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Potentials and caveats with oxygen and sulfur stable isotope analyses in authenticity and origin checks of food and food commodities
Food Control 48 (2015) 143e150
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Potentials and caveats with oxygen and sulfur stable isotope analyses in authenticity and origin checks of food and food commodities Nicole Krivachy (Tanz), Andreas Rossmann, Hanns-Ludwig Schmidt* Isolab GmbH Laboratory for Stable Isotopes, Woelkestrasse 9, D-85301 Schweitenkirchen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 November 2013 Received in revised form 3 June 2014 Accepted 4 June 2014 Available online 12 June 2014
Analyses of stable isotope ratios are officially accepted methods in food authenticity and origin determination. They are routinely practiced by empirical comparison of the unknown samples' d values with those of authentic material. However, as the isotope characteristics of food are influenced by many parameters, it is desirable to also study and use causal correlations of isotope fractionations for the interpretation of experimental data. Corresponding potentials and limits are outlined for oxygen and sulfur stable isotopes. In the natural water cycle, plant leaf and animal cell water are the most important sources for food integrated water and organically bound oxygen. The way from sea water to fruit juice water and to organic matter and the integrated isotope fractionations are shown and the possibilities and limits for the assignment of juices and wine to their geographical origin, history and authenticity are deduced. The oxygen flux and isotopic balance in animals and the sources and drains of animal body water are outlined and the problems and limits for its suitability as a bioindicator for origin assignments of animal food products are discussed. The potential of the d18O value of organically bound oxygen is demonstrated. The sulfate reduction in plants is accompanied by isotope fractionation but as normally no sulfur is excreted, the d34S value of bulk plant matter is identical to that of the primary local source. Small differences in the d34S values of plant compartments are often due to differences in their abundance of main S-containing ingredients, as residual sulfate and cysteine or methionine containing proteins. This is similar with animal tissues and products. Therefore, the sulfur isotope analysis of the bulk matter or of defined fractions of plant and animal samples is an ideal and reliable tool for food origin and authenticity proof and for archaeological and animal migration research. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Oxygen Sulfur Stable isotopes Authenticity Origin check
1. Introduction In recent years the European Community/Union has defined indications for the quality characterization of food and food commodities like “Protected Denomination of Origin (PDO)”, “Protected Geographical Indication (PGI)”, and “Certification of Specific Character (CSC)” (cited in Schmidt, Rossmann, Rummel, & Tanz, 2009; further information see Calderone & Guillou, 2008). The criteria and the methodology to be used for conferring or applying these indications on a food sample are indicated in corresponding regulations. Among the officially recommended methods, stable isotope ratio determinations of the bioelements are occurring in the first
* Corresponding author. Prielhofweg 2, D-84036 Landshut, Germany. Tel.: þ49 871 44497. E-mail addresses: [email protected] (N. Krivachy (Tanz)), [email protected] (H.-L. Schmidt). http://dx.doi.org/10.1016/j.foodcont.2014.06.002 0956-7135/© 2014 Elsevier Ltd. All rights reserved.
range. Factors determining these isotope ratios of a natural sample are those of the primary material in question, thermodynamic and kinetic isotope effects, geographical and climatic conditions of the sample's origin, physiological and anatomic properties of plants and animals' positions in food chains. Vice versa, corresponding parameters can be deduced from the isotopic properties of a sample. Furthermore, most important is the individual dependence of the bioelements on the different external influences, their “indi€ ckigt, & Christoph, 2005). cator function” (Schmidt, Roßmann, Sto And as the informations implied in the isotope ratios of the individual elements are complementary, many recent investigations on food origin and authenticity use multielement isotope ratio determinations (Bahar et al., 2008; Camin et al., 2007; Schlicht, Roßmann, & Brunner, 2006). Carbon isotope ratios are predominantly indicative for food (ingredients) assignments to their origin from the photosynthetic plant types C3, C4 and CAM plants. One application is the proof of an adulteration of beverages of C3 plants with C4 products and vice
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versa. (Jamin, Gonzalez, Remaud, Naulet, & Martin, 1997). In food chains, carbon d values increase by ~2‰ for each trophic level (Michener & Schell, 1994; for fundamentals of isotope abundance and fractionation indications see Box 1). Nitrogen isotope ratios are correlated to agricultural fertilization practices and hence often used to discriminate between compounds from conventional and “organic” production, respectively (Camin et al., 2011; Rapisarda et al., 2010; Schmidt, Roßmann, et al., 2005; Schmidt, Rossmann, et al., 2005; Sturm & Lojen, 2011). The trophic shift of nitrogen isotope indications can attain 2e4‰ per level (Michener & Schell, 1994). Hydrogen and oxygen are preferably originating from plant leaf or animal drinking water, respectively, the isotope characteristics of which depend on local precipitation and climate, but are modulated by biochemical, physiological and anatomical influences (White, 1989). Although hydrogen and oxygen isotope ratios of meteoric water are correlated among each other by the “meteoric water line” (d2H [‰]V-SMOW ¼ 8 d18O[‰]V-SMOW þ 10, Craig, 1961), the indicator function of hydrogen is more important for individual metabolites, as many isotope fractionations of this element in the secondary metabolism lead to characteristic hydrogen isotope patterns of defined plant compounds. This is the basis for origin and authenticity investigations on spices, aromas and fragrances by means of 2H NMR measurements (deuterium
positional or pattern analysis, Schmidt, Werner, Rossmann, Mosandl, & Schreier, 2007). Trophic levels in context with food chains are of minor importance with hydrogen and oxygen, as the primary source water is globally available in form of an infinite pool with large turnover rate. In food analysis, oxygen isotope ratios are often determined on the bulk sample. These d18O values of biomass provide average data of cell water and organically bound oxygen. However, in most cases of food quality investigations, the d18O value of the water is analyzed, even when it is not particularly indicated. The reason is that the corresponding analytic procedure is extremely easy (Horita, Ueda, Mizukami, & Takatori, 1989) and can often, especially with wine and fruit juices, be performed on the untreated sample itself. As the method is based on the isotopic equilibration between the sample water and CO2, the sample should not be in a state of fermentation, producing additional CO2. Further artifacts can occur in context with separation of the water from the sample (e.g. meat). This equilibration method has recently been supplemented by the water isotope ratio analysis by IR methodology (Brand, 2010; Lis, Wassenaar, & Hendry, 2008; West, Goldsmith, Brooks, & Dawson, 2010). In any case, as will be shown in this contribution, the interpretation of the results has to take into account the history of the original primary water. On the other hand, most organically
Box 1 Isotope effects and isotope fractionation in closed and open systems.
The indication of isotope concentrations of bioelements is performed in the d value scale as relative differences to international standards of the International Atomic Energy Agency (IAEA) in Vienna (V). The standard for oxygen is Vienna Standard Mean Ocean Water (V-SMOW) with the isotope ratio R ¼ [18O]/[16O] ¼ 2005.2 106, that for sulfur is Vienna Canyon Diablo Troilite (VCDT) with the isotope ratio R ¼ [34S]/[32S] ¼ 44150.9 106. According to IUPAC rules, delta values are defined as (Coplen, 2011)
. RVSMOW d18 O ¼ RSample RVSMOW
. RVCDT and d34 S ¼ RSample RVCDT
However, for practical reasons and because of the general application in the cited references, we are using in the present paper the old formula with the factor 1000, leading to permill [‰]:
. RVSMOW 1000 d18 O½‰ ¼ RSample RVSMOW
. RVCDT 1000 and d34 S½‰ ¼ RSample RVCDT
For the determination of the d values, organic matter is converted by techniques of the elemental analysis into CO (d18O) and SO2 (d34S), respectively, water is equilibrated with CO2 (d18O). The isotope ratio of the gases is analyzed, relative to that of a (laboratory) standard, in an isotope ratio mass spectrometer (Sieper et al., 2010; Werner, 2003). Thermodynamic isotope effects or fractionation factors a are the ratio of physical properties or equilibrium constants K and K* of 18 isotopologue molecules, e.g. of the vapor pressures of H16 2 O (p) and H2 O (p*): a ¼ p/p*. Kinetic isotope effects akin are the ratio of velocity constants k of such molecules in chemical reactions; the element in question is indicated, e.g. for oxygen as akin ¼ k16/k18. Normally, the “lighter” isotopologues react faster and akin is > 1.0; for the bioelements except hydrogen, it is between 1.00 and 1.07. The basis for kinetic isotope effects on enzyme-catalyzed reactions is the MichaeliseMenten theory. All these considerations concern systems in equilibrium or closed systems, respectively. Most natural systems are open with infinite pools of isotopically constant substrates (e.g. ground water or soil sulfate). This has for consequence a constant isotope fractionation between substrate and product. Thus, the fractionation constant is defined as a ¼ RSubstrate/RProduct and the isotope fractionation or discrimination as D ¼ a 1 ¼ RSubstrate/RProduct 1. The combination of this equation with the above correlation between R and d permits to eliminate RStandard and yields, as dProduct << 1:
D ¼ ðdSubstrate dProduct Þ=ð1 þ dProduct Þ e dSubstrate dProduct This provides the possibility to use directly mass spectrometric results of practical measurements for interpretations. All systems discussed in the present paper are open with the exception that most plants do not excrete sulfur in any form and hence the total assimilated S remains in the system. Yet the following prerequisites concerning S-isotope fractionations have to be taken into account (see Fig. 4). A kinetic isotope effect on a defined reaction becomes only efficient in case of a partial substrate conversion, normally realized by formation of at least two products (metabolic branching, MB), one of them depleted, the other enriched in the heavy isotope (isotopic balance). The reverse is realized by metabolite channeling (MC): In a sequence of reactions the substrate and the subsequent products are quantitatively converted (e.g. by a multi-enzyme-complex). Kinetic isotope effects on any step cannot become efficient.
