Hydroxocobalamin Research Articles

Characterization of a Structurally Distinct ATP-Dependent Reactivating Factor of Adenosylcobalamin-Dependent Lysine 5,6-Aminomutase.

Several anaerobic bacterial species, including the Gram-negative oral bacterium Fusobacterium nucleatum, ferment lysine to produce butyrate, acetate, and ammonia. The second step of the metabolic pathway─isomerization of β-l-lysine to erythro-3,5-diaminohexanoate─is catalyzed by the adenosylcobalamin (AdoCbl) and pyridoxal 5'-phosphate (PLP)-dependent enzyme, lysine 5,6-aminomutase (5,6-LAM). Similar to other AdoCbl-dependent enzymes, 5,6-LAM undergoes mechanism-based inactivation due to loss of the AdoCbl 5'-deoxyadenosyl moiety and oxidation of the cob(II)alamin intermediate to hydroxocob(III)alamin. Herein, we identified kamB and kamC, two genes responsible for ATP-dependent reactivation of 5,6-LAM. KamB and KamC, which are encoded upstream of the genes corresponding to α and β subunits of 5,6-LAM (kamD and kamE), co-purified following coexpression of the genes in Escherichia coli. KamBC exhibited a basal level of ATP-hydrolyzing activity that was increased 35% in a reaction mixture that facilitated 5,6-LAM turnover with β-l-lysine or d,l-lysine. Ultraviolet-visible (UV-vis) spectroscopic studies performed under anaerobic conditions revealed that KamBC in the presence of ATP/Mg2+ increased the steady-state concentration of the cob(II)alamin intermediate in the presence of excess β-l-lysine. Using a coupled UV-visible spectroscopic assay, we show that KamBC is able to reactivate 5,6-LAM through exchange of the damaged hydroxocob(III)alamin for AdoCbl. KamBC is also specific for 5,6-LAM as it had no effect on the rate of substrate-induced inactivation of the homologue, ornithine 4,5-aminomutase. Based on sequence homology, KamBC is structurally distinct from previously characterized B12 chaperones and reactivases, and correspondingly adds to the list of proteins that have evolved to maintain the cellular activity of B12 enzymes.

Vitamin B12 Hypersensitivity: A Retrospective Multicenter Study

Vitamin B12 (Vit B12) deficiency affects approximately 20% of those above the age of 60 years in the United Kingdom and United States. If untreated, it leads to detrimental health outcomes. To investigate a cohort of patients with Vit B12 hypersensitivity (VB12H) referred to 3 UK allergy centers and design a pathway for the investigation of VB12H. A total of 29 patients seen between 2014 and 2022 underwent skin prick testing (1 mg/mL) with cyanocobalamin (CC) and hydroxycobalamin (HC) and intradermal testing (0.1 and 0.01 mg/mL). Patients with negative skin tests underwent a Vit B12 drug provocation test (DPT) with either the index or the alternative drug. Of 29 patients, 18 (62%) presented with immediate VB12H. Eight experienced anaphylaxis (4 to HC and 4 to CC) and had positive skin tests to the index drug. One patient reacted to oral and 7 patients to injectable Vit B12. Seven patients sensitized to one form of Vit B12-tolerated DPT with an alternative Vit B12. One patient with immediate VB12H reacted to polyethylene glycol (PEG) in oral cobalamin. Of 29 patients, 8 presented with delayed hypersensitivity reaction; 4 patients tolerated the intramuscular index formulation, whereas 2 patients tolerated the per oral formulation. One patient presented with symptoms consistent with symmetrical drug-related intertriginous and flexural exanthema. Three patients were referred because of cobalt allergy. Confirmed VB12H is rare. We propose a comprehensive evaluation protocol that includes Vit B12 skin tests and considers PEG allergy in patients presenting with VB12H.

Hydroxycobalamin Administration in the Treatment of Calcium Channel Blocker-Induced Vasoplegia: A Case Report

Calcium channel blocker (CCB)-induced vasoplegia syndrome is an emergent, life-threatening complication that can occur refractory to traditional therapy in the treatment of an acute CCB overdose. We present a case of acute co-ingestion of many prescription medications, including amlodipine and escitalopram. The patient presented with hemodynamic instability that persisted despite conventional therapies. Hydroxycobalamin (HCO) therapy was initiated, resulting in hemodynamic and clinical improvement. We highlight the unique presentation of this case and the potential benefits associated with HCO administration. Key Points: This case demonstrates the potential benefits of HCO administration in CCB-induced vasoplegia syndrome. Given the significant morbidity and mortality associated with CCB toxicity, HCO may provide considerable benefit in patients with hemodynamic instability refractory to standard treatment modalities.

