Production of deuterated beta carotene by metabolic labelling of Spirulina platensis

Biotechnology Letters 23: 447–449, 2001.
447
© 2001 Kluwer Academic Publishers. Printed in the Netherlands.
Production of deuterated ?-carotene by metabolic labelling of Spirulina
platensis
T. Gireesh1, A. Jayadeep1, K.N. Rajasekharan2, V.P. Menon3, M. Vairamany4, G. Tang5, P.P.
Nair6 & P.R. Sudhakaran1,?
1Departments of Biochemistry and 2Chemistry, University of Kerala, Trivandrum, India 695 581
3Department of Biochemistry, Annamalai University, Chidambaram, India
4Indian Institute of Chemical Technology, Hyderabad, India
5Human Nutrition Research Centre on Aging, Tufts University, Boston, USA
6Department of International Health Johns Hopkins University, Baltimore, USA
?Author for correspondence (Fax: +91 471 307158; E-mail: prsbn@md4.vsnl.net.in)
Received 17 October 2000; Revisions requested 24 October 2000/28 November 2000; Revisions received 28 November 2000/12 January 2001;
Accepted 12 January 2001
Key words: ?-carotene, deuterium labelling, Spirulina
Abstract
Method for production of deuterated ?-carotene for the bioavailability studies of vitamin A has been developed us-
ing Spirulina platensis in culture. Suspension cultures of Spirulina in heavy water (99.4% D2O) medium produced
maximum biomass and ?-carotene in 28 to 30 days. Of the total carotenoids, lutein constituted 30 to 35% while
?-carotene was about 24%. MS showed that 60 to 65% H atoms in ?-carotene were deuterated. 100% replacement
of H atom with deuterium was achieved by preventing exchange with atmospheric moisture. The medium could be
used in several cycles for metabolic labelling of carotenoids whereby the cost of production is reduced.
Introduction
ful in the evaluation of the bioavailability and vitamin
A status in humans (Furr et al. 1989).
Vitamin A de?ciency is a common public health prob-
Spirulina platensis is a fresh water microalgae
lem in many parts of the world (Ye et al. 2000).
that has very high nutritive value (Key 1991). Apart
Increasing the availability of dietary provitamin A
from pigments such as chlorophyll and xanthophylls,
carotenoids will improve the vitamin A status among
Spirulina produces ?-carotene which constitutes about
de?cient populations (de Pee et al. 1996). In this
0.5 mg g?1 dry matter. We now report the culture
context, studies on the bioavailability of vitamin A
of Spirulina platensis in D2O enriched water for the
have much signi?cance. Such investigations in animal
production of 2H labelled ?-carotene.
models used radiolabelled vitamin A or precursors of
vitamin A in high dose (Bausch & Rietz 1977, Olson
et al. 1979). These methods are however unacceptable
Materials and methods
in a study involving human subjects. An alternative ap-
proach would be the use of vitamin A rich precursors
Culture of Spirulina
labelled with stable isotope such as 2H or 13C (Wil-
son et al. 1997). The use of such labelled derivatives
Spirulina platensis was grown in Zarouk’s medium
of vitamin A or ?-carotene and subsequent LC-MS
(Becker 1994) using initially H2O and subsequently
analysis can lead to the measurement of the speci?c
D2O enriched water (D2O content 99.4% v/v) pur-
activity of vitamin A in the plasma which could be use-
chased from BARC, Mumbai, India. Cultures were
maintained in conical ?asks at 25 to 30 ?C with light

