Fast analysis of sugars, fruit acids, and vitamin C in sea buckthorn (Hippophae rhamnoides L.) varieties

Article abstract of DOI:10.1021/jf053177r

A fast, one-step gas chromatographic method was developed to analyze trimethylsilyl (TMS) derivatives of sugars, fruit acids, and ascorbic acid in sea buckthorn (Hippohae rhamnoides L.) berries. The method was applied to berry press juice of sea buckthorn of different origins grown in Finland during the 2003 and 2004 seasons. The method gave reliable results for D-fructose, D-glucose, ethyl-D-glucose, and malic, quinic, and ascorbic acids, which are the major sugars and acids in sea buckthorn juice. For the first time in sea buckthorn and evidently in any berry, the presence of ethyl β-D-glucopyranoside is reported. The structure of ethyl glucose was verified by high-performance liquid chromatography (HPLC), gas chromatography (GC), MS, and NMR analyses of both the isolated and the synthesized compounds. In the GC method, vitamin C was analyzed as ascorbic acid only, and dehydroascorbic acid was thus not taken into account.

Full text of DOI:10.1021/jf053177r

2508 J. Agric. Food Chem. 2006, 54, 2508−2513
Fast Analysis of Sugars, Fruit Acids, and Vitamin C in Sea
Buckthorn (Hippophae rhamnoides L.) Varieties
1
KATJA M. TIITINEN,*,† BAORU YANG,† GUDMUNDUR G. HARALDSSON,‡
†,§
SIGRIDUR JONSDOTTIR,‡ AND HEIKKI P. KALLIO
Department of Biochemistry and Food Chemistry and Functional Foods Forum, University of Turku,
FI-20014 Turku, Finland, and Science Institute, University of Iceland, Dunhaga 3,
107 Reykjavik, Iceland
A fast, one-step gas chromatographic method was developed to analyze trimethylsilyl (TMS)
derivatives of sugars, fruit acids, and ascorbic acid in sea buckthorn (Hippohae¨ rhamnoides L.) berries.
The method was applied to berry press juice of sea buckthorn of different origins grown in Finland
during the 2003 and 2004 seasons. The method gave reliable results for D-fructose, D-glucose, ethyl-
D-glucose, and malic, quinic, and ascorbic acids, which are the major sugars and acids in sea buckthorn
juice. For the first time in sea buckthorn and evidently in any berry, the presence of ethyl
â-D-glucopyranoside is reported. The structure of ethyl glucose was verified by high-performance
liquid chromatography (HPLC), gas chromatography (GC), MS, and NMR analyses of both the isolated
and the synthesized compounds. In the GC method, vitamin C was analyzed as ascorbic acid only,
and dehydroascorbic acid was thus not taken into account.
KEYWORDS: Sea buckthorn (Hippophae1 rhamnoides L.); fruit acids; gas chromatography; sugars; ethyl
â-D-glucopyranoside; vitamin C
INTRODUCTION
However, we did not find any examples of applications in which
sugars, fruits acids, and vitamin C would have been analyzed
in a single chromatographic run. We have previously reported
gas chromatographic (GC) analysis of isolated fractions of
sugars and acids and HPLC analysis of vitamin C in the sea
buckthorn fruit (1, 7, 14). The sample preparation for both of
these methods is time- and resource-consuming and requires a
large size of sample.
As the concentration of ascorbic acid (AA) in sea buckthorn
is high, in some samples even close to that of sugars or acids,
it is possible to use one and the same dilution to analyze all of
the compounds in a single procedure. Our goal was to develop
and test a reliable, fast, and easy-to-use GC method in which
sugars, fruit acids, and AA can be simultaneously analyzed as
trimethylsilyl (TMS) derivatives. The method was applied in
the analysis of sea buckthorn berries of different origins grown
in Finland. Also, a sugar compound previously unknown was
identified.
Sea buckthorn (Hippophae¨ rhamnoides L.) is a berry of high
nutritive value with growing commercial importance in western
countries. It has been used since ancient times in traditional
Chinese medicine and nutrition to reduce the risk of numerous
diseases and maintain good health. Especially, the high contents
of vitamin C, carotenoids, tocopherols, sterols, and flavonoids
have increased the interest of western consumers in the fruit
(1-5). Sugars and fruit acids display an important contribution
to the sensory properties of the berry. The ratio of sugars and
organic acids in sea buckthorn is close to 1:1, which is
exceptionally low as compared to other edible fruits and berries
and which gives the products their characteristic sourness. On
the other hand, the vitamin C concentration is in some cases
only 1/10 less than that of sugars or acids in the berry. Fructose
and glucose contribute typically over 90% of sugars and malic
and quinic acids over 98% of all acids in sea buckthorn (6-8).
