by A. Grossmann, H. Timmen, and H. Klostermeyer

Transcription

1 ARCH] Vii:.` z FISHERIES. AND MARINE SERVICE Translation Series No Enzymatic determination of cholesterol in milk fat, - an alternative to convent ional methods by A. Grossmann, H. Timmen, and H. Klostermeyer Original title: Die enzymatische Bestimmung von Cholesterin in Milchfett eine Alternative zu den bisher gebrauchlichen Methoden From: Milchwissenschaft 31(12): , 1976 Translated by the Translation Bureau (AK) Multilingual Services Division Department of the Secretary of State of Canada -Department of Fisheries and the Environment Fisheries and Marine Service Halifax Laboratory Halifax, N.S pages typescript

2 ti I. I DEPARTMENT OF THE SECRETARY OF STATE TRANSLATION BUREAU MULTILINGUAL SERVICES DIVISION CANADA SECRÉTARIAT D'ÉTAT BUREAU DES TRADUCTIONS DIVISION DES SERVICES MULTILINGUES "TRitISLATED FROM - TRADUCTION DE German INTO EN English AUTEUR Anke GROSSMANN, Hermann TIMMEN and Henning KLOSERMEYER TITLE IN ENGLISH - TITRE ANGLAIS Enzymatic Determination of Cholesterol in Milk Fat An Alternative to ConventiOnal Methods TITLE IN FOREIGN LANGUAGE (TRANSLITERATE FOREIGN CHARACTERS) TITRE EN LANGUE ÉTRANGÉRE (TRANSCRIRE EN CARACTLRES ROMAINS) Die enzymatische Bestimmung von Cholesterin in Milchfett - eine Alternative zu den bisher gebrâuchlichen Methoden ie-ference IN FOREIGN LANGUAGE (NAME OF BOOK OR PUBLICATION) IN FULL. TRANSLITERATE FOREIGN CHARACTERS. RiréREnct: EN LANGUE ÉTRANGF.RE (NOM DU LIVRE OU PUOLICATION), Au COMPLET, TRANSCRIRE EN CARACTÈRES ROMAINS. Milchwissenschaft ed.ference IN ENGLISH RÉdF)ENCE EN ANGLAIS Milk Science International ÉDITEuR PLACE OF PuBLICATION LIEU DE PUBLICATION not shown Kempten (Allgnu) YEAR ANNÉE 1976 DATE OF PUBLICATION DATE DE PUF3LICATION VOLUME 31 ISSUE NO. NUMÉRO 12 PAGE NUMBERS IN ORIGINAL NUMÉROS DES PAGES DANS L'ORIGINAL NUMBER OF TYPED PAGES NOMBRE DE PAGES DACTYLOGRAPHIÉES 10 REQUESTING DEPARTMENT mmisrble.glient BRANCHORDPIISION DIRECT;ON OU DIVISION Environment Fisheries - Scientific Information and Publicetions Branch TRANSLATION BUREAU NO. 14,48474 NOTRE DOSSIER N TRANSLATOR (INITIALS) TRADUCTEUR (INITIALES) A K PERSON REQUESTING DEMANDÉ. PAR YOURNUMBER VOTRE DOSSIER N Allan T. Reid, Scientific Documcntation, for Dr. Kovacs, Halifax Lab _ 007- c)_? DATE OF REQUEST OA TE DE LA DEMANDE t o:; -2:.\o i o.$ (.t: v. S/10) t 3 i ZI ishnreill) TRitrr)rA'iU"..A.: cni y tn.z..,.1\ro;,..;

