Blood collection and storage of RBCs:
Blood samples used in this study were obtained from healthy donors of age at least 18 years or above with written informed consent from the National Institute of Health (NIH) Blood Center, Bethesda, Maryland (FDA/CBER, IRB protocol 03084B) [amendment 03-120B (for red cells)]. All research performed in this study are in accordance with relevant guidelines/regulations approved by Research Involving Human Subjects Committee (RIHSC 2021-CBER-041). For RBC storage, approximately 100 ml of whole blood was first passed through a neonatal high efficiency leukocyte reduction filter and then centrifuged at 2500 X g for 10 mins to separate RBCs from platelets rich plasma. After removal of plasma and top-buffy coat, packed RBCs were mixed with 22 ml of AS-3 storage solution and then stored at 4°C in a 100 ml capacity neo-natal red-cell storage bag (Neo Bag) for up to 49 days following a standard blood banking protocol. For some short-term experiments, RBCs were also stored in a dextrose (1gm/L) containing PBS (DPBS). Packed RBCs were mixed with equal volume of DPBS after removing the plasma and stored in 100 ml Neo bags for up to 14 days.
Treatments of RBCs with curcumin-gel formulation:
In this study, we used VAS-101, a novel non-aqueous, biocompatible gel formulation (Vascarta Inc, Summit, NJ, USA), containing a very high concentration of curcuminoids (0.1 M). The curcuminoids (containing high levels of all three naturally occurring curcuminoids) were obtained from a proprietary turmeric root extract (Curcugen®, Dolcas-Biotech Inc, Landing, NJ, USA) dissolved in a PEG 400 based solvent to promote high solubility and stability. The gel formulation without curcuminoids served as the vehicle. Both VAS-101 and the vehicle were developed and provided by Vascarta Inc.
For the short-term study in DPBS, 500 µM curcumin was added to RBCs in storage bags on the first day (0d) of storage. The same volume of a vehicle was added to another bag as the non-treatment control. For the longer-term storage, curcumin (100 µM) or vehicle were added to respective storage bags on day 28, 35, 42 and 49, respectively.
Spectrophotometric analysis:
1ml of the stored blood was taken at day 1, 7 and 14, respectively, and incubated for 1 hour at 37 °C, and then equilibrated at room temperature. The solution was then washed with 2 ml PBS, gently stirred, and then centrifuged for 5 minutes. The supernatant was removed. This process was repeated twice. The RBCs were lysed by adding 3ml of water, gently stirred and left to stand for 10 minutes at room temperature. NaCl (24mg) was added to the lysate and the mixture was centrifuged at 4000xg for 10 minutes. The supernatant was filtered with 0.2 µM to remove RBC membranes. The solution was concentrated and the Hb concentration was measured50.
Autoxidation and hydrogen peroxide-mediated oxidation of RBCs
Spontaneous oxidation of RBCs (60µM/heme) taken from the stored blood in AS3 solution in the presence/absence of curcumin (100 µM) was measured spectrophotometrically for 35 days. Spectral measurements were captured at room temperature for 24 hours. A stock solution of 60 µM of Hb (per heme) was prepared for oxidation experiments. The Hb solution was treated with 20 mM H2O2 for 5 minutes followed by an immediate addition of 2mM Na2S to capture transient ferryl Hb. Spectra were captured at each stage of the Hb transformation (oxyHb, metHb, ferrylHb and sulfHb). Extinction coefficients for each species were used as previously reported by our group50.
Gel electrophoresis and immunoblotting:
Cell lysate proteins were resolved by SDS-PAGE using precast 4-20% NuPAGE bis-tris gels (Thermo Fisher Scientific, Waltham, MA, USA) and then transferred to nitrocellulose membranes (BioRad, Hercules, CA, USA) using standard immunoblotting techniques. Nitrocellulose membranes were processed with different specific primary antibodies [e.g., anti-β actin (ab8227), anti-band3 (ab108414), anti-phospho Y359 band 3 (ab77236) and anti-phospho Y21 band 3 (ab125070) (Abcam, Cambridge, MA, USA)]. Appropriate HRP-conjugated goat anti-mouse IgG (ab97040) and anti-rabbit IgG (ab205718) secondary antibodies were also obtained from Abcam (Cambridge, MA, USA).
