SXHL288 Lab Guide: Lipid quantitation
Lipid quantitation guidance (SXHL288)
You may find it helpful to print this document and have it to hand as you work onscreen with the
spectrophotometer and in the Data Analysis area with your lipid data.
Contents
1. Introduction ................................................................................................................................................... 1
2. The lipid assay................................................................................................................................................ 2
2.1 A quick refresher: overview of the lipid assay technique ....................................................................... 2
2.2 Instructions for using the spectrophotometer ........................................................................................ 2
Familiarisation with the spectrophotometer ............................................................................................ 2
Collecting data for your standard curve .................................................................................................... 4
Drawing your standard curve .................................................................................................................... 5
Determining lipid concentrations for your WAT and BAT samples ........................................................... 6
2.3 Lipid Calculations ..................................................................................................................................... 7
Calculation of lipid per mg of adipose tissue............................................................................................. 7
3 Analysing your data: comparisons between experimental groups ............................................................... 8
(a) Comparison between groups: lipid content per mg tissue ...................................................................... 8
(b) Comparison between groups: lipid content per adipose tissue depot ................................................... 8
(c) Comparison between groups: lipid content per cell ............................................................................... 9
Appendix: Physiology laboratory spectrophotometer guidance .................................................................... 10
1. Introduction
This lab guide covers the use of the interactive onscreen spectrophotometer you will be using to measure
changes in both WAT and BAT composition as a result of cold adaptation, by examining changes in one
biochemical constituent of adipose tissue: lipids. The assay is carried out separately from that for proteins,
although the lipid and protein extractions were conveniently from the same tissue sample, as you will have
seen in the accompanying videos. The tissue sample was homogenised in a reagent that precipitated the
protein and extracted the lipid into the solvent phase. Thus, for this lipid assay you are able to use the
solvent phase while for the protein assay you can use the precipitated residue.
□ Before you start, make an educated guess about what proportion of the BAT or WAT is lipid. Write
down your prediction and later compare it with your results.
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SXHL288 Lab Guide: Lipid quantitation
2. The lipid assay
2.1 A quick refresher: overview of the lipid assay technique
The lipid assay is based on what is termed a turbidimetric analysis. In this technique, the lipids are first
extracted into an organic solvent and then dispersed when the solvent system is broken down by addition
of water, in which lipids are immiscible. Microscopic lipid droplets form micelles within the aqueous layer.
The formation of this emulsion, which has a milky or turbid (opalescent) appearance, is assisted by the
addition of detergent.
The two liquid phases have different refractive properties, and this is the basis for the assay technique.
Light transmitted through the solution is refracted by the micelles (microscopic droplets) and the degree of
refraction is proportional to their number and size; hence the difference between the intensity of the
transmitted light beam and that of the incident light beam is a measure of the amount of dispersed lipid in
the liquid. The assay is carried out by placing the sample (which is in a container called a cuvette) into the
spectrophotometer. The spectrophotometer automatically passes a light beam through the cuvette,
detects how much light has passed through the sample, and reports an absorbance value.
In order to determine the relationship between absorbance and lipid concentration, you first use samples
containing a known amount of lipid. These samples are known as ‘standards’. Once absorbance values are
collected from the standards, the relationship between concentration of lipid and absorbance is drawn as a
graph called a standard curve (don’t expect it to be a curve shape though!).
You then can collect absorbance values for your experimental samples and use the standard curve to
determine how much lipid is present in each sample.
2.2 Instructions for using the spectrophotometer
You will use the spectrophotometer several times in your study.
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In Task 2 you are asked to explore the spectrophotometer
In Task 4 you gather your experimental data
o Standard curve data
o Sample data
In Task 5 you are shown how to analyse your data.
You should use this Lab Guide to help with these tasks, and the instructions below will help you as you
work through them. If you use a printed version of these notes, you might find it helpful to tick off each
section as you complete that stage. Some guidance times are provided to help you plan the online
components of this activity.
Familiarisation with the spectrophotometer (allow up to 30 minutes)
Go to the Physiology lab, navigate the lab panorama to the hotspot on the spectrophotometer image (hint:
it’s on the left hand bench!).
