Urinary c‐peptide creatinine ratio (UCPCR) as a predictor of coronary artery disease in type 1 diabetes mellitus

Abstract Background Elevated C‐peptide has been suggested as a risk factor for coronary artery disease (CAD). Elevated urinary C‐peptide to creatinine ratio (UCPCR) as an alternative measurement is shown to be related to insulin secretion dysfunction; however, data regarding UCPCR predictive value for CAD in diabetes mellitus (DM) are scarce. Therefore, we aimed to assess the UCPCR association with CAD in type 1 DM (T1DM) patients. Methods 279 patients previously diagnosed with T1DM included and categorized into two groups of CAD (n = 84) and without‐CAD (n = 195). Furthermore, each group was divided into obese (body mass index (BMI) ≥ 30) and non‐obese (BMI < 30) groups. Four models utilizing the binary logistic regression were designed to evaluate the role of UCPCR in CAD adjusted for well‐known risk factors and mediators. Results Median level of UCPCR was higher in CAD group compared to non‐CAD group (0.07 vs. 0.04, respectively). Also, the well‐acknowledged risk factors including being active smoker, hypertension, duration of diabetes, and body mass index (BMI) as well as higher levels of haemoglobin A1C (HbA1C), total cholesterol (TC), low‐density lipoprotein (LDL) and estimated glomeruli filtration rate (e‐GFR) had more significant pervasiveness in CAD patients. Based on multiple adjustments by logistic regression, UCPCR was a strong risk factor of CAD among T1DM patients independent of hypertension, demographic variables (gender, age, smoking, alcohol consumption), diabetes‐related factors (diabetes duration, FBS, HbA1C), lipid profile (TC, LDL, HDL, TG) and renal‐related indicators (creatinine, e‐GFR, albuminuria, uric acid) in both patients with BMI≥30 and BMI < 30. Conclusion UCPCR is associated with clinical CAD, independent of CAD classic risk factors, glycaemic control, insulin resistance and BMI in type 1 DM patients.


| INTRODUC TI ON
Type 1 diabetes mellitus (T1DM), which is a deficient production or dysfunction of insulin, has become a worldwide and provincial health concern with a significantly increasing incidence. 1 Cardiovascular diseases (CVD) are recognized as the main etiologic factor for mortality in T1DM patients even in those with appropriate metabolic control. 2 Insulin is an endogenous, anabolic peptide hormone secreted from pancreatic beta cells and regulates blood glucose homeostasis. Preproinsulin is synthesized by pancreatic beta cells and rapidly converts to proinsulin, an 86 amino acid polypeptide which is then cleaved to generate C-peptide and insulin equimolarly, containing 31 and 51 amino ac ids, respectively. [3][4][5] Insulin with a half-life of 3-5 min is metabolized in liver by approximately 50% and has a variable clearance in peripheral circulation. Therefore, its direct measurement is not an accurate method for assessing pancreatic beta cell's function. Also, blood insulin test may have cross-reactivity with exogenous insulin in patients with diabetes who take insulin. Contrarily, C-peptide is slightly extracted by liver but cleared mainly in kidneys and about 5% of total produced C-peptide is excreted into the urine. 6,7 As a result, C-peptide can be spotted in serum and urine. Serum C-peptide test is an inconvenient method due to difficult sample collection and can be measured in a fasted or non-fasted sample and after stimulation. 6,8,9 It is also falsely elevated in patients with renal impairment. 10,11 C-peptide is mostly used to distinguish between insulinoma and factitious hypoglycaemia in nondiabetic individuals. 7 It has been demonstrated that C-peptide is associated with duration of diabetes, age of diagnosis, the need for insulin therapy, predicting glycaemic control, pro-inflammatory condition, and microvascular and macrovascular complications. Elevated Cpeptide level is related to insulin resistance and metabolic syndrome.
Accordingly, higher levels of C-peptide have been suggested as risk factor of coronary artery disease (CAD) and death in patients without type 2 diabetes mellitus (T1DM). [12][13][14][15] Urinary C-peptide is a non-invasive and practical test with shortcomings in patients with renal impairment. 6,7 Urinary C-peptide creatinine ratio (UCPCR) is another simple and reliable test that is correlated with serum C-peptide and 24-h urinary C-peptide even in moderate renal impairment. 9,16,17 However, the amount of UCPCR varies in different genders and weights as a result of alteration in muscle mass and creatinine levels. 18 The role of UCPCR in classification of different types of diabetes has been well established. 7,19 UCPCR has been shown to relate to insulin resistance in patients without diabetes and obese children 20,21 but the relation of UCPCR to CAD in individuals with type 1 diabetes mellitus (T1DM) has not been evaluated. Therefore, in the present study we have aimed to investigate roles of UCPCR in CAD in T1DM patients classified by BMI.

