NUTR 301 Lab Manual

Carbohydrate & Lipid Metabolism in Various States

Background

Carbohydrate Metabolism
Regulation of blood glucose levels is imperative for maintaining proper functioning of tissues
that rely almost exclusively on glucose for fuel (e.g. brain, RBCs) and minimum blood glucose
levels are maintained to ensure proper functioning of these tissues (Stipanuk & Caudill, 2013).
With ingestion of a meal, blood glucose levels rise dramatically above the normal fasting
range. Elevated blood glucose causes the -cells of the pancreas to secrete insulin, which
facilitates cellular glucose uptake and effectively lowers blood glucose levels (McArdle et al.,
2001). In healthy participants, hyperglycemia following a meal normally subsides within 1 to
2 h (Stipanuk & Caudill, 2013). In insulin resistant or diabetic states, the response to dietary
glucose differs vastly from the normal response, with plasma glucose and insulin levels
remaining high 2 h or more after consuming a glucose-containing meal.

Insulin sensitivity refers to the ability of insulin to lower blood glucose levels by stimulating
cells to take up glucose (Pacini & Mari, 2003). Decreased insulin sensitivity indicates an
inability of cells to respond adequately to insulin to increase glucose uptake (McArdle et al.,
2001). This is known as insulin resistance, a state in which the pancreas overproduces insulin
to try to compensate for the high circulating blood glucose levels (McArdle et al., 2001).
Insulin resistance is often present long before clinical manifestations of the Metabolic
Syndrome (MetS) are observed (Monzillo & Hamdy, 2003). The MetS is a common metabolic
disorder that is characterized by glucose intolerance (elevated fasting plasma glucose or type 2
diabetes), insulin resistance, central obesity, dyslipidaemia (high triglycerides and low
concentrations of HDL cholesterol), and hypertension (Eckel et al., 2005). Central obesity and
insulin resistance are thought to be important causative factors in the development of the MetS
(Eckel et al., 2005). Although there is sometimes a genetic susceptibility to insulin resistance,
obesity is strongly correlated, indicating a possible role of higher adiposity and/or positive
energy imbalance in the development of metabolic syndrome (Grundy, 2016). Individuals with
the MetS are at a significantly increased risk of developing type 2 diabetes, atherosclerosis and
cardiovascular disease (Grundy, 2016).

Assessment of carbohydrate metabolism is one of the best methods available to assess insulin
sensitivity and detect insulin resistance. A standard procedure known as the oral glucose
tolerance test (OGTT) was established to estimate insulin sensitivity based on insulin secretion
and disappearance of glucose from the blood (Monzillo & Hamdy, 2003). The OGTT requires
ingestion of a standard amount of glucose followed by venous blood sampling for 2 to 3 h
(generally at 0, 30, 60, 90 and 120 mins) (Pacini & Mari, 2003). Plasma glucose and insulin
concentrations are measured at each time point. Several indices have been derived in an
attempt to quantify insulin sensitivity, such as the Homeostasis Model of Assessment - Insulin
Resistance or HOMA-IR (fasting insulin x fasting glucose/22.5). The resulting value is
compared with normal reference values or ranges (Monzillo & Hamdy, 2003).

Lipid Metabolism
In humans, the size, frequency and composition of meals are often unpredictable. Lipid
metabolism rates must respond rapidly to continuously changing inputs of cholesterol and

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fatty acids. Lipids in excess of immediate needs must be selectively transferred to appropriate
storage sites and released later upon demand (Stipanuk & Caudill, 2013). Following a meal,
the small intestine packages triacylglycerols (or triglycerides; TG), cholesterol and
phospholipids, along with certain apolipoproteins, into large lipoprotein particles called
chylomicrons (CMs) for transport in the circulation. Once absorbed, larger CMs (also known
as triglyceride-rich lipoproteins) are rapidly hydrolyzed to smaller chylomicron-remnant
particles, which are able to be removed from the circulation via the liver. In turn, the liver re-
packages nutrients into other lipoprotein particles of different class and function. These include
high density lipoproteins (HDLs), low density lipoproteins (LDLs) and very low density
lipoproteins (VLDLs) (Stipanuk & Caudill, 2013). Collectively, CMs, VLDLs and LDLs are
responsible for transporting lipids to the peripheral tissues, where the lipid constituents are
used for energy, storage, cell membrane structural integrity, and so on (Stipanuk & Caudill,
2013). The HDL particles play a role in reverse transport of cholesterol from the tissues back to
the liver for eventual excretion (Stipanuk & Caudill, 2013) and thus the cholesterol associated
with HDLs has become known as ‘good cholesterol’.

