Seasonal variations in the concentration of gangliosides and sialic acids in milk from different mammalian species

Int. Dairy Journal 6 (1996) 315-322 Copyright 0 1996 Elsevier Science Limited Printed in Ireland. All rights reserved 09%6946/96/$15.00+0.00 ELSEVIER

0958-6946(95)00013-5

Seasonal Variations in the Concentration of Gangliosides and Sialic Acids in Milk from Different Mammalian Species R. PuenteO, L.A. Garcia-Pardob,

R. Ruedac, A. Gil” & P. Huesoa*

“Departamento

de Bioquimica y Biologia Molecular, Facultad de Biologia, Universidad de Salamanca, 37007 Salamanca, Spain ‘Departamento de Fisiologia, Farmacologia y Toxicologia, Facultad de Veterinaria, Universidad de Leon, 24071 Leon, Spain ‘Departamento de Investigation y Desarrollo, Puleva, 18004 Granada, Spain (Received 21 November 1994; revised version accepted 9 March 1995)

ABSTRACT Seasonal changes in the ganglioside content of cows’, goats’ and ewes’ milk were studied over a one year period. All species considered showed the highest ganglioside content in autumn. The minimum ganglioside content was observed in summer milk for cows and goats but not for ewes. A correlation between the fat and ganglioside content of milk seems to emerge ,from these and other results. Identtfied (selected for milk production) Holstein-Friesian cows had a lower ganglioside content than unidenttfied cows (milk samples from bulk tanks of a dairy factory) Minor changes in the ganglioside pattern of cows’ milk were detected. However, seasonal variations in the total sialic acid content of cows’ and goats’ milk through the year were not detected. The availability of lipid precursors rather than the sialic acid content or sialyltransferases activity seems to be the limiting factor for milk ganglioside biosynthesis. Finally, the ganglioside content of cows’ milk was not affected by mild heat-treatment (thermization, 6S”C, 30 s).

The ganglioside recommendations (NeuAc)z-LacCer;

nomenclature of Svennerholm (1958) and the IUPAC-IUB (1977) are followed: GM~, II3 NeuAc-LacCer; GD3, II3 GT3, II3 (NeuAc)j-LacCer; LacCer, lactosylceramide. INTRODUCTION

The composition stage of lactation,

of milk varies as a function of several factors such as breed, climate, season of the year and feeding. The influence of season

*Author to whom correspondence

should be addressed 315

316

R. Puente et al.

is difficult to separate from climatic and nutritional factors. Additionally, seasonal variations in composition are related to variations in the stage of lactation. Data on these topics have been reported by Gaunt (1973) Ramos & Juarez (1981) and Rodriguez et al. (1985). Lactational changes in the ganglioside and sialic acid contents of milk from cows (Puente et al., 1992), goats (Puente et al., 1994) and ewes (unpublished results) have also been reported. However, the literature lacks data on seasonal variations of these compounds. Gangliosides are sialic acidcontaining glycosphingolipids located mainly on the outer surface of mammalian cell plasma membranes. They are also found in the membrane surrounding fat globules (MFGM) (Keenan et al., 1972) which is derived from the apical plasma membrane of mammary gland secretory cells (Keenan & Dylewski, 1985). It has been postulated that milk gangliosides and sialic acids play a significant role in the defense of neonates against infection (Laegreid et al., 1986). The purpose of the present study was to investigate seasonal influences on the gangliosides and sialic acids of bovine, ovine and caprine milks.

MATERIAL

AND

METHODS

Chemicals Pre-coated thin-layer chromatographic plates (TLC and HPTLC) (silica gel G type 60) were purchased from Merck (Darmstadt, Germany). Dowex 2 x 8 and ZV-acetylneuraminic acid were obtained from Sigma (St Louis, MO, USA). All other products were supplied by Probus (Barcelona, Spain). All chemicals were of analytical grade. Solvents were distilled before use. Milk samples Bulk herd samples from Holstein-Friesian cows (herd of Puleva, Granada) or from bulk tanks at the Puleva factory in Granada (unidentified cows, but mainly Holstein), Murciana-Granadina goats (herd of the dairy farm of the Diputacion of Granada) and ewes (60% churra, 30% merina and others, from Salamanca) were used. Samples were collected over three consecutive days in spring (May), summer (July), autumn (November) and winter (February). The values for each season are the means of the data from these three determinations. Samples were always obtained from the morning milking, frozen immediately at -20°C and lyophilized. Isolation of gangliosides Gangliosides were isolated essentially as described by Puente et al. (1992). Lyophilized whole milk was homogenized with 10 volumes of cold acetone (-20°C) to remove neutral lipids, mainly triacylglycerols. The homogenate was filtered through a sintered glass funnel and the residue homogenized again with 6 volumes of cold acetone (-20°C) and filtered. The solid residue (acetone powder) was successively extracted with 10 volumes each of

