Effect of Carnosine Supplementation as Add-On Therapy With Vitamin B Complex in People With Type 2 Diabetes and Diabetic Neuropathy: A Randomized Controlled Study Free

This was a 12-month, prospective, randomized, open-label, controlled trial that was carried out at the outpatient diabetes and endocrinology clinic of Ain Shams University Hospital from February 2021 to February 2022.

Sixty patients with type 2 diabetes complicated with diabetic neuropathy who matched the inclusion criteria were selected from among the clinic’s regular patients and enrolled in the study. The study protocol was accepted by the Ethical Committee of Beni Suef University (REC-H-PhBSU-21004), and the study was registered on ClinicalTrials.gov (NCT05422352). Informed consent was obtained from all participants at the start of the study. Reporting of the study follows the recommendations of the 2010 Consolidated Standards of Reporting Trials (12).

Inclusion criteria were a diagnosis of type 2 diabetes according to American Diabetes Association criteria (13), diabetic neuropathy, age 50–85 years, disease duration ≥5 years, and taking oral antidiabetic therapy. Active diabetic neuropathy with abnormal nerve conduction readings is sufficient to define diabetic neuropathy, according to the American Academy of Neurology, in addition to the simplified short protocol of the American Academy of Neuro-electrophysiological Medicine (14).

Exclusion criteria included nondiabetic neuropathy caused by exposure to organochlorine pesticides; medications such as sulfa drugs, corticosteroids, and cisplatin; and neurological illnesses such as Parkinson’s disease, epilepsy, and malignancy. Additional exclusion criteria were chronic diseases such as hypothyroidism, renal disease, hepatic disease, or HIV; any chronic infection; or medications that could affect the study results, such as aldose reductase inhibitors, mexiletine, antidepressants, opiates, capsaicin, neuroleptics, antioxidants, and especially methyl cobalamin, anticonvulsants, pyridoxine, or other vitamin B complex preparations. Also, individuals who had a BMI ≥40 kg/m² or were pregnant or breastfeeding were not eligible.

Comprehensive medical histories were taken and physical examinations were performed on all subjects. The examination specifically assessed the age of diabetes onset, disease duration, and presence of chronic diabetes complications, with a particular focus on diabetic neuropathy. Any adverse effects associated with medication administration were also documented. Anthropometric data were collected, and BMIs were calculated. Blood pressure was measured using a mercury sphygmomanometer after a 5-minute resting period in the seated position.

A total of 100 individuals with diabetic neuropathy were assessed for eligibility; of them, 25 did not meet inclusion criteria, 5 declined to participate, and 10 were excluded. Sixty patients were therefore randomized equally into one of two groups based on the therapy they received and followed for 3 months.

Group 1 included 30 individuals who received carnosine capsules in combination with vitamin B complex capsules for 12 weeks. White oval-shaped hard carnosine capsules (15) contained 500 mg of β-alanyl-l-histidine taken orally twice daily. Inactive ingredients included cellulose (for the capsule), as well as magnesium stearate and stearic acid, both from vegetable sources. Vitamin B complex capsules contained vitamins B1 (150 mg), B6 (100 mg), and B12 (1 mg) (Neurovit, European Egyptian Pharmaceutical Industries, Alexandria, Egypt) and were taken twice daily.

Group 2 included 30 patients who received only the vitamin B complex capsules with the same formula described above twice daily for 12 weeks.

All patients completed a questionnaire comprising the 15 yes/no questions of the MNSI before the study began and again at its end. Thirteen of these questions assessed diabetic neuropathy symptoms, one assessed peripheral vascular disease, and one assessed general asthenia. Physical examination assessed the appearance of the feet (deformities, dry skin, callus, infection, and fissure), ulceration of the feet, ankle reflexes, vibration perception at the great toe, and monofilament. The MNSI score directly correlates with the severity of neuropathy. This applies to both the questionnaire and physical examination components of the questionnaire (16). Positive (pain, tingling, and temperature feeling) and negative (numbness) sensory complaints, muscular weakness and cramping, foot ulcers or cracks, and amputation were all covered in the questionnaire (17). All questions were scored as 0 for a negative response or 1 for a positive response (negative replies on items 7 and 13 counted as one point). Diabetic neuropathy was confirmed with seven or more affirmative replies on the MNSI questionnaire (17). Arabic translation of the questionnaire was done by Trjim.com for translation in Cairo and revised by a consultant neurologist. The difference in MNSI score from baseline to 12 weeks was used to assess the effectiveness of the therapy.

