Simultaneous determination of β-alanine betaine and trimethylamine in bacterial culture and plant samples by capillary electrophoresis
© Mohamed Ahmed et al.; licensee Springer. 2014
Received: 3 June 2014
Accepted: 24 July 2014
Published: 22 August 2014
3-N-trimethylaminopropionic acid (β-alanine betaine) and trimethylamine (TMA) are important nitrogenous compounds that perform fundamental roles in biological pathways throughout all kingdoms of life; however, yet their simultaneous determination method is hardly reported.
Capillary electrophoresis method for the simultaneous determination of TMA and β-alanine betaine in microbial culture and plant samples was developed. To increase the sensitivity, TMA and β-alanine betaine in the samples were first derivatized with bromophenacyl bromide and then analyzed by capillary electrophoresis under low pH.
The derivatization was found to be practically useful for the elimination of interfering substances from plant and microbial extracts, as well as giving well resolved peaks for the analytes (β-alanine betaine esters and TMA salt). Analytical features of the developed method showed its respectable performance in terms of linearity (r2 > 0.99), precision (relative standard deviation (RSD) < 5%), and detection limits (0.01 mM).
The developed method allows the quantitative determination of TMA and β-alanine betaine in complex biological samples and assists to study biosynthetic and degradation pathways of these important compounds.
In nature, many plants, bacteria, and marine algae accumulate quaternary ammonium compounds in response to various environmental stresses such as flooding, freezing, heating, drought, and salinity (Rhodes and Hanson ; Gorham ). These compounds form a structurally heterogeneous class of compounds with a unifying character of a polar and fully methyl substituted nitrogen atom, creating a permanent positive charge on the N moiety (Rhodes and Hanson ). Of them, glycine betaine, 3-N-trimethylaminopropionic acid (β-alanine betaine), and proline betaine are known to be the most effective osmoprotectants and are widely distributed in the biosphere (Yancey ). Although glycine betaine is extensively accumulated in many plants in response to various environmental stresses, various members of the highly stress-tolerant plant family Plumbaginaceae accumulate β-alanine betaine instead of glycine betaine (Hanson et al. ). β-Alanine betaine synthesis is not controlled by choline availability, because it is derived from β-alanine by three-step methylation (Rathinasabapathi et al. ). Distinct from glycine betaine synthesis, β-alanine betaine synthesis does not require oxygen, and therefore, it was proposed to be suitable for osmoprotection under saline and hypoxic conditions (Hanson et al. ; Rathinasabapathi et al. ). Consequently, β-Alanine betaine appears to be effective over a broader ecological spectrum than glycine betaine (Rhodes and Hanson ).
In soil microorganisms, our recent reports revealed that β-alanine betaine was accumulated as an intermediate metabolite in the degradation pathway of homocholine by members of the genera Arthrobacter, Rhodococcus, and Pseudomonas (Mohamed Ahmed et al. [2009a], [b]; Mohamed Ahmed et al. ; Mohamed Ahmed et al. ). The potential role of β-alanine betaine in plants' and microorganisms' tolerance to salinity and hypoxia makes its synthetic pathway an interesting target for metabolic engineering. However, the estimation methods of this interesting metabolite are still scarce.