Measurement of ROS, ATP and protein carbonylation in aged RBCs:
Measurement of ROS in RBCs were carried out fluorometrically using a cell-permeant fluorometric probe 2’,7’-dichlorofluorescin diacetate (DCFDA) that detects different reactive oxygen species (ROS) including hydroxyl, peroxyl radicals 51. Upon oxidation, fluorescent DCF was detected by fluorescence spectroscopy with excitation/emission at 495 nm / 530 nm (26). Briefly, RBCs (995 μL 10% v/v suspension in PBS) were incubated with 5 μL of DCFDA (10 mol/L) at 37 °C for 30 min. Following the incubation, the suspension was further diluted 20 times in PBS and the fluorescence was measured using a Synergy-HTX 96-well plate fluorimeter (Biotek Instruments, Winooski, VT, USA). ROS formation was expressed as relative fluorescence units (RUF)/mg Hb.
Intracellular ATP levels in fresh and stored RBCs were measured using a colorimetric ATP-assay kit from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA) following a method previously published51,52. First, fresh or stored RBCs were washed and then resuspended in PBS containing 1% glucose, 170mg/L adenine and 5g/L mannitol for the ATP measurement. ATP concentration was determined by phosphorylating glycerol, resulting in a colorimetric (570 nm) product proportional to the amount of ATP present using a commercial kit from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA).
Protein carbonyl content in RBC lysates was assessed by a dinitrophenyl hydrazine (DNPH) based assay kit (ab126287) as a measure of protein oxidation (Abcam, Cambridge, MA, USA). In these experiments, carbonyl groups in protein side chains are derivatized to DNP-hydrazone following reaction with DNPH. The absorbance of DNP hydrazones formed in this reaction were measured at 375 nm using a BioTek Synergy HTX microplate reader (Agilent, Santa Clara, CA, USA).
Statistical analysis:
Plotting of raw data and all statistical calculations were done using GraphPad Prism 8 software. All values are expressed as mean ± SD and error bars in the bar diagrams are indicative of SD. A p-value of <0.05 was considered statistically significant. The difference between two means were compared using paired Student’s t-test.
Proteomic analysis of RBCs stored for 35 days:
Sample preparation:
Protein extraction from RBC lysates was done using lysis buffer (8M urea, 50 mM Tris HCl pH 8.0, 150 mM NaCL, 1x Roche Complete protease inhibitor). Sonication was done using a QSonica sonic probe with the following settings: Amplitude 50%, Pulse 10 x 1s, 1 on and 1 off. The lysate was then incubated at room temperature for 1 hr with mixing at 1,000 rpm in an Eppendorf Thermomixer. The lysate was clarified by centrifugation at 10K g for 10 min at 25°C.
Proteolysis of extracted protein:
20 µg of each sample was reduced with 14 mM dithiothreitol at 25°C for 30 minutes followed by alkylation with 14 mM iodoacetamide at 25°C in the dark. Proteolysis was done using 2.5 µg trypsin (Promega sequencing grade) at 37°C overnight. The proteolyzed samples were cooled to room temperature. The volume of the sample was brought to 0.5 ml with ammonium bicarbonate. The proteolyzed samples were centrifuged at 10,000 x g and desalted using a Waters HPB solid phase extraction plate. Samples were lyophilized and reconstituted with 0.1 % TFA prior to MS analyses.
Mass spectrometry:
Mass spectrometry experiments were carried out at Bioworks Laboratories (Ann Arbor, MI, USA). The equivalent of 1µg of each digest was analyzed by nano LC-MS/MS with a Waters NanoAcquity HPLC system interfaced to a ThermoFisher Fusion Lumos mass spectrometer. Peptides were loaded on a trapping column and eluted over a 75µm analytical column at 350 nL/min with a 2hr reverse phase gradient; both columns were packed with Luna C18 resin (Phenomenex). The mass spectrometer was operated in data-dependent mode, with the Orbitrap set at a resolution of 60,000 FWHM and 15,000 FWHM for MS and MS/MS, respectively. The instrument was run with a 3s cycle for MS and MS/MS. Advanced Precursor Determination (APD) was employed.
