Visible and Near Infrared Absorption Spectra of Human and Animal Haemoglobin determination and application

Williem.G. Zijlstra, Anneke Buursma, O.W. van Assendelft, Zijlstra, Buursma, van Assendelft

• Publisher: CRC Press
• Year: 2021
• ISBN:
  9781000083019
• Edition: 1
• Topic: Physical Sciences
• Subtopic: Chemistry
• Index: Chemistry

The bright colour of haemoglobin has, from the very beginning, played a significant role in both the investigation of this compound as well as in the study of blood oxygen transport. Numerous optical methods have been developed for measuring haemoglobin concentration, oxygen saturation, and the principal dyshaemoglobins in vitro as well as in vivo.

CHAPTER 1

INTRODUCTION

The light absorbing properties of haemoglobin have, from the very beginning, played a substantial part in the chemical investigation of this compound, as well as in the study of its role in oxygen transport by the blood. Even before 1862, when Hoppe-Seyler isolated the blood pigment and called it haemoglobin, he had concluded from changes in the colour of blood under various circumstances that when carbon monoxide is absorbed by the blood it prevents the blood pigment from binding oxygen, thus impeding oxygen transport [16].

This introductory chapter gives a short survey of the development of spectrophotometry of haemoglobin and its many biological and medical applications in the study of oxygen transport by the blood.

VISUAL SPECTROPHOTOMETRY

Soon after the introduction of spectroscopy in chemical analysis by Bunsen and Kirchhoff [348], Felix Hoppe-Seyler [173] in Germany and George Stokes [377] in England almost simultaneously observed the light absorption bands of haemoglobin in the visible part of the spectrum and the changes which occur when oxygen is added to or removed from the solution. Karl Vierordt was the first (1876) to study not only the spectral changes of haemoglobin in solution, but also in a transilluminated finger. When the circulation through the finger was stopped, the two oxyhaemoglobin bands disappeared and the band of deoxygenated haemoglobin appeared [403]. Although this experiment may be regarded as the beginning of oximetry, it was followed up only a half century later when more suitable equipment became available.

Of more immediate consequence was that Vierordt had also shown the suitability of spectral analysis for biochemical applications [402]. From that time the spectroscopic study of haemoglobin and its derivatives developed rapidly. In 1878 Soret [366, 367] described the strong absorption bands of haemoglobin in the near ultraviolet, and Gustav Hüfner embarked on the development of a spectrophotometer; this resulted in 1889 in an instrument with which quite accurate measurements could be made [178].

In Hüfner’s instrument, the light of a bright kerosene lamp passed through a cuvette of which the lower part was filled with a glass body (Schulzescher Körper); the upper part of the cuvette was filled with the solution of which the absorbance was to be measured. The part of the light which had traversed the glass body, then passed through a nicol prism and thus became polarised. After the two beams had passed through a dispersion prism and slits to isolate small wavebands, they reached a lens system through which both could be seen side by side. In the polarised light beam — the one which had not passed the solution to be measured — was a second nicol prism, which could be rotated in order to attenuate it until its intensity matched that of the beam which had traversed the solution. From the rotation of the second nicol prism, which could be read from a scale, the absorbance of the solution was calculated.

In his well-known investigation of the oxygen binding capacity of haemoglobin [180], Hüfner not only used this spectrophotometer for the determination of the total haemoglobin concentration of the solutions of which he measured the carbon monoxide capacity, but also for checking the purity of these solutions. To this end he used the central part of the region between the α- and the β-peak of oxyhaemoglobin (554–565 nm) and a region around the β-peak (531.5–542.5 nm). Table 1.1 shows some ratios of absorptivities of oxyhaemoglobin, deoxyhaemoglobin and carboxyhaemoglobin in these regions in comparison with the same quantities calculated on the basis of recent measurements [467]. The fair agreement of Hüfner’s results with modern data clearly demonstrates that, at the time, reasonably accurate spectrophotometric measurements already could be made.

Table 1.1
Absorption ratios of HHb, O₂Hb and COHb in two spectral regions according to Hüfner and as calculated from recent measurements

Ratio	Hüfner (1894)	Recent (1991)
${\epsilon }_{\text{O}}{}_{{}_2\text{Hb}}\left(2\right)/{\epsilon }_{\text{O}}{}_{{}_2\text{Hb}}\left(1\right)$	1.581	1.448
εHHb(2)/εHHb(1)	0.761	0.745
${\epsilon }_{\text{O}}{}_{{}_2\text{Hb}}\left(1\right)$/εHHb(1)	0.655	0.705
εCOHb(2)/εCOHb(1)	1.095	1.122
εCOHb(2)/${\epsilon }_{\text{O}}{}_{{}_2\text{Hb}}\left(2\right)$	1.037	1.044

ε(1) = absorptivity in region 1 (554–565 nm); ε(2) = absorptivity in region 2 (531.5–542.5 nm); HHb = deoxyhaemoglobin; O₂Hb = oxyhaemoglobin; COHb = carboxyhaemoglobin. Hüfner’s ratios are from ref. [180]; the recent ratios have been calculated from data given in Chapter 8.

