Simple method for the routine determination of betaine and N,N-dimethylglycine in blood and urine

^a Author for correspondence. Fax 049-0211-811910; e-mail Laryea{at}uni-duesseldorf.de.

	Abstract

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A simple and convenient method using commercially available derivatization reagents is described for the measurement of betaine and N,N-dimethylglycine (DMG) in blood and urine. Precolumn derivatization of plasma or urine is performed directly in acetonitrile without extraction with p-bromophenacyl bromide and crown ether as catalyst. The p-bromophenacyl ester derivatives are then separated by high-performance liquid chromatography, using an isocratic system of acetonitrile and water containing choline. Effluent was monitored at 254 nm. The limit of detection was 5 µmol/L for betaine and 2 µmol/L for DMG. Analytical recovery was >97% for both analytes. Total and within-day CVs were 2.0–4.4% and 0.9–2.2% for DMG. For betaine, the total and within-day CVs were 1.3–5.3% and 0.4–3.8%, respectively. The method is precise and cost-effective and has been used successfully to determine the concentrations of DMG and betaine in human plasma and urine.

	Introduction

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Interest in the medical use of betaine [2(N,N,N-trimethylammonium) acetate] has been generated by the knowledge of its importance as an alternative homocysteine methylating agent. It functions as a substrate for the enzyme betaine-homocysteine methyltransferase (EC 2.1.1.5), which catalyzes the remethylation of homocysteine to methionine. Treatment with betaine was effective in the different forms of homocystinuria caused by cystathionine ß-synthase (EC 4.2.1.22) and 5,10-methylenetetrahydrofolate reductase (MTHFR; EC 1.1.99.15) deficiencies, inborn errors of the transsulfuration, and one-carbon pathways. Betaine previously has been shown to reduce plasma concentrations of homocysteine and to increase methionine (1)(2)(3)(4) . In MTHFR deficiency, treatment with betaine is most effective in reversing demyelination of the brain and spinal cord (5) . Although large amounts of betaine are often given orally to patients with these metabolic disorders, little is known about its absorption in the gut and its metabolism. The extent of inhibition on betaine homocysteine methyltransferase by the formed N,N-dimethylglycine (DMG) is uncertain. To optimize therapies in these patients, a sensitive, specific, and reliable method appears necessary to monitor the concentrations of betaine and DMG in blood and urine.

Problems associated with the isolation, detection, and measurement of quaternary ammonium compounds, including betaines in biological materials, have been reviewed by Gorham (6) . Since then, several methods have been described in the literature for the separation of these naturally occurring compounds. We recently described a method for the determination of betaine and DMG in urine, using ultraviolet absorbance (7) . This method lacks sensitivity, which leads to the need to label the substances with absorbing or fluorescing reagents to improve detection limits. Methods using proton nuclear magnetic resonance (8)(9)(10) require large capital outlay and a high degree of technical expertise. Recently, considerable improvement in the analysis of these compounds was reported by Allen et al. (11) . Their assays are based on an isotope dilution method using gas chromatography–mass spectrometry. However, the method is very laborious because it requires betaine to be converted to DMG by partially purified rat liver betaine homocysteine methyltransferase, which is not commercially available.

Here, we report a simple and sensitive isocratic HPLC-UV method for the determination of DMG and betaine in plasma and urine.

	Materials and Methods

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chemicals
Anhydrous betaine and 18-crown-6 were obtained from Sigma Chemical Co. DMG was purchased from Fluka. p-Bromophenacyl bromide was a product of Pierce. All other reagents and solutions were of analytical grade and purchased from Merck.

standards
DMG and betaine were dissolved in water at a concentration of 1 mmol/L and diluted with water to the final concentrations used during the analysis.

subjects and sample handling
Venous blood from five children with homocystinuria (ages, 1–14 years) and 12 healthy volunteers (ages, 30–50 years) was collected into a Vacutainer Tube containing EDTA. Four patients suffered from MTHFR deficiency and were treated with up to 600 mg/kg betaine monohydrate daily; one patient had cystathionine-ß-synthase deficiency and received 200 mg/kg betaine. Because the blood samples were also used for the determination of total homocysteine, they were placed on ice after collection, and plasma was obtained without delay by centrifuging the blood samples within 30 min after collection at 2000g for 10 min at room temperature.

