Nutritional impact of elevated calcium transport
activity in carrots
Jay Morris*†, Keli M. Hawthorne†, Tim Hotze†, Steven A. Abrams†, and Kendal D. Hirschi*†‡
*Vegetable and Fruit Improvement Center, Texas A&M University, College Station, TX 77845; and†U.S. Department of Agriculture/Agriculture
Research Service, Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030
Edited by Roger N. Beachy, Donald Danforth Plant Science Center, St. Louis, MO, and approved December 10, 2007 (received for review September 21, 2007)
Nutrition recommendations worldwide emphasize ingestion of
plant-based diets rather than diets that rely primarily on animal
products. However, this plant-based diet could limit the intake of
essential nutrients such as calcium. Osteoporosis is one of the
world’s most prevalent nutritional disorders, and inadequate di-
condition. Previously, we have modified carrots to express in-
creased levels of a plant calcium transporter (sCAX1), and these
plants contain ?2-fold-higher calcium content in the edible por-
tions of the carrots. However, it was unproven whether this
change would increase the total amount of bioavailable calcium. In
randomized trials, we labeled these modified carrots with isotopic
calcium and fed them to mice and humans to assess calcium
bioavailability. In mice feeding regimes (n ? 120), we measured
45Ca incorporation into bones and determined that mice required
in sCAX1 carrots. We used a dual-stable isotope method with
42Ca-labeled carrots and i.v.46Ca to determine the absorption of
calcium from these carrots in humans. In a cross-over study of 15
male and 15 female adults, we found that when people were fed
sCAX1 and control carrots, total calcium absorption per 100 g of
carrots was 41% ? 2% higher in sCAX1 carrots. Both the mice and
human feeding studies demonstrate increased calcium absorption
from sCAX1-expressing carrots compared with controls. These
results demonstrate an alternative means of fortifying vegetables
with bioavailable calcium.
absorption ? bioavailability ? radioactive isotope ? stable isotope
and vegetables offer a diverse mixture of nutrients that promote
good health, and it is generally thought that they will be more
beneficial to human health than dietary supplements (3). One
way to increase the nutrient content of some vegetables is to
increase their bioavailable calcium levels (4, 5). Carrots are
among the most popular vegetables in the United States and
and other vitamins and minerals; however, like many vegetables,
they are a poor source of dietary calcium (5, 6). By engineering
carrots and other vegetables to contain increased calcium levels,
we may boost calcium uptake and reduce the incidence of
calcium deficiencies (7).
Previously, we have demonstrated that the calcium (Ca) levels
in plants can be engineered through high-level expression of a
deregulated Arabidopsis calcium transporter. An Arabidopsis
vacuolar calcium antiporter, termed Cation exchanger 1
(CAX1), contains an N-terminal autoinhibitory domain (8, 9).
such as potatoes, tomatoes, and carrots increases the calcium
content in the edible portion of these foods (7, 10, 11). Presum-
ably, these sCAX1-expressing plants have heightened sequestra-
have not established that this modification leads to increased
ow dietary calcium intake can negatively impact health and
increase the risk of diseases such as osteoporosis (1, 2). Fruits
The creation of genetically modified plants with increased
nutritional benefits is an expanding field (12, 13). The term
‘‘nutritional genomics’’ has been used to describe various studies
that implement some form of plant biochemistry, genomics, or
human nutrition. Transgenic plants are frequently analyzed only
for changes in plant metabolism. Ideally, these genetically mod-
ified plants need to be labeled and used in controlled animal and
human feeding studies to assess nutritional impacts. Often, this
type of analysis to assess the nutrient value of transgenic foods
is not reported.
Here, we analyze sCAX1 carrots that express increased levels
of a plant calcium transporter for improved calcium absorption
by using both mice and human feeding trials. The experimental
design provides a rigorous platform to validate the nutritional
impact of engineered foods. Furthermore, our findings offer a
unique mechanism to enhance calcium absorption in numerous
agriculturally important crops.
