Involving men in pregnancy: a cross-sectional analysis of the role of self-efficacy, gender-equitable attitudes, relationship dynamics and knowledge among men in Kinshasa.

BACKGROUND: Although male participation in maternal health has gained increasing recognition and support over the years, little is known about male involvement during pregnancy in the Democratic Republic of the Congo. This paper identified male involvement patterns during pregnancy and evaluated their associations with pregnancy and birth preparedness knowledge, gender-equitable attitudes, self-efficacy, and co-parental relationship factors. Lastly, it explored the moderating effect of gender-equitable attitudes and intimate partner violence on the association between relationship satisfaction and male involvement. METHODS: Data from the 2018 Momentum baseline study were analyzed to determine the predictors of involvement. Factor analysis was used to create male involvement indices for antenatal carebirth preparedness and shared decision making. The sample consisted of 1,674 male partners of nulliparous pregnant women who were 6 months pregnant at baseline. RESULTS: Male involvement in individual pregnancy-related activities was low, ranging from 11% (finding a blood donor) to 49% (saving money during emergencies). Knowledge of the number of antenatal care visits, birth preparedness steps, and newborn danger signs were positively associated with involvement in antenatal care/birth preparedness activities while knowledge of antenatal care benefits was positively associated with involvement in shared decisions. Increasing relationship satisfaction and self-efficacy were associated with antenatal care/birth preparedness involvement and for shared decisions, a positive association with gender-equitable attitude and a negative association with self-efficacy were observed. Moderation effects were also detected. CONCLUSIONS: The findings suggest that male involvement is multifaceted and factors influencing involvement vary depending on the type of involvement. Addressing these factors can improve male participation in maternal health.

Impact of the Momentum pilot project on male involvement in maternal health and newborn care in Kinshasa, Democratic Republic of the Congo: a quasi-experimental study.

BACKGROUND: The World Health Organization recommends that programs that seek to improve maternal and newborn health outcomes actively involve men during pregnancy, childbirth, and postpartum. However, there is little evidence on what strategies work to increase male knowledge of and involvement in antenatal and postnatal care. This study assessed the impact of the Momentum project on male involvement in maternal health and newborn care. The project involved monthly home visits to a cohort of first-time mothers aged 15-24 recruited at six-months gestation and group education sessions for their male partners using the Program P toolkit. Participants were followed-up for 16 months. METHODS: The study used a quasi-experimental design with three intervention and three comparison health zones. Baseline data were collected in 2018 and endline data in 2020. Exploratory factor analysis was used to develop scales of male involvement. We measured the causal influence of Momentum using an intent-to-treat analysis at the health-zone level and a dose-response analysis at the individual level. We used random-effects probit and linear models for outcomes measured at baseline and endline, and treatment effects models with inverse-probability weighting for outcomes measured only at endline. The impact analysis involved 1,204 male partners of first-time mothers with live births. RESULTS: Intervention health zones were associated with an 18.1 percentage point (95% CI [(10.6, 25.6]) increase in knowledge of three or more obstetric danger signs and a 13.9 percentage point (95% CI [6.3, 21.6]) increase in knowledge of newborn danger signs. Significant increases in male involvement in antenatal care (average treatment effect (ATE) = 0.728, 95% CI [0.445, 1.010]), birth planning (ATE = 0.407, 95% CI [0.157, 0.657]), and newborn care (ATE = 0.690, 95% CI [0.359, 1.021]) were found. The magnitude of Momentum's impact increased steadily with the number of prenatal home visits and was statistically significant for all behavioral outcomes except shared decision making. Exposure to both home visits and group education sessions during the prenatal period had a significant impact on all outcomes relative to no exposure. CONCLUSIONS: The study demonstrated the effectiveness of Momentum on male involvement in maternal health and newborn care.

Insights on exclusive breastfeeding norms in Kinshasa: findings from a qualitative study.

