--~--------------------------

Metabolism of Xenobiotics in theIncubated Hen's Egg: Investigations~ith Ethyl 4-hydroxybenzoateLutz Kiep ,D-Brunow

SummaryIt is well known that in vitro preparations of the chick embryocan functionalise and conjugate selected model substances.Based on this biochemical knowledge an ex vivo model was de-veloped to study xenobiotic metabolism in the incubated hen'segg. The xenobiotic is injected into the yolk sac, i.e. the nutri-tional compartment, on day 6 and metabolites are identified inthe excretion medium of the embryonie kidneys (allantoic fluid)on day 11. During this developmental period the embryo lacks01' has very limited sensitivity. Thus the model is in accordancewith the 3R concept.In the present investigation ethyl 4-hydroxybenzoate was chosenas a test substance. Concentrations of this paraben up to 24mg/egg did not affect embryo viability. After inoculation of 6mg/egg, 4-hydroxybenzoic acid and 4,4'-dihydroxy-L-ornithuricacid were identified in theirfree form. The 4-hydroxybenzoic acidwas also eliminated in its conjugatedform (glucuronide and/orsulphate). No unchanged paraben was excreted. 4,4'-dihydroxy-L-omithuric acid [2,5-bis-( 4-hydroxybenzoylamino )pentanoicacid] is a new metabolite. The structure of this amino acid con-jugate was elucidated by synthesis and spectral methods (MS, 'nand 13CNMR).

Zusammenfassung: Metabolismus von Xenobiotica im bebrütetenHühnerei - Untersuchungen mit Ethyl-4-hydroxybenzoatEs ist seit langem bekannt, dass ausgewählte Modellsubstanzenmit in vitro Präparationen des Hühnerembryos funktionalisiertund konjugiert werden können. Auf der Grundlage dieser bio-chemischen Kenntnis wurde ein Modell zur Untersuchung desMetabolismus von Xenobiotica am bebrüteten Hühnerei ent-wickelt. Das Xenobioticum wird am 6. Tag in ein ernährendesKompartiment des Bruteies (Dottersack) injiziert, und dieMetaboliten werden am 11. Tag im Exkretionsmedium der em-bryonalen Nieren (Allantoisflüssigkeit) identifiziert. Innerhalbdieser Entwicklungsperiode ist von einer fehlenden odereingeschränkten Sensitivität des Embryos auszugehen, wodurchsich das Modell in Übereinstimmung mit dem 3R Konzeptbefindet.Für die vorliegende Untersuchung wurde Ethyl-ä-hydroxy-benzoat als Testsubstanz gewählt. Bis zu einer Konzentrationvon 24 mg/Ei beeinträchtigte das Paraben die Lebensfähigkeitder Embryonen nicht. Nach Inokulation von 6mg/Ei wurden dieMetaboliten 4-Hydroxybenzoesäure und 4,4'-Dihydroxy-L-ornithursäure in freier Form identifiziert. Die 4-Hydroxyben-zoesäure wurde auch in konjugierter Form (Glucuronidund/oder Sulfat) eliminiert. Eine Exkretion unveränderter Aus-gangssubstanz erfolgte nicht.Die 4,4'-Dihydroxy-L-ornithursäure [2,5-Bis-(4-hydroxyben-zoylamino)pentansäure] ist bisher nicht beschrieben worden. DieStruktur dieses Aminosäure-Konjugates wurde durch Syntheseund spektrale Methoden (MS, 'nund 13CNMR) aufgeklärt.

Keywords: 3R, hen's egg, chick embryo, xenobiotic metabolism, ethyl4-hydroxybenzoate

1 Introduction

Xenobiotic metabolism studies are per-formed routine1y in laboratory animals.Numerous in vitro systems have beenestablished for this purpose in the pastfew years (Worth and Balls, 2002;Gruber and Hartung, 2004). These in-clude: (i) isolated and cultured hepato-

cytes; (ii) precision-cut liver slices; (iii)subcellular liver fractions; and (iv) het-erologous expression systems. The avianembryo appears to represent another pos-sible tool. In vitro preparations of thechick embryo can functionalise and con-jugate selected model substances. The in-cubated hen's egg has previously been ex-arnined for its ability to ornithine conjuga-

Received 20 April 2005; received in final form and accepted for publication 26 May 2005