Spontaneous urine samples were collected into plastic tubes. Plasma and urine samples were stored at -20 °C until analysis, usually within 14 days. Hemolytic and lipemic plasma were also used in the preliminary study for the assay.

reagent preparation
The derivatizing solution was made by dissolving 66 mg (2.5 mmol) of 18-crown-6 and 1390 mg (50 mmol) of 4-bromophenacyl bromide in 100 mL of acetonitrile.

derivatization procedure
Urine samples were diluted up to 10-fold with distilled water before assay. Plasma was used without dilution for the assay.

To 50 µL of sample or calibrator solution was added 50 µL of 100 mmol/L KH₂PO₄. After the solution was vortex-mixed, 900 µL of derivatizing solution was added, and the mixing continued. The tubes were capped, vortex-mixed, and heated to 80 °C for 60 min. After the mixture was cooled to room temperature, it was again vortex-mixed and centrifuged at 1000g. Fifteen microliters of the supernatant containing the phenacyl esters of DMG and betaine was injected directly into the HPLC.

equipment
The HPLC consisted of a Model 501 pump coupled to a Wisp Model 712 autosampler. Detection was with a 490 Programmable Multiwavelength Detector connected to dual channel monitor (Waters Associates) and a Shimadzu integrator CR3A. The column was a Supelcosil^TM LC-SCX, 5 µm, 25 cm x 4.6 cm (Supelco Inc.).

chromatographic conditions
Sample elution was isocratic over 20 min, using a mobile phase containing 22 mmol/L choline in 900 mL/L acetonitrile and 100 mL/L water. The mobile phase was degassed for 30 min in an ultrasonic bath before use. The flow rate was 1.5 mL/min. The detector was set to monitor the analytes at 254 nm. All chromatography was performed at room temperature.

	Results

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optimization of the assay
The derivatization kinetics were determined at different temperatures, i.e., 20, 40, 60, 80, 90, and 120 °C, for both DMG and betaine. Samples were taken at 10-min intervals for 90 min. The optimum conditions proved to be 60 min at 80 °C. Samples could be derivatized directly without addition of KH₂PO₄; however, DMG gave a double peak, probably from alkylation of its amino group under the reaction conditions.

hplc elution profiles
Careful selection of the ionic strength of choline and the amounts of acetonitrile and water in the mobile phase was found to be effective for the separation of DMG and betaine. We determined that 22 mmol/L choline in 900 mL/L acetonitrile allowed resolution of the phenacyl bromide ester derivatives of DMG and betaine from other compounds present in the mixture. Under the conditions chosen, the retention times of the phenacyl bromide esters of DMG and betaine were 12.7 and 14.8 min, respectively (Fig. 1 A).

View larger version (11K): [in this window] [in a new window]   Figure 1. Elution profiles of DMG and betaine calibrators (A) and plasma (B) and urine (C) from a patient with homocystinuria, caused by 5,10-methylenetetrahydrofolate deficiency, undergoing therapy with betaine. (B) DMG = 7.8 µmol/L; betaine = 101.7 µmol/L; full scale = 0.01 absorbance units. (C) Full scale = 0.02 absorbance units.

Chromatograms of plasma and urine of a patient with homocystinuria caused by MTHFR deficiency are shown in Fig. 1 , B and C; Fig. 2 shows plasma of an unaffected subject with and without added DMG. Betaine and DMG peaks in the plasma samples correspond to the retention of the analytes in the calibrator solution. The peaks are well-resolved, with no extraneous substance interfering with the assay. All of the UV-absorbing compounds eluted within 25 min of injection. Peak 1 in the chromatograms of the urine samples was identified as creatinine by retention time and co-chromatography with an authentic standard. Therefore, it is possible to estimate DMG, betaine, and creatinine in the present system.