Measurements of plasma lipid concentrations in the postprandial period indicate not only the
amount of dietary fat absorbed, but also the efficiency of clearance from the blood. The
measurement of plasma lipid concentrations following a high fat meal is often referred to as a
fat tolerance test. After ingestion of a normal meal containing 1/3 of daily caloric intake,
plasma TG concentration typically peaks at about 3 hours and decreases toward (or below)
fasting levels by 9 h, returning to fasting levels by 12 h (Stipanuk & Caudill, 2013). The ability
to clear postprandial lipids (e.g. triglyceride-rich particles) is thought to correlate with risk of
developing cardiovascular disease (CVD) (Ginsberg and Illingworth, 2001; Lefebvre &
Scheen, 1998). Evidence suggests that sustained elevated postprandial TGs is an independent
predictor of coronary and carotid atherosclerosis (Cohn, 1998; Cohn, 1994). Other
abnormalities in lipid metabolism, including elevated fasting serum total cholesterol, LDL
cholesterol and TGs, are known risk factors for the development of CVD, as is low HDL
cholesterol (Singh & Mehta, 2002). Evidence suggests that postprandial TGs may be superior
to fasting TGs as a predictor of CVD risk (Criqui & Golomb, 1998). As mentioned above,
these lipid abnormalities, in combination with obesity, glucose intolerance and insulin
resistance, characterize the MetS (Eckel et al., 2005).

Animal Model
An animal model is defined (Farlex, 2015) as, “An animal that is an accidental or deliberate
(through selective inbreeding) model of a human disease. Such models are experimental living
systems that are used to study disease mechanisms and provide insight into possible
therapies.” The JCR:LA- corpulent rat is a unique animal model used to study coronary heart
disease, because it exhibits many of the risk factors (obesity, hyperlipidemia, insulin
resistance), as well as developing many features of the disease itself (atherosclerosis, ischemic
lesions of the heart, myocardial infarcts) (Russell & Proctor, 2006).

The corpulent (cp) gene mutation results in defective leptin receptors (Brindley & Russell,
2002). Defective receptors lead to increases in circulating leptin concentration; however the
signal is not recognized. Neuropeptide Y becomes elevated in response, strongly stimulating
feeding behavior and resulting in hyperphagia (Brindley & Russell, 2002). Thus, rats
homozygous for the cp gene (i.e. cp/cp) develop MetS in an extreme form, with marked
obesity, insulin resistance, hyperinsulinemia, hypertriglyceridemia, and atherosclerosis.

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At 3 weeks of age cp/cp rats are detectably obese (Brindley & Russell, 2002), however
hyperinsulinemia and insulin resistance develop as rats mature from 4 to 8 weeks of age
(Russell et al., 1998). Plasma TG is elevated by 4 weeks of age and continues to rise
thereafter, mostly due to marked hepatic hypersecretion of VLDL (Russell et al., 1998).
Importantly, JCR:LA-cp rats heterozygous (cp/+) or homozygous normal (+/+) for the cp
gene are lean and metabolically normal (Russell et al., 1998) and can therefore be used as
‘lean controls’ in nutritional studies. Female rats are less severely affected than male rats
(Russell et al., 1998); thus the male JCR:LA-cp rat provides a valuable model for studying
carbohydrate and lipid metabolism in the ‘normal’ versus insulin resistant state.

Purpose
The purpose of this lab is to characterize differences in carbohydrate and lipid metabolism in
obese animals exhibiting the metabolic syndrome versus metabolically normal control
animals.

Procedures
Data will be provided for plasma TG, glucose (GLC), total cholesterol (TC), and insulin (INS)
concentrations in lean and obese rats – via eClass. Instead of performing these analyses
yourself, you will watch a demonstration video prepared by students in Dr. Proctor’s lab. You
will need to write up your report as if you had performed the analyses and obtained the data
yourself because that is the level of understanding expected for this lab.

