Fractionation of Hydrogen Isotopes in Lipid Biosynthesis

Organic Geochemistry 30 (1999) 1193±1200

Note Fractionation of hydrogen isotopes in lipid biosynthesis

Alex L. Sessions a, Thomas W. Burgoyne b, Arndt Schimmelmann b, John M. Hayes a,*

aDepartment of Geology and Geophysics, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA bBiogeochemical Laboratories, Departments of Chemistry and of Geological Sciences, Indiana University, Bloomington, IN 47405, USA Received 17 April 1999; accepted 16 June 1999 (Returned to author for revision 12 May 1999)

Abstract

Isotopic compositions of carbon-bound hydrogen in individual compounds from eight dierent organisms were measured using isotope-ratio-monitoring gas chromatography±mass spectrometry. This technique is capable of measuring D/H ratios at natural abundance in individual lipids yielding as little as 20 nmol of H2, and is applicable to a wide range of compounds including hydrocarbons, sterols, and fatty acids. The hydrogen isotopic compositions of lipids are controlled by three factors: isotopic compositions of biosynthetic precursors, fractionation and exchange accompanying biosynthesis, and hydrogenation during biosynthesis. dD values of lipids from the eight organisms examined here suggest that all three processes are important for controlling natural variations in isotopic abundance. n-Alkyl lipids are depleted in D relative to growth water by 113±262-, while polyisoprenoid lipids are depleted in D relative to growth water by 142±376-. Isotopic variations within compound classes (e.g., n-alkanes) are usually less than 050-, but variations as large as 150- are observed among isoprenoid lipids from a single organism. Phytol is consistently depleted in D by up to 50- relative to other isoprenoid lipids. Inferred isotopic fractionations between cellular water and lipids are greater than those indicated by previous studies. # 1999 Elsevier Science Ltd. All rights reserved.

Keywords: Hydrogen isotopes; irmGCMS; Isotope fractionation; Lipids; Acetogenic; Isoprenoid; Biosynthesis

1. Introduction DeNiro, 1990). Furthermore, isotope eects for hydrogen are commonly large (Bigeleisen, 1965). Therefore, We have developed an analytical system capable of the ability to examine hydrogen isotopic variation at measuring the D/H ratio of nanogram quantities of in- the natural abundance level in individual organic com- dividual organic compounds with a precision of 5- or pounds should prove to be quite useful. At present, better. Variations in the natural abundance of D in C- with the exception of speci®c analyses of cellulose, bound hydrogen clearly record both environmental almost nothing is known about hydrogen isotopic (Yapp and Epstein, 1982; Sternberg, 1988) and bio- variability in individual compounds. As an initial chemical eects (Estep and Hoering, 1980; Yakir and examination, therefore, we extracted lipids from eight separate organisms and measured the D/H ratio of individual components in those extracts. * Corresponding author. Fax: +1-508-457-2183. The purpose of this study was to examine the natu- E-mail address: [email protected] (J.M. Hayes) ral range of fractionations between cellular water and

