Wirkungsorientierte Analytik von polyzyklischen aromatischen
Kohlenwasserstoffen
in geräucherten Fleischwaren P A K
Transkription
Luciferin Lichtsignal Ah‐Rezeptor
DRE Reportergen
Luciferase
Luminometer
Messung oxidiert
Kerstin Kuhn
Hannover 2009
Wirkungsorientierte Analytik von polyzyklischen aromatischen Kohlenwasserstoffen in geräucherten Fleischwaren
INAUGURAL – DISSERTATION zur Erlangung des Grades einer
Doktorin der Veterinärmedizin - Doctor medicinae veterinariae -
(Dr. med. vet)
Vorgelegt von
Kerstin Kuhn, geb. Fischer aus Heidelberg
Hannover 2009
Abteilung für Lebensmittelsicherheit, Bundesinstitut für Risikobewertung, Berlin
und
Prof. Dr. Bernhard Nowak
Institut für Lebensmittelqualität und –sicherheit Tierärztliche Hochschule Hannover
1. Gutachter: Prof. Dr. Bernhard Nowak
Institut für Lebensmittelqualität und –sicherheit Tierärztliche Hochschule Hannover
und
Prof. Dr. Dr. Alfonso Lampen
Abteilung für Lebensmittelsicherheit, Bundesinstitut für Risikobewertung, Berlin
2. Gutachter: Prof. Dr. Waldemar Ternes
Institut für Lebensmitteltoxikologie und chemische Analytik
Tierärztliche Hochschule Hannover
Tag der mündlichen Prüfung: 15.05.2009
Diese Arbeit wurde durch Mittel der Fritz-Ahrberg-Stiftung, Hannover gefördert.
Für Markus
Determination of Polycyclic Aromatic Hydrocarbons in Smoked Pork by Effect- Directed Bioassay with Confirmation by Chemical Analysis
ist publiziert im Journal of Food Protection, Vol. 71, No. 5, 2008, Seiten 993–999.
Die vorliegende Arbeit
Effect-based and chemical analysis of polycyclic aromatic hydrocarbons in smoked meat – a practical food monitoring approach
ist am 24. Februar 2009 vom Journal Food Additives and Contaminants zur Publikation angenommen worden.
Ergebnisse dieser Dissertation wurden auf folgenden Fachtagungen präsentiert:
47. Arbeitstagung des Arbeitsgebietes Lebensmittelhygiene der Deutschen Veterinärmedizinischen Gesellschaft e.V. (2006):
Nachweis polyzyklischer aromatischer Kohlenwasserstoffe (PAKs) in geräucherten Fleischwaren mit dem CALUX-Bioassay (Poster)
48. Arbeitstagung des Arbeitsgebietes Lebensmittelhygiene der Deutschen Veterinärmedizinischen Gesellschaft e.V. (2007):
Nachweis Polyzyklischer Aromatischer Kohlenwasserstoffe (PAKs) in geräucherten Fleischwaren mittels chemischer Analyse und in-vitro Testmethoden (Vortrag)
49. Arbeitstagung des Arbeitsgebietes Lebensmittelhygiene der Deutschen Veterinärmedizinischen Gesellschaft e.V. (2008):
Einfluss von Räuchertemperatur und –länge auf die Entstehung polyzyklischer aromatischer Kohlenwasserstoffe (PAK) in Fleischwaren - Untersuchungen mit wirkungsbezogener und klassischer Analytik (Poster)
Kapitel 1 Einleitung ... 7
Kapitel 2 Determination of Polycyclic Aromatic Hydrocarbons in Smoked Pork by Effect-Directed Bioassay with Confirmation by Chemical Analysis... 11
Kapitel 3 Effect-based and chemical analysis of polycyclic aromatic hydrocarbons in smoked meat - a practical food monitoring approach ... 19
Kapitel 4 Zusammenfassung der Ergebnisse ... 40
Optimierung des CALUX-Assays ... 44
Praktische Anwendung des CALUX-Assays ... 51
Kapitel 5 Übergreifende Diskussion... 56
Kapitel 6 Zusammenfassung ... 61
Kapitel 7 Summary ... 62
Kapitel 8 Literaturverzeichnis... 63
Kapitel 9 Anhang ... 67
Kapitel 10 Danksagung ... 80
AhR Aryl Hydrocarbon Rezeptor B[a]A Benzo[a]anthracen
B[a]P Benzo[a]pyren
B[b,j,k]F Benzo[b,j,k]fluoranthen
CALUX-assay Chemical Activated Luciferase Gene Expression Assay
CYP Cytochrome P450
DBA Dibenz[a,h]anthracen DMSO Dimethylsulfoxid DRE Dioxin Responsive Element EC25 / EC50 mittlere effektive Konzentration EPA US Environmental Protection Agency EFSA European Food Safety Authority
GC-MS Gaschromatographie-Massenspektroskopie HPLC Hochleistungsflüssigkeitschromatographie
IPCS International Programme on Chemical Safety der WHO IEF Induktionsäquivalent-Faktor
IEQ Induktionsäquivalente
JECFA Gemeinsamer FAO/WHO-Sachverständigenausschuss für Lebensmittelzusatzstoffe
LOD limit of detection (Nachweisgrenze)
LOQ limit of quantification (Bestimmungsgrenze) PAH / PAK polyzyklische aromatische Kohlenwasserstoffe RLU Relative Light Units (relative Lichteinheiten) TCDD 2,3,7,8–Tetrachlorodibenzo-p-Dioxin
WHO World Health Organization
Kapitel 1 Einleitung
Lebensmittel können mit einer Vielzahl unterschiedlicher chemischer Verbindungen kontaminiert sein. Mittels klassischer chemischer Analytik können gezielt ausgewählte Verbindungen in sehr niedrigen Konzentrationen nachgewiesen werden. Das komplexe Spektrum der möglichen Kontaminanten kann dadurch jedoch kaum vollständig und das Gefährdungspotential nicht immer ausreichend erfasst werden. Daher werden in der Umweltforschung seit etwa 25 Jahren integrierte biologisch-chemische Verfahren entwickelt und eingesetzt, die biologische Effekte von Stoffen der Substanzgemische messen (BRACK 2006). Diese werden als wirkungsorientierte Analyse (effect-directed analysis) oder als Toxizitätsidentifizierung und -auswertung (toxicity identification evaluation) bezeichnet. Neben dem Vorteil der Abschätzung des Summeneffektes einer komplex belasteten Probe beschreibt BRACK (2006) die Vorteile durch Senkung der Analysekosten, da hohe Fixkosten über Geräteanschaffungen (GC-MS / HPLC) reduziert werden können.
