Supplemental Data: Angiopoietin-like 3 Is a Potential Biomarker for Retinopathy in Type 2 Diabetic Patients

Contents:

Supplementary Methods

Supplementary Table

Supplementary Figure 1

Supplementary Figure 2

SupplementaryMethods

Study Population

We conducted a cross-sectional study in type 2 diabetic patients who had been diabetic for an average of 9.6 years. The patients with clear diagnosis of type 2 diabetes mellitus (T2DM) were included in the study, who had a plasma glucose of at least 7.0 mmol/L while fasting and/or of 11.0 mmol/L or more 2 hours after an orally administered glucose load of 75 g. The exclusion criteria were type1 diabetic mellitus, gestational diabetes mellitus, and T2DM patients with cancer. The study complied with the Helsinki Declaration for investigation of human subjects. The study involved was approved by the Institutional Review Boards of the Lu He Hospital and Capital Medical University. All the patients provided written informed consent.

From July 2015 until January 2017, 1316 T2DM patients were admitted to the Department of Endocrinology at Lu He Hospital in Beijing. We excluded 124 patients from analysis, because the degree of retinopathy could not be assessed (cataract: n=79; declined retina examination: n=21; acute infection: n=11) or not all required measurements were available on the database (n=10), or because of extreme values of variables exceeding the mean by 3 SD or more (n=4). Thus, the number of diabetic patients statistically analyzed totaled 1192.

Clinical Measurements

In the studies, trained observers measured blood pressure to the nearest 2 mmHg by auscultation of the Korotkoff sounds, using a standard mercury sphygmomanometer. Systolic and diastolic blood pressure were measured and mean arterial pressure was two thirds of the diastolic blood pressure plus one third of the systolic blood pressure. Hypertension was a blood pressure of at least 140 mmHg systolic or 90 mmHg diastolic or use of antihypertensive drugs. The observers measured each participant’s anthropometric characteristics and collected information on medical history, smoking and drinking habits, and intake of medications.Body mass index was weight in kilograms divided by the square of height in meters.

Retina Examination

Retinal photography was performed using a nonmydriatic fundus camera (Topcon, Tokyo, Japan). For each eye, a total of5 images were obtained at 30 degrees and 45 degrees centered on the optic nerve and at 30 degrees centered on the macula, respectively(Supplemental Figure 1).The grade of diabetic retinopathy (DR) was classified by 2 investigators who were masked to the degree of the DR of the patients according to the Early Treatment of Diabetic Retinopathy Study.1 Briefly, mild nonproliferative DR (NPDR) was defined by the presence of microaneurysms; the number of microaneurysms of medium NPDR was more than mild but less than severe NPDR; severe NPDR had >20 intraretinal hemorrhages in each of the 4 quadrants, venous beading in at least 2 quadrants, and intraretinal microvascular abnormalities in at least 1 quadrant in the absence of proliferative DR (PDR); and PDR had vitreous or preretinal hemorrhage.2,3 In case of unclear images, patients underwentfluorescein fundus angiography (FFA) (TRC-50DX;Topcon Corp, Tokyo, Japan) for confirmation.

Biochemical Measurements

Fasting venous blood samples were drawn for measurement of the total and differential white blood cell count, serum total cholesterol, high-density lipoprotein (HDL) cholesterol (HDL-c), triglycerides, and creatinine, plasma glucose, and serum levels of Cpeptide. Glomerular filtration rate (eGFR) was derived from serum creatinine by the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation.3 Low-density lipoprotein (LDL) cholesterol (LDL-c) was computed from serum total cholesterol, HDL-c, and serum triglycerides by the Friedewald equation.4

Enzyme-linked Immunosorbent Assay

Serum levels of angiopoietin-like 3 (ANGPTL3), angiopoietin-like 4 (ANGPTL4), high-sensitive C-reactive protein (CRP), vascular adhesion molecule–1 (VCAM-1); intracellular adhesion molecule–1 (ICAM-1), vascular endothelial growth factor (VEGF) were measured according to the manual instruction. Intra-assay coefficients of variation for ANGPTL3, ANGPTL4, VCAM-1, ICAM-1, VEGF and CRP were 3.03%, 5.67%, 7.3%, 7.3%, 6.5% and 7.3%, respectively. Inter-assay coefficient of variation for ANGPTL3, ANGPTL4, VCAM-1, ICAM-1, VEGF and CRP was 7.37%, 6.96%, 5.2%, 5.4%, 4.6% and 5.8%, respectively.

