ASSESSMENT OF PYRAZINO-PYRIMIDINE COMPOUND AND SOME INFLAMMATORY BIOMARKERS IN PATIENTS WITH TYPE 2 DIABETESHTML Full Text
ASSESSMENT OF PYRAZINO-PYRIMIDINE COMPOUND AND SOME INFLAMMATORY BIOMARKERS IN PATIENTS WITH TYPE 2 DIABETES
Feryal Hashim Rada
Ph.D. Clinical Therapeutic and Biochemistry, Department of Pharmaceutical Chemistry, College of Pharmacy, Al-Nahrain University, Iraq.
ABSTRACT: Neopterin, pyrazino-pyrimidine compound, is a metabolite of guanosine triphosphate and is produced by the activated monocytes, macrophages and dendritic cells upon stimulation by interferon gamma produced by T-lymphocytes. Aim: This study purposed to analyze the serum level of neopterin and to evaluate its correlation with other markers of inflammation in patients with type 2 diabetes at various stages of diabetic nephropathy. Methods: The study done on 80 patients, aged (60 years ±5) with type 2 diabetes, and 60 healthy subjects, aged (50 years±8) recruited from outpatient clinic of nephrology department in Al yarmouk hospital. The serum levels of neopterin, high sensitive C-reactive protein (hs-CRP), interluekin-six (IL-6), tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) were assayed by using enzyme-linked immunosorbent assays (ELISA) kits, and studied. Results: The level of serum neopterin was higher in diabetic patients than in control subjects. There were gradual increases of serum neopterin levels from stage two to stage three and four in diabetic patients. Serum neopterin level correlated positively with serum levels of hs-CRP, IL-6, and IFN-γ and correlated negatively with estimated glomerular ﬁltration rate value. Conclusion: Serum neopterin level is elevated and correlated with the severity of diabetic nephropathy. It may use as a good biomarker for an accurate identification and prognosis of the diseases associated with the activation of cell-mediated immunity.
Neopterin, Diabetic Nephropathy, Inflammatory Markers
INTRODUCTION: Neopterin belonged to pteridine class and formed by fusing pyrimidine and pyrizine rings therefore neopterin sometime called pyrazino-pyrimidine compound. It produced from guanosine triphosphate by activated monocytes, macrophages, dendritic cells, and endothelial cells upon stimulation by interferon gamma (INF-γ) and its release enhanced by tumor necrosis factor 1, 2. After releases, it enhances macrophage cytotoxicity through its interactions with reactive oxygen, nitrogen, and chloride species 2.
Increased neopterin concentration in the urine or blood due to activation of cellular immunity and an endogenous release of INF-γ may affect cellular redox state because of interactions between neopterin or its derivative with reactive oxygen or nitrogen intermediates 3.
Neopterin levels are elevated in several conditions including autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis 4; infections such as hepatitis, human immunodeﬁciency virus, and cytomegalo virus 5. Elevated neopterin level observed in patients with various cancers and in all cancer types investigated, high neopterin levels were signiﬁcantly associated with a poor prognosis 6.
The impact of diabetic nephropathy on the increasing population with chronic kidney disease (CKD) and end-stage renal disease (ESRD) is enormous. Furthermore, prediction and progression of diabetic nephropathy, cardiovascular and renal outcome done by simultaneous evaluation of albuminuria and glomerular filtration rate 7, 8.
The objective of this study is to investigate whether the concentration of neopterin was elevated in serum samples of patients with diabetic nephropathy as compared to control subjects and to evaluate the utility of neopterin as a biomarker of diabetic nephropathy.
MATERIALS AND METHODS: We conducted case-control study among the participants of outpatient clinic of nephrology department in Al yarmouk hospital. This study enrolled 80 patients with diabetic nephropathy and 60 control healthy subjects. Excluded criteria involved patients with known coronary artery disease, heart failure, malignancies, active infections, and patients taking drugs that may effect on inﬂammatory response.
Estimated glomerular ﬁltration rate (eGFR) was calculated using 2009 CKD-EPI creatinine equation and was classified basing on glomerular filtration rate (GFR) category 9, at which patients on stage (G2) have eGFR =(60–89ml/min/ 1.73m2), patients on stage (G3) have eGFR = (30–59 ml/min/1.73m2), and patients on stage (G4) have eGFR = (15–29 ml/min/1.73m2).
