Long term effects on human plasma lipoproteins of a formulation enriched in butter milk polar lipid

High concentrations of plasma cholesterol, especially low density lipoproteins (LDL) and/or triglycerides (TG) and lipoprotein a (Lp (a)) [1] are well known risk factors for cardiovascular disease (CVD) whereas HDL levels are inversely correlated to risk. Plasma lipid and lipoprotein levels are determined by genetic predisposition as well as lifestyle and dietary levels can be influenced by numerous dietary factors acting via several mechanisms. To reduce intestinal cholesterol absorption by dietary factors is an important way to improve blood lipid concentrations. A successful example is that foods containing plant sterols can reduce LDL cholesterol levels by 10-20% [2].

Sphingomyelin (SM) is a polar lipid in the milk fat globule membrane (MFGM) and accounts for 25% of the membrane lipids in bovine milk [3]. SM is also a precursor to the bioactive sphingolipid signaling molecules ceramide, sphingosine and sphingosine-1-phospate [4]. Both dietary and membrane SM interact strongly with cholesterol [5]. Studies on rats have shown that dietary SM inhibits cholesterol absorption and the inhibition is more effective with milk SM than egg SM [6, 7]. Dietary SL decreased both cholesterol and TG concentrations in lipid- and cholesterol fed ApoE Leyden mice [8].

Milk fat contains 2-3% of polar lipids and the Western dietary intake of SM from bovine milk is approximately 4-12 mg/dl [9]. Buttermilk and whey are two liquid remains from manufacturing of butter and cheese, which have in recent years gained more interest as a source of bioactive compounds such as MFGM [10]. During the process of churning and cheese making an excess of polar MFGM membrane material is released in the buttermilk and whey. New technologies for enrichment of MFGM, including the one developed by Arla Foods and used in this study, have emerged. There are no previous studies on long term intake of SL in humans due to lack of suitable formulations.

In this study we examine the effects of a tasty milk drink-like formulation enriched in polar lipids from buttermilk. The effects on plasma cholesterol, TG, HDL, LDL and apolipoproteins A1 (apoA1), B (apoB) and Lp(a) over 4 weeks daily intake of 2-3 times the normal daily intake of SL were examined. The main hypothesis was that the long term intake of SM enriched drink would inhibit intestinal cholesterol uptake with lowered total plasma- and LDL-cholesterol as outcome. We also asked whether TG metabolism is beneficially influenced, as in the apoE Leyden mice [8].

This study investigates whether a daily intake of a SL-enriched milk drink affects fasting blood lipids in 48 subjects (33 men and 15 women) over 4 weeks. The background to this study was that SM was found to inhibit cholesterol absorption in rats [6] and the ability of several dietary SLs to decrease plasma cholesterol and TG and liver accumulation of TG in apoE Leyden mice on a fat- and cholesterol rich diet [8]. Another important factor was that a new formulation, prepared by a dairy technology that does not involve organic solvent extraction and is simple and cost effective, had become available.

The major finding in our study was that no significant plasma lipid lowering effects of the SL rich formulation A was found since there was no decrease in atherogenic plasma lipids or lipoproteins after the four week treatment.

There were, however, trends towards increased concentrations of total plasma cholesterol, LDL, HDL, apo B and TG in group B, which were not seen in group A.

To explain this finding an important aspect is that participants in both groups increased their energy intake and to some extent the intake of saturated fat and cholesterol (Table 6). It is not specified by the participants whether the test drink replaced a regular food item such as another milk product or was added to their normal diet, but according to their food registrations we believe that they mainly added the test drink. One can therefore argue that formulation A may have counteracted an increase in total cholesterol, apoB and TG, related mainly to an increased energy intake which provides some support for a positive effect of the milk polar lipids. ApoB/apoA1 ratios developed, however similar in the two groups (Figure 4).

Comparing the responses for the two formulations in men and women revealed that formulation B consistently increased many of the blood lipids whilst formulation A did not. Only the concentration of TG remained unaffected between the two groups. In group B the increase of TG was accounted for by men and the increased LDL and apoB concentrations were to a larger part accounted for by women. Women in group B were few and happened to have lower average starting concentration of TG than group A. This could make the TG response less sensitive to increased energy intake, which might decrease any difference between group A and B after four weeks. Overall, the variations we observe for the different blood lipids in the placebo groups after four weeks resemble effects that you get from intake of milk lipids rich in saturated fatty acids. This pattern was not observed in group A where, all together, very little effect from the test drink was seen.

