Cytochrome B5 Reductase Research Articles

DEVELOPMENT OF METHEMOGLOBINEMIA IN A PATIENT WITH COVID-19 PNEUMONIA

TOPIC: Critical Care TYPE: Medical Student/Resident Case Reports INTRODUCTION: The World Health Organization officially declared COVID-19 as a pandemic in March 2020. Since then, it has caused more than 1.5 million deaths and has affected more than 68 million people worldwide (1). Methemoglobinemia is a very rare and potentially life-threatening condition. Congenital methemoglobinemia is caused by a deficiency in cytochrome b5 reductase (Cyb5R) while acquired is caused by exposure to an oxidizing agent such as chloroquine or dapsone. We present a case of a critically ill patient with COVID-19 pneumonia who developed methemoglobinemia without an identifiable cause. CASE PRESENTATION: A 64-year-old gentleman with past medical history of hypertension presents to the emergency department (ED) with chief complaints of shortness of breath and myalgias for the past 3 days. In the ED, patient was found to tachycardic at 121 beats/minute, tachypneic at 40 breaths/minute, and febrile at 38.6° Celsius. He was found to have elevated COVID-19 labs which included C-reactive protein, lactate dehydrogenase, ferritin, creatine phosphokinase, d-dimer in addition to elevated BUN 53 mg/dL and creatinine 2.92 mg/dL. His COVID-19 PCR resulted positive and was placed on bilevel positive airway pressure (BPAP) for his respiratory distress and transferred to the medical intensive care unit (MICU) for further monitoring. He was started on broad spectrum antibiotics with vancomycin and cefepime along with dexamethasone and remdesivir. Within 24 hours of admission, patient required mechanical ventilation for worsening hypoxemia. Furthermore, his kidney function continued to decline and required continuous renal replacement therapy. His oxygen requirements improved until hospital day 10;where he developed methemoglobinemia measuring 11.8%. Patient was then given methylene blue 1mg/kg and 1.5 grams of ascorbic acid. Hematology was consulted for emergent exchange transfusion as he developed hemolysis but was deemed too unstable and unlikely to benefit. Patient subsequently went into multisystem organ failure and died on hospital day 12. DISCUSSION: To date, there has only been one reported case of COVID-19 complicated by methemoglobinemia without an identifiable cause (2). Our patient tested negative for glucose-6-phosphate dehydrogenase (G6PD) deficiency and a full medication reconciliation was performed without an identifiable cause. However, there have been 10 cases reported that were either associated with use of an oxidizing agent such as hydroxychloroquine or G6PD deficiency (3–6). Therefore, clinicians should be cognizant of the rising cases of methemoglobinemia and COVID-19. CONCLUSIONS: In the setting of COVID-19 pneumonia requiring mechanical ventilation with refractory hypoxemia, clinicians should be mindful that methemoglobinemia may be a contributing factor. Therefore, in the setting of refractory hypoxemia in COVID-19, measuring methemoglobin levels should be considered. REFERENCE #1: 1) Home - Johns Hopkins Coronavirus Resource Center. Accessed December 6, 2020. https://coronavirus.jhu.edu/2) Lopes DV, Lazar Neto F, Marques LC, Lima RBO, Brandao AAGS. Methemoglobinemia and hemolytic anemia after COVID-19 infection without identifiable eliciting drug: A case-report. IDCases. 2021;23:e01013. doi:10.1016/j.idcr.2020.e01013 REFERENCE #2: 3) Faisal H, Bloom A, Gaber AO. Unexplained Methemoglobinemia in Coronavirus Disease 2019: A Case Report. AA14(9):e01287. doi:10.1213/XAA.00000000000012874) Choo SY. Letters to the Editor in Therapeutic Apheresis and Dialysis Rapidly rising methemoglobinemia in a patient with severe COVID-19 treated successfully with red cell exchange transfusion Running Title: Red Cell Exchange for COVID-19 Methemoglobinemia. doi:10.1111/tap.13598 REFERENCE #3: 5) Naymagon L, Berwick S, Kessler A, Lancman G, Gidwani U, Troy K. The emergence of methemoglobinemia amidst the COVID-19 pandemic. Am J Hematol. 2020;95(8):E196-E197. doi:10.1002/ajh.258686) Palmer K, Dick J, French W, Floro L, Ford M. ethemoglobinemia in patient with g6pd deficiency and sars-cov-2 infection. Emerg Infect Dis. 2020;26(9):2279-2281. doi:10.3201/eid2609.202353 DISCLOSURES: No relevant relationships by Paul Nguyen, source=Web Response No relevant relationships by Jonathan Pickos, source=Web Response No relevant relationships by Aryan Shiari, source=Web Response No relevant relationships by Ayman Soubani, source=Web Response

Inhaled nitric oxide before induction of anesthesia in patients with pulmonary hypertension.