3 X:relary of Sblei TRANfd...ATION OUREM) MULTILINGUAL SERvicEs olvislom LWREAU DES YRADUCTIONS DIVISION DES SERVICES MULTILINGUES v- 0, 41,L 1.0 o u w ort , mi DO DIMEAU Ln PA 11 1 MEN T MI NIS IL ni: Environment LANGUAGE LANGUE invisloviumnfi Fisheries Scientific Information - and Publications Branch. Tnimf,LAyon tun t I ALs) TRAOUCTEUIt (1111 ALES) CITY VILt.r. Ottawa ' German A.K. ficr : 7/ In: Milchwissenschaft 31(12), Enzymatic Determination of Cholesterol in Milk Fat An Alternative to Convention.al Methods by Anke GROSSMANN, Hermann TIMMEN and Henning KLOSTERMEYER From Institut fill' Chemie der Bundesanstalt fur Milchforschung, Kiel (institute for Chemistry of the Federal Institution for Milk Research) 1. Introduction For the determination of cholesterol, a series of principles and a large number of individual specifications have been worked out. This reflects, firstly, the great interest in the knowledge of cholesterol levels and, secondly, the dissatisfaction of the users with the known methods. In fact, all analysis methods for cholesterol require more or less complicated sample preparations or separating operations, the use of highly toxic or caustic chemicals or the use of large instruments. Besides, many methods are not specific for cholesterol; rather, they include also chemically related compounds. With the discovery of cholesterol oxidase, an enzyme which oxidizes the alcohol cholesterol to the ketone A4-cho1estenone, it became possible to develop an enzymatic determination method (1) which is safe f)-25t and easy to perform. The enzyme used accepts as substrate, apart from unedited ITIM.q.UM1011 For infaliol only U.,!MXICrION PON it

4 2. cholesterol, most of the steroids that are hydroxylated in 3--position (3-A-hydroxy-sterol), and oxidizes them to the respective ketones (1,2): The reaction corresponds in its specificity to the precipitation with digitonin. With their help, the cholesterol content of milk fat should be determinable, since milk contains no noteworthy amounts of other 3- hydroxy-steroids (3). However, part of the cholesterol is acylated to the 3-hydroxy group, i.e. as cholesteryl ester (3,4), and can be included only after previous.saponification. The enzymatic cholesterol determination was first used in the clinical laboratory (1,5); subsequently, however, in food analytics, it was also suggested for systems with high cholesterol contents in relation to the total lipids (6), particularly for the determination of the egg content in pasta (7). We now have examined to what extent it may be suited for the determination of the relatively low cholest.erol contents of milk fat. 2. Material and Methods Samples: For the cholesterol determination, one refers to the ( (22) fat obtained from fat determinations according to Rbse-Gottlieb, Schmid- Bondzynski or Weibull-Stoldt; we worked exclusively with butter fat obtained by melting the butter at Q C, decanting the fat phase, and filtering through a dry fluted filter (MN 615 ff 1/)., 715 1/4). Sample Preparation: The determination of the cholesterol jn the presence of the (saponified) total fat, as this is possible for ex. with egg pasta, proved to be impossible for milk fat because of the low cho- lesterol portion in, relation to the fat p6rtion. Of several tested variants for the saponification and extraction of the unsaponifiable portion, the following proved to bo optimal: round-bottomd fissk with an. accuracy of 0.01 g, 5 g of milk fat are weighed into a 250-ml

5 3 cooked for 30 min. at the reflux with 50 ml of 2N methanolic KOH (freshly prepared, without stabilizers), and the still warm soap solution is flushed. with 100 ml H 20 into a 1000-ml separating funnel. After cooling, 100 ml of ether/petroleum ether (1:1) is added, and the whole is intensively, but carefully mixed. After 20 to 30 min., the clear-settled lower phase (soap solution) is drained into the saponifying flask, and the orga- nic phase is introduced into a 500-ml round-bottomed flask. The extraction of the soap solution is repeated twice, whereby the hazard àf the formation of an emulsion is.particularly marked with the third extraction. The collected ether/petroleum-ether phases are concentrated at 35 C in the rotary film evapàrator; the residue, which is unsaponifiable, is brought with propanol-2 quantitatively into a 50-ml measuring flask, and filled up to the mark, at 20 C. 0.4 ml of this solution, with a 5 g sample of milk fat, contains the p.g, of cholesterol required for the most suitable measuring range. Enzymatic Cholesterol Determination (6): A. Principle: Cholesterol is oxidized to cholestenone by cholesterol oxidase. The hydrogen peroxide yielded in this reaction oxidizes methanol, in the presence of catalase, to formaldehyde which then, with acetyl acetone, in the presence of NiJ ions, forms a yellow lutidine dye., The dye concentration, the equivalent of the cholesterol content, is determined photometrically. B. Reagents: Test combination "Cholesterol" of the firm of Boehringer-Mannheim, order No , consisting of Reagent 1: U catalase in about 95 ml of ammonium phosphate buffer 0.8 M, ph 7.0, which consists to 2.6 M of =thanol; Reagent 2: 60 mi of 0.05 M acetyl acetone, 0.3 M methanol, stabilized; Reagent 3: 0.8 mi suspension of li U cliolesterol