After recruitment, participants asked to be in a fasting state. Blood samples were collected; serum was separated and stored at -80 °C until analysis. Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used to measure serum levels of hs-CRP, IL-6,TNF-α, IFN-γ and neopterin and all the samples were analyzed in duplicate. Moreover, spectrophotometric assay kits were used to measure serum levels of albumin, creatinine, and urea.
All eligible participants provided written informed consent to partake in this study. The study protocol conforms to the ethical guidelines and approved by the institution’s ethics committee.
Results are shown as mean ± SD with 95% confidence interval (CI), and P values of (0 <0.05) were regarded to be statistically significant. All statistical analyses performed using series SPSS version 18.
RESULTS: Details and clinical data of the studied groups showed in Table 1. There were no diﬀerences in age and gender distribution observed between control and patients. Of the 80 patients with diabetic nephropathy, 28 were in stage two (G2), 30 in stage three (G3), and 22 in stage four (G4).
TABLE 1: THE DEMOGRAPHIC AND CLINICAL DATA OF THE STUDIED GROUPS
|Age (years)||50 ± 8||60 ± 5|
|Serum Albumin (g/L)||40.2 ± 3.6||32.4 ± 5.7 ***|
|Serum Creatinine (mg/dl)||0.67 ± 0.35||1.58 ± 0.46 ***|
|Serum Urea (mg/dl)||30.7 ± 8.5||68.3 ± 12.4 ***|
|eGFR (ml/min)||97.6 ± 7.5||50.7 ± 12.4 ***|
|Serum Neopterin (nmol/L)||7.5 ± 1.2||35.7 ± 3.36 ***|
|Serum hs CRP (mg/L)||2.25 ± 1.07||43.7 ± 4.8 ***|
|Serum IL-6 (pg/ml)||2.05 ± 1.2||19.2 ± 2.3 ***|
|Serum TNF-α (pg/ml)||3.8 ± 0.98||8.2 ± 2.42 ***|
|Serum INF-γ (pg/ml)||2.13 ± 0.64||8.87 ± 1.5 ***|
Data are presented as mean ±SD (standard deviation) for continuous variables; *** P˂0.001 high significant difference versus control.
Abbreviations: g/L, gram per liter; mg/dl, milligram per deciliter; nmol/L, nanomole per liter; pg/ml, picogram per milliliter; Number, sample size of the participants; hs- CRP, high sensitive C-reactive protein; eGFR, estimated glomerular filtration rate; IL-6, Interluekin-six; TNF-α, tumor necrosis factor-alpha; IFN-γ , Interferon-gamma
In contrast to healthy subjects, serum neopterin level was notably higher in patients with diabetic nephropathy (Table 1) and it is seemed to be 5 folded more than its level in control subjects (Fig. 1).
FIG. 1: BAR GRAPH ELUCIDATED THE NUMBER OF FOLDED INCREASES IN SERUM LEVELS OF INFLAMMATORY BIOMARKERS FOR DIABETIC PATIENTS VERSUS CONTROL LEVELS
Abbreviations: hs- CRP, high sensitive C-reactive protein; IL-6, Interluekin-six; IFN-γ, Interferon-gamma; TNF-α, tumor necrosis factor-alpha As well, there were graded increases in serum neopterin levels from stage two to four as indicated in Fig. 2.
FIG. 2: BAR GRAPH SHOWED THE MEAN SERUM NEOPTERIN LEVELS (nmol/L) AND THE NUMBER (N) OF THE PARTICIPANTS IN HEALTHY CONTROL AND IN DIFFERENT STAGES OF DIABETIC NEPHROPATHY
The serum neopterin level exhibited a high signiﬁcant inverse correlation with eGFR, and had a positive association with hsCRP, IL-6, and IFN-γ in patients with diabetic nephropathy. Whereas a non-significant correlation noted between serum neopterin level and TNF-α in those patients, Table 2.
TABLE 2: CORRELATION COEFFICIENT (r) OF SERUM NEOPTERIN LEVEL WITH THE ESTIMATED GLOMERULAR FILTRATION RATE VALUE AND WITH THE SERUM LEVELS OF OTHER INFLAMMATORY MARKERS IN DIABETIC PATIENTS
|Serum hs-CRP||0.33||P ˂0.01|
|Serum IL-6||0.28||P ˂0.05|
|Serum INF-γ||0.36||P ˂0.001|
|Serum TNF-α||0.21||P ˃ 0.05|
P ˃ 0.05 non-significant correlation; P ˂0.05 significant correlation; P ˂0.01, P ˂0.001 high significant correlation.
