Modeling hemoglobin levels of patients chronically transfused with red blood cells.

BACKGROUND: Good models of chronic red blood cell (RBC) transfusion can improve care by accurately estimating time-dependent hemoglobin (Hb) levels and clinically relevant transfusion parameters. STUDY DESIGN AND METHODS: A previously derived mathematical model based on overall Hb mass balance (HMB model) with input parameters of transfused units, transfusion efficiency, RBC lifespan, endogenous Hb, and transfusion interval was applied to three separate clinical studies spanning six transfusion conditions across patient populations with myelodysplastic syndrome (MDS) or thalassemia. RESULTS: The HMB model accurately predicted mean pretransfusion Hb levels for each ensemble cohort of patients with thalassemia or MDS. Dynamic changes in Hb levels were modeled as a function of changes in key input parameters. Improving the 24-h post-transfusion RBC survival from 72% to 86% can be used to either (1) reduce RBC usage by 15%-20% through longer transfusion intervals or (2) increase pretransfusion [Hb] by 8%-11% while maintaining a constant transfusion interval. DISCUSSION: The endogenous Hb level is introduced in the HMB model which represents the patient's self-contribution to overall Hb levels via the autologous RBC lifecycle and was estimated to be 5.0 g/dL for patients with MDS or thalassemia. Transfusion therapy approaches and complementary therapeutics can target multiple, unique model inputs while monitoring net, overall impact on transfusion efficacy. Applying the HMB model to fit individual patient Hb fluctuations will be explored in the future.

Improved Ion Mobility Separation and Structural Characterization of Steroids using Derivatization Methods.

Steroids are an important class of biomolecules studied for their role in metabolism, development, nutrition, and disease. Although highly sensitive GC- and LC-MS/MS-based methods have been developed for targeted quantitation of known steroid metabolites, emerging techniques including ion mobility (IM) have shown promise in improved analysis and capacity to better identify unknowns in complex biological samples. Herein, we couple LC-IM-MS/MS with structurally selective reactions targeting hydroxyl and carbonyl functional groups to improve IM resolution and structural elucidation. We demonstrate that 1,1-carbonyldiimidazole derivatization of hydroxyl stereoisomer pairs such as testosterone/epitestosterone and androsterone/epiandrosterone results in increased IM resolution with ΔCCS > 15%. Additionally, performing this in parallel with derivatization of the carbonyl group by Girard's Reagent P resulted in unique products based on relative differences in number of each functional group and C17 alkylation. These changes could be easily deciphered using the combination of retention time, collision cross section, accurate mass, and MS/MS fragmentation pattern. Derivatization by Girard's Reagent P, which contains a fixed charge quaternary amine, also increased the ionization efficiency and could be explored for its potential benefit to sensitivity. Overall, the combination of these simple and easy derivatization reactions with LC-IM-MS/MS analysis provides a method for improved analysis of known target analytes while also yielding critical structural information that can be used for identification of potential unknowns.

Modelling haemoglobin incremental loss on chronic red blood cell transfusions.

BACKGROUND AND OBJECTIVES: Understanding the impact of red blood cell (RBC) lifespan, initial RBC removal, and transfusion intervals on patient haemoglobin (Hb) levels and total iron exposure is not accessible for chronic transfusion scenarios. This article introduces the first model to help clinicians optimize chronic transfusion intervals to minimize transfusion frequency. MATERIALS AND METHODS: Hb levels and iron exposure from multiple transfusions were calculated from Weibull residual lifespan distributions, the fraction effete RBC removed within 24-h (Xe ) and the nominal Hb increment. Two-unit transfusions of RBCs initiated at patient [Hb] = 7 g/dl were modelled for different RBC lifespans and transfusion intervals from 18 to 90 days, and Xe from 0.1 to 0.5. RESULTS: Increased Xe requires shorter transfusion intervals to achieve steady-state [Hb] of 9 g/dl as follows: 30 days between transfusions at Xe  = 0.5, 36 days at Xe  = 0.4, 42 days at Xe  = 0.3, 48 days at Xe  = 0.2 and 54 days at Xe  = 0.1. The same transfusion interval/Xe pairs result in a steady-state [Hb] = 8 g/dl when the RBC lifespan was halved. By reducing transfused RBC increment loss from 30% to 10%, annual transfusions were decreased by 22% with iron addition decreased by 24%. Acute dosing of iron occurs at the higher values of Xe on the day after a transfusion event. CONCLUSION: Systematic trends in fractional Hb incremental loss Xe have been modelled and have a significant and calculatable impact on transfusion intervals and associated introduction of iron.

