Uptake rate measurement of some amino acids on normal and treated yeast cells to xenobiotics ...

59 Pages·2004·2.58 MB·English

Uptake rate measurement of some amino acids on
normal and treated yeast cells to xenobiotics using
14C labelled amino acid
Nurhajati Tanudjojo, Pringgo Soedigdo, and Soekeni Soedigdo
Abstract chemical carcinogens [1, 2]. So far many studies on the
physiological influences of BP and DMBA have been
Benzo(α)pyrene (BP) and 7,12—dimethylbenz(α) anth- generally carried out in mammals. Much research has
racene (DMBA) are potent carcinogens for mammals, been done, but the mechanism of carcinogenesis in
which are able to affect the normal metabolic processes. mammals is not clear. The effect of BP and DMBA in
The influence both of BP and DMBA to the transport unicellular eucaryotes has been studied [3]. However,
rate of individual 14C labeled amino acids (14C-lysine; the results from this study could not explain clearly all
14C-valine; 14C-leucine or 14C-tyrosine) in yeast Saccha- the problems found in carcinogenesis.
romyces cerevisiae strain A3 were studied by introducing We studied the influence of BP and DMBA on the
about one µCi (37 kBq) of individual 14C labeled amino uptake rate of some amino acids in yeast. For its growth
acid into 30 ml liquid ethanol media that contained BP and regeneration, uptake of amino acids by the yeast
(0.001% v/v) or DMBA (0.001% v/v), then followed by Saccharomyses cerevisiae from the media is proceeded
inoculating a known concentration of yeast suspension by active transport. Permeases facilitate the active
in such a manner to get the initial optical density (OD) transport of various amino acids [4]. The uptake rate
of new cultures were about 0.10. Uptake rates were of individual amino acid from the media or wort* is
determined at certain intervals after inoculation, using different [4, 5]. Based on the uptake rates, Pierce [6–8]
a liquid scintillation counter. The results show that BP divided the amino acids in wort into four groups. The
had the tendency to increase the uptake rate while DMBA most readily absorbed was the A group that consists
showed a reversed effect on the use of amino acids. It was of glutamic acid, aspartic acid, asparagine, glutamine,
also found that tyrosine was absorbed faster than valine serine, threonine, lysine, and arginine, followed by the
as well as leucine and this was different with the result B group that consists of valine, methionine, leucine,
reported by the former investigators. isoleucine, and histidine, then followed by the C group
that consists of glycine, phenylalanine, tyrosine, tryp-
tophan, alanine, and ammonia and the slowest one was
Key words: uptake rate, amino acid, yeast xenobiotic D group containing only proline.
According to Weisburger et. al. [9] when carcinogens
enter an organism, they are metabolized and become
Introduction the ultimate carcinogens that react with DNA. The
adduct could result in mistranscription in mRNA
Several agents that cause cancer in human beings formation. Since mRNA is a protein template, this
have been studied extensively. Benzo(α)pyrene (BP) matter would further cause the mistranslation in pro-
and 7,12—dimethylbenz(α)anthracene (DMBA) are tein synthesis [10, 11]. The last process could probably
polynucleated hydrocarbons that belong to potent give rise to the change in the structure of either the cell
membrane or the cell wall.
It is supposed that the presence of BP and DMBA
Nurhajati Tanudjojo is affiliated with the Centre for in the culture media of yeast would be able to inhibit
Research and Development of Nuclear Techniques, National or to change the amino acid uptake pattern of yeast.
Nuclear Energy Agency in Bandung, Indonesia. Pringgo Soed- We studied the uptake of some amino acids, which
igdo and Soekeni Soedigdo are affiliated with the Chemistry
Department, Bandung Institute of Technology in Bandung,
Indonesia.
Mention of the names of firms and commercial products does * Wort is a sweet mixture of malt and water, which, when it
not imply endorsement by the United Nations University. is chemically changed by fermentation, will become beer.
Food and Nutrition Bulletin, vol. 23, no. 3 (supplement) © 2002, The United Nations University. 61
62 N. Tanudjojo et al. Uptake rate measurement of some amino acids using 14C labelled amino acid 63
represented each A, B, and C group by yeast using a
Counting and calculation
radioactive tracer method.
