Quantitative Analysis of Methylglyoxal, Glyoxal and Free Advanced Glycation End-Products in the Plasma of Wistar Rats during the Oral Glucose Tolerance Test

Abstract

The purpose of this study was to gain insight into the production behavior of free adducts of advanced glycation end-products (AGEs) in Wistar rats under acute hyperglycemic conditions. Five AGE-free adducts as well as their precursors (i.e., highly reactive carbonyl intermediates of methylglyoxal and glyoxal) in rat plasma were quantitatively determined at greater than nanomolar levels using the liquid chromatography/tandem mass spectrometry method coupled with 2,4,6-trinitrobenzene sulfonate and 2,3-diaminonaphthalene derivatization techniques. An oral glucose (2 g/kg dose) tolerance test to 10-week-old Wistar rats provided evidence that the plasma levels of diabetes-related metabolites did not change acutely within 120 min, irrespective of increasing blood glucose levels.

The increased formation and accumulation of advanced glycation end products (AGEs), which are generated from the modification of proteins with high reactive carbonyl intermediates (α-oxoaldehydes) from glucose, i.e., methylglyoxal (MG) and glyoxal (GO), have been confirmed as a vital risk of developing hyperglycemia, Type 2 diabetes and diabetic complications.^1,2) AGE-free adducts released by proteolysis and/or their carbonyl intermediates in plasma were, thus, expected as competitive biomarkers to predict pre-diabetic and diabetic condition.³⁾ For the study on their plasma levels with diabetic disorders, liquid chromatography/tandem mass spectrometry (LC-MS/MS),⁴⁾ high performance liquid chromatography (HPLC)⁵⁾ and enzyme-linked immunosorbent assay^6,7) have been reported. In previous report, we also proposed an LC-MS/MS method in combination with 2,4,6-trinitrobenzene sulfonate (TNBS) derivatization technique for high-sensitive detection of plasma AGE-free adducts at >0.16 nM.⁸⁾

Although clinical studies revealed high plasma AGE-free adducts in diabetes,^9,10) the lack of evidence on metabolic behavior of the AGE adducts in animal models limits the progression of correlational studies. Besides, the proposed TNBS-MS method⁸⁾ would enable us to investigate their metabolic behavior in living animal models, because of low plasma sample volume (ca. 50 µL) from the tail vein. Thus, in this study we attempted to get an insight of production behavior of AGE-free adducts as well as their precursors (MG and GO) during an oral glucose tolerance test (OGTT) in Wistar rats. Five AGE-free adducts, i.e., glyoxal-derived hydroimidasolone (G-H1), MG-derived hydroimidazolone (MG-H1), N^ε-carboxymethyl-lysine (CML), N^ε-(1-carboxyethyl)-lysine (CEL) and argpyrimidine (AP), were targeted for the proposed TNBS-MS detection. Quantitative measurements of MG and GO were also performed by LC-MS/MS method with 2,3-diaminonaphthalene (DAN)-derivatization technique.¹¹⁾

MATERIALS AND METHODS

Animals

Five male Wistar rats (9 weeks old, 303.2±17.2 g) were purchased from CLEA Japan for the animal study. The rats were acclimatized under laboratory conditions (23±1°C, 55.5±5% humidity, 12 h dark/light cycle) for 1 week before experiments and given free access to a laboratory diet (MF diet; Oriental Yeast Co., Tokyo, Japan) and distilled water. All animal experiments were carried out under the Guidance for Animal Experiments in the Faculty of Agriculture in the Graduate Course of Kyushu University, and in accordance with Law (No. 105, 1973) and Notification (No. 6, 1980 of the Prime Minister’s Office) of the Japanese Government. All experiments were reviewed and approved by the Animal Care and Use Committee of Kyushu University (Permit Number: A24-051). Oral glucose tolerance test (OGTT experiment) was performed at the age of 10 weeks. The rats were fasted overnight (16 h, 6:00 pm to 10:00 am) prior to a single oral administration of 1 mL of 2 g/kg of glucose. At each time point (0, 30, 60, and 120 min) after the administration, an aliquot of blood was taken from the tail vein, being immediately subjected to blood glucose level (BGL) measurement with a disposable glucose sensor (Glutest Pro, Sanwa Chemical Research Co., Tokyo, Japan). Plasma was separated from the remaining blood samples by centrifugation (4°C, 3500×g, 15 min) and stocked at −30°C until LC-MS/MS analyses.