In addition, the presence of an unknown sugar compound has
been reported (7).
MATERIALS AND METHODS
Among the different methods used for the analysis of sugars
and organic acids in fruits and berries, mostly high-performance
liquid chromatography (HPLC) is applied (9-11). HPLC is also
commonly used for the estimation of vitamin C (11-13).
Samples. A professional sea buckthorn farmer in Riihima¨ki, southern
Finland, cultivated sea buckthorn (Hippophae¨ rhamnoides L.) of cvs.
Prevoshodnaya (PRE), Oranzhevaya (ORA), Chuiskaya (CHU), and
Raisa (RAI) in 2003. The varieties Avgustinka (AVG), Botanicheskaya
(BOT), and Trofimovskaya (TRO) were grown in 2003 and 2004, and
the varieties Prozcharachnaya (PRO), Pertsik (PER), and RAI were
grown in 2004 by an association on sea buckthorn in Turku, southwest
Finland. All of the varieties were of the Russian origin, i.e., subspecies
(ssp.) mongolica, except RAI, which is a Finnish variety of the ssp.
* To whom correspondence should be addressed. Tel: +358 2 3336876.
Fax: +358 2 3336860. E-mail: katja.tiitinen@utu.fi.
† Department of Biochemistry and Food Chemistry, University of Turku.
‡ University of Iceland.
§ Functional Foods Forum, University of Turku.
10.1021/jf053177r CCC: $33.50
© 2006 American Chemical Society
Published on Web 03/09/2006

Fast Analysis in Sea Buckthorn Varieties
J. Agric. Food Chem., Vol. 54, No. 7, 2006 2509
rhamnoides. Berries were hand-picked when optimally ripe, frozen
immediately at -20 °C, pooled after freezing, and stored for the
analyses.
Reference Compounds. D-Glucose and L-malic acid were purchased
from Fluka (Buchs, Switzerland), D-fructose and AA were purchased
from Sigma, quinic acid was purchased from Chem Service (West
Chester, PA), and citric acid was purchased from J. T.Baker (Devanter,
Holland). Internal standards L-tartaric acid and D-sorbitol were obtained
from Merck (Darmstadt, Germany). Ethyl â-D-glucopyranoside was
synthesized in this project.
Sample Preparation. Fifty grams of sea buckthorn berries was
thawed in the microwave oven with 225 W twice over 15 s and shaken
out between each thaw and homogenized over 30 s by a Bamix mixer
(Bamix, Metlen, Switzerland). The homogenate was filtered through a
cheesecloth under vacuum conditions. The one and same juice
subsample was used for the fractionation of sugars, acids, and vitamin
C analysis by HPLC and for simultaneous GC analysis of sugars, acids,
and vitamin C. All of the analyses were carried out in triplicate, except
for the variety AVG in 2004, which was analyzed in six replicates.
Chemical Analysis. Vitamin C by HPLC. Vitamin C was analyzed
as AA by HPLC after converting dehydroascorbic acid (DHAA) to
AA with dithiothreitol (DTT, Promega Co., Madison, WI) (1, 12, 14).
The berry juice was diluted 1:40 with DTT in water solution (final
concentration, 80 mg/1 mL juice), and each juice sample was analyzed
in duplicate. The dilution was kept for 2 h in the dark at room
temperature and filtered (0,45 µm) for HPLC analysis. A sample of 20
µL was injected into a Shimadzu SLC-10A system (Shimadzu, Japan)
with a UV detector. The column used was LiChrocharts 250-4
LiChrosphar RP-18, 5 µm (Merck). A buffer of 0.5% KH2PO4,
containing 0.1% DTT with flow rate of 0.4 mL/min, was used as the
mobile phase. Quantification was carried out with an external standard
method.
the unknown compound was isolated with HPLC by Luna NH2 phase
column (Phenomenex, Torrance, CA) over acetonitrile:water (80:20)
with a flow rate of 1.4 mL/min.
