Anal. Chem. 1909, 61, 1755-1758
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Cycling Technique for the Determination of Femtomole Amounts of Sulfite Regina Keil Uniuersitat Tubingen, Institut fur Botanik, Auf der Morgenstelle 1, D- 7400 Tubingen, Federal Republic of Germany
Rudiger Hampp* Uniuersitat Tubingen, Institut fur Botanik, Auf der Morgenstelle 1, D- 7400 Tubingen, Federal Republic of Germany H u b e r t Ziegler
Technische Uniuersitat Munchen, Lehrstuhl fur Botanik, Arcisstrasse 21,0-8000Munchen, Federal Republic of Germany
A micromethod for the fluorometrk determination of sulfite is presented. This method is based on a coupled enzyme assay (sulfite oxidase, NADHdependent peroxidase) the product of which (NAD) is ampllfled by enzymatic cycling. The assay is linear down to 150 fmol (standards, wine samples). With extracts of freezsdrled needle tissue (picea aljles L. (Karst)) the lower limit of detection was 290 fmol.
Sulfite has a long tradition in food chemistry as a preservative, an antioxidant, and an inhibitor of enzymatic and nonenzymatic browning (I).Especially in wine production the use of sulfite was customary for several centuries. The effects caused by SOz (or sulfite, when dissolved) depend not only on the initial levels applied but also on the actual amounts present in the respective tissue or food product during storage. As sulfite is not considered to be harmless to health (2), methods for a sensitive detection of sulfite are necessary. In additon to its role in food chemistry SO2constitutes one of the major air pollutants in industrial and urban areas. I t is now well documented that long-term e x p u r e s to SO2,even a t concentrations that do not result in any visible signs of injury, cause reductions in plant yield (3). Possible interactions of sulfite with metabolic reactions are either deduced from in vitro experiments with cellular organelles or tissue extracts ( 4 ) or from calculations on model systems (5). In order to correlate these observations with in vivo effects the actual concentration of sulfite in polluted tissue has to be known. Methods currently available (and lower limit of detection; l/mL), such as iodometry (7.5 nmol(6)), reaction with 5,5’-dithiobis(2-nitrobenzoicacid) (3 nmol (7))or with 3methyl-l,2-cyclopentanodionedithiosemicarbazone (10 nmol (8)), sulfite oxidase electrode (about 15 nmol(9)), HPLC (30 nmol (10,11),or HPIC (12),are not sufficiently sensitive to assay sulfite levels in plant tissue (4,121.These are obviously very low (12). As we are interested in pollution-dependent alterations in cellular biochemistry (in connection with forest decline) we tried to establish a method for the determination of tissue sulfite with increased sensitivity. In this paper we present an assay which measures sulfite at concentrations of below 200 fmol.