Considerable progress in the spectrophotometric study of haemoglobin was made by Drabkin and Austin, who published an admirable series of Spectrophotometric studies between 1932 and 1946 [94, 95, 96, 97]. Already the first paper of the series [95] gives data on several haemoglobin derivatives (oxyhemoglobin, carboxyhaemoglobin, haemiglobin, haemiglobincyanide), prepared from haemolysed human, dog and rabbit blood, and measured with a König–Martens type spectrophotometer [93]. The total haemoglobin concentration was determined on the basis of the oxygen binding capacity of the solutions. After the absorptivities of haemiglobincyanide at 551, 545 and 540 nm had been determined (ε_HiCN = 11.0, 11.5, 11.5 L · mmol⁻¹ · cm⁻¹, respectively), the total haemoglobin concentration in subsequent studies was determined after converting all haemoglobin in the solution into haemiglobincyanide.

In the second study haemoglobin solutions were produced from washed erythrocytes instead of from haemolysed whole blood, and nitric oxide haemoglobin and sulfhaemoglobin were added to the haemoglobin derivatives studied [96]. An important innovation was the introduction of a flow-through cuvette with a lightpath length of only 0.007 cm [97]. This allowed measurements at a total haemoglobin concentration as is present in whole blood and thus enabled spectrophotometric measurements of the oxygen saturation to be made. Absorptivities of very concentrated solutions of horse haemoglobin were determined, and it was demonstrated that Lambert–Beer’s law is valid for haemoglobin solutions over a concentration range from 0.0001 to 1, where 1 corresponded with a concentration of 25 mmol/L. In a later study, accurate measurements were even made at concentrations around 38 mmol/L. This paper [94] reports mainly crystallographic results, but the ensuing pure solutions of various haemoglobin derivatives of human, horse and dog blood were used for new determinations of absorptivities on the basis of ε_HiCN(540) = 11.5 L · mmol⁻¹ · cm⁻¹. To demonstrate the accuracy which was attained in this advanced stage of visual spectrophotometry, the absorptivities found for human oxyhaemoglobin and deoxyhaemoglobin in solutions of crystallised preparations have been compared with data from Chapter 8, in Table 1.2.

Table 1.2
Absorptivities of oxyhaemoglobin and deoxyhaemoglobin from Drabkin [94] in comparison with recent data

	λ (nm)	ε (Drabkin)	ε (Table 8.1)
O2Hb	578	15.38	15.36
	562	8.47	8.77
	542	14.68	14.52
HHb	555	13.57	13.35

Absorptivities expressed in L·mmol⁻¹ ·cm⁻¹. Drabkin’s values of oxyhaemoglobin have been measured at a total haemoglobin concentration of about 35 mmol/L with a lightpath length of 0.007 cm; the measurements of deoxyhaemoglobin have been made at ct_Hb ≈ 0.1 mmol/L with a lightpath length of 1.0 cm.

PHOTOELECTRIC SPECTROPHOTOMETRY

Horecker was one of the first to use a photoelectric spectrophotometer to record absorption spectra of haemoglobin derivatives and to extend the measurements to 1000 nm, the near infrared spectral region [174]. He found that the absorptivity of oxyhaemoglobin increased again beyond 700 nm, whereas that of carboxyhaemoglobin decreased to near zero. On the basis of these findings Horecker and Brackett [175] developed a spectrophotometric method for the determination of the carboxyhaemoglobin fraction in blood. Combining the method with the principle of Evelyn and Malloy [114] — measuring the absorption change on the addition of a trace of potassium cyanide to the blood sample — they even succeeded in devising a method for measuring the fractions of carboxyhaemoglobin and methaemoglobin in a single blood sample.

The photoelectric spectrophotometer described by Cary and Beckman in 1941 [60], which soon became commercially available, brought accurate and precise spectrophotometric measurements within the reach of the average b...

Table of contents

• Cover
• Half Title
• Title Page
• Copyright Page
• Dedication
• Table of Contents
• Foreword
• Preface
• Chapter 1: Introduction
• Chapter 2: Definitions and terminology
• Chapter 3: Spectrophotometry
• Chapter 4: Total haemoglobin concentration
• Chapter 5: Absorptivity at 540 nm of haemiglobincyanide
• Chapter 6: Preparation of haemoglobin derivatives
• Chapter 7: Determination of absorption spectra
• Chapter 8: Absorption spectra of human HbA and HbF
• Chapter 9: Absorption spectra of dog haemoglobin
• Chapter 10: Absorption spectra of rat haemoglobin
• Chapter 11: Absorption spectra of bovine haemoglobin
• Chapter 12: Absorption spectra of pig haemoglobin
• Chapter 13: Absorption spectra of horse haemoglobin
• Chapter 14: Absorption spectra of sheep haemoglobin
• Chapter 15: Comments on the determination of absorption spectra of haemoglobin
• Chapter 16: Haemoglobinometry
• Chapter 17: Multicomponent analysis of haemoglobin derivatives
• Chapter 18: Oximetry and related techniques
• Chapter 19: The oxygen binding capacity of human haemoglobin
• Chapter 20: The oxygen affinity of human haemoglobin
• References
• Abbreviations and symbols
• Index