6 h. oxidase. nefore use, 3 parts of Reagent 1 and 2 parts of Reagent 2 are put. together, at room temperature, in a brown bottle (= Reagent 4). At' 1 C, ReagentS 1, 2 and 3 may be stored for at least one year, Reagent 4 for three months, according to the manufacturer's indications. C. Determination: 5.00 ml of Reagent 4 are well mixed with 0:: of test solution in the test tube (with screw lock) ml thereofpipetted off and well mixed with 0.02 ml of solution 3. Both test tubes are well screwed and maintained for 60 min. in the water bath, at C, then cooled to room temperature, and the oxtinction difference betwee n and blank value is determined at 412 nm in 1-cm cuvettes (ia.e). sample Calculation of the cholesterol content: At a molar weight of the cholesterol of g/mole, a test volume of 5.4 ml, a dilution factor of (= 2.52/2.50), an extinction coefficient of 7.47 em2hmole, a layer thickness of 1 cm and a. test volume of 0.4 ml, the. cholesterol content is calculated as follows: 386,6. 5,4-1,008 _ 7,47 1 0, ,7043 ie (g cholesterol/liter of test solution). Multiplication of c by 100 x 50/ initial fat weight (in g) results in the cholesterol. content of the milk fat in mg/100 g fat. 3. Results Absorption Maximum, Extinction Coefficient, and Calibration Curve: The absorption maximum of tho'dye that forms in the reaction between methanol, hydrogen peroxide, ammonium+ and acetyl acetone is indicated with 405 nm, 2 the respective, extinetion coefficient with.7.4 cm /imole (6). Our reexamination of the absorption maximum, with various spectrophotometers (Zeiss pm II and Shimadzu UV-200), resulted in 1112 nm: The respective

7 5 extinction coefficient was determined by us with solutions of crystalline cholesterol, purissimum (Roth); it was 7.47 cm 2 //mole. In the enzymatic determination, in the investigated concentration range of Mg of cholesterol/0.4 ml of test solution, the relatidn between absorption and cholesterol amount was linear. The absorption of the lutidine dye that formed during the colourreaction was stable for several hours. Owing to the carotins contained in the milk fat, the auto-absorption at 412 nm must not be disregarded; however, it is eliminated by the fact that one always measures the absorption difference between sample and blank value. Reproducibility:. From a milk-fat sample, 15 parallel determinations of the cholesterol content were carried out; the mean value of the extinction difference was The absolute standard deviation was calculated with , the variation coefficient (relative standard deviation) with %. The conversion of the extinction differences to cholesterol contents, in this case, resulted in a mean value of % of cholesterol in the milk-fat sample, at an absolute standard deviation of %. The reproducibility of the method therefore constitutes the highest possible measuring capacity of photometric methods. Accuracy: In order to determine the accuracy of the method, the recovery rate for the cholesterol added to the samples was determined. Since an addition of cholesterol to milk fai. would have led to absorptions beyond the optimum measuring range, the recovery test was carried out with margarine fat (0.215 % cholesterol'or 3-ft-hydroxy-steroids) in such a manner that to 5 g fat each 12.5 mg cholesterol was added. tests, this cholesterol was recovered up to 97.5 to 98.8 %. In four parallel Labour expended: One worker is capable of carrying out at least.8 determinations per day, whereby about three quarters of the time are spent for preparing the samples.