0146-6380/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S0146-6380(99)00094-7 1194 A.L. Sessions et al. / Organic Geochemistry 30 (1999) 1193±1200 lipids, among lipids of similar and disparate biosyn- irmGCMS (Ricci et al., 1994). The working concen- thetic origin, and among lipids from dierent organ- tration range for this system (to obtain 05- precision) isms. To this end, we examined two cultures of is 20±100 nmoles H2 injected on-column per sample unicellular marine algae (Alexandrium fundyense,a component. dino¯agellate, and Isochrysis galbana, a haptophyte), All values for dD reported here are relative to two species of multicellular marine algae (Fucus vesicu- VSMOW. A laboratory standard containing 15 hom- losis and Ascophyllum sp., both brown algae), a sub- ologous n-alkanes, varying in concentration over a six- mergent higher plant (Zostera marina, a sea grass) and fold range and varying in dD over a 210- range, was an emergent higher plant (Spartina alterni¯ora,a analyzed daily. Values of dD for these alkanes were marsh grass ), a bacterial culture (Methylococcus capsu- determined by oine combustion to a precision of latus, a methanotroph), and the leaves of a carrot 21.6-. The alkane standard was used to (1) determine + plant (Daucus carota ). Aquatic organisms were the H3 factor for the day, (2) normalize dD values to selected preferentially to minimize isotopic dierences the VSMOW scale, and (3) monitor the stability of the + between cellular and growth water due to evapotran- system. The H3 factor is determined as the value of K spiration (Edwards, 1993). Daucus was selected to that minimizes the mean absolute error for the 15 examine isotopic characteristics related to a recently peaks of varying size. Values of K determined using discovered biosynthetic pathway (Schwender et al., this method are generally within 5% of those deter- 1996). mined conventionally (i.e., by observation of i3=i2 at varying values of i2; Friedman, 1953). Regression of dD (irmGCMS) on dD (oine) for these standards 2. Experimental provides a normalization line analogous to that used in batchwise analyses (i.e., so that dD values for 2.1. Isotopic measurements VSMOW and SLAP are 0 and 428-, respectively; Coplen, 1988). Values of R 2 for this regression always Hydrogen isotopic compositions of individual com- exceeded 0.99, and normalization results in corrections pounds were measured using an isotope-ratio-monitor- of <10- (commonly <5-) to any dD value. The ing gas chromatography±mass spectrometry mean precision of measurement of dD for these (irmGCMS) system developed in our laboratory. The alkanes over the 3-month period of this study was system is similar to that described by Scrimgeour et al. 24.2- (np= 33 injections) and the root-mean-square 2 (1999) and Tobias and Brenna (1997), but with several error Sd =n, where d=dDmeasureddDknown and important improvements. The euent from a conven- n = number of values for d ) was 5.3- (n = 491 tional GC using He carrier gas is fed into a graphite- measurements) with no systematic bias due to peak lined alumina tube held at 14008C, at which tempera- size or retention time. ture organic compounds are quantitatively pyrolyzed Values of dD for sample compounds were deter- to graphite, H2, and CO (Burgoyne and Hayes, 1998). mined by reference to coinjected n-alkane standards. An open split transmits 200 ml/min of the resulting gas Where possible, four to six n-alkanes were coinjected stream to a Finnigan MAT 252 mass spectrometer. An with each sample; two of these were used as isotopic electrostatic ®lter in front of the mass-3 Faraday cup reference peaks, with the remaining alkanes providing reduces scattered 4He+ reaching that collector to <75 tests of analytical accuracy. To avoid analyzing isoto- femtoamps at 200 ml/min He. pically exchangeable H from carboxyl and hydroxyl Mass-2 and -3 ion currents are recorded at 4 Hz and positions, and to improve chromatography, alcohols all data are processed using Visual Basic computer and fatty acids were derivatized as trimethylsilyl ethers codes that we have developed. Raw mass-3 ion cur- and esters using bis-(trimethylsilyl)-tri¯uoroacetamide + rents are corrected for contributions from H3 on a (BSTFA). BSTFA is an attractive derivatizing reagent point-by-point basis: each mass-3 data point is cor- because (1) it can be used for both alcohols and acids; rected based on the corresponding mass-2 data point (2) hot, acidic conditions which might promote hydro- 2 using the equation, iHD i3 K i2 , where i2 and i3 gen isotopic exchange are avoided; and (3) all hydro- are the raw mass-2 and -3 ion currents, iHD is the cor- gens in the derivatizing reagent are in the trimethylsilyl + rected ion current, and K is the H3 factor. We have (TMS) groups, so that the dD of the derivatizing H tested this correction scheme extensively, and ®nd that can be measured directly by irmGCMS analysis of the + it reliably corrects for H3 currents for a wide variety BSTFA peak. To test this procedure, we measured the of peak shapes and sizes, retention times, and sample dD of non-exchangeable H in a cholestanol standard + compositions. Following correction for H3 , ion cur- by ®rst measuring the acetate, ketone, and tri¯uoroace- rents are integrated, ion±current ratios are calculated, tate derivatives using conventional, oine techniques, and isotope ratios are computed and standardized then measuring the TMS derivative using irmGCMS. using techniques similar to those used in carbon The three oine measurements produced derivative- A.L. Sessions et al. / Organic Geochemistry 30 (1999) 1193±1200 1195

