Measurements
hypoglycemia, retinopathy, TG, pre-proliferative retinopathy
RETINOPATHY, ADVERSE EVENTS, HYPOGLYCEMIA, BLOOD, PROLIFERATIVE RETINOPATHY
Venous blood and urine samples were collected under fasting conditions at baseline and at 3, 6, and 12 months of treatment and were transferred to the central laboratory (SRL Inc., Tokyo, Japan). Body weight and blood pressure were measured at baseline and at every follow-up visit. Episodes of hypoglycemia or adverse events were collected by medical interviews at the follow-up visits. The findings of Achilles tendon reflex test (classified as normal, reduced, or disappeared) were recorded at baseline and at the 6- and 12-month visits. Ophthalmofundoscopic findings (classified as normal, simple retinopathy, pre-proliferative retinopathy, and proliferative retinopathy) were recorded at baseline and at the 12-month visit. Blood samples were centrifuged to separate the serum for laboratory measurements. The laboratory measurements for efficacy outcomes at 12 months were included only glucose metabolism parameters, serum Cr and CysC, and uACR simply for ease of conducting the study.Plasma glucose levels were determined by the hexokinase UV method using Cicaliquid GLU L (Kanto Kagaku, Tokyo, Japan). HbA1c levels were determined by latex agglutination turbidimetry using RAPIDIA Auto HbA1c (Fujirebio Inc., Tokyo, Japan). GA levels were determined by the visible absorption spectrometry enzymatic method using Lucica GA-L (Asahi Kasei Pharma Co., Tokyo, Japan). Serum insulin and C-peptide levels were measured via chemiluminescent enzyme immunoassay commercial kits (Fujirebio, Inc.), and proinsulin levels were determined via a two-antibody radioimmunoassay (Denis Pharma K.K., Tokyo, Japan). Serum Cr was measured via the visible absorption spectrometry enzymatic method using Detaminer L CRE (Asahi Kasei Pharma Co.). CysC levels were measured using a colloidal gold immunoassay (Alfresa Pharma Co., Osaka, Japan). The eGFR was calculated based on age, sex, and serum Cr or CysC levels. The uACR was measured using an Autokit Micro Albumin (Fujifilm Wako Pure Chemical Co., Tokyo, Japan). Total cholesterol and triglycerides (TG) levels were determined using the cholesterol dehydrogenase UV method and an enzymatic method using Cholestest CHO and Pure Auto S TG-N (Sekisui Medical Co., Tokyo, Japan). High-density lipoprotein (HDL) cholesterol levels were measured via a direct assay using Cholestest N-HDL (Sekisui Medical Co.). Low-density lipoprotein (LDL) cholesterol levels were measured using the Friedewald formula. The non-HDL cholesterol level was calculated the subtracting HDL cholesterol level from the total cholesterol level. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl aminotransferase (GGT), and uric acid levels were determined by standard methods in an external laboratory. Insulin-related parameters and serum lipids were not measured at the 12-month follow-up visit. When the insulin level was > 30 μU/mL, insulin-related parameters, namely FPG and TG, were not included in the analyses because the blood samples were suspected to have been obtained in a non-fasting condition.