BACKGROUND: For optimal growth and development, the World Health Organization recommends that children be exclusively breastfed for the first 6 months of life. However, according to the nationally-representative 2013-2014 Demographic and Health Survey, under 50% of babies in the Democratic Republic of Congo are exclusively breastfed. Although breastfeeding was common in the capital city of Kinshasa, one in five newborns received alternatives to breastmilk during the first 3 days of life. This analysis aimed to identify social norms influencing exclusive breastfeeding, the role of a young first-time mother's (FTM's) social network for her choice to exclusively breastfeed, and perceived social sanctions associated with breastfeeding practices in Kinshasa. METHODS: The qualitative analysis was based on a vignette presented during 14 focus group discussions, with a purposively selected sample (n = 162) of FTMs age 15-24, and the male partners, mothers and mothers-in-law of FTMs age 15-24 in three health zones in Kinshasa in 2017. Thematic content analysis was performed to identify concepts and patterns in the participants' discussions. RESULTS: Overall, community norms were not supportive of exclusive breastfeeding. The main barriers to exclusive breastfeeding were the belief held by most FTMs that exclusive breastfeeding was an uncommon practice; the desire to avoid negative sanctions such as name-calling and mockery for refusal to give babies water in the first 6 months of life; the desire to please key members of their social networks, specifically their mothers and friends, by doing what these influencers expected or preferred them to do; FTMs' own lack of experience with infant feeding; and trust placed in their mothers and friends. CONCLUSION: Social norms can be maintained by the belief about what others do, perceived expectations about what individuals ought to do, the negative sanctions they can face and their preference to conform to social expectations. Thus, addressing cultural beliefs and targeting sensitization efforts to key influencers that provide support to FTMs are needed to promote exclusive breastfeeding in Kinshasa. In doing so, strategies should address the barriers to exclusive breastfeeding including related misconceptions, and improve FTMs' self-efficacy to overcome the influence of others.

Evaluation of a multimedia youth anti-smoking and girls' empowerment campaign: SKY Girls Ghana.

BACKGROUND: Given the long-term health effects of smoking during adolescence and the substantial role that tobacco-related morbidity and mortality play in the global burden of disease, there is a worldwide need to design and implement effective youth-focused smoking prevention interventions. While smoking prevention interventions that focus on both social competence and social influence have been successful in preventing smoking uptake among adolescents in developed countries, their effectiveness in developing countries has not yet been clearly demonstrated. SKY Girls is a multimedia, empowerment and anti-smoking program aimed at 13-16-year old girls in Accra, Ghana. The program uses school and community-based events, a magazine, movies, a radio program, social media and other promotional activities to stimulate normative and behavioral change. METHODS: This study uses pre/post longitudinal data on 2625 girls collected from an interviewer-administered questionnaire. A quasi-experimental matched design was used, incorporating comparison cities with limited or no exposure to SKY Girls (Teshie, Kumasi and Sunyani). Fixed-effects modeling with inverse probability weighting was used to obtain doubly robust estimators and measure the causal influence of SKY Girls on a set of 15 outcome indicators. RESULTS: Results indicate that living and studying in the intervention city was associated with an 11.4 percentage point (pp) (95% CI [2.1, 20.7]) increase in the proportion of girls perceiving support outside their families; an 11.7 pp. decrease (95% CI [- 20.8, - 2.6]) in girls' perception of pressure to smoke cigarettes; a 12.3 pp. increase (95% CI [2.1, 20.7]) in the proportion of girls who had conversations with friends about smoking; an 11.7 pp. increase (95% CI [3.8, 20.8]) in their perceived ability to make choices about what they like and do not like, and 20.3 pp. (95% CI [- 28.4, - 12.2]) and 12.1 pp. (95% CI [- 20.7, - 3.5]) reductions in the proportion agreeing with the idea that peers can justify smoking shisha and cigarettes, respectively. An analysis of the dose-effect associations between exposure to multiple campaign components and desired outcomes was included and discussed. CONCLUSION: The study demonstrates the effectiveness of a multimedia campaign to increase perceived support, empowerment and improve decision-making among adolescent girls in a developing country.

Comprehensive Biochemical and Biophysical Characterization of Hemoglobin-Based Oxygen Carrier Therapeutics: All HBOCs Are Not Created Equally.