ALTEX 22, 3/05

tion and N-acetylation (Tab. 1). Speciesdifferences appear to be quantitativerather than qualitative except for theamino acid conjugation. Based on thisbiochernical knowledge an ex vivo modelof the incubated hen's egg was developedto study xenobiotic metabolism (Kiepand Bekemeier, 1986). The xenobiotic isinjected into the yolk sac, i.e. the nutri-tional compartment, on day 6 (d6) andmetabolites are identified in the excretionmedium ofthe embryonie kidneys (allan-

135

KIEP ~---------------------------------------~---

toic fluid, AF) on day 11. Depending onthe stage of development, the incubatedhen's egg can be used both conservative-ly - in the sense of an animal experiment- and alternatively as a complementarymethod. Up to the end of the first half ofthe incubation period the embryo lacksor has vefy limited sensitivity (Peters etal., 1956). Investigations should be re-stricted to this period to avoid damage tothe embryo (Rosenbruch, 1997). Conse-quently, this refinement is also in accor-dance with the 3R concept (Russel andBureh, 1959). A schematic overview ofthe model to study xenobiotic meta-bolism is presented in Figure 1.Previous investigations (Kiep and

Bekemeier, 1986; Kiep, 1997; Kiep etal., 2002) on this model were carried outwith the slightly water-soluble sub-stances sodium salicylate (metabolie re-actions: aromatic hydroxylation, O-glu-euronidation) and metamizol-sodium(metabolie reactions: aliphatic hydroxy-lation, deamination, N-acetylation, 0-glucuronidation, O-sulphation). Themethod is evidently well suited to thestudy of these hydrophilie compounds.Also the main metabolie pathways thesemodel substances take in the egg corre-

spond to those taken in animals and hu-mans. The fundamental suitability of thismodel was confirmed by investigationswith (+ )-methamphetamine (Neugebauer,1995), 7-ethoxycoumarin (Clement andMladek, 1995), p-nitrophenol (Clementand Mladek, 1996) and SFZ-47 (prodrugof an anti-inflammatory and analgesieagent) (Dong et al., 2003).As the biokinetics of xenobiotics par-

tially depend on their physico-chemicalproperties, the poorly water-soluble ethyl4-hydroxybenzoate was chosen as a testsubstance for the following studies. Thisparaben is widely used as an antimicro-bial preservative in pharmaceuticalpreparations, food products and cosmet-ics. Studies in rats (Derache and Gour-don, 1963; Kiwada et al., 1979), rabbits(Tsukamoto and Terada, 1964), cats(Phillips et al., 1978), dogs (Jones et al.,1956) and humans (Heim et al., 1957)have shown that the substance is ab-sorbed rapidly, metabolised and elimi-nated. Corresponding results were foundin humans with the substrate propyl 4-hydroxybenzoate (Sabalitschka andNeufeld-Crzellitzer, 1954). Urinary ex-cretion of unchanged ethyl 4-hydroxy-benzoate is very low, usually less than

Tab. 1: Biotransformation of selected model substances in the chick embryo

1% of the administered dose. After esterhydrolysis, the main metabolites are the4-hydroxybenzoic acid and 4-hydroxy-hippuric acid in their free form. 4-hy-droxybenzoic acid is also eliminated inits conjugated form (ester glucuronide,ether glucuronide, ether sulphate). Thediglucuronic acid conjugate of 4-hydrox-ybenzoic acid was identified in dog urine(Quick, 1932). Protocatechuic acid(3,4-dihydroxybenzoic acid) was foundin rabbit urine after application of 4-hydroxybenzoic acid (Bray et al., 1950).In rats and rabbits, the protocatechuicacid is partly methylated to vanillic acid(4-hydroxy-3-methoxybenzoicacid) (DeEdset al., 1957). The possible metaboliepathways of ethyl 4-hydroxybenzoate inanimals and humans are summarised inFigure 2.

