Food is any substance organisms metabolize to sustain life. These compounds affect every single part of the human body and although as critical as breathing itself, for practical reasons it hasn’t gotten the attention it deserves. A problem for the ages with no apparent solution.
WRONG, the answer is as simple as finding ways to make nutritional and healthy foods practical and appealing. Therefore the prayer was answered and with antioxidant research functional foods became a reality as the greatest nutritional development in history. They address nutritional efficiency in a pragmatic way by relying on cost efficient scientific research to make nutritional food appealing.
To stay competitive food developers now have the needs to analyze a wide range of properties to find out how their products and ingredients rank in the nutritional scale. From tocopherols to proanthocyanidins, understanding products and the market they cater to is critical to improve the product’s competitiveness but also to better support claims. It is imperative to be able to accurately analyze through proprietary enzymatic assays and cellular assays for preclinical and clinical evaluation which is indeed the way in which results become more meaningful. These assays have been developed to determine the health function and potency of nutraceutical and biopharmaceutical products in a biological environment. Below are the some of the major bioassays in existance for five primary health functions that are critical to functional food and nutraceutical product development:
- Antioxidant: Cellular Antioxidant Analysis
- Anti-inflammatory: Cellular and enzymatic NFkB inhibition, cytokines (TNF-alpha) inhibition, LOX, COX, and PLA2 inhibition analysis
- Anti-aging: Cellular SIRT1 expression enhancement
- Diabetic/obesity concern: cellular glucose uptake enhancement, impact on insulin receptor and carbohydrate digestion
- Oxidative stress: in vivo oxidative stress analysis
For those with more technical interest, you also get the full explanation of what and why this research should be performed as a more detailed description of these biological analyses follows:
1. Antioxidant: Cellular Antioxidant Analysis (CAA)
It defines a material’s intracellular antioxidant capacity and its ability to minimize oxidation damage in cells. It is a preclinical measure of bioavailability of a material that describes the efficiency of the material to be absorbed by human cells as well as its antioxidant effectiveness within the cells.
In the CAA assay, a fluorescent probe is placed inside of representative human cells, whose loss of fluorescence is an indication of the damage extent from oxygen radical. A material to be tested is incubated with the cells to allow its natural absorption into cells. Then, an oxygen radical inducer is introduced into cellular environment which triggers the release of oxygen radicals. Without antioxidant material present inside of the cells oxygen radicals permeate through cell membrane and damage the cells and the marker probe. Such process deters when antioxidant material is present inside of the cells. The cellular antioxidant effect of the test material is then measured by assessing the preservation effect of the marker probe in the presence of the test material absorbed inside of cells.
2. Anti-inflammatory Investigation
Investigation can be furthered through a number of markers for a solid understanding of anti-inflammatory properties of a material. This builds a solid foundation for further investigation in this direction.
NFkB inhibition in cells: NFkB (Nuclear Factor kappa B), a protein complex that is involved in cellular responses to stimuli such as stress and free radicals, has been studied as a biomarker for inflammation. In our Cellular anti-inflammatory NFkB assay, Tumor necrosis factor alpha (TNF-a), a pleiotropic inflammatory cytokine, is introduced to the human cells to trigger cellular inflammation. If an anti-inflammatory material presents in the cellular environment, the material inhibits NFkB activation and the degree of inhibition can be monitored via NFkB expression. NFkB expression level of the human cells, treated with and without test materials, under the stressed condition are therefore monitored and compared, and NFkB inhibition effect of the material will be assessed.
Besides TNFs, other cytokines such as interleukins (IL-1, IL-6, etc.) can also be used as stressor for NFkB-based anti-inflammatory investigation. This would provide information on inhibition effect of tested materials on interleukins induced inflammation.
Cytokines (TNF-alpha) inhibition in cells: TNF-alpha is a pleiotropic inflammatory cytokine that is seen as inflammation trigger and precursor. Similar to TNF-alpha, interleukins that are inflammatory cytokines of keen interests can also be investigated as biomarkers. In this investigation, human cells will be stressed with inflammation inducer that induces cytokine production. If an anti-inflammatory material presents in the cellular environment, the material inhibits cytokine production whose level can be quantified via analysis of its expression level.