The development of hemoglobin (Hb)-based oxygen carriers (HBOCs) has been hampered because of safety concerns in humans. Chemical and/or genetic modifications of the Hb introduce varied structural and conformational constraint on the molecule that resulted in proteins with diverse allosteric responses, nitrosative and oxidative side reactions. Here, we present for the first time a comprehensive biochemical and biophysical comparison of human, bovine, and genetically engineered HBOCs that have been tested in humans. We evaluate oxygen equilibrium and ligand binding kinetics under different experimental conditions as well as their autoxidation kinetics, redox reactions, and heme release. We determined the effects of HBOCs on cellular redox states and mitochondrial respiration. Taken together, these experiments provide a better understanding of the relationship between the structure-function and oxidative reactivity of these proteins. One can therefore select independently among these diverse properties to engineer a safe and effective HBOC with improved biochemical/biophysical characteristics.

Attitudes toward sexual and reproductive health among adolescents and young people in urban and rural DR Congo.

BACKGROUND: In the Democratic Republic of Congo (DRC), onset of sexual intercourse is initiated during adolescence, however only two in ten sexually active unmarried women are using modern contraception. Improving adolescents' and young peoples' knowledge and practices related to sexual and reproductive health (SRH) is necessary to improve health outcomes. However, little is known about the SRH attitudes and needs among young people in the DRC. The study aims to contribute to the available evidence by examining adolescents' and young people's insights on their cultural norms, practices and attitudes towards SRH services. METHODS: Fourteen focus group discussions were conducted with a total of 224 adolescents and young people aged 15-24 years in urban and rural areas of the DRC. The topics discussed and age groups of participants differed somewhat in the urban and rural areas. Data were analyzed to identify themes in the participants' discussion of their attitudes towards SRH. RESULTS: Regardless of age differences, common themes emerged. Both in rural and urban areas premarital sex was largely sanctioned by peers but not adults; adolescents feared pregnancy and had limited knowledge of contraceptive methods. Many were misinformed that certain common pharmaceutical products (e.g., decaris) prevent pregnancy. Key barriers to accessing contraception from health facilities and pharmacies included shame and stigma; urban participants also cited cost and judgmental attitudes of health providers. CONCLUSION: Addressing the SRH needs of adolescents and young people can have life-long protective benefits. Increasingly decision-makers and gatekeepers in the DRC are accepting the concept of providing SRH services and information to young people. This study shows the pressing need for information and services for young people in both urban and rural areas. The continued expansion SRH programming to all health zones and the developed of the National Strategic Plan for Health and Wellbeing of Adolescents and Youth 2016-2020 are steps toward that goal.

Effects of the Momentum project on postpartum family planning norms and behaviors among married and unmarried adolescent and young first-time mothers in Kinshasa: A quasi-experimental study.

This study evaluated the effect of Momentum-an integrated family planning, maternal and newborn health, and nutrition intervention-on postpartum family planning norms and behaviors among ever married and never-married first-time mothers age 15-24 in Kinshasa, Democratic Republic of the Congo. Using data collected in 2018 and 2020, we conducted an intent-to-treat analysis among 1,927 first-time mothers who were about six-months pregnant at enrollment. Difference-in-differences models were run for panel data and treatment effects models with inverse-probability weighting for endline-only outcomes. Average treatment effects (ATE) were estimated. Momentum had positive effects on partner discussion of family planning in the early postpartum period (ever married 15-19: ATE = 0.179, 95% CI = 0.098, 0.261; never married 15-19: ATE = 0.131, 95% CI = 0.029, 0.232; ever married 20-24: ATE = 0.233, 95% CI = 0.164, 0.302; never married 20-24: ATE = 0.241, 95% CI = 0.121, 0.362) and discussion with a health worker, and on obtaining a contraceptive method in the early postpartum period, except among never married adolescents. Among adolescents, intervention effects on modern contraceptive use within 12 months of childbirth/pregnancy loss were larger for the never married (ATE = 0.251, 95% CI = 0.122, 0.380) than the ever married (ATE = 0.114, 95% CI = 0.020, 0.208). Full intervention exposure had consistently larger effects on contraceptive behaviors than partial exposure, except among ever married adolescents. Momentum had no effect on normative expectations about postpartum family planning use among adolescents, and on descriptive norms and personal agency among those who were never married. Results for normative outcomes and personal agency underscored the intersectionality between young maternal age and marital status. Future programs should improve personal agency and foster normative change in support of postpartum family planning uptake and tailor interventions to different age and marital status subsets of first-time mothers.