Labeling Carrots with45Calcium and Stable Isotopes. Previously, we
had demonstrated that sCAX1-expressing carrots contain 2-fold
more calcium than vector control lines (7). These lines are fertile
and display no adverse phenotypes in the greenhouse growth
conditions tested. In hydroponics, because of the shallow nature
of the growth containers, the control and sCAX1-expressing
carrots were both short and wide compared with the soil-grown
carrots. As a first step toward determining bioavailability, we
labeled the edible portions of theses carrots with45Ca and42Ca.
In the labeling growth conditions, we were again able to measure
a 2-fold increase in the Ca2?content of the sCAX1-lines and no
increase in the content of other minerals (Cd2?, Cu2?, Fe2?
Mn2?, and Zn2?) compared with control lines (data not shown).
Both the control and sCAX1 lines had similar mineral profiles to
published values listed in the U.S. Department of Agriculture
(USDA) food composition guide (www.ars.usda.gov/main/
site_main.htm?modecode?12354500). The percentage of45Ca
activity of the administered dose that accumulated in the edible
carrot tissues was 14.86% for control and 30.83% for sCAX1-
expressing carrots (Table 1). Using stable isotopes, we measured
the concentration of42Ca enrichment in the edible portions of
the control carrots at 2.5% and the sCAX1-expressing carrots at
2.4% (Table 1). Thus, calcium labeling by using either radioac-
tive or stable isotopes in the carrots can be used to study calcium
bioavailability in feeding studies.
Mice Fed sCAX1-Expressing Carrots. Previously, we have used both
extrinsic and intrinsic45Ca labeling of diets in mouse feeding
study models to measure tracer incorporation into hind limbs
Author contributions: J.M., K.M.H., S.A.A., and K.D.H. designed research; J.M., K.M.H., and
T.H. performed research; S.A.A. and K.D.H. analyzed data; and J.M., S.A.A., and K.D.H.
wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
‡To whom correspondence should be addressed. E-mail: email@example.com.
© 2008 by The National Academy of Sciences of the USA
February 5, 2008 ?
vol. 105 ?
no. 5 ?
(14). We have validated this mouse model by using various diets,
and our results compared favorably to previous feeding studies
in rats (15). Here, mice were fed both extrinsically and intrin-
sically labeled diets containing control and sCAX1-expressing
carrots. Potentially, sCAX1 expression may alter calcium parti-
tioning within the plant. Initially, we determined whether sCAX1
expression altered oxalic acid concentration, a known inhibitor
? 0.08% mM for the control carrots compared with 1.46% ?
0.10% mM for the sCAX1-expressing carrots. This finding sug-
levels. However, these measurements do not directly address
calcium bioavailability issues.
To study bioavailability, we measured calcium incorporation
by extrinsically labeling the carrots. Extrinsic labeling can give
meaningful bioavailability data for foods like milk (16, 17);
however, it does not work for spinach (15). In general, extrinsic
labeling should be expected to work for any food in which the
various food calcium species are readily exchangeable. Because
we are interested in addressing this exchangeable issue with the
sCAX1-expressing carrots, we made side-by-side tests of the
intrinsically and extrinsically labeled carrots. In the feeding
studies, both control and sCAX1-expressing extrinsically labeled
carrots showed no difference in45Ca absorption. Furthermore,
the sex of the mice did not alter the absorption rate of the labeled
diets. The extrinsically labeled control carrots had incorporation
of 1.42% ? 0.46% in female and 1.47% ? 0.43% in male mice
of the45Ca activity, compared with 1.66% ? 0.55% in females
and 1.70% ? 0.52% in males fed sCAX1–1 carrots and 1.71% ?
0.41% in females and 1.79% ? 0.37% in males fed the sCAX1–2
carrots (Fig. 1A).
Intrinsic labeling is more useful when studying the bioavail-
ability of labeled calcium in vegetable-based diets. Many vege-
tables contain antinutritients that can interfere with calcium
absorption in the gut (18). These compounds can bind the
calcium freely, and some plants also can bind calcium during the
growth process (18–20). In the case of spinach, a poor source of
bioavailable calcium, the incorporation of a45Ca tracer in bone
is reduced by 50% in the intrinsically labeled diets compared
with the extrinsic diets when fed to rats or mice (14, 15).