Comparison of hydroxocobalamin with other resuscitative fluids in volume-controlled and uncontrolled hemorrhage models in swine ( Sus-scrofa ).

Traumatic hemorrhage is the leading cause of preventable death in military environments. Treatment with resuscitative fluids and blood components is based on availability, thus, frequently unavailable in the prehospital setting, due to lack of resources and costs. Hydroxocobalamin (HOC), increases blood pressure via nitric oxide scavenging. We evaluated HOC as a resuscitation fluid, in two swine hemorrhage models. Our objectives were to (1) evaluate whether HOC treatment following hemorrhagic shock improves hemodynamic parameters and (2) determine whether those effects are comparable to whole blood (WB) and lactated ringers (LR). Yorkshire swine (S us scrofa ) (n = 72) were used in models of controlled hemorrhage (CH) (n = 36) and uncontrolled hemorrhage (UH) (n = 36). Randomized animals received treatment with 500 mL of either WB, LR, HOC (150 mg/kg), followed by a six-hour observation (n = 6 each group). Survival, hemodynamics, blood gases (ABGs) and chemistries were collected. Data reported as mean ± standard error of the mean and statistical analysis by ANOVA ( p < 0.05). Blood loss for CH was 41% ± 0.02 versus 33% ± 0.07 for UH. For CH, HOC treatment maintained higher systolic blood pressure (sBP, mm Hg) compared with WB and LR (72 ± 1.1; 60 ± 0.8; 58 ± 1.6; respectively). Heart rate (HR), cardiac output (CO), Sp o2 and vascular resistance were comparable with WB and LR. The ABG values were comparable between HOC and WB. For UH, HOC treatment maintained sBP levels comparable to WB and higher than LR (70 ± 0.9; 73 ± 0.5; 56 ± 1.2). HR, CO, Sp o2 , and systemic vascular resistance were comparable between HOC and WB. Survival, hemodynamics, blood gases were comparable between HOC and WB. No survival differences were found between cohorts. Hydroxocobalamin treatment improved hemodynamic parameters and Ca 2+ levels compared with LR and equivalent to WB, in both models. Hydroxocobalamin may be a viable alternative when WB is not available.

Low methylcobalamin in liver tissues is an artifact as shown by a revised extraction procedure

BackgroundVitamin B12 (cobalamin, Cbl) is represented by several molecular variants distinguished by the exchangeable ligand X coordinated to cobalt ion (XCbl). The most typical XCbl-forms are cyanocobalamin (CNCbl), hydroxocobalamin (HOCbl), methylcobalamin (MeCbl) and 5′-deoxydeoxyadenosylcobalamin (AdoCbl). Cells convert the “inactive” vitamins CNCbl and HOCbl to the two critically important coenzymes AdoCbl or MeCbl. Surprisingly, little or no MeCbl is usually uncovered in the tissue samples, as compared to AdoCbl and HOCbl. We hypothesized that a low level of MeCbl is an artifact of “harsh” extractions, leading to degradation of MeCbl and/or its conversion to other XCbl-forms. MethodsWe designed a “mild” extraction protocol, including homogenization of rat liver in ammonium acetate (pH 4.6), dilution with EtOH (final 60%) and heating for 10 min at 70 °C. The XCbls were separated by HPLC and quantified by isotope dilution assays. ResultsA “mild” extraction revealed the following composition of Cbls: 37% AdoCbl, 35% MeCbl, 15% HOCbl and 13% CNCbl. The usual “harsh” protocol (pH 7, 20 min at 80 °C) changed this balance to 33%, 5%, 43% and 17%, respectively. A model assay revealed that MeCbl underwent demethylation and conversion to HOCbl at pH 3 and pH > 7, when heated with thiols. Other changes included decyanation of CNCbl and destruction of HOCbl. ConclusionsOur procedure reveals a high content of MeCbl in rat liver. General significanceThis result challenges previous data and pinpoints the need for new studies to characterize the endogenous Cbl-forms in health and disease.

Differences in the Formation of Reactive Oxygen Species and Their Cytotoxicity between Thiols Combined with Aqua- and Cyanocobalamins.