There will be several samples representing fasting and postprandial collections at different
time- points. Step-by-step instructions are included on subsequent pages. Plasma samples of
an unknown concentration are prepared by first diluting with double distilled water and each
sample is aliquoted in triplicate onto a 96-well plate, according to a plate layout map provided.
Standards of a known concentration, in triplicate, are also added to the plate according to the
map. This is followed by adding reagent, incubating the plate, and then having the plate “read”
by a spectrophotometer system (SpectraMax® 190 Microplate Reader, Molecular Devices
LLC, USA). The spectrophotometry software compares the absorbances of the known
standards of varying concentrations to the absorbances of the unknown samples on each plate
to back-calculate their concentrations.

The purpose of the discussion lab is to inspire speculation about the results of this experiment
in terms of physiological and metabolic processes most likely resulting in the blood values
observed. There will be an in-depth discussion of carbohydrate and lipid metabolism, and some
application to broader concepts (i.e. implications) and application of this knowledge (i.e. in
humans). Please come to the lab prepared for discussion as you will be asked to participate! It
may help to re-visit relevant information from the course notes and textbook prior to the lab.

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Results
Using the data provided, calculate average GLC, INS and TG concentrations and standard
error (SE=SD/√n) at each time-point for obese and lean rat groups. Present data as a line graph
with SE bars.
Do the same calculations for TC for lean and obese rat groups and present this data as bar
graph (column graph) format including SE bars.
As an index of insulin resistance, calculate the fasting HOMA-IR (Fasting insulin in µIU/ml
x Fasting glucose mol/l / 22.5) values for the lean and obese rat groups. Incorporate the
average HOMA-IR value for each group into the text of your Results section.
In your Appendix, show all raw data + means, standard deviation (SD) and SE at each time
point for each rat group. Show a sample calculation for all unique calculations that require
formulas in Excel (e.g. correct for dilutions and convert to mmol/L).

Discussion
As always, your discussion will primarily consist of observations from your results, whether
these were expected (or not) and why (or why not). Make sure you discuss the physiological
processes likely affecting the shape of the postprandial curves for each graph, and whether you
think the curves were inter-related. Additionally, please consider the following questions:
If the size of the meals is identical between the lean and obese groups, as well as content of
glucose (for MTT) and triglycerides (for FTT) in meals, why is there a difference in
concentration of glucose and triglycerides in the plasma after eating the meals?
Why were differences observed in total cholesterol between lean and obese rats? What is the
major lipoprotein source of this plasma cholesterol?
This lab was an observational comparison of JCR:LA-cp rat groups. Give an example of a
dietary intervention you think has the potential to “normalize” the metabolism of the obese
rats. Discuss.

**Tips for writing your Lab #4 report:

Hypotheses: As always, your hypotheses need to be specific and measurable. But how can
you make it specific if you have not done a rat experiment before and do not know what to
expect? (Hint: check studies using this rodent model). And how do you simplify it enough to
make your hypotheses measurable but concise? Firstly, focus on whether you think obese rats
will differ from lean rats during fasting and fed states (i.e. fasting, peak and end in order to
predict magnitude and duration of response as simply as possible). Remember that lean rats
represent the “normal control” group, so basically, will obese rats have higher levels than
normal at fasting, during and after the tolerance test (i.e. at the start, peak and end)? How
much higher will obese rat group levels be, e.g. <2x, >2x, >5x, >10x the lean rat group?
Experimental Design/Methods: You have been provided with a template for your Methods
section!
Discussion: Your notes from the discussion lab will help with describing the physiological
processes underlying carbohydrate and lipid metabolism during fed and fasted states, however
you will need to cite references that support the physiology and expectations for the rat model.

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PROCEDURES – Previously completed by lab technicians

Study Protocols for Tests Performed on Lean and Obese Rats

All experimental procedures were approved by the University of Alberta Animal
Ethics Committee and conducted in accordance with the Canadian Council on Animal
Care.

JCR:LA-cp rats (cp/cp) were raised in an established breeding colony at the University of
Alberta as previously described (Vine et al., 2007). Male rats (lean and obese) had free access
to water and a standard rat chow diet (see eClass) from weaning until 16 weeks of age, at
which point the diet was supplemented with fat and cholesterol (1%, w/w), to contain 42% of
energy from carbohydrate, 23.7% from protein and 34.3% from fat. When food intake was
measured, lean rats consumed an average of 19.7 g of food per day, whereas obese rats ate
32.4 g.