EXPERIMENTAL SECTION Experimental Rationale. The assay principle is based on the following reactions:
* Author to whom correspondence should be addressed. 0003-2700/89/0361-1755$01.50/0
Hz02+ NADH + H+
NADH pemridaae
2H20 + NAD’
Sulfite oxidation yields H2OZwhich is then reduced by an NADH-dependent peroxidase. The stoichiometricalconsumption of NADH is recorded (13). With W spectroscopy or fluorometry of NADH, the lower limit of detection is between 0.2 and 1nmol. The aensitivity of this assay can be significantlyincreased by using the above reactions as a “specific step” which is coupled to an enzymatic cycling procedure for one of the products, Le., NAD (14, 15). Reagents. Enzymes. SuWe oxidase (Sa-OD, EC 1.8.3.1 from chicken liver, 12 units/mL), NADH peroxidase (NADH-POD, EC 1.11.1.1 from Streptococcus faecalis, 45 units/mL), and horseradish peroxidase (HRP, EC 1.11.1.7 from horseradish, 250 units/mg), as well as the enzymes used for cycling, were obtained from Boehringer Mannheim. Sulf-OD and NADH-POD were dialyzed against 25 mM TRIS buffer. HRP (lyophilized) was dissolved in reaction buffer. Buffers. 2-Amino-2-methyl-1-propanol (AMP), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonicacid (TES), and 3-(N-morpholino)propanesulfonicacid (MOPS) were obtained from Sigma (Deisenhofen, FRG), 2-(N-morpholino)ethanesulfonic acid (MES), imidazole, N-(2-hydroxyethyl)piperazine-N’-2ethanesulfonic acid (HEPES), triethanolamine hydrochloride (TEA), and tris(hydroxymethy1)aminomethane (TRIS) were from Boehringer Mannheim. Stabilizing Reagents. Ethylenediaminetetraaceticacid (EDTA) was obtained from Sigma, and water-insoluble poly(viny1pyrrolidone) (PVP)was obtained from Serva (Heidelberg, FRG). Extraction Medium. MOPS-NaOH (50 mM, pH 7), containing 1.7 mM EDTA, was used for the dilution of sulfite stock solutions or wine samples and for the extraction of lyophilized spruce needle homogenates. Sulfite Solutions. Sodium sulfite (20 mM, Merck, Darmstadt, FRG) was dissolved in doubly distilled water. This stock solution was used for stability tests and for the assay with HRP. A stock solution with higher stability (4 mM sulfite, containing 18 mM acetaldehyde) was prepared according to Beutler (16). Sulfite Reagent. The reagent stock was made from TEANaOH (300 mM, pH 8), NADH (0.1 mM), and EDTA (3 mM). Assay concentrations were obtained by dilution (1:3). Apparatus. Spectrophotometry. Absorption measurements were made with a Uvicon 860 W-visspectrophotometer (Kontron, Munchen, FRG). Fluorometry was with a Farrand ratio-2 fluorometer (Farrand, New York). Narrow-band interference filters with a half-width of about 10 nm were used for excitation (340 nm) and emission (450 nm). Homogenization of frozen needle samples (-80 “C) was under cooling with liquid nitrogen with a Micro Dismembrator I1 (Braun, Melsungen, FRG). This resulted in an average particle size well below 100 pm. 0 1989 American Chemlcal Society
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 15, AUGUST 1, 1989
Sulf - 00
Table I. Effects of Different Buffers on the Recovery of Sulfite Standardsa
NAOH-POD
1
7
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solvent
PH
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7.0 7.0 7.0 7.5 8.0 9.0
55
-\
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\ Drift 2
Figure 1. Kinetics of the photometric determination of sulfite. The difference in extinction (AA ) is determined by extrapolation. Sampling. Spruce needle material (Picea abies L. (Karst.)) was obtained from two open top chambers (University of Hohenheim, FRG) (17). In both chambers young spruce trees (about 5 years old) were permanently fumigated with SO2(between 0.01 and 0.04 ppm) and sprinkled with water of pH 4 (17). Other samples were from spruce trees exposed to environmental levels of SOz (Edelmannshof, Welzheimer Forst, near Stuttgart, FRG; 3 h mean values for SO2 during summer 1986 (May through September) 34-130 pg/m3 air = 0.013 to 0.05 ppm). In April, August, and November 1986, needles (up to 3 years old) were collected and metabolically quenched in liquid nitrogen. Homogenization under cooling with liquid nitrogen (see above, 90 s) was in the presence of freshly distilled acetaldehyde. Acetaldehyde forms a stable adduct