Select the hotspot and read the text in the right hand panel. To access the spectrophotometer, select
Launch Activity. You should launch this activity in a new tab or new window to allow you to return to the
main laboratory more easily.
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SXHL288 Lab Guide: Lipid quantitation
The spectrophotometer (Figure 1) contains various buttons that you can select onscreen to operate the
instrument. It is important to carry out these steps in a particular order.
o First open the help pages by selecting the [Show Help] button. These pages explain the
functions of the various controls on the interactive spectrophotometer.
o You can access these help pages at any stage and you will find them reproduced at the end
of this guide as a handy guide.
o When you have finished reading through, you can close the pages by selecting [Hide help].
Figure 1: The spectrophotometer in the physiology laboratory. You will use this spectrophotometer to collect data from both
lipid and protein assays. After switching on, the spectrophotometer is ready to programme the wavelength when it displays
‘passed’
□ Spend some time exploring the Spectrophotometer, turning it on/off as well as changing and
setting the wavelength.
o After switching on, the instrument will report ‘Passed’ indicating it is ready for you to set
the wavelength
 You should note that the spectrophotometer has a sticky note attached to remind
you which wavelength to use in each assay!
o Follow the instructions to set wavelength to 440 nm
□ The [Samples] button is used to access the samples from which you will collect all your lipid and
protein absorbance data. Open this and note that there are six sets of data that you will be using,
accessible using a pull down menu. Don’t worry about which data set to select at this stage.
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SXHL288 Lab Guide: Lipid quantitation
Collecting data for your standard curve (allow up to 1 hour)
Once you have familiarised yourself with how to operate the Spectrophotometer, you are ready to set-up
the spectrophotometer and collect data. The first task is to calibrate the response of the
spectrophotometer using known concentrations of lipid.
□ Select the [Samples] button to open the list of samples available. From the pull-down menu, select
LIPID CALIBRATION.
□ Draw up a table in your lab notebook in which you will record your data. You should leave enough
space for two readings from each of the 7 standard samples. These are listed by the concentration
of lipid that they contain, which ranges from 0.0 to 5.0 mg/ml. You will also see a sample that is
called ‘Reference’, you will be using this sample, but you do not need to record its absorbance.
□ Ensure that the spectrophotometer is set to the appropriate wavelength (440 nm) for the lipid
assay.
□ You must use the Reference sample first before taking any readings from your standard or
experimental samples.
o Selecting ‘Reference’ from the list of samples to place the reference sample cuvette into
the Spectrophotometer and the sample list will close.
o Use the ‘R’ button to set the reference value, if successful the spectrophotometer will
report back ‘Reference Set’
o Return to the samples listing by selecting the [Samples] button or selecting the cuvette in
the spectrophotometer. Either action will remove the Reference sample from the
spectrophotometer ready for you to select another to measure.
The use of a Reference sample, which calibrates the spectrophotometer for the assay you will be using, is
critical before making any further measurements. You only need to set the reference value once before
taking standards and samples measurements unless you switch the spectrophotometer off or come back to
take repeat readings at another time. Now you are ready to take readings for your standard curve.
□ Take absorbance readings in duplicate (2 separate readings per standard, pressing ‘T’ for each
reading). Record your data directly into the table in your lab book.
o For the first standard sample (labelled 0.0 mg/ml), you should observe an absorbance of
approximately zero (it might be 0.0002, which is fine).
o Now collect absorbance vales for each standard sample ensuring that you record the data
correctly in your table and also ensuring you collect two readings from each sample.
o As you collect your data, do you see any relationship between absorbance and the
concentration of lipid in the standard sample? If so, note this down in your lab notebook.
□ When you have collected all the absorbance values from your standard samples, calculate the
mean value for each pair of absorbance readings for each standard concentration. Using this
average value will increase the accuracy of subsequent analyses.
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SXHL288 Lab Guide: Lipid quantitation
Drawing your standard curve (allow up to 1 hour)
A tool is provided to draw and use your standard curve. This drawing tool is found in the Data Analysis area,
accessed from the lab portal. Again, opening this in a new tab or window of your browser will make it
easier to switch between areas of the laboratory.