Seasonal changes in milk gang&sides and siulic acids

317

chloroform-methanol, 2:1, I:2 and 1:l (by vol). The combined extracts were evaporated to drynesss and taken up in 10 volumes of chloroform-methanol, 2:l (by vol). The extract was subjected to Folch’s partition to obtain the crude ganglioside fraction. The combined upper aqueous phases were partially evaporated and lyophilized. The lyophilizate was dissolved in water and dialyzed exhaustively against distilled water at 4°C for five days. After dialysis, the material was lyophilized and dissolved overnight in chloroform-methanol-water, 60:30:4.5 (by vol). The insoluble material, mainly proteins, was discarded. Because milk contains a high proportion of GMM3,the ganglioside content of the lower organic phase was determined previously but none were found (Puente et al., 1992). Analytical

procedures

Total gangliosides were determined as lipid-bound sialic acid by the resorcinol procedure of Svennerholm (1957). Identification of gangliosides was performed by TLC or high performance TLC (HPTLC) on pre-coated silica gel plates with the following solvent systems: (1) chloroform-methanol-0.5% CaClz 2H20, 55:45:10 (by vol), (2) chloroform-methanol-0.25% aqueous KCI, 60:35:8 (by vol) and (3) chloroform-methanol-2.5M NH40H, 60:30:8 (by vol), as described by Puente et al. (1992, 1994). Gangliosides were visualized by spraying the plates with the resorcinol reagent (Svennerholm, 1957). Individual gangliosides were analysed with a dual-wavelength TLC densitometer (CS-930 Shimadzu, Kyoto) after separation by TLC or HPTLC in solvent system 1. The sialic acid content of milk was determined as follows: A 0.25 g aliquot of lyophilized milk was dissolved in 0.5 mL of water with bath sonication; 2.5 mL of 0.05 M H2S04 were added and the mixture incubated at 80°C for lh. The hydrolysate was cooled to 20-22“C, and purified by ion-exchange chromatography on a column of Dowex 2 x 8, HCO*- form (2 mL). The column was rinsed with 20 mL of water. Adsorbed sialic acids were eluted with 20 mL of 1 M formic acid, lyophilized and quantified by the resorcinol procedure of Svennerholm (1957). Heat treatment (thermization) The milk was cooled (1CrlS’C) at the farm and transported in an insulated container to the laboratory. Milk was heated at 65°C for 30 s and cooled to 2°C. After this, the milk was frozen and lyophilized. Statistical

analysis

Statistical comparisons among data were made by one-way analysis of variance (ANOVA) and the Fisher test, using a standard computerized statistical program, except for comparisons between ID (identified cows) and NID (unidentified cows) samples and HTM (heat-treated milk) and NHTM (non heat-treated milk) samples that were analysed statistically using an unpaired Student’s t-test.

R. Puente et al.

318

RESULTS Ganglioside

AND

DISCUSSION

content

Table 1 shows the ganglioside content (expressed as lipid-bound sialic acid) of milk from cows, goats and ewes over a one year period. The ganglioside content of cows’ milk changed markedly during the year. It was high in autumn for both the ID and the NID samples and the minimum content was found in the spring-summer period. It has been reported (Laben, 1963; Gacula et al., 1968) that most constituents that vary (yield of milk, fat and solid non-fat) are highest in November through January and lowest in May through July. These data are consistent with the seasonal ganglioside profile found by us. It is difficult to account for these seasonal changes. The ID cows received a constant and regulated feed supply and calved throughout the year. The feeding and calving patterns of the NID cows were probably highly variable and very different from the ID cows. However, the seasonal profile of the ganglioside content was very similar. Since gangliosides are complex lipids, a decrease or increase in the milk fat content could be responsible for the variations observed in the ganglioside content. Agabriel et al. (1993) have found that the fat content of bovine milk decreased from October to July and then increased again until October. This profile is very similar to the ganglioside profile obtained by us. In this sense, a correlation between the fat and ganglioside contents of milk has been established in humans (unpublished results). A surprising finding was that the ganglioside content of NID cows was higher than that of ID cows throughout the year (Table 1). As already mentioned, ID samples came from selected Holstein-Friesian cows, whereas NID samples were obtained from bulk tanks containing milk from non-selected animals. It is well documented that