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448
Fig. 1. Kinetics of growth of Spirulina. Suspension cultures of Spir-
ulina platensis enriched in D
Fig. 2. Biomass and ?-carotene content of Spirulina platensis sub-
2O were inoculated in D2O-enriched
medium ( ) and allowed to grow under controlled light and tem-
cultured in D2O enriched water. Cultures of Spirulina platensis
perature. Growth of cells was assessed every 3 days. Cells grown in
enriched in D2O were inoculated and cells were harvested after
normal water ( ) were used for comparison.
28 days and subcultured upto 6th passage. Biomass ( ) and total
carotene content ( ) of each passage were determined. Carotenoids
were isolated and total ?-carotene content was measured. The
phase of 8–10 h (2 klux) under continuous shaking
values given are the average of three experiments. Biomass and
?-carotene were less in the 6th passage compared to 1st and 5th
condition on an orbital shaker. For comparison pur-
(p < 0.05).
pose, cultures were simultaneously grown in H2O
medium. To study the growth of culture under con-
ditions which exclude free contact with atmospheric
Spirulina cultures adapted to growth in D2O enriched
moisture, a bioreactor was designed (1.5 l) with a pro-
water could be subcultured upto 6th cycle. However,
vision for moisture exclusion. The bioreactor was also
after the 5th cycle the biomass production tended to
intended to serve as a model for scale up. The progress
decrease (Figure 2). In order to make optimum use
of the culture growth was monitored spectrophotomet-
of the deuterated medium the growth pro?le of Spir-
rically at 550 nm.
ulina in the spent medium were studied. The amount
of biomass produced was unaffected until the 4th cycle
Extraction and analysis of carotenoids
of the spent medium indicating that the medium pre-
pared in the heavy water was nutritionally suf?cient to
The harvested biomass was lyophilised and extracted
maintain the growth.
repeatedly with acetone/petroleum ether (1:1 v/v). Ex-
Spirulina carotenoids were analysed by LC-MS
tracts were concentrated and dried over anhydrous
and the results are given in Table 1. The amount of
Na2SO4 under N2. The crude extract was reconsti-
carotenoids formed were almost steady upto 5th pas-
tuted in acetone/petroleum ether (1:99 v/v) and chro-
sage in D2O enriched water. The yield of ?-carotene
matographed on a column (5 × 0.5 cm) of neutral alu-
was in the range of 140–175 µg g?1 dry biomass. The
mina (Merk, India) using acetone/petroleum ether (1%
use of spent medium also did not adversely affect the
v/v) as eluent. The fraction containing carotenoids
formation of carotenoids (data not shown).
were evaporated under N2 currents and the residue was
analysed by MS (Waters LC-MS) for carotenoids and
Deuterium incorporation into ?-carotene
deuterium incorporation into ?-carotene.
Carotenoids isolated from Spirulina grown in D2O
enriched water was subjected to LC-MS analysis to
Results and discussion
determine the extent of deuterium incorporation into
?-carotene (Figure 3) MS data showed 60–70% re-
Growth of Spirulina in D
placement of hydrogen by D atoms as indicated by
2O
the molecular mass cluster at around m/z 554. LC-MS
Spirulina platensis grow in D2O enriched water (Fig-
analysis of carotenoids isolated from cells grown in
ure 1). As the growth reached a maximum by 28–
bioreactor under conditions where exchange with at-
30 days, the cells were harvested after 28 days and
mospheric moisture was completely prevented, all the

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449
Table 1. Relative amounts of different carotenoids in
Spirulina platensis grown in D2O enriched medium
in different cycles.
Total carotenoids
Cycles
(µg g?1 dry mass)
1
2
3
4
740
670
560
590
Individual carotenoids
(% total w/w)
Lutein
27
31
29
36
Zeaxanthin
10
8
11
9
Cryptoxanthin
2
1
2
1
?-cis-?-Carotene
1
1
1
1
?-Carotene
0.5
1
1
1
trans-?-Carotene
18
17
21
21
9-cis-?-Carotene
4
4
5
4
S. platensis grown in deuterium enriched medium in
different cycles for 28 days and the biomass analysed
for various carotenoids using liquid chromatography.
Analysis was done in triplicate and the mean values
were expressed as percentage of the total amount of
carotenoids.
Acknowledgement
The ?nancial support from the International Atomic
Energy Agency, Vienna, Austria for this work (Project
No. 8780/R1) is gratefully acknowledged.
Fig. 3. Mass spectrometric analysis of deuterated ?-carotene of
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