Chemistry
by Zeenat Ara Lila, Louis D. Whitesides, Chukudi Weze and Yazmine Thomas
Glycoxidative Modification of Adenosine in Relation to Diabetes and Obesity: Detection of Carboxymethyl-Adenosine in Biological Samples
roteins are modified in residues of lysine, arginine, histidine, etc., due to high concentrations of sugar and fatty acids in the blood- stream in relation to diabetes. This modification occurs because of the presence of reactive –NH2
P tein molecule. The carboxymethyllysine produced,1
groups in the above amino acids of a pro- in the case of lysine
modification, has been used as a marker of diabetic intensity. Ongoing research seeks to understand the complications in diabetes that result from glycoxidative and lipoperoxidation modifications in protein.2,3
Few studies have focused on similar modification of DNA/RNA bases under diabetic conditions. Structurally, DNA/RNA molecules contain –NH2
groups like lysine, and histidine amino acids, which are present in
protein molecules. Reactions involving adenosine present in RNA may also alter RNA functions as a possible consequence of higher levels of sugars in diabetes.
The purpose of this study was to investigate the glycoxidative modi- fication of adenosine with reactive carbohydrates and detection of carboxymethyl-adenosine (CMAd) from the urine samples of obese dia- betic and nondiabetic subjects.
Experimental
Study population The study comprised 48 male and female participants over the age of 18; of these, 20 were healthy (control group), 14 were nondiabetic obese and 14 had type 2 diabetes. Individuals with no background of diabetes, cardiac, respiratory or renal disease and with normal fasting plasma glucose were included in the control group. All participants were recruited through the Regional Medical Center in Orangeburg, S.C. and submitted an informed consent form. The study protocol was approved by the Institutional Review Board of South Carolina State University and the Regional Medical Center.
Clinical and biochemical measurements Body weight and height were measured to the nearest 0.1 kg and 0.1 cm, respectively, with the subjects wearing light clothes and shoeless. Body mass index (BMI), calculated as the weight in kilograms divided by the square of the height in meters, was used as an index of obesity.
Fasting blood and urine samples were collected from the subjects. Blood samples were obtained for the measurements of glucose and glycated hemoglobinA1c (HbA1c). Blood glucose levels (BGL) and HbA1c were
measured at the outpatient laboratory; BGLs were assessed using fast- ing blood samples obtained through venipuncture. HbA1c levels were measured turbidimetrically. Fasting urine samples were obtained from volunteer donors and frozen at –20 °C until use.
Chemicals and methods All chemicals were obtained from Fisher Scientific (Pittsburgh, Penn.) unless otherwise indicated.
1) Incubations of adenosine (A) with D-glucose and D-ribose: A mixture of adenosine (2.67 mg; 0.01 mmol) and D-glucose (1.8 mg; 0.01 mmol) was incubated in 0.2 M phosphate buffer (1 mL, pH 7.4) (with two drops of toluene added to prevent fungal growth) at 37 °C for 30 days. The reaction mixture was kept constant at pH 7.4 during the incubation period by ad- justing the pH as required; 100-μL samples were collected at 2, 4, 6, 8, 10, 20 and 30 days during the incubation period and stored in the refrigerator before analysis. For analysis, a typical sample was adjusted to pH 7.0 and dried (<60 °C) in a rotary evaporator. Methanol (3 mL) was added to the dried residue; the mixture was sonicated, vortexed and centrifuged; and the methanol fraction was collected. The sample was then subjected to HPLC and LC/MS-ESI (electrospray) (Figure 1a).
In a separate experiment, similar incubations of adenosine (2.67 mg; 0.01 mmol) with D-ribose (1.5 mg; 0.01 mmol) were carried out in 0.2 M Pi (phosphate buffer) (1 mL), pH 7.4, at 37 °C for 30 days. The methanol extracts were subjected to HPLC and LC/MS (Figure 1b).
2) Synthesis of CMAd using adenosine (A) and chloroacetic acid: A mix- ture of adenosine (2.67 mg; 0.01 mmol) and chloroacetic acid (0.9 mg; 0.01 mmol) in 0.2 M Pi (1 mL), pH 7.4, at 37 °C, was incubated for 7 days. The pH was adjusted to 7.0, the mixture was dried and the methanol extracts were subjected to HPLC and LC/MS (Figure 1c) as in experiment 1.
3) Identification of CMAd in urine sample: In a typical experiment, 1 mL of fasting urine sample was adjusted to pH 7.0, evaporated in a rotary evaporator and extracted three times with methanol. The residue was analyzed by HPLC and LC/MS (Figures 2b and 3b) as in experiment 1.
Instrumentation
HPLC analysis An HPLC system from Shimadzu Scientific Instruments (Columbia, Md.) included the LC-20AT low-pressure gradient pump with
AMERICAN LABORATORY • 34 • AUGUST 2015
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