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1 ni et. FISHERIES RESEARCH BOARD OF CANADA Translation Series No Carotenoids of human serum and liver. I. By H. Willstaedt and T. Lindqvist Original title: Uber die Carotinoide des Serums und der Leber beim Menschen. I. From: Zeitschrift fur Physiologische Chemie (Journal of Physiological Chemistry), 240 : 10-18, Translated by the Translation Bureau(MV) Foreign Languages Division Department of the Secretary of State of Canada 14 pa Fisheries Research Board of Canada Halifax Laboratory Halifax, N.S. 1970

2 Ftea /4-01 DEPARTMENT OF THE SECRETARY OF STATE TRANSLATION BUREAU FOREIGN LANGUAGES DIVISION CANADA SECRÉTARIAT D'ÉTAT BUREAU DES TRADUCTIONS DIVISION DES LANGUES ÉTRANGÈRES TRANSLATED F.ROM - TRADUCTION DE Perman INTO - EN English AUTHOR - AUTEUR WILLSTAEDT, H. and T. LINDQVIST -TITLE IN ENGLISH - TITRE ANGLAIS 'The carotenoids of human serum and liver. I. Title in foreign language (transliterate foreign charaoters) Über die Carotinoide des Serums and der Leber beim Menschen. I.. R5URENCE IN FOREIGN I,ANGUAGE (NAME OF BOOK OR PUBLICATION) IN FULL. TRANSLITERATE'FOREIGN CHARACTERS. REFERENCE EN LANGUE ETRANGÉRE (NOM DU. LIVRE OU PUBLICATION), AU COMPLET. TRANSCRIRE EN CARACTÉRES PHONETIQUES. Zeitschrift für Physiologische Chemie REFERENCB IN ENGLISH - RÉFÉRENCE EN ANGLAIS Journal of Physiological Chemistry PUBLISH ER - EDITEUR not stated PLACE, OF PUBLICATION LIEU DE PUBLICATION Germany YEAR ANNÉE IBM DATE OF PUBLICATION DATE DE PUBLICATION VOLUME 240 ISSUE NO. NUMÉRO ma. MM. egle PAGE NUMBERS IN ORIGINAL NUMÉROS DES PAGES DANS L'ORIGINAL NUMBER OF TYPED PAGES NOMBRE DE PAGES DACTYLOGRAPHIEES.14 REQUESTING DEPARTMENT MIN IST ERE-CLIENT Fisheries & Forestry TRANSLATION BUREAU NO. NOTRE DOSSIER N BRANCH OR DIVISION DIRECTION OU DIVISION Fisheries Research Board, Halifax PERSON IREQUESTING Dr. W.H. Owen DEMANDE PAR TRANSLATOR (INITIALS) TRADUCTEUR (INITIALES) DATE SOMPLETED ACHEVE LE MV YOUR NUMBER VOTRE DOSSIER N O DATE OF REQUEST DATE DE LA DEMANDE UNEDITED DRAFT TRANSLATION Only for information TRADUCTION NON REVISÉE Information seulement SOi(:...200;10-6 ( R EV. 2/813)

3 it-b lek),.department OF THE SECRETARY OF STATE TRANSLATION BUREAU FOREIGN LANGUAGES DIVISION SECRÉTARIAT D'ÉTAT BUREAU DES TRADUCTIONS DIVISION DES LANGUES ÉTRANGÈRES. CANADA CLIENT'S NO, DEPARTMENT DI VISI ON/BRANCH CITY No DU CLIENT MINISTERE DIVISION/DIRECTION VILLE Fisheries & Forestry Dr. W.H. Owen, FisherieE Research Board Halifax BUREAU NO. LANGUAGE TRANSLATOR (INITIALS) No DU BUREAU LANGUE TRADUCTEUR (INITIALES) DATE 1946 German MV Zeitschrift für Physiologische Chemie, Volume 240, 1936, pp The carotenoids of human serum and - liver. I. By H. WILLSTAEDT and T. LINDQVIST From the Medico-Chemical Institute and the Medical Clinic of Uppsala University (Received for publication March 7, 1936) UNEDITED DRAFT TRANSLATION Only for information TRADUCTION NON REVISÉE Information seulement 22C-10-11