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bound oxygen is correlated to the original primary plant/body water by reactions with defined isotope fractionations, thus presenting a stable and reliable archive for climate identifications and reconstructions. On the other hand, the situation with sulfur is quite different. Sulfur occurs in Nature in multiple oxidation forms and isotopic characteristics. Sulfur assimilation in most plants is an unidirectional process, in which the total originally ingested element remains completely in the plant, independent of (partial) biochemical conversions with isotope fractionation and binding forms. The bulk sulfur isotope ratio thus preserves and reflects the isotopic property of the primary material in question and hence provides an ideal biomarker for the geographical origin of the sample. Nevertheless, isotope fractionations on defined reactions in plants and food chains lead to organic compounds with S from the same source but different d34S values. The consequences for food authenticity and origin assignment will be the topic of the following review. Its aim is to mediate fundamental knowledge for the interpretation of experimental data on oxygen and sulfur isotope ratios, independent on and above the availability and use of data banks from authentic reference material. 2. Oxygen isotope ratios as indicators for beverage and food origin 2.1. Sources and isotope characteristics of food intrinsic water Any water on land originates from the ocean, an infinite reservoir with a globally nearly constant oxygen-18 content (d18O value ¼ 0‰ ¼ Vienna Standard Mean Ocean Water, V-SMOW). An isotope effect on the vapor pressure leads to a preferred evaporation of “light” water isotopologues above the sea. Subsequent partial condensation has for consequence a further 18O-depletion in the vapor phase, and the last rain in the largest distance from the ocean is the “lightest” one (continental effect). This effect is overlapped by influences of local altitude (altitude effect), climatic and seasonal conditions (temperature effect) and precipitation amount (amount effect) (water cycle reviewed by Mook, 2000). From here originate local individual oxygen isotope characteristics of meteoric and ground water. Trees take up this water and transport it via their xylem system to the leaves, where it is, again depending on local climate conditions and with diurnal oscillations (Gessler, Peuke, Keitel, & Farquhar, 2007) enriched in 18O by transpiration. A medium sized apple tree in central Europe needs 30e40 L of water in a normal summer day. The 18O-enriched leaf water is, after partial clet-effect), isotope exchange with countercurrent xylem water (Pe the source for the water of fruit saps (Fig. 1). The overlap of all these geographical, climatic, local, annual and plant physiological influences is normally integrated in data from the analysis of authentic reference samples and the definition of authenticity limits. This is, for example established for juices in Europe by the “Association of the Industry of Juices and Nectars (AIJN) from fruits and vegetables of the European Union” in Brussels. Watering of wine or fruit juices or illegal re-dilution of fruit juice concentrates can be detected due to their lower d18O value relative to authentic
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products (Calderone & Guillou, 2008; Dordevic et al., 2013; Jamin, rin, Re tif, Lees, & Marin, 2003; Wachter, Christoph, & Seifert, Gue 2009). However, one has to take into account that problems can occur from sample processing and/or interference by washing water (Blanch & Ruperez, 2006). Water is also often a main part of animal originating food like milk, eggs and meat, and the d18O values of this water are used for origin assignments of the products. The first question to be discussed in this context concerns the sources and the composition of animal cell or body water and its d18O value. The primary and most important source is drinking water but also the diet of animals can contribute in a variable and not unimportant part. The contributions of the different source and drain factors and their fluxes have originally been compiled by Luz, Kolodny, and Horowitz (1984) and reported by Koch, Fogel, and Tuross (1994). Sources are drinking water, intrinsic water in food and oxidation water, originating from organically bound oxygen and atmospheric dioxygen converted in the respiratory chain. Sinks are urine and water in feces, sweat and breath water, milk and mainly breath CO2, which is highly enriched in 18O via equilibration with water. The correlation of the d18O value of animal body water and drinking water depends on species, the rates of drinking and respiration (Luz, Kolodny, & Horwitz, 1984). For an animal in steady state the above authors find linear relationships between the d18O values of ingested drinking and body water. The slopes of these relationships depend on the amount of drinking, species and its energy expenditure. Generally, whereas the d18O and d2H values of many animal tissues are correlated to each other and determined by the corresponding body and ultimately drinking water, this is especially not the case with carnivores (Pietsch, Hobson, Wassenaar, & Tütken, 2011). For vegetarians, a large contribution from the 18O enriched oxygen atoms in carbohydrates is to be expected. Correspondingly, a dependence of the diet can also be assumed as the reason for the difference found in the blood water of wild and domestic pigs from the same area (Longinelli & Selmo, 2011). Extensive experimental data and model calculations on this topic, in addition also concerning geographical parameters, see Podlesak, Bowen, O'Grady, Cerling, & Ehleringer, 2012. An unpublished own experiment confirmed these correlations for a living human subject. Correlations of body water to the oxygen isotope characteristics of animal tissues and parameters influencing these correlations are compiled by Kirsanow and Tuross (2011). However, in context with the practice of animal originating food investigations, most of these parameters are unknown. Fig. 2 presents a qualitative approximation for the assessment and understanding of the d18O value of water in or from animal products. For practical considerations in the present context, one can assume that the average d18O value of the body water of most domestic animals is about 3 ± 1‰ more positive than that of the drinking water. However, milk water can show large seasonal variations, from þ2‰ in winter to þ6‰ in summer above drinking water (Kornexl, Werner, Rossmann, & Schmidt, 1997), probably due to a larger contribution of grass intrinsic (18O-enriched) water in summer. Even the isotopic composition of silage water can vary in the course of silage preparation and storage (Sun, Auerswald,
Fig. 1. Stages and d18O values from ocean water to fruit juice water and organic matter in plants. d values for central Europe. Adapted after Yakir (1998), data for upper Bavaria, from Werner et al., (2004) and Schmidt et al. (2001). Indications of leaf water diurnal and juice water annual variations.
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Fig. 2. Simplified and exemplary oxygen and water mass and isotopic balances for an omnivore and a lactating cow on summer grass diet, respectively. Oxygen flux fractions in %, for the cow in [ ], d18O values in ( ). Data adapted from Luz, Kolodny, and Horwitz (1984); Koch et al. (1994); Kohn (1996) and Kornexl et al. (1997). The d18O value of milk is estimated assuming a contribution of 95% by milk water with d18O ¼ 8‰ and 5% from organic matter with D ¼ þ 20‰ from here. For further details about the body water of grazing ruminants see Sun, Auerswald, Wenzel, and Schnyder (2013).
Sch€ aufele, & Schnyder, 2014). On this background it is understandable that attempts to assign meat to its geographical origin exclusively on the basis of the d18O value of squeezed tissue water (Hegerding, Seidler, Danneel, Gessler, & Nowak, 2002; Thiem, Lüpke, & Seifert, 2004) were not very promising. Meat water from beef showed shifts relative to local drinking water of >4‰. The corresponding shift for pork meat water was only þ1 to þ2‰, and it was slightly depleted in 18O relative to beef meat water from same area. This difference demonstrates without any doubt an effect of the diet. In both cases, the meat water showed, as expected, a distinct correlation to the local ground water but the variations between individual samples from the same area or even the same litter did not allow unambiguous origin assignments, neither within Germany nor between Germany and Great Britain. Furthermore, significant influences of storage and handling of the samples were observed. Investigations by Renou et al. (2004) confirm the prevalence of diet and age of the animals over the influence of drinking water on beef meat water d18O values. The influence of climate and physiological parameters on the d18O values of wild animal body water is discussed by Kohn (1996). The potential of isotope characteristics, among them the d18O value of egg fractions for traceability and authenticity research of this animal product as well as the influence of storage and sample preparation is compiled in a review by Rock, Rowe, Czerwiec, & Richmond (2013). 2.2. Organically bound oxygen from water Carbonyl (aldehyde and keto) groups of organic compounds can exchange oxygen atoms with surrounding water. This reaction implies a thermodynamic isotope effect with an equilibrium fractionation D18O of the organic functional group of ~þ27‰. As this shift has also been found for carbohydrates, especially cellulose, it has been and is still used for (paleo)climatological and physiological investigations. However, it turned out recently that carbohydrates have a distinct and even variable 18O pattern (Schmidt, Werner, & Roßmann, 2001; Sternberg & Ellsworth, 2011; Sternberg, Pinzon, Anderson, & Jahren, 2006) and that the indicated value is only an average over the six positions of oxygen in glucose. Nevertheless, it can still be used in the above mentioned investigations. However, special bulk and positional d18O values have been found for animal originating carbohydrates, synthesized via gluconeogenesis (Schmidt et al., 2001).
The 18O enrichment and pattern of carbohydrates synthesized via the gluconeogenesis pathway (Fig. 3) starts from oxaloacetate to phosphoenolpyruvate. The subsequent water addition to 2phosphoglycerate, catalyzed by enolase, implies a kinetic oxygen isotope effect of 1.03 (Anderson, 1991). After isomerization of the product to 3-phosphoglycerate, the oxygen atom in position C-3 is protected from oxygen exchange. From its terminal positions C-1 and C-6 of hexoses, only the latter retains permanently its protection. If we assume that the average enrichment of C-1 to C-5 of the disaccharide relative to water is identically correlated to the surrounding water as in “photosynthetic” carbohydrates, we can estimate the shift in position C-6 of the lactose monosaccharides to be 11.4‰. This is, at least qualitatively, as expected from the isotope effect on the enolase reaction. In fact, the bulk discrimination D18O of lactose from cow milk is only 20.6‰ relative to the milk water (Schmidt et al., 2001). The amount of carbohydrates formed by gluconeogenesis from fatty acids in germinating oil seeds is insignificant for most practical questions. More important is the resynthesis of glucose from lactate by glycolysis in animals. However, for ruminants it is the sole pathway to form carbohydrates, starting from propionate, one of the main products of the rumen fermentation. This example of the addition of water to a >C]C< double bond, accompanied by a kinetic isotope effect, is an exception, as most other products of such reactions are intermediates in reaction chains and later on lose again this oxygen atom. Oxygen atoms in hydroxyl groups do not exchange with surrounding water under physiological conditions. However, like carbonyl, carboxyl groups exchange their oxygen atoms with surrounding water via hydrates; the equilibrium isotope effect is ~1.018, leading to a discrimination D18O of the functional groups by þ18 ± 1‰ relative to water (Schmidt et al., 2001). This provides, for example, the possibility to calculate the d18O value of a grape must water from wine tartaric acid (HOOCeCHOHeCHOHeCOOH), which is not affected by the fermentation. The acid contains four carboxyl and two “carbohydrate” O-atoms, yielding an average shift of 21‰ for the discrimination to must water, a result in good agreement with water data of Italian wines (Serra et al., 2005). Esters and amides can have a somewhat higher D18O value for the carbonyl atom (e.g. ~23‰), probably due to an intramolecular isotope effect on the activation of the acid. The (not catalyzed) isotopic equilibration of carbonyl and carboxyl compounds with water is a relatively slow process
Fig. 3. Processes, precursors and reactions of glucose formation by gluconeogenesis and origin of oxygen atom in position C-6 of glucose. Kinetic oxygen isotope effect on enolase reaction k16/k18 ¼ 1.03 (Anderson, 1991). The oxygen atom introduced by the reaction occurs firstly in positions C-1 and C-6 of glucose-6-phosphate (G-6-P) but is finally preserved only in the latter one. OAA ¼ oxaloacetic acid, PEP ¼ phosphoenolpyruvate, PGA ¼ phosphoglyceric acid.
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Fig. 4. Biochemical reactions of the assimilatory sulfate reduction by plants; formation and hydrolysis of glucosinolates. Steps with potential isotope fractionation are indicated by IF. MB ¼ metabolic branching, MC ¼ metabolic channeling (see Box 1). Modified after Tanz and Schmidt (2010), details see Tcherkez and Tea (2013). SS ¼ sulfate sulfur, SO ¼ “organic (reduced) sulfur”, PAPS ¼ 30 -phosphoadenosine-50 -phosphosulfate (“active sulfate”), PAP ¼ 30 -phosphoadenosine-50 -phosphate. XeSH is cysteine. Ss is always relatively enriched, So depleted in 34S.
(t1/2 ¼ min to h) and therefore, the equilibrium with short-lived intermediates is not always attained. For the proof of illegal water addition to fruit juices and must, the d18O value of the water present during fermentation is often determined via that of the ethanol formed. However, the equilibration of the intermediate acetaldehyde with water is only attained under defined condirin, Re tif, Lees, & Marin, tions of the fermentation (Jamin, Gue 2003; Perini & Camin, 2013). Correspondingly, phosphate in living systems equilibrates with water via the multiple formation and hydrolysis of nucleotide oligophosphates (e.g. ATP). The equilibration demands conditions of active and fast metabolism. As these are not always realized in all parts of a plant, the degree of equilibration can differ within different plant organs (Pfahler, Dürr-Auster, Tamburini, Bernsaconi, & Frossard, 2013). The d18O value of phosphate in bioapatite from bone and teeth is often used to identify the place of origin of recent or prehistoric persons on the basis of their drinking water (Chenery, Pashley, Lamb, Sloane, & Evans, 2012). However, uncertainties may arise from the fact that the samples always reflect the isotope content of the body water, which depends only partially from that of the drinking water (see above). The most common application of the oxygen isotope analysis of phosphate in bone and teeth bioapatite is in paleoclimatology; for a review of the methodology and possible artifacts see Grimes and Pellegrini (2012).
