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Correlations of six related pyrimidine metabolites and diabetic retinopathy in Chinese type 2 diabetic patients
Clinica Chimica Acta 412 (2011) 940–945
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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Correlations of six related pyrimidine metabolites and diabetic retinopathy in Chinese type 2 diabetic patients Jian-Fei Xia a, Zong-hua Wang a,⁎, Qiong-Lin Liang b, Yi-Ming Wang b, Ping Li c, Guo-An Luo b,⁎⁎ a b c
College of Chemistry, Chemical Engineering and Environment, Qingdao University, China Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, China Clinical Medicinal Research Institute, Sino-Japanese Friendship Hospital, Beijing, China
a r t i c l e
i n f o
Article history: Received 2 November 2010 Received in revised form 18 January 2011 Accepted 20 January 2011 Available online 26 January 2011 Keywords: Type 2 diabetes Diabetic retinopathy Biomarker Cytosine Cytidine Thymidine
a b s t r a c t Background: Diabetic retinopathy (DR), a kind of diabetic microvascular complication, is the leading cause of visual impairment in adults aged 30 to 65 years. Despite rapid research progress, robust predictors to assess prospectively with high precision the risk for DR in individuals with diabetes are still lacking. We investigated the relationship between pyrimidine metabolites and disease, and find out the potential biomarkers for diagnosis. Methods: The study group consisted of 116 subjects who were divided to 3 groups: control (n = 41), type 2 diabetes without retinopathy (DM, n = 37), and with retinopathy (DR, n = 38). Biochemical and clinical parameters, concentrations of related metabolites, including of cytosine, cytidine, uridine, thymine, thymidine and 2′-deoxyuridine were measured in plasma of all participants. Results: There was a significant increase of concentrations of cytosine (p = 0.010), cytidine (p b 0.001) and thynidine (p b 0.001) with DR compared to DM. The concentration of uridine, thymine and 2′-deoxyuridine did not change. Conclusions: The concentrations of cytosine, cytidine and thynidine may be useful for monitoring the progression of DR and evaluating the treatment. And cytidine has good sensitivity and specificity for diagnosis. © 2011 Elsevier B.V. All rights reserved.
1. Introduction In the recent years, a dramatic increase in the number of people with type 2 diabetes mellitus (T2DM) is being observed. In 2002, it was estimated that 150 million people in the world suffered from diabetes. This number is expected to increase to 300 million by the year 2025; most of these cases will be T2DM [1]. The growing epidemic of diabetes will ultimately affect more people than any other disease. The most important problem in diabetic people is the development of the vascular complications related to microangiopathy and macroangiopathy. Diabetic retinopathy (DR), a serious microvascular complication, is the leading cause of visual impairment in adults aged 30 to 65 years, representing a major public health concern. Almost all patients develop background retinopathy with time, and 40–50% progress to proliferative retinopathy within 25 years of diabetes onset [2]. In addition, the burden of macrovascular morbidity and mortality is also increased in patients suffering from microvascular disease [3– 5]. Thus, DR is a major burden for diabetic patients. Despite intensive ⁎ Corresponding author at: Department of Chemistry, Qingdao University, Qingdao 266071, Shandong, China. Tel./fax: +86 532 85950873. ⁎⁎ Corresponding author. E-mail address: [email protected] (Z. Wang). 0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.01.025
research carried out by numerous groups, the diagnosis of retinopathy still depended on ophthalmoscopy and fluorescein angiography. However, it is generally acknowledged that pathological changes which can be discovered occur at the severe stages of the retinopathy. Therefore, it is of tremendous importance to find out the markers that can be used to predict the retinopathy. It is well established that pyrimidine metabolic pathway may be associated with the development of diabetic microvascular complication. Pyrimidines are the building blocks of DNA and RNA and the basic elements of the cell programming machinery. As the vital components of all living cells, they fulfill a wide range of diverse biological functions, especially the regulation of cell metabolism, energy conservation and transport, formation of coenzymes and of active intermediates of phospholipids and carbohydrate metabolism. Among related metabolites, cytidine is a pyrimidine that besides being incorporated into nucleic acids, can serve as a substrate for the salvage pathway of pyrimidine nucleotide synthesis and as a precursor of the cytidine triphosphate (CTP) needed in the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) biosynthetic pathway. And we have reported that the metabolism of phospholipids is related to DM and its complication in our previous work [6]. Cytidine is partly produced from cytosine and is converted to uridine by cytidine deaminase. The uridine is phosphorylated into uracil,
J.-F. Xia et al. / Clinica Chimica Acta 412 (2011) 940–945
which can be interconverted to each other with 2′-deoxyuridine. Then 2′-deoxyuridine was converted to dUMP, followed by dTMP and thymidine, which are converted to thymine with thymidine phosphorylase. And some researchers have shown that the expression of thymidine phosphorylase was lost or considerably reduced when the organism suffered from microvascular disease. Furthermore, the high concentration of thymidine is a cause of DNA impairment, which is related to diabetes and diabetic retinopathy [7,8]. Metabolic pathway contains these related metabolites and enzymes are depicted in Fig. 1.
2. Materials and methods 2.1. Subjects Plasma samples of 75 patients (41 males and 34 females, age 57.7±8.6 years) in Beijing, China, were collected as cases, and 41 plasma samples of healthy people (23 males and 18 females, age 56.1±7.2 years) in the same area were collected as controls. All study participants had given their informed consent. T2DM was defined in accordance with the criteria of the American Diabetes Association. Details such as age, sex, and, in diabetes subjects, duration of diabetes, and other details of diabetic therapy were recorded; a complete clinical examination was conducted for all subjects. The diabetic retinopathy was determined by ophthalmoscopy and fluorescein angiography through dilated pupils by a retinal specialist prior to the examination of the blood samples, and the patients were classified according to the presence or absence of diabetic retinopathy, regardless of its degrees of severity.