Figure 2: The Standard curves page in the Data analysis room
□ Access the Data Analysis room and select the Standard curves tab (Figure 2)
□ Enter your mean absorbance values for the standards directly into the online table, having selected
the ‘Absorbance values for lipids’ option first.
□ When you have entered values for all of the standards, select ‘Plot graph’ to see the results plotted.
□ Record a copy of your standard curve in your laboratory notebook by taking a screengrab <Ctrl-Prt
Scr> or by using a Snipping tool.
Before moving onto the next section, take a look at your standard curve. Note that as the concentration of
lipid in the sample increases, the graph indicates that the absorbance of the sample increases. This you
would predict, as the assay is based upon the emulsion formed in the Edson’s reagent refracting the light
passed through the cuvette. The graph presents the relationship in the form of a line of ‘best fit’.
A standard curve can only be used if the concentration of the sample you are analysing (or, its absorbance)
is similar to that of the standard on which the calibration was made. Even for set of standard
concentrations, there is a limited range for which the relationship between absorbance and concentration
is linear, and outside this range the standard curve is unreliable.
You should therefore ensure that your measured absorbance value corresponds to a linear region of the
standard curve you choose to use.
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SXHL288 Lab Guide: Lipid quantitation
If you are unfamiliar with using standard curves, try this thought experiment before moving on.
 Imagine you have a sample that gave an absorbance reading of exactly 1.000.
 To use the standard curve to estimate the lipid concentration in that sample, look at your standard
curve and follow the horizontal line from the 1.0 point on the absorbance axis until it hits the
plotted line.
 Now draw a vertical line down from this point to meet the lipid concentration axis, and read this
value. This is the concentration of lipid in the sample with an absorbance of 1.000.
Note that the units of concentration for the lipids are given in mg/ml (milligrams per millilitre) and this can
also be written as mg ml-1.
Determining lipid concentrations for your WAT and BAT samples (allow up to 2 hours)
When you are ready to collect data from your experimental samples,
□ Draw up a table for data recording in your lab notebook.
o
Note that you will need to record both WAT and BAT absorbance values (in duplicate) for
each of your selected animals (5 cold and 5 warm).
o Allow sufficient space in your table to enter the mean of your duplicate absorbance
readings and also space to record the lipid concentration that you will determine from your
standard curve.
□ Use the Reference sample as you did previously to prepare the spectrophotometer
□ Obtain absorbance values for each of your test samples in duplicate (i.e. take two separate readings
per sample).
□ Write these down in your lab notebook and calculate their mean.
You are now ready to use your standard curve to determine the lipid concentration in each sample.
□ Return to your lipid standard curve.
□ Enter each mean absorbance value into the area under the table “Please enter a value for A440
(arbitrary units)”. You will be provided with a reading from the standard curve for each mean
absorbance value converted into lipid concentration (in mg/ml) that you should also record in your
lab notebook. These are the values you will now be using.
You can now record your data in the Results area.
□ Enter your final lipid concentration values into the appropriate lipid (cold or warm) tables under the
Results Tab.
□ Once the table is complete click on the ‘Show statistics’ button to obtain mean lipid concentrations
and the SD and SEM values for each group of animals (the control ‘warm’ group, and the coldadapted group) and each tissue (WAT and BAT).
o Note: if you notice an inputting error and alter any values, you must click on ‘show
statistics’ again to update the values.
□ Make a print out of your final tables with their statistics to stick into your lab notebook.
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SXHL288 Lab Guide: Lipid quantitation
2.3 Lipid Calculations
Once you have obtained all your measurements from the spectrophotometer, produced your standard
curve, and used it to measure the lipid concentrations of all of your samples, there are some further
calculations to perform that will allow you to make comparisons between adipose depots and animals.
Calculation of lipid per mg of adipose tissue
The concentration of lipid that you have calculated using your standard curve is that of the tissue extract
used, not the whole tissue sample. Recall that this tissue extract was made from 30 mg of dissected adipose
material and that the extract was in a final volume of 6 ml of Edson’s reagent.