atmospheric O2 (d18O ¼ þ23.8 ± 0.3‰, Coplen et al., 2002, pp. 36e44), accompanied by a kinetic isotope effect kkin ¼ ~1.018 (Schmidt et al., 2001); from here results a fixed d18O value 7 ± 1‰: ≡CeH þ O2 þ NADPH þ Hþ / ≡CeOH þ H2O þ NADPþ The large difference of this d18O value to that of hydroxyl groups originating from the reduction of carbonyl groups allows unambiguous discriminations between alternative biosynthetic pathways of natural compounds (Werner et al., 2004). It has also been the basis for the development of a method for the identification of the origin of L-tyrosine from a plant or animal source, respectively (Fronza, Fuganti, Schmidt, & Werner, 2002), for assigning animals to a vegetarian, omnivore or carnivore diet, respectively, and to prove the illegal feed of ruminants with meat and bone meal (MBM), basis for their infection with bovine spongiform encephalopathy (BSE, Tanz, Werner, Eisenreich, & Schmidt, 2011). And we postulate that this d18O value also explains why collagens are relatively depleted by 2e3‰ in 18O to other proteins from the same source (Tuross, Warinner, Kirsanow, & Kester, 2008): they are the sole proteins containing ~15% hydroxyproline, formed by hydroxylation of (bound) proline. 3. Sulfur isotope ratios as biomarkers in food origin investigations
2.3. Dioxygen as source of organically bound oxygen
3.1. Sulfur sources, isotope fractionations and d34S values
Hydroxyl groups of phenols, hydroxy fatty acids, steroids and hydroxyproline are introduced by monooxygenase reactions from
Sulfur occurs in Nature as a mixture of four stable isotopes, among which only the main one (32S, 95.020 atom-%) and the
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second frequent one (34S, 4.218 atom-%) are of importance in the present context. The oxidation state of the element varies between þ6 and 2, among which sulfate-S (þ6), elemental S (0) and sulfide or “organic S” (2) are stable. The original form of S on earth and in meteorites were primordial sulfides, and one of them, the Canyon Diablo Troilite (CDT), had been the first international sulfur isotope standard, today replaced by several synthetic ones but still nominally in use as ViennaeCanyon Diablo Troilite (V-CDT). By chemosynthesis and with the occurrence of oxygen in earth history, bacteria produced oxidized forms of sulfur. These were afterward partially deposited as sulfates (evaporites), again (partially) reduced and deposited as sedimentary sulfides or buried in organic matter. The spectrum is completed by primordial and secondary elemental S (Fry, 2006, pp. 43, 255e261; Thode, 1991). All the above mentioned processes imply isotope fractionations, from where sulfur occurs with d34S values between þ40 and 50‰ (Thode, 1991). In addition, in sulfate, also the d18O value may vary, as the oxygen originates from O2, e.g. via combustion processes, or from water via exchange with lower oxidation intermediates (Van Stempvoort & Krouse, 1993). Therefore, in “soil” sulfate and atmospheric SO2, the most important S-sources for plants, d34S and d18O values occur within large limits (Nightingale & Mayer, 2012), basis for discriminations between geographical origins. The sole isotopically nearly constant S-source in the present context is sea water, a pool in steady state between sulfate input from rivers and consumption by reduction. From here originates “sea spray”, an aerosol with at present time d34S ¼ þ23 ± 2‰, contributing to soil sulfate up to 100 km from the ocean. In the dissimilatory sulfate reduction, anaerobic bacteria use the ion as oxidant in their metabolism. Sulfur isotope discriminations (D values) between sulfate and H2S or S of up to 70‰ are observed (Thode, 1991), probably due to the addition of isotope effects on several steps. On the other hand, the assimilatory sulfate reduction of soil sulfate by plants provides “organic S” for S-containing amino acids and descendents, vitamins, coenzymes and redox centers. As the reduction itself proceeds via channeling of intermediates (see Box 1) with quantitative turnover, isotope fractionations can only occur in context with the sulfate activation and the consumption of the active sulfate, both reactions at metabolic branching points with partial turnover of the substrate in different directions (Fig. 4). Most plants store the residual 34S-enriched sulfate in leaf vacuoles. Therefore, the d34S value of the bulk biomass is nearly identical to that of the assimilated sulfate, although an uneven distribution of the reactants may simulate isotope fractionations between different compartments. The first organic S-containing product is cysteine, precursor of any other S-containing compounds, which are all depleted in 34S relative to the precursor (Tcherkez & Tea, 2013). As the concentration of most of them is very low, they do not contribute significantly to the average d34S value of a plant or animal biomass sample. Exceptions are S-containing repellents or attractants as glucosinolates (Brassicaceae) and alliins (Allium sp.), or cysteine and methionine containing proteins. Their relative contents contribute e aside from residual 34S-enriched sulfate e essentially to small differences of d34S values (±2‰) between plant organs and between these and soil sulfate (Tcherkez & Tea, 2013). The sulfur trophic shift between animals and their diet is 0 ± 1‰ (Barnes & Jennings, 2007; Michener & Schell, 1994) but small d34S value differences exist in between animal organs, as methionine prevails in collagens and cysteine in keratins. Connective tissues in animals contain in addition the sulfate esters proteoglykans, the biosynthesis of which implies the sole remarkable sulfur isotope fractionation in animals (Tanz & Schmidt, 2010).
3.2. Assessment and scope of applications The largest field of d34S value application in food origin investigation is in the frame of multi element isotope ratio determinations. Whereas the sulfur content of most plants is far below 1%, Brassicaceae and Allium sp. approach 1% and even more in dry matter, and the differences between sulfate and “organic” d34S values in Brassicaceae attains 6‰; the greatest corresponding D34S value observed for isolated glucosinolates is even 14‰ (Tanz & Schmidt, 2010). These results indicate a remarkable S-isotope fractionation in plants and underline the necessity to use homogeneous bulk material for origin investigations on plants. On the other hand, one has to keep in mind that homogenization of plant material can cause losses of volatile S-containing hydrolysis products, e. g. of mustard oils from glucosinolates liberated by myrosinase (Fig. 4). As early season local asparagus is quite expensive in Germany, often products from other countries are offered as local ones in spring. Their origin can be identified by multi element multi component isotope ratio analysis. As the sulfur content of bulk asparagus is too low, the analysis has been performed on the purified “protein fraction” of the plants, concentrating their “organic” sulfur (Schlicht et al., 2006). Its d34S values were typical for defined cultivation areas and the highest values were found, as expected, for samples from places close to the sea or near salt deposits in bedrocks. Animal samples attain up to 2% sulfur in dry matter but one has to keep in mind that the overwhelming part oft their sulfur originates from the animals' diet, and this can, at least for domestic animals, originate from anywhere and even vary over the seasons (Bahar et al., 2008). Hence, a direct correlation to a defined geographical origin on the basis of a d34S value alone may be quite uncertain, as it depends on the relative amount of local food in the animals' diet. Therefore, most investigations use sulfur isotope characteristics as one parameter among several ones, as shown for origin identifications of lamb meat (Camin et al., 2007) and beef (Bahar et al., 2008). These reservations can be dropped in case of samples from prehistoric or wild subjects. Nevertheless, it has again to be pointed out that the d34S values can be different for individual organs and tissues of animals in dependence of their relative contents of defined proteins and proteoglykans (Tanz & Schmidt, 2010). Most interesting and informative are investigations, in which the d34S values of defined organs or compounds of wild animals are analyzed as the sole biomarkers. Weber, Hutcheon, McKeegan, and Ingram (2002) studied the sulfur isotope characteristics of otolith layers and fry of salmon and found unambiguous correlations to their diet and life history. This example shows that sulfur isotope ratio analysis is a most promising tool in animal migration research, especially because the element's discrimination in food webs and on trophic levels is negligible (Hobson, 2008). Nehlich and Richards (2009) promoted the use of sulfur isotope ratio of bone collagen as a reliable biomarker in archeological research. Not only they could show that e apart from d15N values e d34S values were indicative for the distinction between land animal and fresh water fish as protein source for prehistoric men (Nehlich, Borí c, Stefanovic, & Richards, 2010) but that the latter ones were also useful to identify the capture area of sea fish (Nehlich, Barrett, & Richards, 2013). The latter paper contains several further references dealing with sulfur isotopes as biomarkers for seafood. 4. Conclusions From the above report it turns out that oxygen isotope analyses of juice or wine water alone can often give satisfactory information about the climate of the sample's origin and on potential adulterations by illegal addition of foreign water. Squeeze water from
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vegetables and from animal products may suffer from artifacts introduced by storage and processing. Especially for animal products, one has to keep in mind that body water is a product of several parameters. Organically bound oxygen in non-exchanging functional groups of defined components (OH-groups of carbohydrates, functional O-groups of amino acids and esters) preserves valuable information about origin and history of the sample. The bases are defined chemical and isotopic correlations between the oxygen source and the functional groups in question, allowing to reconstruct the original conditions of the oxygen introduction. Nevertheless, oxygen isotope ratios of water and organic matter alone are e apart from some special questions e not generally sufficient to provide definite information on origin and authenticity of food. They must be combined with additional (isotopic) parameters (examples see Part 2.1). On the other hand, sulfur isotope ratios are most valuable indicators for the origin of plant material, provided average d34S values of bulk biomass are used and/or reference samples are available or the local geological and soil conditions (soil sulfate, sea spray, SO2 pollution, fertilization) are known. Sulfur isotope discriminations in plants and animals together with unequal distributions of the reactants between compartments can simulate fractionations. Therefore, the analysis of defined organs or compounds is necessary. This is especially successful in case of animal migration control, where sulfur isotope analysis alone can provide full information. References Anderson, V. E. (1991). Isotope effects on enzyme-catalyzed b-eliminations. In P. F. Cook (Ed.), Enzyme mechanism from isotope effects (pp. 389e417). Boca Raton: CRC Press. Bahar, B., Schmidt, O., Moloney, A. P., Scrimgeour, C. M., Begley, I. S., & Monahan, F. (2008). Seasonal variation in the C, N and S stable isotope composition of retail organic and conventional Irish beef. Food Chemistry, 106, 1299e1305. Barnes, C., & Jennings, S. (2007). Effect of temperature, ration, body size and age on sulphur isotope fractionation in fish. Rapid Communications in Mass Spectrometry, 21, 1461e1467. Blanch, A. I., & Ruperez, M. (2006). Detection of water addition in NFC juices. In SGF International e.V., W. Rieth, & K. Schulz (Eds.), Results of the Pure Juice Project (pp. 43e51). Nieder-Olm, Germany: SGF. Brand, W. A. (2010). Discrepancies between isotope ratio infrared spectroscopy and isotope mass spectrometry for the isotope analysis of plant and soil waters. Rapid Communications in Mass Spectrometry, 24, 2687e2688. Calderone, G., & Guillou, C. (2008). Analysis of isotopic ratios for the detection of illegal watering of beverages. Food Chemistry, 106, 1399e1405. Camin, F., Bontempo, L., Heinrich, K., Horacek, M., Kelly, S. D., Schlicht, C., et al. (2007). Multi-element (H, C, N, S) stable isotope characteristics of lamb meat from different European regions. Analytical and Bioanalytical Chemistry, 389, 309e320. Camin, F., Perini, M., Bontempo, L., Fabroni, S., Faedi, W., Magnani, S., et al. (2011). Potential isotopic and chemical markers for characterising organic fruits. Food Chemistry, 125, 1072e1082. Chenery, C. A., Pashley, V., Lamb, A. L., Sloane, H. J., & Evans, J. A. (2012). The oxygen isotope relationship between the phosphate and structural carbonate fractions of human bioapatite. Rapid Communications in Mass Spectrometry, 26, 309e319. Coplen, T. B. (2011). Guidelines and recommended terms for expression of stableisotope-ratio and gas-ratio measurement results. Rapid Communications in Mass Spectrometry, 25, 2538e2560. €hlke, J. K., Peiser, H. S., Rieder, S. E., Krouse, H. R., et al. Coplen, T. B., Hopple, J. A., Bo (2002). Compilation of minimum and maximum isotope ratios of selected elements in naturally occurring terrestrial materials and reagents. Water Resources Investigation Report 01-4222. Reston Virginia: US Geological Survey 2002. Craig, H. (1961). Isotopic variations in meteoric waters. Science, 133, 1702e1703. Dordevic, N., Camin, F., Marianella, R. M., Postma, G. J., Buydens, L. M. C., & Wehrens, R. (2013). Detecting the addition of sugar and water to wine. Australian Journal of Grape and Wine Research, 19, 324e330. Fronza, G., Fuganti, C., Schmidt, H.-L., & Werner, R. A. (2002). The d18O-value of the p-OH group of L-Tyrosine permits the assignment of its origin to plant or animal sources. European Food Research and Technology, 215, 55e58. Fry, B. (2006). Stable isotope ecology. New York, USA: Springer ScienceþBusiness Media, LLC. Gessler, A., Peuke, A. D., Keitel, C., & Farquhar, G. D. (2007). Oxygen isotope enrichment of organic matter in Ricinus communis during the diel course as affected by assimilate transport. New Phytologist, 174, 600e613. Grimes, V., & Pellegrini, M. (2012). A comparison of pretreatment methods for the analysis of phosphate oxygen isotope ratios in biophosphate. Rapid Communications in Mass Spectrometry, 27, 375e390.