A 0.5 cm3 sample of supernatant of each culture was
added to the counting bottles each of which contained
Materials and methods 4.5 cm3 of the liquid scintillation cocktail. The mixture
was shaken in order to mix it thoroughly, then counted
The yeast used in this experiment was Saccharomyses using a liquid scintillation counter. Each sample was
cerevisiae strain A3 obtained from Biochemistry Sub- counted for 5 minutes and repeated 3 times. The counts
division, Bandung Instititute of Technology and had obtained in counts per minute (cpm) were transformed
been adapted to grow on 1% liquid ethanol media. BP into desintegrations per minute (dpm).
and DMBA were obtained from Fluka (Buchs, Switzer- The calculation of the uptakes was done as follows:
land), while the 14C labeled amino acids were from the
A – A 1
Radiochemical Center Amersham (Buckinghamshire, A = ——p——l— • —
UK). The A, B, and C groups of amino acids each were u OD – OD T
l p
represented by 14C-lysine, 14C-valine, 14C-leucine, and
14C-tyrosine. Picofluor from Packard (Downer Grove, where:
Ill., USA) was used as a liquid scintillation cocktail. All A = radioactivity of amino acid uptake rate
u
the chemicals used under this experiment were analyti- /0, 10 OD/hour,
cal grade reagents. A = radioactivity of the previous amino acid
p
The Beckman liquid scintillation counter (Beckman, uptake rate /0, 10 OD/hour,
Irvine, Calif., USA, LS 7550, No. Series 7775082) was A = radioactivity of the latter amino acid
l
used to count the radioactivity. Separation of yeast uptake rate /0, 10 OD/hour,
(from residual media) was carried out using a portable OD = the previous optical density of yeast
p
centrifuge. Inoculations and sampling out inoculates at inoculate,
various intervals were done in the sterile Laminair Air- OD = the latter optical density of yeast inocu-
l
flow Bench (Clemco, Australia) and an Edmund Buhler late,
shaker (Dreieich, Germany) was used to activate yeast T = length of time between the previous and
regeneration during incubation. the latter intervals.
Regeneration Results and discussion
The molecular structures of BP and DMBA are shown
BP treated yeast group
in figure 1. Figure 2 shows the ethanol yeast regen-
A sample of 0.3 cm3 of 0.1% BP solution was put into eration curve. The lag phase was the period roughly
an Erlenmeyer flask (125 ml) containing 30 ml of 1% between zero to five hours, the exponential phase was
liquid ethanol media which consisted of 1% KH PO , between 5 to 20 hours, 20 to 30 hours was the early
2 4
1% pepton, and 1% ethanol (v/v). The volume of 10–2 stationary phase and the late stationary phase was the
cm3 (~ 37 kBq or 1 µCurie) of one of the 14C labeled period over 35 hours. Samples were taken from the
amino acids (14C-lysine) was added to the media, fol- yeast culture following the sequence of its regenera-
lowed by inoculation of ethanol yeast which was taken tion growth phase.
from the stock suspension to get the initial optical den- The uptake rate of the 14C-lysine of the three groups
sity of 0.100. The culture was shaken at the speed of 180 of yeast is shown in figure 3. During the lag phase (0
rpm and incubated at room temperature. After 0, 5, 15, to 5 hours of incubation), the decrease of the labeled
25, 35, and 45 hours of inoculation, about 2 ml of the amino acids radioactivity in the residual media was sig-
inoculate was taken from the culture and centrifuged nificant. At the next interval, however, they increased,
to separate the yeast from the media. The supernatant while the yeast population was higher at this interval.
was used to determine the radioactivity found in the
residual media. The same treatment was done for the
2
other 14C-labeled amino acids on the 5th, 10th, and CH 1 3
3
15th regeneration. 10 1112 4
9 5
8 7 6
DMBA treated yeast group CH
3
Benzo(α)pyrene 7,12-dimethylbenz(α)anthracene
The step and the sequence of work was the same as
(BP) (DMBA)
in the BP treated yeast group. In this group DMBA
replaced BP. FIG. 1. Molecular structure of BP and DMBA
62 N. Tanudjojo et al. Uptake rate measurement of some amino acids using 14C labelled amino acid 63
This indicated that during the lag phase the rates of Figure 7 shows the uptake rates of the four 14C-
the uptake were not stable yet. It was supposed that the labeled amino acids on the control yeast. Through the
decreased radioactivity in the media was not simply sum of the all intervals uptake rate it could be stated
caused by the uptake, but that there were other factors that the fastest to be absorbed was 14C-lysine, then
involved. The stickiness of the outer cell wall could followed by 14C-valine, 14C-tyrosine, and 14C-leucine.
lead to the attachment of 14C-lysine molecules onto The same pattern was applied for BP and DMBA yeasts
the cell wall. By further incubating while shaking, the (figs. 8 and 9). As illustrated in the figure 3 to 9, even
molecules were unattached from the media, which though the speed of each amino acid uptake fluctu-
increased the radioactivity of the media, so that at 5 to
15 hours the uptake rates were reduced.