Background:The aim of this study was to examine the action of inhaled nitric oxide in the patients with pulmonary hypertension administered with a face mask before anesthesia induction.Methods:Ten adult patients scheduled for heart surgery with sternotomy were included in this prospective, interventional, single centre study. The inclusion criteria were patients scheduled for heart surgery with sternotomy with cardiopulmonary bypass (CPB), aged >18 years which presents a pulmonary hypertension (PH) (class 2 or 3 according to the Dana Point classification) with systolic pulmonary arterial pressure (PAPS) >40 mmHg diagnosed by preoperative right cardiac catheterization or by transthoracic echocardiography. The exclusion criteria were: heart transplant, PH of type 1, 4, 5, according to the Dana Point classification, methemoglobin reductase deficit, incapacity to understand the protocol and sign the consent.Results:The administration of iNO decrease pulmonary hypertension (P < 0,001 compared to room air; P = 0,01 compared to pure oxygen administration). The iNO administration did not improve arterial blood oxygenation. The hyperoxia, decrease the cardiac index even with right ventricular post charge decrease. The increased blood oxygenation content cause systemic vascular vasoconstriction and decrease the peripheral oxygen extraction showed with VO2 linear increase (P < 0,001).Conclusions:The administration of inhaled nitric oxide with a face mask before anaesthesia induction is safe and effective method to reduce pulmonary hypertension. The oxygen and hyperoxia influences the systemic vascular resistance and peripheral oxygen consumption.

Smooth muscle cell CYB5R3 preserves cardiac and vascular function under chronic hypoxic stress

Chronic hypoxia is a major driver of cardiovascular complications, including heart failure. The nitric oxide (NO) – soluble guanylyl cyclase (sGC) – cyclic guanosine monophosphate (cGMP) pathway is integral to vascular tone maintenance. Specifically, NO binds its receptor sGC within vascular smooth muscle cells (SMC) in its reduced heme (Fe2+) form to increase intracellular cGMP production, activate protein kinase G (PKG) signaling, and induce vessel relaxation. Under chronic hypoxia, oxidative stress drives oxidation of sGC heme (Fe2+→Fe3+), rendering it NO-insensitive. We previously showed that cytochrome b5 reductase 3 (CYB5R3) in SMC is a sGC reductase important for maintaining NO-dependent vasodilation and conferring resilience to systemic hypertension and sickle cell disease-associated pulmonary hypertension. To test whether CYB5R3 may be protective in the context of chronic hypoxia, we subjected SMC-specific CYB5R3 knockout mice (SMC CYB5R3 KO) to 3 weeks hypoxia and assessed vascular and cardiac function using echocardiography, pressure volume loops and wire myography. Hypoxic stress caused 1) biventricular hypertrophy in both WT and SMC CYB5R3 KO, but to a larger degree in KO mice, 2) blunted vasodilation to NO-dependent activation of sGC in coronary and pulmonary arteries of KO mice, and 3) decreased, albeit still normal, cardiac function in KO mice. Overall, these data indicate that SMC CYB5R3 deficiency potentiates bilateral ventricular hypertrophy and blunts NO-dependent vasodilation under chronic hypoxia conditions. This implicates that SMC CYB5R3 KO mice post 3-week hypoxia have early stages of cardiac remodeling and functional changes that could foretell significantly impaired cardiac function with longer exposure to hypoxia.

Structural and functional effects of single nucleotide polymorphism on canine cytochrome b 5 reductase - in silico

The objectives of this study were to investigate the effects of single nucleotide polymorphism in Canine cytochrome b5 reductase using computational methods. Data was obtained from database of National Centre for Biotechnology Information (db SNP) and computational software was used for the analysis. The 3D protein structure was predicted using phyre 2 server. PANTHER analysis predicted the effect of single nucleotide polymorphism (substitution of Isoleucine for Leucine at position 194) as damaging. Analysis using the Mutpred 2 web application also indicated deleterious effects of the amino acid substitution. Molecular mechanisms of structural changes in the amino acid were determined using Mutpred 2 to be altered ordered interface, gain of allosteric sites and altered metal binding. The study indicated that the substitution of Isoleucine by Leucine at position 194 of the amino acid sequence (Ile 194 Leu) resulted in the destabilization of the amino acid structure leading to functional deviation in canine cytochrome b5 reductase.

Pediatric intoxication inducing after dental pain management OTC Benzocaine product.

Background; Methemoglobinemia is a rare disorder of the blood in which there is an increase in the proportion of hemoglobin present in the oxidized form (methemoglobin). It may be inherited, due either to a deficiency of methemoglobin reductase or to a structural abnormality of hemoglobin, or it may be acquired, usually secondary to exposure to drugs or chemicals that oxidize hemoglobin l. Administration of some local anesthetics such as benzocaine at large doses may lead to an acute effect of risk Methemoglobinemia.. Presentation ; A 6-years-old child was suffering from dyspnea, headache, and grayish in appearance. The patient was self-treating with an over-the-counter topical oral benzocaine. Treatment; The diagnosis of methemoglobinemia was made in conjunction with consultation with a medical toxicologist and treatment with oxygen and methylene blue as antidote was given 1mg/kg of 1% solution intravenously over 7minutes. The symptoms improved within 30 minutes and child felt markedly better within 1.5 hour. Conclusion; Methylene blue is the specific antidote, but should be reserved for more severe cases.