149
Hegerding, L., Seidler, D., Danneel, H.-J., Gessler, A., & Nowak, B. (2002). Sauerstoffisotopen-Verh€ altnis-Analyse zur Herkunftsbestimmung von Rindfleisch. Fleischwirtschaft, 82, 95e100. Hobson, K. A. (2008). Applying isotopic methods to tracking animal movements. In K. A. Hobson, & L. I. Wassenaar (Eds.), Terrestrial Ecology Series: Vol. 2. Tracking animal migration with stable isotopes (pp. 45e78). London: Academic Press/Elsevier. Horita, J., Ueda, A., Mizukami, K., & Takatori, I. (1989). Automatic dD and d18O analyses of multi-water samples using H2- and CO2-water equilibration methods with a common equilibration set-up. Applied Radiation and Isotopes, 40, 801e805. Jamin, E., Gonzalez, J., Remaud, G., Naulet, N., & Martin, G. G. (1997). Detection of exogenous sugars or organic acids addition in pineapple juices and concentrates by 13C IRMS analysis. Journal of Agricultural and Food Chemistry, 45, 3961e3967. rin, R., Re tif, M., Lees, M., & Martin, G. J. (2003). Improved detection of Jamin, E., Gue added water in orange juice by simultaneous determination of the oxygen-18 oxygen-16 isotope ratios of water and ethanol derived from sugars. Journal of Agricultural and Food Chemistry, 51, 5202e5206. Kirsanow, K., & Tuross, N. (2011). Oxygen and hydrogen isotopes in rodent tissues: Impact of diet, water and ontogeny. Palaeography, Palaeoclimatology, Palaeoecology, 310, 9e16. Koch, P. L., Fogel, M. L., & Tuross, N. (1994). Tracing the diets of fossil animals using stable isotopes. In K. Lajhta, & R. H. Michener (Eds.), Stable isotopes in ecology and environmental sciences (pp. 63e92). Oxford, UK: Blackwell Scientific Publications. Kohn, M. J. (1996). Predicting animal d18O: accounting for diet and physiological adaptation. Geochimica et Cosmochimica Acta, 60, 4811e4829. Kornexl, B. E., Werner, T., Rossmann, A., & Schmidt, H.-L. (1997). Measurement of stable isotope abundances in milk and milk ingredients e a possible tool for origin asssignment and quality control. Zeitschrift für Lebensmitteluntersuchung und -forschung, 205, 19e24. Lis, G., Wassenaar, L. I., & Hendry, M. J. (2008). High-precision laser spectroscopy D/ H and 18O/16O measurements of microliter natural water samples. Analytical Chemistry, 80(1), 287e293. Longinelli, A., & Selmo, E. (2011). d18O values of Sus scrofa blood water and bone phosphate; a marked discrepancy between domestic and wild specimens. Rapid Communications in Mass Spectrometry, 25, 3732e3734. Luz, B., Kolodny, Y., & Horowitz, M. (1984). Fractionation of oxygen isotopes between mammalian bone-phosphate and environmental water. Geochimica et Cosmochimica Acta, 48, 1689e1694. Michener, R. H., & Schell, D. M. (1994). Stable isotope ratios as tracers in marine aquatic food webs. In K. Lajhta, & R. H. Michener (Eds.), Stable isotopes in ecology and environmental sciences (pp. 138e157). Oxford, UK: Blackwell Scientific Publications. Mook, W. G. (2000). The global cycle of water. In Environmental isotopes in the hydrological cycle (Vol. I); (pp. 1e18). Paris, Vienna: International atomic Energy Agency and United Nations Educational, Scientific and Cultural Organization. Nehlich, O., & Richards, M. P. (2009). Establishing collagen quality criteria for sulphur isotope analysis of archaeological bone collagen. Archaeological and Anthropological Sciences, 1, 59e75. Nehlich, O., Barrett, J. H., & Richards, M. P. (2013). Spatial variability in sulphur isotope values of archaeological and modern cod (Gadus morhua). Rapid Communications in Mass Spectrometry, 27, 2255e2262. Nehlich, O., Borí c, D., Stefanovi c, S., & Richards, M. P. (2010). Sulphur isotope evidence for freshwater fish consumption: a case study from the Danube Gorges, SE Europe. Journal of Archaeological Science, 37, 1131e1139. Nightingale, M., & Mayer, B. (2012). Identifying sources and processes controlling the sulphur cycle in the Canyon Creek watershed, Alberta, Canada. Isotopes in Environmental and Health Studies, 48, 89e104. Perini, M., & Camin, F. (2013). d18O of ethanol in wines and spirits for authentication purposes. Journal of Food Science, 78, C839eC844. Pfahler, V., Dürr-Auster, T., Tamburini, F., Bernasconi, S. M., & Frossard, E. (2013). 18O enrichment in phosphorous pools extracted from soybean leaves. New Phytologist, 197, 186e193. Pietsch, S. J., Hobson, K. A., Wassenaar, L. I., & Tütken, T. (2011). Tracking cats: problems with placing feline carnivores on d18O, d2H isoscapes. PLoS ONE, 6(9), e24601. http://dx.doi.org/10.1371/journal.pone.0024601. Podlesak, D. W., Bowen, G. J., O'Grady, S., Cerling, T. E., & Ehleringer, J. R. (2012). d2H and d18O of human body water: a GIS model to distinguish residents from nonresidents in the contiguous USA. Isotopes in Environmental and Health Studies, 48, 1e21. Rapisarda, P., Camin, F., Fabroni, S., Perini, M., Torrisi, B., & Intrigliolo, F. (2010). Influence of different organic fertilizers on quality parameters and the d15N, d13C, d2H, d34S, and d18O values of orange fruit (Citrus sinensis L. Osbeck). Journal of Agricultural and Food Chemistry, 58, 3502e3506. Renou, J.-P., Bielicki, G., Deponge, C., Gachon, P., Micol, D., & Ritz, P. (2004). Characterization of animal products according to geographic origin and feeding diet using nuclear magnetic resonance and isotope ratio mass spectrometry, Part II: beef meat. Food Chemistry, 86, 251e256. Rock, L., Rowe, S., Czerwiec, A., & Richmond, H. (2013). Isotopic analysis of eggs: evaluating sample collection and preparation. Food Chemistry, 136, 1551e1556. Schlicht, C., Roßmann, A., & Brunner, E. (2006). Anwendung der MultielementMultikomponenten Isotopenverh€ altnismassenspektrometrie (IRMS) zur
150
N. Krivachy (Tanz) et al. / Food Control 48 (2015) 143e150
Prüfung der geographischen Herkunft von Spargel. Journal für Verbraucherschutz und Lebensmittelsicherheit, 1, 97e105. Schmidt, H.-L., Werner, R. A., & Roßmann, A. (2001). 18O pattern and biosynthesis of natural plant products. Phytochemistry, 58, 9e32. €ckigt, D., & Christoph, N. (2005). StabilisotopenaSchmidt, H.-L., Roßmann, A., Sto €t von Lebensmitteln. Chemie in unserer Zeit, nalytik. Herkunft und Authentizita 39, 90e99. Schmidt, H.-L., Rossmann, A., Voerkelius, S., Schnitzler, W. H., Georgi, M., Graßmann, J., et al. (2005). Isotope characterization of vegetables and wheat from conventional and organic production. Isotopes in Environmental and Health Studies, 41, 223e228. Schmidt, H.-L., Werner, R. A., Rossmann, A., Mosandl, A., & Schreier, P. (2007). Stable isotope ratio analysis in quality control of flavourings. In H. Ziegler (Ed.), Flavourings, production, composition, applications, regulations (2nd ed.) (pp. 602e633). Weinheim, Deutschland: Wiley-VCH. Schmidt, H.-L., Rossmann, A., Rummel, S., & Tanz, N. (2009). Stable isotope analysis (Eds.), for meat authenticity and origin check. In L. M. L. Nollet, & F. Toldra Handbook of muscle food analysis (pp. 767e787). Boca Raton: CRC-Press, Taylor and Francis Group. Serra, F., Reniero, F., Guillou, C. G., Moreno, J. M., Marinas, J. M., & Vanhaecke, F. (2005). 13C and 18O isotopic analysis to determine the origin of L-tartaric acid. Rapid Communications in Mass Spectrometry, 19, 1227e1230. Sieper, H.-P., Kupka, H.-J., Lange, L., Rossmann, A., Tanz, N., & Schmidt, H.-L. (2010). Essential methodological improvements in the oxygen isotope ratio analysis on N-containing organic compounds. Rapid Communications in Mass Spectrometry, 24, 2849e2858. Sternberg, L., & Ellsworth, P. F. V. (2011). Divergent biochemical fractionation, not convergent temperature, explains cellulose oxygen isotope enrichment across latitudes. PloS One, 6(11), e28040. http://dx.doi.org/10.1371/ journal.pone.0028040. Sternberg, L., Pinzon, M. C., Anderson, W. T., & Jahren, A. H. (2006). Variation in oxygen isotope fractionation during cellulose synthesis: intramolecular and biosynthetic effects. Plant, Cell & Environment, 29, 1881e1889. Sturm, M., & Lojen, S. (2011). Nitrogen isotopic signature of vegetables from the Slovenian market and its suitability as an indicator of organic production. Isotopes in Environmental and Health Studies, 47, 214e220. €ufele, H., & Schnyder, H. (2014). Oxygen and hydrogen Sun, L., Auerswald, K., Scha isotope composition of silage water. Journal of Agricultural and Food Chemistry, 62(20), 4493e4501. Sun, L. Z., Auerswald, K., Wenzel, R., & Schnyder. (2013). Drinking water intake of grazing steers: the role of environmental factors controlling canopy wetness. Journal of Animal Science, 90, 1e10. Tanz, N., & Schmidt, H.-L. (2010). d34S-Value measurements in food origin assignments and sulfur isotope fractionations in plants and animals. Journal of Agricultural and Food Chemistry, 58, 3139e3146.
Tanz, N., Werner, R. A., Eisenreich, W., & Schmidt, H.-L. (2011). Assessment of enzymatic methods in the d18O-value determination of the L-tyrosine p-hydroxy group for proof of illegal meat and bone feeding to cattle. Journal of Agricultural and Food Chemistry, 59, 9475e9483. Tcherkez, G., & Tea, I. (2013). 32S/34S isotope fractionation in plant sulphur metabolism. New Phytologist, 200, 44e53. Thiem, I., Lüpke, M., & Seifert, H. (2004). Factors influencing the 18O/16O e ratio in meat juices. Isotopes in Environmental and Health Studies, 40, 191e197. Thode, H. G. (1991). Sulphur isotopes in nature and the environment: an overview. In H. R. Krouse, & V. A. Grinenko (Eds.), Stable isotopes: Natural and anthropogenic sulphur in the environment (SCOPE 43) (pp. 1e26). Chichester, West Sussex, GB: John Wiley & Sons Ltd. Tuross, N., Warinner, C., Kirsanow, K., & Kester, C. (2008). Organic oxygen and hydrogen isotopes in a porcine controlled dietary study. Rapid Communications in Mass Spectrometry, 22, 1741e1745. Van Stempvoort, D. R., & Krouse, H. R. (1993). Controls of d18O in sulfate. Review of experimental data and application to specific environments. In C. N. Alpers, & W. W. Blowes (Eds.), Environmental Geochemistry of Sulfide Oxidation, ACS Symposium Series 550 (pp. 446e480). Washington, USA: American Chemical Society. Wachter, H., Christoph, N., & Seifert, S. (2009). Verifying authenticity of wine by Mahalanobis distance and hypothesis testing of stable isotope pattern e a case study using the EU wine Databank. Mitteilungen Klosterneuburg, 59, 237e249. Weber, P. K., Hutcheon, I. D., McKeegan, K. D., & Ingram, B. L. (2002). Otolith sulfur isotope method to reconstruct salmon (Oncorhynchus tshawytscha) life history. Canadian Journal of Fisheries and Aquatic Sciences, 59, 587e591. Werner, R. A. (2003). The online 18O/16O analysis: development and application. Isotopes in Environmental and Health Studies, 39, 85e104. Werner, R. A., Rossmann, A., Schwarz, C., Bacher, A., Schmidt, H.-L., & Eisenreich, W. (2004). Biosynthesis of gallic acid in Rhus typhina: discrimination between alternative pathways from natural oxygen isotope abundances. Phytochemistry, 65, 2809e2813. West, A. G., Goldsmith, G. R., Brooks, P. D., & Dawson, T. E. (2010). Discrepancies between isotope ratio infrared spectroscopy and isotope ratio mass spectrometry for the stable isotope analysis of plant and soil waters. Rapid Communications in Mass Spectrometry, 24, 1948e1954. White, J. W. C. (1989). Stable hydrogen isotopes in plants: a review of current theory and some potential applications. In P. W. Rundel, J. R. Ehleringer, & K. A. Nagy (Eds.), Applications of stable isotopes in ecological research (pp. 142e160). New York, USA: Springer-Verlag. Yakir, D. (1998). Oxygen-18 of leaf water: a crossroad for plant-associated isotopic signals. In H. Griffiths (Ed.), Stable Isotopes, integration of biological, ecological and geochemical processes (pp. 147e168). Oxford, UK: BIOS Scientific Publishers Ltd.