In der Untersuchung von Lebensmitteln werden in den letzten Jahren zunehmend wirkungsorientierte Ansätze im Bereich der Screeningverfahren getestet und eingesetzt.
Im Bereich der Dioxinanalytik regelt die europäische Verordnung VO (EG) Nr. 1883/2006 die Analysemethoden für die amtliche Kontrolle. Sie nennt explizit den Einsatz von Screeningverfahren zur Auswahl der Proben mit einem signifikanten Gehalt an Dioxinen.
In den Niederlanden und Belgien sind solche Verfahren seit einiger Zeit etabliert. Als Vorteil gelten der hohe Probendurchsatz und die dadurch bedingte Zeitersparnis.
Ziel dieser Dissertationsarbeit war es, eine neue wirkungsorientierte Analytik von polyzyklischen aromatischen Kohlenwasserstoffen (PAKs) zu entwickeln, an das Untersuchungsgut Fleisch anzupassen und diese Analytik im praktischen Einsatz in der Lebensmittelkontrolle zu erproben.
PAKs stellen eine große, vielfältige Gruppe von chemischen Stoffen dar. Viele Vertreter gelten als mutagen, teratogen und kanzerogen. Ebenso werden Einflüsse auf die Fruchtbarkeit beschrieben (IARC 2005). Sie werden bei unvollständigen Verbrennungen von Fetten gebildet und kontaminieren Lebensmittel über die Umwelt (Ablagerung aus der Luft) und über Bearbeitungsprozesse wie Räuchern, Braten, Grillen und Rösten (GUILLEN
u. SOPELANA 2003). Geschmolzenes Fett, das in die Glut tropft, erhöht die Kontaminationsmenge (WHO 2005). Die Auswahl des Räuchermaterials, Art der Raucherzeugung (extern oder intern), Verfügbarkeit von Sauerstoff und vor allem die eingesetzte Räuchertemperatur haben großen Einfluss auf die Menge von PAKs, die auf und in der Räucherware gebildet wird (SIMKO 2005).
Die wirkungsorientierte Analytik erfolgte mit dem in vitro CALUX Bioassay (chemical activated luciferase gene expression assay), der damit zum ersten Mal in der Analytik auf PAKs eingesetzt wurde. Dieser Bioassay zeigt die Aktivität aller Substanzen einer Probe, die mit dem Aryl Hydrocarbon Rezeptor (AhR) interagieren. Dieser vermittelt die meisten toxischen Effekte von u.a. Dioxinen, polychlorierten Biphenylen und PAKs (GUILLEN u.
SOPELANA 2003). Aufgrund der Spezifität zum AhR ist es nicht möglich, die exakten Substanzen auszumachen, die ursächlich zur Aktivität einer Probe beitragen. Es wird vielmehr ein generelles biologisches (krebserregendes) Potential einer Probe aufgezeigt, das mit der Wirkung der enthaltenen PAKs korreliert. Dies wurde in vitro von MACHALA et al. (2001) und in vivo von SHIMADA et al. (2002) gezeigt.
In kontaminierten Produkten wird stets eine Vielfalt verschiedener PAKs gebildet. Dennoch wurde viele Jahre lang nur die Leitsubstanz Benzo[a]pyren (B[a]P) auf nationaler und europäischer Ebene reglementiert und untersucht. In den letzten Jahren wuchs das Bewusstsein, dass die Bestimmung von B[a]P alleine für eine komplexe Risikobewertung einer Lebensmittelprobe nicht ausreiche, da festgestellt wurde, dass B[a]P nur mit 1 bis 20% zum gesamten kanzerogenen Potential einer Probe beiträgt (SIMKO 2002). Im Jahre 2005 empfahl die Europäische Kommission 15 ausgewählte PAKs, die vom Wissenschaftlichen Ausschuss „Lebensmittel“ als karzinogen eingestuft wurden, weiter zu untersuchen. In der Folge wurde von der europäischen Behörde für Lebensmittelsicherheit (EFSA) aufgrund einer entsprechenden Bewertung durch den Gemeinsamen FAO/WHO- Sachverständigenausschuss für Lebensmittelzusatzstoffe (JECFA) noch ein weiteres PAK der Liste hinzugefügt (WHO 2005). Auf diese, 15+1 priority PAKs genannte Liste1 fokussiert sich zur Zeit die europäische Forschung, obwohl in einem europaweiten
1 Benzo[c]fluorene, Benz[a]anthracene, Cyclopenta[cd]pyrene, Chrysene, 5-Methylchrysene, Benzo[b]fluoranthene, Benzo[k]fluoranthene, Benzo[j]fluoranthene, Benzo[a]pyrene, Indeno[1,2,3-cd]pyrene, Dibenz[a,h]anthracene, Benzo[ghi]perylene, Dibenzo[a,e]pyrene, Dibenzo[a,h]pyrene, Dibenzo[a,i]pyrene, Dibenzo[a,l]pyrene
Ringversuch 2007 festgestellt wurde, dass noch nicht alle Laboratorien diese neuen Anforderungen umsetzen können und nur ein Viertel aller beteiligten Labore in der Lage waren, zufrieden stellend alle 16 PAKs zu bestimmen (SIMON et al. 2008).
Um die Ergebnisse der wirkungsorientierten Analytik von geräucherten Fleischproben mit denen einer herkömmlichen chemischen Analytik vergleichen zu können, wurden in der vorliegenden Arbeit alle Fleischproben parallel zum Bioassay mittels instrumenteller Analytik (GC-MS) untersucht. Dabei wurde auf insgesamt 31 PAKs untersucht, einschließlich der 15+1 EFSA-priority PAKs. Als Untersuchungsgut kamen zum einen Fleischproben (Bauchspeck des Schweins) zum Einsatz, die selbst unter definierten Bedingungen geräuchert wurden. Zum anderen wurden entsprechende Produkte im Groß- und auf dem Wochenmarkt eingekauft und analysiert.