Pediatric intoxication inducing after dental pain management OTC Benzocaine product.

Background; Methemoglobinemia is a rare disorder of the blood in which there is an increase in the proportion of hemoglobin present in the oxidized form (methemoglobin). It may be inherited, due either to a deficiency of methemoglobin reductase or to a structural abnormality of hemoglobin, or it may be acquired, usually secondary to exposure to drugs or chemicals that oxidize hemoglobin l. Administration of some local anesthetics such as benzocaine at large doses may lead to an acute effect of risk Methemoglobinemia.. Presentation ; A 6-years-old child was suffering from dyspnea, headache, and grayish in appearance. The patient was self-treating with an over-the-counter topical oral benzocaine. Treatment; The diagnosis of methemoglobinemia was made in conjunction with consultation with a medical toxicologist and treatment with oxygen and methylene blue as antidote was given 1mg/kg of 1% solution intravenously over 7minutes. The symptoms improved within 30 minutes and child felt markedly better within 1.5 hour. Conclusion; Methylene blue is the specific antidote, but should be reserved for more severe cases.

Genome-Wide Association Study Using Whole-Genome Sequencing Identifies a Genomic Region on Chromosome 6 Associated With Comb Traits in Nandan-Yao Chicken.

Comb traits have potential economic value in the breeding of indigenous chickens in China. Identifying and understanding relevant molecular markers for comb traits can be beneficial for genetic improvement. The purpose of this study was to utilize genome-wide association studies (GWAS) to detect promising loci and candidate genes related to comb traits, namely, comb thickness (CT), comb weight (CW), comb height, comb length (CL), and comb area. Genome-wide single-nucleotide polymorphisms (SNPs) and small insertions/deletions (INDELs) in 300 Nandan-Yao chickens were detected using whole-genome sequencing. In total, we identified 134 SNPs and 25 INDELs that were strongly associated with the five comb traits. A remarkable region spanning from 29.6 to 31.4 Mb on chromosome 6 was found to be significantly associated with comb traits in both SNP- and INDEL-based GWAS. In this region, two lead SNPs (6:30,354,876 for CW and CT and 6:30,264,318 for CL) and one lead INDEL (a deletion from 30,376,404 to 30,376,405 bp for CL and CT) were identified. Additionally, two genes were identified as potential candidates for comb development. The nearby gene fibroblast growth factor receptor 2 (FGFR2)—associated with epithelial cell migration and proliferation—and the gene cytochrome b5 reductase 2 (CYB5R2)—identified on chromosome 5 from INDEL-based GWAS—are significantly correlated with collagen maturation. The findings of this study could provide promising genes and biomarkers to accelerate genetic improvement of comb development based on molecular marker-assisted breeding in Nandan-Yao chickens.

Congenital Methemoglobinemia Under Diagnosed Condition with Variiable Therapeurtic Options

Methemoglobinemia is a rare overlooked differential diagnosis in patients with cyanosis and dyspnea unrelated to cardiopulmonary causes.Methemoglobin results from oxidation of ferrous iron to ferric iron within the heme moiety of hemoglobin . In healthy individuals, less than 1% of hemoglobin is present in the oxidized form (methemoglobin), which has limited ability to carry oxygen, however with increased oxygen affinity at the remaining binding sites .This results in decrease of oxygen delivery to tissue leading to hypoxaemia and lactic acidosis.Methemoglobin reductase enzyme deficiency is either type I or type II; type I: Cytochrome b5 reductase deficiency, demonstrable only in the erythrocytes, presents as uncomplicated, benign methemoglobinemia, and associated with a normal life expectancy with only fatigue and dyspnea being the most commonly reported symptoms.Hereditary type II methemoglobinemia, is global (affects both red and white blood cells) and is associated with severe neurologic dysfunction and reduced life expectancy . The enzyme's activity is less than 20% of normal.Type II congenital methemoglobinemia constitutes approximately 10% of all cases of congenital methemoglobinemia and usually causes death within the first few years of lifeThere is a significant delay in the onset of symptoms in a patient with type I hereditary methemoglobinemia, as most studies indicate that patients are asymptomatic in infancy and childhood.we report a 29 year old Indian non-smoker male, his story started 6 months prior to presentation when he had generalized fatigue and discoloration of hands. He presented with persistent polycythaemia with elevated hemoglobin level. The patient was misdiagnosed in another center as polycythaemia and treated with Imatinib. The diagnosis of PV was revisited and ruled out in view of negative JAK2, normal erythropoietin level and absence of features of panmyelosis.Clinical cyanosis and low oxygen saturation in the presence of normal arterial oxygen tension was highly suggestive of methemoglobinemia.Arterial blood gas (ABG) revealed a methemoglobin level of 38% (normal: 0-1.5%). Cytochrome B5 reductase (Methemoglobin reductase B) wasdeficient at level of <2.6 U/g Hb) (normal: 6.6-13.3), consistent with methemoglobin reductase (cytochrome b5) deficiency and hence the diagnosis of congenital methemoglobinemia was established.The role of Imatinib in provoking methemoglobinemia is questionable and association between Imatinib and methemoglobinemia never described before. There was no history of other drugs offended in aggravating the patients' symptoms and cyanosis.The patient started on Vitamin C 500 mg for which he responded well with less cyanosis and significant reduction of methemoglobin level.Upon extensive review of English literature for cases of congenital Methaemoglobinaemia due to deficiency of cytochrome b5 reductase, we found 23 cases diagnosed as type I (including the case reported here).17 cases (~74%) of type I and 6 cases (27%) of type II. There is male predominance (73%) versus 26% in females. Almost half of reported cases (12 cases; 52%) are indian, 2 Japanese, 3 English, 2 Arabic, one Spanish and one Italian.For type I, the median calculated age is 31 years with cyanosis and shortness of breath being the most common sign and symptom.For type II: Six cases were reported, all in pediatric age group with median calculated age at presentation is 6 years with neurologic manifestations and mental retardation are the most common type II associated symptoms.Congenital methemogobinemia is a rare underreported hemoglobin disease. Due to lack of systematic epidemiological studies it is usually under investigated and frequently clinically missed especially when presenting in adulthood. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Changes in Metabolite and Protein Expression Profiles of Atypical Dark-cutting and Normal-pH Beef