NMR Analysis of the Sugar DeriVatiVe. 1H and 13C NMR spectra of
the isolated and synthesized sugar derivative were recorded on a Bruker
Avance 400 spectrometer in deuterated water as a solvent at 400.12
and 100.61 MHz, respectively. Chemical shifts (δ) are quoted in parts
per million (ppm), and the coupling constants (J) are in Hertz (Hz).
The following abbreviations are used to describe the multiplicity: d,
doublet; t, triplet; q, quartet; dd, doublet of doublets; ddd, doublet of
doublets of doublets; dt, doublet of triplets; and dq, doublet of quartets.
Two-dimensional (2D) spectra were run as a HHCOSY90 standard 2D
experiment for HH homonuclear correlation and a HCCOW standard
1
2D experiment for CH correlation. H NMR (D2O): δ 4.49 (d, J1,2
)
2
3
8.0 Hz, 1H, H1), 3.99 (dq, J ) 10.0 Hz, J ) 7.1 Hz, 1H, -CH2-
2
3
2
CH3), 3.93 (dd, J6′6 ) 12.3 Hz, J6′5 ) 2.2 Hz, 1H, H6′), 3.75 (dq, J
) 10.0 Hz, 3J ) 7.1 Hz, 1H, -CH2-CH3), 3.73 (dd, 2J66′ ) 12.3 Hz,
3J65 ) 6.0 Hz, 1H, H6), 3.50 (t, J32 ) J34 ) 9.1 Hz, 1H, H3), 3.47 (ddd,
J54 ) 9.6 Hz, J56 ) 6.0 Hz, J56′ ) 2.2 Hz, 1H, H5), 3.39 (dd, J43 ) 9.1
Hz, J45 ) 9.6 Hz, 1H, H4), 3.27 (dd, J23 ) 9.1 Hz, J21 ) 8.0 Hz, 1H,
H2), and 1.25 ppm (t, J ) 7.1 Hz, 3H, -CH3). 13C NMR (D2O): δ
101.8 (C1), 75.9 (C3), 75.8 (C5), 73.1 (C2), 69.6 (C4), 66.2 (-CH2-
CH3), 60.8 (C6), and 14.2 ppm (-CH3).
Synthesis of the Ethyl â-D-Glucopyranoside. After NMR analysis
of the unknown sugar, ethyl â-D-glucopyranoside was synthesized by
dissolving glucose to ethanol:water (9:1) at 50 °C over 10 min with
continuous stirring. The â-glycosidase from almonds was added to the
mixture, and the reaction was performed over 72 h. The mixture was
chromatographed over Celite and washed with ethanol:water (9:1). The
filtrate was evaporated to dryness yielding a yellow crude suspension
(15). Ethyl â-D-glucopyranoside was isolated and purified by HPLC
1
as described above and identified by H and 13C NMR analysis and
Sugar and Acid Fractionations. Sugars and acids were analyzed
according to the method applied earlier in our laboratory (7, 14). The
juice was diluted 1:20 in water, and the internal standards sorbitol and
tartaric acid (Merck) and 2 mL of 0.1 N NaOH were added in the total
volume of 20 mL. One milliliter of the dilution was fractionated by
dual solid-phase extraction, where the lipophilic colors were adsorbed
in the upper nonpolar cyclohexyl Isolute CH (EC) column (100 mg/
mL) (International Sorbent Technology, Hengoed, United Kingdom)
and the acids were trapped in the second, lower anion exchanger Isolute
SAX column (International Sorbent Technology). From the SAX
column, sugars were washed by water and the organic acids were eluted
by 15 N formic acid. Both fractions were diluted, and the sample was
evaporated to dryness and dried in a desiccator overnight. TMS
derivatives of sugars and acids were prepared by adding Tri-Sil (Pierce,
Rockford, IL) reagent for each fraction. The vials were closed with
butyl Teflon septa, shaken vigorously by a Vortex (Vortex-Genie,
Springfield, MA) for 3 min, and incubated at 60 °C for 30 min and at
room temperature overnight.
Juice Preparation for Simultaneous Analysis of Sugars, Acids, and
AA. The juice was diluted 1:20 in water and the internal standards
sorbitol (0.1 g/100 mL in dilution) for sugars and vitamin C and tartaric
acid (Merck) (0.05 g/100 mL) for acids were added in the dilution in
the total volume of 20 mL and filtered (0.45 µm). A 100 µL amount
of filtrate was evaporated to dryness, dried in a desiccator over P2O5
overnight, and silylated as described for fractionated samples.