4 2 Studies on the occurrence and distribution of carotenoids in animal, especially human, organs and fluids were carried on already at an early date. For the literature until 1922, reference should be made to the comprehensive monograph by PALMER [1 ]. However, before 1928 no keen interest was shown in determinations of this type. The discovery of chemical and biochemical relationships between carotene, in particular p-carotene, and vitamin A, fa. which we are specially indebted to v. EULER & KARRER [2], naturally increased the interest in the carotenoids. Since 1929 a number of methods for the quantitative determination of serum carotenoids have been described and used, too, almost all of which assume, however, that the total amount of neutral yellow lipid-soluble pigments extracted from the test material is "carotene" [3 ]. On the basis of results obtained by HYMANS VAN DEN BERGH, MULLER & BROCKMEYER [4 ], some authors attribute 1/3-1/2 of the colour intensity to "xanthophyll" [5 ]. When we began our studies, no experiments on the nature of the serum carotenoids existed. In view of the factsthat animal carotenoids are derived from the food ingested, and that a great variety of carotenoids occur in plants, it did not seem very likely from the very start that only p-carotene and xanthophyll should occur in the serum, especially since ZECHMEISTER & TUZSON C61 had found other carotenoids, too, in human fat and liver. In chromatographic studies on large quantitiès of bovine serum, GILLAM & EL RIDI [71 detected 11 cryptoxanthin in addition to carotene and xanthophyll.

5 3 While this manuscript was being prepared, two investigations on this matter were published. SULLMANN, SZCSÉNYI-NAGY & VERZÀR [8] found "only carotene and xanthophylls" when chromatographing serum samples, but the serum quantities used by them (10 cc) are too small, judging from our own experience, to allow for more detailed differentiation of the pigments. v. DI(NIEL & BÈRES [9] chromatographed 3 litres of serum and found xanthophyll, lycopene, and both a- and p-carotenes, also two unknown carotenoids. In our investigations, we wanted to obtain information on changes in the composition of human serum carotenoids; we therefore studied 12 sera, the sample sizes varying from 140 cc to 250 cc. These quantities were large enough to permit satisfactory chromatographic analysis. Most of the blood samples were taken from patients at the Medical Clinic. In four cases where critically ill patients died shortly after blood-letting, the liver was examined, too. Methods Usually the serum samples were worked up according to the procedure suggested by VAN EEKELEN & EMMERIE [10], but for control purposes we employed the alcohol precipitation method for some sera; this method does not require heating (cases 11 and 12). The ether extracts were evaporated in a nitrogen flow. The residue (which always had a high sterol content) was taken up in petroleum ether, 75,-;:.75 -

6 and partition of this solution between petroleum ether and 90% methanol was allowed to take place. The fraction that had migrated to methanol was sublimated to a benzine-benzene mixture by the addition of water. Then chromatography was carried out. (In sone cases we dispensed with the partition between petroleum ether and methanol and chromatographed directly, but the other procedure is recommended.) The livers were examined according to the procedure by BROCKMANN & TECKLENBURG [11]. The benzine extracts were saponified, concentrated, and worked up like the serum extracts. For chromatography, we used mostly alumina as adsorbent (in addition to the product standardizeà by BROCKMANN, the normal Aluminium oxydatum purissimum by Merck also proved fairly useful). We are fully aware of the advantages of calcium hydroxide in cases where the individual layers of the chromatogram are to be widely 12 separated. We,however, wanting to detect very small quantities of carotenoids, aimed at a sharp distinction of the individual zones on the chromatogram, and for this purpose alumina, according to our experience, is vastly superior to calcium hydroxide. Occasionally we did use calcium hydroxide (Calcium hydricum pulvis alcoholisatus, from the firm Gebr. Siegfried, Zofingen, Switzerland). Elution was carried out as usual. For spectroscopy we used a Zeiss evaluating

7 5 grating spectroscope *. Most quantitative carotenoid determinations - were carried out colorimetrically with a Hellige colorimeter against azobenzene according to KUHN & BROCKMANN [12]. In the first two cases we did not use azobenzene as a colorimetric standard, but determined the relative colour intensities of the carotenoid solutions against each other in a step photometer (filter 430). Results p,carotene was found regularly.in serum. It was. completely -. absent only in one case. (tase 5). The carotene contents relative. t.c1 the total amount of carotenoids, on the other hand, varied widely (extreme values 0%:and 55%, mostly 20-40%). Lycopene was detected with certainty only in one serum sample, which, oddly enough, was the same serum that did not contain any carotene (case 5). It may have occurred also in the serum of case 12. Our studies were carried out at a time when relatively few tomatoes are being eaten in Sweden. The patient in whose serum sample lycopene was found, had eaten a large number of tomatoes one week before the blood sample was taken. In another case where tomatoes had been eaten only ca. 7 hours before the blood-letting, no lycopene was found in the serum (case 6). * We are particularly indebted to Dr. Jan WALDENSTRÔM for kindly putting this instrument at our disposal..

