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Biochimica et Biophysics Acta, 388 (1975) 203-212 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

BBA 56596

THERMAL TRANSITIONS IN THE LOW-DENSITY LIPOPROTEIN AND LIPIDS OF THE EGG YOLK OF HENS

MALCOLM B. SMITH and JOAN F. BACK CSIRO Division of Food Research, North Ryde, New South Wales 2113 (Australia) (Received November 14th, 1974)

Summary 1. Differential scanning calorimetry and light-scattering have been used to investigate temperature-dependent transitions in the low-density lipoprotein and in lipids from hens’ egg yolk. Yolks of different fatty acid composition were obtained by varying the dietary lipid and by adding methyl sterculate to the hens’ diet. 2. Lipoprotein solutions in 50% glycerol/water gave characteristic melting curves between -25°C and 5O”C, and on cooling showed increases in lightscattering between 10” C and -20” C. The temperatures at which major changes occurred depended on the proportions of saturated and unsaturated fatty acids. 3. The thermal transitions in the intact lipoprotein in glycerol solution were reversible, but with marked hysteresis. Lipid extracted from the lipoprotein did not show temperature hysteresis but the transition heats and melting curves were similar to those of the intact lipoprotein. The results support the hypothesis of a “lipid-core” structure for low-density lipoproteins. 4. Scanning calorimetry of egg-yolk lecithins indicated a strong dependence of transition temperature on water content in the range 3%-20% water. A rise in the mid-temperature of the liquid-crystalline to gel transition as the water content is lowered on freezing may be the primary event in the irreversible gelation of egg yolk and aggregation of lipoprotein.

Introduction The physical properties of whole egg yolk and of the constituent lipoproteins are altered by freezing and thawing. Thus, egg yolk that has been stored in the frozen state below a certain temperature and then thawed has a much higher viscosity than unfrozen yolk and may even be a firm gel. Solutions of the major lipoprotein show corresponding increases in viscosity, and decreases in solubility, on freezing and thawing. Investigations of these freezing

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changes which limit the application of frozen storage to egg yolk, have recently been reviewed [ l]. Similar increases in viscosity, but at temperatures above the freezing point, have been observed [2] on cooling yolks from hens that have been fed small amounts of methyl sterculate, which causes an increase in the proportion of saturated fatty acids in the yolk lipid [ 31. It has also been found (Shenstone, F.S. and Smith, M.B., unpublished) that the minimum (“critical”) temperature for storing frozen yolk without thickening may be lowered by feeding sunflower oil and thus increasing the proportion of linoleic acid in the yolk lipid. These observations suggested that there were changes in the interaction of protein and lipid [4,5] at low temperature which were affected by changes in the fatty acid composition of the lipid. In this paper we report the results of measuring, by differential scanning calorimetry and light-scattering, temperature-dependent changes in the lowdensity lipoprotein and lipids from yolks of different fatty-acid composition. Some observations on the influence of water content on a phase transition of egg lecithin and its possible relation to freezing changes in yolk are also presented. Methods Eggs of differing fatty acid composition were obtained from hens fed a basic, fat-free, diet to which was added up to 15% of olive oil, sunflower oil, or hydrogenated beef tallow. Methyl sterculate or crushed Sterculia foetida seed was added to some diets [ 61, and a commercial “laying mash” was also used. Eggs were kept at 22” C for not more than a week before use. All measurements were done on fractions derived from single yolks unless otherwise stated. A low-density lipoprotein fraction was prepared by diluting the yolk with an equal volume of 0.04% NaNJ solution and centrifuging the mixture at 250 000 X g (average) for 5 h at 20°C. The clear yellow layer at the top of the tube was carefully removed and mixed thoroughly with 0.6 of its weight of glycerol, to give a mixture containing 25-30% by weight of lipoprotein in a solvent of approx. 50% glycerol. The glycerol was used primarily to prevent freezing at temperatures above -25” C, but also gave a clear, stable solution which did not change in thermal properties or show any signs of oxidation after 2 weeks at 20” C. The addition of glycerol to aqueous solutions of lipoprotein or of synthetic dimyristoyl-lecithin did not alter their calorimetric transitions above 0°C and further purification of the lipoprotein [4] did not affect its thermal behaviour. Lipid was extracted from 1.6 g of the glycerol solution by adding 1 ml 2 M NaCl, 5 ml methanol and 2.5 ml chloroform. On shaking the mixture, a single phase was obtained, to which was added 2.5 ml chloroform and 2.5 ml HzO. The separated chloroform layer was filtered and 2 ml taken for fatty-acid analysis [ 71. Neutral lipid and lecithin were separated from chloroform-methanol extracts of yolk on an alumina column [8]. Differential scanning calorimetry was carried out with an adiabatic calorimeter specially designed for work with lipids, lipoproteins and membranes [ 91. The lipoprotein was examined in glscerol solution (0.5 g, approx. 0.13 g

