Determination of Amino Acids in Ice Samples

Bulletin of Glaciological Research ,, (,**/) 03ῌ1. 69 ῍Japanese Society of Snow and Ice

Determination of amino acids in ice samples

Fumio NAKAZAWA+, Keiichi OHTA,, Naomi HARADA-, Koji FUJITA. and Masayoshi NAKAWO/ + Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya .0.ῌ20*+, Japan , Department of Ecosystem Studies, School of Environmental Science, University of Shiga Prefecture, ,/** Hassaka-cho, Hikone, Shiga Pref. /,,ῌ2/--, Japan - Japan Agency for Marine-Earth Science and Technology, ,ῌ+/ Natsushima-Cho, Yokosuka-City, Kanagawa ,-0ῌ**0+, Japan . Graduate School of Environmental Studies, Nagoya University, Furo-cho, Chikusa-ku, Nagoya .0.ῌ20*+, Japan / Research Institute for Humanity and Nature, --/ Takashima-cho, Marutamachi-dori Kawaramachi nishi-iru, Kamigyo-ku, Kyoto 0*,ῌ*212, Japan

(Received October ., ,**.; Revised manuscript received November -*, ,**.)

Abstract

This study investigates amino acid concentrations and compositions in ice as a preliminary approach for applying amino acid analysis to ice core study. For the amino acid analysis, *..,/ to *.1/* kg ice samples are used. The ices from Antarctica have +0.2 and +2.- nmol kgῌ+ of amino acids and these concentrations are close to laboratory blank levels, while the ice from the Chongce Ice Cap in China has +0* nmol kgῌ+ of amino acids. Therefore, the analysis for Antarctic ice is estimated to need more than several kg of ice. In contrast, the analysis from the Chongce Ice Cap should be reducible to only several hundred grams of ice because the concentrations in this cap are higher than those in Antarctica. Further, amino acid sources are present in glacier surroundings of temperate regions, whereas polar regions are far from sources. Serine, aspartic acid and alanine are the major components in the samples. However, the amino acid compositions show variation with each sample.

seem to originate mainly from microorganisms, such +.Introduction as snow algae and bacteria, that breed on glacier surfaces in summer season (Kohshima, +321, +323; Ionic species, such as inorganic ions and light Ling and Seppelt, +33*, +33-; Yoshimura et al., +331; carboxylic acids, in ice cores have provided important Takeuchi et al., ,**+), plant products, such as pollen, in information on past climatic and environmental con- glacier surroundings (Koerner et al., +322; Bourgeois, ditions. The chemical species yield data on historical +33*, ,***;Liuet al., +332; Reese et al., ,**-; Nakazawa events including biogenic emission, biomass burning, et al., ,**.) and the bacteria in precipitation (Herlihy et anthropogenic emission and volcanic eruption (e.g., al., +321; Sattler et al., ,**+; Mace et al., ,**-). Thus, the Hammer, +311; Hammer et al., +32*; Legrand and amino acids may have good environmental informa- Angelis, +330; Olivier et al., ,**-). However, there are tion in ice core study. Since these biological activities some other candidates for the source tracers in cases increase in summer season and decrease in winter, when interpretation is di$cult. For example, natural amino acid-rich layers in an ice core may indicate events, such as volcanic eruptions and large forest summer layers. In addition, amino acid concentrations ﬁres, increase concentrations of chloride, nitrate and in each summer layer may be useful as a good marker sulfate ions in the atmosphere. In addition, anthropo- of meteorological conditions such as temperature and genic e#ects such as biomass burning and fossil fuel solar radiation that a#ect these activities. However, combustion can also increase the atmospheric concen- no previous study has reported the amino acids in trations of these constituents. glacier ice. Therefore, as a preliminary approach to ice In contrast, amino acids that build proteins appar- core research, this study aims to survey the level of ently originate from and are abundant in living organ- amino acid concentrations in ice from the Antarctic isms, for example, approximately /*ῌ of the human ice sheet and Chongce Ice Cap in central Asia, and to body’s dry weight is protein (McMurry and Castellion, compare their compositions. +333). Moreover, amino acids contained in glacier ices 70 Bulletin of Glaciological Research

Table +. Detection system for high-performance liquid chromatography (HPLC).