Seven days prior to the test, rats were acclimatized to a reverse light cycle to allow for
metabolic tests to be performed during the active feeding period of the animals.

At 24 weeks of age, the day prior to the test, rats were weighed and fasted for 12 h overnight.

The day of the test, at approximately 8:00 am, a fasting blood sample was taken from each
rat, referred to as the “0” time sample. A meal tolerance test or fat challenge test was
performed (as described in Russell et al., 2007 and Vine at al., 2007, respectively) immediately,
in which rats were placed in individual clean cages and given a 5 g pellet of food. Time was
recorded when half the food was eaten and this was used for subsequent blood draw timings.

For Meal Tolerance Tests (to test carbohydrate metabolism), rats were given a pellet of regular
rat chow (~24% protein w/w, 5% fat w/w, 49% carbohydrate w/w). Blood samples were taken
30 and 60 minutes later.

For Fat Tolerance Tests (to test lipid metabolism), rats were given a pellet of high-fat food
(comprised of ~24% milk fat w/w, prepared by mixing English Double Devon Cream at 47%
milk fat w/w with rat chow). Blood samples were taken 2, 4, 6, 8, and 10 hours later.

For both tests, blood samples were collected in heparinized tubes and put on ice. Samples
were spun in a centrifuge (~3000 rpm for 15 mins) and the plasma (supernatant) was pipetted
into labeled tubes and stored in a -80ºC freezer until analysis by lab technicians.

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PROCEDURES – Select procedures are shown in the demonstration video

Principles of the Assays used in this lab

How do the reagents work?

All assays are enzymatic because enzymes that bring about specific chemical reactions are in
the reagents used. They are colorimetric in that the optical density resulting from the desired
chemical reaction can be measured. In the case of the TG, GLC and TC assays, the amount
of H2O2 produced depends on the amount of substrate (glucose, glycerol, cholesterol,
respectively) in the sample. The coloring agent reacts with H 2O2 in the presence of peroxidase
and the colour intensity generated is directly proportional to the amount of substrate in the
sample.

How does the spectrophotometer determine that a certain color intensity equates to a specific
concentration of substance in a plasma sample?
In order to determine the unknown concentration of a substance in plasma samples (referred to
as “samples”), prepare a range of samples where the concentration of that substance is known
(referred to as “standards”). This way, the colour intensities produced by standards can be
equated to known concentrations. Specifically, you need to prepare standards containing the
substance of interest in amounts chosen to span the range of concentrations you expect to find
in the unknown samples. The spectrophotometer creates and uses a standard curve to equate
the colour intensity of the standards with the known concentrations (as input by the technician).
A standard curve is a graph relating measured optical densities to the standard concentrations
using linear regression. The spectrophotometer calculates a standard curve by plotting
absorbance (Y axis) versus concentration (X axis). The curve can be used to determine
concentrations of the substance in unknown plasma samples as shown in Figure 1. A sample
with an absorbance of 0.250 equates to [TG] of 83 mg/dl.

Figure 1 Example of a triglyceride standard curve. Standards of varying concentration were

prepared (0, 3.125, 6.25, 12.5, 25, 50 and 100 mg/dl) via serial dilutions of a 200 mg/dl (2
mmol/L) standard solution. Standards were assayed and absorbance was read by a
spectrophotometer at a wavelength of 540 nm. (Source: Cayman Chemical Triglyceride
Colorimetric Assay Kit)

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What are the specific reactions involved in each assay?

Glucose Assay Principle

The glucose oxidase/peroxidase method (Genzyme Diagnostics P.E.I. Inc.) used in this lab is
well established for the determination of glucose concentrations in biological fluids. This
method is used for both human and animal samples and is important for diagnosis of diabetes
and hypoglycemia.
Glucose Oxidase
-D-glucose + O2 + H2O  D-gluconic acid + H2O2

Peroxidase
H2O2 + Hydroxybenzoate + 4-aminoantipyrine  Quinoneimine dye (red) + 4 H2O

Triglyceride (TG) Assay Principle

The TG assay (Wako Chemicals USA, Inc.) is an enzymatic method used to determine the
amount of TGs in serum or plasma by detecting the amount of glycerol in the sample (one
glycerol per triacylglycerol molecule). In this two-step process, the TG assay is not affected by
free glycerol in the sample because free glycerol is decomposed in the first step by a series of
enzymes before TG hydrolysis.