Nach der Adaptation des Bioassays an die Untersuchung auf PAK erfolgte der praktische Einsatz in der Untersuchung der geräucherten Bauchspeckproben. Dabei wurde der Einfluss unterschiedlicher Räucherbedingungen (Räuchertemperatur und Räucherdauer) auf die Entstehung kanzerogener PAKs untersucht und bestimmt, wie sensibel die wirkungsorientierte Analytik mittels Bioassay Änderungen der Räucherbedingungen und damit der Kontaminationsmenge anzeigt.
Kapitel 2
Determination of Polycyclic Aromatic Hydrocarbons in Smoked Pork by Effect-Directed Bioassay with Confirmation by Chemical Analysis
published by Journal of Food Protection, Vol. 71, No. 5, 2008, page 993–999
KERSTIN KUHN,1 BERNHARD NOWAK,1* GÜNTER KLEIN,1 ANDREAS BEHNKE,2 ALBRECHT SEIDEL,2 AND ALFONSO LAMPEN3
1 Institute for Food Quality and Food Safety, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, Hannover, D-30173, Germany.
2 Biochemical Institute for Environmental Carcinogens, Prof. Dr. Gernot Grimmer- Foundation, Lurup 4, Großhansdorf, D-22927, Germany.
3 Department of Food Safety, Federal Institute for Risk Assessment, Thielallee 88 – 92, Berlin, D-14195, Germany.
Keywords: smoked pork, PAH, CALUX, bioassay
* Author for correspondence:
Tel: 49-511-856-7319;
Fax: 49-511-856-827319;
E-mail: bernhard.nowak@tiho-hannover.de
Anteil der Erstautorin an der Arbeit:
Versuchsplanung, Probenerstellung und Probenahme, Aufarbeitung und Aufreinigung aller Proben, Durchführung der wirkungsorientierten Analytik, Auswertung der Ergebnisse, Erstellung des Manuskriptes
Copyright䊚, International Association for Food Protection
Determination of Polycyclic Aromatic Hydrocarbons in Smoked Pork by Effect-Directed Bioassay with Confirmation
by Chemical Analysis
KERSTIN KUHN,1BERNHARD NOWAK,1*GU¨ NTER KLEIN,1ANDREAS BEHNKE,2ALBRECHT SEIDEL,2AND
ALFONSO LAMPEN3
1Institute for Food Quality and Food Safety, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, 30173 Hannover, Germany;2Biochemical Institute for Environmental Carcinogens, Prof. Dr. Gernot Grimmer-Foundation, Lurup 4, 22927 Großhansdorf, Germany; and3Department of Food Safety, Federal Institute for Risk Assessment, Thielallee 88-92, 14195 Berlin, Germany
MS 07-303: Received 7 June 2007/Accepted 28 November 2007
ABSTRACT
Polycyclic aromatic hydrocarbons (PAHs) are generated during smoke curing and other heating treatments of food and represent a large class of chemical pollutants including a number of carcinogens. At present, PAHs are frequently detected by costly and time-consuming chemical analysis. Effect-directed in vitro cell–based bioassays of contaminants can offer a rapid, sensitive, and relatively inexpensive alternative for screening of contaminants in comparison to instrumental analysis. They enable estimation of total biological activity of all compounds acting through the same mode of binding. The aryl hydrocarbon receptor as a binding site plays an important role in PAH-induced carcinogenesis. The in vitro chemical-activated luciferase expression assay (using conditions to detect PAH) was investigated for its applicability for effect-directed analysis of PAH levels in smoked meat. There was an intra-assay variability of 0 to 15% and a mean coefficient of variation of 25% (3 to 50%) for the cleanup and bioassay analysis of the smoked pork samples. There was a correlation between the total responses of the bioassay and the individual amounts of the PAHs with a high molecular weight. The comparison of 2,3,7,8-tetrachlo- rodibenzo-p-dioxin and benzo[k]fluoranthene used as standard in the in vitro chemical-activated luciferase expression assay resulted in benzo[k]fluoranthene being able to be used as an alternative, nontoxic standard in the bioassay. This bioassay is an applicable effect-directed functional prescreening method for the analysis of PAHs in smoked meat and appears to have potential in being used for food control in the future.
Polycyclic aromatic hydrocarbons (PAHs) are wide- spread environmental contaminants representing a very im- portant group of carcinogens with the representative com- pound benzo[a]pyrene (B[a]P). Teratogenic, mutagenic, and carcinogenic properties have been reported for many PAHs as well as noncarcinogenic effects such as reproduc- tive toxicity(9). PAHs are formed by an incomplete com- bustion of organic material and can be passed on to foods because of industrial and engine combustion emissions con- taminating air, water, and soil (8). Concerning food tech- nology, PAHs are generated during food processing, such as smoke curing, deep frying, and drying. It is likely that PAHs are formed from melted fat that undergoes pyrolysis when dripping onto the heat source (11). The Scientific Committee on Food of the European Commission reports that heat treatment of food is commonly thought to be the major source of contamination by PAHs(5).For nonsmok- ers, food is the main source of exposure to PAHs(5).The contamination of food in the smoking process is highly var- iable and is caused by the type and composition of wood, type of generator (internal or external), oxygen accessibil- ity, temperature of smoke generation, and smoking time(5, 11).The final report on the tasks for scientific cooperation
* Author for correspondence: Tel: 49-511-856-7319; Fax: 49-511-856- 827319; E-mail: bernhard.nowak@tiho-hannover.de.
(Task 3.2.12) of the European Union collected data in 2004 on the occurrence of PAHs in food in 13 European coun- tries(2).Thirteen percent of the 1,023 samples in the group,
‘‘traditional smoked products,’’ had an amount above 5 ppb B[a]P, which is the threshold in Regulation 208/2005 of the European Union for smoked meat(2, 3). The mean value in this group was 3.27g/kg, and the median was 0.13g/
kg (3).The Scientific Committee on Food determined that a ‘‘virtually safe dose’’ of B[a]P as marker for the mixture of carcinogenic PAHs in food would be in the range of 0.06 to 0.5 ng B[a]P/kg of body weight per day, which can be converted into a maximum daily intake of 0.04 g/kg B[a]P for an adult (5).The Joint FAO/WHO Expert Com- mittee on Food Additives (JECFA) could not establish a tolerable intake for B[a]P in its 64th report(11).