HPLC Shimadzu LC-+*AD Detector Fluorescence detector RF-+*AXL (-/*ῌ.0* nm) Integrator Shimadzu chromatopac GR1Ae plus Column GL Science Intersil ODS-- (+/* mm L, ..0 mm ID, / mm particle size) Guard column GL Science Intersil ODS-- (/* mm L, ..0 mm ID, / mm particle size) Column temperature .*῎

Eluent E+: / mM Na, HPO.ῌ+,H, O// mM NaH, PO.ῌ,H, O(pH1.*/)

E,:E+/CH- CN( ,: +, v/v)

E-:CH- CN/Distilled water (HPLC grade) (.: +, v/v) Flow-rate +.* mL minῌ+

Table ,. Working gradient conditions used for amino ,. Sampling sites and sample preparation acid determination. Time (mim) E+ E, E- Ice core sampling was conducted at two distinct Program + Program , (῍) sites; Asuka camp in East Antarctica (1+῍-+῎S, ,.῍2῎E; *4*+ *4*+ 3-* m a.s.l.) and Chongce Ice Cap in the West Kunlun 3/ / * .4* .4* 2/ +/ * Mountains, China (-/῍+.῎N, 2+῍*1῎E, 0-,1 m a.s.l.). Ice 24* 24* 2* ,* * blocks were taken from the surface of an iceberg o# +04* +04* 1- ,1 * Syowa Station (03῍**῎S, -3῍-/῎E) in the Antarctic +24* +24* 1* -* * Ocean. One section was collected from each ice core; ,/4* ,14* // ./ * +** 2 from the Asuka core, the increment between . and -*4* -,4* /* /* * +*+ - . m depth, while from the Chongce core, the incre- .,4* ..4* -/ 0/ * , 2 - * ment between . and . m depth were used. For the .,4*+ ..4*+ -* 1* * amino acid analysis, *.1/*, *./** and *..,/ kg ice blocks ./4* .14* ,/ 1/ * were used for the Asuka ice, the iceberg ice and the /+4* /-4* ,* 2* * Chongce ice, respectively. /+4*+ /-4*+ * +** * To prevent contamination by laboratory controls, /,4* /.4* * * +** precleaned glasswares, which include filters, were 2.4* 3*4* * +** * used in all laboratory work. The glasswares were 2.4*+ 3*4*+ +** * * prepared by washing in alkaline detergent, placing in 2.4*, (stop) 3*4*, (stop) +** * * +NHCl overnight followed by rinsing with organic- The program + was used for the particulate and free water obtained with a Milli-Q system and then dissolved fractions of the blank sample, the particulate heating in a furnace at ./*῎ for - to . h. Additionally, fraction of the Asuka sample, and the particulate , to eliminate contamination of each sample, about + cm fraction of the Chongce sample. The program was used for the dissolved fraction of the Asuka sample, of the surface was scraped o# with a clean knife in a the particulate and dissolved fractions of the iceberg cold room, and then the sample was melted in a bea- sample, and the dissolved fraction of the Chongce ker. The first melted ice used to wash the surface of sample. the ice samples was discarded. This procedure was repeated - to / times, depending on the sample. The melted ice discarded in this way came to *.+./ to *.++* acid content of the ices. kg. After the ice was completely melted, the melt A laboratory procedural blank were also ana- water was immediately filtrated through .1 mm GF/F lyzed. For particulate amino acids, a new GF/F glass glass fiber filters with a pore size of *.1 mm. fiber filter (diameter: .1 mm; pore size: *.1 mm) was hy- Subsequently, the samples were separated into drolyzed and the following procedure was the same as two portions, a filter sample and filtrate sample. These that for samples. In contrast, for dissolved amino ac- 0 sub-samples (for amino acid analysis) were hydro- ids, .-ml of the organic-free water was concentrated in lyzed with 0NHCl at +*/ ῎ for ,, h. The HCl super- a flask under vacuum, and passed through the cation natant was then transferred to a flask and dried under exchange resin. The following procedure was the vacuum. After drying, the dried residue was desalted same as mentioned above. using cation exchange resin (Dowex /*W-X2), derivat- A detection system for HPLC and working gradi- ized with o-phthalaldehyde and then analyzed by gra- ent conditions used for determination of amino acid dient reversed-phase high-performance liquid chro- are summarized in Table + and , respectively. Twelve matography (HPLC) with UV fluorescence detection amino acids were measured in this study (Table -). in order to determine the total hydrolyzable amino The yield for the purification of amino acids was 3/ to Nakazawa et al. 71