Trimethylamine (TMA) is a volatile low molecular weight tertiary aliphatic amine that has been recognized widely in many animal and plant tissues and is one of the degradation products of nitrogenous organic material such as quaternary ammonium compounds such as choline and homocholine (Craciun and Balskus ; Mohamed Ahmed et al. ). Commonly, the amount of TMA is a useful indicator of spoilage in fresh and lightly preserved seafood as it increases during the breakdown of seafood, such as fish and shrimp (Dalgaard ; Ghaly et al. ). In medical diagnosis, an increase in the concentration of TMA in the breath of patients can be used as a sign of viremic disease (Siminhoff et al. ). Therefore, detection of TMA is of high interests in many fields such environmental protection, food industry, and medical diagnosis. However, one of the challenging aspects of the analysis of β-alanine betaine and TMA lies in their lack of useful chromospheres, and their chemical structures have permanently charged groups that prevent gas chromatographic separation in their intact forms. In the past, analyses of β-alanine betaine and TMA relied on qualitative or semi-quantitative colorimetric tests that employed either thin-layer chromatography and Dragendorff's reagent or reaction with picric acid to form colored complex (Blunden et al.; Grieve and Grattan ). Since these methods are limited in their sensitivity, selectivity and quantitative accuracy, and ability to assay betaines and TMA in one sample, knowledge of the identities and absolute concentrations of β-alanine betaine and TMA in biological materials remained inadequate. Recently, capillary electrophoresis has been applied in many different fields because of its extremely high resolution, its speed, and its applicability to a wide range of molecules whether they are charged or uncharged, or of low or high molecular weight (Shintani and Polonsky ). In the present work, capillary electrophoresis method under low pH (Nishimura et al. 2001; Zhang et al. ) was effectively improved for simultaneous determination of β-alanine betaine and TMA in both plant leaves and microbial culture samples.
β-alanine betaine was synthesized by N-methylation of dimethylaminopropionic acid (Tokyo Kasei Kogyo Co. Ltd, Tokyo, Japan) with methyl iodide as described previously (Mohamed Ahmed et al. ). Briefly, 4 ml of methyl iodide was added to a suspension of dimethylaminopropionic acid (1 g, 6.5 mM) and KHCO3 (1.3 g, 13 mM) in 20 ml of methanol. The mixture was stirred overnight at room temperature and then decanted. Thereafter, the liquid phase was concentrated, and the residue was extracted using 15 ml of mixed solvent (acetonitrile/methanol = 10:1, v/v). The combined extracts were dried under a nitrogen stream to give β-alanine betaine as a colorless powder (1.2 g, 63.2%). The structure and purity of β-alanine betaine were confirmed using proton nuclear magnetic resonance (1H NMR) and capillary electrophoresis. Unless otherwise specified, all other reagents were of analytical grade and were from either Wako (Wako Pure Chemical Industries Ltd, Tokyo, Japan) or Sigma (St. Louis, MO, USA).
Extract preparation from microbial samples
Homocholine-degrading strains were isolated from the soil samples obtained from different locations at Tottori University and around Tottori City, Japan. The bacterial strains were cultivated for 24 h at 30°C on 75 ml of basal homocholine liquid media containing 20 mM homocholine as a sole source of carbon, nitrogen, and energy. The cells were harvested at the exponential phase by centrifugation at 10,000 × g for 20 min at 4°C. The supernatant was collected and preserved at −20°C until used for detection of β-alanine betaine and TMA. The harvested bacterial cells were washed three times with saline solution (8.5 g/l KCl), and re-suspended in 50 mM potassium phosphate buffer (pH 7.5). The resting cell reaction was started by the addition of homocholine (20 mM) to the cell suspension. The suspension was incubated on a shaker at 120 rpm and 30°C. At appropriate time intervals (30 min, 1 h, 2 h, 3 h, and 6 h), aliquots of the cell suspension were withdrawn and boiled for 3 to 5 min to stop the reaction. These extracts were preserved at −20°C until used for sample derivatization.
Extract preparation from plant samples
Plant (Limonium suffruticosum, Phragmites australis, and Elaeagnus oxycarpa) leaf samples, at productive stage, were collected from an area around Aiding Lake in the Turpan Basin, Xinjiang, China, in August 2010. The area of the study site is about 10,000 m2 (100 m × 100 m), and three plots (10 m × 10 m) were established randomly. The samples were collected from five plants of each species and carefully washed with water. The samples were dried in oven at 85°C for 48 h, ground to fine power, and then brought to Arid Land Research Center, Tottori University, Japan, for analysis. For extract preparation, about 100 mg of powdered samples were added to 1.5 ml water, mixed in a plastic tube, incubated at 75°C for 20 min, and then centrifuged at 15,000 × g for 10 min. These samples were preserved at −20°C until used for sample derivatization.