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Potentials and caveats with oxygen and sulfur stable isotope analyses in authenticity and origin checks of food and food commodities Nicole Krivachy (Tanz), Andreas Rossmann, Hanns-Ludwig Schmidt* Isolab GmbH Laboratory for Stable Isotopes, Woelkestrasse 9, D-85301 Schweitenkirchen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 November 2013 Received in revised form 3 June 2014 Accepted 4 June 2014 Available online 12 June 2014
Analyses of stable isotope ratios are officially accepted methods in food authenticity and origin determination. They are routinely practiced by empirical comparison of the unknown samples' d values with those of authentic material. However, as the isotope characteristics of food are influenced by many parameters, it is desirable to also study and use causal correlations of isotope fractionations for the interpretation of experimental data. Corresponding potentials and limits are outlined for oxygen and sulfur stable isotopes. In the natural water cycle, plant leaf and animal cell water are the most important sources for food integrated water and organically bound oxygen. The way from sea water to fruit juice water and to organic matter and the integrated isotope fractionations are shown and the possibilities and limits for the assignment of juices and wine to their geographical origin, history and authenticity are deduced. The oxygen flux and isotopic balance in animals and the sources and drains of animal body water are outlined and the problems and limits for its suitability as a bioindicator for origin assignments of animal food products are discussed. The potential of the d18O value of organically bound oxygen is demonstrated. The sulfate reduction in plants is accompanied by isotope fractionation but as normally no sulfur is excreted, the d34S value of bulk plant matter is identical to that of the primary local source. Small differences in the d34S values of plant compartments are often due to differences in their abundance of main S-containing ingredients, as residual sulfate and cysteine or methionine containing proteins. This is similar with animal tissues and products. Therefore, the sulfur isotope analysis of the bulk matter or of defined fractions of plant and animal samples is an ideal and reliable tool for food origin and authenticity proof and for archaeological and animal migration research. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Oxygen Sulfur Stable isotopes Authenticity Origin check
1. Introduction In recent years the European Community/Union has defined indications for the quality characterization of food and food commodities like “Protected Denomination of Origin (PDO)”, “Protected Geographical Indication (PGI)”, and “Certification of Specific Character (CSC)” (cited in Schmidt, Rossmann, Rummel, & Tanz, 2009; further information see Calderone & Guillou, 2008). The criteria and the methodology to be used for conferring or applying these indications on a food sample are indicated in corresponding regulations. Among the officially recommended methods, stable isotope ratio determinations of the bioelements are occurring in the first
* Corresponding author. Prielhofweg 2, D-84036 Landshut, Germany. Tel.: þ49 871 44497. E-mail addresses: [email protected] (N. Krivachy (Tanz)), [email protected] (H.-L. Schmidt). http://dx.doi.org/10.1016/j.foodcont.2014.06.002 0956-7135/© 2014 Elsevier Ltd. All rights reserved.
range. Factors determining these isotope ratios of a natural sample are those of the primary material in question, thermodynamic and kinetic isotope effects, geographical and climatic conditions of the sample's origin, physiological and anatomic properties of plants and animals' positions in food chains. Vice versa, corresponding parameters can be deduced from the isotopic properties of a sample. Furthermore, most important is the individual dependence of the bioelements on the different external influences, their “indi€ ckigt, & Christoph, 2005). cator function” (Schmidt, Roßmann, Sto And as the informations implied in the isotope ratios of the individual elements are complementary, many recent investigations on food origin and authenticity use multielement isotope ratio determinations (Bahar et al., 2008; Camin et al., 2007; Schlicht, Roßmann, & Brunner, 2006). Carbon isotope ratios are predominantly indicative for food (ingredients) assignments to their origin from the photosynthetic plant types C3, C4 and CAM plants. One application is the proof of an adulteration of beverages of C3 plants with C4 products and vice
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versa. (Jamin, Gonzalez, Remaud, Naulet, & Martin, 1997). In food chains, carbon d values increase by ~2‰ for each trophic level (Michener & Schell, 1994; for fundamentals of isotope abundance and fractionation indications see Box 1). Nitrogen isotope ratios are correlated to agricultural fertilization practices and hence often used to discriminate between compounds from conventional and “organic” production, respectively (Camin et al., 2011; Rapisarda et al., 2010; Schmidt, Roßmann, et al., 2005; Schmidt, Rossmann, et al., 2005; Sturm & Lojen, 2011). The trophic shift of nitrogen isotope indications can attain 2e4‰ per level (Michener & Schell, 1994). Hydrogen and oxygen are preferably originating from plant leaf or animal drinking water, respectively, the isotope characteristics of which depend on local precipitation and climate, but are modulated by biochemical, physiological and anatomical influences (White, 1989). Although hydrogen and oxygen isotope ratios of meteoric water are correlated among each other by the “meteoric water line” (d2H [‰]V-SMOW ¼ 8 d18O[‰]V-SMOW þ 10, Craig, 1961), the indicator function of hydrogen is more important for individual metabolites, as many isotope fractionations of this element in the secondary metabolism lead to characteristic hydrogen isotope patterns of defined plant compounds. This is the basis for origin and authenticity investigations on spices, aromas and fragrances by means of 2H NMR measurements (deuterium
positional or pattern analysis, Schmidt, Werner, Rossmann, Mosandl, & Schreier, 2007). Trophic levels in context with food chains are of minor importance with hydrogen and oxygen, as the primary source water is globally available in form of an infinite pool with large turnover rate. In food analysis, oxygen isotope ratios are often determined on the bulk sample. These d18O values of biomass provide average data of cell water and organically bound oxygen. However, in most cases of food quality investigations, the d18O value of the water is analyzed, even when it is not particularly indicated. The reason is that the corresponding analytic procedure is extremely easy (Horita, Ueda, Mizukami, & Takatori, 1989) and can often, especially with wine and fruit juices, be performed on the untreated sample itself. As the method is based on the isotopic equilibration between the sample water and CO2, the sample should not be in a state of fermentation, producing additional CO2. Further artifacts can occur in context with separation of the water from the sample (e.g. meat). This equilibration method has recently been supplemented by the water isotope ratio analysis by IR methodology (Brand, 2010; Lis, Wassenaar, & Hendry, 2008; West, Goldsmith, Brooks, & Dawson, 2010). In any case, as will be shown in this contribution, the interpretation of the results has to take into account the history of the original primary water. On the other hand, most organically
Box 1 Isotope effects and isotope fractionation in closed and open systems.
The indication of isotope concentrations of bioelements is performed in the d value scale as relative differences to international standards of the International Atomic Energy Agency (IAEA) in Vienna (V). The standard for oxygen is Vienna Standard Mean Ocean Water (V-SMOW) with the isotope ratio R ¼ [18O]/[16O] ¼ 2005.2 106, that for sulfur is Vienna Canyon Diablo Troilite (VCDT) with the isotope ratio R ¼ [34S]/[32S] ¼ 44150.9 106. According to IUPAC rules, delta values are defined as (Coplen, 2011)
. RVSMOW d18 O ¼ RSample RVSMOW
. RVCDT and d34 S ¼ RSample RVCDT
However, for practical reasons and because of the general application in the cited references, we are using in the present paper the old formula with the factor 1000, leading to permill [‰]:
. RVSMOW 1000 d18 O½‰ ¼ RSample RVSMOW
. RVCDT 1000 and d34 S½‰ ¼ RSample RVCDT
For the determination of the d values, organic matter is converted by techniques of the elemental analysis into CO (d18O) and SO2 (d34S), respectively, water is equilibrated with CO2 (d18O). The isotope ratio of the gases is analyzed, relative to that of a (laboratory) standard, in an isotope ratio mass spectrometer (Sieper et al., 2010; Werner, 2003). Thermodynamic isotope effects or fractionation factors a are the ratio of physical properties or equilibrium constants K and K* of 18 isotopologue molecules, e.g. of the vapor pressures of H16 2 O (p) and H2 O (p*): a ¼ p/p*. Kinetic isotope effects akin are the ratio of velocity constants k of such molecules in chemical reactions; the element in question is indicated, e.g. for oxygen as akin ¼ k16/k18. Normally, the “lighter” isotopologues react faster and akin is > 1.0; for the bioelements except hydrogen, it is between 1.00 and 1.07. The basis for kinetic isotope effects on enzyme-catalyzed reactions is the MichaeliseMenten theory. All these considerations concern systems in equilibrium or closed systems, respectively. Most natural systems are open with infinite pools of isotopically constant substrates (e.g. ground water or soil sulfate). This has for consequence a constant isotope fractionation between substrate and product. Thus, the fractionation constant is defined as a ¼ RSubstrate/RProduct and the isotope fractionation or discrimination as D ¼ a 1 ¼ RSubstrate/RProduct 1. The combination of this equation with the above correlation between R and d permits to eliminate RStandard and yields, as dProduct << 1:
D ¼ ðdSubstrate dProduct Þ=ð1 þ dProduct Þ e dSubstrate dProduct This provides the possibility to use directly mass spectrometric results of practical measurements for interpretations. All systems discussed in the present paper are open with the exception that most plants do not excrete sulfur in any form and hence the total assimilated S remains in the system. Yet the following prerequisites concerning S-isotope fractionations have to be taken into account (see Fig. 4). A kinetic isotope effect on a defined reaction becomes only efficient in case of a partial substrate conversion, normally realized by formation of at least two products (metabolic branching, MB), one of them depleted, the other enriched in the heavy isotope (isotopic balance). The reverse is realized by metabolite channeling (MC): In a sequence of reactions the substrate and the subsequent products are quantitatively converted (e.g. by a multi-enzyme-complex). Kinetic isotope effects on any step cannot become efficient.
