The
general public has been aware of protein, carbohydrates, fats, vitamins and
minerals from edible plants for a very long time. The unique mixture of phytochemicals
in specific edible plants may be of equal importance, from the perspective of
health and nutrition. A biomedical researcher describes one of the tools used
by nutritionists to help decipher the web of interactions in which these phytochemicals
are involved, once ingested by human beings.
Introduction
Vitamin
A deficiency is a serious nutritional problem throughout much of the developing
world, especially among poor populations where the consumption of animal
products is minimal and the diet is composed predominantly of plant-derived
foods. Vitamin A is not found in plants; however, green and other colored
vegetables do contain significant concentrations of pro-vitamin A carotenoids,
such as beta-carotene, which can be converted to vitamin A in the human body (Solomons,
2001). At present, we do not fully understand how beta-carotene is absorbed in
the human gut, especially when it is consumed in a plant source, nor do we know
with certainty how much of the vegetable-derived beta-carotene from most foods
is converted to vitamin A (also known as that food’s vitamin A activity). Our group
at the USDA/ARS Children’s Nutrition Research Center at Baylor College of
Medicine in Houston, Texas, along with collaborators at the USDA/ARS Human
Nutrition Research Center on Aging at Tufts University in Boston,
Massachusetts, have utilized a stable isotope method that allows us to directly
measure beta-carotene absorption from specific vegetables or fruit, and to
determine the extent to which this beta-carotene is converted to vitamin A in
the body. The information gained through this methodology is important,
because it helps us understand the potential of various foods to provide
dietary vitamin A. It also provides a scientific basis for establishing
food-based, dietary recommendations to assist people in meeting their daily
vitamin A requirements.
Stable Isotopes in Human
Studies
Stable
isotopes are nonradioactive forms of elements that naturally occur within the
environment. For a given element, such as iron (Fe), different stable isotopes
exist that have differing atomic masses (e.g., Fe-54, Fe-56, Fe-57, and Fe-58),
with each isotope of Fe acting chemically in an identical manner. The various
isotopes of an element can be separated by mass and quantified using an
instrument called a mass spectrometer. Stable isotopes vary in their natural abundance
or percentage in nature, and most nutritionally significant elements have at
least one low-abundance stable isotope (e.g., only 0.28% of all Fe occurs as
Fe-58). In human nutrition studies, low-abundance stable isotopes have been
used to trace the absorption and metabolism of nutrients from a specific food
source. For instance, by growing a plant on an enriched source of a
low-abundance stable isotope, the plant will incorporate the isotope into its
tissues and edible organs and will contain a higher percentage of this isotope,
relative to all other foods (Grusak, 1997). We say that the plant has been
“labeled” with this stable isotope. Following consumption of the labeled plant
food by a human subject, the analysis of isotope amounts in blood, urine, or
fecal samples enables the researcher to determine how much of the nutrient was
absorbed from the specific food in question. It should be noted that because
stable isotopes are nonradioactive, their use is entirely safe in human
studies.
Although
much of the existing stable isotope work has focused on mineral nutrients, it
also is possible to label phytochemicals with stable isotopes of carbon (C) or
hydrogen (H). This is important for a molecule like beta-carotene, which is
entirely composed of C and H atoms. The approach we have taken has been to
generate labeled beta-carotene by replacing several of the H atoms (atomic mass
of 1) with the low-abundance stable hydrogen isotope, H-2 (atomic mass of 2).
This isotope also is called deuterium, and occurs naturally at 0.015% of all
hydrogen atoms (i.e., H-1 occurs at 99.985%). By replacing eight of the 56 H
atoms in a beta-carotene molecule with deuterium atoms, a labeled beta-carotene
with an atomic mass of 544 will be generated. By comparison, a beta-carotene
molecule containing no low-abundance isotopes has an atomic mass of 536. These
two forms of beta-carotene are referred to as isotopomers of the molecule. The
mass-544 beta-carotene also is referred to as the M + 8 isotopomer, where M
equals the mass of the unlabeled molecule. As with the different isotopes of
mineral nutrients, isotopomers of a molecule have similar chemical or
biochemical properties, but they can be easily discerned from one another using
a mass spectrometer.
In
order to label beta-carotene, plants can be grown hydroponically using a
nutrient solution enriched with heavy water. Each molecule of heavy water, or
deuterium oxide, contains two atoms of deuterium instead of two atoms of H-1.
Plants absorb both heavy water and normal water via the root system and
transport the water to their shoot organs, where the hydrogen or deuterium ions
can be utilized in various biochemical reactions. Using this approach, we have
successfully grown spinach, collard (Fig. 1), broccoli, carrots and other
vegetables, and have achieved a labeling of the beta-carotene pool with a high
yield of the M + 8 isotopomer. Because deuterium atoms are inserted randomly
throughout the molecule (Putzbach et al., 2005), a range of isotopomers is
produced (Fig. 2); however, we can quantify any or all of these isotopomers
with a mass spectrometer (Lienau et al., 2003).