Several studies have shown that the level of the independent risk factor Lp(a), although primarily genetically determined, may also be influenced by external factors. In milk context it was earlier reported that casein, in contrast to soy protein significantly decreased Lp(a)[12]. We therefore also compared the response of Lp(a) to the two formulations and found no difference between participants in group A and B. In women Lp(a) decreased, however, in response to the A- and increased to B formulation which was opposite the results in men where the increase was seen in A. Although not conclusive the study indicate sex differences that needs to be further investigated. Future studies should also consider that other components in milk might influence Lp(a).

There are several possible reasons why no clear positive effect of the SL formulation was seen on blood lipids in this study. For one thing, the outcome may reflect that SLs do not affect absorption of endogenous cholesterol which is necessary to achieve an effect on plasma cholesterol in humans. Older studies in which plant sterols were fed in an unesterified form provided little support for a significant cholesterol lowering effect, whereas plant sterol margarines in which sterol esters are mixed into dietary lipids are more effective [13]. Another factor is the dose given. In rodents digestion of SM is extended and has a limited capacity and the physical interaction between SM and cholesterol may be important. In contrast, humans express the key digestive enzyme for SM, alkaline sphingomyelinase also in the liver and secrets it in bile [14]. Sphingomyelin digestion might consequently be more efficient in humans.

In a recent study on ileostomy patients we found that after feeding 50-250 mg milk SM in a breakfast meal the overwhelming part had been digested and the cholesterol/SM ratios in the ileostomy content was high, although some increases in ileostomy output of SM species and ceramide were observed (L Ohlsson et al. submitted). The chosen dose of SL in this study (700 mg SM, 975 mg total, approx 1% of dietary lipids) is 2-3 times a normal Western dietary daily intake, which we believed large enough to test for possible effects. Considering the dose of plant sterols in margarines that is necessary to achieve decreases of total and LDL cholesterol, one may argue that the dose used here is not very high. Another factor is that SLs contain saturated fatty acids and the sphingoid bases are converted mainly to palmitic acid after absorption. They are thus a source of saturated fatty acids that raises LDL cholesterol although the quantity should not be sufficient to have significant influence in this study. Furthermore, both formulations contained a daily dose of 36 mg cholesterol which could be calculated to increase plasma cholesterol by 0,02-0,03 mmol/l [15].

Studies on apo BE receptor gene knockout mice, a model that is very sensitive to dietary cholesterol, suggested that SM-rich diet might raise plasma cholesterol levels and thereby enhance arteriosclerosis [16]. The finding in this study that LDL cholesterol exhibited some increase in the B but not in the A group indicate that these findings may not be relevant to healthy humans. Another important conclusion was that the formulations were both well tolerated and the only type of side effects reported were "Mild abdominal symptoms", which were reported by four persons in group A and one in B. We cannot exclude that the composition of formulation A may have contributed to this symptom, but the relation is uncertain since non specific symptoms like this can be caused by lactose even in individuals with normal lactase levels.

There is rather strong evidence that consumption of milk counteract colon cancer [17]. This effect may in part be ascribed to the calcium and vitamin D content [18]. Since several animal studies have found antitumor effects in the gut with SM or combinations of SL that occur in milk [19–21], there is reason to explore butter milk based formulations high in both calcium, vitamin D and polar lipids for anticarcinogenic and anti-inflammatory effects in human gut.

Polar milk lipids also contain phosphatidylcholine, phosphatidyl serine and smaller amounts of other glycerolipids. Studies with high doses of soy phospholipids have shown an LDL lowering effect, which may be due to high content of linoleic acid [22]. A minor raise in HDL₂ by a high dose (15 g/day) of soy phospholipids was observed [23]. Consequently we do not expect the milk glycerophospholipids to exert any significant effect on the results in this study. However, a study by Sjogren et al [24]showed that milk consumption is associated with a more favourable size of the LDL particles. Further studies of SL enriched milk polar lipids on HDL and LDL composition may thus be motivated.

This study did not provide strong proof of a lipid lowering effects of SL-enriched milk polar lipids, although it supports that formulation A counteracted TG and TDL increases related to an increased energy and fat intake. The SL rich formulation was well tolerated which promotes further studies that consider dose as well as synergies with other factors that may lower cholesterol absorption. Such studies should include hyper lipidemic patients and should consider also possible anti-inflammatory and anti-tumour effects in humans.