TABLE

1

Seasonal Variations in the Ganglioside Concentration

of Cows’, Goats’ and Ewes’ Milk*

cows ID NHTM Spring Summer Autumn Winter

158zt39” 142 i 32” 225 * 39” 221 zt 33”

NID HTM 108 120 266 179

zt f f f

26” 39”’ 43’ 35’

NHTM 292f 11” 323 f 34” 485 f 50’ 367 Y+Z 29”

Goats

Ewes

HTM 371 316 448 342

f * zt f

71” 14” 3ob 21”

63 29 124 61

f 17” f 8’ f 10’ &t8”

57 & 12” 100 +z 18’ 119* 14b 48 f 8”

*Ganglioside concentration is expressed as pg of lipid-bound sialic acid/kg fresh milk. Values are the means&SD of three determinations performed in the milk from three consecutive days. ID, milk from identified Holstein-Friesian cows; NID, bulk tank milk from unidentified cows.

HTM, heat-treated milk (65”C, 30 s); NHTM, non heat-treated milk. u.h,c Means followed by the same letter in the same column do not differ (p > 0.01) according to the Fisher test.

319

Seasonal changes in milk gangliosides and sialic acids

changes in milk composition are possible through selective breeding. Milk yields and the percentages of fat and protein are the most important specific traits from economic and nutritional points of view. Selection for milk production, concomitant with a high production of fat and/or protein, as was the case for the identified Holstein-Friesian cows, could lead to a decrease in the production of milk gangliosides. However, the presence in the unidentified cows of breeds having much elevated milk ganglioside contents cannot be discarded. In this sense, we found (Puente et al., 1992) that cows of Spanish-Brown breed have a milk ganglioside content higher than Holstein-Friesian cows. As reported by Puente et al. (1992) seven different ganglioside species have been detected in bovine milk. One of these, appearing in the monosialoganglioside region on TLC, was not detected in any of the samples analysed and was therefore not considered in this study. Gangliosides were designated Gl-G6, according to their mobility on TLC plates. They were identified by co-migration with authentic standards and according to the ganglioside pattern found by us in cows’ milk (Puente et al., 1992). Gl, G3 and G5 were assumed to be GMM3,GD3 and Grs. G2 was identified as 0-acetyl Go3 since base treatment converted it into ganglioside GDs (Ren et al., 1992). G4 and G6 were tentatively designated on the basis of their mobility on TLC plates and the results reported by Takamizawa et al. (1986) as a mono and a trisialoganglioside, respectively, with a branched oligosaccharide chain. Minor seasonal changes in the ganglioside pattern of cows’ milk were found (Table 2). G3 (GDs) was present at higher levels in ID cows than in NID cows throughout the year. Likewise, G5 (Grs) showed a lower value in ID cows than in NID cows. These changes may be metabolically related since GD3 is the precursor of GT3. Goats’ milk showed the highest ganglioside content in autumn and the lowest in summer (Table 1). These data coincide with those obtained for cows’ milk. As reported by Puente et al. (1994) goats’ milk has a lower ganglioside content than

Effect of Season

on the Distribution

TABLE 2 of Individual Gangliosides Cows’ Milk*

(Ganglioside

ID Spring Gl G2 G3 G4 G5 G6

Summer

Pattern)

of

NID Autumn

17.6f2.2 25.OhO.6 17.7zt2.7 16.4f2.2 8.6zJxO.6 10.9f4.6 49.2f6.1 52.7zt5.6 58.0f3.1 1.8ztO.l 1.8zt1.6 1.8f0.6 13.710.8 6.9f1.6 8.4+5.9 1.3ztO.6 5.0fl.O 3.2&2.1