Abstract The color of meat is an important deciding factor in consumers’ assessment of meat quality. To meat buyers, the bright cherry-red color of meat indicates freshness and wholesomeness. However, atypical dark-cutting beef represents dark-colored meat with a muscle pH between 5.6 and 5.8. Although previous studies have indicated that the ultimate pH of atypical dark-cutting beef is greater than normal, the mechanistic basis for the occurrence is not clear. Therefore, the objective of this study was to identify protein and metabolite profiles of normal and atypical dark-cutting beef. Longissimus thoracic (LT) muscles from 12 different animals (6 atypical dark-cutters and 6 normal-pH beef) were analyzed by comparing changes in metabolites and protein profiles using GC-MS non-targeted metabolomics and LC-MS/MS-based proteomics, respectively. Metabolomics analysis showed down-regulation of glycolytic metabolites such as glucose-6-phosphate, lactose, and glucose, indicating a reduced capacity to accumulate lactate postmortem in atypical dark-cutters compared with normal-pH beef. Mass spectrometry analysis identified 66 proteins with significant changes in protein expression profiles (P &amp;lt; 0.05) between atypical and normal-pH beef. Of these, 22 proteins were up-regulated while 14 were down-regulated in atypical dark-cutting beef. Functional annotation and protein-protein interaction network analyses revealed that up-regulated proteins such as NADH ubiquinone oxidoreductase subunit A7, ATPase Na+/K+ transporting subunit alpha 2, 3-oxoacid CoA-transferase 1, cytochrome b5 reductase 3, actinin alpha 4, tropomyosin 2, heat shock protein beta-7, and DnaJ heat shock protein family (Hsp40) member B4 are involved in energy synthesis, muscle contraction and stress responses. By contrast, down-regulated proteins, including phosphofructokinase, glycogen phosphorylase, amylo-alpha-1,6-glucosidase, phosphorylase kinase regulatory subunit alpha and gamma are involved in glycogen metabolism. In summary, lower glycolytic metabolites and glycogen degradation enzymes are associated with differences in muscle pH and color of atypical dark-cutting beef.

Mechanism of NO Decay in Vascular Smooth Muscle Cells: Role of Cytoglobin and Identification of its Cellular Reducing System