Expanding contraceptive choice among first-time mothers age 15-24 in Kinshasa: The Momentum pilot project.

Introduction: Evidence shows that an expanded range of contraceptive methods, client-centered comprehensive counseling, and voluntary informed choice are key components of successful family planning programs. This study assessed the effect of the Momentum project on contraceptive choice among first-time mothers (FTMs) age 15-24 who were six-months pregnant at baseline in Kinshasa, Democratic Republic of the Congo, and socioeconomic determinants of the use of long-acting reversible contraception (LARC). Methods: The study employed a quasi-experimental design, with three intervention health zones and three comparison health zones. Trained nursing students followed FTMs for 16 months and conducted monthly group education sessions and home visits consisting of counseling and provision of a range of contraceptive methods and referrals. Data were collected in 2018 and 2020 through interviewer-administered questionnaires. The effect of the project on contraceptive choice was estimated using intention-to-treat and dose-response analyses, with inverse probability weighting among 761 modern contraceptive users. Logistic regression analysis was used to examine predictors of LARC use. Results: Project effect was detected on receipt of family planning counseling, obtaining the current contraceptive method from a community-based health worker, informed choice, and current use of implants vs. other modern methods. There were significant dose-response associations of the level of exposure to Momentum interventions and the number of home visits with four of five outcomes. Positive predictors of LARC use included exposure to Momentum interventions, receipt of prenatal counseling on both birth spacing and family planning (age 15-19), and knowledge of LARCs (age 20-24). The FTM's perceived ability to ask her husband/male partner to use a condom was a negative predictor of LARC use. Discussion: Given limited resources, expanding community-based contraceptive counseling and distribution through trained nursing students may expand family planning access and informed choice among first-time mothers.

Changes in hemoglobin oxidation and band 3 during blood storage impact oxygen sensing and mitochondrial bioenergetic pathways in the human pulmonary arterial endothelial cell model.

Red blood cells (RBCs) undergo metabolic, oxidative, and physiological changes during storage, collectively described as the "storage lesion." The impact of storage on oxygen homeostasis, following transfusion, is not fully understood. We show that RBC storage induces changes in oxygen binding that were linked to changes in oxygen sensing (hypoxia-inducible factor, HIF-1α) mechanisms and mitochondrial respiration in human pulmonary arterial endothelial cells (HPAECs). A decrease in oxygen affinity (P50) to approximately 20 from 30 mmHg was seen at the first week but remained unchanged for up to 42 days. This led to the suppression of HIF-1α in the first 3 weeks due to limited oxygen supplies by RBCs. Furthermore, membrane oxidative damage, band 3 alterations, and subsequent microparticle (MP) formation were also noted. Mass spectrometric analysis revealed the upregulation of transitional endoplasmic reticulum ATPase, essential for clearing ROS-damaged membrane proteins and the protein DDI1 homolog, a proteasomal shuttle chaperone. Band 3 complex proteins and superoxide dismutase were among the downregulated proteins. Mitochondrial oxygen consumption rates measured in HPAECs incubated with RBC-derived MPs (14-day and 42-day) showed a rise in maximal respiration. Intervention strategies that target intracellular hemoglobin (Hb)'s redox transitions and membrane changes may lead to the reestablishment of oxygen homeostasis in old RBCs.

Evaluation of the effect of the Momentum project on family planning outcomes among first-time mothers aged 15-24 years in Kinshasa, DRC.

