Pilot Study About the Harmful Effects of Blood Storage on Overweight People and the Role of iNO in This Setting

The objective of this study is to assess effects of the storage of PRBC on endothelial
function, inflammation and platelet activation in overweight people with existing
endothelial dysfunction at baseline.

The present study consists of three different parts, which will be scheduled in a randomized
order on the same subject (crossover study).

During one phase of the study, 14 healthy human volunteers will donate a unit of Packed Red
Blood Cells (PRBC), which will be leukoreduced and stored in Additive Solutions-1 (AS-1),
and then transfused back to the subjects after 3 days of storage at 4º C in the MGH Blood
Bank (Fresh Blood arm). The second part of the study consists in the collection of another
unit of PRBC from the same volunteers which will be transfused back to them after 40 days of
storage (Old Blood arm). Finally in the third part, like in the second one, one unit of PRBC
will be withdraw and stored for 40 days, but 80 ppm (parts per million by volume) Nitric
Oxide in air will be administered together with the transfusion. There will be a 2 weeks
interval after each PRBC transfusion.

We hypothesize that old red blood cells stored under conventional conditions may trigger a
complex, pro-inflammatory, pro-thrombotic and vasoconstriction response. We will compare the
response to PRBC stored for 3 days with the response to PRBC stored for 40 days in the same
healthy volunteers. We also want to test the hypothesis that inhaled nitric oxide may
reverse these adverse effects.

We will monitor/measure the following markers/parameters:

1. Endothelium-mediated changes in vascular (arterial) tone

2. Tissue oxygen saturation will be continuously assessed during and after blood
transfusion

3. Hemolysis as quantified by changes in plasma haptoglobin level, plasma free hemoglobin,
LDH level, bilirubin level, iron level, ferritin, and transferrin

4. Changes of plasma and red blood cell levels of circulating nitrate, nitrite, RXNO,
RNNO, NO-heme

5. Concentration of cytokines, such as IL-6, IL-8, IL-10, IL-12, TNF, IFN-γ

6. Activation of platelets through circulating P-selectin expression on platelets

7. Activation of inflammatory lipid mediators

8. Changes in gene expression profiling analyzing RNA microarray of circulating leukocytes

Inclusion Criteria:

1. Have a photo ID

2. Age>18 years old (required to provide informed consent)

3. Age <60 years old

4. Mild impairment of endothelial function, assessed by post ischemic vasodilation
(L_RHI,<0.7) (38)

5. Body mass index (BMI) >27 kg/m2 and <35 kg/m2

6. Fasting during the days of transfusion.

7. Avoiding intake of high nitrate food (i.e. green leafy vegetables: lettuce, spinach)
on the day prior to the study

8. Feel well on the day of blood donation

9. KG within normal limits

10. Normotensive (systolic blood pressure <140 mmHg and diastolic <90 mmHg)

11. Normal physical exam and blood test results as indicated by:

1. WBC 4.5-11.0 n x103/μL

2. HGB 12.0-17.5 gm/dl

3. PLT 150-400 n x103/μL

4. Plasma Sodium 135-145 mmol/L

5. Plasma Potassium 3.4-4.8 mmol/L

6. Plasma Chloride 98-108 mmol/L

7. Plasma Carbon Dioxide 23.0-31.9 mmol/L

8. Plasma Urea Nitrogen 8-25 mg/dl

9. Plasma Creatinine 0.60-1.50 mg/dl

10. Plasma Glucose 70-110 mg/dl

11. Transaminase-SGPT 10-55 U/L

12. Transaminase-SGOT 10-40 U/L

13. Total Bilirubin < 2 mg/dl

14. Fasting plasma glucose < 110 mg/dl

15. Methemoglobin < 3%

Exclusion Criteria:

1. Psychiatric disturbances such as anxiety, depression, schizophrenia requiring
pharmacological treatment or hospitalization in the last year

2. Systemic disease with or without any functional limitation

3. Pregnancy determined by urine pregnancy test, detecting presence of human chorionic
gonadotropin (hCG), or less than six weeks postpartum

4. Active smoking. Volunteers may be enrolled if they quit smoking for more than 1 year.

5. Excess alcohol use: more than ½ L/day of wine consumption or equivalent

6. Any current use of a medication other than: over-the-counter oral medications, herbal
remedies, nutritional supplements, and oral contraceptives.

7. Antibiotic use within 48 hours of blood donation

8. Use of NSAIDS, corticosteroids, aspirin during the past 7 days

9. Dental work within 24 hours prior to the donation

10. Received or donated blood in the last 4 months

11. Have had any forms of cancer with the exceptions of basal cell skin cancer or
treatment for in situ uterine cervical cancer

12. Currently enrolled in another research study