30 Y-C
For control and BP yeasts, the uptake rates began to Y-BP
YDMBA
appear at 5 to 15 hours and were increasing at 15 to 25 25
hours, while for DMBA yeast, the rate was slower. At
e20
the early stationary phase (25 to 35 hours) the uptake at
rate began to decrease for BP yeast up to the late sta- e r15
k
tionary phase (35 to 45 hours). For control yeast, the a
uptake rate decreased during the late stationary phase, Upt10
however, the uptake rate for the DMBA yeast increased. 5
These phenomena could probably be related to the
metabolic processes of each group of yeasts. 0
0–5 5–15 15–25 25–35 35–45
Similar uptake patterns were indicated for 14C-valine
and 14C-leusine (figs. 4 and 5), however, for 14C-tyro- Period of incubation (hrs)
sine (fig. 6), pyramidal shapes of uptake rate were FIG. 4. Uptake rate of 14C-Val of yeasts at various periods of
shown on the three groups of yeasts, and the peak was incubation (dpm/0.10 OD/h)
found at the incubating interval of 15 to 25 hours.
10 Y-C
Y-BP
YDMBA
III. Stationary phase
B 8
e
at 6
e r
n k
og II. Exponential phase pta 4
L U
2
0
A
0–5 5–15 15–25 25–35 35–45
I. Lag phase
Period of incubation (hrs)
T (hr)
FIG. 5. Uptake rate of 14C-Leu of yeasts at various period of
FIG. 2. Yeast regeneration curve incubation (dpm/0.10 OD/h)
20 Y-C 10 Y-C
Y-BP Y-BP
YDMBA YDMBA
8
15
e e
at at 6
e r10 e r
k k
a a 4
pt pt
U U
5
2
0 0
0–5 5–15 15–25 25–35 35–45 0–5 5–15 15–25 25–35 35–45
Period of incubation (hrs) Period of incubation (hrs)
FIG. 3. Uptake rate of 14C-Lys of yeasts at various periods of FIG. 6. Uptake rate of 14C-Tyr of yeasts at various period of
incubation (dpm/0.10 OD/h) incubation (dpm/0.10 OD/h)
64 N. Tanudjojo et al. Uptake rate measurement of some amino acids using 14C labelled amino acid 65
ated along all intervals, in each group of yeast (control Conclusion
and treated) 14C-tyrosine was absorbed faster than
14C-leucine. But if the uptake rate was considered on The uptake rates of amino acids by yeasts fluctuated
the effective intervals of yeast growth rate (exponential during each generation period. The uptake of amino
up to the early stationary phases) and comparison was acids appeared consistently between the transition
done beyond the uptake rate of 14C-lysine, the tendency and early stationary phases. Tyrosine was absorbed
to increase the uptake of amino acids was shown by relatively faster than valine and leucine which was dif-
BP yeast, while DMBA yeast showed the reverse effect ferent from that stated by Pierce [7–9]. It was supposed
(fig.10). Also, 14C-tyrosine was absorbed faster than that BP and DMBA led to different effects on the cell
14C-valine as well as 14C-leucine. This phenomenon membrane as well as the cell wall, so the uptake rate
differs to that found by Pierce [7–9]. patterns for various amino acids of BP and of DMBA
yeasts were different from that of the control.
16 Lys
Val
10 VLyasl 14 LTyeru
Leu
12
8
e
ate 6 e rat108
ptake r 4 Uptak 46
U
2 2
0
0 0–5 5–15 15–25 25–35 35–45
0–5 5–15 15–25 25–35 35–45
Period of incubation (hrs)
Period of incubation (hrs)
FIG. 9. Uptake rates of 14C-amino acids of treated yeast with
FIG. 7. Uptake rates of 14C-amino acids of control yeast at DMBA at various periods of incubation (dpm/0.10 OD/h)
various periods of incubation (dpm/0.10 OD/h)
14 Y-C
Y-BP
30 LVyasl 12 Y-DMBA
Leu e
25 Tyr at10
e r
e20 k 8
e rat15 upta 6
ptak10 ean 4
U M
2
5
0
0 14C-Lys 14C-Val 14C-Leu 14C-Tyr
0–5 5–15 15–25 25–35 35–45
Amino acid
Period of incubation (hrs)
FIG. 10. Mean uptake rates of 14C-amino acids (dpm/0.10
FIG. 8. Uptake rates of 14C-amino acids of treated yeast with OD/h) of normal and treated yeast to BP and DMBA during
BP at various periods of incubation (dpm/0.10 OD/h) 5–35 hours of incubation
64 N. Tanudjojo et al. Uptake rate measurement of some amino acids using 14C labelled amino acid 65
References
1. Devoret R. Bacterial test for potential carcinogens. Sci 6. Pierce JS. Amino acids in malting and brewing. Process
Am 1971;241;2:28–37. Biochem 1966;1:412–6.