GC. The TMS derivatives of sugar and acid fractions as well as of
the nonfractionated juice samples were analyzed with Varian 3300 GC
equipped with a flame ionization detector (FID) (Varian, Limerick,
Ireland). The analyses were carried out with methyl silicone Supelco
Simplicity-1 fused silica column (30 m, i.d. 0.25 mm; film thickness,
0.25 µm) (Bellefonte, PA). One microliter of sample was injected
manually into the split injector (1:20). The temperature of the injector
was 210 °C, and the detector temperature was 290 °C. The column
temperature was programmed as 2 min at 90 °C, raised to 180 °C at
rate of 8 °C/min, to 215 °C at rate of 4 °C/min and to final temperature
275 °C at rate of 12 °C/min, and held at 275 °C for 6 min.
also as a TMS derivative with GC-MS (electron impact, EI) and with
GC-FID coinjection with sea buckthorn sample.
Statistical Analyses. The statistical analyses were performed using
SPSS (SPSS 12.0.1, SPSS Inc., Chicago, IL) and Unscrambler 9.2
(Camo Process AS, Oslo, Norway). To compare different methods,
principal component analysis (PCA) was used to profile samples based
on the chemical parameters studied.
RESULTS AND DISCUSSION
GC Analysis. A gas chromatogram of TMS derivatives of
diluted sea buckthorn juice compounds is shown in Figure 1.
Malic and quinic acids contributed over 98% of the acids in
the juice. Trace amounts of citric acid were also found. Fructose
and glucose are known to be the main sugars in the berry (6-
8). The three isomeric forms of fructose (R- and â-D-furanose
and â-D-pyranose) and two of glucose (R- and â-D-pyranose)
were the dominating sugar peaks in the chromatogram. Previous
reports indicated the presence of an unknown sugarlike com-
pound (7, 10, 14), which was identified to be ethyl â-D-
glucopyranoside (Figures 1-3). The retention time of ethyl
glucose in GC analysis, 19.2 min, was somewhat longer than
that of AA (Figure 1). In HPLC analysis, the retention time of
4.3 min was noticeably shorter than that of other sugars (Figure
2). The EI-MS spectrum of the TMS derivative of ethyl glucose
is typical for the TMS derivative of a sugar (Figure 3).
Interpretation of the structure was not possible based on the
mass spectrum only. After identification of the compound by
NMR, the mass spectrum is, however, specific enough for
fingerprint identification, when used together with retention time
behavior. The relative amount of ethyl glucose among sugars
varied greatly from 2 to 5% in most varieties to 45% in RAI.
NMR Analyses. The structure of the unknown sugar deriva-
tive was unambiguously determined as ethyl â-D-glucopyrano-
Identification of the Unknown Sugar. Isolation and Purification
of the Compound. The sugar fraction of sea buckthorn was isolated as
described for the analysis of sugars. The sugars were separated, and
1
side by high resolution H and 13C NMR spectroscopy and
confirmed by mass spectrometry. All peaks of the NMR spectra

2510 J. Agric. Food Chem., Vol. 54, No. 7, 2006
Tiitinen et al.
Figure 1. GC-FID chromatogram of sugars, organic acids, and AA as their TMS derivatives from sea buckthorn juice of variety TRO.
CH correlations, respectively. The spectra were identical to those
obtained for purified ethyl glucose synthesized by the method
of Goncalves and co-workers (15).
Reliability of the GC Analyses. The quantitative GC
analyses of sugars and acids, including AA, were calculated
using correction factors based on internal standards and the
analysis of AA by HPLC using an external standard curve (14,
16). The detector response of ethyl glucose was not defined,
and it was estimated to have the same response with glucose.
Reliability of the methods was tested by coefficients of variation
with six subsamples of the variety AVG (Table 1). Both
methods applied showed good repeatability for the acids and
sugars in the berry. The variation in vitamin C content measured
with GC method was slightly high, but it was still in the limits
for reliable results.
Contents of Fruit Acids, Sugars, and Vitamin C. The
contents of fruit acids, sugars, and vitamin C are presented in
Figures 4-6, respectively. Data of acids (Figure 4) and sugars
(Figure 5) are both from the isolated respective fractions and
from the dried, nonfractionated juice. HPLC analysis of vitamin
C covered both AA and DHAA, whereas the GC analysis as
TMS derivatives gave the content of AA only (Figure 6).