Oxidative modifications of glyceraldehyde 3-phosphate dehydrogenase regulate metabolic reprogramming of stored red blood cells.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) plays a key regulatory function in glucose oxidation by mediating fluxes through glycolysis or the pentose phosphate pathway (PPP) in an oxidative stress-dependent fashion. Previous studies documented metabolic reprogramming in stored red blood cells (RBCs) and oxidation of GAPDH at functional residues upon exposure to pro-oxidants diamide and H2O2 Here we hypothesize that routine storage of erythrocyte concentrates promotes metabolic modulation of stored RBCs by targeting functional thiol residues of GAPDH. Progressive increases in PPP/glycolysis ratios were determined via metabolic flux analysis after spiking (13)C1,2,3-glucose in erythrocyte concentrates stored in Additive Solution-3 under blood bank conditions for up to 42 days. Proteomics analyses revealed a storage-dependent oxidation of GAPDH at functional Cys152, 156, 247, and His179. Activity loss by oxidation occurred with increasing storage duration and was progressively irreversible. Irreversibly oxidized GAPDH accumulated in stored erythrocyte membranes and supernatants through storage day 42. By combining state-of-the-art ultra-high-pressure liquid chromatography-mass spectrometry metabolic flux analysis with redox and switch-tag proteomics, we identify for the first time ex vivo functionally relevant reversible and irreversible (sulfinic acid; Cys to dehydroalanine) oxidations of GAPDH without exogenous supplementation of excess pro-oxidant compounds in clinically relevant blood products. Oxidative and metabolic lesions, exacerbated by storage under hyperoxic conditions, were ameliorated by hypoxic storage. Storage-dependent reversible oxidation of GAPDH represents a mechanistic adaptation in stored erythrocytes to promote PPP activation and generate reducing equivalents. Removal of irreversibly oxidized, functionally compromised GAPDH identifies enhanced vesiculation as a self-protective mechanism in ex vivo aging erythrocytes.

Physicochemical stability of lipid injectable emulsions: correlating changes in large globule distributions with phase separation behavior.

Single particle optical sensing (SPOS) and visual inspection were used to characterize a series of lipid injectable emulsions (n=21) featuring three lipid types, two electrolyte conditions, and three pH levels (7.0, 4.75, and 2.5). Seven of the twenty-one sample conditions exhibited phase separation instability by visual inspection within 98 h of emulsion preparation. The phase instability was driven by electrolyte type and pH, and "cracking" phenomena were independent of lipid type despite the base lipids ranging almost two orders of magnitude in PFAT5 levels. Logistic regression analysis showed that the PFAT5 level determined 1h after admixture preparation was not correlated with phase separation behavior. However, PFAT5 measured at later times showed much improved correlations with emulsion instability. PFAT5 was highly correlated with neighboring cumulative distributions termed PFATX where X=2-10 microm. Although the admixtures studied were not clinically relevant, the data demonstrate some limitations of developing empirical correlations between single-point SPOS measurements and emulsion instability. An alternative limit test for emulsion stability based on the rate of change in the large globule counts is proposed to mitigate inherent deficiencies in the current USP Chapter 729 limit test based on single-point determination of PFAT5 values.