Food Chemistry 170 (2015) 316–320
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Methylcobalamin – A form of vitamin B12 identified and characterised in Chlorella vulgaris Anantharajappa Kumudha a, Sagaya Selvakumar b, Pullancheri Dilshad c, Gopal Vaidyanathan c, Munna Singh Thakur b, Ravi Sarada a,⇑ a b c
Plant Cell Biotechnology Department, CSIR-Central Food Technological Research Institute, Mysore 570020, India Fermentation Technology and Bioengineering Department, CSIR-Central Food Technological Research Institute, Mysore 570020, India Waters (India) Pvt. Ltd, Bangalore, India
a r t i c l e
i n f o
Article history: Received 25 April 2013 Received in revised form 30 July 2014 Accepted 10 August 2014 Available online 23 August 2014 Keywords: Chlorella Methylcobalamin MS/MS Gold nanoparticle Aptamer
a b s t r a c t Vitamin B12 is among the most essential biomolecules required for crucial metabolic processes in humans. Vitamin B12 was extracted from Chlorella vulgaris biomass under aqueous conditions, partially purified by passing the extract through amberlite XAD-2, Sep-Pak columns, and further purified by HPLC. The target peak eluent was subjected to characterisation by tandem mass spectrometry (MS/MS), selected ion recording (SIR) and multiple reaction monitoring (MRM) and identified as methylcobalamin (Me-Cbl). Quantification of Me-Cbl was carried out by microbiological and chemiluminescence methods, and found to be 29.87 ± 2 lg/100 g and 26.84 ± 2 lg/100 g dry weight, respectively. The presence of Me-Cbl was further substantiated using gold nanoparticle (AuNPs) based aptamer analysis, and found to be 28.02 ± 2 lg/100 g dry weight. Good similarity was observed among all the methods. Methylcobalamin, a form of vitamin B12 was identified in C. vulgaris and this finding enhances its use as a nutritional supplement. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Vitamin B12 is one of the largest, most structurally complex, non-polymeric biomolecules and an evolutionarily ancient cofactor with carbon–metal bonds (Benner, Ellington, & Tauer, 1989; Eschenmoser, 1988). Vitamin B12 plays an important role in the synthesis of methionine from homocysteine and conversion of methylmalonyl coenzyme A to succinyl coenzyme A in humans. It is, generally, believed that plants neither synthesizes nor utilise cobalamin for growth (Duda, Pedziwilk, & Zodrow, 1967). However some studies have disputed this conclusion (Poston, 1977). Many algae utilise vitamin B12 for growth while a few synthesize vitamin B12. Some algae are known to contain substantial amount of vitamin B12. Porphyra yezoensis is one algae reported to contain as much cobalamin as animal liver (Kanazawa, 1963). Strict Abbreviations: OH-Cbl, hydroxocobalamin; Ado-Cbl, 5-deoxyadenosylcobalamin; Me-Cbl, methylcobalamin; SO-Cbl, sulphitocobalamin; CN-Cbl, cyanocobalamin; CL, chemiluminescence; Urea-H2O2, urea hydrogen peroxide; CLU, chemiluminescence unit; MS/MS, tandem mass spectrometry; SIR, selected ion recording; MRM, multiple reaction monitoring; AuNPs, gold nanoparticle; HPLC, high performance liquid chromatography; SRP, Surface Plasmon Resonance. ⇑ Corresponding author. Tel.: +91 821 2516 501; fax: +91 821 2517 233. E-mail address: [email protected] (R. Sarada). http://dx.doi.org/10.1016/j.foodchem.2014.08.035 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
vegetarians can obtain sufficient bioavailable B12 from some seaweed (Rauma, Torronen, Hanninen, & Mykkanen, 1995). Algal B12 has been found to be bioavailable in B12-deficient rats fed dried purple laver, a type of seaweed (Takenaka et al., 2001). However information on quantitative bioavailability of algal B12 is limited. Humans have consumed Chlorella vulgaris biomass for centuries as health food. Some Chlorella species are grown under open cultivation system and used for the preparation of the Chlorella tablets, and they are a good source of vitamin B12. Since B12 exists in different forms it is important to identify and quantify the forms in C. vulgaris. The forms of vitamin B12 are similar with a cobalt central ion, the four parts of the corrin ring, and a dimethylbenzimidazole group. They mainly differ in the sixth carbon, which may contain a cyano (CN), hydroxyl (OH), methyl (CH3) or 50 -deoxyadenosyl group (C-CO). Although C. vulgaris has been reported to contain vitamin B12 (Katsura, Fujith, Watanabe, & Nakano, 2002), the form of B12 and quantity have not been determined in dry biomass. Some Chlorella species have been reported to accumulate exogenous cobalamin (Watanabe et al., 1997). There is, however, little information available on the forms of vitamin B12 or the physiological roles of the algal cobalamin (Watanabe et al., 1997). The aim of the present study was to identify and quantify the forms of vitamin B12 in C. vulgaris dry biomass.
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2. Materials and methods 2.1. Chemicals and instruments Methylcobalamin (Me-Cbl), adenosylcobalamin (Adl-Cbl), hydroxocobalamin (OH-Cbl), luminol, urea hydrogen peroxide, diethylpyrocarbonate (DEPC), gold (III) chloride, trisodium citrate, and silver nitrate were procured from Sigma Aldrich Co. (St. Louis, MO, USA). Amberlite XAD-2 was obtained from Supelco (SigmaAldrich Co. Bangalore, India). B12 assay medium was obtained from Himedia (Bangalore, India). Vitamin B12 RNA aptamer sequence (50 GGA ACC GGU GCG CAU AAC CAC CUC AGU GCG AGC AA 30 ) was adapted as reported by Lorsch and Szostak (1994). Further, all pyrimidines were 20 -flouro modified and obtained from Trilink Biotechnologies (San Diego CA, USA). All stock solutions were prepared in DEPC treated water and diluted as required. Methanol was of HPLC grade and all other reagents used were of analytical grade. HPLC (SCL-10-AVP) was procured from Shimadzu (Kyoto, Japan). Reverse-phase HPLC Column (4.6 300 mm, l bondpack, particle size 10 lm) and Sep-Pak was procured from Waters Corp. (Milford MA, USA). Luminometer is from Luminoskan TL plus, Thermolab systems (Helsinkin, Finland). Data acquisition was performed with decimal HyperTerminal TL plus software. The column used for ESI-MS was Acquity UPLC HSS T3 2.1 50 mm, 1.8 lm. Positive ion MS/MS experiments performed in product mode on a triple quadrupole Xevo TQD mass spectrometer (Waters Corp., Milford, USA). Triple distilled water was used for the preparation of solvent system for HPLC analysis.
