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Measurement of methylglyoxal in rat tissues by electrospray ionization mass spectrometry and liquid chromatography
Journal of Pharmacological and Toxicological Methods 51 (2005) 153 – 157 www.elsevier.com/locate/jpharmtox
Original article
Measurement of methylglyoxal in rat tissues by electrospray ionization mass spectrometry and liquid chromatography E. W. Randella, S. Vasdevb,*, V. Gillb a
Department of Laboratory Medicine, Faculty of Medicine, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada b Department of Medicine, Faculty of Medicine, Memorial University of Newfoundland, Room 4310, Health Sciences Centre, 300 Prince Philip Drive, St. John’s, Newfoundland, Canada A1B 3V6 Received 18 June 2004; accepted 27 August 2004
Abstract Introduction: Increased methylglyoxal formation due to insulin resistance has been implicated in the development of essential hypertension and in type-2 diabetic complications in animal models. Methylglyoxal is a highly reactive aldehyde, which binds sulfhydryl and amino groups of membrane proteins forming conjugates, advanced glycation end products (AGEs), which alter membrane function, leading to increased blood pressure and oxidative stress. We have shown elevated aldehyde conjugates in tissues of hypertensive rats which may be formed primarily from methylglyoxal. Our objective was to develop a specific method to measure methylglyoxal in rat tissues. Method: This method involves preparation of plasma, blood and tissue homogenates, solid phase extraction of methylglyoxal, derivitization using o-phenylenediamine, further purification of derivatized products by solid phase extraction and quantification by electrospray ionization liquid chromatography mass spectrometry (ESI/LC/MS). Results: Methylglyoxal was highest in aorta followed by heart, liver, kidney and blood in that order in Sprague–Dawley rats. Levels of methylglyoxal in plasma were about an order of magnitude lower than that in tissues, but above the concentration used for the lowest calibration standard. Discussion: We have successfully developed an ESI/LC/MS method for quantification of methylglyoxal in rat tissues. The high selectivity of this method offers an advantage over other methods based on fluorescence. This method will allow the evaluation of methylglyoxal in essential hypertension and type-2 diabetes. D 2004 Elsevier Inc. All rights reserved. Keywords: Tissue methylglyoxal; ESI/LC/MS; Hypertension; Type-2 diabetes
1. Introduction Methylglyoxal is a reactive dicarbonyl compound formed as a by-product of glycolysis and lipid and amino acid metabolism (Thornalley, 1993). Methylglyoxal and other endogenous aldehydes are compounds of unusually high electrophilic reactivity. They react nonenzymatically forming conjugates (AGEs) with free amino (–NH2) groups and sulfhydryl (–SH) groups of membrane proteins, metabolic enzymes and membrane ion channels
* Corresponding author. Tel.: +1 709 777 6375; fax: +1 709 777 6395. E-mail address: [email protected] (S. Vasdev). 1056-8719/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.vascn.2004.08.005
and inhibit their function. Under normal physiological conditions, methylglyoxal is kept at a low level by binding to cysteine and being excreted in bile and urine or through catabolism via the glutathione-dependent glyoxalase system (Schauenstein & Esterbauer, 1979; Schauenstein, Esterbauer, & Zollner, 1977). In insulin resistant states, like essential hypertension and type-2 diabetes, altered glucose metabolism may lead to increased formation of methylglyoxal and other aldehydes. We have shown elevated tissue aldehyde conjugates, which may be formed primarily from methylglyoxal, in hypertensive rats (Vasdev, Ford, Longerich, Parai, & Gadag, 1998; Vasdev, Ford, Parai, Longerich, & Gadag, 2000a, 2000b, 2001; Vasdev, Gill,
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Parai, Longerich, & Gadag, 2002a, 2002b; Vasdev, Gill, Longerich, Parai, & Gadag, 2003; Vasdev, Longerich, & Ford, 1997). The objective of this study was to develop a specific method to measure free methylglyoxal in rat tissues. The methods reported in literature have been developed for quantification of methylglyoxal in plasma and cell culture (McLellan, Phillips, & Thornalley, 1992; Chaplen, Fahl, & Cameron, 1996; Odani, Shinzato, Matsumoto, Usami, & Maeda, 1999). In this report, we describe a procedure which we have successfully applied to the quantification of methylglyoxal from tissues of Sprague– Dawley rats. This method involves preparation of plasma, blood and tissue homogenates, solid phase extraction of methylglyoxal and derivitization using o-phenylenediamine. The derivatized products are further purified by solid phase extraction and quantified by electrospray ionization liquid chromatography mass spectrometry (ESI/LC/MS).
2. Methods 2.1. Chemicals All chemicals and reagents were of analytical grade. Methylglyoxal, 2-methylquinoxaline, 5-methylquinoxaline, o-phenylenediamine and 2,3-hexanedione were purchased from Sigma-Aldrich Canada, Ontario, Canada. Millipore millex 0.22 Am filters were from Fisher Scientific, Ontario, Canada. C-18 solid phase extraction (SPE) columns and reversed phase chromatography columns (C8: 25. Am, 2.1100 mm, WAT058961) were purchased from Waters, Ontario, Canada.