Cobalamin is an essential nutrient required for the normal functioning of cells. Its deficiency can lead to various pathological states. Hydroxocobalamin (HOCbl) and cyanocobalamin (CNCbl) are the forms of vitamin B12 that are most commonly used for supplementation. There is substantial evidence indicating that cobalamins can both suppress and promote oxidative stress; however, the mechanisms underlying these effects are poorly understood. Here, it was shown that the oxidation of thiols catalyzed by HOCbl and CNCbl is accompanied by reactive oxygen species (ROS) production and induces, under certain conditions, oxidative stress and cell death. The form of vitamin B12 and the structure of thiol play a decisive role in these processes. It was found that the mechanisms and kinetics of thiol oxidation catalyzed by HOCbl and CNCbl differ substantially. HOCbl increased the rate of oxidation of thiols to a greater extent than CNCbl, but quenched ROS in combination with certain thiols. Oxidation catalyzed by CNCbl was generally slower. Yet, the absence of ROS quenching resulted in their higher accumulation. The aforementioned results might explain a more pronounced cytotoxicity induced by combinations of thiols with CNCbl. On the whole, the data obtained provide a new insight into the redox processes in which cobalamins are involved. Our results might also be helpful in developing new approaches to the treatment of some cobalamin-responsive disorders in which oxidative stress is an important component.

Cobalamin-Mediated Electrocatalytic Reduction of Ethyl Chloroacetate in Dimethylformamide

The catalytic reduction of ethyl chloroacetate (ECA) by hydroxocobalamin (HOCbl) in dimethylformamide was studied electrochemically and spectroelectrochemically to identify initial steps in the reaction between the electrogenerated Co(I) center of cobalamin (cob(I)alamin) and ECA. Cyclic voltammograms of HOCbl in the presence of ECA show a small increase in current related to reduction of Co(II) to Co(I), and a new peak at more negative potentials related to reduction of an ethyl carboxymethyl-Cbl intermediate. The oxidation state of HOCbl during catalysis was monitored by means of spectroelectrochemical controlled-potential bulk electrolysis. Addition of ECA to electrogenerated cob(I)alamin initially generates the Co(II) form (cob(II)alamin) followed by a gradual formation of an ethyl carboxymethyl-Cbl intermediate. Controlled-potential bulk electrolysis was performed to identify products formed from catalytic reduction of ECA by electrogenerated cob(I)alamin and quantify the number of electrons transferred per molecule of ECA. Product distributions and coulometric results, together with the results of voltammograms and spectroelectrochemical controlled–potential bulk electrolysis, were interpreted to propose a reaction mechanism.

POSB121 Economic Evaluation of the Impact Results of Hydroxocobalamin Use in Victims of Suspected Cyanide Poisoning Caused by Smoke Inhalation

Hydroxocobalamin (HC) has demonstrated clinical effectiveness in victims of suspected Cyanide (CN) poisoning caused by smoke inhalation. Though, the economic aspect remains largely unexplored. This analysis aims to assess the costs associated with clinical benefits and its reduction in intensive care unit (ICU) stays at hospital. Methods: The analysis is based on a 2017 published study (Nguyen L. & al.) where patients receiving HC were compared to patients with a non-antidote intervention. The retained model comprises costs related to HC administration, emergency care, lactate assays and hospitalisation stays in the different units (ICU with mechanical ventilation (MV), progressive care unit (PCU), hospital care).

Intracellular processing of vitamin B12 by MMACHC (CblC).

Vitamin B12 (cobalamin, Cbl, B12) is a water-soluble micronutrient synthesized exclusively by a group of microorganisms. Human beings are unable to make B12 and thus obtain the vitamin via intake of animal products, fermented plant-based foods or supplements. Vitamin B12 obtained from the diet comprises three major chemical forms, namely hydroxocobalamin (HOCbl), methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl). The most common form of B12 present in supplements is cyanocobalamin (CNCbl). Yet, these chemical forms cannot be utilized directly as they come, but instead, they undergo chemical processing by the MMACHC protein, also known as CblC. Processing of dietary B12 by CblC involves removal of the upper-axial ligand (beta-ligand) yielding the one-electron reduced intermediate cob(II)alamin. Newly formed cob(II)alamin undergoes trafficking and delivery to the two B12-dependent enzymes, cytosolic methionine synthase (MS) and mitochondrial methylmalonyl-CoA mutase (MUT). The catalytic cycles of MS and MUT incorporate cob(II)alamin as a precursor to regenerate the coenzyme forms MeCbl and AdoCbl, respectively. Mutations and epimutations in the MMACHC gene result in cblC disease, the most common inborn error of B12 metabolism, which manifests with combined homocystinuria and methylmalonic aciduria. Elevation of metabolites homocysteine and methylmalonic acid occurs because the lack of an active CblC blocks formation of the indispensable precursor cob(II)alamin that is necessary to activate MS and MUT. Thus, in patients with cblC disease, vitamin B12 is absorbed and present in circulation in normal to high concentrations, yet, cells are unable to make use of it. Mutations in seemingly unrelated genes that modify MMACHC gene expression also result in clinical phenotypes that resemble cblC disease. We review current knowledge on structural and functional aspects of intracellular processing of vitamin B12 by the versatile protein CblC, its partners and possible regulators.