Although sCAX1-expressing carrots contained very low concen-
trations of oxalic acid, it is still imperative to measure calcium
bioavailability from intrinsically labeled carrots. In the intrinsi-
cally labeled diets, the incorporation from the control carrots
was 1.96% ? 0.22% in females and 2.04% ? 0.28% in males,
compared with 1.95% ? 0.32% in females and 2.03% ? 0.33%
in males for the sCAX1–1-expressing carrots (Fig. 1B). Again,
these incorporation rates were not sex linked. Thus, in both the
intrinsically and extrinsically labeled diets, the mice get equiv-
alent amounts of bioavailable calcium from half the quantity of
sCAX1-expressing carrots compared with controls.
Humans Fed sCAX1-Expressing Carrots. To determine the bioavail-
ability in humans, we intrinsically labeled a single homozygous
carrot line (sCAX1–1) with stable isotopes and measured the
calcium absorption in 15 males and 15 females from various
ethnicities (Table 2). To ensure adequate calcium intake, the
subjects were asked to report their intake on the day of the study.
Human subjects consumed 796 ? 97 mg of Ca on the first study
day and 797 ? 103 mg of Ca on the second study day, 2 weeks
later. Based on averaged data from the two 24-h dietary recalls,
subjects consumed 1,165 ? 461 mg of Ca. Therefore, subjects
maintained a calcium intake within the enrollment criteria of
600–1,200 mg/day over the 2 weeks between trials.
In the mouse study, we used radioactive isotopes and mea-
sured incorporation into bone; for the human study, fractional
calcium absorption was measured. Using42Ca-labeled carrots,
we found a higher fractional (P ? 0.001) absorption in control
carrots. The fractional absorption of calcium from the control
carrots was 48.8% ? 4.81% for females and 56.9% ? 4.29% for
males, compared with 42.1% ? 4.32% for females and 43.8% ?
3.37% for males (P ? 0.001) (Fig. 2A) for the sCAX1-expressing
carrots. Although fractional calcium absorption is lower in the
sCAX1-expressing carrots, the total calcium absorbed per 100 g
of fresh carrots is 45.9% higher for females and 38.7% higher for
Table 1. Labeling efficiency of radioactive and stable calcium
isotopes in hydroponically grown carrots
45Ca activity, %*
42Ca enrichment, %†
*45Ca isotope activity in edible portion of carrot.
†42Ca in edible portion of carrot.
after a single meal from extrinsically and intrinsically labeled carrot diets. (A)
Thirty mice were fed extrinsically labeled control and sCAX1–1 diets, and 10
mice were fed sCAX1–2 carrots. (B) Thirty mice were fed intrinsically labeled
control and sCAX1–1-expressing carrot-containing diets.
Table 2. Characteristics of human subjects at baseline
Calcium intake, mg/day
24.2 ? 1.6
80.2 ? 12.0
181.0 ? 9.7
24.5 ? 3.2
817 ? 246
25.7 ? 2.8
62.8 ? 7.0
168.3 ? 5.8
22.3 ? 3.0
913 ? 206
All data are mean ? SD. BMI, body mass index.
*W, White; H, Hispanic; A, Asian; ME, multiethnic.
www.pnas.org?cgi?doi?10.1073?pnas.0709005105Morris et al.
males from the sCAX1-expressing carrots (P ? 0.001) (Fig. 2B).
Therefore, the sCAX1-expressing carrots contain more bioavail-
able calcium in both the mouse and human models.
We have modified carrots to express increased levels of a plant
calcium transporter (sCAX1), and these plants contain higher
calcium content in the edible portions of the carrots. Mouse and
human feeding studies demonstrated that sCAX1-expressing
carrots had increased calcium absorption. In the human feeding
studies, calcium absorption efficiency was 42.6% ? 2.8% and
52.1% ? 3.2% (P ? 0.001) for the sCAX1 carrots and control
carrots, respectively. However, total calcium absorption per
100 g of carrots was 41% ? 2% higher in sCAX1 carrots
compared with control carrots (26.50 vs. 15.34 mg of Ca per
100 g) (P ? 0.001).