2.2. Animals Six male Sprague–Dawley rats, body weight 263–272 g, were obtained from the vivarium of Memorial University of Newfoundland, St. John’s, Newfoundland, Canada. Animals were fed rat chow, ad libitum for at least 1 week before experiments. Animals had free access to diet and drinking water and were treated in accordance with guidelines of the Canadian Council on Animal Care. 2.3. Preparation of tissue samples The principle of this method involves derivitization of methylglyoxal with o-phenylenediamine to form 2-methylquinoxaline and of internal standard, 2,3-hexanedione, to produce 1-methyl-2-propylquinoxaline (Fig. 1). A second internal standard, 5-methylquinoxaline, chemically similar to 2-methylquinoxaline and 1-methyl-2-propylquinoxaline, which did not require derivitization was also used for comparison. Animals were anesthetized with phenobarbital (100 mg/ kg body weight) administered intraperitoneally. This anesthetic does not affect glucose metabolism and should not affect methylglyoxal levels (Vasdev et al., 2000a, 2000b). Blood was collected by cardiac puncture in EDTA containing tubes and a sample of plasma was separated immediately by centrifugation at 4 8C. Liver, heart, kidney and aorta samples were obtained under anesthesia. Tissue samples were promptly removed for homogenization. Tissues were immediately weighed and homogenized with a Fisher Dyna-Mix homogenizer over ice for 1–2 min in 5 volumes of ice-cold 10 mM phosphate buffer, pH 7.4. Internal standard, 2.5 nmol of 2,3-hexanedione was added to the homogenates and each were further sonicated, using
Fig. 1. Figure shows the derivitization of a-h dicarbonyl compounds, methylglyoxal and 2,3-hexanedione, using o-pheylenediamine for electrospray ionization liquid chromatography mass spectrometry measurement.
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35-s 10-W bursts. Proteins were precipitated by adding 0.1 volume of 5 M perchloric acid (PCA) and incubated on ice for 10 min. Samples were centrifuged at 10,000g for 10 min to remove the PCA precipitate. Supernatants were passed through C-18 SPE columns that were previously primed using 6 ml acetonitrile and 6 ml of 10 mM phosphate buffer, pH 2.5. The eluate was supplemented with 5-methylquinoxaline, the second internal standard (2.5 nmol) and incubated with 125 nmol of o-phenylenediamine at 4 8C for 18–20 h. The samples were then applied down a prepared C18 SPE column, rinsed twice with phosphate buffer and the retentate eluted in 2 ml of acetonitrile. The elute was further evaporated to dryness under a stream of nitrogen gas and the final residue was reconstituted in 300 Al of methanol. The concentrate was then filtered through 0.22 Am filters into HPLC sample vials for quantitative analysis of methylglyoxal quinoxaline derivative (2-methylquinoxaline) by ESI/LC/MS. 2.4. Preparation of standards Methylglyoxal was quantified using stock solutions of pure methylgloxal (125 nmol/ml) dissolved in 10 mM phosphate (pH 2.5 buffer). Calibrating standards containing 0.125–6.25 nmol methylglyoxal prepared in 5 ml of 10 mM phosphate buffer (pH 2.5) and 2,3-hexanedione was added. Samples were derivatized using o-phenylenediamine as described above for samples and underwent the second extraction on C18 SPE columns only. 2.5. Recovery To determine recovery, two sets of samples (six aliquots each) of rat plasma were prepared. One set of samples was analyzed as is and the second set was spiked with 2 nmol of methylglyoxal. The methylglyoxal in both sets of samples was determined by ESI/LC/MS. Calculation of recovery involved subtraction of the methylglyoxal in the unadulterated plasma sample from that in the spiked sample. The recovery experiment was done using both internal standards,
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2,3-hexanedione and 5-methylquinoxaline, which were added as described above in the preparation of tissue samples. 2.6. Analysis by LC-MS 2-Methylquinoxaline, 5-methylquinoxaline and 1-methyl2-propylquinoxaline were analyzed using a Waters High Performance Liquid Chromatography system (2795 separators module, equipped with a temperature controller) and a Micromass Quattro Ultima PT for ESI/MS. The quinoxaline derivatives were resolved by reverse-phase chromatography on a C8 Column in a two-solvent gradient using 40% methanol, containing 0.2% trifluoroacetic acid (solvent A) and methanol (solvent B). Sample was injected and the column was maintained in solvent A for 0.5 min. Solvent B was then increased to 100% from 0.5 to 4 min and held for an additional 2 min. All flow rates were set at 0.25 ml/min and the column held at a constant temperature of 35 8C. Selective ion monitoring for m/z 145 was used for quantification of 2methylquinoxaline and for monitoring 5-methylquinoxaline, and m/z 173 was used to monitor 1-methyl-2-propylquinoxaline. The MS used positive ion electrospray with capillary spray voltage held at 2.0 kV. The electron multiple voltage was held at 450 V and high and low molecular weight resolutions set at 13. 2.7. Statistical analysis Data are expressed as meanFstandard deviation (S.D.), and were analyzed using analysis of variance (ANOVA) and Tukey honestly significant difference test. Differences between groups were considered statistically significant when pb0.05.