2 Materials and methods

2.1 Instrumentsand chemiealsThe ESI (electrospray ionisation) massspectrum (MS) was obtained with a Mi-cromass LCT spectrometer. NMR (nu-clear magnetic resonance) spectra wererecorded in CD30D on a Bruker AC 300

Reaction Substrate Source Reference

Phase I

Aromatic hydroxylation Aniline Liver microsomes Powis et al., 1976

Aromatic N-heterocyclic Antipyrine Liver microsomes Rifkind et al., 1982hydroxylation

Epoxidation Benzo[a]pyrene Culture of hepatocytes Topp and van Bladeren, 1986

N-dealkylation Aminopyrine Culture of hepatocytes Poland and Kappas, 1971

O-dealkylation o-Nitroanisole Liver microsomes Strittmatter and Umberger, 1969

Oxidative deamination Tryptamine Liver homogenate Ignarro and Shideman, 1968

S-oxidation Chlorpromazine Liver microsomes Brodie and Maickel, 1962

Phase 11

O-glucuronidation o-Aminophenol Liver slices and liver Dutton and Ko, 1966homogenates

O-sulfation 4-Methylumbelliferone Embryo homogenate Collett and Ungkitchanukit, 1979

N-acetylation 0-, m- and Incubated hen's egg Wolfe and Huang, 1959p-Aminobenzoic acid

O-methylation Epinephrine Liver cytosol Ignarro and Shideman, 1968

Amino acid conjugation Benzoic acid Incubated hen's egg Takahashi, 1928(ornithine)

Glutathione conjugation Bromosulfophthalein Liver homogenate Brauer et al., 1963

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~ ~--~--------------------------

spectrometer at 300.13 MHz eH) and75.47 MHz (l3C); solvent signals wereused as internal standards eH: 3.30, 13e:49.0). Ethyl 4-hydroxybenzoate (phar-maceutical grade, purity 99.6%, Lot-NI.0211A153) was purchased from Syn-opharm (Barsbüttel, Germany). Theethyl 4-hydrox,benzoate was dissolvedin 1,2-propanediole to evaluate the sub-strate dose and for metabolism studies.Protocatechuic acid (3,4-dihydroxyben-zoic acid) and vanillic acid (4-hydroxy-3-methoxybenzoic acid) were obtainedfrom Serva (Heidelberg, Germany). 4-Hydroxybenzoic acid, 1,2-propanediole,ß-glucuronidasefarylsulphatase, Extrelut®NT3-prepacked glass columns and PLCplates (silica gel 60 F25J1mm with con-centrating zone) were from Merck(Darmstadt, Germany). 4,4' -Diamino-L-ornithuric acid was synthesised ac-cording to a known methodology (Srnith,1958). All other chemieals were obtainedfrom commercial suppliers and usedwithout further purification. The follow-ing abbreviations are used: CD30D:methanol-da; Et-O: diethyl ether; EtOAc:ethyl acetate; EtMeCO: 2-butanone;HOAc: acetic acid; MeOH: methanol.

2.2 Synthesis of 4,4'-dihydroxy-L-ornithuric acid3 mL of 4 M HCl were chilled to 0° C inan icelNaCl bath. Under stirring, a solu-tion of 185 mg (0.5 mM) 4,4'-diarnino-L-ornithuric acid and 70 mg (1 mM)sodium nitrite in 0.5 M NaOH (1 mL)was added dropwise at a temperature be-low 5° C. Five rninutes after the last ad-dition the reaction rnixture was checkedfor free nitrous acid (iodine-starch paper)and the surplus was removed with asmall amount of urea. Then the rnixturewas heated in a boiling water bath untilnitrogen formation stopped. After cool-ing to room temperature the rnixture wasadjusted with 2 M NaOH to pH 2-3. Sol-id NaCl (1.5 g) was added to the rnixtureand then extracted exhaustively withEtzOlEtOAc (1:1). The organic layer wasdried with Na2S04 and after that the sol-vent was distilled off in vacuo. Theresidue was dissolved in MeOH (500 !1L)and purified with the solvent systemCHCbfMeOHfHOAc (40:10:0.5; byvol.) by preparative TLC (thin layerchromatography).