Poor diets and exercise habits prevent many people from
achieving optimal bone health. In fact, in the United States,
dietary calcium intake has decreased, such that 90% of adoles-
cent girls and 50% of adolescent boys consume less than the
optimal amount of calcium (21). To help compensate for this
deficiency, one strategy is to increase the calcium content of the
foods they do eat. Here, we have shown the ability to improve the
bioavailable calcium content of a staple food; when applied to a
wide variety of fruits and vegetables, this strategy could lead to
more calcium consumption in the diet.
In addition to the nutritional benefits, the use of genetic engi-
neering to increase calcium levels could improve plant productivity
many postharvest issues (22). For example, apples are immersed in
a calcium solution to maintain firmness in shipping and prolong
of pathogen attack on potato tubers (24) and to combat heat stress
(25, 26). All of these measures require the application of calcium-
containing solutions to the soil or fruits. Recently, sCAX1 expres-
sion has been shown to increase calcium level in tomatoes that
increased fruit firmness and prolonged shelf life (10). Aside from
the nutritional impact shown here, using sCAX1 expression in a
variety of fruits and vegetables also could positively impact plant
productivity while decreasing labor costs.
Testing the nutritional qualities of genetically modified foods
is a rigorous process. Any initial mineral nutrition study requires
labeling of the foods with either radioactive isotopes for animal
studies or stable isotopes for human trials. To date, there have
been no reports of stable isotopes used to study the effects of
nutrient availability from genetically modified foods. Most of the
isotope research has been done with existing plants or hybrid
varieties (27, 28). Here, we have developed a labeling protocol
for carrots by using both radioactive and stable isotopes (Table
1). We hypothesize that the 2-fold-higher45Ca activity in the
sCAX1-expressing carrots is a function of using the same activity
42Ca concentration in the solution for the control carrots com-
pared with the modified carrots.
We used a mouse model to initially assess the bioavailability
using this protocol, we demonstrated that mice fed spinach had
absorption values of ?0.24% for intrinsically and ?0.44% for
extrinsically labeled diets. These values are consistent with
similar studies using rats, validating our procedures (14) and
further demonstrating that spinach, which contains high oxalate
levels, is a poor source of bioavailable calcium. The sCAX1-
expressing carrot lines did not contain more oxalate, and this
finding may explain why both intrinsically and extrinsically
labeled carrot diets showed reasonable calcium absorption.
Because of the relative ease in preparing the extrinsic diets, we
were able to analyze two sCAX1-expressing lines to demonstrate
equivalent calcium bioavailability among different lines.
Although animal models provide evidence related to bioavail-
ability, there are fundamental differences in the mechanism of
calcium absorption between humans and small animals. In
particular, humans use a greater proportion of calcium absorp-
tion in the upper small intestine than small animals (29).
Therefore, it was necessary to demonstrate the bioavailability of
the calcium-containing carrot in a human study. We chose young
adults who were healthy and represent a typical population that
might use vegetable sources to obtain a substantial amount of
their calcium intake.
Although both the mouse and human feeding studies demon-
strate that sCAX1-expressing carrots have increased calcium ab-
sorption (Figs. 1 and 2), the experiments measure different end
points. With the mice, we are using a single meal and assessing the
absorption of the tracer into bone. By using this methodology, the
entire increase in calcium in the sCAX1-expressing carrots appears
to be bioavailable. That is, the calcium found in 100 g of normal
carrots can be obtained from 50 g of sCAX1-expressing carrots.
Meanwhile, in the human feeding, we are comparing the fractional
absorption of calcium from each of the carrots. Although there is
a 10% reduction in absorbed calcium from the sCAX1-expressing
carrots, the total concentration of calcium absorbed from the
sCAX1-expressing carrots is 42% ? 2% higher compared with an
equal quantity of control carrots (Fig. 2B). This finding demon-
strates that we have reduced bioavailability as a function of total
calcium content and emphasizes the fact that not all genetic
engineering increases translate into enhanced bioavailability. Plant
biologists often disregard this issue. Our working hypothesis to
all of the calcium sequestered in the vacuole by ectopic expression
of sCAX1 is bioavailable. It may be conjugated to phytates, phos-
meals containing labeled carrots. (A) Fractional absorption of42Ca from
from 100 g of fresh control and sCAX1-expressing carrots.*, P ? 0.001.