3. Results Fig. 1 shows the products of derivitization with ophenylenediamine. The molecular ions (M+1) are 145 m/z
Fig. 2. Representative electrospray ionization liquid chromatography mass spectrometry (ESI/LC/MS) chromatograph of a rat heart sample for methylglyoxal. The concentrations of 2-methylquinoxaline, 5-methylquinoxaline and 1-methyl-2-propylquinoxaline were approximately 2 nmol/ml. A ESI/LC/MS chromatogram showing 1-methyl-2-propylquinoxaline, the derivatization product of the internal standard 2,3 hexanedione using o-phenylenediamine (upper tracing). A ESI/LC/MS chromatogram showing 2-methylquinoxaline, the derivatization product of methylglyoxal using o-phenylenediamine, and 5methylquinoxaline, the second (underivatized) internal standard (lower tracing).
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Table 1 Performance of the LC-MS assay using 2,3-hexanedione as the internal standard No. of aliquots
Added amount (nmol)
Amount detected (nmol)
Recovery (%)
% CV interassay
6 6
0 2.0
0.28F0.12 1.56F0.18
– 63.9F9.0
43 12
Rat plasma sample was spiked with methylglyoxal standard and processed for ESI/LC/MS. Unspiked samples were similarly processed for analysis.
for 2-methylqinoxaline and 173 m/z for 1-methyl-2propylquinoxaline. Fig. 2 shows the typical HPLC chromatograms for 2-methylquinoxaline, 5-methylquinoxaline and 1-methyl-2-propylquinoxaline in a sample extracted from rat heart. The concentrations in the sample correspond to 2.5 nmol for 5-methylquinoxaline, 2.5 nmol of 2,3-hexanedione and about 2 nmol of methylglyoxal. The profile shows good separation of 2-methylquinoxaline, at 4.3 min, from 5-methylquinoxaline, at 5.2 min. The identity of 2-methylquinoxaline and elution times were confirmed using commercially available standards. There was no significant interference between the 1methyl-2-propylquinoxaline at 5.6 min and other components. A small peak migrating beyond 1-methyl-2propylquinoxaline was sufficiently resolved as not to affect quantification. Quantification of 2-methylglyoxal was based on the peak area ratio of 2-methylquinoxaline to the derivatized internal standard (2,3-hexanedione) and determined with reference to a standard curve. A quadratic function best fit the response curve and provided a useful measurement range for tissue methylglyoxal from 0.125 to 6.2 nmol. The minimum detection limit was less than 0.125 nmol. Table 1 shows the calculated levels of methylglyoxal in rat plasma. The recovery of methylglyoxal in the samples
Table 2 Recovery of internal standards in calibration standards and rat plasma No. of aliquots
Sample
2,3-Hexanedione
5-Methylquinoxaline
7
Calibration standards Unspiked rat plasma Spiked rat plasma
23.3F3.7 (100%)
21.3F2.0 (100%)
21.0F1.4 (91%)
15.0F3.0 (71%)*
20.8F9.1 (87%)
7.5F4.6 (36%)*
6 6
2.5 nmol of each internal standard was added to calibration standards and rat plasma samples (unspiked and spiked with 2 nmol of methylgyoxal) and processed for ESI/LC/MS. Comparison between calibration standards and rat plasma was done using area under the curve as a measurement of internal standard recovery. Values are given as arbitrary units (meanFS.D.). Recovery of internal standards in both spiked and unspiked rat plasma was also calculated as a percentage of the recovery in calibration standards, assuming the recovery in the calibration standards was 100%. These values are given in parenthesis. * Indicates values are statistically significant ( pb0.05) using paired ttest as compared to calibration standard.
Table 3 Methylglyoxal content in rat tissues (nmol/g wet weight) determined by ESI/LC/MS using 2,3-hexanedione or 5-methylquinoxaline as an internal standard (IS)
Heart Liver Kidney Aorta Blood
2,3-Hexanedione as IS
5-Methylquoxaline as IS
1.9F0.5 1.6F0.5 1.3F0.3 6.1F1.8* 0.67F0.3
3.4F1.6 2.5F0.8a 2.0F1.00 10.6F6.7* 2.0F1.0a
N=6. Values are given as meanFS.D. a Indicates that the value is statistically significant as compared to the value of the same tissue determined using 2,3-hexanedione ( pb0.05). * Indicates that the aortic methylglyoxal value is statistically significant as compared to all other tissue values determined by the same internal standard using ANOVA ( pb0.05).
was 63.9% using 2,3-hexanedione as the internal standard. This recovery is comparable to that reported by others (Chaplen et al., 1996; McLellan et al., 1992). Recovery of the internal standard itself was gauged by the intensity of the signal received representing area under the curve. Recovery of 2,3-hexanedione was comparable between samples and calibration standards. However, recovery of 5-methylquinoxaline was significantly lower in the both the spiked and unspiked plasma than in the calibration standards (Table 2). This resulted in values for methylglyoxal that were outside the range of the highest calibration standard. Therefore, we were unable to calculate methylglyoxal recovery using this internal standard. Table 3 shows methyglyoxal levels in rat tissues using both internal standards. The highest amounts of methylglyoxal were found in aorta followed by heart, liver, kidney and blood. Levels of methylglyoxal in plasma was about an order of magnitude lower than that in tissues (0.28F0.12 nmol), but were generally above the concentration used for the lowest calibration standard (0.125 nmol). Tissue methylglyoxal results calculated using 5-methylquinoxaline as the internal standard were significantly higher in blood and liver compared with those calculated using 2,3hexanedione ( pb0.05). In all other tissues, the mean values were higher, but did not reach statistical significance due to high standard deviation.