(Click to enlarge)
Figure 1. Collard plants (Brassica oleracea var. acephala)
growing in hydroponic solution containing heavy water. The lid on the growth
container has been raised to reveal the root systems. Plants are four weeks
old.
(Click to enlarge)
Figure 2. Isotopomer profiles for endogenous and
deuterium-labeled beta-carotene. Endogenous beta-carotene is the normal form
found in plants and in our bodies. The M + 1 and M + 2 isotopomers come from
naturally occurring carbon-13 atoms (1.1% in nature) that can be incorporated
into the molecule. The deuterium-labeled isotopomers are derived from random
additions of H-2 atoms. In this figure, they demonstrate a food source in
which the beta-carotene pool has been labeled at approximately 15% (the
predominant isotopomer is M + 8).
Beta-carotene Absorption
and Metabolism
The
ultimate goal of these efforts is to expand our understanding of how dietary
beta-carotene is absorbed from specific foods and to determine how efficiently
the beta-carotene in each food is converted to vitamin A. Once we have
produced a labeled food, it is fed to human subjects as part of a standard meal
and at a normal serving size. Blood samples are then collected over a period
of several weeks, in order to measure total and labeled beta-carotene and
vitamin A in the plasma. A dose of deuterium-labeled retinoic acid also is
given orally, one week after the labeled food. This compound is absorbed at nearly
100% efficiency and is converted to vitamin A in the body, where it serves as
an important reference compound that allows us to calculate the vitamin A
activity of the plant source being studied. It is worth noting that this
isotopic approach can be used with individuals of any nutritional status
(vitamin A replete or deficient) or of any dietary regime (with or without carotenoid
or vitamin A sources), because all pre-existing or subsequently absorbed
beta-carotene or vitamin A in the body will be unlabeled.
The
Food and Nutrition Board of the U.S. Institute of Medicine has recently
proposed a vitamin A conversion factor of 12 to 1, by weight, for food-derived
beta-carotene (this includes all measured trans beta-carotene
equivalents) (Institute of Medicine, 2002). In other words, a food
that contains 12 mg of beta-carotene equivalents is believed to provide 1 mg of
vitamin A to the body. However, this conversion factor is an average value,
and although it is based on an extensive synthesis of the available literature,
it does not represent the actual conversion efficiency of every food source, or
of every food under all conditions. For instance, we recently measured vitamin
A activities [mean (+ S.D.)] of 21 (+ 9) to 1 and 15 (+ 7)
to 1 for spinach and carrots, respectively, when these foods were fed to
healthy, well-nourished adults (Tang et al., 2005). Although these values
overlap the Institute of Medicine value, they also suggest that the
current guidelines may overestimate the conversion efficiencies in some
individuals.
Clearly,
there are many factors that influence vitamin A activity, and much is yet to be
learned. It is known that processing can promote the release of beta-carotene
from the food matrix (Castenmiller et al., 1999) and that dietary oils enhance
its absorption (beta-carotene is a fat soluble molecule) (Brown et al., 2004).
It is still unclear whether other dietary carotenoids can competitively inhibit
the absorption of beta-carotene, whether malnutrition, disease, and/or other stresses
might negatively impact beta-carotene absorption/metabolism, and why only a
portion of absorbed beta-carotene is converted to vitamin A. Thankfully, there
are techniques like the isotopic labeling approach that can help us answer many
of these questions.
Future Possibilities
There
is no doubt that colored fruits and vegetables are important vehicles for
alleviating vitamin A deficiency in at-risk populations. This is one reason
why their consumption is widely promoted. However, if more information were
available on specific plant foods and their use in diverse population groups,
targeted recommendations could be made to the general public and the
health-related community regarding the vitamin A potential of individual fruits
and vegetables. My colleagues and I are attempting to fill this information
gap through the use of isotopically labeled plants. Our current efforts to
assess vitamin A activities include human trials with spinach (a leafy
vegetable), Golden Rice (a transgenic crop that produces beta-carotene in the
rice grain endosperm) (Grusak, 2005; Paine et al., 2005), and spirulina (a cyanobacterium
often used as a high-protein food additive) (Li and Qi, 1997). We will conduct
these studies in both vitamin A-deficient and vitamin A-replete populations.
We also are developing methods to isotopically label sweet potato, maize,
orange cauliflower, and Moringa leaves for future human studies (yet to be
funded).
There
is much work to be done and much information to be gathered, so we are always
interested to learn about novel beta-carotene rich foods, and welcome any
information about new plants that deserve further attention. We also would be
happy to discuss possible carotenoid-related research collaborations and
possibilities for funding, with any interested parties – especially those
focused on developing world problems.
Acknowledgements
The
writing of this article was funded in part by the U.S. Department of
Agriculture, Agricultural Research Service under Cooperative Agreement Number 58-6250-1-001. The contents of this publication
do not necessarily reflect the views or policies of the U.S. Department of
Agriculture, nor does mention of trade names, commercial products, or
organizations imply endorsement by the U.S. Government.
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