ALTEX 22, 3/05

- Air chamber

ChorioallantoicmembraneAllantoic hole

Amnion

- Yolk sac

Albumen sac

Administration 01the test substance ~(1 00 ~U e99) at d6

Sampie prepara-==> tion and isolation

01 metabolites

Sam pie collection~ (appr. 5 mL allantoic

ftuid! e99)

Identilication andquantilication 01metabolites

Fig. 1: Sehematie overview of the model for studying xenobiotie metabolism in theineubated hen's egg

Ester hydrolysis - 4-Hydroxybenzoic acidAmino acid conjugation - 4-Hydroxyhippuric acidGlucuronidation _ Ester glucuronide

j:romatic hydroxylation- ProtocatechuicaCid]

~ethylation _ Vanillic acid

Glucuronidation - EtherglucuronideSulfation _ Ether sulfate

Fig. 2: Proposed metabolie pathways of ethyl 4-hydroxybenzoate in animals andhumans

ESI-MS: mlz (%) = 417 [M-H+2Nat(100),395 [M+Nat (40), 373 [M+Ht (3);IHNMR: 0 (ppm) = 7.74 (d, J= 8.7 Hz, H-2'1H-6,a), 7.70 (d, J = 8.7 Hz, H-2"1H-6"a), 6.83 (d, J = 8.7 Hz, H-3'1H-5'\ 6.81(d, J = 8.7 Hz, H-3"1H-5"b), 4.49 (m, H-2), 3.40 (m, 2H-5); l3CNMR: 0 (ppm) =179.6 (C-1), 170.0 (C_?'c), 169.1 (C_7"c),162.1 (C_4,d), 161.9 (C_4"d), 130.2 (C-

2' fC-6', C-2" fC-6"), 126.6 (C-1'"), 126.5(C-l "e), 116.0 (C-3' fC-5', C-3" fC-5"),56.2 (C-2), 40.6 (C-5), 31.6 (C-3), 26.9(C-4); a-esignals are exchangeable; num-bering according to Fischer's projection.The following abbreviations are used: M:molecular ion; mlz: mass-to-charge ratio;D: chernical shift; J: coupling constant; d:doublet; m: multiplet.

137

KIEP m....-------------~-

2.3 Bioassay2.3.1 Egg incubation, adminis-tration of the test substanceandsam pie collectionFertilised "White Leghorn" eggs fromfree-range hens were obtained from a 10-cal supplier (E. Schmoll, Klein Krams,Germanye, The eggs were first candledand defective eggs were discarded. Theweight of the eggs ranged from 52 g to60 g. Prior to incubation, the eggs werestored at 10° C and 90% relative humidi-ty for 24 h with their large ends up. Theeggs were incubated horizontally in acommercial incubator at 37.8°C (± 0.2"C)and 65% (± 2%) relative humidity andturned every 6 h. At d6 the eggs werecandled and infertile eggs were discard-ed. Using a dentist's drill, a 1-2 mm di-ameter hole was made in the shell direct-1yover the air cell, without damaging thesubjacent shell membrane. Two hoursbefore injection, the eggs were stored inthe incubator in an upright position withthe large ends up. Then, the blunt end ofeach egg was cleaned with ethanol (70%)and 0.1 mL of the test solution (6 mgethyl 4-hydroxybenzoate) was injectedvertically into the yolk sac; controls weretreated with 0.1 mL of the vehicle (1,2-propanediole) only.After sealing the holewith melted paraffin, the eggs were fur-ther incubated in a horizontal positionand candled daily for viability. On dllthe bioassay was terminated by placingthe eggs in a freezer at -18° C for 30 min.After thawing to room temperature, theeggshell over the air chamber was pre-pared off and the inner egg membranewas removed. Then, the chorioallantoicmembrane was penetrated with a syringeand theAF (5.5-7.2 mL/egg) was aspirat-ed (Fig. 1).

2.3.2 Evaluation of thesubstrate doseEmbryonated eggs (d6) were divided intofive experimental groups and one controlgroup, with five eggs in each group. Theeggs in the experimental groups were in-jected with 0.1 mL of ethyl 4-hydroxy-benzoate solutions containing l.5 mg, 3mg, 6 mg, 12mg and 24 mg, respectively.The control group was inoculated with thevehicle (1,2-propanediole) only. After ex-arnination (d11) the toxicological test wasended by freezing the eggs.