8 6 In addition to these pigments, found also by other authors, we detected in the epiphasic layer of almost every sample another two pigments which we shall call I and II, respectively. On the alumina chromatoeam they lie between lycopene and p-carotene, one forming a pink ring below the lycopene (but distinctly separated from the latter by a colourless zone), the other forming a distinct brown-yellow ring below the pink ring. According to their situation on the chromatogram and the differences in their absorption spectra, 1 pigments I and III are not identical with the two unknown carotenoids discovered by v. DANIEL & dres (loc.cit.) in sei-um. Pigment I has the following absorption maxima: in petroleurn ether ' in CS 2 512?? ItS spectrum differs from the lycopene spectrum also in that the first'absorption bànd is less pronounced than : the second one. The third band, too, is comparatively Weak. Pigment II has the following absorption maxima: in petroleum ether in cs (second band rather, blurred) in benzene 487 second band too indistinct for reading off.. Often, complete separation of these two pigments is achieved

9 only by repeated chromatography and vigorous development (benzene: benzine 4:1). It is sometimes altogether impossible to separate very small quantities satisfactorily, and this is why in our tests we frequently had to dispense with pure preparation. Preparative isolation was unfortunately impossible because we did not have sufficient material. The frequent occurrence of these pigments and their relatively high colour intensity make it unlikely that they are mere accidental products. The absorption maxima we found, and the situation and colour hue of the two pigments on alumina chromatograms closely resemble characteristics reported by KUHN & BROCKMANN [13] for two products they obtained by oxidation of carotene under mild conditions, namely p-oxy-semi-carotenone and p-oxycarotene. Of course we do not want to say, on the basis of these similarities, that our pigments and those two substances are identical, especially since KUHN & BROCKMANN describe their products as hypophasic, but will make it the aim of future studies to clarify this issue by preparation on a larger scale and by mixed chromatograms. One thing, however, is certain even now: we are not dealing with genuine carotenoids, but with conversion products which show great similarities to oxidation products of carotene prepared by purely chemical means, especially with regard to chromatographic behaviour and to absorption spectra.

10 In our opinion, the occurrence of such oxidation products may have biochemical significance, for some of the carotene (III) 1 presumably is not broken down to vitamin A in the body, but undergoes a different metabolic process. It is very plausible to assume that in the latter case those positions in the molecule are likely to react which are preferred during purely chemical oxidation, namely the double bonds in the rings: CH, CH, c a%.. HI. *CH, CH, CH,- CH, _ r- --CH CH= CI-1=---CH CH= b :CII-,-CH 6...1H2 11 I P- Carotin III The reaction with the first ring produces p-oxycarotene (IV): CH, CH, CH, Ire <giii _.. ume.. ro» 1 i<oh W... _...-- CHs H, etc. ' CH, CH, etc. 11/3 W. - CH=CH-- C.bH V 0. CH, 8 H, Continued reaction breaks up the second ring of p-oxycarotene, and p-oxy-semi-carotenone (V) is formed., This would explain why the transformation of carotenes to vitamin A in the liver is not quantitative, a fact that has been 779.5"-7

11 known for a long time [14]. (In this respect, compare the numerical data on the effective maximum or minimum doses of carotene and vitamin A given by KUHN, BROCKMANN, SCHEUNERT & SCHIEBLICH [15] and by KARRER & MORF [16]). It would also explain why in growth tests p-carotene does not always prove, twice as active as 11- and Y-carotenes [17], since varying amounts of the carotenes may be broken down in the above-described manner. (However, the differences in the activity of p-carotene and vitamin A may also be explained by differences 15 in their resorptive behaviour.) Moreover, in two cases (2 and 8) we found a carotenoid which, with benzene-benzine development, passes rapidly through the column as a yellow zone. In one case we were able to read off the absorption maxima (473 and 445 in petroleum ether). Perhaps this carotenoid is a-carotene, for we found that with highly dilute solutions the absorption maxima easily occurred at slightly too short wavelengths. In the hypophasic fraction we detected mostly xanthophyll, several times zeaxanthin, sometimes also both of them mixed. The arithmetic mean of all cases for which we had carried out quantitative determinations was 0.06 mg p-carotene per 1 litre serum. v. DANIEL & BfiES found 0.28 mg in '3 litres, which works out to 0.09 mg per litre; in view of the different nutritional situation, the agreement is surprisingly good. The above authors found 0.08 mg

