A Project Report on

Understanding Blood Oxygen Equilibrium using

Combined Mathematical and Experimental Analysis

Submitted by

TRUPTI TANAYA SAHOO

(Univ. Regd. No.: 2001106071)

Under the Supervision of

DR. RANJAN KUMAR PRADHAN

(Head of the Department, Department of Biotechnology)

DEPARTMENT OF BIOTECHNOLOGY
ODISHA UNIVERSITY OF TECHNOLOGY AND RESEARCH
Techno Campus, Mahalaxmi Vihar, Ghatikia, Bhubaneswar-751029
Odisha, India
 ABSTRACT

This project is centered on the study of the biochemistry of blood oxygen equilibrium. It
involves a comprehensive review of various theoretical studies that have explained oxygen
saturation curves in different species. The project aims to implement models of saturation in
MATLAB and design an experiment to bridge the gaps in understanding the shape of the
oxygen curve in both healthy and diseased states. A thorough review was conducted on
research papers that focus on understanding the biochemistry of the oxyhaemoglobin
equilibrium. These papers detailed experiments carried out to determine the exact saturation of
haemoglobin with oxygen, and the implementation of this data using computational
approaches. The empirical equations obtained from these research papers were plotted using
MATLAB to predict the trend of the oxyhaemoglobin equilibrium curve as attempted by
researchers over the years. It was concluded that these equations do not represent the actual
mechanism for oxygen binding to haemoglobin. Consequently, a new approach was adopted
by studying a kinetic model of oxygen saturation. This model considers the actual biochemical
reactions taking place inside Red Blood Cells (RBCs) to provide an expression describing the
relationship between haemoglobin saturation and PO2. This relationship is examined over a
wide range of not only PO2 but also PCO2, pH, 2,3-DPG, and temperature. The experimental
data obtained from the research papers will be further used to analyze and validate this model
of oxyhaemoglobin saturation.

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 INTRODUCTION

Hemoglobin formation involves the binding of succinyl-CoA and glycine to form a pyrrole
molecule, which combines to form protoporphyrin IX. This then combines with iron to form a
heme molecule, which further combines with a globin to form a hemoglobin chain. Four such
chains form a hemoglobin molecule. Variations in these chains result in different types of
hemoglobin, with Hemoglobin A being the most common in adults. Each hemoglobin molecule
can transport four molecules of oxygen. Abnormalities in the chains can lead to conditions like
sickle cell anemia. The key feature of hemoglobin is its ability to bind with oxygen loosely and
reversibly, carrying it as molecular oxygen to the tissues.

Oxygen diffuses from the alveoli into the pulmonary blood due to the higher partial pressure
of oxygen (PO₂) in the alveoli. This diffusion rate is directly proportional to the gas's partial
pressure and solubility coefficient. In other body tissues, a higher PO₂ in the blood than in the
tissues causes oxygen to diffuse into the cells. Oxygen binds with hemoglobin when PO₂ is
high and is released when PO₂ is low, forming the basis for oxygen transport from the lungs to
the tissues.

The oxyhemoglobin dissociation curve graphically represents the relationship between

hemoglobin saturation and blood oxygen tension. It shows how hemoglobin acquires and
releases oxygen, with the percentage of hemoglobin bound with oxygen increasing as blood
PO₂ increases. The curve is non-linear due to the changing affinity of hemoglobin for oxygen
with varying PO₂. The first oxygen molecule binding to hemoglobin changes its shape,
enhancing its ability to bind additional oxygen molecules. The curve serves as a clinical
marker, assessing oxygen transport efficiency and adaptability to varying oxygen needs.

The oxygen-hemoglobin dissociation curve shifts to the right due to increased carbon dioxide
concentration, blood temperature, 2,3-diphosphoglycerate (DPG), and decreased pH. This
shift, known as the Bohr effect, enhances oxygen release in tissues and oxygenation in lungs.
Normal DPG levels slightly shift the curve to the right, but hypoxic conditions increase DPG,
further shifting the curve. This DPG mechanism aids in adapting to hypoxia, especially due to
poor tissue blood flow. During exercise, the curve shifts significantly to the right, delivering
extra oxygen to active muscles.