4. Discussion Previous methods described in the literature use HPLC and fluorescence spectrophotometry to measure methylglyoxal in cell culture or plasma only. One such method by Chaplen et al. (1996) described a procedure for methylglyoxal measurement in cell culture utilizing C-18 SPE cartridges and derivitization using o-phenylenediamine to form quinoxalines. Using elements of this procedure, we developed an ESI/LC/MS method which offers the advantage of greater specificity for measuring methylglyoxal than
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methods based on fluorescence. We then used this method to measure methylglyoxal levels in intact animal tissues. Chaplen et al. (1996) used 5-methylquinoxaline, as an internal standard, added subsequent to the first solid phase extraction. Adding it at this point in the procedure would not account for variable losses encountered during the first step. Use of stable isotope-labeled methylglyoxal to account for any loss of methylglyoxal, which may occur during the first extraction step and/or through the formation of stable covalent linkages with available reactive groups in the tissue homogenates, may improve the accuracy of the determinations. We compared 2,3-hexanedione and 5-methylquinoxaline as internal standards. The 2,3-hexanedione was added at the tissue homogenization step, whereas 5-methylquinoxaline was added subsequent to the first solid phase extraction. Low recovery of 5-methylquinoxaline in samples as compared to calibration standards, resulted in a falsely high response ratio (area under the curve for 2-methylquinoxaline to area under the curve for 5-methylquinoxaline) giving falsely elevated tissue methylglyoxal levels with this internal standard. This low recovery of 5-methylquinoxaline may be due to a matrix effect specific to this compound. To minimize this effect, it would be necessary to use calibration standards with a protein concentration similar to that found in tissues. Recovery of 2,3-hexanedione was similar in samples and calibration standards. This would suggest that the matrix effect is not perceptible using this compound as the internal standard. Also, 2,3-hexanedione, added at the beginning of the procedure, accounts for variable losses encountered during the first step. We suggest that 2,3hexanedione would be a more appropriate internal standard. In this study, we found the highest levels of methylglyoxal in aortic tissue. These high levels of methylglyoxal may explain the sensitivity of vascular tissue to damage as seen in essential hypertension and type-2 diabetes. In conclusion, we have successfully developed an ESI/ LC/MS method for quantification of methylglyoxal in rat tissues. The high selectivity of this method offers an advantage over other methods based on fluorescence. We have used this method to quantify methylglyoxal in rat tissues using 2,3-hexanedione as an internal standard. To the best of our knowledge, this is the first reported method measuring methylglyoxal levels in intact rat tissue. This method may be a useful tool to measure endogenous methylglyoxal in conditions such as essential hypertension and type-2 diabetes. Plasma methylglyoxal may also be useful as one of the important markers for monitoring vascular complications in type-2 diabetes.
Acknowledgements We would like to thank Dr. V. Gadag, Professor of Biostatistics, Division of Community Health, Memorial
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University of Newfoundland, for his assistance with statistical analysis. We would also like to thank the Canadian Institutes of Health Research and the Discipline of Medicine, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada for support to carry out this study.
References Chaplen, F. W. R., Fahl, W. E., & Cameron, D. C. (1996). Method for determination of free intracellular and extracellular methylglyoxal in animal cells grown in culture. Analytical Biochemistry, 238, 171 – 178. McLellan, A. C., Phillips, S. A., & Thornalley, P. J. (1992). The assay of methhylglyoxal in biological systems by derivitization with 1,2-diamino-4,5-dimethoxybenzene. Analytical Biochemistry, 206, 17 – 23. Odani, H., Shinzato, T., Matsumoto, Y., Usami, J., & Maeda, K. (1999). Increase in three a,h-dicarbonyl compound levels in human uremic plasma: specific in vivo determination of intermediates in advanced Maillard reaction. Biochemical and Biophysical Research Communications, 256, 89 – 93. Schauenstein, E., & Esterbauer, H. (1979). Formation and properties of reactive aldehydes. In: Submolecular Biology and Cancer. Excerpta Medica, CIBA Foundation Symposium, 67, 225 – 244. Schauenstein, E., Esterbauer, H., & Zollner, H. (1977). Aldehydes in biological systems. In J.R Lagnado (Ed.), Aldehydes in biological systems, their natural occurrence and biological activities (pp. 1 – 7). London, UK7 Pion. Thornalley, P. J. (1993). The glyoxalase system in health and disease. Molecular Aspects of Medicine, 14, 287 – 371. Vasdev, S., Ford, C. A., Longerich, L., Parai, S., & Gadag, V. (1998). Aldehyde induced hypertension in rats: prevention by N-acetyl cysteine. Artery, 23, 10 – 36. Vasdev, S., Ford, C. A., Parai, S., Longerich, L., & Gadag, V. (2000). Dietary a-lipoic acid supplementation lowers blood pressure in spontaneously hypertensive rats. Journal of Hypertension, 18, 567 – 573. Vasdev, S., Ford, C. A., Parai, S., Longerich, L., & Gadag, V. (2000). Dietary lipoic acid supplementation prevents fructose-induced hypertension in rats. Nutrition, Metabolism and Cardiovascular Diseases, 10, 339 – 346. Vasdev, S., Ford, C. A., Parai, S., Longerich, L., & Gadag, V. (2001). Dietary