View larger version (11K): [in this window] [in a new window]   Figure 2. Elution profiles of the plasma of a healthy subject (A) and plasma from that same subject (B) with 8.0 µmol/L DMG added. Full scale = 0.01 absorbance units. (A) Betaine = 21.6 µmol/L; DMG = 5.7 µmol/L.

limit of detection and sensitivity
The limits of detection for the assay, defined as four times the signal-to-noise ratio, were determined to be 2 and 5 µmol/L for DMG and betaine, respectively. These concentrations are lower than the basal concentrations of DMG and betaine in human plasma and urine. Sensitivity can be increased by using larger quantities of matrix in the case of plasma or by changing the dilution factor of the urine. Sensitivity could also be enhanced by injecting more sample into the HPLC rather than the 15 µL used here.

linearity
The linearity of the method was assessed by analyzing DMG and betaine calibrators ranging in concentration from 2 to 200 µmol/L, using 15 µL samples. DMG and betaine were linearly related to peak height, and this relationship was maintained over the range tested. The regression equations (± SD) were: y = 220 (± 2)x - 348 (± 200) µV (r² = 0.99) for DMG, and y = 121(± 1)x - 185 (± 111) µV (r² = 0.99) for betaine.

recovery
Recoveries of DMG and betaine added to urine and plasma samples were 97–101% (Table 1 ).

imprecision
Total and within-run imprecision (CV) measured on plasma and urine at three concentrations was assessed by analyzing the samples 20 times within 1 day and over 30 separate days (12) . The amounts added to the specimens were chosen to cover the ranges of the calibration curves and to include a specimen of a high value, as encountered in patients being treated with betaine (Table 2 ).

internal standards
No internal standard was used in this assay. Commercially available substances such as sulfonobetaine, trigonelline, and others with similar structures and retention times appeared from proton nuclear magnetic resonance spectrometry to be present in urine and probably plasma samples. Direct derivatization without sample preparation, however, allowed the peak sizes to be monitored efficiently with external standards.

measurement of reference and high values
Plasma.
The concentration ranges of DMG and betaine in the 12 healthy subjects were 4–13 µmol/L and 20–144 µmol/L, respectively. In patients being treated with betaine, the ranges were from 8 to 228 µmol/L for DMG and from 20 to 2680 µmol/L for betaine.

Urine.
The range in the healthy subjects was from 0.8 to 11.6 mmol/mol creatinine for DMG and from 6.4 to 92.7 mmol/mol creatinine for betaine. In urine of betaine-treated patients, the concentrations were increased up to 3.6 mol/mol creatinine for DMG and up to 20.8 mol/mol creatinine for betaine.

	Discussion

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The ability of phenacyl bromide to react with carboxyl groups has been known for some time (13) . Recently, van Kempen et al. (14) and Gorham et al. ((15)) demonstrated that this reagent reacts with quaternary ammonium compounds and can be used for their quantification. We have adapted and modified these methods for the determination of DMG and betaine in plasma and urine.

Our goal was to develop a method for clinical use that would be simple and rapid, would use commercially available reagents, would have a one-step sample preparation, would involve isocratic elution with a single column, would allow simultaneous analyses of DMG and betaine in plasma and urine, and would require only a small sample. The present method appears to have certain advantages over previously reported HPLC methods, principally because most of the interfering substances do not have to be removed before derivatization (7)(14)(15) . This reduces losses of DMG and betaine and the time required for analysis. The absence of this critical step leads to satisfactory criteria of reproducibility and repeatability. The limits of detection were 2 µmol/L for DMG and 5 µmol/L for betaine. Recovery was >97%. Moreover, it is also possible to estimate creatinine in urine samples in the same assay using our system, an advantage for the calculation of urine values related to creatinine. The method also avoids gradient elution, which requires a sophisticated HPLC apparatus. Samples are usually analyzed on the day of derivatization. No detectable losses were found during the course of a working day or in derivatized samples stored at 4 °C for a day or at -20 °C for a week.

This procedure was used to determine betaine and DMG concentrations in plasma and urine samples from a limited number of healthy individuals and from patients receiving betaine monohydrate in doses of 200–600 mg/kg body weight per day. Interestingly, in healthy subjects we found somewhat wider ranges for betaine in plasma and urine than Allen et al. (11) , with 20–144 vs 17.6–73.3 µmol/L and 6.4–92.7 vs 2.3–55.9 mmol/mol creatinine. These differences may reflect methodological differences or different dietary intakes.

Ease of derivative formation, coupled with a simple chromatographic separation, increases the potential of this method for routine analysis of DMG and betaine in plasma and urine.

	Footnotes

 
Heinrich-Heine University, Children's Hospital, Metabolic Unit, Moorenstrasse 5, D-40225 Düsseldorf, Germany.

	References

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