OBJECTIVES: This article presents the effect of Momentum, a nursing student-led community-based service delivery project, on postpartum family planning (FP) outcomes among first-time mothers in Kinshasa, Democratic Republic of the Congo. STUDY DESIGN: We employed a quasi-experimental design, with three intervention and three comparison health zones (HZ). Data were collected through interviewer-administered questionnaires in 2018 and 2020. The sample consisted of 1927 nulliparous women aged 15-24 years who were 6 months pregnant at baseline. Random effects and treatment effects models were used to assess the effect of Momentum on 14 postpartum FP outcomes. RESULTS: The intervention group was associated with a unit increase of 0.6 (95% confidence interval [CI]: 0.4, 0.8)) in contraceptive knowledge and personal agency (95% CI: 0.3, 0.9), a unit decrease of 0.9 (95% CI: -1.2, -0.5) in the number of FP myths/misconceptions endorsed, and percentage point increases of 23.4 (95% CI: 0.2, 0.3) in FP discussion with a health worker, 13.0 (95% CI: 0.1, 0.2) in obtaining a contraceptive method within 6 weeks of delivery, and 13.3 (95% CI: 0.1, 0.2) in modern contraceptive use within 12 months of delivery. Intervention effects included percentage point increases of 5.4 (95% CI: 0.0, 0.1) in partner discussion and 15.4 (95% CI: 0.1, 0.2) in perceived community support for postpartum FP use. The level of exposure to Momentum was significantly associated with all behavioral outcomes. CONCLUSIONS: The study demonstrated the effect of Momentum on increased postpartum FP-related knowledge, perceived norms, personal agency, partner discussion, and modern contraceptive use. IMPLICATIONS: Community-based service delivery by nursing students can potentially lead to improved postpartum FP outcomes among urban adolescent and young first-time mothers in other provinces of the Democratic Republic of the Congo and other African countries.

Association of Male Partners' Gender-Equitable Attitudes and Behaviors with Young Mothers' Postpartum Family Planning and Maternal Health Outcomes in Kinshasa, DRC.

Inequitable gender norms can contribute to rapid repeat pregnancies and adverse maternal health outcomes among adolescent girls and young women. This study examined associations between male partners' gender-equitable attitudes and behaviors and postpartum family planning (FP) and maternal and newborn health (MNH) outcomes among first-time mothers aged 15-24 in Kinshasa, Democratic Republic of the Congo. Participants were 1335 couples who were successfully interviewed in the Momentum project's 2018 baseline and 2020 endline surveys. Multivariable regression models were used to analyze predictors of postpartum FP discussion and use, shared MNH decision making, completion of the maternal health continuum of care, and exclusive breastfeeding. Male involvement in maternal health was significantly associated with FP discussion and shared decision making. Male partners' willingness to be involved in routine childcare and shared decision making were significant positive predictors of exclusive breastfeeding. Postpartum FP outcomes were shaped by the intersection of marital status and male partners' gender-equitable attitudes, intimate partner violence perpetration, and willingness to engage in routine childcare activities to constitute advantage for some outcomes and disadvantage for others. Interventions must use multiple measures to better understand how young mothers' health outcomes are shaped by their male partners' gender-related attitudes and behaviors.

Perceived norms, personal agency, and postpartum family planning intentions among first-time mothers age 15-24 years in Kinshasa: A cross-sectional analysis.

Unintended pregnancy is an important global health problem and frequently occurs during the immediate postpartum period. However, few studies have examined postpartum family planning (PPFP) intentions among adolescent girls and young women. This study assessed whether perceived norms and personal agency predicted PPFP intentions among first-time mothers age 15-24 in Kinshasa, the Democratic Republic of Congo. Data were derived from the 2018 Momentum Project baseline survey. Analysis was based on 2,418 nulliparous pregnant women age 15-24 who were approximately six months pregnant with their first child in six health zones of Kinshasa. Overall PPFP intentions were low and ten to thirteen percent of women stated they were very likely to discuss PPFP next month with (a) their husband/male partner and (b) a health worker, and to (c) obtain and (d) use a contraceptive method during the first six weeks following childbirth. The results of multivariable linear regression models indicated that the PPFP intention index was predicted by description norms, perceptions of the larger community's approval of PPFP, normative expectations, perceived behavioral control, self-efficacy, and autonomy. Rejection of family planning myths and misconceptions was also a significant predictor. Interaction terms suggested that the association of normative expectations with PPFP intentions varied across ethnic groups and that the positive association of injunctive norms with PPFP intentions was significantly increased when the larger community was perceived to disapprove of PPFP use. Normative expectations and PPFP-related self-efficacy accounted for two-thirds of the variance in PPFP intentions. The results suggested that understanding different normative influences may be important to motivate women to use contraception in the immediate postpartum period. In addition to addressing institutional, individual, and social determinants of PPFP, programs should consider integrating norm-based and empowerment strategies.

Functional comparison of hemoglobin purified by different methods and their biophysical implications.