2.2. Biochemical and clinical parameter analysis The biochemical and clinical parameters of 75 patients were reviewed and approved by the Clinical Medicinal Research Institute, Sino-Japanese Friendship Hospital, Beijing, P.R. China. The diabetic duration was defined as the duration from the first diagnosis of type 2 DM to the time of blood sampling. Exclusion criteria included acute myocardial infarction, pregnancy, liver disease, stroke, current use of cholesterol lowering agents, and uncertained diabetic duration. BP was taken in the seated position using standardized sphygmomanometers. A written informed consent has been obtained from all patients and healthy controls. The clinical and biochemical analyses of all the cases were carried out in the years 2007 and 2008. Hemoglobin A1c (HbA1c), fasting glucose, urea nitrogen and lipid concentrations were measured using standard automated assays.
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2.3. Blood collection and preparation Blood samples were collected from 6 to 9 o'clock after an overnight fast. All blood samples were collected into EDTA, centrifuged to obtain plasma in the hospital within 30 min and sent to our laboratory, where they were stored at −80 °C before analysis. All measurements were done on the same sample of blood. Informed consent was obtained from all study subjects, and the institutional ethics committee approved the study. Before analysis, 800 μl of methanol was added to 200 μl aliquots of plasma, vortexed for 2 min, and then centrifuged at 10,000 rpm for 15 min at 4 °C. The clear supernatant was transferred to a 1.5 ml polypropylene tube and dried under a gentle stream of nitrogen at room temperature. The residue was reconstituted with 100 μl of a mixture of methanol–water (1:1, by volume) and stored at 4 °C before analysis.
2.4. Detection of related metabolites in plasma Plasma concentrations of cytosine, cytidine, uridine, thymine, thymidine and 2′-deoxyuridine were simultaneously measured by high-performance liquid chromatography coupled to ultraviolet and tandem mass spectrometry method (HPLC–UV/MS/MS), using an Applied Biosystems Sciex (Toronto, Canada) API 3000 triple quadrupole tandem mass spectrometer, equipped with a Turbo Ionspray interface and an Agilent 1100 binary HPLC system. Among the 6 metabolites, cytidine, thymine, thymidine and 2′-deoxyuridine were detected using UV detector for their good separation and others were detected using MS detector. Samples were separated on an Agilent TC-C18 column (250 mm× 4.6 mm I.D., 5 μm particle size, Agilent) with an Alltech guard column (7.5 mm× 4.6 mm I.D., 5 μm particle size). A mobile phase was used with a flow rate of 0.8 ml/min in which mobile phase A consisted of 10 mmol/l ammonium acetate in Ultra pure water adjusted to pH 5.8 with glacial acetic acid and mobile phase B consisted of 100% methanol. A mobile phase gradient was used starting at 100% A for 5 min, followed by a linear gradient from 100 to 95% A in 5 min, followed by a linear gradient from 95 to 80% A in 10 min, followed by a linear gradient from 80 to 70% A in 20 min, and followed by a linear gradient from 40 to 0% A in 5 min. The flow was reduced to 150 μl/min prior to MS detection using a T-split. The column temperature was maintained at 25 °C, the UV detector was set at 254 nm and the injection volume was 20 μl. The temperature of the turbo ion electrospray was set at 350 °C. The collision gas (nitrogen) was set at 6 mTorr, and nebulizer gas (nitrogen), curtain gas (nitrogen), and assistant drying gas (air) were used at flow rates of 8 l/min, 2 l/min, and 4 l/min, respectively. The ion spray voltage was 5000 V. The method has been validated according to
Fig. 1. The related pyrimidine metabolic pathway. Major components including cytosine, cytidine, uridine, thymine, thymidine and 2′-deoxyuridine were measured in this study.
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the requirement of analytical chemistry [9,10]. Calibration curves suitable for the analysis of plasma were linear (r2 N 0.998) with limits of detection (LOD) from 10 to 80 ng/ml. The recoveries for every metabolites are from 78.9% to 102.9% with CVs of b10%. Intraday and interday CVs were both b10%. Fig. 2 shows the typical UV chromatogram (a) and multiple extracted ion chromatogram (b) of a spiked plasma sample.
Whitney U tests. The Spearman or the Pearson correlations were examined. In this study, a value of p b 0.05 was considered statistically significant. The metabolites, the concentrations of which have statistically significant differences between two groups of the three groups (control, DM, and DR), were defined as potential biomarkers. Receiver operating characteristics (ROC) curve was performed. 3. Results
2.5. Statistical analysis 3.1. Clinical characteristics of type 2 DM The results were analyzed using SPSS statistical package (ver 14.0; SPSS Inc., Chicago, IL). Some of the clinical characteristics of DM and DR subjects were compared with the χ2 test and Fisher's exact test. To achieve normality, concentration data were log-transformed before analysis. Normality of the transformed data was verified by using the Kolmogorov–Smirnov test. The differences between the groups were calculated with Student's t-test or the non-parametric Mann–
Table 1 shows the clinical characteristics of the study groups. As shown, DM patients with retinopathy (DR patients) had significantly (p b 0.05) longer duration of the disease compared to DM patients with retinopathy (DM patients), (13.97 ± 2.40 vs. 10.64 ± 1.66). For blood pressure, systolic blood pressure (SBP) was significantly (p b 0.05) increased in DR patients compared with DM patients
Fig. 2. In a single run, UV chromatogram and multiple extracted ion chromatograms of a spiked plasma sample. (a) UV chromatogram showing 4 compounds; and (b) multiple extracted ion chromatograms of other 2 compounds.