with sulfite, and this was shown to be a prerequisite for a quantitative recovery of added, internal sulfite standards. Reconstitution of needle lyophilizates resulted in a pH of 4. At this pH the equilibrium in aqueous solution between sulfite, bisulfite, and SO2is shifted toward SOz. Subsequent loss of SOz to the gas phase is prevented by acetaldehyde. Two milligrams of the lyophilized powder was mixed with 5 mg of insoluble poly(vinylpyrro1idone)(PVP, Polyclar AT, Serva) and extracted with 1 mL of hot (95 "C)extraction medium (5 rnin). After cooling on ice insoluble material was pelleted (loo00 g, 8 min, 4 "C). Wine samples (white German wines Silvaner Scheurebe, Qualitatswein, Riesling, and Morio-Muskat and rose wine Portugieser ros6) were diluted 1:50 with extraction solution and assayed without further manipulation. Sulfite Determination. Spectrophotometric Method, mol. The assay described by Beutler and Schiitte (13)was used in a modified way. The sulfite reagent contained the following (final concentrations in the assay): TEA-NaOH (pH 8), 0.1 M; NADH, 0.033 mM; EDTA, 1mM. The amounts of enzymes were equivalent to 33 munits (NADH-POD) and 20 munits (Sulf-OD) per milliliter assay volume. A feature of this assay is a background reaction caused by some kind of NADH-oxidase activity which is part of the NADH-POD. This unspecific NADH consumption is independent on the presence of sulfite (Figure 1)and has to be corrected for (compare legend to Figure 1). Efforts to decrease this side reaction (addition of Mn2+as radical scavenger, C U + / ~ + (18,19),ascorbic acid (181,reduced glutathione (20), and cyanide, H.O. Beutler, personal communication) were not successful (21). In order to get a complete recovery of sulfite which, in the presence of cations such as Fe3+and depending on pH, is easily oxidized (22),EDTA was added. EDTA also decreased the unspecific oxidation of NADH to some extent. Indirect Fluorometry, mol (Cycling). In this case the specific step (reactions 1and 2) was combined with an enzymatic amplification of NAD resulting from the NADH-POD reaction. Oxidation of sulfite with stoichiometric formation of NAD was in a total volume of 30 pL and with decreased amounts of enzymes (munits/mL: NADH-POD, 17; Sulf-OD, 10). Typical sample volumes added to the assay were 2 pL. All pipetting steps were on ice. The reaction was started by rapid warming to 25 "C (water bath). Usually, after 15 min of incubation (complete oxidation of sulfite) the reaction was stopped with HCI (final concentration
recovery,
%
81 100 79 77 98
a All solutions were kept at 95 OC for 5 min. Recovery is given in oercent of the content of the standardized solution before heating.
0.2 M) and the samples were kept at 95 "C for 5 min. This causes a quantitative breakdown of NADH without affecting NAD. Two-microliter aliquots were added to 50 fiL of NAD cycling reagent (350 mM ethanol, 2.5 mM @-mercaptoethanol,2.5 mM oxaloacetic acid, 0.025% (w/v) bovine serum albumin (fraction V), 44 units/mL alcohol dehydrogenase (ADH), 15 units/mL malate dehydrogenase (MDH), 125 mM TRIS-HCl (pH 8.0)). In this step, which is performed in fluorometer tubes, the pyridine nucleotide is alternatively reduced (ADH) and oxidized (MDH). The amounts of enzymes added result in about 5OOO cycles/h (25 O C ) . After 1h the cycling step is stopped by heating the samples in a boiling water bath for 3 min. Malate, formed during cycling, is assayed by oxidation (MDH) and stoichiometric formation of NADH (indicator step). The indicator reagent (1mL) consisted of glutamic acid (sodium salt), 10 mM, NAD, 0.2 mM, MDH, 3 units/mL, glutamate oxaloacetate transaminase, 0.5 units/mL, and 2-amino-2-methylpropanol hydrochloride, 50 mM (pH 9.9). For more detailed information see ref 14 and 15. Controls were run either with sample but no SUM-ODor without sample addition (unspecific NADH oxidation). Recovery of sulfite was routinely checked with internal standards. All assays were run with at least three parallels. RESULTS AND DISCUSSION Stability of Sulfite in Different Buffers. In Table I the effects of different buffers (50 mM) on the stability of sulfite are compared. Sulfite solutions (1 mM) were prepared with the different buffers and photometrically