2. Chouroulinkov I, Gentil A, Grover PL, Sims P. Tumour 7. Pierce JS. Amino acids in malting and brewing. Tech
initiating activities on mouse skin of dihydrodiols derived Quart Master Brewers Assoc Am 1966;3:321–6.
from benzo(α)pyrene. Br J Cancer 1976;34:523–32. 8. Pierce JS. The importance on wort nitrogen in brewing.
3. Eckardt F, Muliawan H, de Ruiter N, Kappus H. Rat Tech Quart Master Brewers Assoc Am 1969;6:93–7.
hepatic vinyl chloride metabolites induce gene conver- 9. Weisburger JH, Williams GM. Chemical carcinogenesis:
sion in the yeast strain D7RAD in vitro and in vivo. metabolism of chemical carcinogens. In: Becker FF, ed.
Mutation Res 1981;91:381–90. Cancer a comprehensive treatise. New York: Plenum
4. Suomalainen H, Oura E. Yeast nutrition and solute Press, 1975:185–234.
uptake. In: Rose AH, Harrison JS, eds. The yeast. London 10. Watson JD. The involvement of RNA in the synthesis of
and New York: Academic Press,1971:45–7. protein. Science 1963;140:17–26.
5. Barton-Wright EC, Thorne RSW. Utilization of amino 11. Bosch L, ed. The mechanism of protein synthesis and its
acids during yeast growth in wort. J Inst Brew 1949;55: regulation. New York: American Elsevier, 1972.
383–6.
Selenium determination in whole human blood Selenium determination in whole human blood 67
by radiochemical neutron activation analysis:
preliminary results in Argentina
Sonia Edith Hevia, María Luz Pita Martín de Portela, and Sara Manuela Resnizky
Abstract between different areas in the same country [2]. Hence,
selenium nutritional status differs according to the sele-
In Argentina there are no values regarding the content nium in the food. Often, there is a significant correla-
selenium in food or its effect on nutritional status. Neu- tion between the selenium in the food and the selenium
tron activation analysis and a radiochemical separation status of humans and animals [3].
scheme for selenium has been adjusted for blood samples There are seleniferous areas in Venezuela, but there
from healthy adult individuals. Fasting whole blood was is lack of information from all other countries. Based
collected, lyophilized, packed, and irradiated in a nuclear on indirect studies carried out in cattle it has been
reactor. Selenium was determined by a radiochemical sepa- postulated that selenium deficient areas could exist
ration. The method was validated using certified reference in Buenos Aires [4]. However, there are a few values
materials. The selenium concentrations (mean value ± regarding the selnium content of foods [5], but none
SD) from five pools and from 22 individual samples were regarding human nutritional status or the content of
0.070 ± 0.020 µg/ml and 0.071 ± 0.014 µg/ml, respectively, selenium in the soil.
ranging between 0.047 to 0.105 µg/ml. These preliminary Problems with methodology to determine selenium
results show values close to those published for the popula- account for the lack of information on this subject.
tion living in areas with adequate selenium intake, but Neutron activation analysis is a useful technique for
lower values than those published for the USA population. determining trace elements [6]. A radiochemical
However, it will be necessary to carry out nutritional status separation scheme for selenium, previously devel-
studies in other areas of Argentina, taking into account the oped at our laboratory, has been adjusted for blood
geographic and topographic diversity of the country. samples. Therefore, we applied this method to deter-
mine the level of selenium in healthy humans from
Buenos Aires.
Key words: selenium, neutron activation analysis,
human blood
Objectives
Introduction We evaluated blood selenium levels in a group of
healthy adults living in Greater Buenos Aires to obtain
Selenium is an essential trace element in humans, with information regarding their selenium nutritional
a narrow range between deficiency diseases and toxicity status.
