Practical 2

Blood Lipid Analysis

Introduction
Consumption of excess dietary fat has a number of effects on metabolism and over consumption for
an extended period of time can have pathological consequences. Included in these are
cardiovascular disease, atherosclerosis, obesity, diabetes and cancer, which have all been associated
with the consumption of high fat diets. An important diagnostic tool in determining individuals pre-
disposition to the previously mentioned pathologies is to measure the concentration of a number of
blood-borne lipids. Included in these are; total cholesterol, HDL cholesterol, LDL cholesterol,
triglycerides and non-esterified free fatty acids.

As a model of these changes in this practical class you shall use data from three groups of rats
which have undergone three different feeding regimes and determine how these interventions have
affected body weight and blood lipids. The three groups of animals are:

1. Controls, normal fed animals

2. Fat fed animals, which have been supplemented with additional dietary fat.
3. Low fat fed animals, which have had dietary fat restricted.

Each group of animals has been monitored for a period of 0, 1, 2, 3 or 4 weeks while undergoing
their feeding regime. Blood samples were collected into EDTA coated tubes to prevent blood
clotting and stored at -20oC awaiting analysis.

Aims
1. To determine what effect an altered fat diet has on body weight over a four week period
2. To determine the cholesterol and triglyceride levels in human sera.

Methods

Calculate the mean values for the body weight data contained within the table in the results section.

Analyse the cholesterol and triglyceride contents of the two unknown samples of sera provided.
These sera samples were collected from the patients who have different health conditions. You must
determine their health status based on your results.

1
Cholesterol assay
The following method is summarized in Table 1.

1. Label 5 cuvettes. One for each of the following: reagent blank, standard, control, unknown
sample 1 and unknown sample 2.
2. Add 1 ml of completely dissolved reagent to each cuvette.
3. Add 20 l of distilled water to the cuvette containing reagent blank (remember: 1ml = 1000l).
4. Add 20 l of standard, control and unknown samples (1 and 2) respectively to the labelled
cuvettes and mix gently.

Table 1: Summary of cholesterol or triglyceride assay

Blank Standard Control Test Test

sample 1 sample 2
Water 20 l --- --- --- ---
Standard --- 20 l --- --- ---
Control --- --- 20 l --- ---
Sample --- --- 20 l 20 l
Reagent 1.0 ml 1.0 ml 1.0 ml 1.0 ml 1.0 ml

5. Cover cuvettes with parafilm and incubate them at room temperature for 10 mins.
6. Select a wavelength of 520 nm using up and down arrows, and zero the spectrophotometer with
the reagent blank.
7. Read and record the absorbance of each standard, control and unknown samples.

Triglyceride assay
The following method is summarized in Table 1 above.
1. Label 5 cuvettes. One for each of the following: reagent blank, standard, control, unknown
sample 1 and unknown sample 2.
2. Add 1 ml of completely dissolved reagent to each cuvette.
3. Add 20 l distilled water to the cuvette containing reagent blank.
4. Add 20 l of standard, control and unknown samples (1 and 2) respectively to the labelled
cuvette and mix gently.
5. Cover cuvettes with parafilm and incubate them for 10 minutes at room temperature.

2
6. Select a wavelength of 520 nm and zero the spectrophotometer using up and down arrows with
the reagent blank.
7. Read and record the absorbance of each standard, control and unknown samples.

Cholesterol or Triglyceride calculation

Calculate the results as follows:

Cholesterol
or = Absorbance of Unknown
Triglyceride Absorbance of Standard X Standard value

N.B. standard values will be provided by your demonstrators in the class

3
Results

The following body weight values were recorded for the animals in each group over the four-week
feeding period.

Body Weight (g)

Week
Rat 0 1 2 3 4
1 351 348 350 352 352
2 346 350 353 352 355
Control 3 359 359 360 361 362
4 400 399 405 403 401
5 372 375 375 369 372
Mean
SD
1 400 429 460 493 519
2 392 421 452 483 510
Fat Fed 3 341 366 391 416 444
4 380 407 434 461 490
5 356 383 410 437 463
Mean
SD
1 351 324 298 277 246
2 362 335 308 280 253
Low fat 3 372 336 315 292 260
4 384 347 321 298 269
5 395 347 330 302 277
Mean
SD

SD = Standard Deviation (STDEV)

From the above data determine the effects of each feeding regime over time and represent these
changes graphically.

4
Questions for Discussion
1. How do the results obtained from the two groups of humans (samples 1 & 2) compare with the
guidelines shown in the following table?

Cholesterol Triglycerides
(mg/dL) (mg/dL)
Desirable < 200 < 200
Borderline 200 - 239 200 – 250

High > 240 > 250

2. What pathological condition (if any) may be indicated by the values you obtained for plasma
cholesterol and triglycerides?

3. Using the data collected on body weight, discuss the effects of the feeding regimes, and give
consideration to what consequences may arise if these types of feeding were continued for an
extended period of time.