Understanding Cholesterol as an Essential Nutrient

An international research team led by a Brock University scientist used neutron beams to resolve a scientific controversy over how cholesterol behaves when surrounded by unsaturated fatty acids such as “omega-3’s” and “omega-6’s” within cell membranes.

Source: Canadian Neutron Beam Centre (CNBC)
Contact: cnbc@cnl.ca
Image: CNBC (with images from pixabay and wikimedia)

In popular culture, cholesterol is nearly synonymous with clogged arteries and heart disease. Saturated fats are bad for us, while unsaturated fats are better. Right? Well, not precisely.

While it’s true that an improper balance of these nutrients can cause problems, cholesterol and both types of fats are also essential for human health. For one thing, our bodies convert these fats into lipids, which are the main structural building blocks of cell membranes. Like skin, cell membranes protect what’s inside the cells and have embedded sensors to determine what’s outside them.

Cholesterol, too, is a necessary component of every cell membrane in humans and animals. That’s because cholesterol is instrumental in forming the structures within cell membranes that enable the cells to interact with their surroundings. It also provides membranes with stability over a wide range of temperatures, and is needed for the production of Vitamin D and hormones.

In fact, cholesterol is so important to human health that our systems actually manufacture about 75 percent of the cholesterol found in our bodies from other nutrients (with the rest coming directly from the food we eat). Our bodies even recycle used cholesterol in the liver rather than disposing of it. But the extent of cholesterol’s functions within cell membranes, and exactly how it interacts with lipids and proteins to accomplish those functions, are still open scientific questions that Professor Thad Harroun of Brock University in St. Catharines is trying to answer.

One of the most common ways for scientists to gain insights into how cell membranes function is to make and study lipid bilayers, which are composed of two flat sheets of lipids. Lipid molecules consist of a water-loving ‘head’ and two oil-loving ‘tail’ segments. Types of lipids can be distinguished by the length of the tails, and also whether the tails are composed of unsaturated fatty acids (e.g., omega‑3s and omega‑6s) or saturated ones.

Lipid bilayers serve in scientific experiments as simplified models of living cell membranes. By isolating aspects of interest in these models, scientists seek to build new understandings that can then be applied to real cell membranes in living beings.

Illustration of the lipid bilayers used to model cell membranes in scientific experiments. Lipid heads (blue) form the outside of the model membrane, while their tails (green) stay inside. Each cholesterol molecule (red and white) is usually found upright in one of the two layers (left). Findings in 2016 showed that cholesterol in thinner bilayers is tilted between the two layers, partially intersecting with both (right). (Image: DOI: 10.1039/c6sm01777k)

Neutron beams are one of the few tools that can be used to study the molecules that reside in lipid membranes in realistic conditions. In research published in 2006 and 2008, Harroun used neutron beams at the Canadian Neutron Beam Centre (CNBC) to study the positioning of cholesterol molecules in lipid bilayers—and ended up making some rather controversial discoveries.

The scientific community had generally accepted that cholesterol molecules (which also have a head, and one tail) stand upright in lipid bilayers, with the cholesterol’s head near the heads of the lipids. However, Harroun’s observations suggested that cholesterol actually lies flat between the layers in some lipid bilayers, including in one where the lipid tails were composed of omega‑6 fatty acids. Harroun made similar observations in studies published in 2009 and 2010. These follow-up studies, which also used neutron beams at the CNBC, demonstrated that small changes to the lipid composition could cause the cholesterol to adopt an upright position.

“The idea that cholesterol could lie down flat was met with considerable doubt, and raised questions about the biological significance of this position,” says Drew Marquardt, who joined Harroun’s research team as a graduate student in 2010. “Other scientists made it their goal to reproduce what we saw using computer simulations, but they were unsuccessful.”

In hopes of resolving the controversy, Harroun put together an international research team to further elucidate the behaviour of cholesterol in a variety of lipid bilayers. The researchers decided to use deuterated cholesterol, in which the majority of hydrogen atoms exist in a form called ‘deuterium.’

Illustration of normal hydrogen (left) and deuterium (right). The two types of hydrogen differ in atomic composition, with deuterium having a neutron (green) in addition to a proton (red) and an electron (blue). (Image: IAEA)

Deuterium (a hydrogen atom with a neutron in addition to a proton and an electron) is chemically identical to normal hydrogen for most purposes. While the behaviour of cholesterol molecules is impacted only minimally when deuterium is substituted for normal hydrogen, neutron beams are particularly sensitive to the difference between the two types of atoms. Thus, deuterated cholesterol can be observed more precisely within the lipid bilayers.

Howard Riezman of the University of Geneva, Switzerland, provided a genetically modified yeast strain that produces large amounts of cholesterol. Bob Standaert, a biochemist at Oak Ridge National Laboratory (ORNL) in the U.S., grew the yeast under controlled conditions to ensure the harvested cholesterol was sufficiently deuterated.

In 2014, Marquardt performed neutron beam studies at the CNBC on a variety of lipid bilayers that incorporated the fully deuterated cholesterol molecules. Notably, some of these bilayers were formed with lipids whose tails were composed of either omega‑3 or omega‑6 fatty acids. By comparing his data with earlier results in which only part of the cholesterol molecule (i.e., either its head or its tail) was labelled with deuterium, Marquardt was able to confirm that the cholesterol did indeed reside in the centre of some lipid bilayers—but it didn’t lie flat.

Research team members Professor Roger Koeppe of the University of Arkansas and Professor Stephen Wassall of Indiana University–Purdue University Indianapolis led complementary experiments at their respective facilities to help pinpoint the exact orientation of the cholesterol in these bilayers. The experimental techniques they used were based on ‘magnetic resonance,’ an approach that employs the same physical principles as MRI machines, which doctors use to diagnose trauma to the brain.

In effect, we have put this controversy to rest.

When combined with the data collected using neutrons, the results provided strong evidence for one particular description of cholesterol’s behaviour when it resides in the middle of a lipid bilayer—namely, that it spans across the bilayer’s centre, with its head in one layer and its tail in the other, and is tilted rather than flat. Furthermore, it was determined that the cholesterol can change its orientation by flipping up and down across the centre of a bilayer. Brad Van Oosten, a graduate student working under Harroun, performed computer simulations to reproduce some of these key features regarding cholesterol’s orientation and behaviour.

The international research team’s findings were published as the cover story for the journal Soft Matter in December 2016. Perhaps one of the team’s most significant discoveries was that the lipid bilayer’s thickness—as dictated by the length of the lipids’ tails—is the key feature that determines whether cholesterol stands upright or is tilted in the centre of the bilayer. Specifically, cholesterol resides in the centre only when the bilayer’s thickness is below a certain threshold. Since lipid membranes in living cells are always thicker than that threshold, cholesterol in living cells can still be expected to be oriented in the upright position.

Indeed, by identifying the point at which cholesterol behaves abnormally, the researchers have added assurance that it behaves normally otherwise.

“It turned out to be a case where the exception proves the rule,” explains Marquardt. “In effect, we have put this controversy to rest.”

With more certainty in cholesterol’s position and orientation, scientists can proceed with greater confidence to unravel how it interacts with proteins to form the cell membrane structures that the cell needs to be able to adapt to its environment.

DOI: 10.1039/c6sm01777k