vitamin C supplementation lowers blood pressure in spontaneously hypertensive rats. Molecular and Cellular Biochemistry, 218, 97 – 103. Vasdev, S., Gill, V., Longerich, L., Parai, S., & Gadag, V. (2003). Salt-induced hypertension in WKY rats: Prevention by a-lipoic acid supplementation. Molecular and Cellular Biochemistry, 254, 319 – 326. Vasdev, S., Gill, V., Parai, S., Longerich, L., & Gadag, V. (2002a). Dietary vitamin E supplementation lowers blood pressure in spontaneously hypertensive rats. Molecular and Cellular Biochemistry, 238, 111 – 117. Vasdev, S., Gill, V., Parai, S., Longerich, L., & Gadag, V. (2002b). Dietary vitamin E and C supplementation prevents fructose induced hypertension in rats. Molecular and Cellular Biochemistry, 241, 107 – 114. Vasdev, S., Longerich, L., & Ford, C. A. (1997). Role of aldehydes in hypertension. In: B. K. Sharma, N. Takeda, N. K. Ganguly, & P. K. Singal (Eds.), Adaptation Biology and Medicine, vol. 1. (pp. 326 – 339) New Delhi, India7 Narosa Publishing House.
Original article
Measurement of methylglyoxal in rat tissues by electrospray ionization mass spectrometry and liquid chromatography E. W. Randella, S. Vasdevb,*, V. Gillb a
Department of Laboratory Medicine, Faculty of Medicine, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada b Department of Medicine, Faculty of Medicine, Memorial University of Newfoundland, Room 4310, Health Sciences Centre, 300 Prince Philip Drive, St. John’s, Newfoundland, Canada A1B 3V6 Received 18 June 2004; accepted 27 August 2004
Abstract Introduction: Increased methylglyoxal formation due to insulin resistance has been implicated in the development of essential hypertension and in type-2 diabetic complications in animal models. Methylglyoxal is a highly reactive aldehyde, which binds sulfhydryl and amino groups of membrane proteins forming conjugates, advanced glycation end products (AGEs), which alter membrane function, leading to increased blood pressure and oxidative stress. We have shown elevated aldehyde conjugates in tissues of hypertensive rats which may be formed primarily from methylglyoxal. Our objective was to develop a specific method to measure methylglyoxal in rat tissues. Method: This method involves preparation of plasma, blood and tissue homogenates, solid phase extraction of methylglyoxal, derivitization using o-phenylenediamine, further purification of derivatized products by solid phase extraction and quantification by electrospray ionization liquid chromatography mass spectrometry (ESI/LC/MS). Results: Methylglyoxal was highest in aorta followed by heart, liver, kidney and blood in that order in Sprague–Dawley rats. Levels of methylglyoxal in plasma were about an order of magnitude lower than that in tissues, but above the concentration used for the lowest calibration standard. Discussion: We have successfully developed an ESI/LC/MS method for quantification of methylglyoxal in rat tissues. The high selectivity of this method offers an advantage over other methods based on fluorescence. This method will allow the evaluation of methylglyoxal in essential hypertension and type-2 diabetes. D 2004 Elsevier Inc. All rights reserved. Keywords: Tissue methylglyoxal; ESI/LC/MS; Hypertension; Type-2 diabetes
1. Introduction Methylglyoxal is a reactive dicarbonyl compound formed as a by-product of glycolysis and lipid and amino acid metabolism (Thornalley, 1993). Methylglyoxal and other endogenous aldehydes are compounds of unusually high electrophilic reactivity. They react nonenzymatically forming conjugates (AGEs) with free amino (–NH2) groups and sulfhydryl (–SH) groups of membrane proteins, metabolic enzymes and membrane ion channels
* Corresponding author. Tel.: +1 709 777 6375; fax: +1 709 777 6395. E-mail address: [email protected] (S. Vasdev). 1056-8719/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.vascn.2004.08.005
and inhibit their function. Under normal physiological conditions, methylglyoxal is kept at a low level by binding to cysteine and being excreted in bile and urine or through catabolism via the glutathione-dependent glyoxalase system (Schauenstein & Esterbauer, 1979; Schauenstein, Esterbauer, & Zollner, 1977). In insulin resistant states, like essential hypertension and type-2 diabetes, altered glucose metabolism may lead to increased formation of methylglyoxal and other aldehydes. We have shown elevated tissue aldehyde conjugates, which may be formed primarily from methylglyoxal, in hypertensive rats (Vasdev, Ford, Longerich, Parai, & Gadag, 1998; Vasdev, Ford, Parai, Longerich, & Gadag, 2000a, 2000b, 2001; Vasdev, Gill,
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Parai, Longerich, & Gadag, 2002a, 2002b; Vasdev, Gill, Longerich, Parai, & Gadag, 2003; Vasdev, Longerich, & Ford, 1997). The objective of this study was to develop a specific method to measure free methylglyoxal in rat tissues. The methods reported in literature have been developed for quantification of methylglyoxal in plasma and cell culture (McLellan, Phillips, & Thornalley, 1992; Chaplen, Fahl, & Cameron, 1996; Odani, Shinzato, Matsumoto, Usami, & Maeda, 1999). In this report, we describe a procedure which we have successfully applied to the quantification of methylglyoxal from tissues of Sprague– Dawley rats. This method involves preparation of plasma, blood and tissue homogenates, solid phase extraction of methylglyoxal and derivitization using o-phenylenediamine. The derivatized products are further purified by solid phase extraction and quantified by electrospray ionization liquid chromatography mass spectrometry (ESI/LC/MS).