Background In smooth muscle, cytoglobin (Cygb) functions as a potent nitric oxide (NO) dioxygenase and regulates NO metabolism and vascular tone. However, major questions remain regarding which cellular reducing systems regulate Cygb-mediated NO metabolism. Therefore, we performed studies to assess the intracellular concentrations of Cygb and its reducing systems in vascular smooth muscle cells (SMCs), and their role in the process of NO decay. Methods Cygb and its reducing systems, cytochrome B5 reductase (B5R), cytochrome B5 (B5) and cytochrome P450 reductase (CPR) were measured in aortic SMCs. Murine aortic smooth muscle cells (SMCs) were purchased from American Type Culture Collection (ATCC; CRL-2797™) and passaged. For depleting Cygb, B5R or B5, siRNAs were used. SMCs were transfected with Cygb siRNA, B5R siRNA, B5 siRNA or CPR siRNA using Lipofectamine RNAiMAX (Invitrogen). The rate of NO metabolism by normal SMCs of different cell numbers was assessed using a Clark-type NO electrode (NOCHM-4, WPI) utilizing two ports on the side wall of a four-port water-jacketed electrochemical chamber (NOCHM-4, WPI) and additionally another oxygen electrode was placed through second port on the side wall of the chamber. For protein assays, Cygb, B5R and B5 were expressed and purified using reported procedures. Quantitative immunoblotting was used to estimate the cellular concentrations of Cygb and each reducing system protein from comparison to standards of known concentration for each purified protein. Results Intracellular Cygb concentration was estimated as 3.5 µM, while B5R, B5, and CPR were 0.88, 0.38 and 0.15 µM, respectively. NO decay in SMCs was measured following bolus addition of 1 µM NO to air-equilibrated cells. The NO dioxygenation rate (VNO) with 3.5 x 106 cells•ml-1 was 12.7 ± 0.3 nM•s-1. siRNA-mediated knockdown experiments indicated that ~78% of NO metabolism in SMCs was Cygb-dependent. Of this, ~87 % was B5R- and B5-dependent. CPR knockdown resulted in a small decrease in VNO, while depletion of ascorbate had no effect. Kinetic analysis of VNO for the B5R/B5/Cygb system with variation of B5 or B5R concentrations from their SMC levels showed that VNO exhibits apparent Michaelis-Menten behavior for B5 and B5R. In contrast, linear variation was seen with change in Cygb concentration. Conclusions B5R/B5 was demonstrated to be the major reducing system supporting Cygb-mediated NO metabolism in SMCs and studies were performed to experimentally model and predict the alterations of VNO with changes in cellular B5R/B5/Cygb levels.

CYB5R3 BIASES SOLUBLE GUANYLYL CYCLASE ACTIVATION IN RESISTANCE ARTERIES

Objective: Nitric oxide (NO) regulates blood pressure by binding the reduced heme iron on soluble guanylyl cyclase (sGC) in vascular smooth muscle cells (SMC) which leads to the production of cyclic guanosine 3’,5’-monophosphate (cGMP) and vasodilation. In cardiovascular disease, oxidative stress causes oxidation or loss (apo-) of the sGC heme, impairing NO binding and causing vasoconstriction. Subsequently, new therapeutic compounds termed sGC activators have been developed for use in cardiovascular disease to target oxidized heme, and to a greater extent apo-sGC, to cause irreversible, NO-independent reactivation of cGMP production and vasodilation. However, few studies have defined the impact of NO-independent sGC activation on vascular physiology in non-stressed conditions. Design and method: To address this, we performed vasoreactivity studies on resistance and aortic arteries under non-stressed, physiological conditions and telemetry for in vivo blood pressure recordings. Results: Interestingly, we found that resistance arteries are log-orders more sensitive to sGC activator BAY 58–2667 than conduit aortic arteries in non-stressed conditions. This suggests that resistance arteries may contain a previously unknown, physiologically activatable pool of heme oxidized and/or apo-sGC that can be activated independent of NO and disease. We have previously published that cytochrome b5 reductase 3 (CYB5R3) reduces the oxidized heme sGC to its NO-sensitive, reduced state which confers resilience from systemic hypertension. We hypothesized that CYB5R3 may serve as a rheostat in physiological blood pressure and sGC activator function. In transgenic CYB5R3 overexpression (CYB5R3 OE) mice, we found that resistance arteries, but not aorta have a blunted response to sGC activator induced vasodilation and blood pressure lowering in unstressed conditions (n = 4–8/group). OE CYB5R3 mice also exhibit a ∼7 mmHg decreases in pulse pressure and impaired blood pressure reduction in response to an acute injection of BAY 58–2667 when compared to controls (n = 5/group). Consistent with this, no difference in vasodilation response to NO donor sodium nitroprusside (SNP) was observed between CYB5R3 OE and littermate controls irrespective of vessel type (n = 2–6/group). Conclusions: These data suggest that CYB5R3 is important in regulating resistance artery sensitivity to sGC activator BAY 58–2667 likely via governing sGC redox state in non-stressed, physiological conditions.

Dermatopontin as a potential pathogenic factor in endometrial cancer

The present study aimed to determine the differential expression profiles of proteins in endometrial carcinoma and to screen the proteins associated with the occurrence and development of endometrial cancer (EC). In total, 15 samples of human EC and paracancerous tissues were selected for proteomic analysis using a label-free quantification method based on liquid chromatography-tandem mass spectrometry. The differential proteins were analysed using bioinformatics and verified using reverse transcription-quantitative PCR (RT-qPCR) and western blotting. Finally, the expression of differential proteins in 75 endometrial carcinoma samples and 30 normal endometrial tissue samples were detected using immunohistochemical staining, and the associations between differential protein expression and clinicopathological features were analysed. In total, 579 up-regulated proteins and 346 down-regulated proteins were identified between the two groups and seven proteins with the most significant differences were selected; these proteins included interferon-induced protein with tetratricopeptide repeats 3, poly(ADP-ribose) polymerase family member 9, solute carrier family 34 member 2, cytochrome b5 reductase 1, protein tyrosine phosphatase non-receptor type 1, dermatopontin (DPT) and secretory leukocyte peptidase inhibitor. RT-qPCR and western blotting showed that DPT expression was down-regulated (P<0.001), which was consistent with the mass spectrometry results. The immunohistochemical staining results showed that the positive expression of DPT in EC and normal endometrial tissues was statistically significant (P<0.001). The positive expression of DPT was significantly decreased in poorly differentiated, late stage, lymph node metastasis and myometrial invasion depth ≥1/2 samples (P<0.05). DPT expression was significantly lower in EC, which might play role in the pathogenesis of EC.