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 LITERATURE REVIEW

The oxygen-hemoglobin equilibrium curve (OHEC) curve has been a focal point for
researchers due to its multifaceted significance in clinical and basic research. It helps identify
hemoglobin abnormalities and oxygen transport deficiencies. Having a mathematical
description of the OHEC allows for meaningful comparisons among different individuals.
Although the widely used Hill model (1910) characterizes the OHEC, it is limited to a
saturation range of 20% to 98%. However, other models in the literature may offer a more
comprehensive description of the OHEC, with parameters of greater theoretical significance
than the Hill parameters P50 and n. Adair's stepwise hypothesis of 1925 paved the way for the
modern conception of the hemoglobin molecule. Adair proposed that four oxygen molecules
bind to a single Hb molecule sequentially, giving four equilibrium constants, one for each
sequential reaction. Margaria (1963) simplified the Adair model by equating the first three
equilibrium constants because they were of similar magnitude while the fourth was much
higher. Among the empirical models, the Kelman (1966) model extended the Adair equation
parameters, introducing greater flexibility. However, this extension led to unacceptable
parameter redundancy. Another empirical model, the Severinghaus (1979) model, relies on
arbitrary parameters but is distinct from polynomial ratios due to its derivation from the Hill
equation. Additionally, Easton (1979) employed actuarial mathematics to develop an equation
that fit the OHEC data well.

A list of existing models attempted by researchers to describe the oxyhaemoglobin equilibrium

curve is given below.

Table 1. Existing Models describing Oxyhaemoglobin Equilibrium Curve

Sl. Model Year Description
No.
1 Hill, A. V. 1910 He postulated an nth-order one-step kinetic hypothesis for O2
binding to Hb to derive the simplest model for standard ODC
involving only two parameters. Hill’s equation was found to give
good characterization of the data over the saturation range of 20
to 98%.
2 Adair, G. S. 1925 He postulated a more realistic four-step kinetic hypothesis
(known as the intermediate compound hypothesis) and derived a
more accurate formula for standard ODC involving four distinct
parameters. The model is not analytically invertible.
3 Margaria et al. 1963 They modified Adair’s equation by expressing the four Adair
constants in terms of two distinct parameters, one representing the
O2 affinity for first three heme sites and the other representing an

3
 increased affinity for fourth oxygenation. The model is not
analytically invertible.
4 Kelman G. R. 1966 He proposed an empirical formula for converting O2 tension into
its saturation; it is a little more complicated than Adair’s, using
seven distinct parameters. The model is not analytically invertible
and allows parameter redundancy.
5 Easton D. M. 1979 Easton proposed a new paradigm involving two parameters for
characterizing the standard ODC. His mathematical description
assumed that the formation of HbO2, and hence O2 saturation of
Hb, is exponentially related to the O2 partial pressure.
6 Severinghaus 1979 He developed a simple and accurate empirical formula for
J. W. standard ODC by modifying Hill’s equation. This model was
analytically invertible.
7 Singh et al. 1982 This paper is a study of the simultaneous diffusion of O2 and CO2,
in the presence of haemoglobin is made by considering the
intermediate compound hypothesis of Adair. The fractional
saturation s given by their equation, confirm the Haldane (effect
of the oxygen-haemoglobin reaction on carbon dioxide transport)
and Bohr effects.
8 Sharan et al. 1984 One step kinetics between oxygen and haemoglobin are shown to
be equivalent to Hill’s equation. The results computed from their
comparatively simpler equations based on physical laws, are in
good agreement with those obtained from Kelman’s empirical.
9 Siggaard- 1984 They developed an empirical formula for standard ODC which
Anderson et fits very well to the model and data of Severinghaus. However,
al. their equation is not analytically invertible and requires an
iterative numerical method for inversion.
10 Buerk & 1986 Buerk and Bridges modified Easton’s formula and developed an
Bridges algorithm for computing the nonstandard ODCs with varying pH,
CO2 partial pressure, [DPG]/[Hb] concentration ratio, and
temperature.

Along with theoretical and model-based papers, experimental papers were also reviewed to
study the different approaches undertaken to determine oxygen saturation in vitro. The methods
and techniques used are briefly summarized below.

Table 2. List of previous experimental works on Oxyhaemoglobin Equilibrium Curve

Sl. Author Year Description

No.
1 Naeraa et al. 1963 In this paper, venous blood was drawn from 2 healthy nonsmokers
and heparinized. Seven blood samples with different, artificially
induced metabolic acid-base abnormalities were equilibrated with
8 gas mixtures of known composition at 38°C. Oxygen saturation
and pH were then determined. The gas mixtures were selected to
give two levels of oxygen tension with carbon dioxide tensions of
approximately 20, 45, 60 and 80 mmHg.
2 Hedley-Whyte 1964 In this paper, tonometry of 100 paired samples of blood was
& Laver carried out and PO2 was measured before and after warming to
38°C in a sealed syringe at standard pH and PCO2. Oxygen tensions
were measured with two Beckman oxygen macroelectrodes. This
paper states that, a sample of blood quickly warmed, or cooled, in

