVV-test in comparison with controls (median 200, 207 and 289 vs 63 s, respectively; all p < 0.0005). Only dabigatran elicited a significant increase in CTECA-test compared to controls (median 307 vs 73 s; p < 0.0001). CTECA-test correlated strongly with dabigatran plasma concentration (measured by anti-IIa activity; r = 0.9970; p < 0.0001) and provided 100% sensitivity and 100% specificity for detecting dabigatran. Plasma concentrations (anti-XA activity) of FXa inhibitors correlated with CTRVV-test (r = 0.7998; p < 0.0001), and CTRVV-test provided 83% sensitivity and 64% specificity for detecting FXa inhibitors. Conclusions In emergency situations, ClotPro viscoelastometric assessment of whole-blood samples may help towards determining the presence and type of anticoagulant class that a patient is taking. Trial registration German clinical trials database ID: DRKS00015302.

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