The aryl hydrocarbon receptor (AhR) mediates the ma- jority of the biological effects of PAHs and halogenated aromatic hydrocarbons–like dioxins. Following binding to the AhR, the ligand-receptor complex is activated and translocated to the nucleus, wherein it binds to a dioxin- responsive element and stimulates transcription of target genes like cytochrome P-450 (CYP) 1A1 (4). Numerous studies support a role for the AhR in mediating the toxicity of PAHs and halogenated aromatic hydrocarbons(5, 8).
In this study, the applicability of the in vitro chemical- activated luciferase expression assay (CALUX) for detec-
TABLE 1. Coefficient of determination (R) of the correlation between the amounts of 27 PAHs and the total CALUX-IEQaof 20 smoked pork belly samplesb
PAH
Range via GC-MS
(g/kg) R2
Benzo[e]pyrene 0.01–0.41 0.898
Benzo[b] fluoranthene 0.02–0.55 0.879
Benz[a]anthracene 0.01–1.73 0.874
Sum of benzo[b⫹k⫹j]fluoranthene 0.05–1.15 0.861
Benzo[c]fluorene 0.01–1.05 0.847
Benzo[a]pyrene 0.02–0.73 0.846
Dibenz[a,h]anthracene 0.01–0.08 0.838
Perylene 0.01–0.12 0.83
Indeno[1,2,3-cd]pyrene 0.01–0.52 0.801
Benzo[ghi]perylene 0.01–0.35 0.789
Benzo[c]phenanthrene 0.02–0.47 0.784
Benzo[ghi]fluoranthene 0.02–0.86 0.776
Benzo[j]fluoranthene 0.02–0.36 0.734
Sum chrysene⫹triphenylene 0.07–2.03 0.73
Anthracene 0.46–42.14 0.713
Pyrene 0.80–23.88 0.702
Coronene 0.01–0.1 0.695
Cyclopenta[cd]pyrene 0.02–1.53 0.675
Fluorene 6.02–124.91 0.667
Anthanthrene 0.01–0.23 0.661
5-Methylchrysene 0.02–0.17 0.603
Benzo[k]fluoranthene 0.01–0.43 0.46
Fluoranthene 2.33–55.27 0.447
Acenaphthylene 0.33–172.24 0.321
Phenanthrene 13.37–383.24 0.317
Acenaphthene 2.62–112.62 0.185
Naphthalene 6.77–425.35 0.012
aTCDD induction equivalents; range, 30.4 to 632.1 ng/kg.
bSee Figure 3 for the example B[a]P. Dibenzopyrenes were ana- lyzed in all samples, but were always below the LOQ.
tion and assessment of AhR-mediated activity of PAHs in food probes was evaluated. Such effect-directed analytical methods are widely used in the screening of environmental compounds such as soil, water, and air. With respect to con- sumer safety, the effect-directed analysis is valuable for prescreening food samples because the unknown potency of ingredients and contaminants of meat samples can be determined.
The CALUX cell bioassay uses a rat hepatoma cell line (H4IIE), stably transfected with a luciferase gene contain- ing vector from the firefly, which is under transcriptional control of the dioxin-responsive element(13).Chemical ac- tivation of the AhR results in a production of the enzyme luciferase, generating light on addition of the substrate lu- ciferin. Two types of binding to the AhR are known. Di- oxins and other halogenated aromatic hydrocarbons bind in a relatively stable and persistent manner to the receptor, whereas PAHs are metabolized after some hours, and they can therefore no longer be detected after 24 h of exposure (20).
The potency of a complex mixture such as a meat sam- ple, to cause a defined biological response, is expressed relative to the response of a well-characterized standard or prototypical compound(18).Activities of samples are ex- pressed as bioassay-derived induction equivalents (IEQ) per specified amount of sample (12). In the present study we used 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and ben- zo[k]fluoranthene (B[k]F) as standards. TCDD was used be- cause of its known affinity to the AhR. B[k]F is known to be a good inducer of CYP enzymes(14),has a high affinity to the AhR, and is described as one of the most potent PAH after incubation of 6 h in the CALUX bioassay(12, 19).
The toxicity is lower in comparison to TCDD, and therefore it could be used as an alternative in the effect-directed anal- ysis.
The aim of this study was to investigate whether the CALUX bioassay could be used as a cheap, easy, and fast effect-directed assay as a prescreening test for the detection of contaminants such as PAHs in smoked food matrices like pork belly. To validate the CALUX bioassay in combina- tion with the extraction and cleanup method, a comparison was made between induced equivalents and certain PAHs determined by gas chromatography–mass spectrometry (GC-MS) methodology.
MATERIALS AND METHODS
Chemicals.Cyclohexane (CAS no. 110-82-7), N,N-dimeth- ylformamide (CAS no. 68-122), and acetone (CAS no. 67-64-1) were obtained from Merck (Darmstadt, Germany) in spectroscopy grade. Methanol (CAS no. 67-56-1) was purchased in Pestilyse grade (grade for pesticide analyze) from Roth (Karlsruhe, Ger- many).
TCDD (CAS no. 1746-01-6) was purchased from LGC Promochem (Wesel, Germany). TCDD was dissolved in dimeth- ylsulfoxide (DMSO; CAS no. 67-68-5, spectrophotometric grade, Acros Organics, Geel, Belgium). The final concentration of TCDD was 300 nM. B[k]F (CAS no. 207-08-9, fluorescence grade, Fluka, Steinheim, Germany) was dissolved as 10 mM stock solution in DMSO.
Silica gel 60 (0.06 to 2 mm) was purchased from Roth. All other chemicals used were of high-purity grade.
For the analysis with GC-MS an internal standard mix (d8- naphthalene, d8-acenaphthylene, d10-acenaphthene, 2F-fluorene, d10-phenanthrene, d10-fluoranthene, d10-pyrene, d12-benz[a]an- thracene, d12-benzo[b]fluoranthene, d12-benzo[a]pyrene, indeno- [1,2,3-cd]fluoranthene, andd12-benzo[ghi]perylene) was used for the determination of 16 priority PAHs (U.S. Environmental Pro- tection Agency, method 8310), supplemented with further PAHs named in the 64th Report of JECFA and the European Food Safety Authority (see Table 1). The European Food Safety Authority rec- ommends analyzing the 15 E.U. priority PAHs(15),identified as being genotoxic and carcinogenic (benzo[a]anthracene, benzo- [b⫹j⫹k]fluoranthene, B[a]P, benzo[ghi]perylene, chrysene, cyclo- penta[cd]pyrene, dibenz[a,h]anthracene (DBA), dibenzo- [a,e⫹a,h⫹a,i⫹a,l]pyrene, indeno[1,2,3-cd]pyrene, and 5-methyl- chrysene) along with benzo[c]fluorene recommended by JECFA for future monitoring of food.