Table 1 Table 1 (continued ) dD measurements for individual lipids Species/compound dD(-)a s (-)b Species/compound dD(-)a s (-)b n-C26 alkane 170 11 Cultured specimens n-C27 alkane 166 1 Alexandrium fundyense (dino¯agellate) n-C28 alkane 164 4 Hydrocarbons n-C29 alkane 169 3 d n-C alkane 161 19 C22 alkadiene 204 10 30 Fatty acids n-C31 alkane 160 7 c c 14:0+14:1 232 NA n-C32 alkane 145 NA c 16:0 227 NA n-C33 alkane 155 15 22:4 218 NAc Ketones e Sterols C33 alkanone 178 16 27D5 323 8 Triterpenoids 30(4a,23,24)D22 295 9 lupenone 142 6 d 29(4a,24)D22 311 1 pentacyclic triterpenone 144 5 Other isoprenoids lupenol 171 1 d phytol 357 13 pentacyclic triterpenol 165 3 pentacyclic triterpenold 151 8 Isochrysis galbana (haptophyte) Other isoprenoids Hydrocarbons phytol 278 4 d C31 alkadiene 113 6 Fatty acids Daucus carota (carrot) 14:0+14:1 216 18 Hydrocarbons 16:0 209 10 n-C23 alkane 141 4 18:1 (2 isomers) 181 7 n-C24 alkane 135 18 e Sterols n-C25 alkane 158 6 5,22 28D 258 11 n-C26 alkane 139 7 n-C27 alkane 166 3 Methylococcus capsulatus (methanotroph) n-C29 alkane 162 2 Fatty acids n-C30 alkane 134 15 14:0 144 8 n-C31 alkane 113 21 16:0+16:1 161 5 n-C32 alkane 117 11 Sterolse Alcohols 8(14) 28(4a)D 234 6 n-C22 alkanol 186 11 8(14) 29(4,4)D 234 6 n-C24 alkanol 192 12 n-C26 alkanol 192 6 Native specimens n-C28 alkanol 197 7 Ascophyllum sp. (brown alga) n-C30 alkanol 180 15 Fatty acids Fatty acids 14:0 208 7 12:0 262 8 16:0 210 6 14:0 217 7 16:1 213 8 16:0 172 6 18:1 189 4 16:1 204 17 Sterolse 18:0, 18:1, 18:2, 18:3 190 7 5 29D 219 11 C24 hydroxy-fatty acid 193 12 Sterolse 5,22 Fucus vesiculosis (brown alga) 29D 283 7 5 Fatty acids 29D 292 7 14:0 187 8 Triterpenoids 16:0 167 4 D-amyrin+b-amyrin 252 10 f 18:1 157 10 pentacyclic triterpenols 239 6 d Sterols pentacyclic triterpenol 226 16 d 29D5,24(28) 208 4 pentacyclic triterpenol 231 16 Other isoprenoids Zostera marina (sea grass) caryophyllene (C15H24) 308 6 d Hydrocarbons sesquiterpene (C15H24) 320 7 phytadiene 345 2 n-C17 alkane 167 6 phytol 376 4 n-C19 alkane 159 3 n-C21 alkane 147 6 squalene 241 8 a Corrected for TMS derivatization. Sterolse b Standard deviation of 3 to 5 replicate analyses. 5 27D 253 1 c Only one analysis available. 5,24 27D 205 24 d Speci®c identity could not be determined. 29D5 194 6 e Leading number indicates total C and parenthesized num- Spartina alterni¯ora (marsh grass) bers indicate positions of methyl substituents (where known). Hydrocarbons f Three coeluting triterpenol peaks. n-C25 alkane 150 10 1196 A.L. Sessions et al. / Organic Geochemistry 30 (1999) 1193±1200 corrected dD values of 247, 250, and 253- (all 21.5-), and the irmGCMS measurements produced a corrected dDof25623-.