[1]. The marked geographic variation in concentration
of selenium in soil accounts for the large variation in
the selenium content in food between countries and Materials and methods
Sonia Edith Hevia and Sara Manuela Resnizky are affiliated Subjects
with the Comisión Nacional de Energía Atómica in Buenos
Aires, Argentina. María Luz Pita Martín de Portela is affiliated Blood samples from healthy adults (5 whole blood
with the Facultad de Farmacia y Bioquímica in the Universi-
pools and 22 individual samples from 5 females and 17
dad de Buenos Aires in Buenos Aires, Argentina.
males), aged 18 to 40 years, were collected with EDTA
Mention of the names of firms and commercial products
does not imply endorsement by the United Nations University. and prepared for selenium determination.
66 Food and Nutrition Bulletin, vol. 23, no. 3 (supplement) © 2002, The United Nations University.
Selenium determination in whole human blood 67
Selenium was considered a mere environmental
Sample preparation
toxicant, but since about the mid-twentieth century
Whole blood samples (2 ml) were lyophilized and it has been recognized as an essential micronutrient.
weighed. Selenium standards were prepared by weigh- While selenium is an essential nutrient, it can be toxic,
ing drops of a diluted selenium solution on ash-free therfore it is important to know the selenium status of
filter paper and air dried. The samples and standards soils, animals, and human beings [2].
were pelletized, packed in polyethylene bags and alu- There is a wide range of natural dietary selenium in
minum foil and irradiated for 50 hours at the 40 Kw different parts of the world. In South America, there are
RA-1 reactor (Constituyentes Atomic Center, Buenos seleniferous areas in Venezuela and in the waters of the
Aires, Argentina), at an approximate thermal neutron Orinoco river. There are high-selenium shales in parts
flux of 2 1012 cm–2 seg1. of the Andes mountains accounting for some high sele-
After irradiation and a 2 to 3 week cooling period, the nium foods in Venezuela, but there are no data from
samples and standards were digested with nitric acid other South American countries including Argentina
using a microwave oven and Parr bombs. Then sulfu- [4]. In Chile egg yolks from five different geographic
ric acid and selenium (1 mg) and mercury (100 mg) areas are being analyzed for selenium [7].
carriers were added, and they were heated until nitric Selenium status can be assessed by measuring sele-
acid was entirely removed. Finally, they were diluted nium levels in serum or whole blood or by measuring
to 5 N in sulfuric acid, and HgS was precipitated by the indirect indicator activity of glutathione peroxidase.
additioning hydroxilamine clorhydrate (10%) and Ruksan and Zenelli prepared a map of selenium-defi-
thioacetamide solution (10%). Selenium coprecipi- cient areas in Argentina, based on glutathione peroxi-
tated with HgS. The precipitates were allowed to stand dase levels in cattle grazing in areas where “Enteque
overnight, then filtered and prepared for measurement seco” is endemic [4]. This disease is an enzootic syn-
using adhesive polyethylene foil. drome produced by Solanum malaecoxilum. However,
certain nutritional factors and infections can lead to a
decrease in the glutathione peroxidase activity as do low
Measurement of selenium
selenium levels [8]. Ruksan and Zenelli also believed
Selenium was determined using 75Se (t 1⁄2 = 120 d) with that there are selenium toxic areas in the provinces of
a Hiper Pure Germanium detector (30% efficiency, Córdoba and San Luis [4]. These hypotheses were not
resolution 1.8 keV for the 1332 60 Co peak) coupled to confirmed through the determination of the selenium
a multichannel buffer module. The concentration was content in the soil.
calculated using software developed at our laboratory. There are no national food composition data for
The method was validated using certified reference selenium in Argentina. Hack reported some data on
materials, horse kidney (IAEA H-8) and animal blood the selenium content of wheat grain harvested in five
(IAEA A-13). The results were in accordance with the areas of the Province of Buenos Aires from 1993 to
certified values (table 1). 1995. These results showed a wide variation, with a
mean value and ranges similar to the German wheat
grain [5]. Table 3 shows the values for the provisional
Results and discussion content of selenium in eggs and wheat in the United
States [9], published values for selenium for eggs in
The selenium concentrations of the five whole blood Chile [7] and for wheat in Argentina [5].
pools and 22 individual samples are shown in table 2. Analysis of blood samples from individuals from
The mean value for the five whole blood pool was not different regions showed a good correlation between
different from that of the individual data, nor was there their intake of selenium from food and selenium levels
any difference between mean values for males and in their blood. Whole blood selenium concentration,
females. Ranges were 0.047 to 0.105 µg/ml for males unlike serum selenium levels is an index of long-term
and 0.055 to 0.072 µg/ml for females. selenium status and does not fluctuate from day to day.