The dibenzopyrenes were analyzed in all smoked pork belly samples but were always below the limit of quantification (LOQ).
The internal standard mix was provided by the Biochemical Institute for Environmental Carcinogens, Großhansdorf, Germany, which also performed the GC-MS analyses on an Agilent 6890N GC with an Agilent 5973N MSD (Agilent, Santa Clara, Calif.).
The analyses were performed with slight modifications as de- scribed previously (7). They were carried out in the single-ion
monitoring modus, and the quantitation was obtained by compar- ing the area responses of the target ions in relation to the area responses of the internal standards, after a three-point calibration.
Sample extraction and cleanup procedure.Raw pork bel- lies of grade E (premium grade) were purchased and smoked in an FPC100 smoking chamber (Fessmann, Winnenden, Germany) using beech woods shavings. For a broad spectrum of different stages of PAH accumulation, the samples were smoked with dif- ferent standard smoke programs (hot smoke at 65⬚C and cold smoke at 18⬚C, with time differences). Samples were drawn from both the middle and the margins of the pork belly, since PAH accumulation is known to vary between these zones(17).Three samples of pork belly, prepared in a similar way to ours, were bought in a large supermarket in Hannover, Germany, and ana- lyzed in the same way. The (total) 20 samples of pork belly (100 g each) were minced and blended in a chopper (Rondo 1000, Tefal, Offenbach, Germany) and stored frozen (⫺18⬚C), packed in aluminum boxes until the extractions were done.
Four 10-g subsamples—i.e., three for the bioassay and one for the GC-MS—were collected from each of the 20 pork belly samples and prepared for further analysis. Although one subsam- ple would have sufficed for meat control, three were prepared to ensure the reproducibility of the bioassay. Likewise, three addi- tional subsamples were drawn from each of three pork belly sam- ples in order to verify the reproducibility of the GC-MS analysis.
All subsamples were prepared in the same manner, particularly observing equal reaction and storage times. However one differ- ence in the analysis technique did occur: In those samples intend- ed for GC-MS analysis, an internal standard mix including the deuterated isotopes of the PAHs mentioned above was added at the beginning of the extraction.
For both kinds of samples, extraction was performed as rec- ommended by the German Food and Feed Law (§64 LFGB(1)) via the corresponding instructions of the national reference labo- ratory to determine B[a]P in smoked meat products. This method is also described in the 64th Report of JECFA (11) and recom- mended to be used for fatty foods such as meat.
Analysis commenced with an alkaline hydrolysis by boiling the minced sample under reflux conditions for 1 h in 2 M KOH, which had been dissolved in 90% methanol. When the sample was dissolved, a liquid-liquid partition was performed twice, using cyclohexane. Three washing steps with methanol and water (7:3), and one with pure water concluded the first stage of analysis. After the concentration of the solvent a liquid-liquid partition was per- formed twice with 90% N,N-dimethylformamide. The N,N-di- methylformamide phases were washed with cyclohexane and di- luted with water until the concentration ofN,N-dimethylformam- ide was 50% in the solution. After partition with cyclohexane, the solvent was washed with water. The solvent was concentrated to a volume of approximately 3 ml, and a cleanup with a silica gel column was used (300 mm, 10 mm diameter, 5-g conditioned silica gel). The sample was eluted with 110 ml of cyclohexane and concentrated to a final volume of 1 ml. Prior to use, all glass- ware and equipment was rinsed with acetone and cyclohexane.
The cleaned extract was further evaporated under a gentle N2 stream, and 100l of dimethylsulfoxide was added just prior to the complete evaporation of the solvent. Instead of this, samples dedicated for the analysis with GC-MS were shipped in 1 ml of cyclohexane-toluene (9:1) to the Biochemical Institute for Envi- ronmental Carcinogens, where the GC-MS analysis was per- formed.
Cell culture.Rat hepatoma cells (H4IIE), stably transfected with the luciferase reporter gene plasmid pGudLuc1.1(6), were
provided by Prof. Dr. A. Brouwer (BDS, Amsterdam, The Neth- erlands). The cells were grown in DMEM/F-12 with 10% heat- inactivated fetal bovine serum (Mycoplex, PAA, Co¨lbe, Germany) and 1% glutamine. All media and supplements for cell culture were purchased from PAA. Before each test period gentamicin B1 was added for 10 days to the media at 0.5 mg/ml in order to select cells containing the plasmid, which also contained a neomycin resistance marker.
The CALUX bioassay.Cells were seeded in 96-well culture plates (Nunc, Wiesbaden, Germany) at a density of 22,500 per well (200l per well). The margin wells were filled with medium and were not included in the assay. Twenty-four hours after seed- ing, 50l of culture medium containing the test extracts or the standard dilutions was added to the medium in the wells. Expo- sures were done in quadruplicate. Solvent control and TCDD or B[k]F calibration standards (between 0.3 pM and 300 pM, and 1 pM to 1M for B[k]F, respectively) were included in quadrupli- cate on each well plate. The final concentration of DMSO in each well was 0.1% (vol/vol).
The adequate dilution of the extracts in the range of 0.1 to 0.001% was used, which induces a luciferase response in the eval- uation region between the limit of quantification and 30% of the maximum response. Exposure time to standards and extracts was 6 h, followed by a microscopically examination for abnormalities and viability. The medium with the test compounds was removed, and the cells were harvested in 75l of cell lysis reagent (0.1 M Tris acetate, pH 7.5, 2 mM EDTA, 1% triton). After 5 min of incubation in a refrigerator at 5⬚C, 50l of the reagent including the cell debris in each well was transferred into a white 96-well plate (Luminunc, Nunc). During the transfer steps, the plates were stored on a freezer pack, and luminescence in relative light units was measured directly afterward using the multilabel reader Mith- ras LB 940 (Berthold, Bad Wildbad, Germany). Assay buffer, con- taining gly-gly buffer (25 mM of glycylglycine, 15 mM of MgSO4, and 4 mM EGTA, pH 7.8), 1 mM ATP, and 1 mM di- thiothreitol, was injected automatically just prior to the measure- ment just as 50l of luciferin solution (1 mMD-luciferin in gly- gly buffer). Results were converted into x-fold induction of the mean of the negative samples (the control). The sigmoid dose- response standard curve was fitted: ligand binding,f(x)⫽log 50%
effective concentration [EC50]⫺[1/hillslope⫻log(max⫺min)/
(x⫺min)⫺1], using SigmaPlot 9.01 (Systat, San Jose, Calif.), and the extracts were interpolated on this curve. Results were expressed as TCDD induction equivalents (IEQ) and converted into nanograms per kilogram of meat, respectively, and micro- grams per kilogram for B[k]F IEQ. The limit of detection was set to the DMSO response plus three times the standard deviation and at ten times for the LOQ, respectively.
