Applications of Integration in Biomedical

Science

by
William T. Self
UCF EXCEL Applications of Calculus
Calculus Topic: Defining area under the curve

Topic #1: Approximating rectangles

One possible method for estimating area

under a given curve (or function) is the
use of approximating rectangles

This is a simple method, but has

limitations in its ability to accurately
define the area
Calculus concept # 1
Approximating Rectangles

Section 5.1 #1:

By reading values from the given graph of f (shown on
the next slide) use three rectangles to find a lower
estimate for the area under the given graph of f from
x=0 to x=6. In each case sketch the rectangles that you
use.
 Approximating rectangles

Reminder:
3 rectangles
Lower limit
From x=0 to x=6

Answers:
A) 17
B) 19
C) 21
D) 20
E) 28
Approximating rectangles
The use of this technique is inadequate to
determine the area under a curve since it can
overestimate and underestimate this area

This section of the applications course will

introduce you to concepts and methods in
biomedical science that rely on calculus to
determine the quantity of compounds and
macromolecules
Applications of Integration in Biomedical Science

Some of the future courses (that you may take) that this
will be relevant:

MCB 3020 – General Microbiology

BSC 3403C – Quantitative Biological Methods
MCB 4414 – Microbial Metabolism
BCH 4053 – Biochemistry I
BCH 4054 – Biochemistry II
Life – Its existence on Earth

Time Line for Planet Earth

Prokaryotes
Eukaryotes

Prokaryotes
 involved in formation of the biosphere
 required for plant & animal survival
Life – Cellular level

eukaryotic cell prokaryotic cell

 What are cells made of (E.coli )?

CHNOPS:
Carbon
Hydrogen
Nitrogen
Oxygen
Phosphorus
Sulfur
Adenosine triphosphate - ATP
Biological Macromolecules
Trace Elements
Human composition (complements of Dept. of Energy)

Dry weight %
Carbon 61.7
Nitrogen 11.0
Oxygen 9.3
Hydrogen 5.7
Calcium 5.0
Phosphorus 3.3
Potassium 1.3
Sulfur 1.0
Chlorine 0.7
Sodium 0.7
Magnesium 0.3

Trace amounts of B, F, Si, V, Cr, Mn, Fe, Co, Cu, Zn, Se, Mo, Sn, I.
There are some arguments as to the importance of other trace elements
Biological Cells – Complex mixtures

Basics:
DNA, RNA: Polymers of nucleic acids – encode
proteins

Proteins: Polymers of amino acids – can be structural or

act as enzymes

Lipids: Polymers of carbon – structural components of

cell membranes
Biological Cells – Complex mixtures

A given cell will have thousands of different proteins,

RNA molecules and metabolites present under a
particular growth condition

How do we define the ‘role’ of each individual protein

(for example)?

First we must purify this protein away from all other

components, then study it in a test tube (in vitro)
Protein Purification
Proteins are polymers of
amino acids

Protein sequence defines the

chemical composition

Each protein has unique size,

charge and shape
Chromatography – separation of mixtures

Chromatography in general is the separation of

compounds from mixtures using a Solid phase and a
mobile phase

Typically the solid phase is stationary, and held in

place in a column

The mobile phase (usually aqueous) moves through

the solid phase and carries the sample
Chromatography – separation of mixtures

Samples separate from

each other on the
column due to
differences in their
unique properties:

1.) net charge

2.) hydrophobicity
3.) size
4.) specific affinity
Chromatography – separation of mixtures
Types of chromatography used in protein purification:

1.) Ion Exchange

2.) Gel filtration
3.) Hydrophobic
4.) Affinity
Types of chromatography – Protein separations

1.) Ion exchange:

The solid phase has a strong or weak charged group
(e.g. strong positive charge)

If a protein has a net negative charge (anionic), it will

bind to a column that has a cationic group (positive
charge). Each protein will have a slightly different net
charge and thus mixtures of proteins can be separated
based on net charge
Types of chromatography – Protein separations

2.) Gel filtration

Proteins will separate based on size, due to pores
present in beads in the solid phase. The pores define
the separation capabilities of the media (e.g. 30,000
MW to 3,000,000 MW)
Types of chromatography – Protein separations

3.) Hydrophobic Interaction Chromatography

Based on binding of hydrophobic amino acids (such
as leucine, isoleucine) that are usually buried but
occasionally present on the surface

Common groups on the stationary phase are phenyl

groups or carbon chains
Types of chromatography – Protein separations

4.) Affinity Chromatography

Generally, proteins can be engineered to contain ‘tags’
at their ends that will bind to a certain group (e.g.
metal). This tag is usually unique in the mixture and
thus a ‘tagged’ protein can be purified quite readily
from a cell extract using this procedure.