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bound oxygen is correlated to the original primary plant/body water by reactions with defined isotope fractionations, thus presenting a stable and reliable archive for climate identifications and reconstructions. On the other hand, the situation with sulfur is quite different. Sulfur occurs in Nature in multiple oxidation forms and isotopic characteristics. Sulfur assimilation in most plants is an unidirectional process, in which the total originally ingested element remains completely in the plant, independent of (partial) biochemical conversions with isotope fractionation and binding forms. The bulk sulfur isotope ratio thus preserves and reflects the isotopic property of the primary material in question and hence provides an ideal biomarker for the geographical origin of the sample. Nevertheless, isotope fractionations on defined reactions in plants and food chains lead to organic compounds with S from the same source but different d34S values. The consequences for food authenticity and origin assignment will be the topic of the following review. Its aim is to mediate fundamental knowledge for the interpretation of experimental data on oxygen and sulfur isotope ratios, independent on and above the availability and use of data banks from authentic reference material. 2. Oxygen isotope ratios as indicators for beverage and food origin 2.1. Sources and isotope characteristics of food intrinsic water Any water on land originates from the ocean, an infinite reservoir with a globally nearly constant oxygen-18 content (d18O value ¼ 0‰ ¼ Vienna Standard Mean Ocean Water, V-SMOW). An isotope effect on the vapor pressure leads to a preferred evaporation of “light” water isotopologues above the sea. Subsequent partial condensation has for consequence a further 18O-depletion in the vapor phase, and the last rain in the largest distance from the ocean is the “lightest” one (continental effect). This effect is overlapped by influences of local altitude (altitude effect), climatic and seasonal conditions (temperature effect) and precipitation amount (amount effect) (water cycle reviewed by Mook, 2000). From here originate local individual oxygen isotope characteristics of meteoric and ground water. Trees take up this water and transport it via their xylem system to the leaves, where it is, again depending on local climate conditions and with diurnal oscillations (Gessler, Peuke, Keitel, & Farquhar, 2007) enriched in 18O by transpiration. A medium sized apple tree in central Europe needs 30e40 L of water in a normal summer day. The 18O-enriched leaf water is, after partial clet-effect), isotope exchange with countercurrent xylem water (Pe the source for the water of fruit saps (Fig. 1). The overlap of all these geographical, climatic, local, annual and plant physiological influences is normally integrated in data from the analysis of authentic reference samples and the definition of authenticity limits. This is, for example established for juices in Europe by the “Association of the Industry of Juices and Nectars (AIJN) from fruits and vegetables of the European Union” in Brussels. Watering of wine or fruit juices or illegal re-dilution of fruit juice concentrates can be detected due to their lower d18O value relative to authentic
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products (Calderone & Guillou, 2008; Dordevic et al., 2013; Jamin, rin, Re tif, Lees, & Marin, 2003; Wachter, Christoph, & Seifert, Gue 2009). However, one has to take into account that problems can occur from sample processing and/or interference by washing water (Blanch & Ruperez, 2006). Water is also often a main part of animal originating food like milk, eggs and meat, and the d18O values of this water are used for origin assignments of the products. The first question to be discussed in this context concerns the sources and the composition of animal cell or body water and its d18O value. The primary and most important source is drinking water but also the diet of animals can contribute in a variable and not unimportant part. The contributions of the different source and drain factors and their fluxes have originally been compiled by Luz, Kolodny, and Horowitz (1984) and reported by Koch, Fogel, and Tuross (1994). Sources are drinking water, intrinsic water in food and oxidation water, originating from organically bound oxygen and atmospheric dioxygen converted in the respiratory chain. Sinks are urine and water in feces, sweat and breath water, milk and mainly breath CO2, which is highly enriched in 18O via equilibration with water. The correlation of the d18O value of animal body water and drinking water depends on species, the rates of drinking and respiration (Luz, Kolodny, & Horwitz, 1984). For an animal in steady state the above authors find linear relationships between the d18O values of ingested drinking and body water. The slopes of these relationships depend on the amount of drinking, species and its energy expenditure. Generally, whereas the d18O and d2H values of many animal tissues are correlated to each other and determined by the corresponding body and ultimately drinking water, this is especially not the case with carnivores (Pietsch, Hobson, Wassenaar, & Tütken, 2011). For vegetarians, a large contribution from the 18O enriched oxygen atoms in carbohydrates is to be expected. Correspondingly, a dependence of the diet can also be assumed as the reason for the difference found in the blood water of wild and domestic pigs from the same area (Longinelli & Selmo, 2011). Extensive experimental data and model calculations on this topic, in addition also concerning geographical parameters, see Podlesak, Bowen, O'Grady, Cerling, & Ehleringer, 2012. An unpublished own experiment confirmed these correlations for a living human subject. Correlations of body water to the oxygen isotope characteristics of animal tissues and parameters influencing these correlations are compiled by Kirsanow and Tuross (2011). However, in context with the practice of animal originating food investigations, most of these parameters are unknown. Fig. 2 presents a qualitative approximation for the assessment and understanding of the d18O value of water in or from animal products. For practical considerations in the present context, one can assume that the average d18O value of the body water of most domestic animals is about 3 ± 1‰ more positive than that of the drinking water. However, milk water can show large seasonal variations, from þ2‰ in winter to þ6‰ in summer above drinking water (Kornexl, Werner, Rossmann, & Schmidt, 1997), probably due to a larger contribution of grass intrinsic (18O-enriched) water in summer. Even the isotopic composition of silage water can vary in the course of silage preparation and storage (Sun, Auerswald,
Fig. 1. Stages and d18O values from ocean water to fruit juice water and organic matter in plants. d values for central Europe. Adapted after Yakir (1998), data for upper Bavaria, from Werner et al., (2004) and Schmidt et al. (2001). Indications of leaf water diurnal and juice water annual variations.
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Fig. 2. Simplified and exemplary oxygen and water mass and isotopic balances for an omnivore and a lactating cow on summer grass diet, respectively. Oxygen flux fractions in %, for the cow in [ ], d18O values in ( ). Data adapted from Luz, Kolodny, and Horwitz (1984); Koch et al. (1994); Kohn (1996) and Kornexl et al. (1997). The d18O value of milk is estimated assuming a contribution of 95% by milk water with d18O ¼ 8‰ and 5% from organic matter with D ¼ þ 20‰ from here. For further details about the body water of grazing ruminants see Sun, Auerswald, Wenzel, and Schnyder (2013).
Sch€ aufele, & Schnyder, 2014). On this background it is understandable that attempts to assign meat to its geographical origin exclusively on the basis of the d18O value of squeezed tissue water (Hegerding, Seidler, Danneel, Gessler, & Nowak, 2002; Thiem, Lüpke, & Seifert, 2004) were not very promising. Meat water from beef showed shifts relative to local drinking water of >4‰. The corresponding shift for pork meat water was only þ1 to þ2‰, and it was slightly depleted in 18O relative to beef meat water from same area. This difference demonstrates without any doubt an effect of the diet. In both cases, the meat water showed, as expected, a distinct correlation to the local ground water but the variations between individual samples from the same area or even the same litter did not allow unambiguous origin assignments, neither within Germany nor between Germany and Great Britain. Furthermore, significant influences of storage and handling of the samples were observed. Investigations by Renou et al. (2004) confirm the prevalence of diet and age of the animals over the influence of drinking water on beef meat water d18O values. The influence of climate and physiological parameters on the d18O values of wild animal body water is discussed by Kohn (1996). The potential of isotope characteristics, among them the d18O value of egg fractions for traceability and authenticity research of this animal product as well as the influence of storage and sample preparation is compiled in a review by Rock, Rowe, Czerwiec, & Richmond (2013). 2.2. Organically bound oxygen from water Carbonyl (aldehyde and keto) groups of organic compounds can exchange oxygen atoms with surrounding water. This reaction implies a thermodynamic isotope effect with an equilibrium fractionation D18O of the organic functional group of ~þ27‰. As this shift has also been found for carbohydrates, especially cellulose, it has been and is still used for (paleo)climatological and physiological investigations. However, it turned out recently that carbohydrates have a distinct and even variable 18O pattern (Schmidt, Werner, & Roßmann, 2001; Sternberg & Ellsworth, 2011; Sternberg, Pinzon, Anderson, & Jahren, 2006) and that the indicated value is only an average over the six positions of oxygen in glucose. Nevertheless, it can still be used in the above mentioned investigations. However, special bulk and positional d18O values have been found for animal originating carbohydrates, synthesized via gluconeogenesis (Schmidt et al., 2001).
The 18O enrichment and pattern of carbohydrates synthesized via the gluconeogenesis pathway (Fig. 3) starts from oxaloacetate to phosphoenolpyruvate. The subsequent water addition to 2phosphoglycerate, catalyzed by enolase, implies a kinetic oxygen isotope effect of 1.03 (Anderson, 1991). After isomerization of the product to 3-phosphoglycerate, the oxygen atom in position C-3 is protected from oxygen exchange. From its terminal positions C-1 and C-6 of hexoses, only the latter retains permanently its protection. If we assume that the average enrichment of C-1 to C-5 of the disaccharide relative to water is identically correlated to the surrounding water as in “photosynthetic” carbohydrates, we can estimate the shift in position C-6 of the lactose monosaccharides to be 11.4‰. This is, at least qualitatively, as expected from the isotope effect on the enolase reaction. In fact, the bulk discrimination D18O of lactose from cow milk is only 20.6‰ relative to the milk water (Schmidt et al., 2001). The amount of carbohydrates formed by gluconeogenesis from fatty acids in germinating oil seeds is insignificant for most practical questions. More important is the resynthesis of glucose from lactate by glycolysis in animals. However, for ruminants it is the sole pathway to form carbohydrates, starting from propionate, one of the main products of the rumen fermentation. This example of the addition of water to a >C]C< double bond, accompanied by a kinetic isotope effect, is an exception, as most other products of such reactions are intermediates in reaction chains and later on lose again this oxygen atom. Oxygen atoms in hydroxyl groups do not exchange with surrounding water under physiological conditions. However, like carbonyl, carboxyl groups exchange their oxygen atoms with surrounding water via hydrates; the equilibrium isotope effect is ~1.018, leading to a discrimination D18O of the functional groups by þ18 ± 1‰ relative to water (Schmidt et al., 2001). This provides, for example, the possibility to calculate the d18O value of a grape must water from wine tartaric acid (HOOCeCHOHeCHOHeCOOH), which is not affected by the fermentation. The acid contains four carboxyl and two “carbohydrate” O-atoms, yielding an average shift of 21‰ for the discrimination to must water, a result in good agreement with water data of Italian wines (Serra et al., 2005). Esters and amides can have a somewhat higher D18O value for the carbonyl atom (e.g. ~23‰), probably due to an intramolecular isotope effect on the activation of the acid. The (not catalyzed) isotopic equilibration of carbonyl and carboxyl compounds with water is a relatively slow process
Fig. 3. Processes, precursors and reactions of glucose formation by gluconeogenesis and origin of oxygen atom in position C-6 of glucose. Kinetic oxygen isotope effect on enolase reaction k16/k18 ¼ 1.03 (Anderson, 1991). The oxygen atom introduced by the reaction occurs firstly in positions C-1 and C-6 of glucose-6-phosphate (G-6-P) but is finally preserved only in the latter one. OAA ¼ oxaloacetic acid, PEP ¼ phosphoenolpyruvate, PGA ¼ phosphoglyceric acid.
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Fig. 4. Biochemical reactions of the assimilatory sulfate reduction by plants; formation and hydrolysis of glucosinolates. Steps with potential isotope fractionation are indicated by IF. MB ¼ metabolic branching, MC ¼ metabolic channeling (see Box 1). Modified after Tanz and Schmidt (2010), details see Tcherkez and Tea (2013). SS ¼ sulfate sulfur, SO ¼ “organic (reduced) sulfur”, PAPS ¼ 30 -phosphoadenosine-50 -phosphosulfate (“active sulfate”), PAP ¼ 30 -phosphoadenosine-50 -phosphate. XeSH is cysteine. Ss is always relatively enriched, So depleted in 34S.
(t1/2 ¼ min to h) and therefore, the equilibrium with short-lived intermediates is not always attained. For the proof of illegal water addition to fruit juices and must, the d18O value of the water present during fermentation is often determined via that of the ethanol formed. However, the equilibration of the intermediate acetaldehyde with water is only attained under defined condirin, Re tif, Lees, & Marin, tions of the fermentation (Jamin, Gue 2003; Perini & Camin, 2013). Correspondingly, phosphate in living systems equilibrates with water via the multiple formation and hydrolysis of nucleotide oligophosphates (e.g. ATP). The equilibration demands conditions of active and fast metabolism. As these are not always realized in all parts of a plant, the degree of equilibration can differ within different plant organs (Pfahler, Dürr-Auster, Tamburini, Bernsaconi, & Frossard, 2013). The d18O value of phosphate in bioapatite from bone and teeth is often used to identify the place of origin of recent or prehistoric persons on the basis of their drinking water (Chenery, Pashley, Lamb, Sloane, & Evans, 2012). However, uncertainties may arise from the fact that the samples always reflect the isotope content of the body water, which depends only partially from that of the drinking water (see above). The most common application of the oxygen isotope analysis of phosphate in bone and teeth bioapatite is in paleoclimatology; for a review of the methodology and possible artifacts see Grimes and Pellegrini (2012).