8 6. Determination of Free Cholesterol: The'determination of the cholesterol, after the saponification of the triglycerides, comprises the total of free and acylated cholesterols. We were not able to enzymatically determine the free cholesterol as such, in the presence of the triglycerides; in order to reach the required measuring concentration - at the relatively low cholesterol contents of the milk fat, the large sample amount (i.e. particularly triglycerides) that must be used cannot be maintained in solution by the solvent mixture of the enzymatic determination, and precipitates as turbidity. ( 723 ) 4. Discussion The enzymatic determination of cholesterol, with the use of commercial cholesterol oxidase, proved to be an excellent analysis method for the determination of the total cholesterol content of milk fat, a method which is not likely to be surpassed with regard to the quality of the results. To distinguish between free and esterified cholesberol, the method cannot be used without a special separating operation; however, there is but little interest therefor in food analytics. The method is to be considered as a valuable addition to our analytical possibilities. As to whether it may replace other conventional methods, requires a more -thorough study. The determination method for cholesterol and related sterols (specific for all 3--hydroxy-sterols) -, which is considered a standard for the evaluation of other methods, is the gravimetric determination as digitonide, first described by Windaus, It was applied also tu fat (8). According to a standard.method of the international Dairy milk Federation (9), the digitonin precipitation for the determination of the

9 7 "whole sterol content" takes place after saponification, directly from the soap solution. Since, apart from cholesterol, milk contains no note-: worthy amounts of other sterols, the method is particularly well suited for the cholesterol determination in milk fat. According to literature indications, its precision and accuracy is superior or at least equivalent to other conventional methods; however, it is inferior to the enzymatic method. It is advantageous that the required instrumentation comprises merely a precision. balance and a refrigerator. The labour expended, on the whole, is about equivalent to that of the enzymatic determination; the analysis result, however, is available only after 24 hours at the earliest. A disadvantage is also the high price and the toxicity of the digitonin used. Of highest specificity, and suitable to determine cholesterol, apart from other sterols (for foreign-fat detection), are tile gas-chromatographic methods (e.g. 10). For the.gas-chromatographic sterol analysis in milk fat, there exists a standard of the International Dairy Federation (1l). With regard to labour expended, the method is by no means less time-consuming than the enzymatic method, and requires the use of a suitable gaschromatograph with special column packings. The advantage of high speci- ficity is contrasted by the disadvantage of felatively large variations in the analysis values. In laboratory practice therefore, instead -of the above mentioned methods, refined to standardization, one often works with methods that require less expenditure. Very widespread is the calorimetric method. of Liebermann-Burcbard in the modification of RIFFART and KELLER (12). With this method, cholesterol is converted with concentrated sulfuric acid to. a sterolium salt which, by dehydration with acetanhydri:de, forms a green

10 8. compound. The reaction is very unspecific and responds also to metabolites of cholesterol and to other steroids; moreover, cholesterol esters provide quantitatively different results than free cholesterol. However, since milk fat contains practically only cholesterol (and this cholesterol only to about % as cholesterol esters), and hardly any other steroids, 'the Liebermann-43urchard reaction may be carried out quite specifically. As a comparison to the enzymatic analysis therefore, we investigated a series of milk-fat samples also according to RIFFART and KELEER's method (12). For this purpose, the unsaponifiable portion was isolated, the same as in-the sample preparation for the enzymatic determination. It was taken up in acetic ester; then, aliquot portions were reacted with acetanhydride and concentrated sulfuric acii, and the solutions were determined photometrically at 625 nm, against corresponding blank samples. Since the intensity of the coloration is time-dependent, we always had to measure for one hour, at intervals of about 10 min.; the highest extinction usually appeared after 30 min. The measurements were evaluated by means' of a calibration curve established with cholesterol solutions. The results (mean values) showed very good conformity with those of the enzymatic determination; however, the separate values obtained with the determination method of RnTART and KELLER showed clearly higher variations. With regard to labour expended, both methods are comparable; the chemicals used for the colorimetric determination are cheaper, but more unpleasent to handle. Summarizing, one may say that for milk fat, which contains as steroids almost exclusively cholesterol, the advantage of enzymatic determinations, i.e. the high specificity, is hardly. justifiable. Impressive, however, is the extraordinary reliability of the analysis statements. In this respect, the enzymatic analysis method is definitely superior to the