The use of protein tags has revolutionized the study of

proteins in enzymes in the wake of the era of
molecular biology and cloning.
How does this relate to Calculus???
To find and determine the quantity of a given protein, or
other molecule of interest, we follow the elution of
these molecules using a detector. This pattern is
essentially a continuous function from one time period
to the next as follows:

Samples eluting become a

series of peaks that can be
followed and quantified
by area under the curve
 Calculus concept # 2

Limitations of approximating rectangles

Section 5.1 #2 Use 4 rectangles to find estimates of

each type for the area under the given graph of f from
x = 0 to x =6.

Three questions – left and right endpoints and finally

midpoints
 Calculus Topic: Defining area under the curve

Reminder:
Left endpoints 3

Answers:
A)
B)
C)
D)
E)
 Calculus Topic: Defining area under the curve

Reminder:
Right endpoints 3

Answers:
A)
B)
C)
D)
E)
 Calculus Topic: Defining area under the curve

Reminder:
Midpoints 3

Answers:
A)
B)
C)
D)
E)
Calculus Topic: Defining area under the curve

Which of the three techniques is best?

Why?

Could there be a better way based on your current

knowledge of calculus?
Applications of Integration in Biomedical Science

In addition to protein purification, chromatography

(and area under the curve) has many other uses in
biomedical science
Some issues to discuss:
1.) Arsenic (and other contaminants) in drinking
water
2.) Drug testing (e.g. steroid use)
3.) Bioterrorism – detection of explosives
4.) Pesticides in agriculture and consumer use
Applications of chromatography
How do we determine the presence of a pesticide
present in a lake, river or stream?

How do we quantify such a compound?

Why does this quantization matter?

Drug testing – front lines
Websites for discussion:

http://www.questdiagnostics.com/employersolutions/stand
ard_urine_testing_es.html

http://www.agilent.com/about/newsroom/lsca/background
/2007/bg_sports_drug_testing.pdf
 Drug testing – front lines

Recent article in the journal Nature outlines issues in

drug testing for anabolic steroids
Example of LC-MS analysis
Overview of typical HPLC setup:

Detector is
typically a mass
spectrometer that
can predict the
mass of eluting
compounds
Example of LC-MS analysis
Agilent
Technologies
example of LC
profile of
steroids
Gas chromatography (GC)
Gas chromatography:
Similar to HPLC, with the exception that the mobile phase is a
gas

Sample is either a gas or is derivatized to a volatile form to

allow for separation in a gas mobile phase

Column has a liquid stationary phase which is bound to an

inert support phase that is solid

This form of chromatography is most common in analytical

analysis of pesticides and lipid analysis.
GC – typical set up

Typical GC setup – courtesy of Waters, Inc.

Explosives – GC-MS example

Small amounts of
explosives can be
buried in compounds
that ‘mask’ their
presence in samples
GC-MS can uncover
readily
Calculus concept #3
Fundamental theorem of calculus
The fundamental theorem of calculus
states: (Part 1)

g(x) = ∫ f (t )dt

where f is a continuous function on

[ a,b] and x varies between a and b.
Fundamental Theorem of Calculus
Fundamental Theorem of Calculus
Part 2 states:
If f is continuous on [a,b] then:

∫ f (x ) dx = F (b ) – F (a )
Essentially, for purposes of defining area under the curve, the
difference in the antiderivative of f between two points [a,b ] on
the curve (assuming a continuous function) is equal to the area of
that curve to the x-axis
This is the most critical application (in biological sciences) of
the fundamental theorem
Fundamental Theorem of Calculus

Insert clicker question

Integration (Alvaro figure)
Fundamental Theorem of Calculus
Biomedical Science - review

What are the three most abundant elements in the

human body (dry weight analysis)?