standardized. These solutions were then used to determine a possible loss of sulfite during the extraction procedure (95 "C, 5 min). The large difference in recovery of sulfite between buffered and unbuffered solutions is evident. Rates of recovery were highest with AMP and MOPS. Because of its lower pK, value (7.2) MOPS was used for sample extraction. Substitution of NADH-POD by Horseradish Peroxidase (HRP). Owing to the unspecific NADH oxidation by NADH-POD (Figure 2a) we tested another peroxidase, HRP. Although HRP also exhibits some NADH oxidase activity, this unwanted reaction can be inhibited (18-20). With 0.033 mg/mL HRP direct assay) 1 pM Cu2+completely inhibited the unspecific oxidation of NADH without affecting the peroxidation activity of H R P (Figure 2b). However, the combination of this modified specific step with enzymatic cycling was only possible with sulfite standards but not with extracts from spruce needles. Presumably, residual phenols contained in the needle extract (not completely removed by PVP) are responsible for this effect. According to Yang (23) the peroxidase-H202 enzyme complex promotes the oxidation of phenols to phenoxy radicals. With these radicals sulfite will start a chain reaction which leads to the complete oxidation of sulfite. Extremely reactive intermediates of this process can cause the destruction of NADH without producing NAD. Thus, needle extracts were assayed with the NADHPOD system. Cycling Assay. In order to check for interferences of specific step components with the NAD cycle, the fluores-
ANALYTICAL CHEMISTRY, VOL. 61, NO. 15, AUGUST 1, 1989
SO{-added
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CpmoU
Cycling assay for sulfite. Sulfite standards (0.5 to 1 pmol) were assayed in the absence (0)and presence (0)of spruce needle extract. The difference between both curves (* extract), which is 112 fmol, results from sample sulfite. Flgure 4.
0
.
1
6
0
6
60 1
30
t [minl Kinetics of the sulfite oxidation wlth the NADH-POD system (a) and the HRP system (including 1 I.IM CuCi,) (b). In b there is no unspecific background reaction. Numbers Indicate addition of (1) NADH-POD, (1’) HRP, (2) Sulf-OD, and (3)sulfite standard (10 nmol).
Figure 2.
250
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I
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9 J
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Cycling assay for sulfite, fluorescence yield with sulfite ( 0 ) and NAD standards (0). Figure 3.
cences resulting from sulfite and NAD standards a t identical concentrations were compared. Figure 3 shows that NAD standards (0.5-5 pmol) added to the cycling system without the introduction of a specific step aliquot resulted in a higher fluorescence yield compared to the cycling of NAD resulting from sulfite oxidation (a 2-WLaliquot of the specific step is
introduced into the cycling assay). This decrease in cycling rate (about 40%; compare Figure 3) should thus be caused by a transfer of specific step solutes. However, due to internal standardization of the specific step reaction, this interference is not a problem. Linearity of the Sulfite Determination. Sulfite standards with and without extracts from spruce needles (control needles: from trees showing no signs of pollution effects) were measured in order to check for linearity of the coupled assay. Figure 4 shows that under both conditions the fluorescence increase with increasing concentrations of sulfite was linear, with correlation coefficients higher than 0.999 (slopes, 1.01 (plus extract), 1.0 (without extract); intercepts, 120 fmol (plus extract), 8 fmol (without extract)). The difference in both intercepts (112 fmol) should result from sample sulfite. As in the presence of needle extract the limit of detection (99% confidence interval) was 290 fmol, this sample content cannot be reliably determined. In the absence of needle extract the limit of detection was as low as 130 fmol of sulfite. For wine the lower limit was found to be in the same range as with buffer only (140 fmol). Linearity was maintained up to 10 pmol. Possibly owing to extract constituents, increasing both the volume of the extract added to the specific step or the extract concentration above the values given in this paper resulted