4
 a sealed syringe will exhibit variations in its O2 tension while the
O2 content of the sample will remain virtually constant (Henry's
law).
3 Hlastala et al. 1977 In this paper, heparinized blood was obtained from healthy non-
smoking individuals (N = 93) by venipuncture. Experiments were
performed on fresh whole blood and on blood depleted of DPG;
In this method, blood is first deoxygenated by tonometry with a
N2-CO2 gas mixture. A continuous curve is then inscribed at
constant PCO2 from polarographic measurements as oxygen is
taken up by the deoxygenated blood from a reservoir of pure
oxygen; When whole blood is cooled from 37 to 23°C, the affinity
of hemoglobin for oxygen more than doubles.
4 Winslow et al. 1977 In this paper, oxygen equilibrium curves of fresh, normal human
blood have been measured by new methods (using oxygen
electrode) which allow the control of pH, PCO2, and 2,3-
diphosphoglycerate and which yield higher accuracy at the
extremes of saturation than was possible previously.
5 Reeves, R. B. 1980 In this paper, the oxygen affinity of human blood was measured
with the micro blood film technique over six-degree temperature
intervals from 13 to 43°C. Results are expressed both as standard
curves at pH 7.4 and for conditions of constant carbon dioxide
content.
6 Winslow et al. 1983 In this paper, heparinized blood from two healthy male
nonsmoking donors was obtained by venipuncture.
Concentrations of Hb, carboxyhemoglobin, and methemoglobin
in whole blood were measured with the use of a Microblood
analyzer; They analyzed 56 O2 equilibrium curves of fresh human
blood, each from 0 to 150 Torr PO2. The data were collected over
ranges of values for the 2,3-diphosphoglyceric acid-to-
hemoglobin concentration ratio [DPG]/[Hb] of 0.2-2.7, for pH of
7.0-7.8, and for PCO2 of 7- 70 Torr.
7 Barnhart, 1984 In this paper, a non-spectrophotometric method is described for
M.C. measurement of the O2 dissociation curve and O2 capacity of a
50-ul sample of fluid. PO2 is recorded by a microprocessor as the
sample is oxygenated and then deoxygenated by exposure to
isocapnic gas mixtures across a gas-permeable membrane; For all
values pH = 7.4 and T = 37°C. P10-P90, PO2 at 10-90% saturation
of hemoglobin
8 Zwart et al. 1984 In this paper, heparinized venous blood was obtained from
healthy nonsmoking human donors (N = 6, 3 women and 3 men).
Five complete ODC's were recorded from the blood of each donor
at five different temperatures (22, 27, 32, 37, and 42°C), with pH
= 7.40, PC02 = 40 Torr, and a normal 2,3-DPG content; The PO2
of the blood is measured with a fast-responding Clark-type
electrode. The SO2 of the blood is measured by means of a fiber-
optic reflection oximeter.
9 Winslow et al. 1978 In this paper, oxygen equilibrium curves of 48 healthy adult
subjects have been measured by the H2O2 method. The pH (7.4),
PCO2 (0.3 Torr), and HCO3- are constant during oxygenation. The
system for control of the experiment and data collection and
processing has been automated using a microprocessor.

5
 METHODOLOGY
Model-based Analysis of Oxyhaemoglobin dissociation curve

The various empirical equations along with their parameters obtained from the research papers
reviewed using PubMed are listed below. These are further used to create plots in MATLAB
and study the trend of oxyhaemoglobin equilibrium curve over the years.

Table 3. Equations and Parameters of Existing Models describing Oxyhaemoglobin

Equilibrium Curve
Sl. Model Equation Parameter Values
1 Hill, A. n = 2.7
V. P50 = 26.8 mmHg

2 Adair, a1 = 0.0218
G. S. a2 = 0.000912
a3 = 0.0000038
a4 = 0.00000247
3 Margaria K = 0.0125
et al. m = 154

4 Kelman a1 = -8532.289
G. R. a2 = 2121.401
a3 = -67.073989
a4 = 935960.87
a5 = -31346.258
a6 = 2396.1674
a7 = -67.104406
5 Easton Ym= 0.958
D. M. Ya = -0.0177
K = 0.0691

6 Severing a = 23400
haus J. b = 150
W.

7 Singh et K1 = 0.0263347
al. K2 = 0.03324
K3 = 0.0011424
K4 = 2.6309
m = 0.00002
y6 = 40