A.) Hydrogen, Nitrogen and Calcium

B.) Carbon, Nitrogen and Hydrogen
C.) Magnesium, Carbon and Nitrogen
D.) Carbon, Oxygen and Hydrogen
E.) Carbon, Selenium and Magnesium
Methods of detection in chromatography
After separation (HPLC, GC, etc.) we must identify and
quantify a molecule of interest

Some of the commons ways to find and quantify

molecules:

1.) UV-visible spectroscopy

2.) Mass spectrometry
3.) Flame ionization (FID)
4.) Thermal conductivity (TCD)
Methods of detection in chromatography
These abbreviations lead to the multitude of common
analytical techniques:

LC-MS (Liquid chromatography – detection by mass

spectrometry
GC-MS, etc.

All are still based on the fundamental concepts of

chromatography, and all can use integration of peak
area to determine the quantity of an eluted sample
Methods of detection in chromatography

1.) UV-visible spectroscopy

Functional groups in a
molecule can absorb light at a
given wavelength
Aromatics and metal-complex
ligands are common groups in
biological samples that absorb
light in UV or visible range
Methods of detection in chromatography

DNA absorbs light at

approximately 260
nanometers

Courtesy Biocompare
Methods of detection in chromatography

Proteins absorb light at

approximately 280
nanometers

Due to tryptophan and

tyrosine residues
Methods of detection in chromatography
HPLC analysis of purines

A purine metabolizing enzyme was

tested for its substrate specificity
(which compounds it acts on)
using HPLC analysis

Each substrate and product elutes at

a different time from reverse
phase HPLC (hydrophobic
stationary phase)

Purines followed by UV-vis*

Methods of detection in chromatography

2.) Mass spectrometry

Mass spectrometry determines the overall predicted
molecular weight of a molecule based ‘weighing’ its
charge to mass ratio

Molecules are charged in an ion source, then

accelerated to a high speed. They are then passed
through a magnetic field and their trajectory is
altered by this field, dependent on their charge to
mass ratio
Methods of detection in chromatography

The particles are then detected

and their composition can
be predicted based on this
charge to mass ratio

Other information on the

sample is generally needed
to be able to identify and
confirm the molecule of
interest

Image courtesy of USGS

Methods of detection in chromatography
3.) Flame Ionization Detection (FID)

FID is commonly used in GC applications, and is based on

‘burning’ of the sample
FID is very good at detecting hydrocarbons and other carbon
containing molecules

4.) Thermal Conductivity Detection (TCD)

TCD is commonly used to detect gases (hydrogen) when carried

in an inert gas (argon)
TCD is based on changes in thermal conductivity – useful since it
can detect nearly any compound
Use of calculus in Biomedical Science - Review

What characteristic of proteins is useful in gel filtration

chromatography?

A.) Affinity for ligands

B.) Net charge
C.) Hydrophobicity
D.) Size
E.) Sequence
Proteomics – cutting edge use of chromatography

Cancer diagnosis: Current techniques

Example: Breast cancer
Mammogram
Ultrasound
Biopsy
Genetic screening

Expensive, labor intensive and usually only detect

cancer at later stages (not when first forming)
Proteomics – cutting edge use of chromatography

Proteomics:
The proteome is defined as the set of proteins present in
the cell under a given growth condition
The complement of proteins changes in different cell
types (tissues) and under different conditions (stress,
infection, disease)
Genetic variability also is displayed in the proteome
Proteomics – cutting edge use of chromatography

Proteomics in Cancer diagnosis:

Using reverse phase chromatography to follow the

‘proteome’ of a clinical sample (e.g. serum), one
can obtain a profile of the peptides that are present
in a patient

Analysis of hundreds of patients, both ill and healthy,

allow for patterns to emerge in this analysis
Proteomics – cutting edge use of chromatography

Above is a sample chromatogram of the peptides in

serum of an ovarian cancer patient
Biomarkers of Ovarian Cancer, Gynecologic Oncology 88, S25–S28 (2003)
doi:10.1006/gyno.2002.6679
 Proteomics – cutting edge use of chromatography

Proteomic analysis to diagnose cancer:

In a study published in 2002 using a blinded set of

samples, the proteomic pattern correctly predicted 36
(95%, 95% confidence interval [CI] = 82% to 99%) of
38 patients with prostate cancer, while 177 (78%, 95%
CI = 72% to 83%) of 228 patients were correctly
classified as having benign conditions.

Serum proteomic patterns for detection of prostate cancer.

J Natl Cancer Inst. 2002 Oct 16;94(20):1576-8.