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.