2.2. Sample collection and preparation

Alexandrium clone #GTCA28 was grown in a 1-l culture on f/2 medium at 158C with a 14:10 h light: dark cycle. Isochrysis clone T-ISO was grown in a 20-l culture on f/2 medium at 208C under continuous illu- mination. The f/2 medium for both cultures was prepared from sterile-®ltered Vineyard Sound seawater, which has a salinity of 31.5±32.0-. Both cultures were ®ltered onto precombusted 0.7-mm glass ®ber ®lters (Whatman GF/F) prior to extraction. Ascophyllum and Fucus were collected from Quisset Harbor, Woods Hole, MA. Spartina was collected from a salt marsh on Penzance Point, Woods Hole, and Zostera was collected from Woods Hole Harbor. Daucus with attached Fig. 1. Summary of dD measurements for compound classes. leaves was obtained from a local grocery, and only the Each symbol represents the mean for that class, and the bars leaves were analyzed. The Methylococcus culture ana- represent the range of measured values. lyzed here was the same as that described by Summons et al. (1994) and was obtained in dried form. All native error in the measurement of unknown compounds, we specimens were collected in February 1999. estimate that the dD value measured for each com- Samples were extracted with an Accelerated Solvent pound is accurate to within215-. However, relative Extractor (Dionex) using dichloromethane (DCM)/ dierences between compounds in the same chromato- methanol (90:10 v/v) with three 5-min extraction cycles gram are probably accurate to within25-. at 1008C and 1000 psi. Total lipid extracts from Results are summarized graphically in Fig. 1. Ascophyllum, Spartina, and Daucus were saponi®ed by Several points emerge. First, for any of the eight reacting with methanolic NaOH at 758C for 6 h, then organisms examined, the dierences between com- neutral and acid fractions were extracted into hexane pound classes (e.g., n-alkanes, sterols, fatty acids, etc.) and combined. Lipids were separated into classes by are greater than those within any one class. Second, elution from disposable, solid-phase extraction car- where isotopic compositions of growth water are tridges containing 500 mg of an aminopropyl station- known, all lipids are depleted in D relative to growth ary phase (Supelclean LC-NH2). Fractions collected water by 150- or more. Third, compounds from a were: hydrocarbons (4 ml hexane), ketones (6 ml hex- given class (e.g., sterols) can have substantially dier- ane/DCM 3:1), alcohols (5 ml DCM/acetone 9:1), and ent dD values in dierent organisms, despite growing fatty acids (8 ml DCM/formic acid 4:1). Each fraction in water with the same hydrogen isotopic composition. was analyzed by GCMS to identify compounds of interest. 3.1. Variations of dD within organisms

3. Results and discussion Compound classes exhibit restricted ranges in dD, with individual lipids in each class generally falling Results are summarized in Table 1. Standard devi- within a range of <50-. Variations between classes ations of replicate analyses typically ranged from 3 to are much larger, with dierences of 50±150- being 12-. Worse precision (up to 24-) was obtained for common. Polyisoprenoid lipids are generally depleted some compounds where coeluting or especially small relative to acetogenic (n-alkyl) lipids (compare ®lled peaks were measured. When these were included in and open symbols, Fig. 1), but there are also dier- order to provide a conservative overview of the results, ences between compound classes with biosynthetically the pooled estimate of the standard deviation of a equivalent carbon skeletons (e.g., n-alcohols versus n- single analysis was 9.2- (375 degrees of freedom in alkanes in Daucus ). Based on compounds examined in 155 sets of replicates). The arithmetic mean error for Daucus, the range of variability appears to be some- the coinjected alkanes that served as test compounds what greater within the polyisoprenoid lipids than in these analyses was 0.3-, and the root-mean-square within the acetogenic lipids. error was 7.6-. Considering all possible sources of Three potential sources of isotopic variability in A.L. Sessions et al. / Organic Geochemistry 30 (1999) 1193±1200 1197 lipid hydrogen can be identi®ed: (1) the isotopic com- between classes of polyisoprenoid lipids. In particular, position of biosynthetic precursors, (2) isotope eects phytol is consistently depleted relative to sterols and