The content of malic acid varied from 1.9 to 3.6 g/100 mL,
and that of quinic acid varied from 0.9 to 2.5 g/100 mL juice.
The citric acid content was less than 0.05 g/100 mL in all of
the samples. The fructose content ranged from 0.2 to 3.5 g/100
mL, and that of glucose ranged from 1.5 to 4.2 g/100 mL.
Typically, only glucose and fructose are indicated to be the
sugars of sea buckthorn; the compound often reported as
unknown was identified as ethyl â-D-glucopyranoside. The
content of this sugar varied from 0.3 to 1.0 g/100 mL. Even
Figure 2. HPLC-ELSD chromatogram of sugar fraction in sea buckthorn
variety RAI.
were explicitly assigned by the aid of 2D HHCOSY90 and
HCCOW standard 2D experiments for the HH homonuclear and

Fast Analysis in Sea Buckthorn Varieties
J. Agric. Food Chem., Vol. 54, No. 7, 2006 2511
Figure 3. EI-MS spectrum of the TMS derivative of ethyl
â-D-glucopyranoside.
Table 1. Coefficients of Variation (CV%) for the Methods Used in the Analysis of Sea Buckthorn Juice Variety AVG
malic
acid
quinic
acid
method used
fructose
10.8
glucose
8.0
ethyl
â
-D
-glucopyranoside
11.7
vitamin C
10.4
our previously used
7.2
10.4
methods (n
fast method (n
)
6) (1, 14)
6)
)
5.7
7.4
6.9
3.6
7.8
16.3
Figure 4. Fruit acids in sea buckthorn samples analyzed as TMS derivatives of fractionated juice samples (frac) and as straight juice sample (juice).
Abbreviations: mal, malic acid; quin, quinic acid; and citr, citric acid. Sea buckthorn varieties are abbreviated as indicated in the Materials and Methods.
though in most samples it existed in trace amounts only, it is
worth noticing its abundance in the variety RAI, even up to
45% of the total sugars. Both sugars and acids showed good
repeatability within and between the two methods (Table 1 and
Figures 4 and 5). No statistical differences were seen between
the two methods applied. The high malic acid content together
with low sugars is commonly responsible for the known strong
acidity of sea buckthorn berries (14, 17).
recognized were in some unknown ssp. mongolica clones, 10
mg/100 mL. (1)
The principles of the two methods of vitamin C analysis
applied differ significantly from each other. In the HPLC
method, both AA and DHAA were quantified based on DTT
treatment, whereas in the GC method the conversion was not
done. In all of the samples from 2004, both procedures gave
identical results concerning vitamin C, indicating the common
absence of DHAA at harvesting time by this year. In samples
from 2003, the GC method displayed vitamin C contents
typically 10-20% less than those obtained from HPLC analysis.
The two growing seasons were different. The temperature sum,
i.e., the content of total sunny hours, was 1298 h during the
The content of vitamin C in the juice samples studied was
only moderate, ranging from as low as 29 to 128 mg/100 mL.
In our earlier reports on wild berries of ssp. sinensis from high
altitudes in China, concentrations even as high as 1300 mg/
100 mL have been found. The lowest ones that we have

2512 J. Agric. Food Chem., Vol. 54, No. 7, 2006
Tiitinen et al.
Figure 5. Sugars in sea buckthorn samples analyzed as TMS derivatives of fractionated juice samples (frac) and as straight juice sample (juice).
Abbreviations: fru, fructose; glc, glucose; and et-glc, ethylglucose. Sea buckthorn varieties are abbreviated as indicated in the Materials and Methods.
to AA with DTT and the sample preparation was performed as
previously described, only 60% of DHAA was recognized
according to the GC results. Thus, analysis of DHAA by
converting it to AA was questionable for accurate quantitative
results and the method needs more thorough optimization.
Comparison of the Different Methods. PCA is applicable
when all of the variables collectively characterize each, and all,
of the samples. In this study, PCA is used to see if the samples
are characterized differently due to the method of analysis
(Figure 7) and in order to get an overview of the capability of
different methods to detect the differences between the samples.
