Method optimization of the simultaneous detection of B12 congeners leading to the detection of a novel isomer of hydroxycobalamin in seawater.

RATIONALE: More than half of surveyed microalgae and over 90% of harmful algae have an obligate requirement for vitamin B12 , but methods for directly measuring dissolved B12 in seawater are scarce due to low concentrations and rapid light-induced hydrolysis. METHODS: We present a method to detect and measure the four main congeners of vitamin B12 dissolved in seawater. The method includes solid-phase extraction, separation by ultrahigh-performance liquid chromatography and detection by triple-quadrupole tandem mass spectrometry utilizing an electrospray ion source. This method was applied to coastal field samples collected in the German Bay, Baltic Sea and the Danish Limfjord system. RESULTS: The total dissolved B12 pool ranged between 0.5 and 2.1 pM. Under ambient conditions methyl-B12 and adenosyl-B12 were nearly fully hydrolyzed to hydroxy-B12 in less than 1 h. Hydroxy-B12 and a novel, corresponding isomer were the main forms of B12 found at all field sites. This isomer eluted well after the OH-B12 peak and was also detected in commercially available OH-B12 . Both compounds showed very high similarity in their collision-induced dissociation spectra. CONCLUSIONS: The high instability of the biologically active forms of Me-B12 and Ado-B12 towards hydrolysis was shown, highlighting the importance of reducing the duration of the extraction protocol. In addition, the vitamin B12 pool in the study area was mostly comprised of a previously undescribed isomer of OH-B12 . Further studies into the structure of this isomer and its bioavailability are needed.

Vitamin B12 association with mAbs: Mechanism and potential mitigation strategies.

Process control for manufacturing biologics is critical for ensuring product quality, safety, and lot to lot consistency of therapeutic proteins. In this study, we investigated the root cause of the pink coloration observed for various in-process pools and drug substances in the antibody manufacturing process. Vitamin B12 is covalently bound to mAbs via a cobalt-sulfur coordinate bond via the cysteine residues. The vitamin B12 was identified to attach to an IgG4 molecule at cysteine residues on light chain (Cys-214), and heavy chain (Cys-134, Cys-321, Cys-367, and Cys-425). Prior to attachment to mAbs, the vitamin B12 needs to be in its active form of hydroxocobalamin. During culture media preparation, storage and cell culture processing, cyanocobalamin, the chemical form of vitamin B12 added to media, is converted to hydroxocobalamin by white fluorescence light (about 50% degradation in 11-14 days at room temperature and with room light intensity about 500-1,000 lux) and by short-wavelength visible light (400-550 nm). However, cyanocobalamin is stable under red light (wavelength >600 nm) exposure and does not convert to hydroxocobalamin. Our findings suggests that the intensity of pink color depends on concentrations of both free sulfhydryl groups on reduced mAb and hydroxocobalamin, the active form of vitamin B12 . Both reactants are necessary and neither one of them is sufficient to generate pink color, therefore process control strategy can consider limiting either one or both factors. A process control strategy to install red light (wavelength >600 nm) in culture media preparation, storage and culture processing areas is proposed to provide safe light for biologics and to prevent light-induced color variations in final products.

Multicomponent spectrometric assay of cyanocobalamin and its photoproduct hydroxocobalamin in the presence of ascorbic acid in photolyzed solutions.

The simultaneous determination of cyanocobalamin (CC), hydroxocobalamin (HC) and ascorbic acid (AA) in aqueous solution has been achieved by a multicomponent spectrometric method. CC undergoes photolysis in acidic and alkaline media to form HC and the reaction is enhanced in the presence of AA. The method has been used to evaluate the kinetics of photodegradation reactions of the vitamin. CC, HC and AA present in the photolyzed solutions have been determined by absorbance measurement at 550, 525 and 265 nm at pH 4.0. These wavelengths correspond to the absorption maxima of the three substances and thus provide high specificity and sensitivity to the method. The method has been validated with respect to various parameters relating to the analytical performance characteristics. The recovery of the method for the three compounds ranges from 97.1-103.0% with a RSD value of ±3%. The accuracy of the method is shown by the linearity of the kinetic plots in the concentration range studied. The method is simple, rapid and convenient for the proposed work.

Hydroxocobalamin association during cell culture results in pink therapeutic proteins.

