β-Hydroxy-β-methylbutyrate modifies human peripheral blood mononuclear cell proliferation and cytokine production in vitro
 
 
Journal Title (Medline/Pubmed accepted abbreviation):  Nutrition
Year: 2011
Issue: 1
Page numbers: 92-99
doi (if applicable):  10.1016/j.nut.2009.12.008

Summary of Background and Research Design

Background:The human immune system is known to overreact to exercise or other common situations, causing stress and inflammation. There are certain foods and compounds that can reduce the level of inflammation. Leucine is a branched chain amino acid that is known to regulate the immune system. However, many of its effects are thought to be modulated by its metabolites. β-Hydroxy-β-methylbutyrate (HMB) is a metabolite of leucine and has been shown in both animal and human models to also regulate the immune system but the mechanism is not yet known. Supplementation with HMB yields physiological plasma concentrations of 0.1-0.5 mM.
 
Parameters studied:
 
Lymphocyte proliferation: the division of white blood cells. Lower proliferation means a less active immune system.
 
Cell cycle progression: When cells are in stage G1, cells are growing and performing normal cellular functions. From G1, cells enter S phase (for DNA synthesis), and prepare for division. Then, in stage G2 they grow in further preparation for division. The last stage is M, for mitosis, during which the cell divides into two cells in stage G1. Cells can exit the cell cycle, in which case they are in “G0”. They perform normal cellular functions and do not divide. When a higher proportion of immune cells are in stages S, G2, and M, the immune system is activated. 
 
T-cell-derived cytokine production: T-cells are immune cells and cytokines are cellular signaling compounds that augment the immune response.
CD25: a protein that is expressed on the surface of T-cells, B-cells, and others that functions in signal transduction. Lower expression means a lower inflammatory response.
ERK1/2: a protein that is expressed inside many cells that is involved in intracellular signaling. Lower activity means a lower immune response.

Hypothesis/Research Question:What are the effects of HMB on specific aspects of the immune system?

Subjects:Venous blood was collected from 8 healthy volunteers for use in in vitro experiments.

Experimental design: in vitro

Treatments and protocol:
  • To test the effect of HMB on lymphocyte proliferation, peripheral blood mononuclear cells (PBMCs= any cells that has a spherical nucleus including lymphocytes but also macrophages and monocytes) were labeled with a fluorescent dye and treated with ConA (a compound that stimulates cell division) and HMB at concentrations between 0-10 mM.
  • In order to see if immune cells are preparing for cellular division, thus indicating immune activation, cells were treated with ConA and HMB from 0 to 10 mM.
  • To monitor cytokine production, cells were stimulated with ConA and assayed for concentrations of IL-2, IL-4, IFN-γ, and IL-10.
  • To monitor CD25 expression, cells were stimulated with ConA and CD25 expression was monitored with fluorescence.
  • To monitor pERK1/2 concentrations, ConA was used to stimulate intracellular pERK1/2 and HMB was introduced at various concentrations.

Summary of research findings:
  • HMB at 10 mM decreased proliferation of lymphocytes and increased the percentage of cells in G0 and G1 in the cell cycle. It also significantly decreased the percentage of cells in the S and G2-M phases of the cell cycle.
  • There was not a consistent, dose-related trend in changes in most cytokine concentrations (e.g., IL-2, IL-4, IFN-g) with increasing concentrations of HMB. However, TNF-α production was lower at all concentrations of HMB used.
  • Neither CD25 nor pERK1/2 expression were significantly altered with concentrations up to 10 mM HMB.

Interpretation of findings/Key practice applications:

At physiological concentrations HMB does not appear to regulate immune function according to most of the parameters that were studied. One exception is cytokine TNF-α. Because higher concentrations are required to yield effects, it would be interesting for the authors to examine combinations of compounds that may be able to achieve additive or synergistic effects on the immune system while maintaining practical plasma HMB concentrations.

Limitations:

The authors did not report cell toxicity data from the various doses of HMB. HMB at 10 mM is higher than would be normally present with typical HMB supplementation.    As such, the HMB may have been toxic to the cells, preventing them from cell division or other normal functions.
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