atmospheric O2 (d18O ¼ þ23.8 ± 0.3‰, Coplen et al., 2002, pp. 36e44), accompanied by a kinetic isotope effect kkin ¼ ~1.018 (Schmidt et al., 2001); from here results a fixed d18O value 7 ± 1‰: ≡CeH þ O2 þ NADPH þ Hþ / ≡CeOH þ H2O þ NADPþ The large difference of this d18O value to that of hydroxyl groups originating from the reduction of carbonyl groups allows unambiguous discriminations between alternative biosynthetic pathways of natural compounds (Werner et al., 2004). It has also been the basis for the development of a method for the identification of the origin of L-tyrosine from a plant or animal source, respectively (Fronza, Fuganti, Schmidt, & Werner, 2002), for assigning animals to a vegetarian, omnivore or carnivore diet, respectively, and to prove the illegal feed of ruminants with meat and bone meal (MBM), basis for their infection with bovine spongiform encephalopathy (BSE, Tanz, Werner, Eisenreich, & Schmidt, 2011). And we postulate that this d18O value also explains why collagens are relatively depleted by 2e3‰ in 18O to other proteins from the same source (Tuross, Warinner, Kirsanow, & Kester, 2008): they are the sole proteins containing ~15% hydroxyproline, formed by hydroxylation of (bound) proline. 3. Sulfur isotope ratios as biomarkers in food origin investigations
2.3. Dioxygen as source of organically bound oxygen
3.1. Sulfur sources, isotope fractionations and d34S values
Hydroxyl groups of phenols, hydroxy fatty acids, steroids and hydroxyproline are introduced by monooxygenase reactions from
Sulfur occurs in Nature as a mixture of four stable isotopes, among which only the main one (32S, 95.020 atom-%) and the
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second frequent one (34S, 4.218 atom-%) are of importance in the present context. The oxidation state of the element varies between þ6 and 2, among which sulfate-S (þ6), elemental S (0) and sulfide or “organic S” (2) are stable. The original form of S on earth and in meteorites were primordial sulfides, and one of them, the Canyon Diablo Troilite (CDT), had been the first international sulfur isotope standard, today replaced by several synthetic ones but still nominally in use as ViennaeCanyon Diablo Troilite (V-CDT). By chemosynthesis and with the occurrence of oxygen in earth history, bacteria produced oxidized forms of sulfur. These were afterward partially deposited as sulfates (evaporites), again (partially) reduced and deposited as sedimentary sulfides or buried in organic matter. The spectrum is completed by primordial and secondary elemental S (Fry, 2006, pp. 43, 255e261; Thode, 1991). All the above mentioned processes imply isotope fractionations, from where sulfur occurs with d34S values between þ40 and 50‰ (Thode, 1991). In addition, in sulfate, also the d18O value may vary, as the oxygen originates from O2, e.g. via combustion processes, or from water via exchange with lower oxidation intermediates (Van Stempvoort & Krouse, 1993). Therefore, in “soil” sulfate and atmospheric SO2, the most important S-sources for plants, d34S and d18O values occur within large limits (Nightingale & Mayer, 2012), basis for discriminations between geographical origins. The sole isotopically nearly constant S-source in the present context is sea water, a pool in steady state between sulfate input from rivers and consumption by reduction. From here originates “sea spray”, an aerosol with at present time d34S ¼ þ23 ± 2‰, contributing to soil sulfate up to 100 km from the ocean. In the dissimilatory sulfate reduction, anaerobic bacteria use the ion as oxidant in their metabolism. Sulfur isotope discriminations (D values) between sulfate and H2S or S of up to 70‰ are observed (Thode, 1991), probably due to the addition of isotope effects on several steps. On the other hand, the assimilatory sulfate reduction of soil sulfate by plants provides “organic S” for S-containing amino acids and descendents, vitamins, coenzymes and redox centers. As the reduction itself proceeds via channeling of intermediates (see Box 1) with quantitative turnover, isotope fractionations can only occur in context with the sulfate activation and the consumption of the active sulfate, both reactions at metabolic branching points with partial turnover of the substrate in different directions (Fig. 4). Most plants store the residual 34S-enriched sulfate in leaf vacuoles. Therefore, the d34S value of the bulk biomass is nearly identical to that of the assimilated sulfate, although an uneven distribution of the reactants may simulate isotope fractionations between different compartments. The first organic S-containing product is cysteine, precursor of any other S-containing compounds, which are all depleted in 34S relative to the precursor (Tcherkez & Tea, 2013). As the concentration of most of them is very low, they do not contribute significantly to the average d34S value of a plant or animal biomass sample. Exceptions are S-containing repellents or attractants as glucosinolates (Brassicaceae) and alliins (Allium sp.), or cysteine and methionine containing proteins. Their relative contents contribute e aside from residual 34S-enriched sulfate e essentially to small differences of d34S values (±2‰) between plant organs and between these and soil sulfate (Tcherkez & Tea, 2013). The sulfur trophic shift between animals and their diet is 0 ± 1‰ (Barnes & Jennings, 2007; Michener & Schell, 1994) but small d34S value differences exist in between animal organs, as methionine prevails in collagens and cysteine in keratins. Connective tissues in animals contain in addition the sulfate esters proteoglykans, the biosynthesis of which implies the sole remarkable sulfur isotope fractionation in animals (Tanz & Schmidt, 2010).
3.2. Assessment and scope of applications The largest field of d34S value application in food origin investigation is in the frame of multi element isotope ratio determinations. Whereas the sulfur content of most plants is far below 1%, Brassicaceae and Allium sp. approach 1% and even more in dry matter, and the differences between sulfate and “organic” d34S values in Brassicaceae attains 6‰; the greatest corresponding D34S value observed for isolated glucosinolates is even 14‰ (Tanz & Schmidt, 2010). These results indicate a remarkable S-isotope fractionation in plants and underline the necessity to use homogeneous bulk material for origin investigations on plants. On the other hand, one has to keep in mind that homogenization of plant material can cause losses of volatile S-containing hydrolysis products, e. g. of mustard oils from glucosinolates liberated by myrosinase (Fig. 4). As early season local asparagus is quite expensive in Germany, often products from other countries are offered as local ones in spring. Their origin can be identified by multi element multi component isotope ratio analysis. As the sulfur content of bulk asparagus is too low, the analysis has been performed on the purified “protein fraction” of the plants, concentrating their “organic” sulfur (Schlicht et al., 2006). Its d34S values were typical for defined cultivation areas and the highest values were found, as expected, for samples from places close to the sea or near salt deposits in bedrocks. Animal samples attain up to 2% sulfur in dry matter but one has to keep in mind that the overwhelming part oft their sulfur originates from the animals' diet, and this can, at least for domestic animals, originate from anywhere and even vary over the seasons (Bahar et al., 2008). Hence, a direct correlation to a defined geographical origin on the basis of a d34S value alone may be quite uncertain, as it depends on the relative amount of local food in the animals' diet. Therefore, most investigations use sulfur isotope characteristics as one parameter among several ones, as shown for origin identifications of lamb meat (Camin et al., 2007) and beef (Bahar et al., 2008). These reservations can be dropped in case of samples from prehistoric or wild subjects. Nevertheless, it has again to be pointed out that the d34S values can be different for individual organs and tissues of animals in dependence of their relative contents of defined proteins and proteoglykans (Tanz & Schmidt, 2010). Most interesting and informative are investigations, in which the d34S values of defined organs or compounds of wild animals are analyzed as the sole biomarkers. Weber, Hutcheon, McKeegan, and Ingram (2002) studied the sulfur isotope characteristics of otolith layers and fry of salmon and found unambiguous correlations to their diet and life history. This example shows that sulfur isotope ratio analysis is a most promising tool in animal migration research, especially because the element's discrimination in food webs and on trophic levels is negligible (Hobson, 2008). Nehlich and Richards (2009) promoted the use of sulfur isotope ratio of bone collagen as a reliable biomarker in archeological research. Not only they could show that e apart from d15N values e d34S values were indicative for the distinction between land animal and fresh water fish as protein source for prehistoric men (Nehlich, Borí c, Stefanovic, & Richards, 2010) but that the latter ones were also useful to identify the capture area of sea fish (Nehlich, Barrett, & Richards, 2013). The latter paper contains several further references dealing with sulfur isotopes as biomarkers for seafood. 4. Conclusions From the above report it turns out that oxygen isotope analyses of juice or wine water alone can often give satisfactory information about the climate of the sample's origin and on potential adulterations by illegal addition of foreign water. Squeeze water from
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vegetables and from animal products may suffer from artifacts introduced by storage and processing. Especially for animal products, one has to keep in mind that body water is a product of several parameters. Organically bound oxygen in non-exchanging functional groups of defined components (OH-groups of carbohydrates, functional O-groups of amino acids and esters) preserves valuable information about origin and history of the sample. The bases are defined chemical and isotopic correlations between the oxygen source and the functional groups in question, allowing to reconstruct the original conditions of the oxygen introduction. Nevertheless, oxygen isotope ratios of water and organic matter alone are e apart from some special questions e not generally sufficient to provide definite information on origin and authenticity of food. They must be combined with additional (isotopic) parameters (examples see Part 2.1). On the other hand, sulfur isotope ratios are most valuable indicators for the origin of plant material, provided average d34S values of bulk biomass are used and/or reference samples are available or the local geological and soil conditions (soil sulfate, sea spray, SO2 pollution, fertilization) are known. Sulfur isotope discriminations in plants and animals together with unequal distributions of the reactants between compartments can simulate fractionations. Therefore, the analysis of defined organs or compounds is necessary. This is especially successful in case of animal migration control, where sulfur isotope analysis alone can provide full information. References Anderson, V. E. (1991). Isotope effects on enzyme-catalyzed b-eliminations. In P. F. Cook (Ed.), Enzyme mechanism from isotope effects (pp. 389e417). Boca Raton: CRC Press. Bahar, B., Schmidt, O., Moloney, A. P., Scrimgeour, C. M., Begley, I. S., & Monahan, F. (2008). Seasonal variation in the C, N and S stable isotope composition of retail organic and conventional Irish beef. Food Chemistry, 106, 1299e1305. Barnes, C., & Jennings, S. (2007). Effect of temperature, ration, body size and age on sulphur isotope fractionation in fish. Rapid Communications in Mass Spectrometry, 21, 1461e1467. Blanch, A. I., & Ruperez, M. (2006). Detection of water addition in NFC juices. In SGF International e.V., W. Rieth, & K. Schulz (Eds.), Results of the Pure Juice Project (pp. 43e51). Nieder-Olm, Germany: SGF. Brand, W. A. (2010). Discrepancies between isotope ratio infrared spectroscopy and isotope mass spectrometry for the isotope analysis of plant and soil waters. Rapid Communications in Mass Spectrometry, 24, 2687e2688. Calderone, G., & Guillou, C. (2008). Analysis of isotopic ratios for the detection of illegal watering of beverages. Food Chemistry, 106, 1399e1405. Camin, F., Bontempo, L., Heinrich, K., Horacek, M., Kelly, S. D., Schlicht, C., et al. (2007). Multi-element (H, C, N, S) stable isotope characteristics of lamb meat from different European regions. Analytical and Bioanalytical Chemistry, 389, 309e320. Camin, F., Perini, M., Bontempo, L., Fabroni, S., Faedi, W., Magnani, S., et al. (2011). Potential isotopic and chemical markers for characterising organic fruits. Food Chemistry, 125, 1072e1082. Chenery, C. A., Pashley, V., Lamb, A. L., Sloane, H. J., & Evans, J. A. (2012). The oxygen isotope relationship between the phosphate and structural carbonate fractions of human bioapatite. Rapid Communications in Mass Spectrometry, 26, 309e319. Coplen, T. B. (2011). Guidelines and recommended terms for expression of stableisotope-ratio and gas-ratio measurement results. Rapid Communications in Mass Spectrometry, 25, 2538e2560. €hlke, J. K., Peiser, H. S., Rieder, S. E., Krouse, H. R., et al. Coplen, T. B., Hopple, J. A., Bo (2002). Compilation of minimum and maximum isotope ratios of selected elements in naturally occurring terrestrial materials and reagents. Water Resources Investigation Report 01-4222. Reston Virginia: US Geological Survey 2002. Craig, H. (1961). Isotopic variations in meteoric waters. Science, 133, 1702e1703. Dordevic, N., Camin, F., Marianella, R. M., Postma, G. J., Buydens, L. M. C., & Wehrens, R. (2013). Detecting the addition of sugar and water to wine. Australian Journal of Grape and Wine Research, 19, 324e330. Fronza, G., Fuganti, C., Schmidt, H.-L., & Werner, R. A. (2002). The d18O-value of the p-OH group of L-Tyrosine permits the assignment of its origin to plant or animal sources. European Food Research and Technology, 215, 55e58. Fry, B. (2006). Stable isotope ecology. New York, USA: Springer ScienceþBusiness Media, LLC. Gessler, A., Peuke, A. D., Keitel, C., & Farquhar, G. D. (2007). Oxygen isotope enrichment of organic matter in Ricinus communis during the diel course as affected by assimilate transport. New Phytologist, 174, 600e613. Grimes, V., & Pellegrini, M. (2012). A comparison of pretreatment methods for the analysis of phosphate oxygen isotope ratios in biophosphate. Rapid Communications in Mass Spectrometry, 27, 375e390.
