Submitted to: Lipids Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/1/2004
Publication Date: 12/10/2004
Citation: Moreau, R.A., Hicks, K.B. 2004. The in vitro rates of hydrolysis of phytosterol conjugates and other plant lipids by mammalian digestive enzymes, compared to saponification. Lipids, v.39, No. 8. p.769-776.
Interpretive Summary: In recent years two phytosterol-enriched margarines have received much attention by US consumers and have been granted a health claim by the FDA, confirming their cholesterol-lowering efficacy in humans. The phytosterols in both of these products are bound (esterified) to fatty acids, and clinical evidence indicates that this bond must be broken by digestive enzymes in the small intestine before these products can effectively prevent cholesterol absorption. Although several studies have provided evidence that these chemical bonds are broken during the digestion of these margarines, this current study is the first published report of their breakdown in the test tube (in vitro). We demonstrated that both a mixture of pancreatic enzymes (pancreatin) and a purified commercial pancreatic enzyme (cholesterol esterase) could hydrolyze these bound phytosterols (phytosterol conjugates). In addition, we examined the ability of these two enzymes to hydrolyze several other types of phytosterol conjugates that commonly occur in fruits, vegetables, and grains. This information will be valuable in helping to understand what natural forms of phytosterols are most effective at lowering cholesterol. It will also be valuable to know if these popular pancreatic enzymes can be used in the industrial modification of phytosterols, since phytosterols are commonly used in the synthesis of steroid hormones such as estrogen and progesterone.
Technical Abstract: All fruits, vegetables and grains contain phytosterols. Numerous clinical studies have documented that phytosterols have a significant positive effect on reducing LDL cholesterol levels and thereby reducing the risk of cardiovascular disease. Dietary phytosterols include approximately fifteen common types of sterol molecules that occur either in the "free" (unesterified) form or as conjugates. Most experts believe that the cholesterol lowering mechanism of phytosterols requires that they be in their "free" form. The four common phytosteryl conjugates are: a) fatty acyl esters, b) hydroxycinnamate esters, c) steryl glucosides, and d) fatty acylated steryl glucosides. This study was undertaken to investigate the ability of mammalian digestive enzymes (cholesterol esterase and pancreatin, a mixture of pancreatic enzymes) and for comparison purposes, KOH, to hydrolyze the four common phytosterol conjugates. Phytosteryl fatty acyl esters were hydrolyzed at a moderate rate by both enzyme preparations. One hydroxycinnamate ester (sitostanyl ferulate) was hydrolyzed at a moderate rate by cholesterol esterase and at a low rate by pancreatin, while the other (Oryzanol) was hydrolyzed at a very low rate by both enzymes. Pancreatin had no effect on steryl glucoside but it did hydrolyze the fatty acyl moiety of fatty acylated steryl glucoside, converting it to steryl glucoside. The ability of pancreatin to hydrolyze three other types of lipid conjugates was also evaluated. The first two types (phospholipids and galactolipids) were hydrolyzed, but the third, polyamine conjugates, were not. The rates of hydrolysis of phytosteryl esters by base (saponification) were also studied and it was found that conditions commonly used for the saponification of acyl lipids (1.5 N methanolic KOH, 30 min at 70º C), resulted in a nearly 100% hydrolysis of triacylglycerols but only about 35-45% hydrolysis of the phytosteryl fatty acyl esters or phytosteryl hydroxycinnamate esters.