Process control of protein therapeutic manufacturing is central to ensuring the product is both safe and efficacious for patients. In this work, we investigate the cause of pink color variability in development lots of monoclonal antibody (mAb) and Fc-fusion proteins. Results show pink-colored product generated during manufacturing is due to association of hydroxocobalamin (OH-Cbl), a form of vitamin B12. OH-Cbl is not part of the product manufacturing process; however we found cyanocobalamin (CN-Cbl) in cell culture media converts to OH-Cbl in the presence of light. OH-Cbl can be released from mAb and Fc-fusion proteins by conversion with potassium cyanide to CN-Cbl, which does not bind. By exploiting the differential binding of CN-Cbl and OH-Cbl, we developed a rapid and specific assay to accurately measure B12 levels in purified protein. Analysis of multiple products and lots using this technique gives insight into color variability during manufacturing.

An evaluation of the interference of hydroxycobalamin with chemistry and co-oximetry tests on nine commonly used instruments.

The administration of hydroxocobalamin (OHCob), alone or with sodium thiosulfate, is a standard therapy for cyanide poisoning. OHCob is a red chromophore, and its interference with co-oximetric and colorimetric laboratory measurements has been evaluated in a few conflicting reports. The interference of OHCob was investigated in samples spiked with 10 different concentrations of OHCob (0-1500 mg/L). The concentration of 73 different analytes was measured using nine different analysers (ABL 800 Flex, Advia 1800, Advia Centaur Xp, Architect ci8200, Immulite 2500, Konelab 30i, Modular Analytics SWA, Synchron LX 20 and Vitros 5.1). All instruments yielded some results that were affected by OHCob at concentrations equivalent to a single therapeutic dose. Of the 73 different analytes, 64% showed interference on at least one instrument. Of all 187 tests performed, 47% were biased with more than 10%. Interference was generally limited to photometric assays, whereas immunological and ion-selective electrode measurements were unaffected. OHCob present in the blood after treatment for cyanide poisoning interfered with many laboratory assays in an unpredictable way, making some results invalid. Some affected tests are important in the treatment of cyanide poisoning. The interference is not solely due to wavelength, but also to chemical interaction. Without delaying the administration of OHCob, blood should, preferably, be drawn in advance, or, at least, the laboratory should be informed about the OHCob treatment. If the laboratory receives OHCob-containing samples, methods and instruments should be selected to minimize bias, and the manufacturer of the OHCob should recommend relevant precautions to customers in the package insert.

Spectrophotometry of hydroxocobalamin and hemoglobin reveals production of an unanticipated methemoglobin variant.

INTRODUCTION: Cyanide-poisoned patients often require pulse oximetry and co-oximetry to measure oxyhemoglobin, deoxyhemoglobin, carboxyhemoglobin, and methemoglobin. These and other critical laboratory measurements can be confounded by the cyanide antidote hydroxocobalamin. The postulated mechanism of this confounding is direct optical interference. METHODS: The spectra of swine hemoglobin with and without hydroxocobalamin were measured from 450-800 nm. The resulting complex spectrum was divided into hemoglobin, hydroxocobalamin, and a remainder spectrum. RESULTS: The remainder spectrum appears to be a methemoglobin variant quantitatively dependant on the amount of hydroxocobalamin added to the hemoglobin solution and the presence of oxygen. The Pearson's correlation coefficient comparing the known swine methemoglobin spectrum with the remainder spectrum reveals a very high degree of correlation (r(2) = 0.986). CONCLUSION. This is the first study to document methemoglobin formation caused by hydroxocobalamin. Further studies are needed in vitro and in vivo to assess this previously unreported methemoglobin variant.

Analysis of corrinoids in ovine tissues.

Corrinoids from various ovine tissue samples (liver, blood, small intestinal fluid and faeces) were analysed using a combination of high-performance liquid chromatography (HPLC) and a radioisotope dilution assay (RIDA) to estimate the distribution of corrinoids--the cobalamins hydroxocobalamin (OH-cbl), methylcobalamin (me-cbl) and 5'-deoxyadenosylcobalamin (ado-cbl), and cobalamin analogues--in these tissues. Samples were taken from either cobalt-deficient or cobalt-replete ewes, and ruminant and pre-ruminant lambs. In liver, ado-cbl predominated, followed by analogues, OH-cbl and me-cbl. Supplementation with either cobalt (ruminant) or vitamin B12 injections (pre-ruminant) increased the amount of ado-cbl and decreased analogues. In blood, OH-cbl predominated, followed by ado-cbl, analogues and me-cbl, respectively. In small intestinal fluid, the distribution from largest to smallest percentage was analogues, ado-cbl, OH-cbl and me-cbl. In faeces, analogues constituted the greatest proportion, followed by OH-cbl, ado-cbl and me-cbl, respectively. Owing to the small sample sizes only cautionary interpretations can be made. In contrast to humans, where me-cbl constitutes the highest proportion of corrinoids in plasma and ado-cbl in the liver, in sheep the amount of ado-cbl was consistently higher than me-cbl in all tissues. This may be due to the higher metabolic need of sheep for ado-cbl due to gluconeogenesis. Analogues and OH-cbl were found in each tissue, contrary to previous postulations. The much higher amount of vitamin B12 in small intestinal fluid compared with faeces indicates that a large proportion of the vitamin is absorbed by the gastro-intestinal tract.