Hemoglobin (Hb) that is purified from red blood cells (RBCs) is commonly subjected to harsh processing conditions, such as high temperatures and extensive column separation, which may damage the Hb by altering the heme prosthetic group and/or the Hb protein structure. In this study, bovine and human Hb purified by tangential flow filtration (TFF) was compared to commercial preparations of human Hb (Hemosol, Inc., Toronto, Canada) and bovine Hb (Biopure, Inc., Cambridge, MA). Purified Hbs were characterized by measuring their overall purity (SDS-PAGE, SEC, and ESI-MS), susceptibility to oxidation (k(ox)), responses to physiological conditions (pH, [Cl(-)], [IHP], and T), and ligand binding kinetics (O(2), NO, and CO). All Hbs evaluated possessed comparable biophysical properties, however, a small amount of catalase was detected in the TFF-purified Hbs that reduced the rate of autoxidation. Mass changes observed by mass spectrometry suggest that structural alterations may be introduced into Hbs that are purified by extensive chromatographic separations. These results demonstrate that TFF is a suitable process for the purification of Hb from RBCs with a quality equivalent to that of commercial Hb preparations that employ more extensive purification strategies. This work also shows that TFF can yield highly pure Hb which can be used for Hb-based O(2) carrier synthesis.

Antisickling Drugs Targeting ßCys93 Reduce Iron Oxidation and Oxidative Changes in Sickle Cell Hemoglobin.

Sickle cell disease is a genetic blood disorder caused by a single point mutation in the ß globin gene where glutamic acid is replaced by valine at the sixth position of the ß chain of hemoglobin (Hb). At low oxygen tension, the polymerization of deoxyHbS into fibers occurs in red blood cells (RBCs) leading to an impaired blood vessel transit. Sickle cell hemoglobin (HbS), when oxidized with hydrogen peroxide (H2O2), stays longer in a highly oxidizing ferryl (Fe4+) form causing irreversible oxidation of ßCys93 to a destabilizing cysteic acid. We have previously reported that an antisickling drug can be designed to bind specifically to ßCys93 and effectively protect against its irreversible oxidation by H2O2. Here, we report oxygen dissociation, oxidation, and polymerization kinetic reactions for four antisickling drugs (under different preclinical/clinical developmental stages) that either site-specifically target ßCys93 or other sites on the HbS molecule. Molecules that specifically bind to or modify ßCys93, such as 4,4'-di(1,2,3-triazolyl) disulfide (TD-3) and hydroxyurea (HU) were contrasted with molecules that target other sites on Hb including 5-hydroxymethyl-2-furfural (5-HMF) and L-glutamine. All reagents induced a left shift in the oxygen dissociation curve (ODC) except L-glutamine. In the presence of H2O2 (2.5:1, H2O2:heme), both TD-3 and HU reduced the ferryl heme by 22 and 37%, respectively, which corresponded to a 3- to 2-fold reduction in the levels of ßCys93 oxidation as verified by mass spectrometry. Increases in the delay times prior to polymerization of HbS under hypoxia were in the following order: TD-3 > HU > 5-HMF = L-glutamine. Designing antisickling agents that can specifically target ßCys93 may provide a dual antioxidant and antisickling therapeutic benefits in treating this disease.

Electron paramagnetic resonance oximetry as a novel approach to monitor the effectiveness and quality of red blood cell transfusions.

BACKGROUND: The goal of red blood cell transfusion is to improve tissue oxygenation. Assessment of red blood cell quality and individualised therapeutic needs can be optimised using direct oxygen (O2) measurements to guide treatment. Electron paramagnetic resonance oximetry is capable of accurate, repeatable and minimally invasive measurements of tissue pO2. Here we present preclinical proof-of-concept of the utility of electron paramagnetic resonance oximetry in an experimental setting of acute blood loss, transfusion, and post-transfusion monitoring. MATERIALS AND METHODS: Donor rat blood was collected, leucocyte-reduced, and stored at 4 °C in AS-3 for 1, 7 and 14 days. Red blood cell morphology, O2 equilibrium, p50 and Hill numbers from O2 binding and dissociation curves were evaluated in vitro. Recipient rats were bled and maintained at a mean arterial pressure of 30-40 mmHg and hind limb muscle (biceps femoris) pO2 at 25-50% of baseline. Muscle pO2 was monitored continuously over the course of experiments to assess the effectiveness of red blood cell preparations at different stages of blood loss and restoration. RESULTS: Red blood cell morphology, O2 equilibrium and p50 values of intra-erythrocyte haemoglobin were significantly altered by refrigerated storage for both 7 and 14 days. Transfusion of red blood cells stored for 7 or 14 days demonstrated an equivalently impaired ability to restore hind limb muscle pO2, consistent with in vitro observations and transfusion with albumin. Red blood cells refrigerated for 1 day demonstrated normal morphology, in vitro oxygenation and in vivo restoration of tissue pO2. DISCUSSION: Electron paramagnetic resonance oximetry represents a useful approach to assessing the quality of red blood cells and subsequent transfusion effectiveness.