Step 1:
GK, GPO Catalase
Free glycerol  2 H2O2  2 H2O + O2
ATP

Step 2:
LPL
Triglyceride  Glycerol + 3 FFAs

GK
Glycerol  Glycerol-3-Phosphate
ATP

GPO
Glycerol-3-Phosphate + O2  Glycerone phosphate + H2O2

Peroxidase
H2O2 + HDAOS + 4-aminoantipyrine  Blue pigment (450 nm)

Note: GK, Glycerol Kinase; GPO, Glycerol-3-Phosphate Oxidase; LPL, Lipoprotein Lipase; FFAs, Free
Fatty Acids

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Cholesterol Assay
The cholesterol assay (Wako Chemicals USA, Inc.) is an enzymatic method for the
quantitative determination of total cholesterol (TC) in serum or plasma. To determine the
amount of TC in the sample (free cholesterol + cholesterol in cholesterol esters), cholesterol
esters first need to be hydrolyzed to free cholesterol.

Cholesterol Ester Hydrolase

Cholesterol ester  Free Cholesterol + FFAs

Cholesterol Oxidase
*Free Cholesterol  H2O2

Peroxidase
H2O2 + 4-aminoantipyrine + DAOS  Blue Color Complex + 2 H2O (600 nm)

*Note: This is total free cholesterol (free cholesterol in plasma + free cholesterol hydrolyzed from cholesterol
esters)

Low Density Lipoprotein (LDL) Cholesterol (note: FYI, not done in lab or included ethods)
LDL cholesterol (LDLc) concentration is determined in a similar way to TC in plasma,
however a two- step process is needed. In the first step, added reagent protects the LDLs while
all non-LDLs are oxidized. The sample has to read by the spectrophotometer at this point to
establish a baseline color. Reagent is added in a second step, whereby remaining LDLs are
oxidized and a blue color complex is formed in proportion to the amount of LDLc in the
plasma sample.

In outpatient laboratories and some human research labs, plasma LDLc concentration is often
calculated, rather than measured. The TC, HDLc and TG concentrations can be determined
by an automated enzymatic method and then the Friedewald (1972) equation used to calculate
LDLc: LDLc = TC - HDLc - TG/5.0 (g/dl). In this equation, TG/5 is used to estimate VLDL
cholesterol concentration in plasma (VLDLs carry most of the TGs in fasting plasma and
cholesterol from this source can be estimated in this way).

It should be noted that cholesterol metabolism in rats and mice differ from humans in that
cholesterol transport is primarily based on HDLs, rather than on LDLs (Russell & Proctor,
2006).

Insulin Assay Principle

Insulin sensitivity testing can be diagnostically important in humans for monitoring diabetes
control and for insulin levels in diabetic patients taking insulin to control their blood sugar
concentration. This procedure is quite lengthy and complicated, and therefore insulin analyses
will not be done as part of the lab. Data will be provided to you for your report and therefore
the insulin assay must be included in your Methods section.

The ultrasensitive rat insulin enzyme-linked immunosorbent assay (ELISA) provides a

method for the quantitative determination of insulin in rat serum or plasma. The ELISA kit
(ALPCO Immunoassays) is a solid phase two-site enzyme immunoassay based on the

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sandwich technique in which two monoclonal antibodies are directed against separate
antigens on the insulin molecule. Insulin in the plasma sample binds to anti-insulin antibodies
coated on the wells of a 96 well plate and then to anti- insulin peroxidase conjugated
antibodies. A washing step removes unbound enzyme labeled antibody. Bound conjugate
antibody is detected by reaction with the enzyme substrate. The reaction is stopped by adding
acid to give a colorimetric endpoint. If this seems confusing, see this 2 minute explanation
developed by Cary Engleberg at the University of Michigan: https://youtu.be/RRbuz3VQ100

PREPARATION OF STANDARD SOLUTIONS (note: standards have been prepared for you)

Preparation of Glucose Standards

Standards of different concentrations are required so the spectrophotometer can make a
standard curve of known glucose concentrations. Two glucose standard stock solutions 400
mg/dl (Stock 1) and 90 mg/dl (Stock 2) have been provided in the glucose analysis kits. The
table below shows a series of diluted glucose standards prepared using the stock solutions and
double distilled water (ddH2O) as a diluent.

