Effect of Cocoa Supplementation Peripheral and Autonomic Diabetic Neuropathy

Study Overview
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Brief Summary
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Type 2 diabetes mellitus is a high incidence disease in Mexico and is associated with the development of chronic degenerative complications such as diabetic neuropathy. The latter manifests itself as a set of disorders that occur as a consequence of a chronic hyperglycemic state that can induce oxidative stress and inflammation, resulting in damage to the autonomic and peripheral nervous system. In Mexico, it has been reported that this complication usually occurs between 29% and 90% of patients with diabetes. Cocoa is a food with a high content of flavonoids, which are phenolic compounds with antioxidant and anti-inflammatory effects. Additionally, its consumption has been associated with a decrease in hyperglycemia and insulin resistance, improvement in mitochondrial function, and, based on the above, an effect on diabetic complications has been suggested; This has been demonstrated in in vivo and in vitro models, but not in the human population. Once the symptoms of diabetic neuropathy have started, palliative treatments are prescribed, and to date there are no pharmacological compounds that have been shown to reverse the consequences of diabetic peripheral and autonomic neuropathy. Additionally, clinical trials of compounds with antioxidant properties have only performed subjective evaluations based on questionnaires on the perception of the improvement of diabetic neuropathy and some biochemical markers or nerve conduction tests, however, the results shown have not been conclusive. This is why a double-blind, randomized controlled clinical trial is proposed, with the objective of evaluating the effect of cocoa supplementation in patients with type 2 diabetes mellitus and peripheral and autonomic diabetic neuropathy on a) the biochemical profile, which includes the evaluation of the glycemic and lipid profile, quantification of pro-inflammatory cytokines and oxidative stress markers; b) the clinical profile through the application of standardized questionnaires, anthropometric measurements and blood pressure, and c) somatosensory processing through the paired pulse H reflex test. The hypothesis of this study is that cocoa supplementation will have a beneficial effect on the biochemical and clinical profile and somatosensory processing of peripheral and autonomic diabetic neuropathy.

Detailed Description
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Type 2 diabetes mellitus (T2DM) is a high incidence disease in Mexico and is associated with the development of chronic degenerative complications such as diabetic neuropathy. The latter manifests itself as a set of disorders that occur as a consequence of a chronic hyperglycemic state that can induce oxidative stress and inflammation, resulting in damage to the autonomic and peripheral nervous system. In Mexico, it has been reported that this complication usually occurs between 29% and 90% of patients with diabetes. Cocoa is a food with a high content of flavonoids, which are phenolic compounds with antioxidant and anti-inflammatory effects. Additionally, its consumption has been associated with a decrease in hyperglycemia and insulin resistance, improvement in mitochondrial function, and, based on the above, an effect on diabetic complications has been suggested; This has been demonstrated in in vivo and in vitro models, but not in the human population. Once the symptoms of diabetic neuropathy have started, palliative treatments are prescribed, and to date there are no pharmacological compounds that have been shown to reverse the consequences of diabetic peripheral and autonomic neuropathy. Additionally, clinical trials of compounds with antioxidant properties have only performed subjective evaluations based on questionnaires on the perception of the improvement of diabetic neuropathy and some biochemical markers or nerve conduction tests, however, the results shown have not been conclusive. This is why a double-blind, randomized controlled clinical trial is proposed, with the objective of evaluating the effect of cocoa supplementation in patients with type 2 diabetes mellitus and peripheral and autonomic diabetic neuropathy on a) the biochemical profile, which includes the evaluation