Table -. Concentration of amino acids in the ice (nmol kgῌ+). Abbreviation: Glu (glutamic acid), Ala (alanine), Val (valine), Gly (glycine), Ile (isoleucine), Ser (serine), Thr (threonine), Arg (arginine), Asp (aspartic acid), Met (methionine), Phe (phenylalanine), Tyr (tyrosine).

Sample Type sample Glu Ala Val Gly Ile Ser Thr Arg Asp Met Phe Tyr Total Blank Particulate metter +.0 +.- +.+ *.0 -.3 ,.+ +./ *./ +./ +.. n.i. *.3 +0 Dissolved matter *.. *./ n.i. *., n.i. *.. ,.* n.i. +.. *.- n.i. n.i. /., Asuka Particulate metter +.0 +.0 +.2 *.+ n.i. -.* +., +.+ *.3 *./ n.i. n.i. +,.* Dissolved matter *.+ *.0 *.- *., *.0 +.* *.2 *., *.3 n.i. n.i. *.+ ..2 Iceberg Particulate metter *.. +., *.- *.. +., *.2 +.* n.i. *.3 n.i. n.i. *.+ 0.. Dissolved matter +.2 +.+ *.1 *./ +.. +.3 +./ *., +.. +., n.i. *., ++.3 Chongce Particulate metter -* ,. +/ ,.1 1.3 ,* +1 /./ ,* -.- ,.3 -.* +/, Dissolved matter *./ *.3 +.1 *.. n.i. *.3 *.3 *.0 *.3 *./ n.i. *., 1./ The n.i. is “not identiﬁed”, meaning that it was impossible to identify the peak of amino acid because amounts were below the detection limit or inseparable from adjacent peaks.