317
Sep-Pak column for a second time. The Sep-Pak cartridge was washed with 75% ethanol solution and then equilibrated with distilled water. The corrinoid compound was eluted with 25% ethanol. The fractions were concentrated under reduced pressure. The purified sample was analysed by HPLC. 2.4. High performance liquid chromatography The concentrated sample was analysed on a reverse phase
lbondpack HPLC column (4.6 300 mm, particle size 10 lm) procured from Waters Corp. Elution of the vitamin B12 compound was carried out with a linear gradient of methanol [from 0% to 90% of a 50% (v/v) methanol solution containing 0.1% (v/v) acetic acid] for 40 min, with a flow rate of 1 mL/min (Watanabe et al., 2000). The absorbance was measured at 361 nm and 546 nm (Hudson, Subramanin, & Allen, 1984). The retention times of authentic standards of methylcobalamin, adenosylcobalamin, hydroxocobalamin and samples were recorded. 2.5. Estimation of vitamin B12 by microbiological assay Lactobacillus delbrueckii MTCC 911 was used for microbiological assay of vitamin B12. Standards were prepared in the range of 0.01–0.2 lg/mL. Purified fractions from HPLC were collected, pooled and concentrated before being analysed. The turbidity (%T) of L. delbrueckii test cultures were measured at 600 nm (Watanabe, Takenaka, Abe, Tamara, & Nakano, 1998). 2.6. Chemiluminescence
2.2. Organism and culture C. vulgaris strain was obtained from National Environmental Engineering Research Institute (Nagpur, India) and grown aseptically in BBM medium at 25 °C with 40 rpm. The medium contained 0.25 g of NaNO3, 0.074 g of K2HPO4, 0.175 g of KH2PO4, 0.073 g of MgSO4, 0.024 g of CaCl22H20, 0.025 g of NaCl, 0.005 g of FeSO47H2O, 0.05 g of EDTA and 1 mL of trace element (0.3100 g/ 100 mL of H3BO3, 0.2230 g of MnSO44H20, 0.0287 g of ZnSO4 7H2O, 0.0088 g of (NH4)6MoO244H20, 0.0146 g of (CoNO3)24H2O, 0.0033 g of Na2WO42H2O, 0.0119 g of KBr, 0.0083 g of KI, 0.0154 g of Cd(NO3)24H2O, 0.0198 g of NiSO4(NH4)2SO46H2O, 0.0020 g of VOSO42H2O and 0.00237 g of AlCl36H2O). The culture was grown at ambient temperature in a raceway pond. The culture growth was monitored by measuring the absorbance at 560 nm. The biomass was harvested after the culture reaches stationary phase by centrifugation and washed twice with distilled water, lyophilized and stored at 80 °C until use. 2.3. Extraction of vitamin B12 Lyophilized C. vulgaris biomass (100 g) was used for vitamin B12 extraction, suspended in triple distilled water (300 mL) and autoclaved at 121 °C for 10 min. The homogenate was centrifuged at 10,000g for 10 min. The cooled supernatant containing vitamin B12 was adjusted to pH 6 and used for B12 analysis. The whole extraction process was carried out in dark. For purification, the sample was loaded on to an Amberlite XAD-2 column (2 25 cm) and eluted with 80% methanol. Amberlite XAD-2 was prepared for column chromatography by making a slurry using absolute methanol. The column was filled with methanol, and the resin slurry added to a bed height of 15–16 cm. The column was equilibrated with water. Further purification was carried out by passing the samples through a Sep-Pak column. The fractions, eluted from Amberlite XAD-2 with 80% methanol, were pooled and passed through a
Chemiluminescence (CL) reactions were monitored using luminometer in polystyrene cuvette, and the signals plotted at 10-s intervals for a period of 10 min. An optimised concentration of luminol and vitamin B12 was added to the cuvette followed by the addition of urea-H2O2. Signals were measured in terms of chemiluminescence units (CLU) and are generated by a reaction between luminol and urea hydrogen peroxide, and vitamin B12 (Kumar, Chouhan, & Thakur, 2009). An increase in CLU can only be observed in the presence of vitamin B12. 2.7. Gold nanoparticle and RNA aptamer detection RNA aptamer-based colorimetric sensor, for the detection of vitamin B12 using gold nanoparticles (AuNPs), was used as described elsewhere (Selvakumar & Thakur, 2012). An aqueous solution of mono-dispersed quasi-spherical AuNPs was prepared using a modified method (Turkevich, Stevenson, & Hillier, 1951; Xia, Bai, Hartman, & Wang, 2010). Briefly, a total of 45 mL of Milli-Q water in a reaction flask was refluxed for 10 min with 5 mL of 0.1% tetrachloroauric acid, 2 mL 1% trisodium citrate, and 42.5 lL of 0.1% silver nitrate. Tetrachloroauric acid, trisodium citrate and silver nitrate were mixed together in a separate beaker and incubated for 5 min before being added drop wise to the reaction flask. The reduction of gold metal ions (Au3+) to yield AuNPs (Au0) was confirmed by the appearance of a dark cherry red colour. Colloidal AuNPs were stored at 4 °C. The size and concentration of AuNPs were determined using an ultraviolet-visible (UV-vis) spectrophotometer in a wavelength range from 400 nm to 700 nm (Haiss, Thanh, Aveyard, & Fernig, 2007). A mixture consisting of an optimised concentration of AuNPs and aptamer (300 lL) was shaken gently for 10 min at room temperature, and incubated for 10 min with 100 lL HPLC purified fraction of C. vulgaris. After slow addition of an optimised concentration of NaCl (1 mol/L) to the sample, colour and spectra changes were measured using a UV–vis spectrophotometer in a wavelength range from 400 nm to 700 nm.