138

2.4 Bioanalysis2.4.1 Sampie preparation andisolation of metabolitesFor the extraction of metabolites, the AFof 5 eggs was combined. Five millilitresof the pooled AF were adjusted to pH 2-3 with 1 M HCl and extracted exhaus-tively with EtzOlEtOAc (1:1). The com-bined organic layers were dried withNa2S04 and evaporated to dryness invacuo. For TLC (2.4.2) the residue wasdissolved in 100 flL MeOH. The aqueouslayer was neutralised with 1 M NaOHand hydrolysis was performed for thedetection of conjugated metabolites un-der the following conditions: (i) After ad-dition of 500 ul, of acetate buffer (pH5.5), the solution (5 mL) was stirred andincubated with ß-glucuronidase/arylsul-phatase (100 flL) at 37.5- 38.5° C for24 h. (ii) After addition of 500 ul, ofHCl (37%), the solution (5 mL) wasstirred and heated for 1 h in a boilingwater bath. The hydrolysed fluids wereadjusted to pH 2-3 and injected ontoExtrelut® NT3-columns. The elutionof the metabolites was performed withEt20lEtOAc (1:1) according to the man-ufacturer's instructions. Then, the eluateswere worked up as described before.Sampies of control AF (5 mL) from un-injected eggs were treated the same.

2.4.2 Identification ofmetabolitesThe identification of metabolites oc-curred by comparative TLC with the au-thentic reference substances in differentsolvent systems (Tyman, 1996; Wiltshire,2000). TLC analyses were carried out onsilica gel 60 F254aluminium sheets (Mer-ck). I-SilL of sample (2.4.1) were ap-plied to a length of 5-15 mm and thechromatogram developed over a path of10 cm in a paper lined chamber, previ-ously left to equilibrate for at least 30min at room temperature. The spots werevisualised by UV absorption at 254 nrn.

3 Results

Inoculation of ethyl 4-hydroxybenzoatein concentrations up to 24 mg/egg didnot affect the viability of the embryos. Adose of 6 mg/egg was chosen for themetabolism studies and two compounds

in their free form were detected in theAF. Excretion of unchanged paraben wasnot detected. The metabolites could beidentified unambiguously as 4-hydroxy-benzoie acid (major) and 4,4' -dihy-droxy-L-ornithuric acid (minor) by com-parative TLC with the authentiereference substances in three differentsolvent systems (Tab. 2). Regarding theresults of enzymatic hydrolysis withß-glucuronidase/arylsulphatase, the 4-hydroxybenzoic acid is also eliminatedin its conjugated form (glucuronideand/or sulphate). There were no indica-tions for the presence of protocatechuicacid and vanillic acid. The metabolicprofile of ethy14-hydroxybenzoate in thechick embryo is shown in Figure 3.The 4,4' -dihydroxy-L-ornithuric acid

[2,5-bis-(4-hydroxybenzoylamino )pen-tanoic acid] is a new metabolite. Depart-ing from 4,4' -diarnino-L-ornithuric acid(Smith, 1958), the synthesis was per-formed by diazotation of the primaryaromatic amino groups and subsequenttransformation of the diazonium salt todihydroxyornithuric acid. The structurewas confirmed by mass spectrometry andNMR spectroscopy. The ESI-MS showsa [M-H+2Nar ion at mfz 417. The IHand 13C NMR data are in agreementwith the structure of 4,4' -dihydroxy-L-ornithuric acid (2.2).

4 Discussion

The urinary excretion of unchanged ethyl4-hydroxybenzoate is very low in ani-mals and humans since the ester linkageis hydrolysed readily. This is also thecase in the incubated hen's egg, in whichunchanged paraben was not detected.The metabolie profile of ethy14-hydrox-ybenzoate in the chick embryo corre-sponded to that in animals and humans(Fig. 2 and Fig. 3). Consequently, themodel seems to be suitable for the inves-tigation of the metabolism of thelipophilic and poorly water-soluble testsubstance. However, species differencesexist in the conjugation of aromatic car-boxylic acids with endogenous aminoacids (Williams, 1967).In mammals, the favoured conjugation