The expression and purification of human steroid hormone processing enzyme CYP7B1 and the characterization of its interactive network

The cytochrome P450 monooxygenase CYP7B1 is an integral membrane protein at the endoplasmic reticulum (ER) membrane and catalyzes the hydroxylation of 7-alpha position of the steroid backbone, generating a series of important steroids such as 7α-hydroxy dehydroepiandrosterone and 7α-hydroxy pregnenolone. The disruption of CYP7B1 function could lead to abnormal oxysterols levels in neurons and the liver, and thus result in diseases such as hereditary spastic paraplegia and congenital bile acid synthesis defect. CYP7B1 was found to play a critical role in the biochemical network for biosynthesis/modification of steroids which involves hydrogenation/dehydrogenation, hydroxylation, oxygenation reaction at various carbon positions of the steroid backbone. Although a biologically-related enzyme, CYP7A1, has been successfully purified and structurally characterized, little was known about the structure and catalyzing mechanism for CYP7B1. To understand the structural basis of CYP7B1 functions, recombinant expression systems were screened for high-level expression and purification. Humans CYP7B1 was successfully expressed in the baculovirus/insect cell system and purified with its redox cofactor, indicating a natural folding for CYP7B1 samples. When challenged with detergents of various lengths of hydrophobic side chains, purified CYP7B1 proteins showed moderate stability but failed to maintain the natural folding upon the “harsh” detergent such as octyl glucoside. To explore the interactive proteins for CYP7B1, a pulldown-mass spectrometry assay using purified CYP7B1 was conducted and identified multiple ER-resident enzymes including cytochrome P450 reductase CYPOR as interactive proteins for CYP7B1, as well as other redox enzymes such as cytochrome b5 reductase and cytochrome b5 family members, indicating the existence of a physical interactive network at ER compromising cytochrome P450 and b5 family members and their reductases. Further efforts were conducted to confirm molecular interaction from mass-spectral assay and the in vitro macromolecular interaction assay showed the association between CYP7B1 and CYPOR. The CYP7B1-CYPOR protein complex was purified upon the optimization of co-expression system and in vitro assembly protocols, implying a catalytic complex might exist in ER membrane comprising CYP7B1, CYPOR, and other redox enzymes. Our results showed that human cytochrome P450 protein CYP7B1 could be purified in its cofactor-bound form and bind to cytochrome P450 reductase CYPOR to form a stable complex resistant to solubilization by detergent. Further studies should be conducted to map the components and interactions in the redox-enzyme complex at ER and determine the structural basis for the electron transfer therein.

Abstract P726: Cytochrome B5 Reductase 3 Modifies The Brain-Blood Axis Response To Ischemic Stroke In Mice With Sickle Cell Disease

Introduction: Stroke and silent infarcts are serious complications of sickle cell disease (SCD), occurring frequently in children. Decreased nitric oxide bioavailability and responsiveness contribute to neurovascular disease. Cytochrome b5 reductase 3 (Cyb5R3) is a heme iron reductase that reduces oxidized soluble guanylate cyclase heme iron (Fe 3+ --&gt; Fe 2+ ) to preserve nitric oxide signaling. A loss-of-function Cyb5R3 missense variant (T117S) occurs with high frequency (0.23 minor allele) in persons of African ancestry. Hypothesis: We hypothesized that impaired reductase function of T117S Cyb5R3 exacerbates brain damage after ischemic stroke in SCD. Methods: Bone marrow transplant was used to create male SCD mice with wild type (SS/WT) or T117S (SS/T117S) Cyb5R3. Blood was sampled before and after middle cerebral artery occlusion (55 minutes occlusion, 48 hours reperfusion). Infarct volume (IV) was determined by 2,3,5-triphenyltetrazolium chloride. Intravascular hemolysis and correlation (Pearson’s R) of hematology changes with IV were determined. Baseline Walk-PHaSST (NCT00492531) data were analyzed for stroke occurrence. Results: Brain IV (63 vs 27 cm 3 , P=0.003) and mortality (3/6 vs 0/8) were greater in SS/T117S vs SS/WT. Red blood cells, hemoglobin and hematocrit declined as IV increased. Plasma oxyhemoglobin increased in parallel with IV (r = 0.74, P=0.09). There were different signatures to hematologic changes that occurred with IV in SCD. Relative to wild type, T117S contracted the erythroid compartment (red blood cell: -13% vs 13%, P=0.003; hematocrit: -20% vs 1%, P=0.008; hemoglobin: -18% vs 2%, P=0.007). Mean platelet volume correlated with IV in SS/T117S (r = 0.87, P=0.06), while the inverse occurred in SS/WT (r = -0.63, P=0.09) Monocytes increased in parallel with IV in SS/T117S (r = 0.73, P=0.16), but followed the opposite trajectory in SS/WT (r = -0.77, P=0.04). WalkPHaSST participants with T117S Cyb5R3 self-reported more ischemic stroke (7.4% vs 5.1%) relative to wild type. Conclusion: Cyb5R3 is an important modifier of the evolution and outcome of ischemic brain injury in SCD and its hematologic consequences. Our findings indicate a bidirectional relationship between stroke and anemia in SCD that may axially turn on Cyb5R3 activity.

