867

Journal of Food Protection, Vol. 50, No. 10, Pages 867-87J (October 1987) Copyright" International Association of , Food and Environmental Sanitarians

Oxidation of Cholesterol in Commercially Processed Cow's Milk

MAE Z. CLEVELAND and NATHOLYN D. HARRIS*

Department of Nutrition and Food Science, Florida State University, Tallahassee, Florida 32306

(Received for publication March 26, 1987) Downloaded from http://meridian.allenpress.com/jfp/article-pdf/50/10/867/1651105/0362-028x-50_10_867.pdf by guest on 23 September 2021

stant nonfat dry milk (NDM) were purchased from local super­ ABSTRACT markets and analyzed the same day. NDM was hydrated at 50% Pasteurized whole milk, ultra-high temperature heated milk, with distilled water and refrigerated 30 min before lipid extrac­ canned evaporated milk, skim milk and instant nonfat dry milk tion. Fresh , taken from a Guernsey cow on a local were analyzed for presence of oxidized cholesterol compounds. farm, was collected into a clean glass-stoppered flask which had Effects of heating whole milk and storage of whole milk lipid been flushed with nitrogen and placed on dry ice. The flask extracts were also examined. Analytical thin-layer chromato­ with freshly drawn milk was immediately stoppered and kept graphy data indicate that cholesterol in liquid milk was stable on dry ice. Lipids were extracted within 15 min after the milk during commercial , sterilization and evaporation. was drawn from the cow. However, instant non-fat dry milk contained 7-hydroxy-choles- Whole milk and rehydrated NDM were used for determina­ terol. Heating whole milk for 12 h at 85°C did not produce tion of the influence of heating on cholesterol oxide formation. oxysterols, but GC-MS analysis indicate that storage of whole The were heated at 85°C for 12 h uncoverd with continu­ milk lipids may have produced steroidal ketones. ous stirring. To study the effect of storage on development of oxidized cholesterol compounds, whole milk lipid extracts that had been stored in the dark at 5°C for 2 months before analysis were examined. The role of cholesterol in health and disease is being scrutinized and questioned as investigators bring new evi­ Extraction of lipids dence to light. Of recent concern are the conditions under A modified Radin method (18) was used for extracting lipids. which cholesterol oxidizes and the effect that these A 5-ml milk sample was added to 12 ml of HPLC glass- oxidized products have on diet and health. Over 60 reac­ distilled grade isopropanol/hexane (Burdick & Jackson, Muske­ tion products of cholesterol oxidation have been isolated gon, MI) (2:1, v/v) mixture in a glass centrifuge tube with tef­ and identified and a number of these products have dem­ lon lined cap, mixed for 60 sec and allowed to stand 20 min. onstrated biological activity in various cell and organ cul­ An additional 8 ml of hexane was added and the mixture was tures and intact animal tissues (1,4,5,9,10,12,13,17,22). centrifuged for 15 min at 2500 x g at 20°C. The lipid portion The most common oxidized cholesterol compounds are was transferred quantitatively to an evaporation flask and the products from A or B ring or side chain reactions (20). solvent was evaporated under vacuum. The lipid extract was transferred to a glass test tube with hexane/isopropanol (10:1, In recent years, researchers have reported the presence v/v) solvent and dried under nitrogen. of oxidized cholesterol compounds in some foods, nota­ bly spray-dried egg products (2,8,15), beef tallow Thin-layer chromatography (16,19), French fried potatoes (14,16) and milkfat (3). High performance analytical silica gel Sl-HPF, 200-jjim thin- To date only one group has reported cholesterol ketones layer chromatography (TLC) plates (J. T. Baker Chemicals, in stored milk (6), and no reports have appeared in which Phillipsburg, NJ) were used. Extracts from the whole milk sam­ milk was studied as purchased from the supermarket. ples were dissolved in 300 JJLI chloroform and a 2-(JL1 sample Various types of processing such as sterilization, spray was applied 2 cm from the bottom of the plate. Nonfat dry drying or evaporation are done to provide milk in conve­ milk extractions were dissolved in 100 JJLI of chloroform and nient forms for the consumer. The purpose of this study 2 jjul were applied to the chromatoplate. Standards including cholesterol, cholest-4-en-3-one, cholest-4,6-dien-3-one, 20a-hy- was to investigate the presence of certain oxidized choles­ droxy-cholesterol, cholesta-5a,6a-epoxide and 7-ketocholesterol terol products in four types of milk and to study the ef­ (Sigma, St. Louis, MO) and cholest-3,5-dien-7-one, 7a-hydro- fects on cholesterol after heating whole milk or after stor­ xycholesterol, 7B-hydroxycholesterol, 25-hydroxycholesterol age of isolated milk sterol fractions. and cholestane-3B,5,6B-triol (Steraloids, Wilton, NH) were ap­ MATERIALS AND METHODS plied for identification purposes. Plates were developed first in ethylene dichloride (17 cm) to separate sterol esters, trig­ Sample lycerides, fatty acids and phospholipids from free sterols and Whole pasteurized milk, ultra-high temperature (UHT) heated second in ethyl acetate (5 cm) for further separation of the whole milk, canned evaporated whole milk, skim milk and in­ sterols. Plates were viewed under UV light at 266 and 254 nm,