Site-directed mutagenesis of cysteine residues alters oxidative stability of fetal hemoglobin.

Redox active cysteine residues including ßCys93 are part of hemoglobin's "oxidation hotspot". Irreversible oxidation of ßCys93 ultimately leads to the collapse of the hemoglobin structure and release of heme. Human fetal hemoglobin (HbF), similarly to the adult hemoglobin (HbA), carries redox active γCys93 in the vicinity of the heme pocket. Site-directed mutagenesis has been used in this study to examine the impact of removal and/or addition of cysteine residues in HbF. The redox activities of the recombinant mutants were examined by determining the spontaneous autoxidation rate, the hydrogen peroxide induced ferric to ferryl oxidation rate, and irreversible oxidation of cysteine by quantitative mass spectrometry. We found that substitution of γCys93Ala resulted in oxidative instability characterized by increased oxidation rates. Moreover, the addition of a cysteine residue at α19 on the exposed surface of the α-chain altered the regular electron transfer pathway within the protein by forming an alternative oxidative site. This may also create an accessible site for di-sulfide bonding between Hb subunits. Engineering of cysteine residues at suitable locations may be useful as a tool for managing oxidation in a protein, and for Hb, a way to stave off oxidation reactions resulting in a protein structural collapse.

Oxidative instability of hemoglobin E (ß26 Glu→Lys) is increased in the presence of free α subunits and reversed by α-hemoglobin stabilizing protein (AHSP): Relevance to HbE/ß-thalassemia.

When adding peroxide (H2O2), ß subunits of hemoglobin (Hb) bear the burden of oxidative changes due in part to the direct oxidation of its Cys93. The presence of unpaired α subunits within red cells and/or co-inheritance of another ß subunit mutant, HbE (ß26 Glu→Lys) have been implicated in the pathogenesis and severity of ß thalassemia. We have found that although both HbA and HbE autoxidize at initially comparable rates, HbE loses heme at a rate almost 2 fold higher than HbA due to unfolding of the protein. Using mass spectrometry and the spin trap, DMPO, we were able to quantify irreversible oxidization of ßCys93 to reflect oxidative instability of ß subunits. In the presence of free α subunits and H2O2, both HbA and HbE showed ßCys93 oxidation which increased with higher H2O2 concentrations. In the presence of Alpha-hemoglobin stabilizing protein (AHSP), which stabilizes the α-subunit in a redox inactive hexacoordinate conformation (thus unable to undergo the redox ferric/ferryl transition), Cys93 oxidation was substantially reduced in both proteins. These experiments establish two important features that may have relevance to the mechanistic understanding of these two inherited hemoglobinopathies, i.e. HbE/ß thalassemia: First, a persistent ferryl/ferryl radical in HbE is more damaging to its own ß subunit (i.e., ßCys93) than HbA. Secondly, in the presence of excess free α-subunit and under the same oxidative conditions, these events are substantially increased for HbE compared to HbA, and may therefore create an oxidative milieu affecting the already unstable HbE.

Evaluation of Stem Cell-Derived Red Blood Cells as a Transfusion Product Using a Novel Animal Model.