+**ῌ (Harada, +33+). The precisions (RSD, n῍,) were acid concentrations in Antarctic ice should be gener- +- to +ῌ except for valine (Val), isoleucine (Ile), me- ally low. thionine (Met), phenylalanine (Phe) and tyrosine (Tyr). The Chongce sample shows that Glu, Ala, Ser and The precisions of the ﬁve amino acids were not deter- Asp are the major components of the particulate frac- mined. tion and comprise 0,ῌ of the total. In contrast, Val, Ala, Ser, Thr and Asp are the major components of the -. Results and discussion dissolved fraction and are 1+ῌ of the total. The total concentration in the particulate in the Chongce ice is Total concentrations of particulate and dissolved much higher than those of the Asuka and the iceberg amino acids in the ice samples from the three sites samples, speciﬁcally, +- times that of the Asuka and ,. range between +0.2 to +0* nmol kgῌ+. Table - lists the times that of the iceberg, even though the dissolved concentrations of twelve amino acids in the blank and matter in the ices from the three sites have similar and - ice samples. The blank values are not subtracted blank levels. from the sample values because we did not measure The di#erence in particulate amino acid concen- amino acid concentrations in the organic-free water trations between the Antarctica and Chongce ices used for the blank sample and the blank values were seems to be attributable to the di#erence in the source close to the detection limits of , to - pmol. Therefore, strength in two regions. The ice samples in lower it would be safe here to use the blank values as latitudinal ice caps and glaciers (between about 0*῎N references. and 0*῎S) typically contain higher concentrations of The total concentrations in the Asuka core and nutrients, such as water-soluble chemical species, iceberg samples are at similar levels. Moreover, the than that from the polar ice sheets (e.g., Wagenbach, concentration of each amino acid is at the blank level +323; Clausen and Langway, +323; Lyons et al., +33+). in both the particulate and dissolved fractions. In the The higher concentrations play the more important Asuka sample, serine (Ser), Val, glutamic acid (Glu) role in the breeding of snow algae. Moreover, on gla- and alanine (Ala) are the major components in the cier surfaces in lower latitudes, some surface melting particulate fraction, and constitute 01ῌ of the total occurs in the summer season, and snow algae grow in amino acids. Ser, aspartic acid (Asp), Tyr and Ala are the meltwater (Pollock, +31*; Yoshimura et al., ,***). the major components in the dissolved fraction, and However, the surface of polar ice sheets is virtually constitute 03ῌ of the total. In the iceberg sample, Ala, lifeless because of the lack of meltwater, even though Ile, threonine (Thr) and Asp are the major components viable microbial cells and spores are present (Catranis in the particulate fraction, and constitute 01ῌ of the and Starmar, +33+). Thus, the local growth of snow total. Ser, Glu, Thr, Ile and Asp are the major compo- algae may cause the higher concentration of particu- nents in the dissolved fraction, and are 01ῌ of the late amino acids in Chongce ice compared to that in total. The Asuka ice section is estimated to have been Antarctic ice. hundreds of years old based on an average accumula- In addition, the wind-blown-particulate amino ac- tion rate of *.+3 maῌ+ of ice between +322 and +33+ ids such as in pollen, may also contribute to the Cho- (Azuma et al., +33.). In addition, the ice from the ice- ngce Ice Cap. Ice cores from lower latitude ice caps berg was much older than the Asuka ice because it and glaciers that are typically within a few tens of had once been a part of the Antarctic ice sheet. Amino kilometers from vegetation sources contain more than acids seem to be well preserved in glacier ice because +*** grains Lῌ+ of pollen (Ambach et al., +300; Haeberli of its low temperature. Therefore the level of amino et al., +32-;Liuet al., +332, Reese et al., ,**-; Nakazawa 72 Bulletin of Glaciological Research