J.-F. Xia et al. / Clinica Chimica Acta 412 (2011) 940–945 Table 1 Clinical parameters of diabetic patients with (DM) and without retinopathy (DR). Parameters
DM
DR
p
N (male/female) Age (years) Duration of DM (years) BMI (kg/m2) HbA1c (%) Fasting blood glucose (mmol/l) Triglycerides (mmol/l) Total cholesterol (mmol/l) HDL-cholesterol (mmol/l) LDL-cholesterol (mmol/l) SBP (mm Hg) DBP (mm Hg) Urea nitrogen (mmol/l)
37(21/16) 56.56 ± 7.27 10.64 ± 1.66 25.04 ± 2.85 8.09 ± 1.84 8.02 ± 2.95 2.39 ± 1.08 5.31 ± 0.56 1.36 ± 0.10 2.96 ± 0.30 132.96 ± 4.36 77.04 ± 2.83 8.11 ± 1.63
38(20/18) 57.69 ± 5.83 13.97 ± 2.40 25.20 ± 4.05 8.86 ± 1.97 7.69 ± 2.23 2.06 ± 1.37 5.13 ± 1.08 1.22 ± 0.14 2.98 ± 0.31 142.21 ± 6.75 78.86 ± 3.32 14.86 ± 3.42
NS NS 0.033 NS NS NS NS NS NS NS 0.012 NS b 0.001
(142.21 ± 6.75 vs. 132.96 ± 4.36) whereas diastolic blood pressure (DBP) was not changed. In addition, urea nitrogen was significantly (p b 0.001) increased in DR patients compared with DM patients (14.86 ± 3.42 vs. 8.11 ± 1.63). Fasting blood glucose and HbA1c were not changed between DM patients and DR patients. Lipid profiles such as total cholesterol, triglyceride, HDL-cholesterol and LDL-cholesterol of DM patients were not significantly changed compared to DR patients. 3.2. The concentrations of related pyrimidine metabolites in plasma The concentrations of cytosine and cytidine in the group of DR were significantly higher as compared with DM (p = 0.010 and p b 0.001) and control groups (p b 0.001 and p b 0.001). Additionally, also, the group of DM had significantly higher concentration of cytosine as compared to the healthy controls (p b 0.001). For concentrations of uridine and 2′-deoxyuridine, no statistically significant differences between the groups were observed (Table 2). The concentrations of thymidine in the group of DR were significantly higher as compared with DM (p b 0.001) and control groups (p b 0.001). But no statistically significant differences of concentration of thymine between the group of control and DM were observed. The concentrations of the metabolites with significant changes were shown in Fig. 3. 3.3. Relationship between correlative metabolites and other parameters Table 3 shows the relationship between concentrations of cytosine, cytidine, uridine, thymine, thymidine, 2′-deoxyuridine and clinical parameters including age, BMI, SBP, HbA1c, and various other variables in all patients of both the DM and DR groups. Cytidine correlated positively with SBP and urea nitrogen. Especially, the Pearson correlation coefficient between cytidine and urea nitrogen was above 0.5 (r = 0.617). In addition, thymidine correlated positively with urea nitrogen (r = 0.456).
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elevation of urea nitrogen. Epidemiologic studies have shown that DR is strongly clustered in families and that race has a major effect on DR susceptibility and rate of progression, firmly establishing the importance of genetic risk factors in the development of DR [11]. Despite rapid research progress, robust predictors to assess prospectively with high precision the risk for DR in individuals with diabetes are still lacking. Thus, it is necessary to set out the study of biomarker discovery, especially for the low-molecular weight metabolites, which are important and easy to be measured. In the present study, three potential biomarkers (cytosine, cytidine and thymidine) of retinopathy were showed. For the groups DM and DR, the ROC curve of these three markers was calculated (Fig. 4). As results, the ROC curve of cytidine showed better than other potential markers. The area under curve (AUC) of cytidine was 0.849 ± 0.048, and significantly higher than that of the null hypothesis (true area was 0.5, p b 0.01). This means that the plasma cytidine concentration could serve as a potential diagnostic marker for DM to DR. To find an optimal cutoff point to dichotomize ‘diseased’ or ‘healthy’, Youden's index was used in this study. According to Youden's index, the optimal operating point of cytidine was 0.076 mg/l. At this cutoff point, the sensitivity was 73.7% and specificity was 91.9%. In this study, the concentration of cytidine in the group of DR was significantly higher as compared with DM (p b 0.001) and control (p b 0.001). In addition, cytidine correlated positively with urea nitrogen and SBP. And we suppose that it may be a potential biomarker for the diagnosis of diabetic retinopathy and evaluation of treatment. Cytidine, a nucleoside that is composed of the base cytosine linked to the fivecarbon sugar D-ribose, is considered as the precursor of the cytidine triphosphate (CTP), needed in the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) biosynthetic pathway. On the one hand, the changes of cytidine concentration induce the changes of CTP, which influence the metabolism of phospholipids. On the other hand, as well as cytidine, cytosine can serve as the substrate for the salvage pathway of pyrimidine nucleotide. The significant changes of cytosine and cytidine induce the abnormality of the salvage pathway of pyrimidine nucleotide, followed by the dysfunction of phospholipid metabolism. Phospholipids are the main constituents of biological membranes and have important structural and functional properties. The changes of composition and concentration can affect the function of membrane and induce related disease, including diabetes and its complications [14–16]. In our previous work, we found out that the concentrations of phospholipids decreased with the development of
Table 2 Related metabolites in diabetic patients with retinopathy (DR), without retinopathy (DM), and control groups. Compounds
DM (n = 37)
DR (n = 38)
Cytosine (mg/l)
0.11 ± 0.01
0.21 ± 0.03
0.28 ± 0.04
4. Discussion
Cytidine (mg/l)
0.044 ± 0.01
0.052 ± 0.01
0.21 ± 0.06
To better understand the role played by specific components leading to DM and DR, their quantification in the current study is dependent on an integrated technical platform composed of hyphenated LC–UV/MS/MS analysis, conventional clinical assays, and classical statistical analysis. Compared with routine biochemical approaches, the highly selective and sensitive LC–UV/MS/MS method permits simultaneous determination of six relevant components. Current evidence suggests that both genetic and environmental factors determine susceptibility to develop DR [11,12]. Hypertension, poor glycemic and lipid control and smoking increase the risk for developing diabetic microangiopathy [13]. In this work, we showed that the patients with DR had higher SBP than those without DR. And DR patients may be associated with renal impairment, which results in the
Uridine (mg/l)
1.28 ± 0.10
1.31 ± 0.10
1.23 ± 0.14
Thymine (mg/l)
0.037 ± 0.007
0.033 ± 0.004
0.045 ± 0.010
2′-Deoxyuridine (mg/l)
0.17 ± 0.04
0.14 ± 0.02
0.14 ± 0.03
0.033 ± 0.003
0.046 ± 0.008
0.21 ± 0.07
Thymidine (mg/l)
a b c
Control (n = 41)
p value from t-test between control and DM. p value from t-test between control and DR. p value from t-test between DM and DR.