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lipid), or after freeze-drying an aqueous solution directly in the sample cup (0.15 g dry weight). Solutions of lipid in chloroform were also dried in the sample cup under Nz at 40” C. Lecithin solutions were dried under vacuum in the cup at 35°C and re-hydrated to defined water contents by adding known weights of water, sealing the cup and equilibrating for 24 h at 25” C. The sample cup and a reference cup containing 50% glycerol or silicone oil were cooled in the calorimeter overnight to -25” C and then heated at rates between 8 and 12” C * h-l. The differential power required to maintain the two cups at the same temperature, and the cup temperature, were recorded as a function of time. The total heat of transition between two temperatures was obtained by planimetry, after correcting for the small base-line drift. Calibration was carried out electrically and from the known heat capacity of a 1 g aluminium block. Changes in light-scattering in the temperature range -20” C to 30” C were measured with 2.5% (w/v) solutions of lipoprotein in 50% glycerol, 0.1 M NaCl. The apparatus consisted of an insulated Perspex box, supplied with 30% ethylene glycol from a refrigerated bath, and fitted with a rotating holder for four rectangular cells. Collimated light from a low-pressure mercury lamp passed through each cell in turn, and the light scattered at 90” to the incident light was measured with a photomultiplier, high-tension battery and galvanometer. For each set of readings, the temperature of the circulating liquid was changed by 2” C, and then held constant for 30 min. The effects of freezing on whole yolk and solutions of low-density lipoprotein were studied with the aid of a “temperature-gradient bar”. This consisted of a 50 cm length of aluminium bar (5 cm X 12.5 cm) with a double row of 1.2 cm diameter holes bored in it. The bar was heavily insulated, and the temperatures at the ends were controlled by circulating liquid from a refriger-

Fig. 1. The method for observing the effect of freezing temperature on viscosity of egg yolk. Samples (1 ml) were frozen in the temperature gradient bar for 16 h. with temperature gradient -1O’C to -2O’C (from left to right). thawed at 25OC. and photographed after laying the rack on its side for 10 s.

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ated bath through a hollow block at one end, and by means of an electric heater and thermostat at the other end. The temperature gradient was linear to 0.2” C when the temperatures at the ends differed by 15” C. Glass tubes, containing yolk (1 g) or lipoprotein (0.5 g of 20% w/v solution), were cooled in the bar for 1 h, and the contents then seeded with ice crystals. After freezing for 16 h, the tubes were placed in a water bath at 25” C for 1 h, and the viscosity change in the yolk was observed by laying the rack of tubes on its side for 10 s (Fig. 1). The lipoprotein samples were diluted with 5 ml of 50% glycerol, 0.1 M NaCl, and the absorbance at 600 nm measured in a calorimeter. Results Fatty-acid composition of lipoprotein samples The total lipid extracted from each lipoprotein preparation (in most cases from a single yolk) was analyzed for fatty acids, and the results are given in Table I. Because of the small number of hens placed on each diet, no attempt was made to relate dietary fatty acids or sterculate to yolk-lipid composition; however, it is convenient to consider all the results in three groups according to the broad effects of diet on the fatty acid composition of the yolk lipid. In both Tables I and II, group A represents samples of yolks from hens fed sunflower oil; B comprises samples from “normal” diets and the fat-free and TABLE I FATTY ACID COMPOSITION OF TOiAL SITY LIPOPROTEIN AND FROM YOLK:

LIPID AND OF LECITHIN AND LIGHT-SCATTERING

EXTRACTED FROM LOW DENTEMPERATURES

Designation of yolk sample; single yolks except for lecithin preparations A4 and B6. Groups A. B. and C described in text. Values for percentage of total methyl esters recovered from gas-liquid chromatogram. ‘I’,= temperature (‘C) at which significant change in light-scattering occurred on cooling (see text). Sample

Fatty acids (%) Ts

14:o

16:0

18:0

16:l

18:l

18:2

20:4

Lipoprotein, total lipid AI A2 A3

0.2 0.4 0.4

19.7 21.3 23.0

7.8 7.4 10.1

2.3 2.2 2.2

48.5 37.6 37.9

20.1 29.8 24.8

1.1 1.0 1.3

-14.0 -18.3 -19.0

Bl B2 B3 B4 B5

0.5 1.0 0.5 0.2 0.6

21.6 22.8 29.0 23.0 30.2

8.7 8.0 5.3 9.6 9.4

3.0 3.8 5.7 3.4 3.8

55.6 53.3 41.3 54.0 42.0

8.7 9.8 17.3 9.4 13.5

0.9 1.1 0.7 0.2 0.6

-11.4 -9.7 -10.2 -11.4 4.3

Cl c2 c3 c4

0.7 0.9 0.7 1.5

34.0 35.3 30.2 32.6

20.2 18.8 16.6 23.1

2.4 1.7 2.4 1.0

28.5 30.9 30.8 20.9

13.3 12.3 17.9 19.2

0.6 0 1.2 1.4

Lecithin A4 B3 B4 B6 Cl

0.1 1.7 1.2 0.4 2.4

29.3 34.0 29.0 27.1 41.0

16.2 13.4 14.7 13.5 9.9

1.7 3.0 1.0 2.3 1.7

24.7 25.9 31.2 39.5 20.2

23.4 16.6 14.3 11.7 20.5

3.6 5.1 4.8 5.2 3.9

9.3 0.5 6.3 7.0

207 TABLE II CALORIMETRIC

HEATS OF TRANSITION

(H) OF LIPOPROTEINS

AND LIPIDS

Designation of single yolk used in preparation. as in Table I. f.d., freeze-dried lipoprotein; lip, total lipid; other samples are lipoprotein in 50% glycerol solution. O”, cooled only to 0°C. Sample

H(caI/g lipid. in intervals) Total H -lw-O°C

&-10°C

lo-2o”c

20-3

Al A2 A2 f.d.

2.2 1.7 1.2

4.2 4.1 3.6

2.1 1.7 3.2

0.1 0 0.4

0 0 0

0 0 0

Bl B2 B3 B3 B3 B4 B4 B5 B5

1.5 1.0 0.7 0.3

4.4 3.9 3.3 2.4

0.4 0.7 0.3 1.0

3.0 2.3 1.9 0.7

7.3 1.4 8.1 7.8 5.5 6.2 5.3 7.9 5.9

0.9 1.4 0.9 1.2 1.9 1.9 2.5 3.1 3.4

0 0 0 0.2 0.3 0.2 0.7 1.0 0.7

0 0 0 0 0 0 0 0 1.1

14.1 13.7 13.0 11.9 7.7 11.7 11.5 14.8 12.8

0.1 1.1 0.5 0.4 0.6 0.4

1.1 1.0 2.3 1.9 1.7 1.1

2.3 1.8 5.8 4.1 3.6 1.6 0.8

5.1 4.3 4.3 4.2 2.8 4.2 3.0

3.6 1.0 1.9 2.5 2.4 4.6 3.7

5.3 1.3 1.1 1.9 3.3 3.3 3.7

17.5 16.5 15.9 15.0 14.4 15.2 11.2

lip. lip. O” lip. f.d.

Cl Cl lip. c2 c3 C3 f.d. c4 c4 on

0” C

>30°c