Cycloooxygenase (COX) Inhibition: It determines the inhibition potential of a material on COX-1 and COX-2 activity via enzymatic analysis. Cyclooxygenases-2 (COX-2) inhibitors are among the important targets for treatment of inflammation related diseases. COX has two well-known isoforms, COX-1 and COX-2, which are similar in their amino-acid sequences and identity. COX-2 predominates at sites of inflammation, while COX-1 is constitutively expressed in the gastrointestinal tract. It is reported that selective COX-2 inhibitors can target inflammation and pain with reduced risk of chronic ulceration and acute injury. Through COX Inhibition investigation, it can assess the inhibition capability of a material by monitoring its impact on the activity of a COX 1 and COX 2.
Lipooxygenase (LOX) Inhibition: It determines the inhibition potential of a material on LOXs activity via enzymatic analysis. Inhibition of three main LOX enzymes, LOX-5, LOX-12, and LOX-15, can be investigated via these analyses.
Lipoxygenases (LOXs) are a family of nonheme iron-containing dioxygenases enzymes distributed in animals, plants, and fungi. These enzymes catalyze distinct cellular reactions and produce fatty acid hydroperoxides throughout the reaction processes. These fatty acid hydroperoxides products have been identified as mediators of a series of inflammatory diseases including rheumatoid arthritis, inflammatory bowel disease, atherosclerosis and certain types of cancer. Therefore, LOX inhibitors have been investigated as modulators of the inflammatory processes and promising therapeutic targets. LOX Inhibition investigation can assess the inhibition capability of a material by monitoring its impact on the activity of a three forms of LOXs: LOX 5, 12, and 15.
Secretory Phospholipases A2 (sPLA2) Inhibition: Secretory Phospholipases A2 (sPLA2) are a subfamily of Phospholipases A2 enzymes that catalyze the hydrolysis of phospholipids yielding precursors of pro-inflammatory lipid mediators including bioactive eicosanoids and platelet-activating factor (PAF). sPLA2 inhibitors hold an established role in inflammation treatment, since inhibition of sPLA2 in theory would prevent the formation of inflammatory eicosanoids prior to the cyclooxygenase (COX) reaction. Therefore, theoretically, sPLA2 inhibitors eliminate the need for selective COX-2 versus COX-1 inhibitors in anti-inflammatory therapeutics.
3. Anti-aging properties via cellular analysis
Investigation of potential anti-aging effects of a material via cellular anti-aging analysis using SIRT1 as a biomarker. SIRT1 is a protein that is believed to play important roles in longevity and age-related diseases. Previous studies have shown that when cells age, SIRT1 expression decreases, while induction and activation of SIRT1 has been associated with extended life span. These studies have triggered the search for SIRT1 activators that may be used as dietary supplements to promote health and longevity.
Via SIRT1 investigation in cells, it determines the ability of a material in stimulating SIRT1 protein expression or activity in human cells, which translates to its anti-aging potential. Here, SIRT expression of cells treated with and without a material are monitored as they age, and anti-aging properties are assessed.
4. Anti-diabetic function and weight control property via cellular analysis
Investigation of potential anti-diabetic/weight control properties of a material in a biological system is carried out via determining the impact of material on glucose uptake, insulin receptor activity, and carbohydrate digestion capacity of human cells.
Glucose regulation is a functional process that under degenerate conditions may result in various health issues including obesity, diabetes, and cancer. Metabolically, the insulin receptor, a transmembrane receptor that is activated by insulin and insulin-like growth factors, plays a key role in the glucose regulation. Under decreased insulin receptor activity condition, the cells are unable to take up glucose, resulting hyperglycemia (an increase in circulating glucose), leading to diabetes mellitus type 2 (commonly called Type 2 diabetes). Therefore, materials that are able to enhance the activation of glucose uptake or insulin receptor are helpful to improve diabetic condition.
