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© 2004 The American Society for Nutritional Sciences J. Nutr. 134:1110-1113, May 2004

Nutrient Metabolism
Research Communication

Lymphatic Fat Absorption Varies among Rats Administered Dairy Products Differing in Physiochemical Properties1

Maj-Britt Fruekilde2 and Carl-Erik Høy3

BioCentrum-DTU, Biochemistry and Nutrition Group, and Centre for Advanced Food Studies, The Technical University of Denmark, Lyngby, Denmark

2To whom correspondence should be addressed. E-mail: mbf{at}biocentrum.dtu.dk.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
We examined in rats the intestinal absorption of fat from dairy products differing in physiochemical properties. Five dairy products (cream cheese, cream, sour cream, butter, and mixed butter) with minor differences in fatty acid composition were administered by gavage to rats, and lymphatic fat absorption was examined. Absorption was followed for 8 h after administration of 300 mg fat from the dairy products. Administration of cream and sour cream resulted in faster lymphatic fat absorption than cream cheese, butter, and mixed butter, and at 8 h the accumulated absorption of fat was significantly higher. The lymphatic absorption of fat after cream cheese administration was similar to the absorption after butter and mixed butter administration up to the 4-h time point; then it increased to a level between that of rats administered cream or sour cream and butter or mixed butter. Overall, these results demonstrated different lymphatic absorption patterns of fat from dairy products differing in physiochemical properties. Because the fatty acid composition of the dairy products differed only slightly, other factors such as viscosity, type of emulsion, particle size, and likely also protein content may have contributed to the differences in absorption.

KEY WORDS: • lymphatic absorption • milk fat • butter • physiochemical properties • rats

Gastrointestinal lipid digestion and absorption consist of several sequential steps that include physiochemical and enzymatic events (1). Digestion of dietary triglycerides starts in the stomach with the action of lingual and gastric lipases (2) and continues in the duodenum by pancreatic lipase (3). Then, the products that were generated by lipolysis and that accumulated at the fat globule surface are transferred into structures made of phospholipids and bile salts, forming mixed micelles in the aqueous phase (1). After absorption into the enterocytes, fatty acids are activated into CoA-esters and reacylated with 2-monoglycerides to form a new population of triglycerides (4), packaged as chylomicrons (5) and secreted into the lymph (6).

Milk products provide nutrients, including calcium, proteins, riboflavin, retinol, and vitamin B-12, but dairy products based on whole milk also contain substantial amounts of SFA and cholesterol (7), which increase blood cholesterol (8,9) and thus the risk of coronary heart disease. On the other hand, it was observed that milk and milk products may have hypocholesterolemic effects (10–13). Although different dairy products have similar fatty acid compositions, they may affect the plasma cholesterol level differently. Therefore, factors in addition to the fatty acid composition may influence the lipemic response.

Dairy products have different physiochemical properties as follows: 1) some are water in fat emulsions (butter), others are fat in water emulsions (milk, yogurt, cream and sour cream); and 2) some are consumed fresh (milk, cream), and others are consumed after fermentation (yogurt, buttermilk, and sour cream). The fermentation changes the pH, consistency, and lactose content.

In vitro experiments with complex emulsions showed that the degree of lipid emulsification affects the activity of digestive lipases (14,15). The viscosity of the dairy products also influences the gastric emptying time (16). Furthermore, fermented milk was observed to reduce the absorption of cholesterol compared with low-fat milk in ileostomy subjects (17).

In the present study, we examined the effects of the physiochemical properties of dairy products on lymphatic fat absorption in rats. The dairy products examined differed with respect to fat, protein, and calcium contents as well as viscosity, particle size, culture applied for fermentation, and type of emulsion (oil in water or water in oil emulsions). It is possible that the physiochemical properties of the dairy products affect lipid digestion and absorption and thus the plasma response. The following dairy products were examined: cream cheese, cream, sour cream, butter, and mixed butter.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Animals and diets. The following experiments were approved by the Danish Committee for Animal Experiments. Male albino Wistar rats were purchased from Møllegaard Breeding and Research Centre. They were fed a standard pellet diet (Altromin no 1324, Chr. Petersen A/S), containing 190, 40, and 60 g/kg of protein, fat and fiber, respectively. The rats weighed 250–300 g at the time of surgery, and 8 rats were assigned to each treatment. The number of rats in the group receiving cream was increased to 12 rats (6 rats catheterized by 2 different people) because the surgeon was replaced shortly before the experiment ended. No significant interpersonal differences were observed.