Determination of hydroxocobalamin and cyanocobalamin by derivative spectrophotometry in cyanide poisoning.

Hydroxocobalamin (OHCo) and cyanocobalamin (CNCo) are determined directly in biological media, without extraction, by using first derivative spectrophotometry. We diluted 200 mL of plasma, urine, or standards with 1.8 mL of pH 6 buffer (boric acid, potassium dihydrogen orthophosphate, and potassium hydroxide). The first derivative spectra of the dilutions were plotted between 320 and 400 nm. At the exact zero-crossing point for hydroxocobalamin, the derivative values of cyanocobalamin concentration were determined. The same procedure was followed for hydroxocobalamin at the zero-crossing point for cyanocobalamin. The derivative values of the concentration curves are linear in the range 5-100 microM. The minimum detection limit is approximately 5 microM for hydroxocobalamin of cyanocobalamin on the determination of hydroxocobalamin or vice versa, although the spectra strongly overlap. The method is fast and simple to use, thus making it easy to assess the in vivo transformation of hydroxocobalamin into cyanocobalamin after the administration of high doses of hydrocobalamin in cyanide poisoning.

Photolysis of cyanocobalamin in aqueous solution.

Cyanocobalamin is photolysed in aqueous solution to produce hydroxocobalamin. The kinetics of photolysis has been studied at pH 1-12 using a newly developed spectrophotometric method for the simultaneous determination of the two compounds at 550 and 525 nm or 361 and 351 nm. Cyanocobalamin follows zero-order kinetics at the pH values studied and the rate is catalysed by both hydrogen and hydroxyl ions. The log k-pH profile indicates that cyanocobalamin has maximum stability near pH 7. The cationic species appears to be more susceptible to photolysis than the neutral form. The rate constant for the reaction at pH 1 is 1.32 x 10(-7) mol l-1 min-1 compared with 0.050 x 10(-7) mol l-1 min-1 at pH 7.

The use of hydroxocobalamin in the Schilling test.

Hydroxocobalamin and cyanocobalamin have been compared as the 'flushing dose' in the Schilling test. In healthy, haematologically normal subjects excretion of the test dose was greater following a hydroxocobalamin flushing dose than following a cyanocobalamin flushing dose, and to a lesser extent this was also true in patients requiring investigation. There were occasional discrepant results, but in general it appears that, although reference values differ, hydroxocobalamin is a suitable replacement for cyanocobalamin in the Schilling test.

Conversion of hydroxo(aquo) cobalamin to sulfitocobalamin in the absence of light: a reaction of importance in the identification of the forms of vitamin B12, with possible clinical significance.

During determinations of the forms of vitamin B12 in foods and human tissues several samples yielded a growth zone on bioautography which was distinct from those due to methylcobalamin, adenosylcobalamin, hydroxocobalamin, or cyanocobalamin. The material responsible for this growth zone was identified as sulfitocobalamin and the mechanisms in its formation from hydroxocobalamin involve absence of light and the presence of bisulphite ions derived from atmospheric sulfur dioxide or indigenous sulfite ions. The formation of sulfitocobalamin during the extraction of cobalamins from foods and tissues can be prevented by reception and homogenization of the material in an ammonia buffer instead of water; the hydroxocobalamin is then converted to ammonia cobalamin, which is more resistant to attack by (bi)sulfite ions. The conversion of hydroxo(aquo)cobalamin to sulfitocobalamin in the dark can be rapid, and materials for analysis should be placed in the ammonia buffer before darkroom work is begun. The rapid conversion of hydroxo(aquo)cobalamin to sulfitocobalamin in the dark raises the possibility that hydroxocobalamin in foods may be converted to the less well absorbed sulfitocobalamin in the upper gastrointestinal tract.

The forms of vitamin B12 in foods.

1. The forms of vitamin B12 were determined in foods, most of which had been prepared for consumption. 2. Five forms were detected: adenosylcobalamin, hydroxocobalamin, methylcobalamin, cyanocobalamin and sulphitocobalamin. Adenoxylcobalamin and hydroxocobalamin were the predominant forms. 3. The intestinal absorption of [57Co]sulphitocobalamin was estimated and found to be lower than that of [58Co]cyanocobalamin.