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2.8. UPLC–ESI-MS
3.3. Chemiluminescence analysis of vitamin B12
UPLC–ESI-MS was carried out in a UPLC/Xevo TQD Tandem quadrupole with nominal mass measurement in positive mode. The cone and desolvation gas were set to 28 and 1000 L/h, respectively. Sample source conditions were as follows: capillary voltage, 3.00 kV; sample cone voltage, 10 V; extraction cone voltage, 2 V; source temperature, 135 °C; and desolvation temperature, 45 °C; cone gas flow (L/h) 10; collision gas flow (mL/min) 0.21; LM 1 resolution 15.00; HM 1 resolution 15.00; ion energy 1, 0.50; MS mode entrance, 50.00; MS mode collision energy, 1.00; MS mode exit 50.00; MSMS mode entrance, 1.00; MSMS mode collision energy, 1.00; MSMS mode exit, 0.5; LM 2, resolution, 15.00; HM 2, resolution, 15.00; ion energy 2, 1.0; gain, 1.00 and multiplier 522.98. Samples were introduced into the mass spectrometer through a direct-flow injection UPLC system for solvent delivery at the flow rate of 0.6 mL/min. The column used was Acquity UPLC HSS T3 2.1 50 mm, 1.8 lm. A linear gradient of 10 mmol/L ammonium formate and 0.1% formic acid in water (A) and 10 mmol/L ammonium formate and 0.1% formic acid in methanol (B) was used. Column temperature was set at 35 °C.
HPLC fractions were collected and tested for cobalt-enhanced CL. It is reported that Co2+ enhances the photon production arising from the reaction between luminol and H2O2 (Kumar et al., 2009). During chemiluminescence, numbers of photons produced are directly proportional to vitamin B12 concentration. The samples were quantified and found to contain 26.84 ± 2 lg of Me-Cbl per 100 g dry biomass of C. vulgaris.
2.9. MS/MS experiments Positive ion MS/MS experiments were performed in product mode on a triple quadrupole TQD mass spectrometer (Waters Corp.). The instrument was operated with the following instrumental conditions: calibration dynamic 1, capillary (kV) 3.00; cone (V) 10.00; extractor (V) 2.00; RF (V) 0.10; source temperature (°C)135; desolvation temperature (°C) 450; cone gas flow (L/h) 10; desolvation gas flow (L/h) 1100; collision gas flow (mL/min) 0.21; LM 1, resolution 15.00; HM 1, resolution 15.00; ion energy 1, 0.50; MS mode entrance 50.00; MS mode collision Energy, 1.00; MS mode exit, 50.00; MSMS mode entrance,1.00; MSMS mode collision energy 1.00; MSMS mode exit 0.50; LM 2, resolution 15.00; HM 2, resolution 15.00; ion Energy 2, 1.00; gain, 1.00; multiplier 522.98. 3. Results and discussion 3.1. Extraction of vitamin B12 and partial purification using Amberlite XAD-2 and Sep-Pak columns In the present study, water (aqueous) extraction method was used to identify forms of vitamin B12 in C. vulgaris dry biomass. Lyophilized samples exhibited better stability for the biologically active vitamin B12 and hence were used for the extraction (Watanabe et al., 2000). The purification of cobalamins on Amberlite XAD-2, and further through a Sep-Pak column, ensured efficient binding of vitamin B12 from the complex matrix (Fenton & Rosenberg, 1977). The eluant was analysed by HPLC.
3.4. Microbiological assay of vitamin B12 This method is one of the most frequently used for routine analysis of vitamin B12 (Kralova, Rauch, & Cerna, 1982). The turbidity, caused by cell growth, is proportional to vitamin B12 concentration. The purified sample was found to contain 29.87 ± 2 lg of Me-Cbl per 100 g dry biomass of C. vulgaris, based on the standard plot for Me-Cbl. The quantification of Me-Cbl by microbiological assay and chemiluminescence assay were similar, which substantiate the presence of Me-Cbl as the predominant form of vitamin B12 in C. vulgaris. Few algae are known to have the ability to take up and accumulate the exogenous vitamin B12. C. vulgaris is one such species that can take up as well as synthesize the coenzyme (Watanabe et al., 1997). 3.5. Quantification of vitamin B12 using gold nanoparticle (AuNPs) and RNA aptamer Aptamers are short DNA or RNA sequences with high affinity towards a particular analyte, and AuNPs have been considered as an on-site detection method with high specificity and sensitivity because of their easy preparation, simple operation, and detection of colorimetric signal with the naked eye. Solutions of AuNPs are red in colour due to their specific and size-dependent Surface Plasmon Resonance (SPR) absorption at 520 nm. The addition of salt creates electrostatic repulsion between negatively charged AuNPs, and results in aggregation of AuNPs leading to a red-to-purple colour shift, and absorption above 600 nm. AuNPs were treated with vitamin B12 RNA aptamer both in the presence, and in the absence, of vitamin B12. On addition of the salt, the solution changed colour from red to purple due to the presence of vitamin B12, whereas the latter retained its original colour. UV-vis studies provided quantitative results that clearly showed absorption at 520 nm gradually decreased while absorption at 640 nm increased. This blue shift in the SPR absorption suggested the formation of large AuNPs aggregates. A significant colour change was visualised at concentration as low as 0.1 lg/mL vitamin B12, suggesting AuNPs are sensitive probes for aptamer structures. But, in our case, the colour
0.35 0.3
The solvent system used for the HPLC analysis of vitamin B12 separated all forms i.e., OH-Cbl, Adl-Cbl and Me-Cbl and their retention times (RTs) were 20.1, 29.2 and 35.2 min, respectively. Purified samples were injected into HPLC to identify the forms of vitamin B12 present, based on RT for standards. It was observed the peak at 35.2 min was identical to that of Me-Cbl indicating the samples contained Me-Cbl. There were no other prominent peaks in the chromatograms of the purified samples. Although vitamin B12 absorption at 361 nm is about three times greater than at 546 nm, co-eluting metabolites interfered with the signal at 361 nm but not at 546 nm, and hence the absorption at both the wavelengths was recorded (Hudson et al., 1984).