Hydroxocobalamin

Hydroxocobalamin

Efficacy of Intravenous Hydroxocobalamin for Treatment of Sodium Methanethiolate Exposure in a Swine Model (Sus scrofa) of Severe Methanethiol Toxicity.

Methanethiol is a highly toxic chemical present in crude oil and natural gas. At high concentrations, methanethiol causes metabolic acidosis, seizures, myocardial infarction, coma, and death. Occupational Health and Safety Administration lists methanethiol as a potential terrorist weapon. Methanethiol blocks the electron transport chain, resulting in lactic acidosis and acidemia. There is no specific treatment for methanethiol. Our objective was to measure the efficacy of intravenous (IV) hydroxocobalamin (HOC) versus no treatment (control) in methanethiol-induced apnea in a swine model. Sixteen anesthetized swine received IV sodium methanethiolate to apnea and were randomized to receive either IV HOC or no treatment. Physiologic and laboratory parameters were monitored throughout the study. Power analysis indicated that 8 animals per group would be sufficient to find a moderate effect (f = 0.24) with 2 groups, α = 0.05, and 80% power. Both groups were similar in baseline characteristics. Following treatment, the HOC group had significantly higher heart rate and blood pressure at 5-10minutes post-apnea, higher systemic vascular resistance at 5minutes post-apnea, higher tidal volume, higher end-tidal carbon dioxide, and lower end-tidal oxygen 10-15minutes post-apnea compared with controls. None of the animals survived to the end of the study (60minutes). The Kaplan-Meier survival curves were significantly different between cohorts (log-rank p = 0.0321), with the HOC group surviving longer than controls (32.4 ± 7.3 vs. 25.8 ± 1.0minutes). In our model of intravenous methanethiolate poisoning, IV HOC administration resulted in a transient improvement in vital signs and prolonged time to death; however, it did not improve survival.

Assessment of cellular cobalamin metabolism in Gaucher disease

BackgroundGaucher disease (GD) is a lysosomal disorder caused by biallelic pathogenic mutations in the GBA1 gene that encodes beta-glucosidase (GCase), and more rarely, by a deficiency in the GCase activator, saposin C. Clinically, GD manifests with heterogeneous multiorgan involvement mainly affecting hematological, hepatic and neurological axes. This disorder is divided into three types, based on the absence (type I) or presence and severity (types II and III) of involvement of the central nervous system. At the cellular level, deficiency of GBA1 disturbs lysosomal storage with buildup of glucocerebroside. The consequences of disturbed lysosomal metabolism on biochemical pathways that require lysosomal processing are unknown. Abnormal systemic markers of cobalamin (Cbl, B12) metabolism have been reported in patients with GD, suggesting impairments in lysosomal handling of Cbl or in its downstream utilization events.MethodsCultured skin fibroblasts from control humans (n = 3), from patients with GD types I (n = 1), II (n = 1) and III (n = 1) and an asymptomatic carrier of GD were examined for their GCase enzymatic activity and lysosomal compartment intactness. Control human and GD fibroblasts were cultured in growth medium with and without 500 nM hydroxocobalamin supplementation. Cellular cobalamin status was examined via determination of metabolomic markers in cell lysate (intracellular) and conditioned culture medium (extracellular). The presence of transcobalamin (TC) in whole cell lysates was examined by Western blot.ResultsCultured skin fibroblasts from GD patients exhibited reduced GCase activity compared to healthy individuals and an asymptomatic carrier of GD, demonstrating a preserved disease phenotype in this cell type. The concentrations of total homocysteine (tHcy), methylmalonic acid (MMA), cysteine (Cys) and methionine (Met) in GD cells were comparable to control levels, except in one patient with GD III. The response of these metabolomic markers to supplementation with hydroxocobalamin (HOCbl) yielded variable results. The content of transcobalamin in whole cell lysates was comparable in control human and GD patients.ConclusionsOur results indicate that cobalamin transport and cellular processing pathways are overall protected from lysosomal storage damage in GD fibroblasts. Extending these studies to hepatocytes, macrophages and plasma will shed light on cell- and compartment-specific vitamin B12 metabolism in Gaucher disease.