In weight management, a proven successful approach has been the disruption of nutrient digestion. Investigation has been driven towards nutraceuticals that inhibit the breakdown of complex carbohydrates and fats within the gut. Materials inhibit activity of α-amylase, an enzyme that catalyses the hydrolysis of starch into sugars, deter carbohydrate digestion therefor have been used in humans with promising results relating to weight loss.
It investigates the impact of a material on glucose uptake, insulin receptor activity, or carbohydrate digestion capacity of human cells to provide insight of its anti-diabetic and weight control properties.
5. Oxidative Stress Reduction Analysis
Mostly carried out in conjunction with a clinical study or a preclinical (animal) study. Blood (plasma or serum), urine, or tissue samples are taken from participating subjects and oxidative stress analyses are performed to assess the impact of a material on the oxidative stress condition of the participating subjects.
For initial stage of investigation, four key areas of the investigation are recommended: in vivo antioxidant, lipid peroxidation, DNA damage, and protein damage level. In vivo antioxidant indicates the defense level in a biological system, while lipid peroxidation, DNA damage, protein damage are key areas that assess damage level in a biological system.
Although one can assess the oxidative stress level in a biological system from one key area, for instance, in vivo antioxidant level alone, assessing these four key areas can provide a better and more concrete evaluation of oxidative stress condition in a biological system. One or more biomarkers from each key area can be assessed as the first step of investigation:
5-1 In vivo antioxidant: a number of in vivo antioxidants have been observed in biological system:
a. Super Oxide Dismutase (SOD): One of the most important antioxidant enzymes in vivo. SOD catalyzes the dismutation of the superoxide anion into hydrogen peroxide and molecular oxygen. SOD has been used as a key indicator of the in vivo antioxidant level in biological systems.
b. Catalase: An ubiquitous enzyme that destroys hydrogen peroxides formed during oxidative stress. Catalase activity has served as a biomarker for in vivo antioxidant level.
c. Glutathione / oxidized glutathione ratio (GSH/GSSG): Glutathione (GSH) is a major tissue antioxidant that helps protecting cells from free radical damage by providing reducing equivalents for the reduction of lipid hydroperoxides. During this process, oxidized glutathione (GSSG) forms as a reaction product. When a biological system is exposed to increased levels of oxidative stress, GSSG accumulates and the ratio of GSH to GSSG decreases. GSH level, mostly importantly the GSH/GSSG ratio, have been used as indicative biomarkers of in vivo oxidant and oxidative stress level in cells and tissues.
For specific interests, other markers are also identifiable:
d. Glutathione Peroxidase (GPx)
e. In vivo ORAC: Using above biomarkers would provide confirming information on in vivo antioxidant level. GSH/GSSG analysis also provides initial information on oxidative stress level, which further provides complementary information of the antioxidant status in vivo.
5-2 Lipid peroxidation is a well-defined mechanism of cellular damage in animals and plants. Isoprostanes (F2-isoprostanes, F2-isoPs) is a recommended initial biomarker for this key area. Isoprostanes can be assessed in both LCMSMS technique and ELISA/spectrophotometric approach with LCMSMS method provides more reliable results.
Other biomarkers can also be monitored for this area:
- TBARS (Thiobarbituric Acid Reactive Substances)
- Malondialdehyde (MDA)
- 4-Hydroxynonenal (4-HNE)
- Lipid hydroperoxide (LOP)
5-3 For DNA oxidation, 8-hydroxydeoxyguanosine (8-OHdG) is a recommended initial biomarker. 8-OHdG has been known as a ubiquitous and sensitive marker of oxidative stress for oxidative DNA damage. RNA oxiation damage can be monitored via 8-Hydroxyguanosine (8-OHG).
5-4 Protein damage: oxidative damage of proteins occurs in vivo during aging and in certain disease conditions. Direct measurements of oxidative damage to proteins is recommended with 3-nitrotyrosine as a main biomarker. Measures of protein oxidation by-products such as carbonyl content (POC) can also be used as a complementary approach. Another biomarker can be monitored for this area is Advanced Glycation End Products (AGE).