12 10 of hypophasic pigments per litre, compared to our 0.04 mg. Our findings on several livers (cases 3,5,10, and 11) confirmed the results obtained by ZECHMEISTER and TUZSON on the occurrence of lycopene in addition to carotene. The order of magnitude of our carotene values is the same as ZECHMEISTERts, but our values are slightly lower. (DIETEL [181 did not find any carotenoids in human liver, which is rather strange.) Our lycopene values, however, are considerably lower, which seems natural in view of the fact that in our, cases the food did not contain much lycopene. In addition to lycopene and carotene, we found in the epiphasic fraction also the carotenoids I and II already mentioned in the discussion of the serum results; these carotenoids lie between lycopene and carotene in the chromatogram. During the working up of one liver (case 5), the chromatogram showed a zone above p-carotene proper which had the same colour as the latter and after elution had the same absorption bands as p-carotene. We did not find this pigment in any of the other cases, and we cannot say whether it was cryptoxanthin. In the hypophasic fraction of the liver extracts we found mostly zeaxanthin and xanthophyll, in one case (3) in petroleum ether a pigment with the bands 474 and 446 above xanthophyll. The wavelengths of the absorption bands were slightly too long for violaxanthin or taraxanthin, but perhaps the pigment was not completely free of xanthophyll. (Usually, taraxanthin does not occuehe Swedish diet,

13 11 f Table I. Serum findings The pigments are given in the descending sequence of the chromatogram. In order to obtain comparable results, all Y values have been converted to the content of 1 litre serum. The % values give the percentage of the respective carotenoid in the total carotenoids.i-means that the carotenoid was present, but could not be determined quantitatively..2. Fall Diagnose _ ',.., re 0.) a c, 0 r.." T.,_ ,:i cl... a a -7. il a 3 e gig rel.. -0 tzt q9 ',.-.4' > rà) 7; >1 "PeL cem Zi." j5 r 9/0 r 0/0 r 0/0 r / r Jo r 0/o r /o 7 /o Y 0/o - I Vitium organ. cordis Oedema pulmonum 160? 17 I + II /0 II IVIyoearditis ehroniea, Oedema pulmonum 230 ( III. Bronchopneumonia 150? IV desgt V Glomerulonephrit. chroniea. Uraemia 200 5' V I Gesuncl ? VII llypertonia, leichte Herzinsuffizienz ti 200*) VIII Gesund.1Q 200 cli IX Bronchopneumonia X desgl. LI 140 -I-,..-- -I- + + XI desgl. 1.2, Zusammen 180? XII desgl. 1.2, 200? (?) **) I I 1 - case 2 - diagnosis 3 serum quantity used, in cc 4 sex of serum donor 5 unknown phytoxanthin 6 lycopene 7 unknown epiphasic pigments 8 0-carotene 9 unknown epiphasic pigment 10 - healthy 11-1-lnertonia, slight cardiac insufficiency 12 - ditto "zusammen" - total * - combined serum of two patients with the same diagnosis ** - II was contaminated by an unknown pigment

14 12 A whereas violaxanthin is abundant in the bog berries (Rubus chamae- 17 morus [191 which are very popular in Sweden.) It is also possible that this pigment was a transformation product of xanthophyll that had been produced in the body. A case in point would be the finding by BROCKMANN & VÔLKER [20], according to which a pigment was formed from xanthophyll in the body of canaries which had the same absorption bands as taraxanthin or violaxanthin. Table II. Liver findings Y converted to 1,000 g fresh liver. The cases bear the same designations as in Table I. - c). ' P ^"" = 4 d. 0 t. I Fall F,cà.) 5..: ri I sc.:1, II r 0 G :1 j 1/41 e' ' 'Pi, fln F.., 4 eta) li pà c..) 44e, g r 010 r % r 0/0 r 0/0 r lb 70/0 r io r P/0 III ± V ? + (?) : + X XI case 2 - quantity of liver used 3 - phytoxanthin with 4 - lycopene short-wave absorption (violaxanthin, taraxanthin) 5 - unknown epiphasic pigment with p-carotene spectrum 6 - i-carotene 7 - unknown epiphasic pigment Unfortunately, we have colorimetric carotenoid determinations in both serum and liver for only one case, namely for case 11. If