Determination of MG/GO and AGE-Free Adducts in Rat Plasma

Determination of AGE-free adducts by TNBS-LC-MS/MS method was performed according to our previous report.⁸⁾ LC separation was performed on a Waters 3 µm Atlantis T3 column (2.1 mm×100 mm) (Waters, Milford, MA, U.S.A.). A linear elution gradient of 20–100% methanol (MeOH) containing 0.1% formic acid (FA) over 20 min at 0.20 mL/min was performed at 40°C. Sample preparation was carried out following the procedure below: To 50 µL plasma, 10 µL of internal standard (IS) mixture (50 nM MG-H1-d₃ and 500 nM CEL-d₄, PolyPeptide Laboratories France SAS, Strasbourg, France) and 150 µL of 20% acetonitrile (CH₃CN) containing 0.1% FA was added, followed by centrifugation at 14000×g for 30 min at 4°C using a Millipore Amicon Ultra-0.5 centrifugal filter with a molecular weight cut of <3000 Da (Billerica, MA, U.S.A.). After evaporation to dryness, 50 µL of 150 mM TNBS solution (pH 10.0) was added to the sample, and incubated at 30°C for 30 min. After adding 50 µL of 0.2% FA, an aliquot (20 µL) of the solution was injected into an LC-MS/MS system (Agilent1200 HPLC (Agilent Technologies, Waldbronn, Germany) coupled to an Esquire6000 ESI-Ion Trap mass spectrometer (Bruker Daltonics, Bremen, Germany)). Transition conditions for MS/MS-multiple reaction monitoring (MRM) analysis in an ESI-positive ionization mode were the same as previous report.⁸⁾ Monoisotopic isolations (m/z) at the width of m/z 1.5 were 426.0>152.1, 440.2>184.1, 416.0>142.0, 430.0>156.1, 466.0>192.0, 443.1>187.1, and 434.0>160.0 for trinitrophenyl (TNP)-G-H1, TNP-MG-H1, TNP-CML, TNP-CEL, TNP-AP, TNP-MG-H1-d₃ and TNP-CEL-d₄, respectively.

Measurement of MG/GO by the LC-MS/MS was performed using the same rat plasma samples as aforementioned, independent on the plasma AGE assay. DAN derivatization technique was conducted for the measurement with a slight modification of the report by Yamada et al.¹⁰⁾ Briefly, to 30 µL of plasma, 10 µL of 500 nM 3,4-hexanedione as IS and 150 µL of 20% CH₃CN containing 0.1% FA was added, followed by centrifugation at 14000×g for 30 min at 4°C using the Millipore Amicon Ultra-0.5 centrifugal filter. Then, 10 µL of 30 mM DAN solution (in MeOH) was added to the obtained filtrate, and incubated at 30°C for 60 min. After evaporating to dryness, 100 µL of 0.1% FA was added to the dried sample, and an aliquot (20 µL) of the solution was injected into the LC-MS/MS at the same LC-MS conditions as AGE-free adduct assay. Monoisotopic isolations (m/z) at the width of m/z 1.5 were 195.0>167.9, 181.0>153.9, and 236.9>221.9 for DAN-MG, DAN-GO, DAN-hexanedione, respectively. Under the present DAN-derivatization LC-MS/MS-MRM conditions good linear calibration curves (r>0.996) were obtained in the range of 0.05–10 µM for both DAN-MG and DAN-GO (data not shown).

Statistical Analyses

Data are expressed as the mean±S.D. Statistical significance was analyzed using one-way ANOVA followed by Tukey’s post hoc test and a 0.05 level probability was used as the criterion for significance.

RESULTS AND DISCUSSION

Previous clinical studies provided evidence that the production of AGE-free adducts was closely associated with diabetic conditions, and the plasma levels of some AGEs (e.g., MG-H1 and CEL) in diabetic patients were much higher than those in normal subjects.^3,9) However, no systematic studies on the relationship between the production behavior of diabetes-related metabolites and the progression of diabetes or acute BGL rise, because of the lack of high-sensitive detection assays. In this study, thus, to get useful information on effect of acute BGL rise on the production of metabolites, 120 min-OGTT experiments in Wistar rats were performed by proposed high-sensitive MS assays that can detect analytes at ca. 50 µL of plasma sample in living animals.⁸⁾