| Study design and participants
The present research has enrolled 279 patients with a T1DM diagnosis at Shahid Modarres Hospital affiliated to Shahid Beheshti University of Medical Sciences in Tehran, Iran, from November 2020 to November 2021 ( Figure 1). The exclusion criteria were as follows:

| Data measurements
The individuals' demographic features, habits, and past medical histories were collected using the hospital's computerized patient record system. The body mass index (BMI) was calculated by the weight divided by the square of the height (kg/m2). Blood pressure (BP) has been recorded three times consecutively, on the left arm following the seated condition of patient lasting for at least 5 min.
Consequently, the average of three recorded BPs was employed for evaluations. Blood samples have been taken after 8-hour fasting and before the breakfast. Fasting glucose, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides (TGs), total cholesterol (TC), creatinine (Cr), and uric acid (UA) were quantified. High-performance liquid chromatography was used to determine HbA1C levels. A chemiluminescence immunoassay analyser has been used to detect C-peptide and blood insulin levels. An immunoturbidimetric test analyser has been employed to detect albuminuria, and the e-GFR has been determined by the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation and creatinine levels. Patients collected urine specimen at the time of blood sampling and the urinary C-peptide and creatinine were analysed using electrochemiluminescence immunoassay and Roche P800 platform, respectively, to calculate UCPCR (nmol/mmol).

| Definitions
A BMI ≥30 kg/m 2 has been described as obesity, based on the criteria of the World Health Organization (WHO) obesity classification 22 ; hence, the categorization of patients based on BMI was performed into 2 groups of non-obese patients (BMI ≤30 kg/m 2 ) and obese (BMI ≥30 kg/m 2 ). Diabetic cases who had already been identified were discovered by an examination of their health records using the WHO criteria from 1999: random blood glucose levels of more than 200 mg/dL (11.1 mmol/L), Fasting blood glucose levels of more than 126 mg/dL (7.0 mmol/L), with clinical manifestations (diabetic ketoacidosis, polydipsia polyuria), or an Angiography-shown coronary artery blockage of 50% or more, coronary revascularization, or a history of myocardial infarction (MI) verified by Q waves evident in the ECG or medical records was considered hard CAD.

| Statistical analysis
Categorical variables and continuous data having a normal distribution have been presented as percentages (%) and the mean ± standard deviation (SD), respectively. The chi-squared test and Student's t test were utilized to evaluate differences between the two groups, while one-way ANOVA has been utilized for three groups or more. To compare continuous data, the Mann-Whitney Utest was utilized. In T1DM patients, the risk variables for CAD were determined using binary logistic regression in 4 designed models:  Table.1 demonstrates the baseline characteristics of the study patients. The mean age of CAD and non-CAD patients was similar between groups (54.8 and 54.7 years, respectively). Regarding the sex ratio, male patients made up 57.1% of the CAD group and 47.2% of the non-CAD group (p = .12). The BMI (p = .01) and history of smoking (p = .04) were also greater in the CAD group, while the history of alcohol consumption did not show a contrast between the two groups. Regarding the clinical history, prevalent hypertension F I G U R E . 1 Flow chart of the studied groups. BMI, body mass index; CAD, coronary artery disease; DM, diabetes mellitus; eGFR, estimated glomerular filtration rate; MODY, maturity onset diabetes of young; T1DM, type 1 diabetes mellitus; T2DM, type 2 diabetes mellitus.  As the focal point of our study is to assess the effect of UCPCR,

