The study was a randomized controlled trial comparing the efficacy between oral carnosine supplementation and placebo groups, as an additional treatment from conventional therapy. The study was conducted among patients with T2DM treated at Phramongkutklao Hospital between 1 April 2018 and 31 March 2019, with all subjects selected by inclusion criteria. Patients were randomized in blocks of four and allocation concealment, then divided in two groups at a ratio of 1 : 1, as shown in Fig. 1. Group 1 constituted the group receiving oral carnosine, having a powdery appearance in gelatin capsules of 500 mg (two capsules each time) after breakfast and dinner. The patients required a daily dose for 90 consecutive days. Group 2 comprised the group receiving the placebo, which had a powdered appearance in gelatin capsules, similar to carnosine in all respects. The patients had to take 2 capsules each time, after breakfast and dinner for 90 consecutive days, the same as group 1. The study complies with the Declaration of Helsinki (1964). The study was registered at Thai Clinical Trials Registry (TCTR) (TCTR20200724002).
The inclusion criteria included T2DM, age of 18 years old or older, urine albumin-creatinine ratio (UACR) of 30 to 299 mg/g creatinine (Cr) at least two in three within three to six months, stable dose of ACEIs or ARBs for blood pressure control at least three months before enrolling, and stable hemoglobinA1C (HbA1C) within three months before the study. The exclusion criteria comprised active infections, CKD from nondiabetic cause, advanced malignancy, history of hypersensitivity to protein nutrients, problems with nutrient absorption of the gastro-intestinal tract and liver disease.
The data we collected before and after in this study, were relevant information on diabetic nephropathy, including diagnostic criteria, duration of the disease and complications of diabetes mellitus such as diabetic retinopathy and diabetic neuropathy. Also, other underlying diseases, including hypertension, heart disease, liver disease, infectious diseases and malignancy were recorded. The history of medication including antihypertensive drugs and lipid lowering agents were recorded. Physical examination data including height, weight, blood pressure and body mass index (BMI) were collected. The laboratory tests including fasting plasma glucose (FPG), HbA1C, blood urea nitrogen, Cr, calculation of estimated glomerular infiltration rate using the 2009 Chronic Kidney Disease Epidemiology Collaboration Equation, total cholesterol, triglyceride, low density lipoprotein and high density lipoprotein were noted. Participants would receive urine testing for urinary TGF-β level by enzyme linked immunosorbent assay. UACR by immunonephelometric assay method, before and after receiving carnosine or placebo for a period of 90 days is shown in Fig. 1.
Follow-up study results
The researcher verified consistent carnosine intake by asking for the remaining tablets and followed up the side effects of carnosine intake by using the adverse effects assessment form (Naranjo’s algorithm). The primary outcome was the change of urinary TGF-β level after 12 weeks in the oral carnosine supplementation group, compared with that of the placebo group. The secondary outcome was the improving level of UACR after 12 weeks in the oral carnosine supplementation group compared with that of the placebo group.
Statistical software was used for statistical analysis. Descriptive statistics were used to present general information, laboratory results and urinary substances measurement level, including percentages, averages and standard deviations in the case of normal distributed continuous data. Inferential statistics was used to compare between general information, laboratory results and the percentage changes of variables in the oral carnosine supplementation and placebo groups, based on Student’s test statistics. Pearson chi-square test or Fisher's exact test was used for discrete or categorical variables. Paired-sample t tests was used for the continuous variables and presented by the relative risk of 95% confidence intervals with p-value less than 0.05, regarded as statistically significant.