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Paper Synopsis of:

Localization of Low Density Lipoprotein receptors on plasma

membrane of normal human fibroblasts and their absence in cells from

Familial Hypercholesterolemia homozygote

By: Cody Mitchell

SC 291

Professor Owen

2/23/12

Word count: 734

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Low density Lipoprotein (LDL) is one of 5 groups of major lipoproteins that

facilitate the transport of fat molecules such as cholesterol throughout the bloodstream.

LDL deposits itself on the walls of blood vessels, causing blockages also known as

atherosclerosis. Numerous health issues are attributed to high level LDL. In normal

humans cells, an average of 55 Ferritin marked LDLs were shown to bind to each

millimeter of coated regions on non mutant plasma membranes. Although, in humans

who have the disease familial hypercholesterolemia the LDL did not bind to the cells,

regardless of equal numbers of coated regions. Data shows that in humans with

familial hypercholesterolemia, the receptor site for this LDL is corrupt and

malfunctioning, leading to the lack of LDL binding. Also data shows that in normal cells,

LDL binding is limited to the indented Coated regions of the plasma membrane.

The methods associated with this experiment (Anderson, Goldstein, and et al 2434-

2438) were rigorous and time consuming for the scientists. It began with collection of

mature Fibroblasts from skin biopsies of healthy subjects, or subjects with receptor

negative homozygous familial hypercholesterolemia. Once these cells were procured

they were grown in a monolayer and observed after 48 hours of incubation in LDL

deficient growth medium. The LDL and LDL deficient serum were both procured from

healthy subjects, and then spun down via centrifuge to separate the components

according to density. In order to bind the Ferritin to the LDL, Glutaraldehyde was used

to activate the Ferritin. Once the Ferritin was active, the Glutaraldehyde was separated

from the Ferritin, and the Ferritin was added into purified LDL in a ratio of 0.6. This

mixture was then incubated for 60-72 hours at 4 degrees Celsius. The chilled mixture

was then centrifuged for 18 hours to separate the free Ferritin and free LDL from the

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LDL/Ferritin mix. This LDL/Ferritin mix was then aspirated out of the tube, purified with a

.5 M solution of NaCl, and examined. Examination of this sample showed that over 90%

of the LDL particles had been bound with between one to three Ferritin cores. The

Fibroblast monolayer was then placed into a 4 degree Celsius cold room, and left to set

for .5 hours, after when the medium was replaced with 2 mL of ice-cold growth medium

containing 5% LDL deficient serum and the LDL/ Ferritin mix. The solution was left to

incubate for 2 hours in 4 degrees Celsius, after which it was washed five times with a

phosphate buffered saline and fixed in the cold for one hour with 2% Glutaraldehyde in

.1 M sodium phosphate buffer. The sample was dehydrated and set in epon,

sandwiched together into the correct orientation, and prepared for microscopy with a

micro-grid for quantitative purposes.

The experiment showed that Fibroblasts, when incubated at 4 degree Celsius

and exposed to LDL/ Ferritin for 2 hours, would bind to the Ferritin cores at the

invagination. This occurred in equal amounts on the upper and lower portion of the cell.

Infrequently the Ferritin cores were found to be bound to places not associated with the

invaginations. In samples with familial hypercholesterolemia, treated with the same

conditions, no Ferritin cores were found to bind to the coated regions. After a

randomization of data, and a third party to prevent observer bias, results of a

quantitative study shown that In the familial hypercholesterolemia samples, no Ferritin

was found to bind, where as in the non mutant samples 49 and 61 cores were found to

bind to the coated regions.

This experiment was quite thorough, with extensive steps to prevent bias, and

promote accurate results. The researchers on this study seem to have thorough

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knowledge on the topic, and a wide range of previous research concerning LDL and

Ferritin binding. One aspect I thought was reassuring about this research study is that

the researchers brought in a third party to analyze randomized, coded results to

thoroughly prevent any form of bias.

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Citations

Anderson, Richard, Joseph Goldstein, et al. "Localization of Low Density Lipoprotein receptors

on plasma membrane ofnormal human fibroblasts and their absence in cells from Familial

Hypercholesterolemia homozygote." Proc. Natl. Acad. Sci. USA. 73.7 (1976): 2434-2438. Print.