Absorbance
3.2. HPLC analysis of vitamin B12
0.25 0.2 0.15
Control
0.1 Vitamin B12 from purified chlorella
0.05 0 400
450
500 550 600 Wavelength (nm)
650
700
Fig. 1. UV-vis absorption spectrum for the standard methylcobalamin and the purified Chlorella vulgaris extract using RNA aptamer and AuNPs.
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Fig. 2A. MS spectrum of Chlorella vulgaris extract.
Fig. 2B. MS/MS spectrum of Chlorella vulgaris extract.
difference was not observed due to the lower concentration of vitamin B12 in the purified sample fractions. Therefore, the UV-Vis spectrum was required to estimate the amount of vitamin B12 in
the sample (Fig. 1). Based on the spectral data, vitamin B12 in the sample was found to be 28.02 ± 2 lg/100 g dry weight. For the convenient analysis of the spectral and colour changes, the absorbance
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ratios at A520/A640 were plotted, and found to be well matched with the spectral observation. The higher the vitamin B12 concentration, the greater the change in colour and higher the A520/A640 ratio.
encouragement. Kumudha A. is grateful to the Council of Scientific and Industrial Research for providing senior research fellowship. References
3.6. LC–MS and MS/MS of the sample The LC–MS analysis of purified samples, along with standard, were carried out. The MS TIC (total ion chromatogram) observed for methylcobalamin standard showed the ionisation of methylcobalamin in ESI positive mode. The UV data and the corresponding full scan (MS) data of standard were compared with the purified samples to confirm the presence of methylcobalamin in the ESI positive mode. The mass of Me-Cbl is 1344.38 m/z. Spectra of the purified sample showed it is doubly charged and, thus, a mass 673.93 m/z was observed (Fig. 2A). Since the intensity observed in the sample was low, selected ion recording (SIR), MS/MS and multiple reaction monitoring (MRM) of the samples were carried out. The MS/MS of the standard Me-Cbl and purified sample also showed the doubly charged ion of Me-Cbl (Fig. 2B). The mass of the ionised peak confirmed the presence of Me-Cbl in C. vulgaris sample. Schreiner, Razzazi, and Luf (2003) and Chen and Jiang (2008) proposed a capillary electrophoresis method for determining vitamin B12. However, these methods focus on the presence of cobalamin or cyanocobalamin and not the forms of the vitamin B12 in the C. vulgaris. Very few algae have been studied for the identification of vitamin B12 forms. P. yezoensis, P. suborticulate and Spirulina platensis are some of the algae which are reported to contain biologically active vitamin B12 compound (Kumudha, Kumar, Thakur, Ravishankar, & Sarada, 2010; Takenaka et al., 2001; Yamada et al., 1996). In this study, we found Me-Cbl at 29.87 ± 2 lg/ 100 g, and 26.84 ± 2 lg/100 g and 28.02 ± 2 lg/100 g dry biomass of C. vulgaris using microbiological, chemiluminescence and AuNPs based RNA aptamer assays, respectively. The bioavailability or biological activities of microalgal vitamin B12 are yet to be explored. 4. Conclusion In the present study LCMS, MSMS, SIR, MRM and RNA aptamer along with other conventional analytical techniques such as HPLC, microbiological assay, and chemiluminescence were used to confirm the presence of methylcobalamin, a form of vitamin B12 in C. vulgaris. This is the first report where several methods are used to confirm the form of vitamin B12. Among the methods MRM is highly selective and specifically look for the fragments of interest and RNA aptamer that binds to vitamin B12 are very sensitive and rapid. In the study it was concluded that the C. vulgaris biomass contains Me-Cbl, a biologically active form of vitamin B12. This finding enhances the use of C. vulgaris as a nutritional supplement owing to its rich content of various nutrients as well as vitamin B12. Acknowledgements The authors thank the Director of the Central Food Technological Research Institute (Mysore, India) for providing constant
Benner, S. A., Ellington, A. D., & Tauer, A. (1989). Modern metabolism as a palimpsest of the RNA world. Proceedings of the National Academy of Science USA, 86, 7054–7058. Chen, J. H., & Jiang, S. J. (2008). Determination of cobalamin in nutritive supplements and Chlorella foods by capillary electrophoresis–inductively coupled plasma mass spectrometry. Journal of Agricultural and Food Chemistry, 56, 1210–1215. Duda, J., Pedziwilk, Z., & Zodrow, K. (1967). Studies on vitamin B12 content of the leguminous plant. Acta Microbiological Polonica, 6, 233–238. Eschenmoser, A. (1988). Vitamin B12: Experiments concerning the origin of its molecular structure. Angewandte Chemie International Edition, 27, 5–39. Fenton, W. A., & Rosenberg, L. E. (1977). Improved techniques for the extraction and chromatography of cobalamins. Analytical Biochemistry, 90, 119–125. Haiss, W., Thanh, N. T. K., Aveyard, J., & Fernig, D. G. (2007). Determination of size and concentration of gold nanoparticles from UV-vis spectra. Analytical Chemistry, 79, 4215–4221. Hudson, T. S., Subramanin, S., & Allen, R. J. (1984). Determination of pantothenic acid and vitamin B12 in nutritional products. Journal – Association of Official Analytical Chemists, 67, 994–998. Kanazawa, A. (1963). Vitamins in algae. Bulletin of Japanese Society of Scientific Fisheries, 29, 713–731. Katsura, H. K., Fujith, T., Watanabe, F., & Nakano, Y. (2002). Purification and characterization of a corrinoid compound from Chlorella tablets as an algal health food. Journal of Agricultural and Food Chemistry, 50, 4994–4997. Kralova, B., Rauch, P., & Cerna, J. (1982). Use of vitamin B12 radioassay in the analysis of biological materials, mainly of foods. Food/Nahrung, 26, 803–810. Kumar, S. S., Chouhan, R. S., & Thakur, M. S. (2009). Enhancement of chemiluminescence for vitamin B12 analysis. Analytical Biochemistry, 388, 312–316. Kumudha, A., Kumar, S. S., Thakur, M. S., Ravishankar, G. A., & Sarada, R. (2010). Purification, identification and characterization of methylcobalamin from Spirulina platensis. Journal of Agricultural and Food Chemistry, 58, 9925–9930. Lorsch, J. R., & Szostak, J. W. (1994). In vitro selection of RNA aptamers specific for cyanocobalamin. Biochemistry, 33, 973–982. Poston, J. M. (1977). Leucine 2,3-aminomutase: A cobalamin-dependent enzyme present in bean seedlings. Science, 195, 301–302. Rauma, A. L., Torronen, R., Hanninen, O., & Mykkanen, H. (1995). Vitamin B12 status of long-term adherents of a strict uncooked vegan diet (‘‘living food diet’’) is compromised. Journal of Nutrition, 125, 2511–2515. Schreiner, M., Razzazi, E., & Luf, W. (2003). Determination