p b 0.001a b 0.001b = 0.010c NSa b 0.001b b 0.001c NSa NSb NSc NSa NSb NSc NSa NSb NSc NSa b 0.001b b 0.001c
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Fig. 3. Comparison of plasma potential risk factor concentrations among the groups of control (Con), DM and DR. DR subjects had significantly higher mean plasma concentrations of cytosine (p b 0.001 and p = 0.010), cytidine (p b 0.001 and p b 0.001) and thymidine (p b 0.001 and p b 0.001) compared to the groups of Con and DM. The group of DM had significantly higher mean plasma concentrations of cytosine (p b 0.001) compared to the group of Con.
diabetic microvascular complications [6]. Additionally, cytidine can be hydrolytic deaminated to uridine with cytidine deaminase (CDA) [17]. Because the concentration of uridine didn't change significantly when the diabetic retinopathy happened, the increase of cytidine may be due to the decrease of CDA activity, which is related to many diseases, such as abnormal pregnancy. Therefore, the research on the activity of CDA may be useful in understanding the pathogenesis of diabetic retinopathy. Thymidine may be another potential biomarker for diagnosis of retinopathy and evaluation of treatment. In our study, the concentration of thymidine in the group of DR was significantly higher as compared with DM (p b 0.001) and control (p b 0.001). And thymidine correlated positively with urea nitrogen. Thymidine, the DNA base T, is a naturally occurring compound that exists in all living organisms and DNA viruses. It is reported that persistently elevated concentrations of blood thymidine may unbalance nucleotide pools in mitochondria, leading to DNA impairment [8]. For the diabetic patients, the DNA impairments in endothelial cell may lead to vascular dysfunction and induce vascular complications. Thereby, one possible clue for the prevention and therapy retinopathy will be to reduce or, if possible, to virtually eliminate plasma thymidine. In addition, it is reported that at diabetic state, 5′-nucleotidase, the enzyme catalyzing dTMP to thymidine in pyrimidine metabolic pathway, was markedly decreased [18]. At the same time, thymidine kinase, the enzyme catalyzing thymidine to dTMP, was markedly elevated [19]. So we hypothesized that the elevated concentration of thymidine resulted from the decreased activity of thymidine phosphorylase (TP), which catalyzes the dephosphorylation of
thymidine to thymine and 2-deoxy-D-ribose-1-phosphate, a process that helps to maintain the nucleotide pool. In the present paper, we calculated the ratio of thymine and thymidine (thymine:thymidine), which can reflect the activity of TP. We found that there was a significant decrease of this ratio with the DR group compared to the control group (p b 0.01). Sengupta et al. [20] reported that TP, similar to vascular endothelial growth factor (VEGF), might be related to angiogenesis, which increases vascular permeability and promotes angiogenesis in response to ischemia or hypoxia, resulting in visual impairment. Sivridis et al. [21] have reported that TP expression was lost or considerably reduced from tubular cells of idiopathic membranous nephropathy (IMN), a principal disease of microvascular. So the changes of TP activity may be an important factor in understanding the pathogenesis of retinopathy. Acknowledgements The authors acknowledge the support of the Sino-Japanese Friendship Hospital (Beijing, China) who offered samples and clinical and biochemistry parameters. The studies have been supported by a grant from the National Basic Research Development Program of China (973 Program, No. 2005CB523503).
Table 3 Correlation between metabolites and clinical parameters.
Age Duration BMI Fasting blood glucose HbA1c Urea nitrogen Cholesterol Triglyceride HDL LDL SBP DBP
Cytosine
Cytidine
Uridine
Thymine Deoxyuridine Thymidine
− 0.189 0.108 0.067 0.073
0.107 0.111 0.171 − 0.129⁎
0.007 − 0.230⁎ − 0.130 0.097
0.177 0.010 0.102 − 0.118 0.018 − 0.072 − 0.006 0.037
0.024 0.076 − 0.151 − 0.096
− 0.072 0.030 − 0.028 0.072 − 0.054 − 0.058 − 0.069 − 0.026
0.102 0.117 0.223 0.296⁎ − 0.100 − 0.056 − 0.167 − 0.119 0.008 0.097 − 0.048 0.012 0.149 0.042 0.060 − 0.040
− 0.069 0.456⁎⁎
− 0.099 − 0.173 0.235⁎ 0.617⁎⁎ − 0.016 − 0.036 0.023 − 0.138 − 0.032 0.125 − 0.031⁎ − 0.059 0.201 0.397⁎⁎ 0.119 0.032
⁎ Correlation is significant at the 0.05 level (2-tailed). ⁎⁎ Correlation is significant at the 0.01 level (2-tailed).
0.023 − 0.006 − 0.082 0.060 0.167 − 0.049
Fig. 4. ROC curves of cytosine, cytidine and thymidine in groups of DM and DR. Cytidine shows more sensitivity and specificity for diabetic retinopathy diagnosis than other potential biomarkers.