Formula: Ci * Vi = Cf * Vf
Ci = initial stock concentration Cf = final concentration
Vi = initial stock volume Vf = final volume

Sample Calculation: How would you prepare 100 l of a 45 mg/dl glucose standard solution
if you were given a 90 mg/dl glucose standard stock solution?
Vi = (Cf * Vf)/Ci
(200 mg/dl *100 l)/400 mg/dl = 50 l of glucose standard stock solution
Therefore you would need 50 l of the glucose standard stock solution and 50 l of ddH20.
This is known as a 1 in 2 dilution as you have one volume (50 l) of glucose standard stock
solution and two volumes of total solution (100 l).

Table of Glucose Standards

Glucose Standards Standard Stock Diluent Volume Total Volume
(mg/dl) Volume (l) (ddH20, l) (l)
400 Stock 1
200 50 50 100
100 25 75 100
90 Stock 2
67.5 75 25 100
45 50 50 100
18 20 80 100
0 0 100 100

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NUTR 301 Laboratory Manual

Triglyceride (TG) Standards

The triglyceride standard solutions have been prepared for you from a TG kit that provides a
400 mg/dl standard stock solution. You will need to provide the calculations for how much
TG stock solution and diluent (ddH20) was used to make 200 l of each TG standard solution.
Fill in the table for your report and include in the Appendix of your report. Refer to example
calculation from preparation of diluted glucose standards for the formula.

Table of Triglyceride Standards

[Triglyceride] Stock Volume Volume Final Volume
(mg/dl) (l) ddH2O (l) (l)
64
32
16
8
4
2
0

Preparation of Cholesterol Standards

The cholesterol kit provides a 200 mg/dl total cholesterol standard stock solution. This is an
example table and filling it out is not required for your reports.

Table of Cholesterol Standards

[Cholesterol] Stock Volume Volume ddH2O Total Volume
(mg/dl) (μl) (l) (μl)

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PROTOCOL FOR GLUCOSE ANALYSIS

 Rat plasma samples: labeled either A or B (representing lean or obese animal groups),
and plasma collection time point (0, 30, or 60 min), i.e. A-60 is A group (lean), at the 60 min
collection time-point. Note that because some of the samples have elevated glucose
concentrations, you will need to run a 1 in 2 dilution of the original plasma samples.
 Glucose standard solutions: Stock 1 (400 mg/dl) and Stock 2 (90 mg/dl).
 Empty tubes: for diluted plasma and prepared standard solutions.

Glucose Analysis Procedure (Based on the Instruction Manual for the Diagnostic
Chemicals Limited Glucose Assay Kit).

Preparation of Standards and Samples

1. Label all empty tubes.
2. Vortex all samples before transfers.
3. Standards have been prepared according to the Table of Glucose Standards (6
standard solutions from 2 stock solutions to make 8 total standards).
4. Prepare dilute plasma samples (1:2 dilution) by adding 4 l of ddH2O to an
empty tube and adding 4 l of sample (at 8 l total volume each, so you can plate 2 l of
each in triplicate).
5. Vortex prepared standards and sample tubes (and centrifuge liquid to bottom,
if needed).

Plating and Analyzing Standards and Samples

6. Pipette 2 l of each standard into plate wells, in triplicate, as indicated on the
plate layout sheet.
7. Pipette 2 l of each diluted plasma sample, in triplicate, as indicated on the
plate layout sheet.
8. Take your plate and plate layout sheet to the reagent station. Using a multi-
channel pipette, add
200 l of glucose reagent to each used well.
9. Incubate the plate for 3 min at 37˚C (write your group name on cover if
needed). Remove and eliminate bubbles that have formed (blow lightly across surface of
wells).
10. Take your plate to the spectrophotometer to be read by a lab technician. The
absorbance is measured at a wavelength of 505 nm. Obtain a printout of your results.

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PROTOCOL FOR INSULIN ANALYSIS

NOTE: This will not be done during the lab, instead you will be provided with insulin data to
analyze. However, you will need to include insulin analysis information in your Methods.

Preparation of Insulin Standards

Insulin standards (calibrators 0, 0.02, 0.05, 0.15, 0.4, 1, 3 and 5.5 g/l) are provided in the kit.