Fig. +. Amino acid composition in ice samples. (a) Particulate amino acids in the Antarctic Asuka core at a depth of +**.2 to +*+.- m; (b) Dissolved amino acids in the Antarctic Asuka core; (c) Particulate amino acids in the Antarctic iceberg; (d) Dissolved amino acids in the Antarctic iceberg; (e) Particulate amino acids in the Chinese Chongce ice at a depth of ,.2 to -.* m; (f) Dissolved amino acids in the Chinese Chongce ice. Abbreviation: Glu (glutamic acid), Ala (alanine), Val (valine), Gly (glycine), Ile (isoleucine), Ser (serine), Thr (threonine), Arg (arginine), Asp (aspartic acid), Met (methionine), Phe (phenylalanine), Tyr (tyrosine). et al., ,**.), whereas the pollen of polar ice sheets blank level. This fact seems to be due to few sources of mostly originates from sources hundreds to thou- amino acids in situ and in the surroundings. It is sands of kilometers away. Thus, the pollen concentra- therefore concluded that more than several kg of ice tion in ice is typically only +* to +** grains Lῌ+ (McAndrews, samples would be needed for analysis in order to be +32.; Short and Holdsworth, +32/; Koerner and well above the blank level. However, ice core samples Bourgeois, +322; Bourgeois, +33*, ,***). Judging from are typically limited, and it is therefore essential to the above, the ice from lower latitudinal ice caps and secure enough ice volume for ice core study in the glaciers can be expected to contain enough particu- polar ice sheets of such as Antarctica. late amino acids for analysis. Thus, the sample volume Ser, Asp and Ala are the major components in / may be reduciable to about several hundred grams out of the 0 sub-samples (Fig. +). However the amino considering the laboratory procedural blank if the ice acid compositions show variation with each sample. from lower latitude ice caps and glaciers has a concen- The three amino acids were also found to be major tration similar to that of the Chongce ice sample. compounds in soil organic matter, pollen, marine al- In contrast, the various ice from the polar ice gae and marine bacteria (e.g., Auclair and Jamieson, sheets may be di$cult to analyze using only several +3.2; Bieberdorf et al., +30+; Gupta and Reuszer, +301; hundred grams of ice samples. This study used the Simon and Azam, +323; Cowie and Hedges, +33,;Cao *.1/* and *./** kg ice samples for the analysis of Ant- and Xiang, +33/; Senwo and Tabatabai, +332), al- arctic ices, thereby showing that concentrations of though the amino acid composition di#ers by type. both particulate and dissolved amino acids are at the The exact cause of the di#erences in amino acid com- Nakazawa et al. 73 position in the ice is not yet known. In particular, the Azuma, N., Goto-Azuma, K. and Nakawo, M. (+33.): Photogra- +** amino acid composition in the particulate fraction of metric analysis of a m ice core from Asuka Camp, East Antarctica: Preliminary results. Bulletin of Glacier the Chongce sample may relate to some origin of Research, +,, 03ῌ1/. amino acids. Thus, further research is required to Bieberdorf, F. W., Gross, A. L. and Weichlein, R. (+30+): Free ascertain whether these di#erences are great enough amino acids content of pollen. Annals of Allergy, +3, 203 ῌ210 to have practical significance. . Bourgeois, J. C. (+33*): Seasonal and annual variation of pollen content in the snow of a Canadian high Arctic ice ..Conclusion cap. Boreas, +3, -+-ῌ-,,. Bourgeois, J. C. (,***): Seasonal and interannual pollen vari- ability in snow layers of arctic ice caps. Review of Pa- This study investigates the concentrations and laeobotany and Palynology, +*2, +1ῌ-0. compositions of amino acids in ice samples from three Cao, S. and Xiang, B. (+33/): A comparative study on protein sites. We conclude that a large ice sample (more than content and amino acid composition of eight species of several kg) would be needed for analysis of Antarctic Unicellular marine feed algae. Acta Oceanologica Sinica, +., +.+ῌ+.0. ice, such as the Asuka core and iceberg. Consequently, Catranis, C. and Starmer, W. T. (+33+): Microorganisms en- it is essential to secure enough sample volume for trapped in glacial ice. Antarctic Journal of the United continuous analysis in ice core studies. In contrast, we States, ,0, ,-.ῌ,-0. +323 conclude that the amount of several hundred grams of Clausen, H. B. and Langway, Jr., C. C. J. ( ): The ionic de- posits in polar ice cores. In The Environmental Record in ice is needed for amino acid analysis of the lower Glaciers and Ice Sheets. (H. Oeschger and C. C. J. Lang- latitudinal ice caps and glaciers, like the Chongce Ice way, Jr. Ed.), pp. ,,/ῌ,.1, John Wiley & Sons, New York. Cap of the West Kunlun Mountains, due to the short Cowie, G. L. and Hedges, J. I. (+33,): Sources and reactivities of amino acids in a coastal marine environment. Limnol- distance from amino acid sources, such as snow algae, ogy and Oceanography, -1, 1*-ῌ1,.. bacteria and pollen. Therefore, amino acid analysis in Gupta, U. C. and Reuszer, H. W. (+301): E#ect of plant species ice cores from lower latitude regions seems to be on the amino acid content and nitrification of soil or- +*0 0 -3/ῌ.** easier than that in those from the polar regions. ganic matter. Soil Science, ( ), . Haeberli, W., Schotterer, U., Wagenbach, D., Haeberli- On the other hand, the cause of the di#erent Schwitter, H. and Bortenschlager, S. (+32-): Accumula- amino acid compositions among the samples is an tion characteristics on a cold, high-alpine firn saddle unsettled question. The amino acid composition, espe- from a snow-pit study on Colle Gnifetti Monte Rosa, ,3 ,0*ῌ,1+ cially that in the particulate fraction of the Chongce Swiss Alps. Journal of Glaciology, , . Hammer, C. U. (+311): Past volcanism revealed by Greenland sample, may relate to the sources of amino acids, and ice sheet impurities. Nature, ,1*, .2,ῌ.20. allow us to more easily provide further information to Hammer, C. U., Clausion, H. B. and Dansgaard, W. 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