(including exchange of organic H with H2O) associated pentacyclic triterpenoids by 50±140-. Part of this with biosynthetic reactions (Martin et al., 1986), and large variability may be due to isotopically dierent (3) the isotopic composition of hydrogen added, com- pools of NADPH in the cytosol, where triterpenols are monly from NADPH, during biosynthesis (Smith and synthesized, and the chloroplasts, where phytol is syn- Epstein, 1970; Luo et al., 1991). This third control, thesized. In plants, NADP+ is reduced to NADPH by which is not related to the ¯ow of substrates used in two processes: electron-transport chains associated assembly of the carbon skeleton, is notably dierent with photosynthesis in the chloroplasts, and during from those encountered in carbon-isotopic studies oxidation of sugars in the pentose-phosphate pathway (Hayes, 1993). Below we discuss evidence that isotopi- in the cytosol (Abeles et al., 1992). The sources of cally dierent pools of NADPH may exist within cells; hydrogen for NADPH are very dierent in these two this suggests that compound-speci®c hydrogen isotopic cases (H2O in photosynthesis, and C-bound H from analyses will provide information about compartmen- sugars in the pentose-phosphate pathway), so it is talization of biosynthesis in cells, as well as about bio- plausible that isotopically distinct pools of NADPH synthetic pathways. might exist within these dierent cellular compart- The depletion of D in isoprenoid lipids relative to ments. acetogenic lipids was ®rst described by Estep and Isotopic dierences between phytol and triterpenols Hoering (1980). Our results con®rm and extend, but may also be due in part to dierent biosynthetic path- do not explain, this ®nding. Acetogenic lipids, sterols, ways. Schwender et al. (1996) have recently described a and pentacyclic triterpenols and all use acetyl-CoA Ð novel pathway for polyisoprenoid synthesis, in which either directly or via the formation of mevalonic acid glyceraldehyde phosphate and pyruvate Ð rather than (MVA) Ð as the biosynthetic precursor (Abeles et al., three molecules of acetyl-CoA Ð condense to form I- 1992). Slightly more hydrogen in acetogenic lipids de- deoxy-D-xylulose phosphate (DOXP), the immediate rives from NADPH (050%) as compared to isopre- precursor of isopentenyl phosphate. The phylogenetic noid lipids derived from the MVA pathway (037%). If distribution of this novel pathway is still under investi- the acetogenic-versus-isoprenoid contrast (roughly gation. Of the species included here, only Daucus has 200- versus 250-) were attributed to this dier- been examined. Daucus uses the well-known MVA ence alone, it would require that NADPH-derived pathway to synthesize cytosolic isoprenoids, including hydrogen is substantially enriched (dD 1 0) and acet- sterols, while using the novel DOXP pathway to syn- ate-derived hydrogen is highly depleted (dD 1 400). thesize plastidic isoprenoids, such as phytol More plausibly, as noted by Estep and Hoering (1980), (Lichtenthaler, 1999). In general, the DOXP pathway the depletion of D in polyisoprenoids relative to n- appears to be closely related to chloroplasts, and it is alkyl lipids is due to isotope eects associated with the presumed to be a relict biochemical pathway preserved two pathways. from the endosymbiotic origin of chloroplasts (Disch In Daucus, fatty acids and n-alcohols have similar et al., 1998). If dierences between the DOXP and dD values, but are depleted in D by 050- relative to MVA pathways are responsible for the observed de- n-alkanes. Estep and Hoering (1980) also found sub- pletion of phytol relative to triterpenols, this would stantial hydrogen isotopic dierences (up to 54-) require either that DOXP and pyruvate are D-depleted between fatty acids and hydrocarbons separated from relative to acetate, or that there is a larger isotope three higher plants and from Trichodesmium. In that eect associated with the DOXP pathway. case, however, the pattern was reversed from that Based on the data presented here, we cannot dis- observed here, with fatty acids in the higher plants D- tinguish the source of isotopic variability in isoprenoid enriched relative to the hydrocarbons. Given that the lipids. Diering biosynthetic precursors, isotope eects carbon skeletons of fatty acids, n-alcohols, and n- and exchange associated with biosynthesis, isotopically alkanes are all formed via the same biosynthetic path- distinct pools of NADPH, or all three may be respon- way, these dierences must be due to changes in the sible for the observed pattern. If dierent isotope isotopic composition of acetate starting material, or of eects can be positively associated with the DOXP and NADPH, or both. These changes could occur through MVA pathways, compound-speci®c D/H measure- time (e.g., as growth rate changes), or could exist ments would become a valuable tool for studying the spatially within the cell or plant at any given time. distribution and importance of this novel biosynthetic There appears to be a slight trend toward D-enrich- pathway. ment with longer chain length in the fatty acids. If real, this may also re¯ect synthesis in dierent parts of 3.2. Isotopic fractionation the plant. Substantial hydrogen isotopic variations occur We calculate values for e (isotopic fractionation) by 1198 A.L. Sessions et al. / Organic Geochemistry 30 (1999) 1193±1200

Table 2 most D-enriched lipid compounds observed in this Hydrogen isotopic fractionationsa study and fatty acids were in most cases the most abundant lipid components; it therefore appears unli- b Compound-speci®c measurements, this study kely that the smaller fractionations measured by previous studies are due solely to dierences in the Sample e e e e fa/w ak/w st/w ph/w relative abundance of these components. A possible ex- Alexandrium 226 310 357 planation is that components of the bulk extracts Isochrysis 202 258 which do not appear in GC analyses (e.g., lipoproteins) Ascophyllum 205 219 are D-enriched relative to the lipids we examined. In Fucus 170 208 addition, bulk lipid measurements may incorporate

Zostera 158 223 hydrogen from H2O or exchangeable oxygen-bound Spartina 162 278 positions, which would be expected to produce anoma- lously large dD values (Schimmelmann, 1991).