JOURNAL OF FOOD PROTECTION, VOL. 50, OCTOBER 1987 868 CLEVELAND AND HARRIS

TABLE 1. TLC data on some oxidized cholesterol compounds. Mobilities uv H?S04 Compound (254nm) (color) cm Rchol Cholest-4-en-3-one + light pink 4.0 1.05 Cholest-4,6-dien-3-one + pink 3.9 1.02 Cholesterol magenta 3.8 1.00 Cholest-3,5-dien-7-one weak beige 3.75 .98 20a-hydroxy cholesterol gray-green 3.25 .85 25-hydroxycholesterol blue-gray 2.95 .77 Cholesta-5a,6a-epoxide yellow 2.85 .75 7-Ketocholesterol + 2.8 .74 7[J-hydroxycholesterol blue 2.6 .68 7a-hydroxycholesterol blue 2.5 .65

Cholestane-3p ,5,6p-triol light yellow 1.5 .26 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/50/10/867/1651105/0362-028x-50_10_867.pdf by guest on 23 September 2021

then sprayed with 50% aqueous H2S04, air-dried, and heated at 110°C up to 5 min for color development. The coloration

and Rchoi values for the sterols are given in Table 1. Plates were heated an additional 30 min for complete charring of the compounds. Preparative PK6F silica gel 500-u,m TLC plates (Whatman, Hillsboro, OR) were used for isolation of cholesterol and oxidized cholesterol compounds. Two bands were marked, scraped and eluted for further analysis. The first band, Fraction 1, between 3.5 and 4.5 cm included cholesterol, cholest-3, 5- dien-7-one, cholest-4-en-3-one and cholest-4,6-dien-3-one.

Some of these compounds have similar Rf values but were sepa­ rated with gas chromatography. Fraction 2, between 2.2 and 3.5 cm from the sample application, contained oxycholesterols 7a- and 7p-hydroxycholesterols, 20a-hydroxycholesterol, 25- hydroxycholesterol, 7-ketocholesterol and cholesta-5,6-epoxide.

Although Rf values were similar for the 7-ketone and the epoxide in this group, the ketone was detected under UV light Figure 1. TLC comparison of lipids from several types of milk. but did not give a color reaction to sulfuric acid. The epoxide, A. Mixture of (I) cholest-4-en-3-one & cholest-4,6-dien-3-one: on the other hand, was not detected under UV light but dis­ (2) cholesterol; (3) 25-hydroxycholesterol; (4) cholestaStx, played a yellow color after spraying with H2S04 and then heat­ 6a-epoxide (5) 7-ketocholesterol; (6) 7$-hydroxycholesterol; ing. and (7) cholestane-3$,5,6$-triol. B Nonfat dry milk lipid sample. Gas chromatography-mass spectrometry C. Raw milk lipid sample. Samples were analyzed on a Varian 3740 gas chromatograph D. UHT milk lipid sample. (Varian Associates, Palo Alto, CA) equipped with a flame ioni­ E. Canned evaporated milk lipid sample. zation detector and methyl silicone capillary column, SP-2100 F. Whole pasteurized milk lipid sample. (Supelco, Inc., Bellefonte, PA) 30 m long. Column temperature was programmed to start at 220°C, increasing 4°C/min to 260°C. Injection port and detector temperatures were 200°C and 330°C, respectively. Nitrogen was the carrier gas. Capillary column gas chromatography-mass spectrometry (GC-MS) was performed with a Finnigan 4510 GC mass spec­ trometer equipped with an INCOS data reduction system. A 30 203.2 m DB-5 fused silica capillary column (J & W Scientific, Inc., ill. h ,„ I ;,. Jill lllllllll,|l|li,.i:l.l ,,iii , i ,hli.i..i, J..,. In, lllllllLl.lilllni.il.lllllll Cordova, CA) which was passed directly through a vacuum in­ 60 80 100 120 140 160 terlock into the ion source was used. The electron energy was 70 eV and the ion source pressure was less than 5 x 10"6 torr for the electron ionization experiments.

