Role of Carnosine in Combination With Vitamin B Complex in Preventing the Progression of Diabetic Neuropathy in People With Type 2 Diabetes Free

This 12-month prospective randomized, open-label, controlled trial was conducted in the outpatient diabetes and endocrinology clinic of Ain Shams University Hospital in Cairo, Egypt, from February 2021 to February 2022. Drug administration was conducted according to a computer-generated 1:1 randomized schedule. The randomization sequence was kept in sealed, sequentially numbered envelopes stored in the investigational drug pharmacy to maintain allocation concealment.

Sixty patients with type 2 diabetes complicated by diabetic neuropathy who matched the inclusion criteria were engaged in the diabetes clinic at Ain Shams University. The study protocol was accepted by the Beni Suef University Ethics Committee (REC-H-PhBSU-21004) and conformed to the Helsinki Declaration of 2008, and the trial was registered at ClinicalTrials.gov (NCT05422352). Before participating, each participant provided informed consent. Study reporting follows the Consolidated Standards of Reporting Trials 2010 statement (19). No funding was received for the study.

The sample size was calculated using Med Calc, v. 11.3.0.0, software. A previous study (9) found that neopterin level could significantly detect the occurrence of DPN at the cutoff point of 32 nmol/L with a sensitivity of 100%, specificity of 96.7%, and area under the curve of 0.989. With a 95% CI and the power of the test set to 90%, the minimum sample size needed per group was found to be 12 participants.

Individuals who were diagnosed with type 2 diabetes according to American Diabetes Association criteria, were 50–85 years of age, had a diabetes duration ≥5 years, and were on oral antidiabetic therapy were eligible for the trial. According to the American Academy of Neurology, active diabetic neuropathy with aberrant nerve conduction measurement is sufficient to define DPN using the streamlined brief protocol of the American Academy of Neuro-electrophysiological Medicine (20,21).

Exclusion criteria included nondiabetic neuropathy such as may be caused by organochlorine pesticide exposure or drugs (e.g., sulfa drugs, corticosteroids, cisplatin, or taxol), severe neurological diseases (e.g., Parkinson’s disease or epilepsy), malignancy, chronic illnesses (e.g., hypothyroidism, renal dysfunction, hepatic disease, existing chronic infection, or HIV infection), and BMI ≥40 kg/m², as well as current pregnancy or breastfeeding.

All participants underwent a thorough clinical examination including a complete medical history, with particular attention on the age at diabetes diagnosis, diabetes duration, and chronic diabetes sequelae, primarily peripheral neuropathy, as well as adverse effects of therapy. BMI and anthropometric measurements were assessed. Blood pressure was measured using a mercury sphygmomanometer (Granzia Italy, Corso Europa, Italy) after 5 minutes in a seated position.

A total of 100 individuals with diabetic neuropathy were assessed for eligibility; 25 did not meet inclusion criteria, 5 declined to participate, and 10 were excluded. Sixty individuals were randomized into one of two arms (30 patients per arm) and were followed for 3 months (Figure 1). Those in group 1 received L-carnosine capsules in combination with vitamin B complex capsules for 12 weeks. The L-carnosine capsules included 500 mg of β-alanyl-L-histidine (Now Foods, Bloomingdale, IL) and were taken orally twice daily. Other inactive substances included cellulose and magnesium stearate and stearic acid, both of which were derived from vegetables. The supplements were in the form of white, oval-shaped hard capsules (22). The vitamin B complex capsules (Neurovit, European Egyptian Pharmaceutical Industries, Cairo, Egypt) contained 1 mg each of B6 + B1+ B12 taken twice daily. Participants in group 2 received only the vitamin B complex capsules twice daily for 12 weeks.

In sterilized vacutainer tubes, peripheral blood samples were taken on potassium-ethylene diamine tetra-acetic acid 1.5 mg/mL (Beckton Dickinson, Franklin Lakes, NJ) to measure A1C. Blood samples that had been clotted were taken for biochemical examination of neopterin and MDA levels, and the serum was separated by centrifugation for 15 minutes at 1,000g. The serum was kept at −80°C (−112°F) until it was used. Blood was drawn at the start of the study and at the end, at week 12. When the blood samples were collected, participants were free of potentially conflicting or interfering factors such as fever or illness.

The Cobas Integra 800 analyzer (Roche Diagnostics, Mannheim, Germany) was used to evaluate fasting blood glucose (FBG) levels and assess fasting lipid profiles. Serum total cholesterol ≥200 mg/dL indicated dyslipidemia.