One of the challenging aspects of analysis of β-alanine betaine and TMA lies in their lack of useful chromophores and thus could not be detected in ultraviolet-visible (UV/vis) light range. To overcome this limitation, the samples were derivatized with 4-bromphencyl bromide before analysis with capillary electrophoresis. Esterification was carried out following the methods of Nishimura et al. () with some modifications. Briefly, 0.1 ml of the sample extract and/or authentic standards of β-alanine betaine and TMA were placed in a micro-tube and mixed with 0.05 ml of buffer solution (100 mM KH2PO4/distilled water/acetonitrile = 1:1:4). To the mixture, 0.3 ml of 4-bromophenacyl bromide (20 mg/ml in acetonitrile) was added. The tube was capped and heated at 90°C for 90 min. The reaction mixture was evaporated to dryness with a centrifugal evaporator (CVE-200D; Tokyo Rikakikai, Tokyo, Japan). The residue was dissolved in 300 μl of 50 mM sodium phosphate buffer (pH 3.0), mixed well, and centrifuged at 10,000 × g for 20 min at 4°C. The supernatants, which contained ester and salt of the metabolites β-alanine betaine and TMA, were filtered using 45-μm filter (Millex Millipore, Billerica, MA, USA) to remove the micro-particles that might block the flow through the capillary tube. The filtered samples were then analyzed by capillary electrophoresis.
Capillary electrophoresis analysis
Capillary electrophoresis analysis was conducted using a capillary electrophoresis system model Photal CAPI-3300 (Otsuka Electronics. Co. Ltd., Osaka, Japan) equipped with a fused silica capillary of 75-μm i.d. with a total length of 80 cm (effective length of 68 cm). Before starting the analysis, the capillary was conditioned with 0.1 M NaOH for 5 min followed by conditioning with distilled water for 3 min and electrolyte buffer for 3 min (50 mM sodium phosphate buffer, pH 3.0). Between each run, the capillary was flushed with distilled water (1 min) and electrolyte buffer (3 min). The temperature of the capillary was set at 25°C and then the samples and/or the authentic standards (β-alanine betaine and TMA) were injected hydrostatically (25 mm, 60 s). During the run and in order to avoid sample carry-over into the electrophoresis buffer, the capillary was dipped twice in distilled water and washing buffer (same electrophoresis buffer that set in other tubes). The applied potential was 20 kV, and the peaks of TMA-salt and β-alanine betaine-ester were monitored at 262 nm.
Statistical analyses were performed with the SPSS v. 18.0 software (SPSS Inc., Chicago, IL, USA). One-factor ANOVA was performed to identify statistically significant differences among treatments, followed by Tukey's HSD test (P ≤ 0.05).
Results and discussion
Repeatability, linearity, and detection limit of TMA and β-alanine betaine
Application of the method to microbial and plant samples
Trimethylamine and β-alanine betaine content in bacterial culture and plant samples
Bacterial culture (mmol/l)
Arthrobacter sp. strain E5
Pseudomonas sp. strain A9
Rhodococcus sp. strain A2
Rhodococcus sp. strain A4
Plant (μmol/g DW)
Elaeagnus oxycarpa (200 mM NaCl)
A capillary electrophoresis method for the simultaneous determination of TMA and β-alanine betaine was developed. The method described here has generally wide detection range suitable for analysis of TMA and β-alanine betaine in microbial and plant samples. The advantages of the current method are its low cost, low detection limit, simple operation, rapid, and high sensitivity.
Financial assistance from the Ministry of Education, Culture, Sports, Science, and Technology of Japan in the form of a scholarship for the first and second authors is gratefully acknowledged.
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