of water soluble vitamins in soft drinks and vitamin supplements using capillary electrophoresis. Food/ Nahrung, 47, 243–247. Selvakumar, L. S., & Thakur, M. S. (2012). Nano RNA aptamer wire for analysis of vitamin B12. Analytical Biochemistry, 427, 151–157. Takenaka, S., Sugiyama, S., Ebara, S., Miyamoto, E., Abe, K., Tamura, et al. (2001). Feeding dried purple laver (nori) to vitamin B12 deficient rats significantly improves vitamin B12 status. British Journal of Nutrition, 85, 699–703. Turkevich, J., Stevenson, P. C., & Hillier, J. (1951). A study of the nucleation and growth process in the synthesis of colloidal gold. Discussions of the Faraday Society, 11, 55–75. Watanabe, F., Abe, K., Takenaka, S., Tamura, Y., Maruyama, I., & Nakano, Y. (1997). Occurrence of cobalamin coenzymes in the photosynthetic green alga, Chlorella vulgaris. Bioscience Biotechnology and Biochemistry, 61, 896–897. Watanabe, F., Takenaka, S., Abe, K., Tamara, Y., & Nakano, Y. (1998). Comparison of a microbiological assay and a fully automated chemiluminescence system for the determination of vitamin B12 in food. Journal of Agricultural and Food Chemistry, 46, 1433–1436. Watanabe, F., Takenaka, S., Katsura, H., Miyamoto, E., Abe, K., Tamura, Y., et al. (2000). Characterization of a vitamin B12 compound in Edible purple laver, Porphyra yezoensis. Bioscience Biotechnology and Biochemistry, 64, 2712–2715. Xia, H., Bai, S., Hartman, J., & Wang, D. (2010). Synthesis of monodisperse quasispherical gold nanoparticles in water via silver (I)-assisted citrate reduction. Langmuir, 26, 3585–3589. Yamada, S., Shibata, Y., Takayama, M., Narita, Y., Sugawara, K., & Fukuda, M. (1996). Content and characteristics of vitamin B12 in some seaweeds. Journal of Nutritional Science and Vitaminology, 42, 497–505.
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Methylcobalamin – A form of vitamin B12 identified and characterised in Chlorella vulgaris Anantharajappa Kumudha a, Sagaya Selvakumar b, Pullancheri Dilshad c, Gopal Vaidyanathan c, Munna Singh Thakur b, Ravi Sarada a,⇑ a b c
Plant Cell Biotechnology Department, CSIR-Central Food Technological Research Institute, Mysore 570020, India Fermentation Technology and Bioengineering Department, CSIR-Central Food Technological Research Institute, Mysore 570020, India Waters (India) Pvt. Ltd, Bangalore, India
a r t i c l e
i n f o
Article history: Received 25 April 2013 Received in revised form 30 July 2014 Accepted 10 August 2014 Available online 23 August 2014 Keywords: Chlorella Methylcobalamin MS/MS Gold nanoparticle Aptamer
a b s t r a c t Vitamin B12 is among the most essential biomolecules required for crucial metabolic processes in humans. Vitamin B12 was extracted from Chlorella vulgaris biomass under aqueous conditions, partially purified by passing the extract through amberlite XAD-2, Sep-Pak columns, and further purified by HPLC. The target peak eluent was subjected to characterisation by tandem mass spectrometry (MS/MS), selected ion recording (SIR) and multiple reaction monitoring (MRM) and identified as methylcobalamin (Me-Cbl). Quantification of Me-Cbl was carried out by microbiological and chemiluminescence methods, and found to be 29.87 ± 2 lg/100 g and 26.84 ± 2 lg/100 g dry weight, respectively. The presence of Me-Cbl was further substantiated using gold nanoparticle (AuNPs) based aptamer analysis, and found to be 28.02 ± 2 lg/100 g dry weight. Good similarity was observed among all the methods. Methylcobalamin, a form of vitamin B12 was identified in C. vulgaris and this finding enhances its use as a nutritional supplement. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Vitamin B12 is one of the largest, most structurally complex, non-polymeric biomolecules and an evolutionarily ancient cofactor with carbon–metal bonds (Benner, Ellington, & Tauer, 1989; Eschenmoser, 1988). Vitamin B12 plays an important role in the synthesis of methionine from homocysteine and conversion of methylmalonyl coenzyme A to succinyl coenzyme A in humans. It is, generally, believed that plants neither synthesizes nor utilise cobalamin for growth (Duda, Pedziwilk, & Zodrow, 1967). However some studies have disputed this conclusion (Poston, 1977). Many algae utilise vitamin B12 for growth while a few synthesize vitamin B12. Some algae are known to contain substantial amount of vitamin B12. Porphyra yezoensis is one algae reported to contain as much cobalamin as animal liver (Kanazawa, 1963). Strict Abbreviations: OH-Cbl, hydroxocobalamin; Ado-Cbl, 5-deoxyadenosylcobalamin; Me-Cbl, methylcobalamin; SO-Cbl, sulphitocobalamin; CN-Cbl, cyanocobalamin; CL, chemiluminescence; Urea-H2O2, urea hydrogen peroxide; CLU, chemiluminescence unit; MS/MS, tandem mass spectrometry; SIR, selected ion recording; MRM, multiple reaction monitoring; AuNPs, gold nanoparticle; HPLC, high performance liquid chromatography; SRP, Surface Plasmon Resonance. ⇑ Corresponding author. Tel.: +91 821 2516 501; fax: +91 821 2517 233. E-mail address: [email protected] (R. Sarada). http://dx.doi.org/10.1016/j.foodchem.2014.08.035 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
vegetarians can obtain sufficient bioavailable B12 from some seaweed (Rauma, Torronen, Hanninen, & Mykkanen, 1995). Algal B12 has been found to be bioavailable in B12-deficient rats fed dried purple laver, a type of seaweed (Takenaka et al., 2001). However information on quantitative bioavailability of algal B12 is limited. Humans have consumed Chlorella vulgaris biomass for centuries as health food. Some Chlorella species are grown under open cultivation system and used for the preparation of the Chlorella tablets, and they are a good source of vitamin B12. Since B12 exists in different forms it is important to identify and quantify the forms in C. vulgaris. The forms of vitamin B12 are similar with a cobalt central ion, the four parts of the corrin ring, and a dimethylbenzimidazole group. They mainly differ in the sixth carbon, which may contain a cyano (CN), hydroxyl (OH), methyl (CH3) or 50 -deoxyadenosyl group (C-CO). Although C. vulgaris has been reported to contain vitamin B12 (Katsura, Fujith, Watanabe, & Nakano, 2002), the form of B12 and quantity have not been determined in dry biomass. Some Chlorella species have been reported to accumulate exogenous cobalamin (Watanabe et al., 1997). There is, however, little information available on the forms of vitamin B12 or the physiological roles of the algal cobalamin (Watanabe et al., 1997). The aim of the present study was to identify and quantify the forms of vitamin B12 in C. vulgaris dry biomass.