Mechanism of Electron Transport in Mammalian Stearoyl-Coa Desaturase 1

Mammalian stearoyl-CoA desaturase 1 (SCD1) is a membrane-embedded metalloenzyme that catalyzes the formation of a double-bond on a saturated acyl-CoA. SCD1 has a diiron center coordinated by 9 histidine residues. The proper function of SCD1 requires an electron transport chain composed of membrane-bound cytochrome b5 reductase (b5R) and cytochrome b5 (cyt b5). Crystal structures of human and mouse SCD1 were reported recently. However, two zinc ions occupied the diiron center and the enzymes were inactive.

Multiple putative methemoglobin reductases in C. reinhardtii may support enzymatic functions for its multiple hemoglobins

The ubiquitous nature of hemoglobins, their presence in multiple forms and low cellular expression in organisms suggests alternative physiological functions of hemoglobins in addition to oxygen transport and storage. Previous research has proposed enzymatic function of hemoglobins such as nitric oxide dioxygenase, nitrite reductase and hydroxylamine reductase. In all these enzymatic functions, active ferrous form of hemoglobin is converted to ferric form and reconversion of ferric to ferrous through reduction partners is under active investigation. The model alga C. reinhardtii contains multiple globins and is thus expected to have multiple putative methemoglobin reductases to augment the physiological functions of the novel hemoglobins. In this regard, three putative methemoglobin reductases and three algal hemoglobins were characterized. Our results signify that the identified putative methemoglobin reductases can reduce algal methemoglobins in a nonspecific manner under in vitro conditions. Enzyme kinetics of two putative methemoglobin reductases with methemoglobins as substrates and in silico analysis support interaction between the hemoglobins and the two reduction partners as also observed in vitro. Our investigation on algal methemoglobin reductases underpins the valuable chemistry of nitric oxide with the newly discovered hemoglobins to ensure their physiological relevance, with multiple hemoglobins probably necessitating the presence of multiple reductases.

Defining the reducing system of the NO dioxygenase cytoglobin in vascular smooth muscle cells and its critical role in regulating cellular NO decay

In smooth muscle, cytoglobin (Cygb) functions as a potent nitric oxide (NO) dioxygenase and regulates NO metabolism and vascular tone. Major questions remain regarding which cellular reducing systems regulate Cygb-mediated NO metabolism. To better define the Cygb-mediated NO dioxygenation process in vascular smooth muscle cells (SMCs), and the requisite reducing systems that regulate cellular NO decay, we assessed the intracellular concentrations of Cygb and its putative reducing systems and examined their roles in the process of NO decay. Cygb and the reducing systems, cytochrome b5 (B5)/cytochrome b5 reductase (B5R) and cytochrome P450 reductase (CPR) were measured in aortic SMCs. Intracellular Cygb concentration was estimated as 3.5 μM, while B5R, B5, and CPR were 0.88, 0.38, and 0.15 μM, respectively. NO decay in SMCs was measured following bolus addition of NO to air-equilibrated cells. siRNA-mediated knockdown experiments indicated that ∼78% of NO metabolism in SMCs is Cygb-dependent. Of this, ∼87% was B5R- and B5-dependent. CPR knockdown resulted in a small decrease in the NO dioxygenation rate (VNO), while depletion of ascorbate had no effect. Kinetic analysis of VNO for the B5/B5R/Cygb system with variation of B5 or B5R concentrations from their SMC levels showed that VNO exhibits apparent Michaelis–Menten behavior for B5 and B5R. In contrast, linear variation was seen with change in Cygb concentration. Overall, B5/B5R was demonstrated to be the major reducing system supporting Cygb-mediated NO metabolism in SMCs with changes in cellular B5/B5R levels modulating the process of NO decay.