Statistical Analysis

For database management and statistical analysis, we used the SAS system, version 9.4 (SAS Institute Inc., Cary, NC). We normalized the distributions of fasting blood glucose, -glutamyltransferase, serum creatinine, triglyceride, fasting Cpeptide, Urinary albumin-to-creatinine ratio, ANGPTL3, ANGPTL4, CRP, VCAM-1, ICAM-1 and VEGF by a logarithmic transformation. For untransformed and logarithmically transformed variables, we expressed the central tendency and spread of the distributions by the arithmetic mean and SD and the geometric mean and interquartile range, respectively.

We compared means by the large-sample ztest and proportions by Fisher’s exact test. We searched for covariables of ANGPTL3 or ANGPTL4 using a stepwise regression procedure with the Pvalues for variables to enter and stay in the models set at .15. In multivariable-adjusted logistic analysis,log10 probability plots were constructed. The covariables considered included sex, age, body mass index, waist-to-hip ratio, HbA1c, mean arterial pressure, -glutamyltransferase, total cholesterol, serum triglyceride, serum creatinine, urinary albumin-to-creatinine ratio, smoking, history of hypertension and cardiovascular disease, and use of antidiabetic drugs (insulin, insulin secretagogues, metformin, and glucosidase inhibitors), lipid-lowering drugs (statins), and antihypertensive drugs (diuretics, vasodilators [blockers and calcium-channel blockers], and inhibitors of the renin-angiotensin system [angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and beta blockers]). Significance was a two-tailed Plevel of .05 or less.

References

1. Grading diabetic retinopathy from stereoscopic color fundus photographs--an extension of the modified Airlie House classification. ETDRS report number 10. Early Treatment Diabetic Retinopathy Study Research Group. Ophthalmology 1991;98:786-806.

2. Lee B, Novais EA, Waheed NK, et al. En face doppler optical coherence tomography measurement of total retinal blood flow in diabetic retinopathy and diabetic macular edema. JAMA Ophthalmol 2017;135:244-251.

3.Levey AS, Stevens LA, Schmid CH, et al. A new equation to estimate glomerular filtration rate. Ann Intern Med 2009;150:604-612.

4. Johnson R, McNutt P, MacMahon S, Robson R. Use of the Friedewald formula to estimate LDL-cholesterol in patients with chronic renal failure on dialysis. Clin Chem 1997;43:2183-2184.

SupplementalTableCovariables Selected by Stepwise Regression

Serum ANGPTL3(μg/mL) / Serum ANGPTL4(ng/mL)
Age (y) / … / 0.004d
Waist-to-hip ratio / -0.304a / …
Mean arterial pressure (mmHg) / 0.002 / …
HbA1c (%) / … / 0.027b
glutamyltransferase / … / 0.029
Total cholesterol (mmo/L) / 0.054d / …
Serum triglyceride (mmol/L) / … / 0.042a
Serum creatinine(μmol/L) / … / 0.221d
UACR(mg/mmol) / 0.014a / 0.034d
Medications (0,1)
RAAS / 0.067a / …
Diuretics / 0.130a / …
Statins / -0.081c / …
Insulin / 0.087b / -0.154c
insulin secretagogues / 0.074a / -0.189d
Metformin / -0.073b / …

ANGPTL3 = angiopoietin-like 3; ANGPTL4 = angiopoietin-like 4,RAAS = renin-angiotensin-aldosterone system; UACR = urinary albumin-to-creatinine ratio.

The variables considered for entry into the models included sex, age, body mass index, waist-to-hip ratio, mean arterial pressure, HbA1c, serum total cholesterol, serum triglyceride, serum creatinine, glutamyltransferase (as index of alcohol intake), smoking, years of diabetes, history of coronary heart disease or hypertension, treatment with diuretics (thiazides, loop diuretics, and aldosterone antagonists), RAAS (inhibitors of the renin-angiotensin system, including angiotensin-converting enzyme inhibitors or angiotensin type1 receptor blockers or blockers), vasodilators (calcium channel blockers and blockers), statins, insulin, metformin, insulin secretagogues, and glucosidase inhibitors.

Pvalues for variables to enter and stay in the regression models were set at .15. Significance of the association: a.05 <P ≤ .15; bP ≤ .05; cP ≤ .01; dP ≤ .001. An ellipsis indicates not entering the model (P .15).

Supplemental Figure 1. The representative collection of retinal images of type 2 diabetes mellitus patients without retinopathy (Left) and with nonproliferative retinopathy (Right).

Supplemental Figure 2. Serum levels of angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 3 (ANGPTL4) by sex. Serum levels of ANGPTL3 (Top) and ANGPTL4 (Bottom) in male and female type 2 diabetic patients are shown, categorized by the grade of diabetic retinopathy (DR). NPDR = nonproliferative diabetic retinopathy; PDR = proliferative diabetic retinopathy.

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