Abbreviations: eGFR, estimated glomerular filtration rate; hs-CRP, high sensitive C-reactive protein; IL-6, Interluekin-six; IFN-γ, Interferon-gamma; TNF-α, tumor necrosis factor-alpha
There were high significant increases in mean serum levels of other inflammatory biomarkers found in patients with diabetic nephropathy as compared to control subjects, at which hsCRP increased 19 folded , IL-6 increased 9.4 folded, TNF-α increased 2 folded and IFN-γ increased 4 folded , Fig. 1.
DISCUSSION: The current study showed that the patients with diabetic nephropathy exhibited high signiﬁcant increase in serum level of neopterin compared to healthy subjects, and this increase correlated positively with the increased circulating levels of several inflammatory biomarkers (hsCRP, IL-6, and IFN-γ).
Serum level of neopterin reveal continuous increases with the decreasing GFR, these ﬁnding is consistent with previous studies 10, 11 that proposed the decreased renal elimination and/or increased inflammatory reaction on renal function are behind the progressive increased in neoptrin levels.
Other study showed that the elevated activity of the pteridine pathway leads to the generation of singlet oxygen, hydroxyl radical and nitric oxide 12, which may affect renal function and decreased renal elimination.
Grossmann et al. in 2015, noted that neopterin level was positively associated with the prevalence of the diabetes disease 13. As well, neopterin considered as a marker of diabetic progression and complication 14.
The diabetic patients in the current study showed inflammatory activation and increases in the inflammatory biomarkers (CRP, IL-6, IFN-γ, and TNF-α) levels which are consistent with other study done on patients with chronic kidney disease 11.
Conversely, other study reveal weak association of CRP level with diabetes prevalence after adjusting other cardiovascular risk factors and comorbidities and this occur may be due to the early activation of the immune system 13.
CRP is one of the inflammatory biomarkers for type 2 diabetes–associated cardiovascular diseases 15, 16. The regulatory functions of CRP include enhancement of leukocyte reactivity, complement fixation, and modulation of platelet activation 17.
Furthermore, cytokines such as IL-6, or TNF-α that may contribute to islet cell autoimmunity in type 2 diabetes through activation of T cells, B cells, and macrophages may increase expression of β-cell antigens, and subsequent β-cell apoptosis 18.
In 2004, Bodlaj et al. documented that the presence of type 2 diabetes mellitus in addition to end stage renal disease (ESRD) was not associated with the further increases in serum levels of the inflammatory parameters, i.e. the deteriorated prognosis of diabetic ESRD patients is probably not associated with the activation of inflammatory processes 19.
Many studies have proven a strong association of neopterin with cardiovascular disease 20, increased cardiac events rates 21 and atheromatous plaque activity and progression in patients with coronary artery disease 22. In vascular smooth muscle cells, elevated neopterin level cause subsequent increase in nitric oxide (NO) produsction 23 and induce programmed cell death by activated the transcriptional nuclear factor (NF)-κB 24.
Glucocorticoids drugs may cause suppression of cell-mediated immunity and consequently result in decreased neopterin levels. As well, statin drug, which has anti-inflammatory effects by inhibiting HMG-CoA (hydroxyl methyl glutaryl – CoA) can decrease serum level of neopterin 25. Therefore, patients taking these drugs excluded in this study.
CONCLUSION: As the neopterin level serves as an indirect indicator for oxidative stress thereby impaired renal elimination and increased inflammatory reaction that occur during diabetic nephropathy might be associated with the stepwise increase in serum neopterin level. More studies needed to confirm the association between the neopterin elevation and the clinical events accompanied diabetic nephropathy.
CONFLICT OF INTERESTS: There is no conflict of interests to declare.
ACKNOWLEDGMENTS: I thank the doctors and other medical staffs for data collection and analyses and I appreciate all the patients and subjects participating in this research.
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How to cite this article:
Rada FH: Assessment of pyrazino-pyrimidine compound and some inflammatory biomarkers in patients with type 2 diabetes. Int J Pharm Sci Res 2017; 8(6): 2691-95.doi: 10.13040/IJPSR.0975-8232.8(6).2691-95.
All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
Feryal Hashim Rada
Ph.D. Clinical Therapeutic and Biochemistry, Department of Pharmaceutical Chemistry, College of Pharmacy, Al-Nahrain University, Iraq
14 November, 2016
08 January, 2017
02 February, 2017
01 June, 2017