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Contents lists available at ScienceDirect
Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Correlations of six related pyrimidine metabolites and diabetic retinopathy in Chinese type 2 diabetic patients Jian-Fei Xia a, Zong-hua Wang a,⁎, Qiong-Lin Liang b, Yi-Ming Wang b, Ping Li c, Guo-An Luo b,⁎⁎ a b c
College of Chemistry, Chemical Engineering and Environment, Qingdao University, China Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, China Clinical Medicinal Research Institute, Sino-Japanese Friendship Hospital, Beijing, China
a r t i c l e
i n f o
Article history: Received 2 November 2010 Received in revised form 18 January 2011 Accepted 20 January 2011 Available online 26 January 2011 Keywords: Type 2 diabetes Diabetic retinopathy Biomarker Cytosine Cytidine Thymidine
a b s t r a c t Background: Diabetic retinopathy (DR), a kind of diabetic microvascular complication, is the leading cause of visual impairment in adults aged 30 to 65 years. Despite rapid research progress, robust predictors to assess prospectively with high precision the risk for DR in individuals with diabetes are still lacking. We investigated the relationship between pyrimidine metabolites and disease, and find out the potential biomarkers for diagnosis. Methods: The study group consisted of 116 subjects who were divided to 3 groups: control (n = 41), type 2 diabetes without retinopathy (DM, n = 37), and with retinopathy (DR, n = 38). Biochemical and clinical parameters, concentrations of related metabolites, including of cytosine, cytidine, uridine, thymine, thymidine and 2′-deoxyuridine were measured in plasma of all participants. Results: There was a significant increase of concentrations of cytosine (p = 0.010), cytidine (p b 0.001) and thynidine (p b 0.001) with DR compared to DM. The concentration of uridine, thymine and 2′-deoxyuridine did not change. Conclusions: The concentrations of cytosine, cytidine and thynidine may be useful for monitoring the progression of DR and evaluating the treatment. And cytidine has good sensitivity and specificity for diagnosis. © 2011 Elsevier B.V. All rights reserved.
1. Introduction In the recent years, a dramatic increase in the number of people with type 2 diabetes mellitus (T2DM) is being observed. In 2002, it was estimated that 150 million people in the world suffered from diabetes. This number is expected to increase to 300 million by the year 2025; most of these cases will be T2DM [1]. The growing epidemic of diabetes will ultimately affect more people than any other disease. The most important problem in diabetic people is the development of the vascular complications related to microangiopathy and macroangiopathy. Diabetic retinopathy (DR), a serious microvascular complication, is the leading cause of visual impairment in adults aged 30 to 65 years, representing a major public health concern. Almost all patients develop background retinopathy with time, and 40–50% progress to proliferative retinopathy within 25 years of diabetes onset [2]. In addition, the burden of macrovascular morbidity and mortality is also increased in patients suffering from microvascular disease [3– 5]. Thus, DR is a major burden for diabetic patients. Despite intensive ⁎ Corresponding author at: Department of Chemistry, Qingdao University, Qingdao 266071, Shandong, China. Tel./fax: +86 532 85950873. ⁎⁎ Corresponding author. E-mail address: [email protected] (Z. Wang). 0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.01.025
research carried out by numerous groups, the diagnosis of retinopathy still depended on ophthalmoscopy and fluorescein angiography. However, it is generally acknowledged that pathological changes which can be discovered occur at the severe stages of the retinopathy. Therefore, it is of tremendous importance to find out the markers that can be used to predict the retinopathy. It is well established that pyrimidine metabolic pathway may be associated with the development of diabetic microvascular complication. Pyrimidines are the building blocks of DNA and RNA and the basic elements of the cell programming machinery. As the vital components of all living cells, they fulfill a wide range of diverse biological functions, especially the regulation of cell metabolism, energy conservation and transport, formation of coenzymes and of active intermediates of phospholipids and carbohydrate metabolism. Among related metabolites, cytidine is a pyrimidine that besides being incorporated into nucleic acids, can serve as a substrate for the salvage pathway of pyrimidine nucleotide synthesis and as a precursor of the cytidine triphosphate (CTP) needed in the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) biosynthetic pathway. And we have reported that the metabolism of phospholipids is related to DM and its complication in our previous work [6]. Cytidine is partly produced from cytosine and is converted to uridine by cytidine deaminase. The uridine is phosphorylated into uracil,
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which can be interconverted to each other with 2′-deoxyuridine. Then 2′-deoxyuridine was converted to dUMP, followed by dTMP and thymidine, which are converted to thymine with thymidine phosphorylase. And some researchers have shown that the expression of thymidine phosphorylase was lost or considerably reduced when the organism suffered from microvascular disease. Furthermore, the high concentration of thymidine is a cause of DNA impairment, which is related to diabetes and diabetic retinopathy [7,8]. Metabolic pathway contains these related metabolites and enzymes are depicted in Fig. 1.
2. Materials and methods 2.1. Subjects Plasma samples of 75 patients (41 males and 34 females, age 57.7±8.6 years) in Beijing, China, were collected as cases, and 41 plasma samples of healthy people (23 males and 18 females, age 56.1±7.2 years) in the same area were collected as controls. All study participants had given their informed consent. T2DM was defined in accordance with the criteria of the American Diabetes Association. Details such as age, sex, and, in diabetes subjects, duration of diabetes, and other details of diabetic therapy were recorded; a complete clinical examination was conducted for all subjects. The diabetic retinopathy was determined by ophthalmoscopy and fluorescein angiography through dilated pupils by a retinal specialist prior to the examination of the blood samples, and the patients were classified according to the presence or absence of diabetic retinopathy, regardless of its degrees of severity.