of the glycemic and lipid profile, quantification of pro-inflammatory cytokines and oxidative stress markers; b) the clinical profile through the application of standardized questionnaires, anthropometric measurements and blood pressure, and c) somatosensory processing through the paired pulse H reflex test. Hypothesis: The hypothesis of this study is that cocoa supplementation will have a beneficial effect on the biochemical and clinical profile and somatosensory processing of peripheral and autonomic diabetic neuropathy. Statistical analysis: For the evaluation of the intragroup variables, a statistical analysis will be carried out with ANOVA for repeated samples with Tukey's post hoc, or, where appropriate, Friedman with Dunn's post hoc, as well as Student's t for dependent groups, or in its case, with Wilcoxon. The intergroup comparison will be made with Student's T for independent samples, or if applicable, with Mann Whitney's U, considering p <0.05 as statistical significance and using the statistical software GraphPad Prism version 5. The H reflex test will be performed by electrical stimulation through disposable surface electrodes connected to a constant current bipolar electrical stimulator (Digitimer DS8R). The recording of the electrophysiological signals will be carried out using surface electrodes connected to the signal acquisition and amplification system (LabChart and PowerLab 8/35, ADInstruments). The signals obtained will be sampled at 10 kilohertz (KHz) with a 0.5- 500 Hz band-pass filter. The signals will be stored in a computer for later analysis. The placement of electrodes for stimulation will be carried out as follows: the active electrode (anode) at the level of the Achilles tendon, the positive electrode (cathode) above the inverted v between the calf muscles (gastrocnemius). Subsequently, the reference electrode will be placed at the level of the gastrocnemius heads. It will be stimulated behind the knee where the tibial nerve has its anatomical path. The test will start with an intensity of 0 milliamp (mA) and then pulses will be given every 0.5 millivolts (mV) until the evoked potential (H reflex) is observed in a consistent and clearly identifiable way as a function of latency (35-45 ms). The electrical stimulus consists of the application of 1 square pulse (1 ms duration each pulse) every 10 seconds (10 pulses in total). The maximum intensity of the applied current will be according to the sensitivity and tolerance of the individual in both lower limbs during the tests on the sensory and motor nerves. The applied electrical pulse should not cause a painful sensation, but it can cause a tingling sensation. The test will be suspended if the individual reports pain or does not wish to continue with the research protocol. The H reflex test will be done in two parts. The first part of the protocol consists of determining the stimulus intensity vs. amplitude of motor responses from the appearance of the M wave and the H wave, for which the electric current will be increased in steps of 0.5 µA until the appearance of the waves. For this part, only one electrical pulse (1 ms duration) will be given every 10 seconds. The intensity of electrical current that will be used for the second part of the protocol will be that whose value in the amplitude curve of the H wave-electrical current intensity reaches 60% of the maximum amplitude. This stimulation value guarantees the reproducibility and minimum variability of this wave, which also prevents muscle contraction that contaminates the electrical register. The second part constitutes the paired electrical stimulation test in which two electrical pulses (1 ms in duration) will be produced at different frequencies between the pulses: 0.1, 1, 5 and 10 Hz. The interval between the paired pulses will be 10 s, until completing 10 series. The electrophysiological recordings will be analyzed with the Clampfit 10.0 software. The latency and amplitude of the evoked potentials H1 and H2 will be determined for each electrical pulse and at all stimulation frequencies, taking the stimulus artifact as a reference. Subsequently, the ratio of the amplitude of the paired H2/H1 pulses will be determined to establish the modulation of spinal excitability. A ratio ≥0.6 for any stimulation frequency will be considered as an indicator of dysfunction in somatosensory processing according to Marshall et al.