in a decrease in yield. Recovery of internal sulfite standards added to needle homogenates with the extraction buffer was about 90%. Sulfite in Spruce Needle Material. None of the samples (1-to 3-year-old needles) contained amounts of sulfite sufficiently high to permit its detection within the 99% confidence interval. With a level of probability of 95% sulfite was detected in 1-year-old needles from the open top chambers and in two samples (1-year-old and 3-year-old needles) taken from spruce trees at their natural location (Welzheimer Forst). The amount of sulfite detected in these samples was 1.1 to 1.3 nmol/mg dry weight (needle dry weight was about 45% of fresh weight). This amount corresponds to a cell sap concentration of about 1 mM. In spite of the high sensitivity of the assay, tissue levels of sulfite of less than 1 nmol/mg dry weight cannot be reliably assayed. However, tissue concentrations of sulfite are more likely to be in the micromolar range (12). Thus the results given here for needle extracts indicate that only under increased levels of SO2 pollution (or slow
Anal. Chem. 1989, 61, 1758-1762
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decomposition of SO2within the tissue) sulfite can be detected with minor significance. Sulfite in Wine. With wine samples "sulfite cycling" worked without problems. Using this technique we were able to detect significant amounts of sulfite in different proveniences of wine which were declared as sulfite free (according to current determination methods). These wines contained between 50 and 90 pM sulfite. A comparison between the limits of detection of sulfite in the original method (15 nmol) and in the cycling assay (130 fmol) shows that the method reported in this communication offers an increase in sensitivity by a factor of lo5. However, the degree to which this increase in sensitivity can be utilized depends on the sample properties. The determination of SO3*in spruce needle extracts is restricted to values of more than 1 nmol/mg dry weight. This is possibly due to phenolic compounds or other ingredients. Nevertheless with this method the amount of sulfite in SOz-stressed plants can be determined. This may open access to other complex sample matrices.
ACKNOWLEDGMENT We wish to thank Dr. T. Benz for constructive advice. Registry No. SOS2-,14265-45-3. LITERATURE CITED (1) (2) (3) (4)
Joslyn, M. A.; Braverman, J. B. S. Adv. Food Res. 1954, 5 . 96. Baker, G. J.; Collett, P.: Allen, D. H. M .J. Aust. 1981, 2. 614. Roberts. T. M.; Danall, N. M.; Lane, P. Adv. Appl. 810l. 1984, 9 , 1. Ziegler, I . Res. Rev. 1975, 56, 79.
Pfanz, H.; Martinoh, E.; Lange, 0.-L.; Heber, U. Plant fhysiol. 1987, 85, 928. Rebelein, H. Chem. Mlkroblol. Techno/. Lebensm. 1973, 2(4), 112-121. Humphrey, R. E.: Ward, M. H.; Hinze, W. Anal. Chem. 1970, 42(7), 698-702. Laila, A. Anal. Lett. 1987, 20(9), 1333-1345. Smith, V. J. Anal. Chem. 1987, 59, 2256-2259. Schwedt, G.; Baurle, A. Fresenius' 2. Anal. Chem. 1985. 322, 350-353. Grill, D.; Esterbauer, H. Phyton (Austrla) 1973, 15(1-2), 87-101. Wellburn. A. R. New phytol. 1985, 700. 329-339. Beutler, H. 0.:Schiitte. I . Deutsche Lebensm. Rundsch. 1983, 79(10), 323-330. Lowry, 0. H.; Passonneau, J. V. A Flexible System of Enzymatlc Analysis; Academic Press: New York, 1972. Kato, T.; Berger, S. J.; Carter, J. A,; Lowry, 0. H. Anal. Biochem. 1973, 53,86-97. Beutler, H. 0 . "Sulfit. UV-Test". I n Methoden der biochemkchen Analytk und LebensmllteIanalyNk mif Test-Combinationen; Boehrlnger Mannheim GmbH: Mannhelm, 1986; pp 116-117. Seufert, G.; Arndt. U. AFZ, A/&. Forst Zeltschrlfi 1985, 112, 13-18. Klebanoff, S. J. J. Biol. Chem. 1959, 234(9), 2480-2485. Akazawa, T.; Conn, E. E. J. Blol. Chem. 1958, 232. 403-415. Klebanoff, S. J.; Yip, C.; Kessler, D. Blmhlm. Blophys. Acta 1962, 58, 563-574. Keil, R. "Biochemische Untersuchungen an gesunden und geschiidigten Fichtennadeln (Picea abies) in AbGngigkeit von Nadelalter und Vegetationsperiode: Sulfit, Malat. PyrMinnukleotide", Dissertation, Fakuitat f . Bioiogie der Eberhard-Karls-Universitiit, Tublngen, F. R.G., 1988, pp 52-54. Cotton, F. A.; Wiikinson, G. Advanced Inorganlc Chemlstry; Wiley & Sons: New York, 1980; pp 532-533. Yang, S. F. Arch. Biochem. Blophys. 1967, 122,481-487.