2. Methods 2.1. Chemicals All chemicals and reagents were of analytical grade. Methylglyoxal, 2-methylquinoxaline, 5-methylquinoxaline, o-phenylenediamine and 2,3-hexanedione were purchased from Sigma-Aldrich Canada, Ontario, Canada. Millipore millex 0.22 Am filters were from Fisher Scientific, Ontario, Canada. C-18 solid phase extraction (SPE) columns and reversed phase chromatography columns (C8: 25. Am, 2.1100 mm, WAT058961) were purchased from Waters, Ontario, Canada.
2.2. Animals Six male Sprague–Dawley rats, body weight 263–272 g, were obtained from the vivarium of Memorial University of Newfoundland, St. John’s, Newfoundland, Canada. Animals were fed rat chow, ad libitum for at least 1 week before experiments. Animals had free access to diet and drinking water and were treated in accordance with guidelines of the Canadian Council on Animal Care. 2.3. Preparation of tissue samples The principle of this method involves derivitization of methylglyoxal with o-phenylenediamine to form 2-methylquinoxaline and of internal standard, 2,3-hexanedione, to produce 1-methyl-2-propylquinoxaline (Fig. 1). A second internal standard, 5-methylquinoxaline, chemically similar to 2-methylquinoxaline and 1-methyl-2-propylquinoxaline, which did not require derivitization was also used for comparison. Animals were anesthetized with phenobarbital (100 mg/ kg body weight) administered intraperitoneally. This anesthetic does not affect glucose metabolism and should not affect methylglyoxal levels (Vasdev et al., 2000a, 2000b). Blood was collected by cardiac puncture in EDTA containing tubes and a sample of plasma was separated immediately by centrifugation at 4 8C. Liver, heart, kidney and aorta samples were obtained under anesthesia. Tissue samples were promptly removed for homogenization. Tissues were immediately weighed and homogenized with a Fisher Dyna-Mix homogenizer over ice for 1–2 min in 5 volumes of ice-cold 10 mM phosphate buffer, pH 7.4. Internal standard, 2.5 nmol of 2,3-hexanedione was added to the homogenates and each were further sonicated, using
Fig. 1. Figure shows the derivitization of a-h dicarbonyl compounds, methylglyoxal and 2,3-hexanedione, using o-pheylenediamine for electrospray ionization liquid chromatography mass spectrometry measurement.
E.W. Randell et al. / Journal of Pharmacological and Toxicological Methods 51 (2005) 153–157
35-s 10-W bursts. Proteins were precipitated by adding 0.1 volume of 5 M perchloric acid (PCA) and incubated on ice for 10 min. Samples were centrifuged at 10,000g for 10 min to remove the PCA precipitate. Supernatants were passed through C-18 SPE columns that were previously primed using 6 ml acetonitrile and 6 ml of 10 mM phosphate buffer, pH 2.5. The eluate was supplemented with 5-methylquinoxaline, the second internal standard (2.5 nmol) and incubated with 125 nmol of o-phenylenediamine at 4 8C for 18–20 h. The samples were then applied down a prepared C18 SPE column, rinsed twice with phosphate buffer and the retentate eluted in 2 ml of acetonitrile. The elute was further evaporated to dryness under a stream of nitrogen gas and the final residue was reconstituted in 300 Al of methanol. The concentrate was then filtered through 0.22 Am filters into HPLC sample vials for quantitative analysis of methylglyoxal quinoxaline derivative (2-methylquinoxaline) by ESI/LC/MS. 2.4. Preparation of standards Methylglyoxal was quantified using stock solutions of pure methylgloxal (125 nmol/ml) dissolved in 10 mM phosphate (pH 2.5 buffer). Calibrating standards containing 0.125–6.25 nmol methylglyoxal prepared in 5 ml of 10 mM phosphate buffer (pH 2.5) and 2,3-hexanedione was added. Samples were derivatized using o-phenylenediamine as described above for samples and underwent the second extraction on C18 SPE columns only. 2.5. Recovery To determine recovery, two sets of samples (six aliquots each) of rat plasma were prepared. One set of samples was analyzed as is and the second set was spiked with 2 nmol of methylglyoxal. The methylglyoxal in both sets of samples was determined by ESI/LC/MS. Calculation of recovery involved subtraction of the methylglyoxal in the unadulterated plasma sample from that in the spiked sample. The recovery experiment was done using both internal standards,
