The cross-sectional study finally enrolled 1465 confirmed or newly diagnosed T2DM patients in our inpatient department between August 2012 and September 2015, who completed the measurement of serum albumin and DPN screening. Inclusion criteria were long-term residence (≥ 5 years) in Sichuan province of China and age 16–89 years old. The exclusion criteria were as follows: (1) presence of other endocrine disorders, acute diabetic complications including diabetic ketoacidosis, hyperosmolar hyperglycemic nonketotic coma, and hypoglycemia, non-diabetic neuropathy; (2) any evidence of severe respiratory and cerebrovascular disease, severe congestive heart failure (New York Heart Association functional class IV), severe renal failure (serum creatinine ≥ 221 µmol/L), liver disease or abnormal liver function; (3) presence of inflammatory disease, autoimmune disease such as rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, and synovitis, acute infectious disease, cancer, hematological diseases and cognitive dysfunction; (4) alcoholism, pregnancy or lactation; (5) use of immunosuppressant, antioxidant, anti-inflammatory drugs, analgesics, systemic corticosteroids; (6) use of possible or known drugs that may affect peripheral nerve function and sympathetic system; (7) Subjects with missing or incomplete data.
The study was performed in accordance with the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the human research ethics committee of the Affiliated Hospital of Southwest Medical University, and informed consent was obtained from all patients after full explanation of the purpose and nature of our study.
Clinical and biochemical measurements
Body weight and height were taken using standard protocols with the subjects in light clothing and without shoes. Body mass index (BMI) was calculated by dividing weight in kilograms by height in metres squared. Systolic and diastolic blood pressures were measured in all subjects on the right arm using a standard mercury sphygmomanometer . All measurements were operated by trained study personnel.
In the morning, after an overnight fast of 8 h or longer, venous blood samples were collected from study participants to determine fasting blood glucose (FBG), glycated hemoglobin A1C (HbA1c), lipid profiles, including total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), cystatin C (CysC), uric acid (UA), serum albumin, creatinine (Cr), neutrophil and lymphocyte counts, neutrophil to lymphocyte ratio (NLR), and red cell distribution width (RDW) at a certified central laboratory. FBG and HbA1c levels were measured by the glucose- oxidase method and anion exchange high performance liquid chromatography, respectively (arkray ELUENT 80A, Japan). Lipid profiles, ALT, AST, GGT, CysC, UA, serum albumin and Cr were analyzed using a 7060 full-automatic biochemical analyzer (Hitachi, Tokyo, Japan). Serum albumin level was determined using the bromocresol green method according to the manufacturer’s instruction, and its normal range was 40–55 g/L. The neutrophil and lymphocyte counts, NLR, and RDW were determined using an automated blood cell counter (Mindray BC-6800, Shenzhen, China). Estimated glomerular filtration rate (eGFR) was calculated according to serum Cr, gender, age, and race [19,20,21,22]. During the morning, spot urine samples were collected to measure microalbumin with immunoturbidimetric tests, and creatinine enzymatically. The urinary microalbumin to creatinine ratio (ACR; mg/g creatinine) was calculated by dividing urine microalbumin by urine creatinine.
Measurements of ankle-brachial index (ABI) and diagnosis of DPN
Ankle-brachial index (ABI) values were measured by a continuous-wave Doppler ultrasound probe (Vista AVS, Summit Co.) and vibration perception thresholds (VPT) value was performed by a neurothesiometer (Bio-Thesiometer; Bio-Medical Instrument Co., Newbury, OH, USA) as previously described [19, 20]. Touch sensation was assessed by a 10 g Semmes–Weinstein monofilament. All measurements were operated by the same experienced physician. Well-trained professionals used a standard questionnaire and face-to-face interviews to obtain information on participants’ neuropathy symptoms, including feelings of burning, numbness, tingling, pain, fatigue, and paresthesia, and performed the clinical examinations. DPN was defined as VPT values of 25 V or more and/or inability to feel the monofilament .
All statistical analyses were performed using the SPSS 20.0 software (Chicago, IL). All data were first analyzed for normality of distribution using the Kolmogorov–Smirnov test of normality, and homogeneity of variance. Normally distributed continuous variables are presented as mean ± standard deviation (SD) and nonparametric distributed continuous variables are presented as median (25th percentile–75th percentile). Categorical variables are express as numbers (percentages).
Serum albumin quartiles were categorized as follows: Q1 (21.70–37.60 g/L), Q2 (37.70–41.30 g/L), Q3 (41.40–44.40 g/L), and Q4 (44.50–57.60 g/L).
Moreover, all participants were divided into DPN group (n = 231) and no DPN group (n = 1234). A previous work has proposed that the mean ± SD of serum albumin in T2DM patients with and without DPN is 42.51 ± 2.88 g/L and 44.28 ± 3.56 g/L, respectively . We set type I error α at 0.05 and type II error β at 0.1, and assume that the sample size ratio between the T2DM patients with DPN group and the T2DM patients without DPN group is 1: 5.5. Based on these assumptions and a previous work, sample size calculations indicated that a total of 269 patients (41 T2DM patients with DPN, and 228 T2DM patients without DPN) were required for the study. In other words, this study would provide 90% statistical power as long as the sample size in the T2DM patients without DPN group is ≥ 228 and the sample size in the T2DM patients with DPN group is ≥ 41.
Differences in clinical and biochemical parameters between two groups or among three or more groups were compared by Student’s t-test, Mann–Whitney U test, One-way analysis of variance (ANOVA), Kruskal–Wallis test, or χ2 test. Associations between serum albumin and other variables were tested by the Spearman’s correlation analysis and also partial correlation analyses controlling for gender, age, BMI and diabetic duration. A binary logistic regression analysis was conducted to estimate the odds ratios (ORs) for DPN according to quartiles of serum albumin in type 2 diabetes. Model 1 was unadjusted. Model 2 was adjusted for age, gender, and BMI, and diabetic duration. Model 3 was additionally adjusted for SBP, DBP, FBG, HbA1c, TC, TG, HDL-C, LDL-C, NLR, RDW, ALT, AST, GGT, UA, serum Cr, CysC, eGFR, ACR, and ABI. The Q1 served as the reference group, and Odds ratios (OR) and 95% confidence intervals (CI) were estimated. Receiver operating characteristic (ROC) curve analysis was performed to identify the optimal cutoff values for serum albumin as indicators of DPN.
In all statistical tests, a two-sided P < 0.05 was considered statistically significant.