partner is glycine (Steventon and Hutt,2002). Most birds on the other hand con-

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jugate aromatic carboxylic acids with or-nithine (Jaffe, 1877; McGilvery and Co-hen, 1950; Baldwin et al., 1960; Marshalland Koeppe, 1964; Bridges et al., 1970;Igarashi et al., 1992). This difference inthe conjugation behaviour appears to bedue to the difference in the major endproduct of nitregen metabolism in mam-mals, i.e. urea, and birds, i.e. uric acid(Williams, 1967; Jondorf, 1981; Steven-ton and Hurt, 2002). Uric acid is charac-teristicaIly produced by terrestrialspecies that develop within a sheIl inwhich nitrogenous waste products mustbe stored in an insoluble form. Since thebiosynthesis of uric acid involves glycineas aprecursor, it is conceivable that thenon-proteinic amino acid ornithineserves as an alternative conjugation part-ner. Furthermore, the connection be-tween the form of the N-excretion andthe deamination of the proteolytic aminoacids is striking. Mammals form ammo-nia by transdeamination, birds by oxida-tive deamination. The deamination ofamino acids and the biosynthesis of uricacid during the ontogenesis of the chickembryo in the incubated hen's egg areweIl described (Drel, 1965; Emmanueland Gilanpour, 1978).The conjugation of benzoic acid

(Takahashi, 1928) and of the aminoben-zoic acid isomers (Wolfe and Huang,1959) with ornithine is already possiblein the chick embryo. Ornithine conjuga-tion was detected on d6, just one day af-ter uric acid excretion begins (Wolfe andHuang, 1959). This amino acid conjuga-tion with a hydroxybenzoic acid isomerhas not been described previously in thechick embryo. Recently, 2,2' -dihydroxy-ornithuric acid was identified in theexcreta of broiler chickens after applica-tion of 2-hydroxybenzoic acid (Hillaertet al., 2003). The occurrence of this or-

nithuric acid in the AF after inoculationof salicylic acid into the incubated hen'segg has been discussed previously (Kiepand Bekemeier, 1986). Taken together,next to glucuronidation the conjugationof aromatic carboxylic acids with anamino acid is an important phase 11reac-tion of xenobiotic metabolism in thechick embryo. Also, the further phase 11reactions (Tab. 1) indicate that enzymeactivities already appear in the first halfof the incubation period. Moreover, theability of the chick embryo to performoxidative biotransformation prematurelyis evident (Hamilton et al., 1983; Brun-ström, 1986; Heinrich-Hirsch et al. 1990).In summary, the model of the incubat-

ed hen's egg is a highly organised testsystem for xenobiotic metabolism stud-ies. The crucial advantage of the modelexists in the consideration of biokinetic

aspects. After inoculation of the xenobi-otic into the nourishing compartment ofthe egg, absorption, distribution,metabolism and excretion (ADME) oc-cur as in a mature organism. The modelincludes both phase I and phase 11 reac-tions of metabolism and only thebioavailable foreign compound ismetabolised. In spite of interspecies dif-ferences, indications of the metabolicprofile ofaxenobiotic can be gainedfrom the model. Consequently, thismodel could lead to the reduction of an-imal experiments in this area of xeno-biochemistry.

ReferencesBaldwin, B. C., Robinson, D. andWiIliams, R. T. (1960). Studies indetoxication - 82. The fate of benzoic

Ethyl4-hydroxybenzoate

~H

OB

Glucuronidation/Sulfation

Conjugates

4-Hydroxybenzoic acid

j Amino acidconjugation

d;LzXHO ~

OH

4,4'-Dihydroxy-L-ornithuric acid

Fig. 3: Metabolie profile of ethyl 4-hydroxybenzoate in the chick embryo

Tab. 2: TLC data (RF values) of ethyl 4-hydroxybenzoate and its metabolites in different solvent systems (silica gel 60 F254,visualisation by UV absorption)

CHC 13/EtOAc/ HOAc CHCI3/MeOH/ HOAc CHCI3/EtMeCO/ HOAc/H20(10:8:2; by vol.) (16:4:0.2; by vol.) (8:8:4:1; by vol.)

Ethyl4-hydroxybenzoate 0.63 0.67 0.79

4-Hydroxybenzoic acid 0.53 0.56 0.68

4,4'-Dihydroxy-L- ornithuric acid 0.06 0.13 0.32

ALTEX 22, 3/05 139

KIEP m.....-------------~-

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AcknowledgementThe author is thankful to Prof. Dr. Karl-heinz Seifert, Organic Chemistry, NW II,University of Bayreuth, Germany, forproviding spectral data (MS, lH and l3CNMR) of the 4,4' -dihydroxy-Lvor-nithuric acid and for helpful discussions.

Correspondence toDr. Lutz KiepRingstr.4D-19372 BrunowlMecklenburgGermanye-mail: lutz.kiep@t-online.de

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