Preparation of Enzyme Conjugate (for one 96 well plate)

Mix 600 l of enzyme conjugate 11x with 6 ml of enzyme conjugate buffer.

Preparation of Wash Buffer (for one 96 well plate)

Add 40 ml of wash buffer 21X to 760 ml of ddH2O in a 1L beaker or plastic container.

Preparation of Insulin Samples

Rat plasma samples corresponding to 3 different plasma collection time points (0, 30 and 60 min)
are labeled either A or B (lean or obese animal group). Because some of the samples will have
elevated insulin and fat concentrations and may be turbid, you will need to run plasma samples
and a 1 in 10 dilution of your original plasma samples for B rats only.

Insulin Analysis Procedure: Step-by-Step (Based on Alpco Diagnostics Ultrasensitive Rat

Insulin ELISA kit procedures).
1. Label your sample tubes and 96 well plate.
2. Dilute samples with ddH2O and vortex to ensure standards and samples are mixed.
3. Pour calibrator 0 into a reagent reservoir. Using the multi-channel pipette, add 25 l of
calibrator 0 to all wells on the plate.
4. Pipette 5 l of standard, and sample in duplicate into the appropriate wells.
5. Pour prepared enzyme conjugate into a reagent reservoir. Add 75 l of enzyme
conjugate mixture using a multi-channel pipette to all wells.
6. Put the plate on an orbital shaker for 2 h at room temperature. Record the time in your
lab note book and inform the lab instructor.
7. Remove and discard the solution from all wells and wash 6 times with 350 l of prepared
wash buffer. After the last wash the wells will be empty.
8. Pour substrate TMB solution into a reagent reservoir. Using the multi-channel pipette,
add 200 l of substrate TMB solution to all wells and incubate for 30 minutes at room
temperature. Cover the plate with aluminum foil to protect the light sensitive substrate.
9. To stop the reaction, add 50 l of stop solution to all wells with a multi-channel pipette.
Avoid making bubbles.
10. Put the plate on a shaker for 5 seconds to ensure mixing of substrate and stop solution.
11. Measure the absorbance at a wavelength of 450 nm with a plate reader
(spectrophotometer). Obtain a printout of the results.

PROTOCOL FOR TOTAL CHOLESTEROL ANALYSIS (note: not done in lab)

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 Rat plasma Samples: labeled either A or B (lean or obese animal group), a number to
distinguish individual samples, and plasma collection time point (0 only = fasting), i.e. A3-0
is sample #3, A group, at the 0 hour time-point.
 Empty tubes: for diluted plasma (1:3).
 Cholesterol Standard solutions.

Total Cholesterol Analysis: Step-by-Step

Preparation of Standards and Samples

1. Label all empty tubes for standard dilutions and diluted samples before you begin.
1. Vortex all tubes containing standards and samples (centrifuge liquid to bottom if
needed).
2. Dilute cholesterol standards according to the Table of Cholesterol Standards.
3. Dilute plasma samples; A and B rats: 1 in 3 dilutions (15 l plasma + 30 l ddH2O).
4. Once transferred to new tubes, vortex all samples (centrifuge liquid to bottom if
needed).

Plating and Analyzing Standards and Samples

5. Note the plate layout sheet provided, indicating which wells will contain your
standards and which will contain samples (label the 96-well plate if helpful).
6. Add 10 μl of standards and samples, in triplicate, to the appropriate wells.
7. Bring your plate and plate layout sheet to the cholesterol reagent station and using a
multi- channel pipette, add 200 μl of color solution to used wells only.
8. Mix shortly by lightly tapping the edge of the plate.
9. Put the lid on, label with group name (if needed) and incubate for 5 mins at 37 C.
10. Let the plate cool for 5 min.
11. The lab technician will read the absorbance at 600 nm on a spectrophotometer.
Obtain your results printout.

PROTOCOL FOR TRIGLYCERIDE ANALYSIS

Each student pair or triplet will be given tubes containing:
 Plasma samples: labeled A or B (representing lean or obese animal groups) and plasma
collection time point (0, 2, 4, 6, 8, 10 h), i.e. A-6 is the ‘A’ sample from the 6 h time-point.
Samples may be turbid or have concentrations outside the analytical range of the
spectrophotometer  A samples are run at 1:3 dilution, and B at 1:10 dilution.
 Triglyceride standard solutions: already prepared at concentrations indicated.