For example, the closer the corresponding samples between the
two methods lay on the plot, the more similarly the methods
describe the samples. Respectively, the farther the samples lay
from each other, the more different they are with respect to the
measured parameters. The first two principal components (PCs)
explained 74% of the variance of the data. The first PC explains
the differences in sugar content. There is a strong positive
correlation between fructose and glucose contents (Pearson’s
correlation ) 0.650, p < 0.01), as well as between glucose and
AA (Pearson’s correlation ) 0.710, p < 0.01). On the other
hand, the content of ethyl glucose has a strong negative
Figure 6. Vitamin C in sea buckthorn juice samples analyzed with HPLC
(AA and DHAA) and as TMS derivatives (AA). Sea buckthorn varieties
are abbreviated as shown in the Materials and Methods.
season 2003, whereas in 2004 it was 1065 h only, equaling on
average two sunny hours more per day in 2003. This difference
could increase the proportion of DHAA in 2003, because of
oxidative stress induced by the abundant sunshine.
Tri-Sil reagent used in the GC method does not silylate the
oxo groups of DHAA, and the derivative will not be volatile
enough for GC analysis. When DHAA in juice was converted
Figure 7. Differences in PCA between sea buckthorn varieties and methods used. MAL, malic acid; QUIN, quinic acid; FRU, fructose; GLC, glucose;
f, fractionation or HPLC in vitamin C; and j, juice.

Fast Analysis in Sea Buckthorn Varieties
J. Agric. Food Chem., Vol. 54, No. 7, 2006 2513
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correlation with glucose (Pearson’s correlation ) -0.510, p <
0.01) and AA (Pearson’s correlation ) -0.647, p < 0.01)
contents. The second PC explains the differences in acid content
in the samples, the samples with high malic acid content laying
in the upper part of the PCA biplot. The two methods studied
profiled the samples identically in 2004, which are seen as
fractionated (f) and direct GC measured (j) samples next to each
other in the plot. On the other hand, different samples are
grouped around the plot showing the overall difference between
the samples. In 2003, the difference in vitamin C content moved
the samples analyzed by HPLC (f) toward AA attributes as the
contents measured by HPLC were higher.
On the basis of the results of this study, the direct GC analysis
of sugars and acids of sea buckthorn juice as TMS esters and
ethers is a reliable and repeatable method. It is easy to apply,
and it is time-saving as there is no need for complex sample
preparation steps. As the variation in sea buckthorn berries in
a bush is limited, large sample amounts are not usually required
to perform representative analysis. The direct GC method is
suitable also in breeding operations, where often a large number
of samples need to be screened and typically only small amounts
of sample are available.
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composition of sea buckthorn juice from Hippophae¨ rhamnoides
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ABBREVIATIONS USED
AA, ascorbic acid; DTT, dithiothreitol; DHAA, dehydroascor-
bic acid; ssp., subspecies; PC, principal component; PCA,
principal component analysis; TMS, trimethylsilyl.
(12) Go¨kmen, V.; Acar, J. A simple HPLC method for the determi-
nation of total vitamin C in fruit juices and drinks. Fruit Process.
1996, 5, 198-201.
ACKNOWLEDGMENT
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foodstuffs and beverages by high-performance liquid chroma-
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of sea buckthorn (Hippophae¨ rhamnoides) varieties. J. Agric.
Food Chem. 2005, 53, 1692-1699.
(15) Goncalves, P. M. L.; Roberts, S. M.; Wan, P. W. H. Regiose-
lective acylation of carbohydrate derivatives using lipases leading
to a facile two-step procedure for the separation of some alpha-
and beta-glucopyranosides and galactopyranosides. Tetrahedron
2004, 60, 927-932.
(16) Kallio, H.; Hakala, M.; Pelkkikangas, A.-M.; Lapvetela¨inen, A.
Sugars and acids of strawberry varieties. Eur. Food Res. Technol.
2000, 212, 81-85.
We are grateful to Hannu Lappalainen from Sammalma¨en
tyrniseura and Seppo and Anja Huuhtanen for providing the
berries of sea buckthorn for the study.
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Daya Publishing House: New Delhi, India, 2005; Vol. 2., pp
70-97.
Received for review December 20, 2005. Revised manuscript received
February 9, 2006. Accepted February 9, 2006. ABS graduate school,
Employment and Economic Development Centre in Lapland, and
Huovin Sora are acknowledged for the financial support.
JF053177R