GenBio1 - Lipids 1 Flashcards
True or False: Literature lacks consistency in extraction method details
True
True or false: Existing extraction methods are well-documented for blood serum but not for cerebrospinal fluid (CSF).
True
Why should we study lipidomics?
To discover new biomarkers
True or false: lipid extraction methods aren’t well-documented for blood plasma and serum, but they are for cerebrospinal fluid (CSF).
False: lipid extraction methods are well-documented for blood plasma and serum, but not for cerebrospinal fluid (CSF).
true or false: CSF has much lower lipid concentrations than serum, making specialized extraction methods necessary.
True
Recent methods use safer solvents like…
MTBE or heptane
Classic lipid extraction methods involve…
chloroform
____ is important for studying neurological disorders
CSF
The study focuses on three types of lipids:
glycerolipids, glycerophospholipids, and sphingolipids
MTBE
Methyl-tert-butyl ether
What was added to the CSF pool?
Stable isotopically labeled lipid standards
What is CSF?
is the clear liquid around your brain and spinal cord.
The study evaluated four liquid-liquid extraction (LLE) methods and one protein precipitation method in an initial screening experiment. Name these methods.
LLE: Folch, Bligh & Dyer, MTBE, BUME
Protein precipitation: MMC
All extractions were conducted at what temperature for initial screening?
room temperature
Lipid extracts were dried using…
Nitrogen gas
Mixing was done using a
Thermal shaker
Folch method
This method involves mixing CSF with a chemical called chloroform to get the lipids out. It’s like shaking up a bottle to mix oil and water, and then collecting the oil (lipids) that separate out.
Bligh & Dyer method
Similar to Folch, but they use a different mixture of chemicals (chloroform and methanol) and more steps to separate the lipids.
MTBE method
In this method, they mix CSF with a chemical called Methyl tert-Butyl Ether (MTBE). It’s a bit like using a special liquid to dissolve the lipids and then separating them from the rest.
BUME method
They use butanol and methanol to mix with CSF. It’s a bit like shaking CSF with these chemicals to pull out the lipids.
MMC method
Here, they mix CSF with a mixture of three chemicals (methanol, MTBE, chloroform) to get the lipids.
Liquid-liquid extraction
a method by which a compound is pulled from solvent A to solvent B where solvents A and B are not miscible.
Protein precipitation
process of separating a protein from a solution as a solid by altering the protein solubility with addition of a reagent.
What was used to replace chloroform in the Folch and Bligh & Dyer methods?
dichloromethane (DCM)
Design of Experiments (DoE)
DoE strategies are designed to provide maximum relevant information with a minimal number of experimental runs.
How many extractions occurred?
30
Peak areas
refer to measurements of how much of a specific lipid is present in a sample. They are used to determine the concentration or abundance of lipids during mass spectrometry analysis.
To confirm the effectiveness of the optimized Folch and BUME methods in extracting endogenous lipids from CSF, pooled CSF samples were intentionally enriched with an ______ solution
isotopically labeled internal standard (ISTD)
The lipid extracts prepared in the study were analyzed using ….
HPLC-MS techniques.
Peak areas were subjected to _____
z-score normalization
High positive z-score indicates…
high relative extraction efficiency, and vice versa.
Which methods showed positive z-scores
Folch, Bligh & Dyer, and BUME methods
Which method had the lowest peak area and highly negative z-scores
MTBE
What were the 3 most promising candidates?
the Folch, Bligh & Dyer, and BUME methods were identified as the most promising candidates
What did some authors recommend?
recommend using ice-cold solvents and maintaining sample/solvent mixtures at low temperatures, while others perform lipid extraction at room temperature.
Temperature and mixing time
Temperature was investigated within a range of 10–22 °C, and mixing times were varied between 10 and 30 minutes.
Lowering the temperature from 22 °C to 10 °C resulted in a 4-fold increase in peak areas for…
dCer
True or false: It is advisable to work with hot solvents
False: It is advisable to work with ice-cold solvents
Setting mixing time ____ minutes generally resulted in substantially higher peak areas for most analytes.
2 to 30 mins
True or false: Longer mixing times during mixing time 1 led to lower peak areas
True
This is the first round of mixing, where the liquids are combined and stirred. During this step, the goal is usually to make sure all the ingredients are evenly mixed. Think of it like stirring a cake batter to make sure all the flour and sugar are well blended.
Mixing time 1
After the first mixing, there’s a second round of stirring. This step is often done for a specific purpose, like separating certain substances from one another. Imagine you’re making a fruit smoothie, and after the initial blend, you need to mix it again to make sure the ingredients are perfectly smooth and no chunks remain.
Mixing Time 2
True or false: the use of DCM instead of chloroform should generally be avoided
True: DCM is less hazardous but not as effective for analyzing lipids
_______ has been suggested to improve extraction efficiencies in classical liquid-liquid extraction (LLE) methods
Acidification
True or false: it is recommended to use weak and low-concentration acids.
True: Acidification can help get more lipids during extraction.
But too much acid can break down the lipids and mess up the results. So, acidification was used to improve the extraction of certain lipids, but its effectiveness varied depending on the type of lipid and the extraction method. It was a way to optimize the process for better results in lipid analysis.
Repeated extraction cycles purpose
The aim of this experiment was to evaluate the potential benefit of a second and a third extraction cycle
True or false: BUME method was able to recover all investigated target lipids better or at least comparably to the Folch method.
False: Folch method was able to recover all investigated target lipids better or at least comparably to the BUME method.
Data processing revealed a total of ___ features in the batch comprising three replicates of modified Folch and BUME extracts.
466
____ is highly efficient for extracting a wide range of lipid classes from cerebrospinal fluid (CSF)
Folch method
_____ performed poorly
MMC
This change improved recoveries of acidic glycerophospholipids.
Substitution of water with 5% acetic acid
Use of ice-cold solvents and cooled equipment
Maintaining low temperatures throughout the extraction process was critical for efficient lipid recovery.
Adjusted mixing times
Evaluating and optimizing mixing times was essential to achieve high extraction efficiencies while preventing degradation of target lipids due to excessively long mixing.