Calcium absorption in female and male human subjects fed single
Morris et al. PNAS ?
February 5, 2008 ?
vol. 105 ?
no. 5 ?
maintaining a positive gain in total calcium despite reduced bio-
availability will be critical when modifying plants by using sCAX1.
uct be safe. Here, we have presented preliminary human nutrition
studies by using a single carrot line grown under controlled labo-
ratory conditions. Future work needs to be done testing multiple
lines and various growth regimes. Previously, using biochemical
approaches, we have shown that sCAX1 can transport a wide range
of substrates (9, 30). Potentially, CAX-expressing crops could be
used for enrichment of other nutrients (e.g., CAX-mediated Zn2?
accumulation). However, these types of manipulations will require
different growth conditions and modifications in CAX transport
properties (31). Given that the ionic radius of Ca2?is almost
identical to that of Cd2?, sCAX1-expressing plants also can poten-
tially sequester increased levels of cadmium. Here, we used
cadmium-free hydroponic solutions to avoid any adverse metal
accumulation in the carrots. Careful monitoring of the growth
conditions and nutrient composition of the food will have to be
taken with any crop expressing CAX transporters.
Our findings directly evaluate the nutritional consequence of
transgenic foods in animal and human feeding studies. We
establish unequivocally that modifying a single plant calcium
transporter improves plant calcium absorption. Although this
work represents initial studies toward understanding the nutri-
tional impact of transgenic foods, the technology may be even-
tually applied to various crops because it involves the overex-
pression of a gene found in all plants. Additionally, the approach
in this work can serve as a paradigm for related similar hypoth-
of nutrients contained within the plant matrix.
Materials and Methods
Mice Feeding Study. Animal protocols were approved by the Baylor College of
Medicine Institutional Animal Care and Use Committee. The C57BL/6 (Charles
River Laboratories) mice were housed in cages with ad libitum access to water
and food (AIN93G diet; ref. 32), and the feeding protocols have been de-
scribed previously (14). The mice ate ?3.5–4 g of diet per day at 6–7 weeks of
age (n ? 120). The mice were stratified and held without food for 24 h and
separated into treatment groups by using a randomized block design. After
24 h, 3.5 g of the carrot diets was placed in a glass food dish and placed into
the cage. The amount of diet fed to each mouse represented 14.0 kcal, which
is similar to the 14.4–16 kcal/day that control mice of identical age consume
and ensured that the mice ate the majority of the meal in 24 h. All mice were
remaining diet, if any, was placed in a bag and saved for analysis.
Growth of Carrots and Preparation of45Ca-Labeled Plants. Carrots (Daucus
carota L. var. Danver 60) and single-copy homozygous sCAX1-expressing
were subsequently transferred to hydroponic growth containers (33, 34).
Carrot plants were grown in hydroponics for 60 days, and then the solution
was supplemented with 1?Ci45Ca per liter. The carrots were then grown an
additional 30 days.
The plant material was harvested and dried at 25°C. Once dried, the material
hydroponically (14). After 90 days, the solution was supplemented with 5 mg
42Ca per liter for the control carrots and 2.5 mg42Ca per liter for the sCAX1-
expressing carrots. This label was added to 21 liters of hydroponic solution,
and the carrots were grown until all of the solution was absorbed by the
plants. A second round of42Ca labeling was done in 10 liters of hydroponic
solutions. This two-part labeling took 14–18 days. Fresh carrots were har-
vested, weighed, sliced, and stored at ?20°C.
One 0.25- to 0.50-inch latitudinal slice from control and sCAX1-expressing
Furnace, Barnsted International). These carrot pieces were then dissolved in 3
M HCl, neutralized with 1 M NaOH, dried for 24 h at 70°C, and resuspended in
0.1 M HCl. Total calcium was determined by inductively coupled plasma-
atomic emission spectrometry (ICP) (35), and42Ca isotopic enrichment was
42Ca-Labeled Plants. Carrots were germinated and grown
determined with the use of a magnetic sector thermal ionization mass spec-
trometer (Finnigan MAT 261; Finnigan) (36).
determined by methods outlined in Nakata and McConn (37).
