This study was conducted according to the guidelines described in the Declaration of Helsinki, and all procedures involving human subjects/patients were approved by the Institutional Review Boards of the University of South Carolina and Palmetto Health Hospital. Written informed consent was obtained from all the subjects. The trial is registered with the National Institutes of Health Clinical Trials (NCT01195077) (Teas 2010). Subjects were given an honorarium of $2/day to take the supplements and $15/blood draw to cover the cost of transportation to the clinic.
Subjects were all treatment naïve, and were referred by physicians to our study because of declining CD4 cell counts or increasing viral loads. Other study criteria included no allergies to seaweed, Spirulina, shellfish, or iodine and an interest in participating in the study. Participants were instructed to continue to eat their normal diets and vitamins, supplements, and medications during the study. None of the subjects consumed seaweed or Spirulina in either their normal diets or as supplements. Subjects were randomly assigned to a supplement treatment arm, and arms were balanced by supplement type. At each clinic visit, supplements were provided in prefilled 7-day plastic containers with separate compartments for morning and evening doses.
Blood collection and handling
Blood samples were drawn from fasting participants between 0630 and 1030 hours by venipuncture at a consistent time for each subject at baseline and at the end of each treatment period. Blood samples were analyzed by the Richland Palmetto Hospital lab. Metabolic panel included: glucose, blood urea nitrogen (BUN), creatinine, BUN/creatinine ratio, sodium, potassium, chloride, carbon dioxide, calcium, protein, albumin, globulin, A/G ratio, bilirubin, alkaline phosphatase (ALP), aspartate transaminase (AST), and alanine transaminase (ALT). Lipid panel included total cholesterol, triglycerides, high-density cholesterol (HDL), low-density cholesterol (LDL), and very low-density cholesterol (VLDL). Complete blood counts (CBC) with differential included: white blood cells (WBC), red blood cells (RBC), hemoglobin (Hb), hematocrit (Hct), mean cell volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), platelets, absolute lymphocytes, percent lymphocytes, percent neutrophils, and percent monocytes. HIV-specific measurements included percent CD4 positive of all lymphocytes, CD4 cells (microliters), HIV-1 RNA by PCR copies (milliliters), and log10 HIV-1 RNA.
Undaria and Spirulina supplements
Seaweeds are consumed regularly by millions of people, particularly in Japan and Korea. They have been approved by the United States Food and Drug Administration and are on the list of foods Generally Regarded as Safe (Food and Drug Administration 1982). Spirulina received a class A safety rating by the Dietary Supplements Information Expert Committee of the United States Pharmacopeial Convention (Marles et al. 2011).
Undaria pinnatifida (Harvey) Suringar was harvested from Bahia Bustamante on the Patagonian coast of Argentina (Soriano SA). The sporophylls were removed from the stipes, shade dried, and pulverized before encapsulation (Vicrofer SRL, Buenos Aires, Argentina) into 500-mg capsules. The powder was tested by Soriano SA, and the nutritional analysis is presented in Table 1. Independent testing of the powder for iodine was conducted at the Iodine Research Laboratory at the Boston University School of Medicine (Teas et al. 2009a, b); fucoidan content was determined following the Stevenson analytical method for determining algal sulfated galactans at the Industrial Research Limited, Crown Research Institute in Wellington, NZ (Stevenson and Furneaux 1991). Spirulina was cultivated and harvested by Earthrise (Calipatria, CA). It was spray dried and encapsulated into 600-mg capsules. Earthrise® nutrient composition and bioactive component data for dried Spirulina were based on the Earthrise® label. Nutrient compositions of the supplements are presented in Table 1.
Medical Outcomes Study-HIV quality of life questionnaire
The Medical Outcomes Study-HIV (MOS-HIV) quality of life questionnaire has 30 items clustered around 13 dimensions of quality of life for people with HIV and has been validated in several populations (Wu et al. 1997). The questionnaire scores were calculated using the specified scaling and scoring program (Mapi Research Trust, Lyon, France) and represent percent change from baseline. Subjects were interviewed by the same project coordinator who administered the MOS-HIV quality of life questionnaire while waiting for the phlebotomist. Any comments or health effects also were noted at this time.
A total of 13 subjects were recruited; data from one subject were excluded because of inconsistent attendance at the clinic visits and complaints about the burden of taking ten capsules/day. Another subject dropped out of the study after the first 2 weeks, and her data also were excluded from analysis, leaving a final sample of 11 evaluable subjects, 5 in phase I and 6 in phase II. Nine of the subjects met the criteria of being healthy and not yet needing ARV; two people in the phase II trial just met the Centers for Disease Control and Prevention (CDC) criteria for ARV and had been placed on a waiting list for ARV. The referring physician felt that participation in our study would possibly benefit her patients until the medications were available. Subjects in phase II were followed for periods ranging from 5 to 14 months. Of these participants, one moved out of state after 13 months, one was lost to follow-up after 14 months, one chose a different treatment, and the two subjects who had CDC-defined AIDS were removed from the study when their CD4 cells again decreased to below 350 cells mL−1, and funding for ARV became available. Although we had hoped to recruit 12 subjects in each arm, the phase II study ended early because the referring physician left the practice and interest in our study ceased.
Although all the study participants had HIV-related lab tests performed, lab requests for metabolic panel, lipid profile, and CBC with differential counts were not standardized until later in the study. The number of evaluable patients for these additional lab results therefore varied. The number for each test is noted in Tables 2, 3, 4, 5, and 6. Lab values are reported as means ± standard error and compared with lab-specific reference values for each endpoint. Repeated measures analyses of variance with posttest were used to determine linear trend (GraphPad Prism 5, USA). Statistical tests were all two-tailed, and significance was set at p < 0.05.