2.2. Biochemical and clinical parameter analysis The biochemical and clinical parameters of 75 patients were reviewed and approved by the Clinical Medicinal Research Institute, Sino-Japanese Friendship Hospital, Beijing, P.R. China. The diabetic duration was defined as the duration from the first diagnosis of type 2 DM to the time of blood sampling. Exclusion criteria included acute myocardial infarction, pregnancy, liver disease, stroke, current use of cholesterol lowering agents, and uncertained diabetic duration. BP was taken in the seated position using standardized sphygmomanometers. A written informed consent has been obtained from all patients and healthy controls. The clinical and biochemical analyses of all the cases were carried out in the years 2007 and 2008. Hemoglobin A1c (HbA1c), fasting glucose, urea nitrogen and lipid concentrations were measured using standard automated assays.
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2.3. Blood collection and preparation Blood samples were collected from 6 to 9 o'clock after an overnight fast. All blood samples were collected into EDTA, centrifuged to obtain plasma in the hospital within 30 min and sent to our laboratory, where they were stored at −80 °C before analysis. All measurements were done on the same sample of blood. Informed consent was obtained from all study subjects, and the institutional ethics committee approved the study. Before analysis, 800 μl of methanol was added to 200 μl aliquots of plasma, vortexed for 2 min, and then centrifuged at 10,000 rpm for 15 min at 4 °C. The clear supernatant was transferred to a 1.5 ml polypropylene tube and dried under a gentle stream of nitrogen at room temperature. The residue was reconstituted with 100 μl of a mixture of methanol–water (1:1, by volume) and stored at 4 °C before analysis.
2.4. Detection of related metabolites in plasma Plasma concentrations of cytosine, cytidine, uridine, thymine, thymidine and 2′-deoxyuridine were simultaneously measured by high-performance liquid chromatography coupled to ultraviolet and tandem mass spectrometry method (HPLC–UV/MS/MS), using an Applied Biosystems Sciex (Toronto, Canada) API 3000 triple quadrupole tandem mass spectrometer, equipped with a Turbo Ionspray interface and an Agilent 1100 binary HPLC system. Among the 6 metabolites, cytidine, thymine, thymidine and 2′-deoxyuridine were detected using UV detector for their good separation and others were detected using MS detector. Samples were separated on an Agilent TC-C18 column (250 mm× 4.6 mm I.D., 5 μm particle size, Agilent) with an Alltech guard column (7.5 mm× 4.6 mm I.D., 5 μm particle size). A mobile phase was used with a flow rate of 0.8 ml/min in which mobile phase A consisted of 10 mmol/l ammonium acetate in Ultra pure water adjusted to pH 5.8 with glacial acetic acid and mobile phase B consisted of 100% methanol. A mobile phase gradient was used starting at 100% A for 5 min, followed by a linear gradient from 100 to 95% A in 5 min, followed by a linear gradient from 95 to 80% A in 10 min, followed by a linear gradient from 80 to 70% A in 20 min, and followed by a linear gradient from 40 to 0% A in 5 min. The flow was reduced to 150 μl/min prior to MS detection using a T-split. The column temperature was maintained at 25 °C, the UV detector was set at 254 nm and the injection volume was 20 μl. The temperature of the turbo ion electrospray was set at 350 °C. The collision gas (nitrogen) was set at 6 mTorr, and nebulizer gas (nitrogen), curtain gas (nitrogen), and assistant drying gas (air) were used at flow rates of 8 l/min, 2 l/min, and 4 l/min, respectively. The ion spray voltage was 5000 V. The method has been validated according to
Fig. 1. The related pyrimidine metabolic pathway. Major components including cytosine, cytidine, uridine, thymine, thymidine and 2′-deoxyuridine were measured in this study.
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the requirement of analytical chemistry [9,10]. Calibration curves suitable for the analysis of plasma were linear (r2 N 0.998) with limits of detection (LOD) from 10 to 80 ng/ml. The recoveries for every metabolites are from 78.9% to 102.9% with CVs of b10%. Intraday and interday CVs were both b10%. Fig. 2 shows the typical UV chromatogram (a) and multiple extracted ion chromatogram (b) of a spiked plasma sample.
Whitney U tests. The Spearman or the Pearson correlations were examined. In this study, a value of p b 0.05 was considered statistically significant. The metabolites, the concentrations of which have statistically significant differences between two groups of the three groups (control, DM, and DR), were defined as potential biomarkers. Receiver operating characteristics (ROC) curve was performed. 3. Results
2.5. Statistical analysis 3.1. Clinical characteristics of type 2 DM The results were analyzed using SPSS statistical package (ver 14.0; SPSS Inc., Chicago, IL). Some of the clinical characteristics of DM and DR subjects were compared with the χ2 test and Fisher's exact test. To achieve normality, concentration data were log-transformed before analysis. Normality of the transformed data was verified by using the Kolmogorov–Smirnov test. The differences between the groups were calculated with Student's t-test or the non-parametric Mann–
Table 1 shows the clinical characteristics of the study groups. As shown, DM patients with retinopathy (DR patients) had significantly (p b 0.05) longer duration of the disease compared to DM patients with retinopathy (DM patients), (13.97 ± 2.40 vs. 10.64 ± 1.66). For blood pressure, systolic blood pressure (SBP) was significantly (p b 0.05) increased in DR patients compared with DM patients
Fig. 2. In a single run, UV chromatogram and multiple extracted ion chromatograms of a spiked plasma sample. (a) UV chromatogram showing 4 compounds; and (b) multiple extracted ion chromatograms of other 2 compounds.