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2. Materials and methods 2.1. Chemicals and instruments Methylcobalamin (Me-Cbl), adenosylcobalamin (Adl-Cbl), hydroxocobalamin (OH-Cbl), luminol, urea hydrogen peroxide, diethylpyrocarbonate (DEPC), gold (III) chloride, trisodium citrate, and silver nitrate were procured from Sigma Aldrich Co. (St. Louis, MO, USA). Amberlite XAD-2 was obtained from Supelco (SigmaAldrich Co. Bangalore, India). B12 assay medium was obtained from Himedia (Bangalore, India). Vitamin B12 RNA aptamer sequence (50 GGA ACC GGU GCG CAU AAC CAC CUC AGU GCG AGC AA 30 ) was adapted as reported by Lorsch and Szostak (1994). Further, all pyrimidines were 20 -flouro modified and obtained from Trilink Biotechnologies (San Diego CA, USA). All stock solutions were prepared in DEPC treated water and diluted as required. Methanol was of HPLC grade and all other reagents used were of analytical grade. HPLC (SCL-10-AVP) was procured from Shimadzu (Kyoto, Japan). Reverse-phase HPLC Column (4.6 300 mm, l bondpack, particle size 10 lm) and Sep-Pak was procured from Waters Corp. (Milford MA, USA). Luminometer is from Luminoskan TL plus, Thermolab systems (Helsinkin, Finland). Data acquisition was performed with decimal HyperTerminal TL plus software. The column used for ESI-MS was Acquity UPLC HSS T3 2.1 50 mm, 1.8 lm. Positive ion MS/MS experiments performed in product mode on a triple quadrupole Xevo TQD mass spectrometer (Waters Corp., Milford, USA). Triple distilled water was used for the preparation of solvent system for HPLC analysis.
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Sep-Pak column for a second time. The Sep-Pak cartridge was washed with 75% ethanol solution and then equilibrated with distilled water. The corrinoid compound was eluted with 25% ethanol. The fractions were concentrated under reduced pressure. The purified sample was analysed by HPLC. 2.4. High performance liquid chromatography The concentrated sample was analysed on a reverse phase
lbondpack HPLC column (4.6 300 mm, particle size 10 lm) procured from Waters Corp. Elution of the vitamin B12 compound was carried out with a linear gradient of methanol [from 0% to 90% of a 50% (v/v) methanol solution containing 0.1% (v/v) acetic acid] for 40 min, with a flow rate of 1 mL/min (Watanabe et al., 2000). The absorbance was measured at 361 nm and 546 nm (Hudson, Subramanin, & Allen, 1984). The retention times of authentic standards of methylcobalamin, adenosylcobalamin, hydroxocobalamin and samples were recorded. 2.5. Estimation of vitamin B12 by microbiological assay Lactobacillus delbrueckii MTCC 911 was used for microbiological assay of vitamin B12. Standards were prepared in the range of 0.01–0.2 lg/mL. Purified fractions from HPLC were collected, pooled and concentrated before being analysed. The turbidity (%T) of L. delbrueckii test cultures were measured at 600 nm (Watanabe, Takenaka, Abe, Tamara, & Nakano, 1998). 2.6. Chemiluminescence
2.2. Organism and culture C. vulgaris strain was obtained from National Environmental Engineering Research Institute (Nagpur, India) and grown aseptically in BBM medium at 25 °C with 40 rpm. The medium contained 0.25 g of NaNO3, 0.074 g of K2HPO4, 0.175 g of KH2PO4, 0.073 g of MgSO4, 0.024 g of CaCl22H20, 0.025 g of NaCl, 0.005 g of FeSO47H2O, 0.05 g of EDTA and 1 mL of trace element (0.3100 g/ 100 mL of H3BO3, 0.2230 g of MnSO44H20, 0.0287 g of ZnSO4 7H2O, 0.0088 g of (NH4)6MoO244H20, 0.0146 g of (CoNO3)24H2O, 0.0033 g of Na2WO42H2O, 0.0119 g of KBr, 0.0083 g of KI, 0.0154 g of Cd(NO3)24H2O, 0.0198 g of NiSO4(NH4)2SO46H2O, 0.0020 g of VOSO42H2O and 0.00237 g of AlCl36H2O). The culture was grown at ambient temperature in a raceway pond. The culture growth was monitored by measuring the absorbance at 560 nm. The biomass was harvested after the culture reaches stationary phase by centrifugation and washed twice with distilled water, lyophilized and stored at 80 °C until use. 2.3. Extraction of vitamin B12 Lyophilized C. vulgaris biomass (100 g) was used for vitamin B12 extraction, suspended in triple distilled water (300 mL) and autoclaved at 121 °C for 10 min. The homogenate was centrifuged at 10,000g for 10 min. The cooled supernatant containing vitamin B12 was adjusted to pH 6 and used for B12 analysis. The whole extraction process was carried out in dark. For purification, the sample was loaded on to an Amberlite XAD-2 column (2 25 cm) and eluted with 80% methanol. Amberlite XAD-2 was prepared for column chromatography by making a slurry using absolute methanol. The column was filled with methanol, and the resin slurry added to a bed height of 15–16 cm. The column was equilibrated with water. Further purification was carried out by passing the samples through a Sep-Pak column. The fractions, eluted from Amberlite XAD-2 with 80% methanol, were pooled and passed through a