    Surgery. Rats that had not been food deprived were anesthetized i.m. with ~0.055 mL Zoletil-mixture/100 g body weight (The Royal Veterinary and Agricultural University, Copenhagen, Denmark). The main mesenteric lymph duct was cannulated (18) and the catheter was led subcutaneously to the abdomen. A collection tube was attached to the abdomen of the rat (19), and a plastic collar was placed around its neck to prevent it from eating the catheter. After surgery, rats were given 0.05 mL Antisedan (Farmos) i.m. to antagonize the anesthetic, and 5 mL of physiological saline (9 g/L NaCl) was given subcutaneously to prevent dehydration. After surgery (4–6 h) rats were administered 0.2 mL of analgesic (Torbugesic) diluted 1:10 with sterile water. Rats were placed in individual cages. A glucose solution (55 g/L glucose, H2O) and tap water were freely available, but no food was given.

    Test lipids. Lymph collection experiments were performed with 5 different dairy products. In each experiment, 300 mg of fat was administered; this is equivalent to 0.37 mL melted butter or mixed butter, 0.79 mL cream or sour cream, or 1.43 g cream cheese. All dairy products were purchased in local supermarkets. Cream cheese was manufactured by Nørup Mejeri, and the other products were produced by Arla Foods.

    Lymph collection. After 20–24 h recovery (postoperative d 1), the experiment was initiated by the collection of a baseline fraction of lymph from –1 h to 0 h. At time "zero," a dairy product was administered as a bolus into the stomach with a gavage needle. Subsequently, lymph was collected into tubes at 1-h intervals for the next 8 h. The tubes contained 100 µL of 100 g/L Na2-EDTA-2H2O (Merck). The samples were immediately frozen and stored at –20°C until analyzed. After sampling, the rats were killed by an overdose of sodium pentobarbital.

    Analytical methods. Tritridecanoin (Nu-Chek-Prep) was added to lymph samples and dairy products as an internal standard, and lipids were extracted using the method of Folch (20). Lipid extracts were transmethylated, catalyzed by KOH in methanol (21), and the FAME were dissolved in heptane and analyzed by GLC. Using a modification of the GC methods of Vistisen et al. (22), the injections were run with a split ratio of 1:16 and the initial oven temperature was 50°C (3 min). Temperature programming was as follows: 15°C/min to 160°C, 1°C/min to 180°C, 0.5°C/min to 190°C, 20°C/min to 200°C, which was maintained for 10 min.

    Statistical analysis. Results are expressed as means ± SEM. Differences after 8 h were tested using one-way ANOVA. Intergroup comparisons were performed using the Student-Newman-Keuls method. Differences were considered significant at P < 0.05. The statistical analyses were carried out using Graphpad Prism version 3.0 (Graphpad Software). Comparisons of the shape of the curves were performed by repeated measurement two-way ANOVA (SAS version 8.2, SAS Institute). To stabilize variance over time, this analysis was carried out on data that were square-root transformed.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
    Composition of the dairy products. The contents of fat, protein, carbohydrate, calcium, and the cultures used for fermentation varied in the different dairy products (Table 1). The fatty acid compositions of the dairy products were similar except that mixed butter was a mixture of 75% milk fat and 25% rapeseed oil.4 Mixed butter had a lower SFA content and a higher content of 18:1(n-9), 18:2(n-6) and 18:3(n-3) than the other dairy products.


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  		TABLE 1 Composition of dairy products and the culture used for fermentation1

 
    Accumulated lymphatic absorption. Administration to rats of the dairy products resulted in major differences in the accumulated lymphatic absorption of fatty acids (Fig. 1). The rats fed cream and sour cream had higher accumulated absorption at 8 h than those administered butter, mixed butter (P < 0.001) or cream cheese (P < 0.01). Feeding cream cheese to rats resulted in greater accumulated absorption compared with butter and mixed butter (P < 0.05). Three hours after administration of cream and sour cream, the accumulated lymphatic absorption of fatty acids was higher than that after cream cheese, butter, and mixed butter, whereas the lymphatic absorption of fatty acids after administration of cream cheese was similar to that after butter and mixed butter up to the 4-h time point. The absorption patterns after administration of the 5 dairy products differed (P < 0.001). Administering cream and sour cream to rats resulted in a rapid increase in fat absorption within the first few hours, whereas administration of cream cheese resulted in delayed lymphatic fat absorption.