DAN-MS¹⁰⁾ and TNBS-MS methods⁸⁾ were applied to evaluate the production of α-oxoaldehydes and AGE-free adducts for acute hyperglycemic experiment (OGTT) in Wistar rats. As shown in Fig. 1, both derivatization LC-MS/MS-MRM methods were allowed to make successive detections of MG and GO, and five AGE-free adducts in Wistar rat plasma. Analytes were quantified using IS-guided calibration curves. For MG and GO quantification, their peak area ratios of MG and GO against 3,4-hexanedione showed good linearity (r>0.996) as a function of concentration (MG in 0.05–10 µM; y=0.0582x+0.0403, GO in 0.05–10 µM; y=0.0565x+0.035); the limits of detection for MG and GO were estimated to be 0.17 µM and 0.07 µM, respectively. For AGE-free adducts quantification, their peak area ratios of MG-H1 and AP against MG-H1-d₃, and G-H1, CML and CEL against CEL-d₄ showed good linearity (r>0.997) as a function of concentration (MG-H1 in 5.0–100 nM; y=0.3109x−0.1198, AP in 0.1–50 nM; y=1.3138x+0.1053, G-H1 in 5.0–500 nM; y=0.0216x−0.2926, CML in 5.0–500 nM; y=0.0100x−0.0932, CEL in 5.0–500 nM; y=0.0076x−0.0542); the limits of detection for MG-H1, AP, G-H1, CML, and CEL were estimated to be 1.0 nM, 0.16 nM, 1.7 nM, 5.3 nM, and 2.7 nM, respectively. Where, y is the peak ratio of analyte against IS and x is a concentration.

Fig. 1. LC-MS Detections of MG, GO and AGE Free-Adducts in Wistar Rats

Plasma sample from fasted 10 week-Wistar rats was subjected to DAN- and TNBS-LC-MS/MS/-MRM analyses. Concentrations of 3,4-hexanedione, MG-H1-d₃ and CEL-d₄ as internal standards were 100, 10, and 100 nM, respectively. MRM analysis was performed at respective mass-transitions denoted in Materials and Methods. LC separations were performed on a Waters Atlantis T3 column (2.1 mm×100 mm, 3 µm) with 20–100% MeOH in 0.1% FA at a flow rate of 0.20 mL/min at 40°C.

According to the above-mentioned LC-MS/MS-MRM methods, we evaluated the metabolic behavior of each analyte in Wistar rat during OGTT protocols (2 g/kg glucose) at fixed intervals up to 120 min. As summarized in Table 1, fasting BGL of 10-week Wistar rats was 68.7±4.6 mg/dL. Maximal response to glucose challenge was at 30 min after gavage (122.3±7.4 mg/dL). Endogenous MG, GO, G-H1, MG-H1, CML, CEL, and AP at 0 min or fasting state were quantified to be 13.3×10³, 4.3×10³, 35.8, 16.7, 97.7, 380.3, and 9.6 nM, respectively. The levels of five AGE-free adducts in 10-week Wistar rats (Table 1 at 0 min) were comparative to those in 8-week Sprague Dawley rats,⁸⁾ indicating that the metabolic behavior of AGE-free adducts might be similar in normal and young rat species. The plasma MG level obtained in this study (13.3±2.3 µM) was in good agreement with the reported level by Wang et al.⁵⁾ (11.2±0.4 µM), suggesting the validity of the present DAN-MS method for α-oxoaldehydes assay.

Table 1. Plasma Levels of Methylglyoxal (MG), Glyoxal (GO), and AGE Free-Adducts in 10 Week-Old Wistar Rats during Oral Glucose Tolerance Test (2 g/kg)

Time after administration (min)	0	30	60	90	120
Blood glucose (mg/dL)	68.7±4.6a	122.3±7.4b	104.2±14.0b	92.2±10.4a	87.3±12.8a
MG (μM)	13.3±2.3	14.1±3.2	11.0±3.2	14.1±5.4	13.0± 4.0
GO (μM)	4.3±1.4	3.1 ±0.6	4.0±1.9	3.3±1.4	3.9±1.0
G-H1 (nM)	35.8±1.6	36.0±0.5	33.0±1.6	36.2±3.3	39.7±6.1
MG-H1 (nM)	16.7±6.8	16.6±4.5	17.5±3.4	17.0±4.8	19.6±5.9
CML (nM)	97.7±14.8	91.5±7.7	73.0±6.4	85.5±0.2	111.8±11.2
CEL (nM)	380.3±58.5	271.0±116.3	303.1±26.1	265.6±75.2	357.6±133.8
AP (nM)	9.6±6.0	8.5±3.6	6.0±3.8	6.0±2.9	6.9±4.8

Data are expressed as the mean±S.D. (n=3). Means without a common letter for blood glucose indicate statistical difference by Tukey–Kramer’s t-test for post-hoc analysis at p<0.05. MG, GO, and five AGE free-adducts did not show significant difference during the 120 min-OGTT by one-way ANOVA analysis.