A RARE CAUSE OF NEONATAL CYANOSIS - CONGENITAL METHEMOGLOBINEMIA — A CASE REPORT

Cyanosis can occur at any age but it is a great challenge when it is in the newborn. Multiple causes have been established, and it usually is an ominous sign, especially when it occurs with neonatal sepsis or cyanotic congenital heart disease. Cyanosis caused by abnormal forms of hemoglobin can be life threatening, and early recognition is mandatory to prevent unnecessary investigations and delay in management. Abnormal hemoglobin newborn screening is a useful tool for the diagnosis. Acquired methemoglobinemia caused by environmental oxidizing agents, is common, but congenital deficiency of the innate reducing enzyme is so rare that only a few cases are documented in the medical literature around the world. Here I am presenting a case of a neonate with cyanosis as a result of congenital deficiency of cytochrome b5 reductase enzyme. This neonate was found to have cyanosis and appeared blue at newborn follow-up visit. Sepsis, congenital heart disease, prenatal administration, and ingestion of oxidant dyes were excluded as causes of the cyanosis by history, examination and appropriate tests. Chocolate discoloration of arterial blood provided a clue to the diagnosis. A normal newborn screen and hemoglobin electrophoresis made the diagnosis of hemoglobin M unlikely as the cause of the methemoglobinemia (Hb A 58.9%, A2 1.9%, and F 39.2%). Red blood cell enzyme activity and DNA analysis revealed a homozygous form of the cytochrome b5 reductase enzyme deficiency. She responded very well to daily methylene blue and ascorbic acid administration, and she has normal growth and developmental parameters, although she shows an exaggerated increase in her methemoglobin level.

The Identification of Cytochrome B5 Reductase 2 and Fructose-1,6-Bisphosphatase 1 Proteins in Non-Small Cell Lung Cancer: Proteomics Approach

Background: Lung cancer is one of the major causes of death worldwide, with more than a million deaths a year. Despite advanced surgical techniques and combined therapies, lung cancer is still a disease with poor prognosis. According to available reports, the number of patients with lung cancer among Iranian men and women is increasing. In a study, 4,361 deaths from lung cancer have been recorded in Iran, which are 9.80% and 6.07% of all cancer deaths among men and women, respectively. Objectives: This study aimed at investigating the proteomes in patients with non-small cell lung cancer (NSCLC) and comparing them with those of the healthy individuals to detect protein markers for the diagnosis of the disease in the early stages. Methods: According to the sample size estimation, the tissue samples of 30 patients with NSCLC were compared with 30 healthy tissues. Proteomics and reverse transcriptase polymerase chain reaction (RT-PCR) methods were used. First, the tissue samples were collected under sterile conditions and then protein was extracted. Next, 2-dimensional electrophoresis and mass spectrometry were performed. Finally, with proteomic analysis in patients and healthy individuals, differences in the proteomic pattern of healthy and cancerous tissue were examined. Results: More than 40 differences were revealed in the proteomics pattern of the healthy and cancerous tissues, and 2 different spots were submitted for liquid chromatography-mass spectrometry (LC-MS/ MS). Then cytochrome b5 reductase 2 (CYB5R2) and fructose-1,6-bisphosphate1 were detected. It has been shown that these proteins down-regulated in cancer tissue compared to healthy tissue. It was also shown molecularly that the expression of fructose-1,6-bisphosphatase 1 messenger ribonucleic acid (mRNA) and cytochrome b5 reductase 2 messenger ribonucleic acid (mRNA) in the cancerous tissue was 0.8 and 0.64 times less than their expression in healthy tissues, respectively. Conclusions: The result of this study, which deals with tumor markers of NSCLC, can be considered as a screening diagnostic test for diagnosis of the disease in the early stages. This research could provide the basis for further research to identify the critical effects of these 2 molecules cytochrome b5 reductase 2 and fructose-1,6-bisphosphate on tissue carcinogenesis. It is recommended to do more thorough research.

An inherent dysfunction in soluble guanylyl cyclase is present in the airway of severe asthmatics and is associated with aberrant redox enzyme expression and compromised NO-cGMP signaling

A subset of asthmatics develop a severe form of the disease whose etiology involves airway inflammation along with inherent drivers that remain ill-defined. To address this, we studied human airway smooth muscle cells (HASMC), whose relaxation drives airway bronchodilation and whose dysfunction contributes to airway obstruction and hypersensitivity in severe asthma. Because HASMC relaxation can be driven by the NO-soluble guanylyl cyclase (sGC)-cGMP signaling pathway, we questioned if HASMC from severe asthma donors might possess inherent defects in their sGC or in redox enzymes that support sGC function. We analyzed HASMC primary lines derived from 17 severe asthma and 16 normal donors and corresponding lung tissue samples regarding sGC activation by NO or by pharmacologic agonists, and also determined expression levels of sGC α1 and β1 subunits, supporting redox enzymes, and related proteins. We found a majority of the severe asthma donor HASMC (12/17) and lung samples primarily expressed a dysfunctional sGC that was NO-unresponsive and had low heterodimer content and high Hsp90 association. This sGC phenotype correlated with lower expression levels of the supporting redox enzymes cytochrome b5 reductase, catalase, and thioredoxin-1, and higher expression of heme oxygenases 1 and 2. Together, our work reveals that severe asthmatics are predisposed toward defective NO-sGC-cGMP signaling in their airway smooth muscle due to an inherent sGC dysfunction, which in turn is associated with inherent changes in the cell redox enzymes that impact sGC maturation and function.