J.-F. Xia et al. / Clinica Chimica Acta 412 (2011) 940–945 Table 1 Clinical parameters of diabetic patients with (DM) and without retinopathy (DR). Parameters
DM
DR
p
N (male/female) Age (years) Duration of DM (years) BMI (kg/m2) HbA1c (%) Fasting blood glucose (mmol/l) Triglycerides (mmol/l) Total cholesterol (mmol/l) HDL-cholesterol (mmol/l) LDL-cholesterol (mmol/l) SBP (mm Hg) DBP (mm Hg) Urea nitrogen (mmol/l)
37(21/16) 56.56 ± 7.27 10.64 ± 1.66 25.04 ± 2.85 8.09 ± 1.84 8.02 ± 2.95 2.39 ± 1.08 5.31 ± 0.56 1.36 ± 0.10 2.96 ± 0.30 132.96 ± 4.36 77.04 ± 2.83 8.11 ± 1.63
38(20/18) 57.69 ± 5.83 13.97 ± 2.40 25.20 ± 4.05 8.86 ± 1.97 7.69 ± 2.23 2.06 ± 1.37 5.13 ± 1.08 1.22 ± 0.14 2.98 ± 0.31 142.21 ± 6.75 78.86 ± 3.32 14.86 ± 3.42
NS NS 0.033 NS NS NS NS NS NS NS 0.012 NS b 0.001
(142.21 ± 6.75 vs. 132.96 ± 4.36) whereas diastolic blood pressure (DBP) was not changed. In addition, urea nitrogen was significantly (p b 0.001) increased in DR patients compared with DM patients (14.86 ± 3.42 vs. 8.11 ± 1.63). Fasting blood glucose and HbA1c were not changed between DM patients and DR patients. Lipid profiles such as total cholesterol, triglyceride, HDL-cholesterol and LDL-cholesterol of DM patients were not significantly changed compared to DR patients. 3.2. The concentrations of related pyrimidine metabolites in plasma The concentrations of cytosine and cytidine in the group of DR were significantly higher as compared with DM (p = 0.010 and p b 0.001) and control groups (p b 0.001 and p b 0.001). Additionally, also, the group of DM had significantly higher concentration of cytosine as compared to the healthy controls (p b 0.001). For concentrations of uridine and 2′-deoxyuridine, no statistically significant differences between the groups were observed (Table 2). The concentrations of thymidine in the group of DR were significantly higher as compared with DM (p b 0.001) and control groups (p b 0.001). But no statistically significant differences of concentration of thymine between the group of control and DM were observed. The concentrations of the metabolites with significant changes were shown in Fig. 3. 3.3. Relationship between correlative metabolites and other parameters Table 3 shows the relationship between concentrations of cytosine, cytidine, uridine, thymine, thymidine, 2′-deoxyuridine and clinical parameters including age, BMI, SBP, HbA1c, and various other variables in all patients of both the DM and DR groups. Cytidine correlated positively with SBP and urea nitrogen. Especially, the Pearson correlation coefficient between cytidine and urea nitrogen was above 0.5 (r = 0.617). In addition, thymidine correlated positively with urea nitrogen (r = 0.456).
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elevation of urea nitrogen. Epidemiologic studies have shown that DR is strongly clustered in families and that race has a major effect on DR susceptibility and rate of progression, firmly establishing the importance of genetic risk factors in the development of DR [11]. Despite rapid research progress, robust predictors to assess prospectively with high precision the risk for DR in individuals with diabetes are still lacking. Thus, it is necessary to set out the study of biomarker discovery, especially for the low-molecular weight metabolites, which are important and easy to be measured. In the present study, three potential biomarkers (cytosine, cytidine and thymidine) of retinopathy were showed. For the groups DM and DR, the ROC curve of these three markers was calculated (Fig. 4). As results, the ROC curve of cytidine showed better than other potential markers. The area under curve (AUC) of cytidine was 0.849 ± 0.048, and significantly higher than that of the null hypothesis (true area was 0.5, p b 0.01). This means that the plasma cytidine concentration could serve as a potential diagnostic marker for DM to DR. To find an optimal cutoff point to dichotomize ‘diseased’ or ‘healthy’, Youden's index was used in this study. According to Youden's index, the optimal operating point of cytidine was 0.076 mg/l. At this cutoff point, the sensitivity was 73.7% and specificity was 91.9%. In this study, the concentration of cytidine in the group of DR was significantly higher as compared with DM (p b 0.001) and control (p b 0.001). In addition, cytidine correlated positively with urea nitrogen and SBP. And we suppose that it may be a potential biomarker for the diagnosis of diabetic retinopathy and evaluation of treatment. Cytidine, a nucleoside that is composed of the base cytosine linked to the fivecarbon sugar D-ribose, is considered as the precursor of the cytidine triphosphate (CTP), needed in the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) biosynthetic pathway. On the one hand, the changes of cytidine concentration induce the changes of CTP, which influence the metabolism of phospholipids. On the other hand, as well as cytidine, cytosine can serve as the substrate for the salvage pathway of pyrimidine nucleotide. The significant changes of cytosine and cytidine induce the abnormality of the salvage pathway of pyrimidine nucleotide, followed by the dysfunction of phospholipid metabolism. Phospholipids are the main constituents of biological membranes and have important structural and functional properties. The changes of composition and concentration can affect the function of membrane and induce related disease, including diabetes and its complications [14–16]. In our previous work, we found out that the concentrations of phospholipids decreased with the development of
Table 2 Related metabolites in diabetic patients with retinopathy (DR), without retinopathy (DM), and control groups. Compounds
DM (n = 37)
DR (n = 38)
Cytosine (mg/l)
0.11 ± 0.01
0.21 ± 0.03
0.28 ± 0.04
4. Discussion
Cytidine (mg/l)
0.044 ± 0.01
0.052 ± 0.01
0.21 ± 0.06
To better understand the role played by specific components leading to DM and DR, their quantification in the current study is dependent on an integrated technical platform composed of hyphenated LC–UV/MS/MS analysis, conventional clinical assays, and classical statistical analysis. Compared with routine biochemical approaches, the highly selective and sensitive LC–UV/MS/MS method permits simultaneous determination of six relevant components. Current evidence suggests that both genetic and environmental factors determine susceptibility to develop DR [11,12]. Hypertension, poor glycemic and lipid control and smoking increase the risk for developing diabetic microangiopathy [13]. In this work, we showed that the patients with DR had higher SBP than those without DR. And DR patients may be associated with renal impairment, which results in the
Uridine (mg/l)
1.28 ± 0.10
1.31 ± 0.10
1.23 ± 0.14
Thymine (mg/l)
0.037 ± 0.007
0.033 ± 0.004
0.045 ± 0.010
2′-Deoxyuridine (mg/l)
0.17 ± 0.04
0.14 ± 0.02
0.14 ± 0.03
0.033 ± 0.003
0.046 ± 0.008
0.21 ± 0.07
Thymidine (mg/l)
a b c
Control (n = 41)
p value from t-test between control and DM. p value from t-test between control and DR. p value from t-test between DM and DR.