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  		FIGURE 1 Accumulated lymphatic transport of total fatty acids in rats after the administration of various dairy products containing 300 mg fat. Values are means ± SEM, n = 6–12. The absorption patterns differed. Means (8 h) without a common letter differ, P < 0.05.

 
    Fatty acid composition of lymph lipids. Two hours after administration of the dairy products, lymphatic fat absorption peaked for most of the dairy products examined (data not shown). The fatty acid profiles of the administered dairy products were reflected in the fatty acid composition of total lymph lipids 2 h after administration.4 Fatty acids with a chain length shorter than 8 carbon atoms were not detected in the lymph lipids. Lymph collected 2 h after administration of cream cheese, cream, sour cream, and butter had similar fatty acid compositions, whereas lymph from rats administered mixed butter had a significantly lower SFA content and higher contents of 18:1(n-9), 18:2(n-6) and 18:3(n-3), reflecting the fatty acid composition of mixed butter. Eight hours after administration of dairy products, the content of fatty acids typical of dairy products [especially 14:0, 16:0 and 18:1(n-9)] decreased, and endogenous fatty acids [18:2(n-6), 20:4(n-6) and 22:6(n-3)] increased compared with lymph collected at 2 h, indicating that the rats were in the postabsorptive state. Furthermore, the differences in fatty acid composition of lymph collected after administration of mixed butter or butter, cream and sour cream were much smaller at 8 h than at 2 h. On the other hand, lymph collected 8 h after administration of cream cheese had significantly higher contents of SFA (10:0–16:0) and a lower content of 18:2(n-6) than lymph collected after administration of the other products. This indicated that the absorption of fat from cream cheese continued to occur.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
We examined the intestinal absorption of fat from different dairy products that varied in fat, protein and calcium contents, viscosity, particle size, culture applied for fermentation, as well as type of emulsion (oil in water and water in oil).

The observed differences in accumulated fat absorption to the lymphatic system 8 h after administration of different dairy products indicated differences in absorption patterns. The absorption of dietary triglycerides in humans and animals is very efficient under normal circumstances (18,23,24). It is therefore possible that the accumulated absorption would have been similar for the dairy products if the examination had been carried out for a prolonged time. On the other hand, milk fat is not absorbed solely into the lymphatic system because milk fat has a high content of short- and medium-chain fatty acids, which are mainly absorbed directly into the blood through the portal vein (25,26). Furthermore, long-chain SFA may be absorbed less efficiently by the enterocytes and possibly also reesterified less efficiently into triglycerides than the corresponding unsaturated fatty acids (27). For these reasons, not all of the administered fat will enter the lymphatic system. The dairy products examined had similar fatty acids compositions (except mixed butter); thus, factors other than fatty acid composition must be involved in creating the different absorption patterns. Differences in viscosity, type of emulsion, particle size, calcium, and protein content could influence the absorption of fat.

The accumulated lymphatic absorption of fat 8 h after administration of 300 mg fat was between 100 and 200 mg for the 5 dairy products examined. This is in agreement with the results of Porsgaard and Høy (18), who reported an accumulated lymphatic absorption of ~100 mg fat 8 h after the administration of 270 mg butter. Degrace et al. (24) recovered 91 mg triglyceride in lymph 6 h after administration of 0.65 mL butter, and this increased to 346 mg after 24 h.