Both groups of patients underwent nerve conduction studies (NCSs) in left and right lower limbs using the Nihon Kohden X1 MEB-2300 device. In the common peroneal and posterior tibial nerves, motor nerve conduction characteristics such as compound muscle action potential amplitudes, distal latency, and motor conduction velocity were assessed. Peak latency and amplitude of sensory nerve action potential were measured as part of sensory conduction research. To establish enough uniformity in the experimental protocol, all participants’ stimulation and recording conditions were kept consistent. According to the criteria shown in Table 1, changes in NCS results from baseline in week 12 were regarded as an improvement in diabetic neuropathy.

Neurological assessments were performed by a qualified and certified neurologist who was not aware of patients’ randomization throughout clinical examinations and conduction of the NCSs.

Peripheral blood samples were obtained in sterile vacutainer tubes containing potassium-ethylene diamine tetra-acetic acid (final concentration 1.5 mg/mL) for A1C determination. For biochemical examination, clotted blood samples of NGF were obtained, and the serum was separated by centrifugation at 1,000 RPM for 15 minutes. The serum was kept at 80°C until it was used. Blood was collected at the beginning of the study (day 0) and at 12 weeks. Patients were free of any potentially interfering or confounding factors such as fever or infections at the time of blood collection. FBG level, fasting lipid profile, and serum creatinine were estimated using Cobas Integra 800 (Roche Diagnostics, Mannheim, Germany). To assess the regulation of growth, maintenance, and proliferation of neurons, a human NGF enzyme-linked immunosorbent assay kit was provided by the bioassay technology laboratory (Shanghai Crystal Day Biotech Co.), according to manufacturer instructions.

It was reported in previous studies that the appropriate interval of carnosine treatment was 3 months (18), and this time period was used to reveal the maximum effect of carnosine as add-on therapy to vitamin B complex on NGF levels, MNSI score, and NCS results. All patients were thoroughly and clinically followed up monthly during the study duration for assessment of effects and compliance with both vitamin B complex and carnosine treatment and for monitoring of adverse effects. Assessment of compliance with treatment was based on the amount of dispensed and returned capsules. According to the literature, noncompliance was defined as taking <80% of the study medicine or of the vitamin B complex.

Data were gathered, reviewed, and entered into IBM SPSS, v. 23, statistical software. Quantitative data were presented as mean ± SD and ranges when found to be parametric and median with interquartile range (IQR) when found to be nonparametric. Qualitative variables were presented as numbers and percentages.

Comparisons between groups on qualitative variables were done by using the χ² test. Comparisons between groups on quantitative data with parametric distribution were done using an independent t test, whereas the comparison between groups on quantitative data with nonparametric distribution was done by using a Mann-Whitney U test. Comparisons between two paired groups on quantitative data with parametric distribution were done using a paired t test, and comparisons between two paired groups on quantitative data with nonparametric distribution were done using a Wilcoxon test. Spearman correlation coefficients were used to assess the correlation between two quantitative parameters in the same group. The CI was set to 95%, and the margin of error accepted was set to 5%. P values <0.05 were considered significant, and those <0.01 were highly significant.

There were no significant differences in baseline demographic and laboratory characteristics of the two groups, as shown in Table 2.

The MNSI questionnaire was used as a subjective assessment of progress. There was a significant difference in MNSI score after 12 weeks of supplementation with carnosine in the treatment group (P < 0.01), as shown in Figure 1A and Table 3. In contrast, no difference in scores was found in the control group from baseline (8.07 ± 1.20) to 12 weeks (8.37 ± 1.22) (P = 0.071).

After 12 weeks of carnosine and vitamin B complex treatment, supplementation showed a significant increase in NGF levels in the carnosine group and alternatively no significant difference in levels of NGF in the control group between baseline and 12 weeks (Figure 1B and Table 3).