RESULTS AND DISCUSSION 2*7.3 369.3 ,51.4 . .lli.ll ,.,, I .1. , I.Hlll. Liquid whole milk 220 240 260 280 300 320 340 360 380 The major sterol in milkfat is cholesterol (11). The Figure 2. Mass spectrum identified as cholest-4-en-3-one iso­ major TLC spot (spot 2, Chromatograms C through F, lated from whole milk lipid extracts that stored at 5°C for 2 Fig. 1) for raw, UHT, evaporated and whole milk, re- months.

JOURNAL OF FOOD PROTECTION, VOL. 50, OCTOBER 1987 OXIDIZED CHOLESTEROL IN PROCESSED MILK 869

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Figure 3. Mass spectrum identified as cholest-3,5-dien-7-one isolated from whole milk lipid extracts stored at 5°C for 2 mo. Figure 4. TLC comparison of lipid samples from unheated and heated milk. spectively, displayed color and Rf value of cholesterol. A. Mixture of (I) cholest-4-en-3-one & cholest-4,6-dien-i-one; The spot above cholesterol in the chromatograms corres­ (2) cholesterol; (3) 25-hydroxycholesterol; (4) cholestanSa, ponds with Rf values for a sterol ketone. However, the 6a-epoxide; (5) 7-ketocholesterol; (6) 7fi-hydroxycholes- spot was not detected with UV light as would be ex­ terol; and (7) cholestane-3^,5,6^-triol. pected for a ketone nor did it give the color reaction of B. Unheated whole milk lipid sample. a ketone, as indicated in Table 1, when the chromatoplate C. Unheated nonfat dry milk lipid sample. D. Unheated skim milk lipid sample. was sprayed with H2S04 and heated. E. Heated whole milk lipid sample. Oxidized cholesterol compounds which are more polar F. Heated nonfat dry milk lipid sample. than the parent sterol can be separated, detected and ten­ G. Heated skim milk lipid sample. tatively determined by analytical TLC (7). Chromatog­ rams for the fresh raw milk and commercial liquid milks 4-en-3-one and cholest-3,5-dien-7-one. Figure 2 shows did not indicate presence of oxidized compounds when the high resolution mass spectral measurements of compared with R values and color reactions of the stan­ f cholest-4-en-3-one with a molecular weight of 384.3. dard compounds. Chromatograms C, D, E and F (Fig. This is consistent with the elemental composition 1) for raw, UHT, evaporated and whole milk samples D27H44O. The intense ion at m/e 124 (base peak) and respectively displayed a beige spot with similar R values f one at 342 are indicators of a 4 ene, 3 keto moiety (6). for 7-hydroxycholesterol in Chromatogram A (spot 6 in The mass spectral measurements of the compound iden­ Fig. 1), which is a mixture of oxidized sterols. However, tified as cholest-3,5-dien-7-one (Fig. 3) show a molecular spot 4 from the liquid milk samples (Fig. 1, Chromatog­ weight of 382.3 consistent with the elemental composi­ rams C, D, E, F) did not display the immediate distinct tion C27H42O. The rearrangement ion at m/e 174 (base blue color of the diol after H S0 treatment and heating. 2 4 peak) and those at m/e 187 and m/e 161 are consistent The TLC analysis showed consistent results in 5 different for the spectrum of the standard compound, cholest-3,5- samples of each type of milk studied. dien-7-one (6). GC chromatograms for the TLC sterol fractions were It is suggested from this investigation that the steriod similar for all types of liquid milk. The major component ketones were formed during storage of the isolated of this fraction was cholesterol. Some minor peaks which sterols. Analysis of raw milk and commercial milk sam­ represented compounds e luted after cholesterol did not ples did not indicate the presence of these ketones, which agree with retention times of oxidized cholesterol nor suggests that they are not a natural constitutent of milk were they identified by MS analysis as cholesterol nor are they formed under normal processing procedures ketones or other common cholesterol derivatives. Flana­ for commercial sales. Both of these ketones have been gan et al. (6) analyzed anhydrous milkfat after it had been identified among products of cholesterol autoxidation. stored for 18 months at 5°C and isolated two steroidal The dienone is a well-recognized product of cholesterol ketones, cholest-4-en-3-one and cholest-3,5-dien-7-one, oxidation (21,24) and cholest-4-en-3-one is an oxygen-in­ by GC-MS. In the current study, these ketones were not dependent isomerization product of cholest-5-en-one, an identified in raw milk or milk purchased from the super­ initial reaction product of cholesterol dehydration (20). market. To determine the effect of storage of the sterol fraction of these milks, the whole milk extract was stored in the dark for 2 months at 5°C in a glass vial with teflon Nonfat milk cap and then analyzed in the same manner as the other The nonfat dry milk chromatogram B (Fig 1) demon­ samples. Mass spectra of two substances (Fig. 2, 3) were strated a small blue spot (spot 6) below the cholesterol in excellent agreement with spectra of authentic cholest- band after spraying with 50% aqueous H2S04. This spot