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2,3-hexanedione and 5-methylquinoxaline, which were added as described above in the preparation of tissue samples. 2.6. Analysis by LC-MS 2-Methylquinoxaline, 5-methylquinoxaline and 1-methyl2-propylquinoxaline were analyzed using a Waters High Performance Liquid Chromatography system (2795 separators module, equipped with a temperature controller) and a Micromass Quattro Ultima PT for ESI/MS. The quinoxaline derivatives were resolved by reverse-phase chromatography on a C8 Column in a two-solvent gradient using 40% methanol, containing 0.2% trifluoroacetic acid (solvent A) and methanol (solvent B). Sample was injected and the column was maintained in solvent A for 0.5 min. Solvent B was then increased to 100% from 0.5 to 4 min and held for an additional 2 min. All flow rates were set at 0.25 ml/min and the column held at a constant temperature of 35 8C. Selective ion monitoring for m/z 145 was used for quantification of 2methylquinoxaline and for monitoring 5-methylquinoxaline, and m/z 173 was used to monitor 1-methyl-2-propylquinoxaline. The MS used positive ion electrospray with capillary spray voltage held at 2.0 kV. The electron multiple voltage was held at 450 V and high and low molecular weight resolutions set at 13. 2.7. Statistical analysis Data are expressed as meanFstandard deviation (S.D.), and were analyzed using analysis of variance (ANOVA) and Tukey honestly significant difference test. Differences between groups were considered statistically significant when pb0.05.
3. Results Fig. 1 shows the products of derivitization with ophenylenediamine. The molecular ions (M+1) are 145 m/z
Fig. 2. Representative electrospray ionization liquid chromatography mass spectrometry (ESI/LC/MS) chromatograph of a rat heart sample for methylglyoxal. The concentrations of 2-methylquinoxaline, 5-methylquinoxaline and 1-methyl-2-propylquinoxaline were approximately 2 nmol/ml. A ESI/LC/MS chromatogram showing 1-methyl-2-propylquinoxaline, the derivatization product of the internal standard 2,3 hexanedione using o-phenylenediamine (upper tracing). A ESI/LC/MS chromatogram showing 2-methylquinoxaline, the derivatization product of methylglyoxal using o-phenylenediamine, and 5methylquinoxaline, the second (underivatized) internal standard (lower tracing).
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Table 1 Performance of the LC-MS assay using 2,3-hexanedione as the internal standard No. of aliquots
Added amount (nmol)
Amount detected (nmol)
Recovery (%)
% CV interassay
6 6
0 2.0
0.28F0.12 1.56F0.18
– 63.9F9.0
43 12
Rat plasma sample was spiked with methylglyoxal standard and processed for ESI/LC/MS. Unspiked samples were similarly processed for analysis.
for 2-methylqinoxaline and 173 m/z for 1-methyl-2propylquinoxaline. Fig. 2 shows the typical HPLC chromatograms for 2-methylquinoxaline, 5-methylquinoxaline and 1-methyl-2-propylquinoxaline in a sample extracted from rat heart. The concentrations in the sample correspond to 2.5 nmol for 5-methylquinoxaline, 2.5 nmol of 2,3-hexanedione and about 2 nmol of methylglyoxal. The profile shows good separation of 2-methylquinoxaline, at 4.3 min, from 5-methylquinoxaline, at 5.2 min. The identity of 2-methylquinoxaline and elution times were confirmed using commercially available standards. There was no significant interference between the 1methyl-2-propylquinoxaline at 5.6 min and other components. A small peak migrating beyond 1-methyl-2propylquinoxaline was sufficiently resolved as not to affect quantification. Quantification of 2-methylglyoxal was based on the peak area ratio of 2-methylquinoxaline to the derivatized internal standard (2,3-hexanedione) and determined with reference to a standard curve. A quadratic function best fit the response curve and provided a useful measurement range for tissue methylglyoxal from 0.125 to 6.2 nmol. The minimum detection limit was less than 0.125 nmol. Table 1 shows the calculated levels of methylglyoxal in rat plasma. The recovery of methylglyoxal in the samples
Table 2 Recovery of internal standards in calibration standards and rat plasma No. of aliquots
Sample
2,3-Hexanedione
5-Methylquinoxaline
7
Calibration standards Unspiked rat plasma Spiked rat plasma
23.3F3.7 (100%)
21.3F2.0 (100%)
21.0F1.4 (91%)
15.0F3.0 (71%)*
20.8F9.1 (87%)
7.5F4.6 (36%)*
6 6
2.5 nmol of each internal standard was added to calibration standards and rat plasma samples (unspiked and spiked with 2 nmol of methylgyoxal) and processed for ESI/LC/MS. Comparison between calibration standards and rat plasma was done using area under the curve as a measurement of internal standard recovery. Values are given as arbitrary units (meanFS.D.). Recovery of internal standards in both spiked and unspiked rat plasma was also calculated as a percentage of the recovery in calibration standards, assuming the recovery in the calibration standards was 100%. These values are given in parenthesis. * Indicates values are statistically significant ( pb0.05) using paired ttest as compared to calibration standard.