Clinical analysis and outcome of patients with c. 482G >A variant of MMACHC gene in cblC type methylmalonic acidemia

Objective To explore the clinical manifestations, treatment and outcomes of patients with c. 482G>A(p.R161Q)variant of MMACHC gene in cblC type methylmalonic acidemia(MMA). Methods The clinical manifestations, mass spectrometry results, genotypes, treatment and outcomes of 75 patients with cblC type MMA carrying c. 482G>A(p.R161Q)variant were retrospectively analyzed. Results Of the 75 patients, 57(76%)were from newborn screening and one of them had an onset. Among the rest 18 unscreened patients, 2 were diagnosed after their full sisters′ or brothers′ diagnosis, the others were clinical patients. There were 17 clinical patients, with the medium age of onset 12 years old(10 days~26 years old). 12 late onset patients(70.6%)presented with poor academic performance, memory loss, poor expression, and decreased exercise capacity, while 5 early onset patients(29.4%)presented with convulsion and delay of development. All patients were vitamin B12-responsive. The levels of blood propionylcarnitine, the ratio of propionylcarnitine to acetylcarnitine, urinary methylmalonic acid and methyldecanoic acid, and plasma homocysteine were significantly decreased after treatment(P< 0.01). All patients diagnosed from newborn screening had normal development. However, only 3 clinical patients had a rather normal outcomes and the others remained different levels of intelligence and(or)motor dysfunction after treatment. Conclusion The c. 482G>A(p.R161Q)variant of MMACHC gene is associated with late onset cblC type MMA. Patients with this variant have a better response to hydroxycobalamin than other variants. The outcome of patients diagnosed from the newborn screening is good. When symptoms occur, the disability rate is often high. Therefore, newborn screening is a recommended method to prevent this disease. Key words: Methylmalonic acidemia; MMACHC gene; Methylmalonic acid; Propionylcarnitine

High-dose hydroxocobalamin in end-stage liver disease and liver transplantation.

Distributive shock is a serious complication in patients with chronic or end-stage liver disease, and can be exacerbated by vasoplegia in this patient population. Vasoplegic syndrome (VS) is a state of shock refractory to catecholamines and vasopressin that is often multifactorial in liver failure patients, and can occur in any phase of liver transplantation (LT) [i.e., pre-transplantation, intraoperative, and post-transplantation]. Methylene blue (MB) has been a well-established pharmacologic therapy for VS. However, it has been known to cause dose-related toxicity. Hydroxocobalamin (HXC) is not currently FDA approved for the management of VS, but studies have demonstrated its ability to cause an increase in systolic blood pressure by hypothesized mechanisms with only minimal side effects. To date, only three other reports have demonstrated the use of HXC in LT patients, which highlighted its use both intraoperatively and post-transplantation. Our report illustrates the utility of HXC in four LT patients with VS. Two of these cases illustrate the usefulness of HXC in the pre-transplantation period, which has never been previously reported. HXC is a useful pharmaceutical agent in the management of VS, especially if contraindications to MB exist or in cases of MB-resistant vasoplegia. Further studies with large sample sizes are necessary to ascertain the optimal dosage of HXC in LT patients.

Hydroxocobalamin

Hydroxocobalamin

Hydroxocobalamin

Hydroxocobalamin

Off to the Races: Comparison of Excited State Dynamics in Vitamin B12 Derivatives Hydroxocobalamin and Aquocobalamin.

Ultrafast time-resolved spectroscopy was used to study the photochemistry of hydroxocobalamin (HOCbl) and aquocobalamin (H2OCbl+) in solution. Spectroscopic measurements and TD-DFT simulations provide a consistent picture of the spectroscopy and photochemistry. Excitation of H2OCbl+ results in formation of an excited state followed by rapid internal conversion to the ground state (0.35 ± 0.15 ps) through an S1/S0 seam at a slightly elongated Co-O bond length and a significantly elongated Co-NIm bond length. In contrast, the initial elongation of the axial bonds in HOCbl is followed by contraction to an excited state minimum with bonds slightly shorter than those in the ground state. Internal conversion to the ground state follows on a picosecond time scale (5.3 ± 0.4 ps). For both compounds, photodissociation forming cob(II)alamin and hydroxyl radicals (∼1.5% yield) requires excitation to highly excited states. Dissociation is mediated by competition between internal conversion to the S1 surface and prompt bond cleavage.

WITHDRAWN: Other implications of hydroxocobalamin as an alternate vasoplegia therapeutic

WITHDRAWN: Other implications of hydroxocobalamin as an alternate vasoplegia therapeutic

Hydroxocobalamin

Hydroxocobalamin

Hydroxocobalamin

Hydroxocobalamin