Proteomic data analysis:
Raw files from the mass spectrometric analysis were converted to .mgf file format prior to searching against the Swiss Prot Database for protein identification. The database search was done with the following parameters: two missed cleavages, peptide tolerance 10 ppm, MS/MS tol. +/- 0.1 Dalton, variable modification (C) carbamidomethylation, (M) oxidation, (M) deoxidation, (C) trioxidation, (H W) oxidation, peptide charge = 1+,2+,3+. The data files from Mascot were then submitted to Scaffold for peptide and protein validation using “Peptide Prophet” and “Protein Prophet”. Probabilities were set to 95% for peptide identification and 90% for protein identifications. Label free quantitation was done using Scaffold’s “weighted spectral counting method”. The Volcano plot was generated by Scaffold 5 (mass spectrometry software).
Post Translational Modifications (PTMs) were identified by searching the initial results obtained from the Mascot Search of the raw mass spectrometry data using an error tolerant search. The results of the error tolerant search were then searched using Scaffold v 5 for file conversion and peptide and protein validation. These results were then submitted to Scaffold PTM for validation of the PTM assignment, quantitation and statistical analysis.
Circulation lifetime and post-transfusion recovery of curcumin treated RBCs:
Animal preparations:
Animal handling and care followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the University of California San Diego Institutional Animal Care and Use Committee approved the experimental protocol. All methods were carried out in accordance with the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments). Guinea pigs weighing between 300–400 g were used in this study.
Blood collection and preparation:
Guinea pigs were anesthetized with isoflurane (Drägerwerk AG, Lübeck, Germany) in compressed room air (flow rate 1.0 LPM) slowly, by increasing the isoflurane 0.4% every 3 min until a surgical depth of anesthesia was achieved, typically 3%. Under anesthesia, a femoral artery catheter was implanted. Each donor bled freely into 1.4 mL of CP2D taken from an AS-3 blood preparation kit (Haemonetics Corporation, Braintree, MA, USA) until 50% of blood volume was lost. Donor blood was then pooled, and CP2D concentration was adjusted to 14%. Pooled blood was centrifuged at 1000 g for 7 minutes, and the supernatant removed. AS-3 (22%/vol) was then added, and the blood was mixed gently by inverting the bag for 1 minute. Pooled blood was then passed through a neonatal leukocyte reduction filter (Haemonetics Corporation, Braintree, MA, USA). RBC units intended for storage received 500 µM curcuminoids on the first day (0d) of storage. The same volume of a vehicle (no curcuminoids) was added to another bag to serve as the vehicle control. At the 2nd and 3rd weeks of storage, blood was radiolabeled with Technetium-99 (Tc99) as described by Zink et. al53. Briefly, RBC samples (1.0 mL) were added to a sterile reaction vial and gently mixed to dissolve the lyophilized UltraTag-RBC (UltraTag-RBC, Mallinckrodt, St. Louis, MO, USA), and allowed to react for 5 to 7 minutes. Then, UltraTag pH buffers were added to adjust pH, by gently mixing them into the reaction vial and by inverting the container. Samples were washed with sterile PBS twice and centrifuged to remove unreacted Tc99, and labeled RBCs were injected. 200 μL of Tc99 radiolabeled blood (approximately 2% of blood volume) was delivered I.V. to male anesthetized guinea pigs and 65 μL samples were drawn at 5 minutes, 30 minutes, and 24 hours post-injection. Samples were all run for detection of radioactivity on a Cobra II gamma counter (Packard Instrument Co., Meriden, CT, USA) at the same time so that the reported counts were independent of sample time and only representative of the still-circulating radio-labeled RBCs.
ATP and 2,3DPG: Aliquots of RBCs were mixed with cold trichloroacetic acid (DiaSys Deutschland, Flacht, Germany) and vortexed for 60 seconds. Aliquots sat on ice for 5 minutes and were then centrifuged at 3600g at 4°C for 10 minutes. The supernatant was removed and frozen at -80°C for later analysis. Supernatants were assayed enzymatically with commercially available kits. DPG was measured with the Roche 2,3-Diphosphoglycerate kit (Roche Diagnostics, Indianapolis IN, USA) according to manufacturer’s instruction. ATP was measured by DiaSys ATP Hexokinase FS kit (DiaSys Diagnostic Systems GmbH, Holzheim, Germany) according to manufacturer’s instruction.