RESULTS AND DISCUSSION
Statistical evaluation. The repeatability of the CA- LUX bioassay (variability of extraction samples analyzed in the same run), performed in quadruplicate was 0 to 15%.
Only curve fittings were used with aR2greater than 0.98.
For quality assurance, three parameters in every assay were calculated and evaluated: the reported value of the 3 and 10 pM dilution, and of one known meat extraction sample.
Three times the standard deviation was defined as exclusion criterion. The mean of the extraction samples was calcu- lated from at least four assays in independent runs (different days, different plates), with a variation coefficient between these plates averaging 20% (2.6 to 34.5%). Deviations of
FIGURE 1. Dose-response curves for luciferase induction in H4IIE cell, stably transfected with GudLuc1.1, for 2,3,7,8-tetra- chlorodibenzo-p-dioxin (TCDD) and for benzo[k]fluoranthene (B[k]F). Cells were incubated for 6 h, with increasing concentra- tions of TCDD or B[k]F at 37⬚C. Luciferase activity in cell lysates was determined as described in ‘‘Materials and Methods.’’ The x-fold induction concerning the activity of cell lysates incubated with medium and solvent is shown. See Figure 2 for the corre- lation of the bioassay results of dioxin and B[k]F.
TABLE 2. Spiking of unsmoked pork belly samples with B[k]F and determination with CALUX bioassay
Spiked with B[k]F (g/kg)
B[k]F CALUX IEQ
(g/kg) Mean⫾SD
1,000 807.5 866.6⫾83.6
925.7
100 94.9 97.4⫾7
105.3 92
10 9.9 10.7⫾1.7
12.6 9.5
TABLE 3. Spiking of unsmoked samples with B[k]F and deter- mination of the amount of B[k]F with GC-MSa
Spiked with B[k]F (g/kg)
B[k]F amount
g/kg)
Recovery of d12-B[b]F (%)
100 81.9 88.8
10 7.9 89.3
aFor comparison, only the recovery of the adequate PAH in the internal standard is shown.
more than twice the standard deviation were defined as out- lier.
The pork belly samples were extracted four times, and the reproducibility (mean coefficient of variation of the CA- LUX IEQ) was 25% (3.2 to 50.3%).
LOQ of the individual PAHs in the GC-MS analysis were in the region of 0.01 to 0.04g/kg. For results under the LOQ, we used half of the LOQ as the value for cal- culations, as those recommended in the SCOOP report(3).
All the calculations were performed using Microsoft Excel 11.0 (Microsoft, Redmond, Wash.) and SAS 8.02 (SAS Institute Inc., Cary, N.C.) software.
Preliminary tests.The responses of the CALUX bio- assay were sensitive and reproducible for exposure to TCDD and to B[k]F (Fig. 1). However, the AhR is saturated faster by TCDD binding. The LOQ averaged 1.8 pM TCDD (5.1 ng TCDD/kg) and 92 pM B[k]F (0.23 g B[k]F/kg). The mean maximum induction of the H4IIE cells was 18.7 ⫾ 4.57–fold of the mean of the negative samples (the controls) for TCDD and 11.3⫾3.18 for B[k]F.
Villeneuve et al. (19)compiled for B[k]F a maximum re- sponse at 92% of TCDD in the CALUX assay. The EC50 yielded 52.2⫾16.25 pM (148.7⫾ 46.3 ng/kg) for TCDD and 1,719 ⫾ 934 pM (4,215 ⫾ 2,290 ng/kg) for B[k]F, respectively. This resulted in an induction equivalent factor of 0.0304 for B[k]F (EC50of TCDD divided by the EC50 of B[k]F).
Verifying of the cleanup method.The extraction and cleanup method was verified in two steps. First the recovery of the internal standard used for the samples analyzed with GC-MS was determined. The mean recovery of the heavier deuterated PAHs was 75% (d12-B[a]A), 83% (d12-benzo- [b]fluoranthene), and 96% (d12-B[a]P and d12-benzo-
[ghi]perylene). The recovery of PAHs of lower relative mo- lecular mass was 40 to 60%.
Furthermore, some unsmoked samples were spiked with B[k]F (10, 100, and 1,000 g/kg), extracted as de- scribed and analyzed in parallel with GC-MS and the CA- LUX bioassay. B[k]F was used as calibration standard in these assays. The recovery of B[k]F with GC-MS was near- ly as high as the recovery of the corresponding deuterated internal standard, and the effect in the bioassay is compa- rable (Tables 2 and 3).
Comparison of the results of CALUX using TCDD or B[k]F as standard. The CALUX bioassay was per- formed with TCDD and with B[k]F as standard substance for the dose-response curve, using the same extraction sam- ple. There was a reasonable correlation between the IEQ of TCDD and B[k]F IEQ (see Fig. 2), with a coefficient of determination at R2 ⫽ 0.957. The B[k]F IEQ yielded 0.0306 of the TCDD IEQ according to the induction equiv- alent factor of B[k]F, based on the EC50of 0.0304 described above. Hence, both substances are applicable as standard substance for calculating the dose-response curve and there- fore useful for determining the IEQs of unknown food sam- ples.
Comparison of CALUX results versus GC-MS anal- ysis.We calculated the correlation of the analyzed CALUX- IEQ of 20 meat samples with the amount of 27 PAHs mea- sured by GC-MS for these samples. Figure 3 shows the correlation between analyzed B[a]P levels and the IEQ to TCDD. TheR2of the correlations of all analyzed PAHs are shown in Table 1.