Our results demonstrated that 3 h after administration of cream and sour cream, the accumulated lymphatic absorption of fat increased compared with butter, mixed butter, and cream cheese, indicating a more rapid absorption of fat after administration of cream and sour cream. This may be due to a more rapid hydrolysis of the triglycerides in cream and sour cream than in butter and mixed butter, because cream and sour cream enter the stomach as emulsions (oil in water) and do not need further emulsification before lipolysis. It was shown that emulsion droplet size affected the lipolysis rate of triglycerides in humans (28) and rats (15). Intragastric feeding of fine emulsions (0.7 µm) resulted in faster lipolysis than infusion of coarse emulsions (10 µm), indicating that the larger initial surface area of the particles increased the hydrolysis rate in the stomach (15,28). On the other hand, a delayed occurrence of chylomicron triglycerides in plasma for the fine emulsions compared with the coarse emulsions and a slower gastric emptying rate of fine emulsions than coarse emulsions were observed. The increased accumulated absorption of fat 8 h after the administration of cream and sour cream, compared with butter and mixed butter, may be explained by the type of emulsions, oil in water vs. water in oil emulsion. The absorption of fat from cream cheese was similar to that of butter and mixed butter for the first 4 h, and then increased to a level between cream or sour cream and butter or mixed butter. The lymph collected 8 h after administration of cream cheese contained higher levels of 12:0, 14:0, and 16:0 than lymph collected after administration of the other products. This indicated a delayed absorption of fat from cream cheese. The cream cheese has a higher viscosity and protein content than cream and sour cream, which possibly affected the gastric emptying rate, thereby delaying the absorption of fat. Strandhagen et al. (16) observed longer gastric emptying time for fermented milk than for regular milk, which they attributed to the higher viscosity of the fermented milk. Borel et al. (15) observed that a complex fine lipid emulsion containing protein had a decreased rate of lipolysis and gastric emptying compared with a fine lipid emulsion without protein. These findings support the hypothesis that the delayed lymphatic absorption of cream cheese fat compared with cream and sour cream was caused by slower gastric emptying.

This study demonstrated different lymphatic absorption patterns of dairy products with different physiochemical properties. This indicates that viscosity, type of emulsion, particle size, and likely also protein content influence the digestion and absorption of dairy fat, and possibly affect the lipemic response.


   ACKNOWLEDGMENTS
 
The authors thank Karen Jensen, Lillian Vile, and Egon Christensen for technical assistance and Lene Theil Skovgaard, Department of Biostatistics, University of Copenhagen for statistical support.


   FOOTNOTES
 
1 Supported by the Danish Dairy Research Foundation and the Danish Research Development Program for Food Technology (FØTEK 2). Back

3 Carl-Erik Høy is deceased. Back

4 Data are available with the online posting of this paper at www.nutrition.org. Back

Manuscript received 3 October 2003. Initial review completed 13 November 2003. Revision accepted 9 February 2004.


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

1. Carey, M. C. & Hernell, O. (1992) Digestion and absorption of fat. Semin. Gastrointest. Dis. 3:189-208.

2. Hamosh, M. (1994) Gastric and lingual lipases. Johnson, L. R. eds. Physiology of the Gastrointestinal Tract 3rd ed. 1994:1239-1253 Raven Press New York, NY. .

3. Mattson, F. H. & Beck, L. W. (1956) The specificity of pancreatic lipase for the primary hydroxyl groups of glycerides. J. Biol. Chem. 219:735-740.[Free Full Text]

4. Lehner, R. & Kuksis, A. (1996) Biosynthesis of triacylglycerols. Prog. Lipid Res. 35:169-201.[Medline]

5. Hussain, M. M. (2000) A proposed model for the assembly of chylomicrons. Atherosclerosis 148:1-15.[Medline]

6. Sabesin, S. M. & Frase, S. (1977) Electron microscopic studies of the assembly, intracellular transport, and secretion of chylomicrons by rat intestine. J. Lipid Res. 18:496-511.[Abstract]

7. Jensen, R. G., Ferris, A. M. & Lammi-Keefe, C. J. (1991) Symposium: Milk Fat-Composition, Function, and Potential for Change. The Composition of Milk Fat. J. Dairy Sci. 74:3228-3243.[Abstract/Free Full Text]

8. Howard, A. N. & Marks, J. (1977) Hypocholesterolaemic effect of milk. Lancet 30:255-256.

9. Kris-Etherton, P. M., Derr, J., Mitchell, D. C., Mustad, V. A., Russell, M. E., McDonnell, E. T., Salabsky, D. & Pearson, T. A. (1993) The role of fatty acid saturation on plasma lipids, lipoproteins, and apolipoproteins: I. effects of whole food diets high in cocoa butter, olive oil, soybean oil, dairy butter, and milk chocolate on the plasma lipids of young men. Metabolism 42:121-129.[Medline]