149
Hegerding, L., Seidler, D., Danneel, H.-J., Gessler, A., & Nowak, B. (2002). Sauerstoffisotopen-Verh€ altnis-Analyse zur Herkunftsbestimmung von Rindfleisch. Fleischwirtschaft, 82, 95e100. Hobson, K. A. (2008). Applying isotopic methods to tracking animal movements. In K. A. Hobson, & L. I. Wassenaar (Eds.), Terrestrial Ecology Series: Vol. 2. Tracking animal migration with stable isotopes (pp. 45e78). London: Academic Press/Elsevier. Horita, J., Ueda, A., Mizukami, K., & Takatori, I. (1989). Automatic dD and d18O analyses of multi-water samples using H2- and CO2-water equilibration methods with a common equilibration set-up. Applied Radiation and Isotopes, 40, 801e805. Jamin, E., Gonzalez, J., Remaud, G., Naulet, N., & Martin, G. G. (1997). Detection of exogenous sugars or organic acids addition in pineapple juices and concentrates by 13C IRMS analysis. Journal of Agricultural and Food Chemistry, 45, 3961e3967. rin, R., Re tif, M., Lees, M., & Martin, G. J. (2003). Improved detection of Jamin, E., Gue added water in orange juice by simultaneous determination of the oxygen-18 oxygen-16 isotope ratios of water and ethanol derived from sugars. Journal of Agricultural and Food Chemistry, 51, 5202e5206. Kirsanow, K., & Tuross, N. (2011). Oxygen and hydrogen isotopes in rodent tissues: Impact of diet, water and ontogeny. Palaeography, Palaeoclimatology, Palaeoecology, 310, 9e16. Koch, P. L., Fogel, M. L., & Tuross, N. (1994). Tracing the diets of fossil animals using stable isotopes. In K. Lajhta, & R. H. Michener (Eds.), Stable isotopes in ecology and environmental sciences (pp. 63e92). Oxford, UK: Blackwell Scientific Publications. Kohn, M. J. (1996). Predicting animal d18O: accounting for diet and physiological adaptation. Geochimica et Cosmochimica Acta, 60, 4811e4829. Kornexl, B. E., Werner, T., Rossmann, A., & Schmidt, H.-L. (1997). Measurement of stable isotope abundances in milk and milk ingredients e a possible tool for origin asssignment and quality control. Zeitschrift für Lebensmitteluntersuchung und -forschung, 205, 19e24. Lis, G., Wassenaar, L. I., & Hendry, M. J. (2008). High-precision laser spectroscopy D/ H and 18O/16O measurements of microliter natural water samples. Analytical Chemistry, 80(1), 287e293. Longinelli, A., & Selmo, E. (2011). d18O values of Sus scrofa blood water and bone phosphate; a marked discrepancy between domestic and wild specimens. Rapid Communications in Mass Spectrometry, 25, 3732e3734. Luz, B., Kolodny, Y., & Horowitz, M. (1984). Fractionation of oxygen isotopes between mammalian bone-phosphate and environmental water. Geochimica et Cosmochimica Acta, 48, 1689e1694. Michener, R. H., & Schell, D. M. (1994). Stable isotope ratios as tracers in marine aquatic food webs. In K. Lajhta, & R. H. Michener (Eds.), Stable isotopes in ecology and environmental sciences (pp. 138e157). Oxford, UK: Blackwell Scientific Publications. Mook, W. G. (2000). The global cycle of water. In Environmental isotopes in the hydrological cycle (Vol. I); (pp. 1e18). Paris, Vienna: International atomic Energy Agency and United Nations Educational, Scientific and Cultural Organization. Nehlich, O., & Richards, M. P. (2009). Establishing collagen quality criteria for sulphur isotope analysis of archaeological bone collagen. Archaeological and Anthropological Sciences, 1, 59e75. Nehlich, O., Barrett, J. H., & Richards, M. P. (2013). Spatial variability in sulphur isotope values of archaeological and modern cod (Gadus morhua). Rapid Communications in Mass Spectrometry, 27, 2255e2262. Nehlich, O., Borí c, D., Stefanovi c, S., & Richards, M. P. (2010). Sulphur isotope evidence for freshwater fish consumption: a case study from the Danube Gorges, SE Europe. Journal of Archaeological Science, 37, 1131e1139. Nightingale, M., & Mayer, B. (2012). Identifying sources and processes controlling the sulphur cycle in the Canyon Creek watershed, Alberta, Canada. Isotopes in Environmental and Health Studies, 48, 89e104. Perini, M., & Camin, F. (2013). d18O of ethanol in wines and spirits for authentication purposes. Journal of Food Science, 78, C839eC844. Pfahler, V., Dürr-Auster, T., Tamburini, F., Bernasconi, S. M., & Frossard, E. (2013). 18O enrichment in phosphorous pools extracted from soybean leaves. New Phytologist, 197, 186e193. Pietsch, S. J., Hobson, K. A., Wassenaar, L. I., & Tütken, T. (2011). Tracking cats: problems with placing feline carnivores on d18O, d2H isoscapes. PLoS ONE, 6(9), e24601. http://dx.doi.org/10.1371/journal.pone.0024601. Podlesak, D. W., Bowen, G. J., O'Grady, S., Cerling, T. E., & Ehleringer, J. R. (2012). d2H and d18O of human body water: a GIS model to distinguish residents from nonresidents in the contiguous USA. Isotopes in Environmental and Health Studies, 48, 1e21. Rapisarda, P., Camin, F., Fabroni, S., Perini, M., Torrisi, B., & Intrigliolo, F. (2010). Influence of different organic fertilizers on quality parameters and the d15N, d13C, d2H, d34S, and d18O values of orange fruit (Citrus sinensis L. Osbeck). Journal of Agricultural and Food Chemistry, 58, 3502e3506. Renou, J.-P., Bielicki, G., Deponge, C., Gachon, P., Micol, D., & Ritz, P. (2004). Characterization of animal products according to geographic origin and feeding diet using nuclear magnetic resonance and isotope ratio mass spectrometry, Part II: beef meat. Food Chemistry, 86, 251e256. Rock, L., Rowe, S., Czerwiec, A., & Richmond, H. (2013). Isotopic analysis of eggs: evaluating sample collection and preparation. Food Chemistry, 136, 1551e1556. Schlicht, C., Roßmann, A., & Brunner, E. (2006). Anwendung der MultielementMultikomponenten Isotopenverh€ altnismassenspektrometrie (IRMS) zur
150
N. Krivachy (Tanz) et al. / Food Control 48 (2015) 143e150
Prüfung der geographischen Herkunft von Spargel. Journal für Verbraucherschutz und Lebensmittelsicherheit, 1, 97e105. Schmidt, H.-L., Werner, R. A., & Roßmann, A. (2001). 18O pattern and biosynthesis of natural plant products. Phytochemistry, 58, 9e32. €ckigt, D., & Christoph, N. (2005). StabilisotopenaSchmidt, H.-L., Roßmann, A., Sto €t von Lebensmitteln. Chemie in unserer Zeit, nalytik. Herkunft und Authentizita 39, 90e99. Schmidt, H.-L., Rossmann, A., Voerkelius, S., Schnitzler, W. H., Georgi, M., Graßmann, J., et al. (2005). Isotope characterization of vegetables and wheat from conventional and organic production. Isotopes in Environmental and Health Studies, 41, 223e228. Schmidt, H.-L., Werner, R. A., Rossmann, A., Mosandl, A., & Schreier, P. (2007). Stable isotope ratio analysis in quality control of flavourings. In H. Ziegler (Ed.), Flavourings, production, composition, applications, regulations (2nd ed.) (pp. 602e633). Weinheim, Deutschland: Wiley-VCH. Schmidt, H.-L., Rossmann, A., Rummel, S., & Tanz, N. (2009). Stable isotope analysis (Eds.), for meat authenticity and origin check. In L. M. L. Nollet, & F. Toldra Handbook of muscle food analysis (pp. 767e787). Boca Raton: CRC-Press, Taylor and Francis Group. Serra, F., Reniero, F., Guillou, C. G., Moreno, J. M., Marinas, J. M., & Vanhaecke, F. (2005). 13C and 18O isotopic analysis to determine the origin of L-tartaric acid. Rapid Communications in Mass Spectrometry, 19, 1227e1230. Sieper, H.-P., Kupka, H.-J., Lange, L., Rossmann, A., Tanz, N., & Schmidt, H.-L. (2010). Essential methodological improvements in the oxygen isotope ratio analysis on N-containing organic compounds. Rapid Communications in Mass Spectrometry, 24, 2849e2858. Sternberg, L., & Ellsworth, P. F. V. (2011). Divergent biochemical fractionation, not convergent temperature, explains cellulose oxygen isotope enrichment across latitudes. PloS One, 6(11), e28040. http://dx.doi.org/10.1371/ journal.pone.0028040. Sternberg, L., Pinzon, M. C., Anderson, W. T., & Jahren, A. H. (2006). Variation in oxygen isotope fractionation during cellulose synthesis: intramolecular and biosynthetic effects. Plant, Cell & Environment, 29, 1881e1889. Sturm, M., & Lojen, S. (2011). Nitrogen isotopic signature of vegetables from the Slovenian market and its suitability as an indicator of organic production. Isotopes in Environmental and Health Studies, 47, 214e220. €ufele, H., & Schnyder, H. (2014). Oxygen and hydrogen Sun, L., Auerswald, K., Scha isotope composition of silage water. Journal of Agricultural and Food Chemistry, 62(20), 4493e4501. Sun, L. Z., Auerswald, K., Wenzel, R., & Schnyder. (2013). Drinking water intake of grazing steers: the role of environmental factors controlling canopy wetness. Journal of Animal Science, 90, 1e10. Tanz, N., & Schmidt, H.-L. (2010). d34S-Value measurements in food origin assignments and sulfur isotope fractionations in plants and animals. Journal of Agricultural and Food Chemistry, 58, 3139e3146.
Tanz, N., Werner, R. A., Eisenreich, W., & Schmidt, H.-L. (2011). Assessment of enzymatic methods in the d18O-value determination of the L-tyrosine p-hydroxy group for proof of illegal meat and bone feeding to cattle. Journal of Agricultural and Food Chemistry, 59, 9475e9483. Tcherkez, G., & Tea, I. (2013). 32S/34S isotope fractionation in plant sulphur metabolism. New Phytologist, 200, 44e53. Thiem, I., Lüpke, M., & Seifert, H. (2004). Factors influencing the 18O/16O e ratio in meat juices. Isotopes in Environmental and Health Studies, 40, 191e197. Thode, H. G. (1991). Sulphur isotopes in nature and the environment: an overview. In H. R. Krouse, & V. A. Grinenko (Eds.), Stable isotopes: Natural and anthropogenic sulphur in the environment (SCOPE 43) (pp. 1e26). Chichester, West Sussex, GB: John Wiley & Sons Ltd. Tuross, N., Warinner, C., Kirsanow, K., & Kester, C. (2008). Organic oxygen and hydrogen isotopes in a porcine controlled dietary study. Rapid Communications in Mass Spectrometry, 22, 1741e1745. Van Stempvoort, D. R., & Krouse, H. R. (1993). Controls of d18O in sulfate. Review of experimental data and application to specific environments. In C. N. Alpers, & W. W. Blowes (Eds.), Environmental Geochemistry of Sulfide Oxidation, ACS Symposium Series 550 (pp. 446e480). Washington, USA: American Chemical Society. Wachter, H., Christoph, N., & Seifert, S. (2009). Verifying authenticity of wine by Mahalanobis distance and hypothesis testing of stable isotope pattern e a case study using the EU wine Databank. Mitteilungen Klosterneuburg, 59, 237e249. Weber, P. K., Hutcheon, I. D., McKeegan, K. D., & Ingram, B. L. (2002). Otolith sulfur isotope method to reconstruct salmon (Oncorhynchus tshawytscha) life history. Canadian Journal of Fisheries and Aquatic Sciences, 59, 587e591. Werner, R. A. (2003). The online 18O/16O analysis: development and application. Isotopes in Environmental and Health Studies, 39, 85e104. Werner, R. A., Rossmann, A., Schwarz, C., Bacher, A., Schmidt, H.-L., & Eisenreich, W. (2004). Biosynthesis of gallic acid in Rhus typhina: discrimination between alternative pathways from natural oxygen isotope abundances. Phytochemistry, 65, 2809e2813. West, A. G., Goldsmith, G. R., Brooks, P. D., & Dawson, T. E. (2010). Discrepancies between isotope ratio infrared spectroscopy and isotope ratio mass spectrometry for the stable isotope analysis of plant and soil waters. Rapid Communications in Mass Spectrometry, 24, 1948e1954. White, J. W. C. (1989). Stable hydrogen isotopes in plants: a review of current theory and some potential applications. In P. W. Rundel, J. R. Ehleringer, & K. A. Nagy (Eds.), Applications of stable isotopes in ecological research (pp. 142e160). New York, USA: Springer-Verlag. Yakir, D. (1998). Oxygen-18 of leaf water: a crossroad for plant-associated isotopic signals. In H. Griffiths (Ed.), Stable Isotopes, integration of biological, ecological and geochemical processes (pp. 147e168). Oxford, UK: BIOS Scientific Publishers Ltd.