11 9. gas-chromatographic methods which, in any case, owing to the high expenditure required for instrumentation, are of interest only for eries analyses. The digitonin method, e.g. in th è form of the IDF standard (9), has the advantage of requiring but little instrumentation; however, the handling of digitonin solutions is a drawback. We therefore would recommend the use of the enzymatic cholesterol determination whenever a medium or large number of cholesterol determinations in milk fat have to be carried out. For the investigation of separate samples, the Liebermann-Burchard method may be recommended, and if quality of the results is of special importance, the digitonin precipitation. If the investigational material is not definitely pure milk fat, gas-chromatographic investigation should also be used. BibliograpPv (I) RICHMOND, W.: Clin. Chem. 19. (12) (1973). (2) SMITH, A. G., BROOKS, *C. J. W.: J. Chromatogr (1974).. (3) Wril?.,B, B. H., JOHNSON, A. H., ALFORD, J. A.: Fundamentals of Dairy Chemistry'. The AVI Publishing Company, Inc., Wes'rport, Conn S. 185f. (4) MULDER, H, ZUIDHOF. T. A.: Neth. N1ilk Dairy J. 12 (3) 173 "A 79 (1958). (5) POSŒHLAII, P., BE.RNT, E., GRUBER, W.: Z. Klin. Chem. KIIXL ibiochern. 12. (9) (1974). (6) Boeirringer Mannheim GmbH: Methoclen der en7.yrnatischen Lebensmittelanalytik 75/76. Firrnen-Prospc,kt. (7) BEUT.E.;iR, H. O., NECHAL, G.: Getreicle, Mehl, Brot (1976). (8) I IERDER, P. C. den.: Neth. Milk Dairy J i ( (J ) Internationaler N4ilchwirtschaftsverbanci: Nachweis biler lette in N4ilch1ett mit linty des Phytosteroieceta?.,--::-s. Intern: tionaler Standard FiL-IDF 32: Milchwi,: enschaft ; (1966). (10) Pllt\P;eiAl2,.I. K.: J. Assn. Off. Anal. Chern (1975). (11) International Dairy Federation: Detection of vegetable fat in fdt by gas:liquid chromatography of sterols. fitternatonal Stanilaid FIL-IDE 54: International Ddiry Federation, Square Verclote 41,1040 Brussel, Belgien, (12) RIFEA.RT, H., KfiLLER, H.: Z. Linters. Lebensmittel 68, (2) (1934). Translation of non-ennish Bibliographic Entries: (6) (9) Methods of enzymatic food analytics, 75/76. Company prospectus. int.ernational Dairy FederaUion: Detec -É.ion of vegetable fats in milk fat by mens of the phytostcrol-acetate test.

12 Surnma.rv - GROSSMANN, A., TIMMEN, 11., KLOSTERMEYER, H.: Enzymatic e. st Li-nation of cholesterol ln milli fat an alternative to the methoils In current use. Milchwissenschaft 31. (12) ( Cholesterol (enzymatic determination), milk fat (cholesterol determinat ion). The enzymatic detennination of cholesterol introduced for the anal ysis of Wood serum and egg-yolk-containing foods was examine(' tor its aiiplicability to milk fat. The fat svas saponified and the lut id in dye, de veloped after addition of the test combinat ion "cholesterol" (Boehringer Mannheim) in the unsaponifiable, was esti- > mated fiholometrically 412 ren, E = 7.47 cra =4t Mol). With a relative standard deviation of ± 0.50%, (mean cholesterol content. = 0.313%, n = 15) and a recovery rate of % (12.5pg cholesterol added to 5 g fat, ri = 4) the enzymatic 'procedure proved lobe a method which may hardlybe exceeded as regards the. qua- 1 ity of its results for total cholesterol. As comparer' to cligitonin piecipitation and the Lieberinann-Burchard proceduce, the enzyinatic method is recommended when mimerons determinations are to be performed. If the material to be analyzed is not re.sliably pure milk fat, it may be necessary to differentiate between cholesterol and other sterols by gas chromatography. GROSSMAI\IN, A., TIMIvIEN, H., ICLOSTERMEYER, H.: Détermination enzymatique du cholestérol dans la matière grasse du lait procédé alternatif pour les méthodes d'emploi courant. Milchwissenschaft 31. (12) (1976). 44 Cholestérol (dé.tennination enzymatique), matièr e grasse du lait (détermination du cholestérol).