Nov. 19, 1998 Using a 'glowing twin' of cholesterol and other bile components, chemical engineers at the University of Delaware--working with a doctor at the University of Washington School of Medicine--have characterized the behavior of an enzyme known to speed the growth of gallstones, which affect some 20 million Americans.
The system for studying bile chemistry may someday help geneticists pinpoint and perhaps even turn off the genes responsible for gallstone formation, a disorder associated with more than $5 billion in medical costs each year, says Eric Kaler, chairperson of UD's Department of Chemical Engineering and the University?s Elizabeth Inez Kelley Professor of Chemical Engineering.
And, the UD technique may suggest new strategies for investigating a host of other problems involving cholesterol--particularly cardiovascular ailments, says Kaler's graduate student, Steven Parker Wrenn.
"Within a synthetic bile system, we can pinpoint the onset of nucleation--a preliminary step in gallstone formation, which no one has previously been able to measure," Wrenn reported Nov. 18 during the American Institute of Chemical Engineers (AIChE) 1998 annual meeting in Miami. "Our technique is not intended to be used by doctors as a diagnostic tool for treating patients," Wrenn emphasizes, "but rather, as a way to identify the proteins that speed or slow the formation of crystalline gallstone precursors."
In collaboration with Sum P. Lee, M.D., of the University of Washington School of Medicine--one of "The Best Doctors in America," according to a 1998 survey by Woodward/White, Inc. of South Carolina--Kaler and Wrenn devised a system for detecting changes in the fluorescence energy of 'twins' or analogs for cholesterol and lecithin, two components of bile.
The research team has so far investigated the enzyme, phospholipase C, known to be a key stone-forming factor. (A forthcoming article in a peer-reviewed journal will describe the group's research in detail, Wrenn says.)
Gallstones affect an estimated 10 to 15 percent of the population, according to the National Institutes of Health. Most gallstones, composed mainly of hardened cholesterol clusters, are smaller than 2 centimeters in diameter--slightly smaller than an inch--and some patients never develop symptoms. But gallstones can cause serious problems, ranging from chronic pain and pancreas damage to blocked ducts requiring surgery and an increased risk of gallbladder cancer, the NIH has reported.
How do gallstones form? After a meal, Wrenn explains, hormones prompt the gallbladder to release bile into the gastrointestinal tract. Produced in the liver and stored downstream in the gallbladder, this yellow-brown fluid breaks down fats, and it helps the body absorb fat-soluble vitamins such as A, D, E and K.
Because water can't dissolve cholesterol, it must be delivered to the gastrointestinal tract inside carrier structures--mainly fluid-filled spheres or "vesicles" made from lecithin. A waxy substance found in egg yolks, lecithin "self-assembles," or curls up to form tiny spheres within bile, Wrenn says.
A second cholesterol carrier within bile is a rod-like cluster of bile salt molecules known as a micelle. Unfortunately, micelles prefer to dissolve lecithin, and tend to dissolve more lecithin than cholesterol. The action of micelles, therefore, results in cholesterol-enriched vesicles, which are prone to nucleation. Various proteins also affect the rate at which cholesterol "nucleates" or leaves the lecithin spheres, Wrenn says.
"Once cholesterol clusters reach a critical size," Wrenn notes, "they are considered to be a nucleus. At that point, the nucleus can only grow larger. It will never again dissolve back to free cholesterol. It will grow and eventually crystallize, sometimes resulting in macroscopic gallstones."
Pinpointing growth factors
Within the rich, chemical broth of bile, a number of proteins are believed to either promote or slow cholesterol nucleation and, therefore, gallstone growth. Clinicians describe these enzymes as "pro-nucleating" or "anti-nucleating" factors, Wrenn says.
To identify the key players in gallstone formation, UD researchers developed a system for observing the interactions of cholesterol and lecithin within artificial bile, a complex soup synthesized in Kaler's laboratory. Specifically, they "tagged" the two molecules with a pair of naturally glowing (fluorescent) analogs: dehydroergosterol, a chemical twin for cholesterol; and dansylated lecithin, which resembles the lecithin molecule found in bile. ("Dansyl" is an acronym for DimethylAminoNaphthalene-Sulfonyl.)
Whenever these glowing, analog molecules come within 50 nanometers of each other--a distance equivalent to the diameter of a typical lecithin sphere--they undergo a process of energy transfer, Wrenn explains. Using a spectrometer to detect ultraviolet light, the UD researchers could "see" cholesterol leaving lecithin vesicles, based on changes in the extent of the energy transfer between the two analog molecules. An increase in the dehydroergosterol signal, coupled with a decrease in the dansylated lecithin signal, told the researchers that cholesterol had nucleated.
As expected, introducing phopholipase C to the sample dramatically increased nucleation speeds. The enzyme seems to accelerate the process, through a chemical reaction involving calcium, which forms diacylglycerol on the outer layer of the lecithin vesicle. Because diacylglycerol doesn't dissolve in water, resulting vesicles more rapidly form cholesterol-rich clusters.
Phospholipase C has long been known to promote gallstone growth, Wrenn says, but the new UD technique made it possible to better understand the enzyme's chemical function and behavior. And, the UD technique gave researchers their first glimpse of nucleation events in process.
Support for this research was provided by Grant RO1 DK41678 from the National Institutes of Health. Lee's work also is supported in part by the Medical Research Service of the Department of Veterans Affairs.
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