Famine From Feast: Linking Vitamin C, Red Blood Cell Fragility, and Diabetes

Diabetes type two is a debilitating disease that leads to chronic morbidity such as accelerated microvascular disease. Accelerated microvascular disease may produce blindness, end stage renal disease, myocardial infarction, stroke, and limb ischemia. Strategies to prevent or delay microvascular disease have the potential to improve the lives of millions and prevent catastrophic illness. The major focus of prevention of microvascular disease in diabetes has been on the endothelium and its role in protection of blood vessels. An unexpected means to prevent microvascular disease in diabetes may be coupled to the function of vitamin C in red blood cells (RBCs) of diabetic subjects. Based on new and emerging data, vitamin C concentrations in RBCs may be inversely related to glucose concentrations found in diabetes. Based on animal data, we hypothesize that RBCs with low vitamin C levels may have decreased deformability, leading to slower flow in capillaries and microvascular hypoxia, the hallmark of diabetic microangiopathy. Low vitamin C concentrations in RBCs of diabetic participants may be able to be increased, by using vitamin C supplements. Findings in animals may not accurately reflect effects in humans because of species differences in mechanisms of vitamin C entry into RBCs. Therefore, clinical research is essential to characterize vitamin C physiology in RBCs of diabetics. In this protocol, we will investigate physiology of vitamin C in RBCs of diabetic participants as a function of glycemia, without vitamin C supplementation (baseline) and with vitamin C supplementation (min 8-week follow-up). We will screen type II diabetic participants on insulin and/or oral hypoglycemic medication(s) and select those with hemoglobin A1C concentrations of <= 12%. To investigate how response to the nutritional interventions in individuals with diabetes varies from normal, nondiabetic controls will also be recruited and studied. Selected participants will be hospitalized twice, each time for approximately one week at the baseline and 8-week follow-up time points. The primary objective of the first hospitalization (baseline) will be to evaluate the effect of hyperglycemia on vitamin C RBC physiology regardless of baseline vitamin C concentrations (without any vitamin C supplementation). The second hospitalization (min 8-week follow-up) investigates the effect (if any) of vitamin C supplementation to changes in RBC physiology during periods of normal (euglycemic) and elevated (hyperglycemic) glucose concentrations. As inpatients, participants will have two venous sampling periods each of approximately 24 hours.

On admission, participants may be fitted with continuous glucose monitors (CGMs), participants will be transitioned to an individualized inpatient diabetes regimen determined by investigators, based on pre-admission diabetes regimen and glycemic control. For participants with diabetes, the inpatient diabetes regimen will be titrated to achieve euglycemia (fasting and pre-meal glucoses <140mg/dl) prior to the first sampling period (euglycemic sampling). The first sampling period will be performed under conditions of euglycemic control for approximately 24 hours. The second sampling period will be performed under controlled hyperglycemia induced by decreasing doses of the diabetes regimen and providing a high carbohydrate load diet (70-75% carbohydrate). Correction-scale insulin will be provided for glucoses >350-400mg/dl. For nondiabetic controls, an oral glucose tolerance test (75 grams dextrose) will be administered on admission. Controls will receive the same metabolic diets and undergo the sampling schedule as the cohort with diabetes. During the two hospitalizations, samples will be withdrawn via venous catheter for RBC deformability, vitamin C concentrations and other related research studies. Following completion of baseline measurementts, subjects will be provided a prescription for vitamin C 500mg twice daily. Given that vitamin C and vitamin E are related antioxidants, and that both vitamins appear to be associated with RBC rigidity, diabetic participants may also be given a prescription for 400 international units (IU) of vitamin E (RRR alpha tocopherol) daily. Participants will continue vitamin C and E supplementation for a minimum of 8 weeks depending on RBC vitamin C concentrations. To evaluate any effect of vitamin E supplementation, plasma and RBC vitamin E levels may be measured concurrently with vitamin C levels during the hospitalizations. All participants will be seen as outpatients at biweekly or monthly intervals with regular measurement of plasma and/or RBC vitamin C concentrations. Target RBC vitamin C concentration >30uM is required prior to the minimum 8-week follow-up inpatient sampling. Vitamins C and E supplementation will be discontinued upon the follow-up inpatient admission. Risk of both vitamin supplements are minimal as both supplementation doses are safe. Outcomes are to measure RBC rigidity and vitamin concentrations before and after supplementation. After a minimum of 8 weeks (depending on RBC vitamin C levels), participants will be hospitalized again, and sampling repeated as described. In this manner, each particpant serves as his/her own control, and deformability of red blood cells can be determined in relation to glycemia and to vitamin C concentrations in RBCs and plasma. Participants will be required to consume standardized meals during inpatient stays. To avoid obscuring plasma vitamin C changes that may result from hyperglycemia, dietary vitamin C content will be approximately 30-35 mg per meal. Additionally, to avoid confounding vitamin E measurements, diets will provide approximately 6 mg alpha tocopherol per day. Standardized meals at the 2nd inpatient admission will be provided to match what was consumed by the subject at their 1st inpatient admission.

• INCLUSION CRITERIA:

Study Initiation

• Male or female 18-65 years old, able to give informed consent.
• Diabetes type 2 HgA1C <= 12% on insulin and/or oral hypoglycemic agents or nondiabetic without any prior history or diagnosis of diabetes.
• In general good health with no other significant illness.
• Mild concomitant disease such as mild hypothyroidism (TSH <10) is acceptable.
• Blood pressure with or without medication <160/90 mmHg with no known significant target organ damage (end organ damage includes the following: proliferative retinopathy, serum creatinine >1.5 or EGFR < 55 mL/min, symptomatic ischemic heart disease, severe congestive heart failure, advanced peripheral vascular disease.
• Willingness to use effective contraceptive methods such as barrier method for the duration of study (female subjects).

8-week follow-up

Above criteria with addition of RBC vitamin C concentration >30 uM prior to inpatient studies.

Exclusion criteria

Entire duration of study

• Diabetic type 1 subjects will be excluded due to the possibility of ketosis and hemodynamic instability with lack of insulin.
• Any subjective or objective evidence of microangiopathy such as history of claudication, symptomatic peripheral vascular disease, symptomatic coronary artery disease, stroke, retinopathy, nephropathy (serum creatinine >1.5 or EGFR < 55 mL/min).
• Diabetic subjects with retinopathy to avoid accelerated retinopathy with hyperglycemia.
• Concomitant disease such as severe heart failure, severe liver disease (transaminases > 3 times normal), or severe systemic disease of any sort.
• Pregnancy, breastfeeding.
• History of diabetic ketoacidosis or hyperosmolar coma.
• Subjects with clear evidence of non-compliance with protocol/study instructions.
• Subjects who are unwilling or lack capacity to provide informed consent.