RECEIVED for review January 25,1989. Accepted May 1,1989. This project was financed by grants from the -"Projekt Europaisches Forschungszentrum fur Massnahmen zur Luftreinhaltung" (PEF) and the European Community.
Determination of Lead in Antarctic Ice at the Picogram-per-Gram Level by Laser Atomic Fluorescence Spectrometry Michail A. Bolshov,' Claude F. Boutron,*g2and Aleksandr V. Zybin' Institute of Spectroscopy, USSR Academy of Sciences, Troitzk, 142092 Moscow Region, USSR, and Laboratoire de Glaciologie et Ggophysique de 1'Enuironnement d u CNRS, Domaine Uniuersitaire, 2, rue MoliBre, B.P. 96, 38402 St. Martin d'H8res Cedex, France
We present here prellmlnary results of the measurement of Pb In ancient Antarctic Ice down to the subpg/g level by laser exclted at& fluorescence spectrometry wlth electrothermal atomization. Detailed caltbratlon of the spectrometer was successtully achleved down to the sub-pg/g level by using ultralow concentration Pb standards. The Ice core samples, which had prevlously been mechanically decontaminated, were dlrectly analyzed for Pb by using very mall volumes (20 pL only), without any preconcentration step or chemical treatment. The results are In very good agreement with those previously obtalned for the same Ice samples by Isotope dllutlon mass spectrometry.
1. INTRODUCTION For 20 years there has been a growing interest in the investigation of the occurrence of Pb (and of several other heavy
*Author to whom correspondence and reprint requests should be addressed. *Institute of Spectroscopy. Laboratoire de Glaciologie et GBophysiquede YEnvironnement.
metals such as Cd, Cu, Zn, Hg,...) in the well-preserved dated snow and ice layers deposited in .the central areas of the remote Antarctic and Greenland ice sheets (1-14). This is indeed a unique way to reconstruct the past natural tropospheric fluxes of this highly toxic heavy metal on a global scale and to determine to what extent these fluxes are now influenced by human activities. Such investigation has unfortunately proved to be very difficult because of the extremely low concentrations to be measured. As an illustration, P b concentrations in Holocene Antarctic ice have recently been shown to be as low as about 0.4 pg of Pb/g (6, 9). First of all, it is mandatory to decontaminate the snow or ice samples before final analysis, since most available samples are more or less contaminated on their outside, regardless of the precautions taken to collect them cleanly in the field (2-4,6-9,11-14). Ultrasensitive analytical techniques must then be used. Due to the extremely low concentrations involved, ultraclean procedures are required throughout the entire analytical procedure, from sample decontamination to final analysis (11, 14-16). Up to now, the analytical techniques that have been used for the final analysis of the decontaminated samples are either
0003-2700/89/0361-1758$01.50/00 1989 American Chemical Society