TABLE 1. Selenium concentrations in certified reference
TABLE 2. Selenium levels in the blood of the study popu-
materials
lation
Mean selenium concentration ± SD
Mean values ± SD µg/ml
(ppm)
Whole blood pools (n = 5) 0.070 ± 0.020 (0.050–0.100)
Experimental Certified value
Total group (N = 22) 0.071 ± 0.014 (0.047–0.105)
IAEA H-8 4.4 ± 0.2 4.67 (4.37–4.97)
horse kidney Males (n = 17) 0.072 ± 0.016 (0.047–0.105)
IAEA A-13 0.22 ± 0.06 0.24 (0.15–0.31) Females (n = 5) 0.066 ± 0.008 (0.055–0.072)
animal blood
66 Food and Nutrition Bulletin, vol. 23, no. 3 (supplement) © 2002, The United Nations University. Number in brackets are ranges.
68 S. E. Hevia et al.
Longnecker et al. demonstrated that selenium blood methodology will allow to determine the selenium
levels reflect usual dietary selenium intake. They found nutritional status of humans and cattle in differ-
a basal blood selenium level of 0.196 µg/ml in healthy, ent parts of Argentina. Blood selenium levels in this
white males aged 22 to 64 years, with a selenium intake group of Argentinian healthy adults, living in Greater
of 80 µg/day; after feeding them a piece of bread con- Buenos Aires, were similar to those published for the
taining 206 µg of selenium daily for two weeks, their population living in areas with an adequate selenium
selenium blood level significantly increased and intake. However, it will be necessary to carry out col-
remained fairly constant for the 52 weeks of treatment laborative studies to determine the nutritional status
and returned to the baseline level 12 weeks after the in other areas of Argentina, taking into account the
treatment ended [10]. Hence, selenium concentrations geographic and topographic diversity of the country.
in whole blood may reflect usual selenium intake. Therefore, these results should not be extrapolated to
The mean values of the Argentinian group studied, the whole country.
those for the areas with a high incidence of Keshan’s
disease, and those with variable (high, low, or normal)
selenium intakes are shown in figure 1 [3, 8]. Argentina
From this preliminary study we concluded that this
USA, Ohio
USA, South Dakota
TABLE 3. Selenium content of eggs and wheat in the United
States, eggs in Chile, and wheat in Argentina Canada
Moderate selenium
Selenium (µg/100 g) (Beijing)
USA Chile Argentina Egypt
Low selenium
Egg white 17.6 ± 7.60 79 ± 41
(China)
Egg yolk 45.2 ± 10.7 81 ± 43 Keshan’s disease
Wheat grain 70.7 ± 14.2 42 ± 23 0 0.05 0.10 0.15 0.20 0.25 0.30
(1994/95 harvest)
Blood selenium level (µg/ml)
29 ± 40
(1993/94 harvest)
FIG. 1. Selenium levels in blood from Argentina and
Sources: ref. 5, 7, 9. worldwide
References
1. Levander OA, Burk RF. Selenium. In: Brown ML, ed. 6. Resnizky S, Dallorso ME, Pawlak E. Neutron activation
Present knowledge in nutrition, 6th ed. Washington, analysis of Se in bovine plasma samples. Biol Trace Elem
D.C.: Nutrition Foundation,1990:268–78. Res 1999;71(2):343–7.
2. Standing Committee on the Scientific Evaluation of 7. Ruz M, Codoceo J, Hurtado S, Muñoz L, Gras N. Char-
Dietary References Intakes, Food and Nutrition Board, acterization of the regional distribution of selenium in
and Institute of Medicine. Antioxidants’ role in chronic Chile using selenium in hens’ eggs as a monitor. J Trace
disease prevention still uncertain; huge doses considered Elem Med Biol 1995;9:156–9.
risky. Setting new dietary recommendations. Washing- 8. Diplock AT. Indexes of selenium status in human popu-
ton, D.C.: National Academy of Sciences, 2000. lations. Am J Clin Nut.1993;57(suppl):256S–8S.
