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
subjects 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 diabetic subjects. In this protocol we will investigate physiology of
vitamin C in RBCs of diabetic subjects as a function of glycemia, without vitamin C
supplementation (arm 1) and with vitamin C supplementation (arm 2). We will screen type II
diabetic subjects on insulin and/or oral hypoglycemic medication(s) and select those with
hemoglobin A1C concentrations of 7-12%. Selected subjects may be hospitalized twice, each
time for approximately one week. The primary objective of the first hospitalization (arm 1)
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 (arm 2) investigates the effect (if any) of vitamin C supplementation to
changes in RBC physiology during euglycemic and hyperglycemic states. As inpatients, subjects
will have two venous sampling periods each of approximately 24 hours. On admission, oral
hypoglycemic agents will be discontinued, and basal-bolus insulin regimen initiated. Insulin
doses will be clinically determined and 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 24 hours. The
second sampling period will be performed under controlled hyperglycemia induced by
withholding basal and bolus insulin and providing a high carbohydrate load diet (70-75%
carbohydrate). Correction-scale insulin will be provided for glucoses >350-400mg/dl.
Hyperglycemia will not exceed 9 hours, and will be reversed by reinstituting insulin.

During the two sampling periods, samples will be withdrawn via venous catheter for RBC
deformability and vitamin C concentrations. Following completion of arm 1, subjects
considered for participation in arm 2 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 subjects may also be given a
prescription for 200 mg of vitamin E (RRR alpha tocopherol) daily. Subjects 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 various phases of arms 1 and
2. All subjects will be seen as outpatients at weekly or biweekly intervals with regular
measurement of plasma and RBC vitamin C concentrations. Target RBC vitamin C concentration
>30uM is required prior to arm 2 inpatient sampling studies. Vitamins C and E supplementation
will be discontinued upon inpatient admission for arm 2. 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), subjects will be hospitalized again, and sampling
repeated as described. In this manner, each subject 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.

Subjects will be required to consume standardized meals during inpatient stays. All meals
will be prepared by the NIH Clinical Center Metabolic Kitchen. To avoid obscuring plasma
vitamin C changes that may result from hyperglycemia, dietary vitamin C content will be
restricted to less than 10 mg per meal. Additionally, to avoid confounding vitamin E
measurements, diets will provide less than 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:

Arm 1

- Male or female 18-60 years old, able to give informed consent.

- Diabetes type 2 HgA1C 7-12 percent on insulin and/or oral hypoglycemic agents.

- 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).

Arm 2

Above criteria with addition of RBC vitamin C concentration greater than micrM prior to
inpatient studies.

EXCLUSION CRITERIA (Arm ! and 2):

- 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).

- 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.

- Participation in each protocol delineated evaluation procedure will be judged on a
case by case basis with patient safety as the paramount consideration.

- Pregnancy, breastfeeding.

- History of diabetic ketoacidosis or hyperosmolar coma.

- Subjects with clear evidence of non-compliance with protocol/study instructions.