p b 0.001a b 0.001b = 0.010c NSa b 0.001b b 0.001c NSa NSb NSc NSa NSb NSc NSa NSb NSc NSa b 0.001b b 0.001c
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Fig. 3. Comparison of plasma potential risk factor concentrations among the groups of control (Con), DM and DR. DR subjects had significantly higher mean plasma concentrations of cytosine (p b 0.001 and p = 0.010), cytidine (p b 0.001 and p b 0.001) and thymidine (p b 0.001 and p b 0.001) compared to the groups of Con and DM. The group of DM had significantly higher mean plasma concentrations of cytosine (p b 0.001) compared to the group of Con.
diabetic microvascular complications [6]. Additionally, cytidine can be hydrolytic deaminated to uridine with cytidine deaminase (CDA) [17]. Because the concentration of uridine didn't change significantly when the diabetic retinopathy happened, the increase of cytidine may be due to the decrease of CDA activity, which is related to many diseases, such as abnormal pregnancy. Therefore, the research on the activity of CDA may be useful in understanding the pathogenesis of diabetic retinopathy. Thymidine may be another potential biomarker for diagnosis of retinopathy and evaluation of treatment. In our study, the concentration of thymidine in the group of DR was significantly higher as compared with DM (p b 0.001) and control (p b 0.001). And thymidine correlated positively with urea nitrogen. Thymidine, the DNA base T, is a naturally occurring compound that exists in all living organisms and DNA viruses. It is reported that persistently elevated concentrations of blood thymidine may unbalance nucleotide pools in mitochondria, leading to DNA impairment [8]. For the diabetic patients, the DNA impairments in endothelial cell may lead to vascular dysfunction and induce vascular complications. Thereby, one possible clue for the prevention and therapy retinopathy will be to reduce or, if possible, to virtually eliminate plasma thymidine. In addition, it is reported that at diabetic state, 5′-nucleotidase, the enzyme catalyzing dTMP to thymidine in pyrimidine metabolic pathway, was markedly decreased [18]. At the same time, thymidine kinase, the enzyme catalyzing thymidine to dTMP, was markedly elevated [19]. So we hypothesized that the elevated concentration of thymidine resulted from the decreased activity of thymidine phosphorylase (TP), which catalyzes the dephosphorylation of
thymidine to thymine and 2-deoxy-D-ribose-1-phosphate, a process that helps to maintain the nucleotide pool. In the present paper, we calculated the ratio of thymine and thymidine (thymine:thymidine), which can reflect the activity of TP. We found that there was a significant decrease of this ratio with the DR group compared to the control group (p b 0.01). Sengupta et al. [20] reported that TP, similar to vascular endothelial growth factor (VEGF), might be related to angiogenesis, which increases vascular permeability and promotes angiogenesis in response to ischemia or hypoxia, resulting in visual impairment. Sivridis et al. [21] have reported that TP expression was lost or considerably reduced from tubular cells of idiopathic membranous nephropathy (IMN), a principal disease of microvascular. So the changes of TP activity may be an important factor in understanding the pathogenesis of retinopathy. Acknowledgements The authors acknowledge the support of the Sino-Japanese Friendship Hospital (Beijing, China) who offered samples and clinical and biochemistry parameters. The studies have been supported by a grant from the National Basic Research Development Program of China (973 Program, No. 2005CB523503).
Table 3 Correlation between metabolites and clinical parameters.
Age Duration BMI Fasting blood glucose HbA1c Urea nitrogen Cholesterol Triglyceride HDL LDL SBP DBP
Cytosine
Cytidine
Uridine
Thymine Deoxyuridine Thymidine
− 0.189 0.108 0.067 0.073
0.107 0.111 0.171 − 0.129⁎
0.007 − 0.230⁎ − 0.130 0.097
0.177 0.010 0.102 − 0.118 0.018 − 0.072 − 0.006 0.037
0.024 0.076 − 0.151 − 0.096
− 0.072 0.030 − 0.028 0.072 − 0.054 − 0.058 − 0.069 − 0.026
0.102 0.117 0.223 0.296⁎ − 0.100 − 0.056 − 0.167 − 0.119 0.008 0.097 − 0.048 0.012 0.149 0.042 0.060 − 0.040
− 0.069 0.456⁎⁎
− 0.099 − 0.173 0.235⁎ 0.617⁎⁎ − 0.016 − 0.036 0.023 − 0.138 − 0.032 0.125 − 0.031⁎ − 0.059 0.201 0.397⁎⁎ 0.119 0.032
⁎ Correlation is significant at the 0.05 level (2-tailed). ⁎⁎ Correlation is significant at the 0.01 level (2-tailed).
0.023 − 0.006 − 0.082 0.060 0.167 − 0.049
Fig. 4. ROC curves of cytosine, cytidine and thymidine in groups of DM and DR. Cytidine shows more sensitivity and specificity for diabetic retinopathy diagnosis than other potential biomarkers.
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