From the group of positive correlating PAHs, B[a]P and DBA seem to be good markers for comparing the GC- MS result with the bioassay. Both substances have a rea-
FIGURE 2. Comparison of 2,3,7,8-tetra- chlorodibenzo-p-dioxin (TCDD) and ben- zo[k]fluoranthene (B[k]F) each used as standard substance in the CALUX bioas- say for calculating the response of 20 smoked pork belly samples, measured in induction equivalents (IEQ).
FIGURE 3. Correlation between the in- duction equivalents (IEQ) to 2,3,7,8-tetra- chlorodibenzo-p-dioxin (TCDD) analyzed in the CALUX-assay in 6 h of incubation of 20 samples of pork belly, with their lev- els of benzo[a]pyrene (a) and DBA (b) de- termined by GC-MS.
sonable reproducibility measured in the GC-MS (variation
⬍10% by extracting the same pork belly sample four times). They are important PAHs, being classified as car- cinogens (International Agency for Research on Cancer
(16) and the International Program on Chemical Safety, World Health Organization(21)); a carcinogenicity of these compounds after oral uptake is described (5). Therefore, they are important for the risk assessment of smoked meat.
The Scientific Committee on Food examined B[a]P as a marker of occurrence and effect of the carcinogenic PAHs in food. Thus, they reported that B[a]A, benzo- [b⫹j⫹k]fluoranthene, indeno[1,2,3-cd]pyrene and DBA have a low variability to B[a]P. European Regulation 208/
2005 allows these to be used as a marker of the total amount of carcinogenic PAHs in a food sample (3). Jira (10)describes B[a]P dominating the toxicity equivalents of meat samples, with an amount of more than 0.15 g/kg B[a]P.
Therefore, we compared the induction equivalents of the bioassay results with the amount of B[a]P and DBA:
Figure 3a for B[a]P and Figure 3b for DBA. The correlation is positive with a coefficient of determination ofR2⫽0.85 for B[a]P andR2 ⫽ 0.84 for DBA. This indicates a com- parability of the result of the CALUX bioassay, with the amounts of these individual important carcinogenic PAHs determined by chemical analysis.
The AhR related specificity of the bioassay does not allow identification of the specific nature of the chemicals that are responsible for the enhanced signal. The assay does not analyze individual substances, but those compounds binding to the AhR. Thus, it results in a statement about the effect of the specific compounds in a meat sample onto the receptor.
Applied as a prescreening method, it serves to deter- mine whether the contaminants in an unknown meat sample are above a given threshold. This assessment is possible by comparing the bioassay results with those of positive con- trols with a known amount of contaminants.
The activation of the AhR by other ligands like dioxin and dioxin-like compounds could be distinguished from the activation by PAHs. Vondracek et al.(20)reported that the PAH-induced luciferase production decreases after 6 h of exposure and is no longer detectable after 24 h, whereas the dioxin-induced luciferase production is still detectable.
Therefore, a distinction of the receptor activation by bio- degradable compounds such as PAHs is possible from the response of relatively persistent compounds like dioxins (20).Therefore, this method is recommended for samples which may contain dioxins.
Calculating the theoretical IEQ of the food samples by using the induction equivalent factors (IEFTCDD) described by Machala et al.(12),a reasonable correlation to the total measured CALUX IEQ of the samples was found. The co- efficient of determination yielded R2 ⫽ 0.91 for the cal- culation using the EC50values, andR2⫽0.92 for the cal- culation with EC25 values (12)—another indication for comparability of results of the two investigated methods.
The CALUX bioassay can be an applicable, effect- directed, and functional prescreening method for analyzing PAHs in smoked meat. The bioassay enables an estimation of the biological activity of all compounds acting by bind- ing to the AhR. As the bioassay is based on a biologically relevant mechanism of action, it can provide an indication of the mechanism-specific, biological potency of a sample (18).The laborious further chemical analyses would be lim- ited to those samples showing results above a certain value in the bioassay. This threshold should be established in fur-
ther research, including a full validation using intra- and interlaboratory ring trials. This would be an advantage for food control laboratories in the future. B[k]F and TCDD are both useful as standards for calculating the dose- response curve. This would be beneficial because the toxic TCDD may not been necessary for screening tests. The extraction and cleanup method described in this study is a useful method and the recovery rate of the included internal standard is more than satisfying. However, further research is necessary to simplify and accelerate the extraction and cleanup method, since this part of analyzing the samples is still time-consuming.
ACKNOWLEDGMENT
Our special thanks go to the Fritz-Ahrberg Foundation, Hannover, Germany, for its generous financial support.
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Kapitel 3
Effect-based and chemical analysis of polycyclic aromatic
hydrocarbons in smoked meat - a practical food monitoring approach
accepted on 24th February 2009 by Journal Food Additives and Contaminants
KERSTIN KUHN1∗, BERNHARD NOWAK1, ANDREAS BEHNKE2, ALBRECHT SEIDEL2 AND ALFONSO LAMPEN3
1 Institute for Food Quality and Food Safety, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, Hannover, D-30173, Germany
2 Biochemical Institute for Environmental Carcinogens, Prof. Dr. Gernot Grimmer- Foundation, Lurup 4, Großhansdorf, D-22927, Germany
3 Department of Food Safety, Federal Institute for Risk Assessment, Thielallee 88 – 92, Berlin, D-14195, Germany
Anteil der Erstautorin an der Arbeit:
Versuchsplanung, Probenerstellung und Probenahme, Aufarbeitung und Aufreinigung aller Proben, Durchführung der wirkungsorientierten Analytik, Auswertung der Ergebnisse, Erstellung des Manuskriptes
∗ Corresponding author Kerstin Kuhn, E-mail: ker.kuhn@gmail.com
Abstract
Polycyclic aromatic hydrocarbons (PAHs), which are generated by heat treatment and smoke curing of meat, pose a risk to human health. At present, the determination of these unwanted contaminants requires costly, time-consuming chemical analysis of smoked meat. An alternative is effect-directed high throughput bioassays, which could also be used as a prescreening method. We recently adapted the in vitro chemical-activated luciferase expression (CALUX) assay as a rapid, sensitive and inexpensive screening technique for compound such as dioxins, polychlorinated biphenyls and PAHs. The aim of the present study was to apply a practical approach under realistic conditions. Custom- made meat samples produced under defined conditions with different PAH levels were analysed using this bioassay and gas chromatography-mass spectrometry to determine the influence of different smoking conditions (temperature and duration) on PAH levels.