Supplementation with carnosine was planned to last for 12 weeks, as previously reported (23–25), to reveal the maximum apparent effects of carnosine supplementation on neopterin and MDA. During the trial, all patients underwent a 4-week follow-up visit, including FBG and lipid profile measurements, to assess compliance, evaluate the effects of both vitamin B complex and carnosine, and monitor for indicators of potential side effects. Compliance was assessed by counting the returned capsules. Based on the literature, noncompliance was defined as taking <80% of the study medicine and vitamin B complex capsules.

The primary study end point was the difference in neopterin level after 12 weeks of treatment in participants receiving carnosine plus vitamin B complex supplementation versus those receiving only the vitamin B complex supplementation. At 12 weeks, the secondary end point was the difference in MDA levels between the two groups. The safety end point was the incidence of any adverse events during the study.

To assess nerve function, all participants completed the brief, 15-item Michigan Neuropathy Screening Instrument (MNSI) at the beginning and end of the study. The MNSI uses a 15-point scoring system. Thirteen questions assess symptoms of DPN, including positive sensory experiences (pain, tingling, and temperature changes) and negative sensory experiences (numbness), along with muscular weakness, cramping, and foot issues such as ulcers, cracks, or amputations. A separate question addresses peripheral vascular disease, and another evaluates general fatigue (asthenia). Higher scores indicate greater severity of neuropathy (26). Most MNSI items are scored using a simple scoring system in which “no” = 0 and “yes” = 1. However, for questions 7 (temperature sensitivity) and 13 (foot sores/amputation), a “no” answer indicates a potential neuropathy symptom and is therefore scored as 1 point. A score ≥7 suggests the presence of neuropathy (27).

Data are presented as mean ± SD. All experimental data were statistically analyzed using SPSS, v. 23, statistical software. Unpaired Student t tests were used to determine statistical significance. Results were considered significant at P <0.05.

Table 1 shows no significant between-group differences in baseline demographic and laboratory variables.

Carnosine supplementation significantly reduced mean FBG and A1C levels in group 1 compared with group 2 (P <0.001), as shown in Tables 2 and 3. It also significantly lowered mean systolic blood pressure levels (P <0.018) but did not affect diastolic blood pressure levels.

No significant between-group differences were found concerning total cholesterol and triglyceride levels; however, a significant difference was found in the mean difference between groups (P <0.001).

After 12 weeks of carnosine supplementation, MDA levels were significantly decreased in group 1 compared with group 2 (P <0.001). Neopterin levels decreased in group 1 compared with group 2 (P <0.001), as shown in Table 2.

Neopterin and MDA were tested for association with other studied variables, and a significant positive correlation was found between neopterin and MDA (r = 0.775, P = 0.000) (Figure 2).

After 12 weeks of carnosine supplementation, MNSI scores improved significantly in group 1 (P <0.01) (Figure 3). In contrast, group 2 showed no change in MNSI score after 12 weeks of vitamin B complex supplementation alone (baseline 8.07 ± 1.20 vs. post-therapy 8.37 ± 1.22, P = 0.071).

Supplementation with carnosine had no side effects and was well tolerated.

DPN is considered the most painful and disabling vascular complication of diabetes and affects quality of life for many people (28). Many processes contribute to the progression of diabetic neuropathy, including increases in polyol pathway flux and sorbitol buildup, which lead to fructose depletion and a reduction in sodium-potassium pump activity (5). Moreover, disturbance in fatty acid metabolism causes a modulation in nerve membrane construction, and buildup of advanced glycation end products (AGEs) results in nerve dysfunction, demyelination of nerve fibers that are susceptible to phagocytosis, and stimulation of macrophages to secrete protease (4). Glycation interactions between reducing sugars and free amine groups result in the creation of AGEs. A series of additional events follow, resulting in changes in membrane function and proteome damage, such as crosslinking of proteins (29).

Carnosine has been shown to have many beneficial effects in humans, including on neurodegenerative diseases such as Alzheimer’s disease, aging, exercise performance, and prevention of type 2 diabetes and complications such as nephropathy (24,30–32).

In this study, group 1, who received supplementation with both carnosine and vitamin B complex, showed significantly lower neopterin and MDA levels than group 2, who received only vitamin B complex supplementation. Also, individuals in group 1 experienced better glycemic control and lower blood pressure than those in group 2. These findings reflect the ability of carnosine supplementation to decrease inflammation and ROS, which usually accompany diabetic neuropathy, by decreasing neopterin and MDA levels and ultimately reducing the progression of diabetic neuropathy.