Chemiluminescence (CL) reactions were monitored using luminometer in polystyrene cuvette, and the signals plotted at 10-s intervals for a period of 10 min. An optimised concentration of luminol and vitamin B12 was added to the cuvette followed by the addition of urea-H2O2. Signals were measured in terms of chemiluminescence units (CLU) and are generated by a reaction between luminol and urea hydrogen peroxide, and vitamin B12 (Kumar, Chouhan, & Thakur, 2009). An increase in CLU can only be observed in the presence of vitamin B12. 2.7. Gold nanoparticle and RNA aptamer detection RNA aptamer-based colorimetric sensor, for the detection of vitamin B12 using gold nanoparticles (AuNPs), was used as described elsewhere (Selvakumar & Thakur, 2012). An aqueous solution of mono-dispersed quasi-spherical AuNPs was prepared using a modified method (Turkevich, Stevenson, & Hillier, 1951; Xia, Bai, Hartman, & Wang, 2010). Briefly, a total of 45 mL of Milli-Q water in a reaction flask was refluxed for 10 min with 5 mL of 0.1% tetrachloroauric acid, 2 mL 1% trisodium citrate, and 42.5 lL of 0.1% silver nitrate. Tetrachloroauric acid, trisodium citrate and silver nitrate were mixed together in a separate beaker and incubated for 5 min before being added drop wise to the reaction flask. The reduction of gold metal ions (Au3+) to yield AuNPs (Au0) was confirmed by the appearance of a dark cherry red colour. Colloidal AuNPs were stored at 4 °C. The size and concentration of AuNPs were determined using an ultraviolet-visible (UV-vis) spectrophotometer in a wavelength range from 400 nm to 700 nm (Haiss, Thanh, Aveyard, & Fernig, 2007). A mixture consisting of an optimised concentration of AuNPs and aptamer (300 lL) was shaken gently for 10 min at room temperature, and incubated for 10 min with 100 lL HPLC purified fraction of C. vulgaris. After slow addition of an optimised concentration of NaCl (1 mol/L) to the sample, colour and spectra changes were measured using a UV–vis spectrophotometer in a wavelength range from 400 nm to 700 nm.
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2.8. UPLC–ESI-MS
3.3. Chemiluminescence analysis of vitamin B12
UPLC–ESI-MS was carried out in a UPLC/Xevo TQD Tandem quadrupole with nominal mass measurement in positive mode. The cone and desolvation gas were set to 28 and 1000 L/h, respectively. Sample source conditions were as follows: capillary voltage, 3.00 kV; sample cone voltage, 10 V; extraction cone voltage, 2 V; source temperature, 135 °C; and desolvation temperature, 45 °C; cone gas flow (L/h) 10; collision gas flow (mL/min) 0.21; LM 1 resolution 15.00; HM 1 resolution 15.00; ion energy 1, 0.50; MS mode entrance, 50.00; MS mode collision energy, 1.00; MS mode exit 50.00; MSMS mode entrance, 1.00; MSMS mode collision energy, 1.00; MSMS mode exit, 0.5; LM 2, resolution, 15.00; HM 2, resolution, 15.00; ion energy 2, 1.0; gain, 1.00 and multiplier 522.98. Samples were introduced into the mass spectrometer through a direct-flow injection UPLC system for solvent delivery at the flow rate of 0.6 mL/min. The column used was Acquity UPLC HSS T3 2.1 50 mm, 1.8 lm. A linear gradient of 10 mmol/L ammonium formate and 0.1% formic acid in water (A) and 10 mmol/L ammonium formate and 0.1% formic acid in methanol (B) was used. Column temperature was set at 35 °C.
HPLC fractions were collected and tested for cobalt-enhanced CL. It is reported that Co2+ enhances the photon production arising from the reaction between luminol and H2O2 (Kumar et al., 2009). During chemiluminescence, numbers of photons produced are directly proportional to vitamin B12 concentration. The samples were quantified and found to contain 26.84 ± 2 lg of Me-Cbl per 100 g dry biomass of C. vulgaris.
2.9. MS/MS experiments Positive ion MS/MS experiments were performed in product mode on a triple quadrupole TQD mass spectrometer (Waters Corp.). The instrument was operated with the following instrumental conditions: calibration dynamic 1, capillary (kV) 3.00; cone (V) 10.00; extractor (V) 2.00; RF (V) 0.10; source temperature (°C)135; desolvation temperature (°C) 450; cone gas flow (L/h) 10; desolvation gas flow (L/h) 1100; collision gas flow (mL/min) 0.21; LM 1, resolution 15.00; HM 1, resolution 15.00; ion energy 1, 0.50; MS mode entrance 50.00; MS mode collision Energy, 1.00; MS mode exit, 50.00; MSMS mode entrance,1.00; MSMS mode collision energy 1.00; MSMS mode exit 0.50; LM 2, resolution 15.00; HM 2, resolution 15.00; ion Energy 2, 1.00; gain, 1.00; multiplier 522.98. 3. Results and discussion 3.1. Extraction of vitamin B12 and partial purification using Amberlite XAD-2 and Sep-Pak columns In the present study, water (aqueous) extraction method was used to identify forms of vitamin B12 in C. vulgaris dry biomass. Lyophilized samples exhibited better stability for the biologically active vitamin B12 and hence were used for the extraction (Watanabe et al., 2000). The purification of cobalamins on Amberlite XAD-2, and further through a Sep-Pak column, ensured efficient binding of vitamin B12 from the complex matrix (Fenton & Rosenberg, 1977). The eluant was analysed by HPLC.