3. Diplock AT. Trace elements in human health with 9. Gebhardt SE, Holden JM. Provisional table on the sele-
special reference to selenium. Am J Clin Nutr 1987;45: nium content of foods. Human Nutrition Information
1313–22. Service, HNIS/PT-109. Washington, D.C.: United States
4. Oldfield JE. Selenium world atlas. Grimbergen, Belgium: Department of Agriculture, 1992.
Yves Palmieri Ed., 1999. 10. Longnecker MP, Stampfer MJ, Morris SJ, Spate V, Baskett
5. Hack A, Bruggemann J. Contenido de selenio en trigo C, Mason M, Willet WC. A 1-y trial of the effect of high-
argentino. La Alimentación Latinoamericana 1996; selenium bread on selenium concentrations in blood
214:36–8. and toenails. Am J Clin Nutr 1992;57:408–13.
Stable isotope techniques in human nutrition research:
concerted action is needed
Thomas Walczyk, Andy Coward, Dale A. Schoeller, Tom Preston, Jack Dainty,
Judith R. Turnlund, and Venkatesh Iyengar
Abstract nutrition research. Their use now permits researchers
to explore aspects of basic and applied nutrition in vivo
Use of stable isotopes in applied nutrition has been a that are otherwise impossible to assess. Stable isotope
developing field for over 60 years. The past 20 years has labels can be used to trace the absorption and utili-
seen improvements in computer and mass-spectrometer zation of minerals and trace elements in the human
technology that has opened up even greater possibilities in body [1]. With isotopically labeled water we can assess
the understanding of human metabolism. While improve- parameters of body composition and energy expendi-
ments in technology can bring great opportunities, it can ture [2] and substrates labeled with 2H or 13C allow us
also cause problems if there is no consensus among the to measure the activity of selected enzyme pathways
stable isotope user-community on standardization of and gastrointestinal function to determine functional
new techniques and methods. Users of stable isotopes consequences of altered nutrition [3]. As valuable as
have traditionally been split into two groups; those who such information is for basic and applied nutrition
work with heavy isotopes (e.g., 58Fe, 70Zn) and those who research, it is often challenging to obtain meaningful
work with light isotopes (e.g., 2H 18O). Standardization data using these techniques. Specialists from various
2
issues have been addressed by the light isotope users and scientific disciplines including nutritionists, food scien-
awareness of this type of problem is starting to emerge tists, analytical chemists, and mass spectrometrists have
within the heavy isotope community. to work together, each contributing their expertise.
In 1999 and 2000 meetings were held at the Interna-
tional Atomic Energy Agency (IAEA, Vienna, Austria)
Key words: nutrition, stable isotopes, mass spec- to assess the current situation and future directions
trometry, harmonization, SIGN for the application of isotopic techniques in nutrition
research. IAEA plays an active role in the promotion
and dissemination of isotopic techniques in applied
Background research, including human nutrition research. IAEA
projects, managed by both the Department of Nuclear
In the latter part of the twentieth century, stable iso- Applications and the Department of Technical Coop-
tope techniques became indispensable tools for human eration, in the field of human nutrition range from
small research projects, through training to national
and regional projects especially focused on monitoring
Thomas Walczyk is affiliated with the Laboratory of
nutritional interventions.
Human Nutrition, Institute of Food Sciences, Swiss Fed-
At the 2000 consultants meeting, the consensus was
eral Institute of Technology (ETH) in Zurich, Switzerland.
Andy Coward is affiliated with the MRC Human Nutrition that there is a need to improve data quality [4]. Com-
Research, Elsie Widdowson Laboratory in Cambridge, UK. parability of data between laboratories is often limited
Dale A. Schoeller is affiliated with the Department of Nutri- as techniques are sophisticated and sample analysis, as
tional Sciences, University of Wisconsin in Madison, Wisc.,
well as calculations, are challenging. Sources of bias are
USA. Tom Preston is affiliated with the Isotope Biochemistry
Laboratory, Scottish Universities Environmental Research multiple and require more than simple underpinning
Centre in East Kilbride, UK. Jack Dainty is affiliated with of the work with quality control by the use of refer-
the Institute of Food Research (IFR) in Norwich, UK. Judith ence materials. Considering the likely expansion of
R. Turnlund is affiliated with the Western Human Nutrition
stable isotope techniques, concerted action is needed
Research Center, University of California in Davis, Calif.,
USA. Venkatesh Iyengar is affiliated with the International to ensure comparability of results and data quality.