Bulk lipid measurements, previous studies 3.3. Variations in dD between organisms Sample e e e Referencec l/w sl/w nsl/w Dierences in dDofupto80- that cannot be Cyanobacteria 175 1 reconciled as stemming from dierences in cellular Macrophytes 152 232 1 water exist between the same compounds in dierent Red algae 139 2 organisms (e.g., compare ephytol/water in Spartina and Brown algae 178 2 Alexandrium in Table 2). These dierences appear to Green algae 132 2 be most pronounced between the cultured organisms Macrophytes 125 3 (Alexandrium, Isochrysis, and Methylococcus ) and Daucus versus the native specimens (Ascophyllum, a Abbreviations: water (w), alkanes (ak), fatty acids (fa), Fucus, Spartina, and Zostera ). Values of e sterols (st), phytol (ph), bulk lipids (l), saponi®able lipids (sl), sterol/fatty acid range from 70 to 118- in the former group, and non-saponi®able lipids (nsl); ea=b 1000 da 1000= db 10001. from 18 to 46- in the latter. etriterpenol/alkane is b Calculated using the mean dD for each compound class, 112- in Daucus and +8- in Spartina, and ephytol/ and assuming dDw=0-. alkane is 274- in Daucus and 138- in Spartina. c (1) Estep and Hoering (1980); (2) Sternberg et al. (1986); Thus, the depletion of D in isoprenoid relative to (3) Sternberg (1988). acetogenic lipids is greater in the actively growing cultures and in Daucus than in the native specimens collected in winter. If the diering extents of isotopic estimating that the dD value of the cellular water for fractionation observed here represent a more general all samples except Daucus and Methylococcus was pattern, they should be useful for understanding the close to 0-. For those organisms that were submerged underlying mechanisms, which could involve dier- in ocean water (Alexandrium, Isochrysis, Ascophyllum, ences in photosynthetic activity, growth rate, water Fucus, and Zostera ) this is straightforward. Cellular temperature, light intensity, nutrient supply, or other water in Spartina may have been enriched relative to parameters. the growth water by evaporation, but the nearly identi- A correlation between D-depletion in lipids and cal dD values for alkanes from Zostera and Spartina photosynthetic activity would not be surprising, as a suggest that this was minimal. The isotopic compo- similar situation is observed for carbohydrate metab- sitions of water in the Daucus and Methylococcus cul- olism. Photosynthesis produces C-bound H in carbo- tures are unknown. hydrates which is D-depleted relative to tissue water Fractionation factors are de®ned and summarized in by 100±170- (Estep and Hoering, 1981; Yakir and

Table 2. Values for efatty acid/water range from 170 to DeNiro, 1990). In contrast, ``heterotrophic processing'' 226- (mean=200-), values for ealkane/water range of carbohydrates within plants (such as isomerization from 158 to 162- (mean=160-), and values for of triose phosphates and interconversion of fructose-6- esterol/water range from 208 to 310 (mean=244-). phosphate and glucose-6-phosphate) results in the By comparison, fractionations between bulk lipids and exchange of up to 50% of C-bound H with tissue water measured by oine techniques range from 125 water, with those exchanged positions becoming D- to 178- (mean=150-). The observed fraction- enriched by up to 158- relative to the tissue water ation between water and alkanes is indistinguishable (Yakir and DeNiro, 1990). Luo and Sternberg (1991) from that previously observed between water and bulk found that starch from chloroplasts was highly lipids, but fractionations between fatty acids and water depleted relative to cytoplasmic cellulose in the same are nearly 50- larger on average. n-Alkanes were the plant, due probably to this eect. Thus, the dD value A.L. Sessions et al. / Organic Geochemistry 30 (1999) 1193±1200 1199 of carbohydrates in plants is thought to represent a References balance between autotrophic and heterotrophic metabolism, and can potentially be used as an indicator of Abeles, R.H., Frey, P.A., Jencks, W.P., 1992. Biochemistry. photosynthetic status in plants (Yakir, 1992). Our Jones and Bartlett, Boston. results suggest that a similar pattern may exist in Bigeleisen, J., 1965. Chemistry of isotopes. Science 147, 463± lipids. 471. Burgoyne, T.W., Hayes, J.M., 1998. Quantitative production

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