3.4. Microbiological assay of vitamin B12 This method is one of the most frequently used for routine analysis of vitamin B12 (Kralova, Rauch, & Cerna, 1982). The turbidity, caused by cell growth, is proportional to vitamin B12 concentration. The purified sample was found to contain 29.87 ± 2 lg of Me-Cbl per 100 g dry biomass of C. vulgaris, based on the standard plot for Me-Cbl. The quantification of Me-Cbl by microbiological assay and chemiluminescence assay were similar, which substantiate the presence of Me-Cbl as the predominant form of vitamin B12 in C. vulgaris. Few algae are known to have the ability to take up and accumulate the exogenous vitamin B12. C. vulgaris is one such species that can take up as well as synthesize the coenzyme (Watanabe et al., 1997). 3.5. Quantification of vitamin B12 using gold nanoparticle (AuNPs) and RNA aptamer Aptamers are short DNA or RNA sequences with high affinity towards a particular analyte, and AuNPs have been considered as an on-site detection method with high specificity and sensitivity because of their easy preparation, simple operation, and detection of colorimetric signal with the naked eye. Solutions of AuNPs are red in colour due to their specific and size-dependent Surface Plasmon Resonance (SPR) absorption at 520 nm. The addition of salt creates electrostatic repulsion between negatively charged AuNPs, and results in aggregation of AuNPs leading to a red-to-purple colour shift, and absorption above 600 nm. AuNPs were treated with vitamin B12 RNA aptamer both in the presence, and in the absence, of vitamin B12. On addition of the salt, the solution changed colour from red to purple due to the presence of vitamin B12, whereas the latter retained its original colour. UV-vis studies provided quantitative results that clearly showed absorption at 520 nm gradually decreased while absorption at 640 nm increased. This blue shift in the SPR absorption suggested the formation of large AuNPs aggregates. A significant colour change was visualised at concentration as low as 0.1 lg/mL vitamin B12, suggesting AuNPs are sensitive probes for aptamer structures. But, in our case, the colour
0.35 0.3
The solvent system used for the HPLC analysis of vitamin B12 separated all forms i.e., OH-Cbl, Adl-Cbl and Me-Cbl and their retention times (RTs) were 20.1, 29.2 and 35.2 min, respectively. Purified samples were injected into HPLC to identify the forms of vitamin B12 present, based on RT for standards. It was observed the peak at 35.2 min was identical to that of Me-Cbl indicating the samples contained Me-Cbl. There were no other prominent peaks in the chromatograms of the purified samples. Although vitamin B12 absorption at 361 nm is about three times greater than at 546 nm, co-eluting metabolites interfered with the signal at 361 nm but not at 546 nm, and hence the absorption at both the wavelengths was recorded (Hudson et al., 1984).
Absorbance
3.2. HPLC analysis of vitamin B12
0.25 0.2 0.15
Control
0.1 Vitamin B12 from purified chlorella
0.05 0 400
450
500 550 600 Wavelength (nm)
650
700
Fig. 1. UV-vis absorption spectrum for the standard methylcobalamin and the purified Chlorella vulgaris extract using RNA aptamer and AuNPs.
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Fig. 2A. MS spectrum of Chlorella vulgaris extract.
Fig. 2B. MS/MS spectrum of Chlorella vulgaris extract.
difference was not observed due to the lower concentration of vitamin B12 in the purified sample fractions. Therefore, the UV-Vis spectrum was required to estimate the amount of vitamin B12 in
the sample (Fig. 1). Based on the spectral data, vitamin B12 in the sample was found to be 28.02 ± 2 lg/100 g dry weight. For the convenient analysis of the spectral and colour changes, the absorbance
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ratios at A520/A640 were plotted, and found to be well matched with the spectral observation. The higher the vitamin B12 concentration, the greater the change in colour and higher the A520/A640 ratio.
encouragement. Kumudha A. is grateful to the Council of Scientific and Industrial Research for providing senior research fellowship. References
3.6. LC–MS and MS/MS of the sample The LC–MS analysis of purified samples, along with standard, were carried out. The MS TIC (total ion chromatogram) observed for methylcobalamin standard showed the ionisation of methylcobalamin in ESI positive mode. The UV data and the corresponding full scan (MS) data of standard were compared with the purified samples to confirm the presence of methylcobalamin in the ESI positive mode. The mass of Me-Cbl is 1344.38 m/z. Spectra of the purified sample showed it is doubly charged and, thus, a mass 673.93 m/z was observed (Fig. 2A). Since the intensity observed in the sample was low, selected ion recording (SIR), MS/MS and multiple reaction monitoring (MRM) of the samples were carried out. The MS/MS of the standard Me-Cbl and purified sample also showed the doubly charged ion of Me-Cbl (Fig. 2B). The mass of the ionised peak confirmed the presence of Me-Cbl in C. vulgaris sample. Schreiner, Razzazi, and Luf (2003) and Chen and Jiang (2008) proposed a capillary electrophoresis method for determining vitamin B12. However, these methods focus on the presence of cobalamin or cyanocobalamin and not the forms of the vitamin B12 in the C. vulgaris. Very few algae have been studied for the identification of vitamin B12 forms. P. yezoensis, P. suborticulate and Spirulina platensis are some of the algae which are reported to contain biologically active vitamin B12 compound (Kumudha, Kumar, Thakur, Ravishankar, & Sarada, 2010; Takenaka et al., 2001; Yamada et al., 1996). In this study, we found Me-Cbl at 29.87 ± 2 lg/ 100 g, and 26.84 ± 2 lg/100 g and 28.02 ± 2 lg/100 g dry biomass of C. vulgaris using microbiological, chemiluminescence and AuNPs based RNA aptamer assays, respectively. The bioavailability or biological activities of microalgal vitamin B12 are yet to be explored. 4. Conclusion In the present study LCMS, MSMS, SIR, MRM and RNA aptamer along with other conventional analytical techniques such as HPLC, microbiological assay, and chemiluminescence were used to confirm the presence of methylcobalamin, a form of vitamin B12 in C. vulgaris. This is the first report where several methods are used to confirm the form of vitamin B12. Among the methods MRM is highly selective and specifically look for the fragments of interest and RNA aptamer that binds to vitamin B12 are very sensitive and rapid. In the study it was concluded that the C. vulgaris biomass contains Me-Cbl, a biologically active form of vitamin B12. This finding enhances the use of C. vulgaris as a nutritional supplement owing to its rich content of various nutrients as well as vitamin B12. Acknowledgements The authors thank the Director of the Central Food Technological Research Institute (Mysore, India) for providing constant
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