Atomic Energy Agency in Vienna, Austria. This refers in particular to applications in which data
Food and Nutrition Bulletin, vol. 23, no. 3 (supplement) © 2002, The United Nations University. 69
70 T. Walczyk et al. Stable isotope techniques in human nutrition research 71
generated by stable isotope techniques may influence A history of interdisciplinary collaboration
political decisions, e. g., within the framework of gov-
ernmental led dietary advice and food fortification Stable isotope research in nutritional sciences serves as
programs. The workshop “Stable isotope users group an example of how collaboration between individual
in nutrition: opportunities, perspectives and harmo- scientists in diverse disciplines can lead to significant
nization,” held on Monday 27th August 2001 at the scientific advances. Few examples of this are more
17th International Conference on Nutrition under the illustrative than the development and application of
sponsorship of IAEA tackled this problem. the doubly-labeled water method to the measurement
of human energy expenditure.
The doubly-labeled water method was born out of
Concept of the workshop a collaboration between Nathan Lifson, a physiolo-
gist, and Alfred O. Nier, a physicist. This collaboration
Organized by Dr. Thomas Walczyk and Dr. Andy spanned a period of 15 years beginning in the late
Coward with the support of IAEA, this workshop 1940s. Both were on the faculty of the University of
brought together experts and other stable isotope users Minnesota, but members of different departments
for both light isotope techniques (macronutrients and with no obvious interdepartmental ties. Dr. Lifson was
vitamins) and heavy isotope techniques (mineral and investigating the source of oxygen in respired CO . Dr.
2
trace elements). Experts from both fields were asked to Nier, a pioneer of mass spectrometry, was investigating
reflect on the current situation to initiate discussions the isotopic nature of elements and designing instru-
among the participants to identify specific needs for ments for isotopic purification and measurement.
future action. Neither alone could readily perform the studies to
Dr. Andy Coward reported on the current status of determine the source of oxygen in respired CO , but
2
standardization for the different light isotope tech- Dr. Nier could obtain and even produce 18O for use as
niques that are in routine use and stressed the need for a tracer and measure the 18O abundance in CO , while
2
harmonization of these techniques to ensure data com- Dr. Lifson provided the timely scientific question and
parability in the literature. Dr. Dale Schoeller described designed the animal experiment. This collaborative
some recent advances in light isotope techniques and demonstrated that the oxygen in respired CO came
2
pointed out the need for interdisciplinary collabora- from isotopic equilibration of the body water of the
tion between nutritionists and mass spectrometrists/ mice used in the study [5], through what is now known
analytical chemists in this field of research. Dr. Tom to be the action of carbonic anhydrase.
Preston described the existing SIGN network (Stable The results of this simple collaborative experiment,
Isotopes in Gastroenterology and Nutrition) that was however, led to a far more interesting idea. Dr. Lifson
established in Europe as a forum for consensus build- hypothesized that isotopic equilibration between the
ing and standardization of light isotope techniques. oxygen in CO and that in water would produce a
2
For the mineral and trace element techniques, Dr. differential turnover of the oxygen and hydrogen in
Thomas Walczyk highlighted the different sources of body water. The oxygen turnover would be a measure
bias in the design and conduction of stable isotope stud- of water and CO flux through the body, while the
2
ies, sample preparation, mass spectrometric analysis, hydrogen turnover would be a measure of water flux
and data evaluation techniques. Dr. Jack Dainty looked through the body. The difference would therefore be
critically at current practice in the transformation of a measure of CO flux and this would provide a non-
2
mass spectrometric data into nutritionally meaningful invasive measure of energy expenditure through the
results, the lack of uncertainty estimates in generated application of the principles of indirect calorimetry.
data, and the need for an agreed set of defined terms Continued interdisciplinary collaboration soon
in stable isotope research. Finally, Dr. Judith Turnlund proved the hypothesis and validated it under a variety
presented available techniques for isotopic analysis of of conditions [6–8] but application to human studies
minerals and trace elements in biological samples and came only 30 years later [9, 10]. It is unlikely that the
compared the use of inductively coupled plasma mass delay was at all related to limitations on the availability
spectrometry (ICP-MS) and thermal ionization mass of 18O labeled water or isotope ratio mass spectrom-
spectrometry (TIMS) for copper isotopic analysis in eters that were capable of measuring small 18O enrich-
human experiments. ments. Both items were commercially available during
The Vienna workshop was the first meeting to bring most of the period between when Lifson proposed the
together these two groups and was attended by more human studies and when they were finally performed
than 80 delegates. Here we report the main points that by others. Rather, the delay appears to be due to the
were addressed by the experts and the outcomes of the absence of a collaboration between persons with the
discussions from the floor. expertise in mass spectrometry and persons with an
expertise in human energy metabolism.