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 NUTR 301 Lab Manual

Triglyceride Analysis Procedure: Step-by-Step

Preparation of Standards and Samples (Note: this has been completed for you, proceed to step 5)
1. Label all empty tubes for diluted plasma samples before you begin.
2. Vortex all standard and plasma sample tubes.
3. Dilute plasma samples for all time-points; for A rats: 1 in 3 dilutions (60 l plasma +
120 l)
4. ddH2O) and for B rats: 1 in 20 dilutions (9 l plasma + 171 l ddH2O)
5. Once transferred to new tubes - vortex. Centrifuge liquid to bottom of tube if needed.

Plating and Analyzing Standards and Samples

6. Note the plate layout sheet indicating which wells will contain your standards and which
wells will contain samples (if helpful, label the 96-well plate accordingly).
7. Carefully pipette 4 l of each standard, in triplicate, into the appropriate plate wells.
8. Pipette 4 l of each diluted sample, in triplicate, into the appropriate plate wells.
9. At the TG reagent station, add 90 l of Enzyme Color A, using a multi-channel pipette
from a clean reagent reservoir into only the used wells.
10. Incubate at 37 C (label your plate with group initials). Set timer; remove after 15 min.
11. *Get your plate read by the lab technician using a spectrophotometer (absorbance
against the blank at 600/700 nm). Make sure bubbles are removed as they may
interfere with readings.
12. Return to reagent station and add 30 l of Enzyme Color B from the reagent reservoir
into the used wells using a multi-channel pipette.
13. Incubate a second time at 37 C for 30 min. After retrieving plate, check for bubbles
(remove bubbles by blowing lightly across surface of the wells).
14. Get your plate read by lab technician using the spectrophotometer at 600 nm - obtain
results printout.

*Note: Normally absorbance readings obtained following Enzyme Color A are subtracted
from absorbance readings after Enzyme Color B (using the SoftMax® Pro GxP Software), to
correct for the presence of free glycerol in plasma samples. However with time constraints of
the lab, this step may be omitted.

Calculations for All Analyses

15. On your results printout, cross out disparate ‘Result’ values with large CVs (>15) and
recalculate the ‘MeanResult’ values if needed (you may need input from the instructor).
1. Correct results for sample dilutions (e.g. if 1:3 dilution, multiply result by 3).
2. For TG, to convert from mg/dL to mmol/L, multiply by conversion factor of 0.0113.
Retrieve the data from all analyses as posted in a spreadsheet on eClass. You will need to
correct for dilutions and convert to the appropriate units before graphing it. For insulin,
convert g/L to pmol/L by multiplying by 172.1. (To convert pmol/L to µIU/ml - for
calculation of HOMA-IR - divide by 6.945). For other analytes, convert from mg/dL to
mmol/L using conversion factors; GLC = 0.0555, TC = 0.02586 and TG = 0.0113.

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 NUTR 301 Lab Manual
NUTR 301 Laboratory Manual

96-Well Plate Layout for Assays

An example of a 96-well plate layout plan for a triglyceride assay is shown below.

1 2 3 4 5 6 7 8 9 10 11 12
A 0 0 0 A-0 A-0 A-0 B-0 B-0 B-0

B 2 2 2 A-2 A-2 A-2 B-2 B-2 B-2

C 4 4 4 A-4 A-4 A-4 B-4 B-4 B-4

D 8 8 8 A-6 A-6 A-6 B-6 B-6 B-6

E 16 16 16 A-8 A-8 A-8 B-8 B-8 B-8
F 32 32 32 A-10 A-10 A-10 B-10 B-10 B-10
G 64 64 64
H
Standards and plasma samples are plated in triplicate. Standards are pipetted into columns 1 - 3 and
rows A - G in order of increasing concentration (note that ‘0’ is a concentration of 0 mg/dl, therefore
only ddH2O). Absorbance readings from these values are used to make a standard curve from which
the concentration of plasma samples can be determined by the spectrophotometer. Diluted plasma
samples from lean (A) and obese (B) rats are pipetted into columns 4 – 9, rows A - F. To conserve
supplies, do not fill empty wells with reagent.

Figure 1. A 96-well plate is

shown as an example. This
one was used in an insulin
analysis, after addition of
substrate TMB solution to
some of the wells.

Photo courtesy of MCVD lab,

University of Alberta.

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