| DISCUSS ION
In the present study, we have attempted to assess UCPCR levels in CAD patients and figure out whether CAD could be predicted in patients based on their UCPCR levels. Our analysis showed that UCPCR levels were significantly greater in CAD patients as a whole, and as sub-grouped based on having a BMI ≥30. Also, further modellings conducted on the patients indicated that CAD cases, when adjusted for a variety of different variables, still had remarkably greater levels of UCPCR, regardless of their BMI.
Aggravation of systematic inflammation, 23 oxidative stress condition, 24 tissue hypoxia, 25 microcirculatory damage 26 and as a result, the formation of atherosclerotic plaques in T1DM may be the cause of the co-occurrence of CAD. Nonetheless, in patients with T1DM, the involvement of various variables other than the mentioned wellacknowledge risk factors for CAD has yet to be discovered. T1DM is recognized by autoimmune demolition of pancreatic beta cells.
Recently, studies have shown that this destruction is not an ongoing process since some individuals with long-standing T1DM still have functional residual pancreatic beta cells that produce C-peptide as a co-product of insulin biosynthesis. [27][28][29] It has been long believing   In this study, we compared CAD and non-CAD patients based on the clinical and laboratorial characteristics and also categorized by BMI. We also considered well-acknowledged risk factors of CAD including hypertension, dyslipidaemia, treatment with statins, mild to moderate reduced eGFR, diabetes-related factors and sociodemographic properties. Similar to previous studies, those with CAD had significantly higher incidence of hypertension, albuminuria, TC, LDL, HbA1C, longer T1DM duration, as well as declined levels of eGFR and HDL. It is noteworthy that treatment with statins was generally high (>85% in both groups) in the participants of this study; thus, its effect on lowering the risk of coronary artery disease may have been diluted. This might explain why there is not a clear link between statins and coronary artery disease in this study.
A considerable number of diabetic individuals are obese with elevated BMI as the independent risk factor of CAD. Moreover, urinary Cpeptide, levels of C-peptide and UCPCR could be influenced by weight.
Therefore, following investigating the role of UCPCR on clinical CAD, we decided to analyse whether UCPCR have different effect in different BMI groups of T1DM patients. We obtained that among obese TA B L E 3 Binary logistic regression analysis for risk factors of CAD among patients with type 1 diabetes mellitus. (BMI≥30) as well as the non-obese (BMI < 30) group, UCPCR was related to CAD regardless of conventional CAD risk factors. These findings imply that UCPCR may be linked to or implicated in the production of atherosclerotic plaques within the coronary arteries of T1DM patients, both obese and non-obese. Herein, UCPCR was suggested as a risk factor of CAD with the adjusted OR of more than 1, greater in nonobese patients in comparison with obese patients. This may suggest the greater implication of UCPCR measurement in non-obese patients.
One of the reasons for the importance of this study is that measuring UCPCR is simple, cost-effective and practical in comparison with serum assays of C-peptide and insulin. 43 To the most precise of our knowledge, we have been the first to assess UCPCR as a risk factor of CAD have not been assessed, which may have resulted in a lack of explanatory power for C-role peptides as the atypical inflammatory marker of CAD. Seventh, we excluded patients with eGFRs of less than 45 mL/ min/1.73 m2; however, it is plausible this exclusion decreased the potential of our study to recognize a correlation between CAD, diabetic nephropathy and UCPCR. Last but not least, since the individuals in this study have been drawn from a single facility, there were inevitable biases in patient selection, acquired data and confounding factors.
In conclusion, this is a novel topic regarding the predictive role of UCPCR in patients with T1DM with lack of previous research studies. UCPCR is a practical and non-invasive biomarker that has shown its effectiveness in differentiating T1DM from other DM groups, guiding the individual's treatment regarding poor-or wellcontrolled DM, and as we showed, the subsequent CV risk prediction. We discovered that in T1DM patients, whether obese or not, UCPCR is associated with clinical CAD, independent of established CAD risk variables such as insulin resistance and glycaemic control.
Meanwhile, further studies are required to evaluate the expansion of clinical implications and cost-effectiveness of this marker in both fields of medical management and biomedicine.

PATI ENT CO N S ENT S TATEM ENT
Written informed consent has been acquired from patients to publish this study according to the journal's patient consent policy.

FU N D I N G I N FO R M ATI O N
There is no funding to present study.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data supporting the findings of the study are available on request from the corresponding author.

E TH I C S A PPROVA L S TATEM ENT
The authors all declare that this manuscript is not published elsewhere.