The statistical analysis by Tukey’s t-test as summarized in Table 1 revealed that the levels of MG, GO and five AGE-free adducts did not show any significant changes during the 120 min-OGTT protocol, irrespective to significant increase in BGL. As Thornalley et al.¹¹⁾ reported, a long-term (or 3-week) period was required for the production of α-oxoaldehydes from lysyl residue and protein glycation by glucose in vitro. A gradual increase in α-oxoaldehydes with chronic progress of diabetes was also reported in diabetic human blood system.¹²⁾ Our study, on the other side, demonstrated that acute BGL rise in 120 min-OGTT protocol in young Wistar rats did not induce rapid and tentative increase in α-oxoaldehydes and AGE-free adducts in rat blood systems, indicating that acute increase in blood glucose may not cause serious production of diabetes-related metabolites due to their slow production speed in blood system.^11,12) Though data were not shown, no significant change in the metabolites for 10-week diabetic animal model, Goto-Kakizaki rats (e.g., MG-H1 at 0, 30, 60, 90, and 120 min were 13.2, 11.2, 13.3, 12.0, and 10.0 nM, respectively) also suggested that the production of diabetes-related metabolites was not affected by diabetic conditions as well as acute BGL rise.

In conclusion, we have demonstrated in this study that the rat plasma levels of metabolites related to diabetes such as α-oxoaldehydes and AGE-free adducts were not influenced by acute BGL rise in non-diabetic young Wistar rats. Further experiments are now in progress, regarding long-term protocols from pre- to diabetic stages of spontaneously hyperglycemic rats to clarify the diabetes-related metabolic biomarkers including AGEs or glycated proteins.

Acknowledgments

This study was in part supported by a Fukuoka Prefectural Support Fund for Creating New Products or New Technology (Development stage) to TM. SJ Chen thanks the support of China Scholarship Council (No. 201206350185).

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Fosmark DS, Torjesen PA, Kilhovd BK, Berg TJ, Sandvik L, Hanssen KF, Agardh CD, Agardh E. Increased serum levels of the specific advanced glycation end product methylglyoxal-derived hydroimidazolone are associated with retinopathy in patients with type 2 diabetes mellitus. Metabolism, 55, 232–236 (2006).
• 2) Rabbani N, Sebekova K, Sebekova K Jr, Heidland A, Thornalley PJ. Accumulation of free adduct glycation, oxidation, and nitration products follows acute loss of renal function. Kidney Int., 72, 1113–1121 (2007).
• 3) Ahmed N, Babaei-Jadidi R, Howell SK, Thornalley PJ, Beisswenger PJ. Glycated and oxidized protein degradation products are indicators of fasting and postprandial hyperglycemia in diabetes. Diabetes Care, 28, 2465–2471 (2005).
• 4) Thornalley PJ, Battah S, Ahmed N, Karachalias N, Agalou S, Babaei-Jadidi R, Dawnay A. Quantitative screening of advanced glycation endproducts in cellular and extracellular proteins by tandem mass spectrometry. Biochem. J., 375, 581–592 (2003).
• 5) Wang X, Desai K, Clausen JT, Wu L. Increased methylglyoxal and advanced glycation end products in kidney from spontaneously hypertensive rats. Kidney Int., 66, 2315–2321 (2004).
• 6) Boehm BO, Schilling S, Rosinger S, Lang GE, Lang GK, Kientsch-Engel P, Stahl P. Elevated serum levels of N^ε-carboxymethyl-lysine, an advanced glycation end product, are associated with proliferative diabetic retinopathy and macular oedema. Diabetologia, 47, 1376–1379 (2004).
• 7) Zhu W, Li W, Silverstein RL. Advanced glycation end products induce a prothrombotic phenotype in mice via interaction with platelet CD36. Blood, 119, 6136–6144 (2012).
• 8) Hashimoto C, Iwaihara Y, Chen SJ, Tanaka M, Watanabe T, Matsui T. Highly-sensitive detection of free advanced glycation end-products by liquid chromatography-electrospray ionization-tandem mass spectrometry with 2,4,6-trinitrobenzene sulfonate derivatization. Anal. Chem., 85, 4289–4295 (2013).
• 9) Tan KCB, Shiu SWM, Wong Y, Tam X. Serum advanced glycation end products (AGEs) are associated with insulin resistance. Diabetes Metab. Res. Rev., 27, 488–492 (2011).
• 10) Yamada H, Miyata S, Igaki N, Yatabe H, Miyauchi Y, Ohara T, Sakai M, Shoda H, Oimomi M, Kasuga M. Increase in 3-deoxyglucosone levels in diabetic rat plasma. Specific in vivo determination of intermediate in advanced Maillard reaction. J. Biol. Chem., 269, 20275–20280 (1994).
• 11) Thornalley PJ, Langborg A, Minhas HS. Formation of glyoxal, methylglyoxal and 3-deoxyglucosone in the glycation of proteins by glucose. Biochem. J., 344, 109–116 (1999).
• 12) Lu J, Randell E, Han YC, Adeli K, Krahn J, Meng QH. Increased plasma methylglyoxal level, inflammation, and vascular endothelial dysfunction in diabetic nephropathy. Clin. Biochem., 44, 307–311 (2011).