In vitro studies - Nutrition Research Methodology

In vitro studies

Scientific research involves studies across a reduction-ist spectrum. As studies become more reductionist, more and more confounding factors are stripped away. In vitro studies represent part of the reduction-ist approach in nutrition research. The range of tech-niques used is large.

●Chemical analysis studies provide data on nutrient and nonnutrient content of foods.

●Digestibility techniques, in which a substrate is exposed to enzymes capable of digesting the sub-strate, help to refine the gross chemical analytical data to predict nutritional potential.

●Intact organs such as the liver of experimental animals can be used in studies such as perfused organ studies. In such studies, the investigator can control the composition of material entering an isolated organ and examine the output. Sections of organs can also be used, such as the everted gut sac technique. A small section of the intestine is turned inside out and placed in a solution containing some test material. Uptake of the test material into the gut can be readily measured.

●Another approach is the construction of mechani-cal models that mimic an organ, usually the gut (in nutrition research). Many of these models success-fully predict what is observed in vivo and have advantages such as cost and flexibility in altering the experimental conditions with great precision. System biology is a recently launched platform to integrate metabolic pathways using computational biology.

The application of molecular biology techniques to tissue and cell culture systems has provided research-ers with powerful strategies to evaluate and establish metabolic pathways and regulatory roles of nutrient and nonnutrient components of food. Thus, North-ern, Southern and Western blotting techniques to quantitate specific RNA, DNA, and proteins in tissues in response to nutrients are common tools in the nutrition laboratory. The influence of some nutrients or nutritional conditions on ribosomal dynamics as well as on cell hyperplasia or hypertrophy processes has been estimated through RNA, DNA, or protein/ DNA values, respectively.

Furthermore, molecular biological approaches have allowed numerous in vitro discoveries that have aided our understanding of the genetic basis of nutrient functions and metabolic states in vivo. The polymerase chain reaction (PCR) can be used for DNA and/or messenger RNA (mRNA) amplification to determine the genetic background and/or gene expression in very small cellular samples. Transfec-tion studies allow the insertion of DNA into cells to examine nutrient function. Thus, cell lines that usually lack the expression of a particular gene can be trans-fected with DNA containing the gene promoter, as well as all or part of the transfected gene of interest, to study the interactions of various nutrients with the expression of a particular gene. Conversely, knockout cell lines allow us to investigate the consequences of losing a specific gene. In either case, nutrient function at the cell level and the cell–gene level may be studied and provide definitive results. Gene regulation by nutrients has been assessed in different isolated cells and tissues using appropriate indicators and markers of gene expression RNA levels.

The integration of biochemical and molecular technologies into nutrition research allows the poten-tial for an integrated systems biology perspective examining the interactions among DNA, RNA protein, and metabolites. Following the completion of the human genome sequence, new findings about individual genes functions and their involvement in body homeostasis is emerging. Thus, technologies to achieve a simultaneous assessment of thousands of gene polymorphisms, the quantitation of mRNA levels of a large number of genes (transcriptomics) as well as proteins (proteomics), or metabolites (metab-olomics) is rapidly progressing. Advances in DNA and RNA microarray-based tools as well in the application of classic two-dimensional gel electrophoresis, various Liquid chromatography-mass spectrometry (LC-MS) techniques, image scanning, or antibody arrays is contributing to unraveling the intimate mechanisms involved in nutritional processes. Epigenetics studies constitute a rising methodology to be applied in nutritional research.