Previous studies have reported that low-dose carnosine supplementation improves glucose tolerance, reducing both plasma glucose and insulin resistance (24). Additionally, a growing body of evidence from animal research suggests that carnosine supplementation may be beneficial in protecting against diabetes complications (31,33). Carnosine reduced plasma glucose levels and insulin resistance and increased insulin secretion and β-cell mass, as well as decreasing blood pressure, markers of advanced glycation, and chronic inflammation in diabetic rats (34,35).

The mechanism of reduction in blood glucose and blood pressure results from a decrease in the sympathetic nerve activity of the liver, adrenal glands, and pancreas (34). In this study, we observed a lower systolic blood pressure but no change in diastolic blood pressure in group 1. The effect of carnosine on blood pressure has been reported in previous studies and attributed to its vasodilatory effect and relaxation of smooth muscle through cGMP generation (36). This study also found a significant decrease in the neopterin levels in group 1 compared with group 2. High levels of neopterin are commonly related to inflammation and oxidative stress and are almost twice as high in people with type 2 diabetes than in those with type 1 diabetes (11). Oxidative stress is also elevated in people with diabetes complications more so than in those without complications (36). It also signals an activation of the cell-mediated immune response and an increase in ROS (37).

Previous studies have found that neopterin is a good inflammatory marker for assessing diabetes progression because its levels increase from prediabetes to type 2 diabetes (37). Also, DPN is accompanied by inflammatory and immune processes and elevated neopterin levels (9). This study showed a significant decrease in MDA levels in group 1 compared with group 2. This finding agrees with previous studies showing that carnosine decreased MDA in combination with free radical scavenging and metal chelation effects (38).

This study also demonstrated a significant decrease in MNSI scores, suggesting alleviation of neuropathy symptoms and a positive neuropathy assessment. Group 1 participants reported relief from burning sensations and touch sensitivity, fewer leg cramps, and reduced worsening of nighttime symptoms. These findings align with previous research suggesting the effectiveness of carnosine in improving diabetic neuropathy and especially abnormal sensory perception (39).

This study found a strong positive correlation between neopterin and MDA, indicating that oxidative stress affects neopterin levels, as previously described (40). In a previous study, carnosine was shown to have neuroprotective actions through oxidative stress prevention, decrease of intraneuronal amyloid β, and modulation of nitric oxide production (41). A vast body of literature supports the impact of oxidative stress in the development of neurodegenerative disorders (42).

DPN is one of the complications of diabetes in which neurons are highly subjected to oxidative stress, inflammation, hyperglycemia, and activation of the polyol pathway, and all pathways play an important role in the development of diabetic neuropathy (43).

ROS are a significant cause of chronic low-grade inflammation and are created in excess by hyperglycemia-induced oxidative stress caused by an imbalance between the peroxidation and antioxidant defense systems (44). Carnosine has been shown to have antioxidant, anti-glycating, metal ion chelating, and anti-inflammatory properties (45). AGEs that result from hyperglycemia damage mechanisms of repair, weaken nerve blood flow, impair the integrity of neurons, and reduce neurotrophic support (43).

Data from animal studies suggest that supplementation with carnosine prevents the progression of diabetic neuropathy in diabetic rodents (46). Moreover, the preventive effect of carnosine on lipid peroxidation and decreasing the levels of MDA and cytokines have also been reported from rat studies (47).

Limitations of this study are its small sample size, single study site, and limited duration. Larger and multicenter trials will be needed to confirm our findings over longer time periods.

Oxidative stress plays a significant role in the development of diabetic neuropathy. Supplementation with carnosine as adjuvant therapy with vitamin B complex supplementation in people with type 2 diabetes can play a significant role in preventing the progression of diabetic neuropathy. Carnosine supplementation holds promise for protecting neurons from further oxidative damage and, in a dose of 500 mg twice daily, has no adverse effects and is well tolerated.

No potential conflicts of interest relevant to this article were reported.

M.H.H. conceived of and implemented the study and contributed to its design; conducted the practical work of the research, including recruiting patients, collecting samples, and administering questionnaires; and wrote the manuscript. H.E.D.M.A. supervised the practical work and contributed to the study design. N.A.I. participated in the practical work and revised the manuscript. H.R. contributed to the study design and revised the manuscript. H.F.S. supervised the development of the manuscript. M.H.H. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.