Mouse Diet Preparation. Initially, we determined the nutritional composition
of carrots for the following constituents: fiber, total protein, calcium, potas-
sium, and magnesium (35, 38, 39). Two standard diet mixes were then ob-
the addition of 1.0 g of dried ground control carrot and 0.5 g of dried ground
sCAX1-expressing carrots to 2.5–3.0 g of prepared diet mix. This 3.5 g of
composite diet was nutritionally equivalent (including 5.0 mg/g calcium) to
the standard AIN93G diet eaten by mice during a given 24-h period (32).
Before feeding, diets were mixed to homogeneity with 2 ml of dH2O. For the
extrinsically labeled diet, the45Ca label was a component in the water.
Mice Bioavailability Analysis. The mice bones were analyzed as described
and the bones ashed in a muffle furnace (Thermolyne Furnace, Barnsted
International). The activity of45Ca incorporated in the bones was determined
as described previously (14).
it was processed and analyzed as previously described. Then, the45Ca activity in
The percent dose of45Ca in the hind limbs was calculated by using Excel
(Microsoft), and statistical differences were calculated by using ANOVA in SPSS.
advertising and word of mouth. The Institutional Review Board of Baylor
College of Medicine and Affiliated Hospitals approved this protocol, and
informed written consent was obtained from all subjects. Subjects were
eligible for enrollment if they had no underlying health conditions that may
affect calcium absorption, had an average Ca intake of 600–1,200 mg/day
less than the 95th percentile for age and gender (Table 2).
Isotope Preparation and Mineral Absorption Study. We procured42Ca (94%
for human use as the chloride salt by the Investigational Pharmacy Service of
the General Clinical Research Center of TCH (National Institutes of Health Grant
water since midnight. Subjects were randomized to receive either the control or
modified carrots at visit 1 (with the alternate being consumed at visit 2). Diets
were controlled so that each breakfast provided approximately one third of the
daily calcium intake of the subjects’ mean intake (?900 mg of Ca on the study
day). The remaining intake was divided between lunch and dinner, with one
optional snack of negligible calcium content. Subjects consumed either 65 g of
the modified carrot or 120 g of the control carrot to provide ?38 mg of Ca.
Subjects also consumed 170 g of calcium-fortified orange juice (Minute Maid,
given 15 ?g of46Ca i.v. This process was repeated at visit 2 to ensure that the
Dinner was provided in the form of a fast-food gift card from one of three
locations. Subjects were instructed by the study dietitian as to what they were
allowed to eat at each location to maintain the 300 mg of Ca per meal require-
ment. Subjects notified the study dietitian the following day as to what they
consumed. After the morning test meal, subjects were instructed to collect their
urine in 8-h pools for 24 h. Subjects were contacted between the two visits for
24-h dietary recalls on two nonconsecutive days to ensure that their calcium
intake was maintained during the study.
Calculation of Mineral Absorption. Urine samples were prepared for mass spec-
analyzed for isotopic enrichment with the use of a magnetic sector thermal
ionization mass spectrometer (Finnigan MAT 261; Finnigan). Each sample was
analyzed for the42Ca/48Ca and46Ca/48Ca ratios with correction for fractionation
to the reference44Ca/43Ca. The accuracy and precision of this technique for
natural abundance samples compared with those of standard data are 0.15%
www.pnas.org?cgi?doi?10.1073?pnas.0709005105Morris et al.
thei.v.isotopeduringthe24hafterisotopeadministration(fromtimeofthefirst Download full-text
oral dose until 24 h after the last oral dose).
ACKNOWLEDGMENTS. We thank Drs. Ian Griffin, Paul Nakata, and Dennis
Bier for critical reading and helpful comments with this manuscript; Gloria
Orozco, Adrianne Morse, Sevahn Allahverdian, Maria Hamzo, and Zhen-
sheng Chen for subject assistance and sample analysis; Jenell Dancy and
staff for help with subject assistance during their time at the General
Clinical Research Centers; and Adam Gillum for graphic design assistance.
This work was supported by National Institutes of Health Grant IR01 DK
062366 and U.S. Department of Agriculture Grant CSRESS#2005-34402-
16401 Designing Foods for Health (to K.D.H.).
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