Official Title
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Evaluation of the Effect of Cocoa Supplementation on Biochemical and Clinical Profile and Sensory-motor Processing of Peripheral and Autonomic Diabetic Neuropathy: Randomized Clinical Trial

Conditions
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Diabetic Neuropathies

Intervention / Treatment
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* Dietary Supplement: Cocoa
* Other: Placebo


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Adults aged 40-60 years with a diagnosis of type 2 diabetes mellitus and diabetic neuropathy Minimum time of diagnosis of T2DM of 5 years Who have a Michigan Neuropathy Screening Instrument (MNSI) score ≥2 Male and Female Have them sign the informed consent letter Exclusion Criteria: Subjects who modify their pharmacological treatment during the study Subjects who do not attend one of the intermediate consultations

Ages Eligible for Study
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Minimum Age: 40 Years
Maximum Age: 60 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Treatment
Allocation: Randomized
Intervention Model: Parallel Assignment
Masking: Triple


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Intervention group<br>Diet for patients with diabetes + 4 capsules of cocoa powder, 500 mg each, daily for 12 weeks. | Dietary Supplement: Cocoa<br>* Each capsule of cocoa powder contains 12.5 mg of flavonoids, providing a total of 50 mg per day.<br>|
| Placebo Comparator: Control group<br>Diet for patients with diabetes + 4 capsules of methylcellulose 500 mg each, daily for 12 weeks. | Other: Placebo<br>* Each capsule contains 500 mg of methylcellulose<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Rate-dependent depression on the frequency of stimulation of the H reflex | The ratio of the amplitude of the pulses Hn/H1 ≥ 0.6 for stimulation frequencies 1, 5 and 10 Hz, will be considered as an indicator of dysfunction in somatosensory processing. | At baseline and after 12 weeks | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Toronto Clinical Scoring System | It is a system of clinical evaluations carried out by the researcher to identify peripheral neuropathy, assigning a score to symptomatology, reflexes and sensory tests. 6-8 points: mild diabetic neuropathy, 9-11 points: moderate diabetic neuropathy, 12-19 points: severe diabetic neuropathy. | At baseline and after 12 weeks | 
| BEST questionnaire | The questionnaire includes 4 questions that refer to gastrointestinal symptoms. This questions are measured in a scale from 0 (better health status) to 100 (worse health status) and it is related to gastrointestinal autonomic diabetic neuropathy. | At baseline, after 4, 8 and 12 weeks | 
| Bristol stool form scale | It is composed of categories that include an image and an explanation, ranging from 1 to 7, being 1 separate hard pieces, which pass with difficulty and 7 watery stools. It is related to gastrointestinal autonomic diabetic neuropathy. | At baseline, after 4, 8 and 12 weeks | 
| Weight | Weight of an individual in kg determined by the scale. The measurement is done without shoes and with as little clothing as possible. The subject must be placed in the center and remain still during the measurement. | At baseline, after 4, 8 and 12 weeks | 
| Waist and abdominal circumference | Waist circumference in cm: The measuring tape is placed in a horizontal plane around the waist, taking the midaxillary line as a reference, locating the midpoint between the lower costal margin and the highest lateral border of the iliac crest. Abdominal circumference in cm: The top of the hip bone and the top of the right iliac crest are located and the measuring tape is placed horizontally around the abdomen, at the level of the iliac crest, at the end of a normal expiration. | At baseline, after 4, 8 and 12 weeks | 
| Systolic and diastolic blood pressure | A sphygmomanometer is used to obtain blood pressure with the technique specified in the Clinical Practice Guidelines for the diagnosis and treatment of arterial hypertension at the first level of care, it is measured in mmHg. | At baseline, after 4, 8 and 12 weeks | 
| Glucose | Blood glucose concentration and is measured as mg/dL. | At baseline and after 12 weeks | 
| Triglycerides | Blood triglycerides concentration and is measured as mg/dL. | At baseline and after 12 weeks | 
| High-density lipoprotein cholesterol | Blood high-density lipoprotein cholesterol concentration and is measured as mg/dL. | At baseline and after 12 weeks | 
| Low-density lipoprotein cholesterol | Blood low-density lipoprotein cholesterol concentration and is measured as mg/dL. | At baseline and after 12 weeks | 
| Triglycerides/HDL ratio | It is obtained after dividing the serum concentration of triglycerides in mg/dL by the serum concentration of HDL in mg/dL. It does not have units. | At baseline and after 12 weeks | 
| Glycated hemoglobin A1c | Value of the fraction of hemoglobin that has glucose attached and is reported in percentage (%). | At baseline and after 12 weeks | 
| Serum insulin | Blood insulin concentration and is measured as μU/mL. | At baseline and after 12 weeks | 
| Homeostasis Model Assessment (HOMA) | It is performed after multiplying the serum insulin concentration in μU/ml by the serum glucose concentration in mg/dL, dividing by 405. It does not have units. | At baseline and after 12 weeks | 
| C Reactive Protein (CRP) | Blood CRP concentration and is measured as mg/dL. | At baseline and after 12 weeks | 
| Tumor necrosis factor alpha | Blood tumor necrosis factor alpha concentration and is measured as pg/mL. | At baseline and after 12 weeks | 
| Interleukin-10 | Blood Interleukin-10 concentration and is measured as pg/mL. | At baseline and after 12 weeks | 
| Interleukin-1 beta | Blood Interleukin-1 beta concentration and is measured as pg/mL. | At baseline and after 12 weeks | 
| Interleukin-6 | Blood Interleukin-6 concentration and is measured as pg/mL. | At baseline and after 12 weeks | 
| Malondialdehyde | Physiological ketoaldehyde produced by decomposition of unsaturated lipids from the metabolism of arachidonic acid, measured as μmol/mg. | At baseline and after 12 weeks | 
| Carbonyl | Free radical composed of one carbon atom and one oxygen atom, measured as nmol/mg. | At baseline and after 12 weeks | 
| Total antioxidant capacity | Antioxidant response to aggressors oxidative, measured as nmol/L. | At baseline and after 12 weeks | 
| Diabetes 39 Instrument | It is a self-administered instrument that allows patients to describe how their QOL was affected during the previous month in five domains: energy and mobility (15 questions), diabetes control (12 questions), anxiety and worry (4 questions), social impact (5 questions), and sexual behavior (3 questions). Responses are scored on a seven-point scale that ranged from not affected at all (score = 1) to extremely affected (score = 7). All responses are summed and it is applied a linear transformation to a 0-100 scale. Lower scores indicated a better QOL. | At baseline and after 12 weeks |