Winter

Spring

11.5Yt1.9 24.7f7.2 ll.Of0.5 3.9f0.9 53.9f4.4 54.1&10.0 2.8f0.6 2.3ztO.l 13.6f2.4 8.7zt1.9 7.2f0.9 6.3zt2.3

Summer

Autumn

Winter

20.9f7.8 8.2f1.9 59.lflO.O 0.5f0.2 7.3f3.0 4.0f0.9

10.7f1.4 11.3f2.1 64.8f1.8 l.lf0.2 8.1f1.7 4.0*1.1

13.1zt2.3 8.2f0.5 67.Ozt2.0 0.6&0.1 7.6f2.6 3.5kl.4

*Percentage of each ganglioside (expressed as lipid-bound sialic acid) in the total lipidbound sialic acid. Gangliosides were designated Gl to G6 according to their mobility on TLC plates. Mobility decreases from Gl to G6. ID and NID, see Table 1. Values are the means f SD of three consecutive days.

determinations

performed

in milk

from

three

R. Puente et al.

320

cows’ milk. It has been observed (Morand-Fehr et al., 1986) that goats produce more fat in autumn than in summer, regardless of the stage of lactation. As mentioned above, gangliosides are complex lipids and high milk fat levels may be accompanied by higher contents in gangliosides. Ewes’ milk also showed the maximum ganglioside content in autumn (Table 1). However, the content of summer milk was not the minimum. It is very difficult to explain these variations, although Ramos & Juarez (1981) reported that seasonal variations in the composition of ewes’ milk are related mainly to the stage of lactation. Sialic acid contents No changes in the sialic acid content of cows’ and goats’ milk throughout the year (Table 3). Neither were any differences observed ID and NID samples. Infuence of heat treatment (thermization)

on milk ganglioside

were found between the

content

The influence of heat treatment was determined only in cows’ milk because this type of milk is the most important in the dairy industry. Thermization (heat treatment at 65”C, 30 s) was used since this is a pre-treatment of milk normally used by the Spanish dairy company Puleva. Heat-treatment did not alter the ganglioside content of cows’ milk (Table 1) (Student’s t-test, P> 0.05). Pasteurization or UHT-treatment did not alter the sialic acid in either the lipid- or non-fat fractions of bovine milk (Neeser et al., 1991). Arumughan et al. (1978) reported that heating bovine milk to pasteurization temperature (7l”C, 15 s) does not affect the carbohydrate content of k-casein, including its sialic acid content. It seems that the mild heat treatments employed in the dairy industry produce no appreciable changes in the TABLE 3

Seasonal Variations in the Sialic Acid Content of Cows’ and Goats’ Milk* cows

Spring Summer Autumn Winter

Goats

ID

NID

57&3” 58*6” 65~t7” 64f8*

57&S” 54f3” 69&l” 61~t2”

229f28” 235+13” 242& 17” 233*18”

*Sialic acid content is expressed as mg of sialic acid kg-’ fresh milk. ID and NID, see Table 1. OMeans followed by the same letter in the same column do not differ (P> 0.05) according to the Fisher test. Values are the mean&SD of three determinations performed in milk from three consecutive days.

Seasonal

changes

in milk gangliosides

321

and sialic acids

carbohydrate content of milk, probably because the glycosidic linkages involved in glycoconjugate structure are resistant to such treatments. However, significant decreases in the sialic acid and hexosamine contents of rc-casein were found when severe heat treatments (boiling, sterilization) were applied (Arumughan et al., 1978)

CONCLUSIONS Milk from cows, goats and ewes has a higher ganglioside content in autumn than in other seasons. The total sialic acid content was very similar throughout the year in all three species studied. Since gangliosides are complex lipids, it appears that seasonal effects are reflected in milk fat content. Furthermore, the availability of lipid precursors, rather than the sialic acid content or sialyltransferases activity, seems to be the limiting factor for milk ganglioside biosynthesis. The ganglioside content was not altered by the mild heat treatments currently used in the dairy industry (thermization, 65°C 30 s).

ACKNOWLEDGEMENTS This work was supported by a grant from Puleva-Union Industrial y Agroganadera S.A. in collaboration with the Centro para el Desarrollo Tecnologico Industrial (CDTI) and Ministerio de Industria, Comercio y Turismo (MICYT), no 900020. We are gratefully indebted to Mr N. Skinner for revising the English version and Miss M.J. Ruano for help and encouragement.

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