The assessment of electrophysiological nerve conduction showed a significant change in the latency and conduction velocity and amplitude of sensory nerves and motor nerves after 12 weeks of supplementation of carnosine plus vitamin B complex compared with group 2, who received vitamin B complex only (Figure 2).

NGF and MNSI scores were tested for association with other studied variables in patients with diabetic neuropathy. A negative correlation was found between NGF and MNSI (r = −0.472, P = 0.009).

Diabetic neuropathy is highly prevalent around the world and has devastating consequences, with major impacts on quality of life and morbidity. About one-third of all people with diabetes suffer from diabetic neuropathy. There are no approved pharmacotherapies for the prevention of diabetic neuropathy (2).

In diabetic neuropathy, both large myelinated fibers and small, thinly myelinated fibers are involved. Diabetes neuronal abnormalities can be caused by experimental reduction of particular neurotrophic factors, their receptors, or their binding protein. In experimental diabetes models, there is a decrease in the obtainability of these growth factors, which may result in metabolic abnormalities or may be independent of glycemic control. These neurotrophic factors are crucial for neuronal maintenance, apoptosis resistance, and regeneration potential (19).

Increasing evidence suggests that the accumulation of advanced glycation end products (AGEs) plays a major role in the pathogenesis of diabetic neuropathy (20). It has been postulated that AGE-modified peripheral nerve myelin is susceptible to macrophage phagocytosis, which induces macrophages to release protease, which in turn may help demyelination in diabetic neuropathy (21).

There is also increasing evidence of NGF deficiency in diabetes. NGF plays a significant role in the pathogenesis of diabetic neuropathy, and increasing its levels may help to prevent the progression of diabetic neuropathy (5). Low NGF levels in diabetes might be the result of decreased NGF synthesis, transport, or both, most likely as a consequence of glucose-induced oxidative stress.

In the current study, the supplementation effect of carnosine together with vitamin B complex compared with vitamin B complex alone was evaluated and showed a significant improvement in NGF levels after 12 weeks of follow-up. Previous studies have shown that carnosine may stimulate neuronal cells by increasing neurotrophin synthesis in glial cells (22). Other studies have shown the effect of carnosine in the prevention of diabetes complications, mainly diabetic nephropathy, and in decreasing the oxidative stress that usually accompanies diabetes (23).

Carnosine is considered an ideal antioxidant with unique properties such as being nontoxic, water soluble, and heat inactive; additionally, it is a nutrient with neuroprotective properties (10). Carnosine has been found to prevent the formation of AGEs by reducing blood glucose levels, thereby preventing early glycation and even reversing previously formed AGEs (24). Moreover, carnosine can inhibit the oligomerization of proteins that play an important role in the degeneration of neurons in many neurodegenerative illnesses (e.g., Alzheimer’s and Parkinson’s diseases) (25). Additionally, the results of the current study showed a significant reduction in MNSI score, which is considered a noninvasive method of measuring improvement in diabetic neuropathy. Patients experienced relief in burning sensations and touch sensitivity, fewer leg cramps, and a reduced worsening of symptoms at night. These findings were in line with a previous study, in which carnosine was found to improve diabetic neuropathy, particularly abnormal sensory perception (25).

The current study used NCSs as a parameter to assess the function of both motor and sensory peripheral nerves. We found a significant improvement in latency and conduction velocity for both sensory and motor nerves and a significant improvement in the amplitude of sensory nerves. Previous studies have shown improvement in autonomic and somatic symptoms with a decrease in the signs of diabetic neuropathy after supplementation of methylcobalamin (26). To the authors’ knowledge, this is the first study to confirm the ability of carnosine in combination with vitamin B complex to alleviate symptoms of diabetic neuropathy as assessed with NCSs.

One limitation of this study is its small sample size. Thus, larger, multicenter randomized trials will be needed to confirm our findings of the effects of carnosine supplementation in diabetic neuropathy and NCS results for extended periods of time.

NGF plays a role in the pathogenesis of diabetic neuropathy, and supplementation of carnosine in combination with vitamin B complex was found to enhance NGF levels and to improve diabetic neuropathy symptoms as assessed by the MNSI and NCSs.

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.F.S. supervised the development of 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 of the study and revised the manuscript. H.R. contributed to the study design and revised 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.