We found that cold smoking for up to six hours did not result in strong PAH contamination, whereas hot (65 °C) and longer smoking caused a considerable increase in both the bioassay response and the levels of 31 individually determined PAHs. The response in the effect-based bioassay was in good accordance to the values of chemical analysis and the bioassay made it possible to determine accurately the degree of contamination. Our results show that this assay is suitable for high throughput screening for unknown levels of toxicologically relevant PAHs in meat samples and is sensitive enough to differentiate between different PAH levels generated under various smoking conditions. Effect-based screening techniques therefore provide a new instrument for official food monitoring.
Keywords
Smoked meat, polycyclic aromatic hydrocarbons (PAHs), CALUX, bioassay, effect- directed analysis
Introduction
Polycyclic aromatic hydrocarbons (PAHs) are a widespread group of environmental contaminants, many of which, including the representative compound benzo[a]pyrene (B[a]P), are known to exert teratogenic, mutagenic and carcinogenic effects (Guillen and Sopelana 2003; Shaw and Connell 1994). PAHs in food may result from their uptake from a contaminated environment or from food processing (Gomaa et al. 1993). In general, PAHs are formed as complex mixtures during heat treatment processes such as smoke curing, barbecuing, deep frying, or drying and they contaminate food by direct contact with these combustion products. Contamination of food during the smoking process is highly variable and depends on the composition of the wood, the type of generator (internal or external), oxygen accessibility, the temperature of smoke generation and smoking time (European Union 2002; Simko 2005).
At the moment, PAHs in smoked meat samples are determined by costly, time-consuming chemical analysis. Toxic equivalence factors (Nisbet and LaGoy 1992) or potency equivalency factors (Collins et al. 1998) for individual PAHs as to their carcinogenic potency relative to B[a]P are used for risk assessment of PAH mixtures in smoked meat and other heat-treated food items. We adapted an effect-based screening assay using the in vitro chemical-activated luciferase gene expression (CALUX) assay (Kuhn et al. 2008).
This bioassay can evaluate the total activity of the sample compounds that interact with the aryl hydrocarbon receptor (AhR). The AhR mediates most of the toxic effects of dioxins, polychlorinated biphenyls and PAHs (Guillen and Sopelana 2003). The total carcinogenic potential is in good accordance with AhR-mediated activities of individual PAHs both in vitro (Machala et al. 2001) and in vivo (Shimada et al. 2002). Following the binding of the ligand to the receptor, the resulting ligand-receptor complex is activated and translocated to the nucleus, where it binds to a dioxin-responsive element (DRE) and stimulates the transcription of target genes including those of PAH-metabolising enzymes such as cytochrome P450 1A1 (Denison and Heath-Pagliuso 1998). The CALUX assay is based on a genetically engineered cell line (rat hepatoma H4IIE cells) which contains a stable transfected vector including the firefly luciferase gene under the transcriptional control of several DREs. This transgenic cell line responds to ligand exposure with the induction of the luciferase, which in the presence of its substrate produces a luminescent
signal proportional to the cell's response. Because of its specifity to AhR, this bioassay does not allow identification of the chemicals that are responsible for the enhanced signal but it does provide information about the effect of the specific compounds in a meat sample on the receptor.
Effect-directed analysis (EDA) is routinely used for screening of environmental contaminants occurring in soil, water and air (Giesy et al. 2002, Vondracek et al. 2001) and it should also be possible to use it as a pre-screening method to test smoked meat samples, contaminants and the potency of both known and unknown ingredients. Such a pre-screening method can handle a high sample throughput and analyse large numbers of samples for the presence of potentially toxic compounds, thus improving food monitoring and consumer safety.
In a previous study we showed that higher molecular weight PAHs, particularly B[a]P and dibenzo[a,h]anthracene (DBA), could be detected by the CALUX assay (Kuhn et al. 2008).
The potency of the meat samples to cause a defined biological response mediated by the AhR activity of the PAHs present in the sample was expressed relative to the response of the two standards, benzo[k]fluoranthene or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Kuhn et al. 2008). The biological potency of the contaminated meat samples was expressed as induction equivalents (IEQs).
The aim of our present study was to apply a practical approach using an effect-based screening assay under real-life conditions with custom-made meat samples with different degrees of PAH accumulation. The robustness of this method was investigated both in terms of the influence of smoking conditions (temperature and duration) on PAH levels and in regard to their distribution in the smoked meat sample. The robustness of the bioassay was confirmed by the analysis of 31 individual PAHs which were determined in every meat sample by gas chromatography-mass spectrometry (GC-MS).
Material and methods
Samples
Grade E (premium) raw pork bellies were purchased from a local slaughter house and smoked in an FPC100 smoking chamber (Fessmann, Winnenden, Germany) using beechwood shavings. Five types of samples were produced: unsmoked products, products smoked cold (for 3 or 6 h) and products smoked hot (for 1 or 3 h). The raw pork bellies were deboned and the meat was rubbed with a mixture of curing salt and white pepper to yield final concentrations in the meat of 2% and 0.2%, respectively. The bellies were packed in polyamide-polyethylene foil under vacuum and stored for two weeks at 7 °C.
The salt was removed by washing and the bellies were dried for two days at 7 °C. Two types of indirect smoking were applied: cold (18 °C) and hot (65 °C). Smoke was generated in a separate chamber from which the smoke was passed onto the meat. The pork bellies were then stored at 7 °C for four days before sampling. The unsmoked pork bellies were prepared (deboned, rubbed, packed, washed) and stored in the same way and served as negative controls.
According to Toth (1971), PAH accumulation takes place in the outer part of a meat sample. We therefore used two sampling regions, the "middle" and "edge". The middle samples were taken more than 1 cm from the edge of the pork belly. The smoke thus reached the pork belly only from the top and the bottom, but not from the sides. The edge samples were taken from the outer 1 cm of the pork belly.
All 30 samples of pork belly (100 g each) were minced and blended in a chopper (Rondo 1000, Tefal, Offenbach, Germany), packed in aluminium boxes, frozen and stored at -18 °C until the extractions were performed.
Five 10-g aliquots (four for the bioassay and one for the GC-MS) were collected from each of the 30 pork belly samples and prepared for further analysis. Although a recently published study of ours showed that one aliquot would be sufficient for meat monitoring, we prepared more to ensure reproducibility of the bioassay (Kuhn et al. 2008). All aliquots were prepared in the same manner, with particular care taken to assure equal reaction and storage times.
