Chemistry: analytical and immunological testing – Lipids – triglycerides – cholesterol – or lipoproteins
Reexamination Certificate
2000-10-19
2004-08-31
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Lipids, triglycerides, cholesterol, or lipoproteins
C436S161000, C436S162000, C436S172000, C422S070000, C210S198200, C210S198300, C210S656000, C210S658000
Reexamination Certificate
active
06783988
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods for quantitative and qualitative analyses of phospholipids. Specifically, the present invention provides methods of using one-dimensional thin layer chromatography to detect and quantify phospholipids. More specifically, the present invention provides one-dimensional thin layer chromatography methods used to detect and quantify the phospholipid content of mammalian tissues.
DEFINITION OF TERMS
The specification that follows will be more easily understood by reference to the following definitions. Ordinary terms have been used in manners that preserve their traditional meanings. However, there are numerous references to technical aspects of chromatography that require an understanding of specific terms. While these definitions do not deviate substantially for the meanings accepted by those of ordinary skill in the chemical arts, there may be multiple interpretations of the terms used herein. It such cases the following definition of terms prevails.
a. Analyte(s):
Compound(s) to be detected in a sample (e.g., lipids
present in a tissue extract).
b. Column:
A glass or metal tube that is filled with an inert matrix
used in high pressure chromatography, gas-liquid
chromatography or column chromatography.
c. Elution
Solvent used to separate components in a mixture.
Solvent:
d. Extraction
Solvent used to extract the mixture to be analyzed from
Solvent:
the naturally occurring milieu.
e. Partition:
The process of separating compounds into various phases
based on solubility. For example, separating water
soluble materials (water soluble phase) from compounds
soluble in organic compounds (organic phase) by
allowing the respective phases to separate after mixing.
f. Resolved:
The process of separating components from a mixture
using a chromatographic technique. In thin-layer
chromatography resolved refers to the process of
separating components from a mixture into discrete,
detectable spots.
g. Sensitivity:
An assay's limit of detection for a given analyte.
h. Specificity:
The assay's ability to discriminate between similar
analytes in a sample.
i. Spotting:
The process of applying a small amount (microliter
quantities) of sample on the chromatographic support's
base edge.
j. Support:
A thin-layer chromatography plate (glass or plastic) that
has been coated with an inert matrix or a sheet of blotter
paper in the case of paper chromatography.
BACKGROUND OF THE INVENTION
Bioprosthetic heart valves (BPHV) have been used since the early 1970s as replacements for diseased human cardiac valves. Originally, the reduced thrombogenicity associated with BPHV made them attractive alternatives to mechanical heart valves. However, BPHV fashioned from bovine pericardium and porcine aortic valves are susceptible to dystrophic calcification. Calcification is associated with approximately 40 to 50 percent of all BPHV failures necessitating re-operation and valve replacement in 10% to 20% of all adult recipients. The calcification rate in children, young adults and acute pathologic conditions is greatly accelerated, consequently, the use of BPHV is limited in these patients.
Recent studies have demonstrated that tissue lipid extraction can significantly reduce calcification, and mineralization generally, in gluteraldehyde cross linked bovine pericardial tissues. This had led some authorities to conclude that lipids act as initiators and/or promoters of tissue mineralization. Therefore, various techniques have been developed to remove lipids from BPHVs prior to implantation. However, in order to monitor and fully understand the role lipids play in minerization of preserved tissues, it is necessary to quantify and qualitate tissue lipid content before, during and after processing. This requires a thorough understanding of lipid chemistry and the problems associated with lipid analytical methods.
Lipids are biological molecules (biomolecules) that are insoluble in water, soluble in organic solvents and are essential components of the plasma membranes that envelop and compartmentalize all living cells. The lipid content of living membranes regulate molecular entry and egress at the cellular and sub-cellular levels. They are found in cell membranes and sub-cellular organelles such as mitochondria, chloroplasts, Golgi bodies, the endoplasmic reticulum and the cell nucleus. In addition to their role as structural components of membranes, lipids serve as energy reserves (triacetylglycerol, also known as neutral fats) and participate in cellular recognition and cell signaling.
There are five major categories of lipids found in biological systems: fatty acids, triacylglycerols, sterols, glycerophopholipids, and sphingolipids. Fatty acids rarely occur in un-complexed, or free forms, in nature. Rather, fatty acids are components of other lipids such as glycerophospholipids, triacylglycerols, and sphingophospholipids. Triacyiglycerols are non-polar (uncharged, hence “neutral fats”) fatty acid triesters of glycerol that are synthesized and stored in adipocytes. Adipocytes are “fat cells” that make up the fatty, or adipose, tissue abundant in the subcutaneous tissues of animals that serve as stored energy reserve and provide thermal insulation. Sterols include cholesterol, which are major components of animal cellular and sub-cellular membranes. Sterols occur in much lower concentration in plants and have not been identified in bacterial (prokaryotic) cell membranes. Moreover, cholesterol is an essential precursor to steroid hormones.
Glycerophospholipids (phosphoglycerides) are the most common lipids associated with cell membranes. The simplest phosphoglyceride is phosphatidic acid (A) which is relatively scarce in cell membranes; however, phosphatidic acid can serve as a precursor for all major phosphoglycerides including phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and diphosphatidylglycerol (also known as cardiolipin and found primarily in mitochondrial membranes).
Biochemistry
Voet and Voet;
Lipid Metabolism
; pages 663-726; John Wiley and Sons, New York, 2000. Sphingophospholipids include sphingomyelins (SM) and are a principle component of nerve cell myelin sheaths. Sphigophospholipids (SP) have similar conformations and charge distribution to glycerophospholipids and include the phosphorylated head group associated with glycerophospholipids. Consequently, there is considerable chemical similarity between glycerophospholipids and sphigophospholipids; as a result they will be referred to herein collectively as “phospholipids” for convenience.
Phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, cardiolipin and sphingophospholipids are generally the most abundant phospholipids found in pericardial tissues. It has been reported that phosphatidylserine, cardiolipin and phosphatidylinositol play significant roles in mineralization (D)alas, E. Ioannou, P. V. and P. G. Koutsoukos, In Vitro
Calcification: Effect of Molecular Variables of the Phospholipid Molecule
. 1990. American Chemical Society. Vol 6:3 535-538). Therefore, it is essential that any analytical method used to assess tissue lipid content have equal sensitivity and specificity for all phospholipids. Phospholipids possess many chemical similarities that make their physical separation from tissue extracts challenging. One of the most effective techniques for separating complex mixtures of large organic compounds, including phospholipids, is chromatography.
Mikhail Semenovich Tswett (“Tswett”), a Russian botanist, developed chromatography (from the Latin for color writing) shortly after the turn of the twentieth century. Tswett was studying plant pigments and developed the technique of column adsorption chromatography by passing plant extracts through glass columns packed with calcium carbonate (chalk). The different pigments (each a separate organic compound) separated into discrete bands within the packed calcium carbonate column
Cabiling Christine May
Cunanan Crystal M.
Dinh Tan Thanh
Tremble Patrice
Condino Debra D.
Edwards Lifesciences Corporation
Wallenhorst Maureen M.
Yadav Rajiv
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