Reliance on volunteer blood donors can lead to transfusion product shortages, and current liquid storage of red blood cells (RBCs) is associated with biochemical changes over time, known as 'the storage lesion'. Thus, there is a need for alternative sources of transfusable RBCs to supplement conventional blood donations. Extracorporeal production of stem cell-derived RBCs (stemRBCs) is a potential and yet untapped source of fresh, transfusable RBCs. A number of groups have attempted RBC differentiation from CD34+ cells. However, it is still unclear whether these stemRBCs could eventually be effective substitutes for traditional RBCs due to potential differences in oxygen carrying capacity, viability, deformability, and other critical parameters. We have generated ex vivo stemRBCs from primary human cord blood CD34+ cells and compared them to donor-derived RBCs based on a number of in vitro parameters. In vivo, we assessed stemRBC circulation kinetics in an animal model of transfusion and oxygen delivery in a mouse model of exercise performance. Our novel, chronically anemic, SCID mouse model can evaluate the potential of stemRBCs to deliver oxygen to tissues (muscle) under resting and exercise-induced hypoxic conditions. Based on our data, stem cell-derived RBCs have a similar biochemical profile compared to donor-derived RBCs. While certain key differences remain between donor-derived RBCs and stemRBCs, the ability of stemRBCs to deliver oxygen in a living organism provides support for further development as a transfusion product.

Oxygen binding and oxidation reactions of human hemoglobin conjugated to carboxylate dextran.

Human hemoglobin (Hb) conjugated to benzene tetracarboxylate substituted dextran produces a polymeric Hb (Dex-BTC-Hb) with similar oxygen affinity to that of red blood cells (P(50)=28-29 mm Hg). Under physiological conditions, the oxygen affinity (P(50)) of Dex-BTC-Hb is 26 mm Hg, while that of native purified human HbA(0) is 14 mm Hg, but it exhibits a slight reduction in cooperativity (n(50)), Bohr effect, and lacks sensitivity to inositol hexaphosphate (IHP), when compared to HbA(0). Oxygen-binding kinetics, measured by rapid mixing stopped-flow method showed comparable oxygen dissociation and association rates for both HbA(0) and Dex-BTC-Hb. The rate constant for NO-mediated oxidation of the oxy form of Dex-BTC-Hb, which is governed by NO entry to the heme pocket, was reduced to half of the value obtained for HbA(0). Moreover, Dex-BTC-Hb is only slightly more sensitive to oxidative reactions than HbA(0), as shown by about 2-fold increase in autoxidation, and slightly higher H(2)O(2) reaction and heme degradation rates. Dextran-BTC-based modification of Hb produced an oxygen-carrying compound with increased oxygen release rates, decreased oxygen affinity and reduced nitric oxide scavenging, desirable properties for a viable blood substitute. However, the reduction in the allosteric function of this protein and the lack of apparent quaternary T-->R transition may hinder its physiological role as an oxygen transporter.

Cross-linking with O-raffinose lowers oxygen affinity and stabilizes haemoglobin in a non-cooperative T-state conformation.

O-R-polyHbA(0) is an intra- and intermolecularly O-raffinose cross-linked derivative of deoxygenated human haemoglobin developed as an oxygen therapeutic. When compared with its native protein (HbA(0)), O-R-polyHbA(0) was found to be locked in the T (tense) quaternary conformation with a lower oxygen affinity, a reduced Bohr effect (50% of HbA(0)) and no measurable cooperativity (h=1). The kinetics of oxygen and CO binding to the protein indicate lower 'on' rates and faster 'off' rates than HbA(0) and the absence of effects of inositol hexaphosphate (IHP) on the kinetics. Other properties consistent with a T-like conformation are inaccessibility of the betaCys-93 thiol group of O-R-polyHbA(0), the hyperfine splitting from nitrogen in the EPR spectrum of the Fe(II)NO complex of O-R-polyHbA(0) and decreased flexibility in the distal haem pocket, as indicated by low-spin bis-histidine complexes detected by EPR of oxidized chains. A comparison of the properties of O-R-polyHbA(0) with those of HbA(0) with and without IHP, as well as the reaction of nitrite with deoxygenated haemoglobin, provide additional insights into the variations in the conformation of T-state haemoglobin in solution (modifications of the T state produced by adding organic phosphates, like IHP and 2,3-diphosphoglycerate). Although the physiological ramifications of locking HbA(0) in the T conformation with the O-raffinose are still unknown, valuable insights into haemoglobin function are provided by these studies of O-R-polyHbA(0).