Trial Information

Micronutrient Supplemented Probiotic Yogurt for HIV/AIDS and Other Immunodeficiencies: A Randomized, Placebo-Controlled Trial

Drs. Reid and Hekmat have recently developed a fermented milk product (yogurt) containing
Lactobacillus GR-1 and RC-14 and concluded that such dairy products are suitable vehicles
for these beneficial microorganisms [Hekmat, S. & Reid, G. 2006].

The strains Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 have a proven
efficacy to treat and protect against gastrointestinal and urogenital infections [Anukam, K.
et. al. 2006; 2007]. In one study, the supplementation of yogurt with these probiotics was
shown to eradicate diahrea and increase the CD4 T-lymphocyte counts in HIV subjects [Anukam,
K. et.al. 2006]. HIV infected subjects often suffer diarrheal episodes, and consequently
experience malabsorption of certain nutrients [Cunningham-Rundles, 2000]. A study conducted
by Cunningham et. al [2000] found that probiotic Lactobacillus plantarum 299 had a positive
impact on the immunity of 10/17 children and on the growth of all of the children who were
HIV-positive.

It is estimated that 75 percent of immune function is localized in the gastrointestinal
tract, which may explain why oral administration of probiotics is an excellent tool for
immunomodulation [Olah, A. et. al. 2002]. In a study of 45 patients with acute pancreatitis,
22 were given probiotic Lactobacillus plantarum 299 and 10g of oat fibre with regular
enteral nutrition; and 23 were given a placebo in the form of regular enteral nutrition.
Significantly fewer (n=1 versus n=7) patients in the probiotic/fibre group experienced
septic complications requiring surgery, highlighting the safety and potential hospitalized
benefits that could be accrued for critical care nutrition.

A study has recently shown in 40 HIV-infected patients taking a stavudine and/or
didanosine-based HAART regimen plus micronutrients twice daily for twelve weeks, that the
absolute CD4 count increased by an average of 24% versus 0% in the placebo group (p=0.01).
The mean HIV-1 RNA decreased in the micronutrient supplementation group [Kaiser, et.al.
2006]. This is significant for the HIV/AIDS pandemic in Africa and for the many people in
Canada living with HIV/AIDS. In addition, this may be related to other immunodeficiencies in
hospitalized patients from chemotherapeutic and radiological treatments, organ
transplantation, controlling diabetes, and other chronic ailments.

Clinical malnutrition is a heterogenous group of disorders including macronutrient
deficiencies that lead to body cell mass depletion and micronutrient deficiencies, and these
often coexist with infectious and inflammatory processes [Hughes,S. & Kelly, P. 2006].
Diverse factors affect bioavailability, such as the nutritional status of individuals, the
presence in the diet of substances which facilitate or inhibit its absorption, interactions
among micronutrients, illnesses, and chemical characteristics of the compound used for
fortification. There is good evidence that specific micronutrients, particularly zinc and
vitamin A, influence immunity, while iron supplementation is often not effective in Africa
[Hughes, S. & Kelly, P. 2006].

More than 20% of older adults may have marginal or frank vitamin B12 deficiency [Park,
et.al. 2006], while folic acid is often deficient in children and reproductive aged females.
Glutamine, arginine, fatty acids, and vitamin E provide additional benefits to
immunocompromised persons or patients who suffer from various infections, while long-chain
polyunsaturated n-3 fatty acids, vitamin E, vitamin C, selenium, and nucleotides can
modulate immunity to fight infection [Field, et.al. 2002]. In order to produce energy, the
use of glucose by nervous tissue requires vitamin B1; this vitamin modulates cognitive
performance, especially in the elderly. Vitamin B9 preserves brain during its development
and memory during ageing. Vitamin B6 is likely to benefit in treating premenstrual
depression. Vitamins B6 and B12, among others, are directly involved in the synthesis of
some neurotransmitters [Bourre, JM. 2006]. Thus, three formulations added to L. rhamnosus
GR-2 supplemented yogurt, will be tested at the Brescia College Tasting Centre where
volunteers will score the products for taste and texture [Hekmat, S. & Reid, G. 2006]