To determine the amount of lipid per mg of adipose tissue for each individual animal, you therefore need
to adjust the values obtained for each sample (in mg/ml) by taking into account the volume of the sample
and the amount of tissue it was derived from.
This is given by the equation:
For example:
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If 30 mg of tissue was extracted in 6 ml of Edson’s reagent, and the resulting extract has a lipid
concentration of 2.4 mg ml-1 then the total amount of lipid obtained from that 30 mg of tissue is
2.4 mg x 6 = 14.4 mg of lipid.
This is then divided by the amount of tissue used (30 mg): 14.4 / 30 = 0.48 mg of lipid.
This is then expressed as 0.48 mg lipid / mg tissue or 0.48 mg (mg tissue)-1
Note that 0.48 mg is the same as 480 µg (micrograms), because 1 mg is the equivalent of 1000 µg.
Perform the calculation using the formula above for each sample. Keep a record of your workings and
results in your lab notebook. You will use these answers in the following calculations.
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SXHL288 Lab Guide: Lipid quantitation
3 Analysing your data: comparisons between experimental groups
You now need to collate your data so that you can make suitable comparisons between your experimental
groups. To do this you first need to organise them into their separate tissue groups i.e. WAT or BAT, and
then further separate them by experimental groups i.e. control (warm) or cold-adapted. You can then
compare groups in two ways, as detailed below.
(a) Comparison between groups: lipid content per mg tissue
For each experimental group and tissue, you need to arrange your data into the appropriate groups (BAT,
WAT, warm or cold-adapted for each) and then for each of these enter you can calculate:
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
the mean value for lipid content per mg tissue
the standard deviation and the standard error of the mean (SEM). SEM = Standard Deviation
divided by the square root of n (where n is your sample size). You may make use of the appropriate
table under the Results tab to do this or use another method.
You can now collate your data into a table for use in reports and consider how a graph might to best
present your data.
To perform statistical analysis of your results, you may make use of the t-test to assess whether any
differences you observe are significant.
(b) Comparison between groups: lipid content per adipose tissue depot
You have a record of the masses of the adipose tissue depots that were removed from each of your
experimental rats. Use your results obtained so far (from above) and these adipose tissue depot masses
(from the Results tab or the Cell Counts table in the online Data Analysis tool) to produce a comparison
between groups for the adipose depots.
Note that the data on the mass of tissue is given in grams (g), so you will need to convert these values from
g to mg by multiplying each by 1000 (e.g. 0.90 g x 1000 = 900 mg).
So the steps to follow are:
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
Convert the depot masses into mg values for each rat.

This value is the amount of lipid that is present in each depot. Make sure to use the correct units
with your final values.
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Carry out a comparison between groups
For each depot in each animal, multiply its mass (in mg) by the concentration of lipid in that depot
(lipid per mg, from section 2.3).
You can now collate your data into a table for use in reports, consider how a graph might best
present your data and how you might use a statistical test to assess the significance of any
difference you have observed.
Do your results still show the same pattern as the comparison in (a)?
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SXHL288 Lab Guide: Lipid quantitation
Look back at the prediction about the proportion of WAT and BAT made up of lipid that you wrote down in
your lab notebook before carrying out this activity. How does your prediction compare with the results that
you have obtained?
(c) Comparison between groups: lipid content per cell
Once you have determined the lipid content per mg for each tissue sample (in step a, above), you can use
your data obtained from the histological analysis of BAT and WAT tissues to calculate the lipid content per
cell in BAT and WAT.
Basically, if you know how much lipid is contained within one mg of tissue and you know how many cells
are in that mg of tissue, then dividing the amount of lipid by the cell number will give you an estimate of
how much lipid is contained within one cell.
Record your calculations and results (with appropriate units) in your lab notebook. Now do a comparison
between groups, as before. Do your results still show the same pattern as in (a) and (b)?
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SXHL288 Lab Guide: Lipid quantitation
Appendix: Physiology laboratory spectrophotometer guidance
These instructions are available from within the spectrophotometer’s Help pages.
Remember that for lipid data collection you need to use a wavelength of 440 nm.
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