Table 3 Methylglyoxal content in rat tissues (nmol/g wet weight) determined by ESI/LC/MS using 2,3-hexanedione or 5-methylquinoxaline as an internal standard (IS)
Heart Liver Kidney Aorta Blood
2,3-Hexanedione as IS
5-Methylquoxaline as IS
1.9F0.5 1.6F0.5 1.3F0.3 6.1F1.8* 0.67F0.3
3.4F1.6 2.5F0.8a 2.0F1.00 10.6F6.7* 2.0F1.0a
N=6. Values are given as meanFS.D. a Indicates that the value is statistically significant as compared to the value of the same tissue determined using 2,3-hexanedione ( pb0.05). * Indicates that the aortic methylglyoxal value is statistically significant as compared to all other tissue values determined by the same internal standard using ANOVA ( pb0.05).
was 63.9% using 2,3-hexanedione as the internal standard. This recovery is comparable to that reported by others (Chaplen et al., 1996; McLellan et al., 1992). Recovery of the internal standard itself was gauged by the intensity of the signal received representing area under the curve. Recovery of 2,3-hexanedione was comparable between samples and calibration standards. However, recovery of 5-methylquinoxaline was significantly lower in the both the spiked and unspiked plasma than in the calibration standards (Table 2). This resulted in values for methylglyoxal that were outside the range of the highest calibration standard. Therefore, we were unable to calculate methylglyoxal recovery using this internal standard. Table 3 shows methyglyoxal levels in rat tissues using both internal standards. The highest amounts of methylglyoxal were found in aorta followed by heart, liver, kidney and blood. Levels of methylglyoxal in plasma was about an order of magnitude lower than that in tissues (0.28F0.12 nmol), but were generally above the concentration used for the lowest calibration standard (0.125 nmol). Tissue methylglyoxal results calculated using 5-methylquinoxaline as the internal standard were significantly higher in blood and liver compared with those calculated using 2,3hexanedione ( pb0.05). In all other tissues, the mean values were higher, but did not reach statistical significance due to high standard deviation.
4. Discussion Previous methods described in the literature use HPLC and fluorescence spectrophotometry to measure methylglyoxal in cell culture or plasma only. One such method by Chaplen et al. (1996) described a procedure for methylglyoxal measurement in cell culture utilizing C-18 SPE cartridges and derivitization using o-phenylenediamine to form quinoxalines. Using elements of this procedure, we developed an ESI/LC/MS method which offers the advantage of greater specificity for measuring methylglyoxal than
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methods based on fluorescence. We then used this method to measure methylglyoxal levels in intact animal tissues. Chaplen et al. (1996) used 5-methylquinoxaline, as an internal standard, added subsequent to the first solid phase extraction. Adding it at this point in the procedure would not account for variable losses encountered during the first step. Use of stable isotope-labeled methylglyoxal to account for any loss of methylglyoxal, which may occur during the first extraction step and/or through the formation of stable covalent linkages with available reactive groups in the tissue homogenates, may improve the accuracy of the determinations. We compared 2,3-hexanedione and 5-methylquinoxaline as internal standards. The 2,3-hexanedione was added at the tissue homogenization step, whereas 5-methylquinoxaline was added subsequent to the first solid phase extraction. Low recovery of 5-methylquinoxaline in samples as compared to calibration standards, resulted in a falsely high response ratio (area under the curve for 2-methylquinoxaline to area under the curve for 5-methylquinoxaline) giving falsely elevated tissue methylglyoxal levels with this internal standard. This low recovery of 5-methylquinoxaline may be due to a matrix effect specific to this compound. To minimize this effect, it would be necessary to use calibration standards with a protein concentration similar to that found in tissues. Recovery of 2,3-hexanedione was similar in samples and calibration standards. This would suggest that the matrix effect is not perceptible using this compound as the internal standard. Also, 2,3-hexanedione, added at the beginning of the procedure, accounts for variable losses encountered during the first step. We suggest that 2,3hexanedione would be a more appropriate internal standard. In this study, we found the highest levels of methylglyoxal in aortic tissue. These high levels of methylglyoxal may explain the sensitivity of vascular tissue to damage as seen in essential hypertension and type-2 diabetes. In conclusion, we have successfully developed an ESI/ LC/MS method for quantification of methylglyoxal in rat tissues. The high selectivity of this method offers an advantage over other methods based on fluorescence. We have used this method to quantify methylglyoxal in rat tissues using 2,3-hexanedione as an internal standard. To the best of our knowledge, this is the first reported method measuring methylglyoxal levels in intact rat tissue. This method may be a useful tool to measure endogenous methylglyoxal in conditions such as essential hypertension and type-2 diabetes. Plasma methylglyoxal may also be useful as one of the important markers for monitoring vascular complications in type-2 diabetes.
Acknowledgements We would like to thank Dr. V. Gadag, Professor of Biostatistics, Division of Community Health, Memorial
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University of Newfoundland, for his assistance with statistical analysis. We would also like to thank the Canadian Institutes of Health Research and the Discipline of Medicine, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada for support to carry out this study.
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