Thirty test subjects will be recruited during the first four months of the study, for
inclusion in a trial starting at the 4-6 month time-point, with completion by 10-12 months.
A crossover study will be performed in London in which 10 HIV positive volunteers with CD4
counts between 200 and 400 on their standard treatment (including Highly Active
Antiretroviral Therapy), and 10 subjects with Cancer who are in the recovery stages and not
currently receiving chemotherapy or radiation therapy, and 10 healthy adults (aged 18-30)
will randomly be assigned to receive one cup of Yogurts 1, 2 or 3 and a control
unsupplemented yogurt 4, daily for one month, followed by a two week washout where no yogurt
will be consumed, then one month on the second yogurt, two weeks off etc for 162 days.
Subjects will not be receiving antibiotics, not have any mental illness that would affect
compliance, or not be requiring surgery during the study. No restrictions will be placed on
subjects continuing prescribed or other remedies, but none will consume any other probiotic
product during the study. The volunteers will be gender matched and a randomization scheme
will ensure that subjects are blinded and different yogurts are distributed at each time
(thus, for example two subjects will receive yogurt 1, two yogurt 2, three yogurt 3 and
three yogurt 4 on day sample day one). The two week washout period will be used, as
probiotic organisms do not persist for longer than that. Various measurements will be taken
on the day of consuming the first and last batches of yogurt (days 0, 30, 44, 74, 88, 118,
132, 162). These will include weight, height, and from blood samples the CD4 count, CBC,
levels of TNFα, IL-12, IL-10 and G-CSF [Kim, et.al. 2006], liver function test (AST, ALT),
serum albumin, total protein, blood urea, serum zinc levels, serum selenium levels and HIV-1
RNA. A linear analogue self-assessment tool will be used to assess the subject's energy
levels, daily activities and overall quality of life [Kaiser, et.al. 2006].

The present project is an important step in developing new yogurts, supplemented with
micronutrients and probiotic lactobacilli for malnourished children, adults, and seniors in
Canada, as well as surgical, transplant, and hospitalized patients, and HIV/AIDS subjects in
Canada and in Africa. The following formulations will be used to supplement the yogurt:

1. The Kaiser micronutrient formula [14]: N-Acetyl cysteine (NAC) 300 mg, Acetyl
L-carnitine 250 mg, Magnesium 92.5 mg, Alpha lipoic Acid 100 mg, Selenium 13.8 µg,
Beta carotene + Vitamin A 5666 IU, Zinc 2.4 mg, Vitamin C 21 mg, Copper 0.225 mg,
Vitamin B1 0.3 mg, Boron 0.5 mg, Vitamin B2 0.3 mg, Potassium 1200 mg, Pantothenic acid
1.3 mg, Iron 3.3 mg, Niacinamide 3.8 mg, Manganese 0.5 mg, Inositol 15 mg, Biotin 7.5
µg, Vitamin B6 0.3 mg, Chromium 7.5 µg, Vitamin B12 0.6 µg, Molybdenum 11.3 µg,
Vitamin D 52 IU, Choline 121.9 mg, Vitamin E 5.7 IU, Bioflavonoid complex 75 mg, Folic
acid 100 µg, L-Glutamine 25 mg, Betaine HCL 37.5 mg;

2. A multi-component formula containing zinc 30mg, vitamins A + beta carotene 5666 IU , C
21mg and E 5.7 IU, selenium 13.8µg, iron 3.3mg, folic acid 100µg, vitamin B12 0.6 µg,
vitamin B6 0.3 mg, vitamin B1 0.3mg

3. A basic supplement containing zinc 30mg, vitamins A 666 IU, vitamin C 21mg and E 5.7
IU, and folic acid 100µg and 16 mg docosahexaenoic acid (DHA-Omega 3 Fatty Acid) and
24mg eicosapentaenoic acid (EPA-Omega 3 Fatty Acid) [Austin, J. et.al 2006 & Alan DD,
et.al 2006].