JOURNAL OF FOOD PROTECTION. VOL. 50, OCTOBER 1987 870 CLEVELAND AND HARRIS

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Separa­ tion and identification of cholesterol oxidation products in dried egg No evidence was found of oxidized sterols in whole preparations. J. Food Sci. 50:595-598, 646. pasteurized milk, UHT milk, canned evaporated milk and 16. Park, S. W., and P. B. Addis. 1985. HPLC determination of C-7 skim milk as purchased from the supermarket. After heat­ oxidized cholesterol derivatives in food. J. Food Sci. 50:1437- ing whole milk or liquid milk at 85°C for 12 h choles­ 1441. terol oxides were not detected. However, storage of the 17. Peng, S. K., P. Tham, C. B. Taylor, and B. Mikkelson. 1979. Cytotoxicity of oxidation derivatives of cholesterol on cultured aor­ sterol fraction of whole milk at 5°C for 2 months pro­ tic smooth muscle cells and their effect on cholesterol biosynthesis. duced cholest-4-en-3-one and cholest-3,5-dien-7-one. Am. J. Clin. Nutr. 32:1033-1042. TLC chromatograms indicated presence of 7-hydro­ 18. Radin, N. S. 1981. Extraction of tissue lipids with a solvent of xycholesterol in spray dried instant nonfat milk. The low toxicity. Meth. in Enzymol. 72:5-7. spray drying process, which introduces oxides in addition 19. Ryan, T. C, J. I. Gray, and 1. D. Morton. 1981. Oxidation of to heat to the dry milk particles, may catalyze cholesterol cholesterol in heated tallow. J. Sci. Food Agric. 32:305-308. 20. Smith, L. L. 1981. Cholesterol autoxidation. Plenum Press, NY. oxidation. 21. Smith, L. L., J. I. Teng, M. J. Kulig, and F. L. Hill. 1973. Sterol metabolism. XXIII. Cholesterol oxidation by radiation-induced pro­ ACKNOWLEDGMENTS cesses. J. Org. Chem. 38:1763-1765. 22. Sunde, M. L., and J. J. Lalich. 1985. Renal and blood vessel re­ Research was supported by a Sigma Xi Grant in Aid of Research sponse to oxidized cholesterol in chicks and quail. Fed. Proc. and the Florida State University E. N. Whitney Fund. 44:938 (Abstr.).

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