10. Mann, G. V. (1977) A factor in yogurt which lowers cholesteremia in man. Atherosclerosis 26:335-340.[Medline]

11. Hepner, G., Fried, R., Jeor, S., Sr., Fusetti, L. & Morin, R. (1979) Hypocholesterolemic effect of yogurt and milk. Am. J. Clin. Nutr. 32:19-24.[Abstract/Free Full Text]

12. Agerbæk, M., Gerdes, L. U. & Richelsen, B. (1995) Hypocholesterolaemic effect of a new fermented milk product in healthy middle-aged men. Eur. J. Clin. Nutr. 49:346-352.[Medline]

13. Steinmetz, K. A., Childs, M. T., Stimson, C., Kushi, L. H., McGovern, P. G., Potter, J. D. & Yamanaka, W. K. (1994) Effect of consumption of whole milk and skim milk on blood lipid profiles in healthy men. Am. J. Clin. Nutr. 59:612-618.[Abstract/Free Full Text]

14. Armand, M., Borel, P., Ythier, P., Dutot, G., Melin, C., Senft, M., Lafont, H. & Lairon, D. (1992) Effects of droplet size, triacylglycerol composition, and calcium on the hydrolysis of complex emulsions by pancreatic lipase: an in vitro study. J. Nutr. Biochem. 3:333-341.

15. Borel, P., Armand, M., Pasquier, B., Senft, M., Dutot, G., Melin, C., Lafont, H. & Lairon, D. (1994) Digestion and absorption of tube-feeding emulsions with different droplet sizes and compositions in the rat. J. Parenter. Enteral Nutr. 18:534-543.[Abstract/Free Full Text]

16. Strandhagen, E., Lia, Å., Lindstrand, S., Bergström, P., Lundström, A., Fondén, R. & Andersson, H. (1994) Fermented milk (ropy milk) replacing regular milk reduces glycemic response and gastric emptying in healthy subjects. Scand. J. Nutr. 38:117-121.

17. Andersson, H., Bosaeus, I., Ellegård, L., Grahn, E., Tidehag, P., Hallmans, G., Holm, S. & Sandberg, A.-S. (1995) Effects of low-fat milk and fermented low-fat milk on cholesterol absorption and excretion in ileostomy subjects. Eur. J. Clin. Nutr. 49:274-281.[Medline]

18. Porsgaard, T. & Høy, C.-E. (2000) Lymphatic transport in rats of several dietary fats differing in fatty acid profile and triacylglycerol structure. J. Nutr. 130:1619-1624.[Abstract/Free Full Text]

19. Hanberg, A. & Trossvik, C. (1995) In situ collection of intestinal lymph in the non-restrained rat. Scand. J. Lab. Anim. Sci. 22:329-334.

20. Folch, J., Lees, M. & Sloane Stanley, G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509.[Free Full Text]

21. Christopherson, S. W. & Glass, R. L. (1969) Preparation of milk fat methyl esters by alcoholysis in an essentially nonalcoholic solution. J. Dairy Sci. 52:1289-1290.[Abstract/Free Full Text]

22. Vistisen, B., Mu, H. & Høy, C.-E. (2003) Recoveries of rat lymph FA after administration of specific structured 13C-TAG. Lipids 38:903-911.[Medline]

23. Jones, P.J.H., Pencharz, P. B. & Clandinin, M. T. (1985) Absorption of 13C-labeled stearic, oleic, and linoleic acids in humans: application to breath tests. J. Lab. Clin. Med. 105:647-652.[Medline]

24. Degrace, P., Caselli, C., Rayo, J. M. & Bernard, A. (1996) Intestinal lymph absorption of butter, corn oil, cod liver oil, menhaden oil, and eicosapentaenoic and docosahexaenoic acid ethyl esters in rats. Lipids 31:405-414.[Medline]

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28. Armand, M., Pasquier, B., André, M., Borel, P., Senft, M., Peyrot, J., Salducci, J., Portugal, H., Jaussan, V. & Lairon, D. (1999) Digestion and absorption of 2 fat emulsions with different droplet sizes in the human digestive tract. Am. J. Physiol. 70:1096-1106.





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