15 13 we assume that an adult has 3 litres of serum and his liver weighs li kg, we arrive at values of 0.03 mg (3-carotene in the total serum and 0.45 mg p-carotene in the entire liver for case 11. Thus the amount of carotene stored in the liver was approximately 15 times the amount circulating in the blood. Further studies will have to establish the extent to which this ratio is characteristic. We would also like to mention that in this case the absolute values were unusually low. Summary The carotenoids from fairly large quantities of serum ( cc) from 12 patients and the carotenoids from the liver of four of those patients were separated chromatographically. The quantitative colorimetric determinations made with these samples are reported. In addition to lycopene and carotene, two hitherto unknown pigments were found in the epiphasic fraction. Their nature is discussed. In the hypophasic fraction we usually found zeaxanthin or in one liver sample possibly also. violaxanthin. xanthophyll, Non-chromatographic determinations of the content of yellow pigments in serum do not give any definite indications as to the actual amount of (3-carotene present.

16 14 References 1. Palmer, Carotenolds and related pigments, New York Vgl. die Monographien von H. Willstaedt, Carotinoide, Bakterien- und Pilzfarbsteffe, Stuttgart 1934; L. Zechmeister Carotinoide, Berlin 1934, und E. Lederer, Lee caroténoides des plantes, Paris 1934, Les caroténoides des animaux, Paris 1935, sowie den übersichtsartikel von H. Willstaedt, Wschr. 14, I, 841, z. B. Wendt, Kiln. Wschr. 14, I, 9, 1935; Schneider und Widmann, ebenda 14,1, 670, 1935; Rachewsky, Bull. Soc. Chirn. biol. 17, 1187, Biochem. Z. 108, 279, Z. B. van Eekelen und Menken, Acta brev. ncerl. 4, 78, Diese Z. 231, 259, 1935; Bull. Soc. Chim. biol. 17, 1110, 1935; Diese Z. 234, 241, 1935; Nahum'. 23, 680, Biochemic. J. 29, 2465, É. Biochem. Z. 283, 263, Diese Z. 238, 160, Acta brev. neerl. 4, 171, 1935; vgl. such van Eekelen, Kiln. Wschr. 14, 829, 1935, 11. Diese Z. 221, 117, Diese Z. 206, 41, Liebigs Ann. 516, 95 und zw. S. 121, Vgl. z. B. Moore, Biochernic. J. 2, 275, Diese Z. 221, 129, 1938; man vgl. such die Tabelle bel Bomskow, Die Methodik der Vitaminforschung, Leipzig 1935, S Helvet. chirn. Acta 16, 615, Vgl. v. Euler, Karrer und Zubrys, Flelvet. chim. Acta 17, 24, 1934; Kuhn :uud Brockmaun, Klin. Wschr. 12, 972, 1933; fanden demgegeniiber ig-carotin stets doppelt so stark im Tierversuch wirksam wie a- und y-carotin. 18. Kiln. Wschr. 12, 601, Willstaedt, unverliffentlichte Versuche. 20. Diese Z. 224, 193, 1934, u. zw. S Translation of nonenglish entries: 2. Cf. the following monographs: H. WILLSTAEDT, Carotenoids, Bacterial and Fungal Pigments, Stuttgart 1934; L. ZECHMEISTER, Carotenoids, Berlin 1934; E. LEDERER, Plant carotenoids, Paris 1934; E. LEDERER, Animal carotenoids, Paris 1935; and the review article by H. WILLSTAEDT 3. "ebenda" - in the same journal. 10. "vgl. auch" - see also see also the table by BOMSKOW: The methods of vitamin research KUHN & BROCKMANN, K1in.Wschr.12, 972, 1933, on the other hand, found in animal tests that p-carotene. was always twice as active as a- and Y-carotenes unpublished experiments.... "Diese Z." - this Journal

FISHERIES AND MARINE SERVICE. Translation Series No Investigation of the lipid composition of several species of fish

FISHERIES AND MARINE SERVICE. Translation Series No Investigation of the lipid composition of several species of fish FISHERIES AND MARINE SERVICE ARCHIVES Translation Series No. 3637 Investigation of the lipid composition of several species of fish by A.G. Rudenko, and V.I. Lisovskaya Original title: Issledovanie sostava