The Analysis of Biological Materials. The Proceedings of a Conference Held in Pretoria, South Africa, October 1977, by the Spectroscopic Society of South Africa

102 Pages·1979·2.41 MB·English

by  L. R. P. Butler (Eds.)

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THE ANALYSIS
OF BIOLOGICAL MATERIALS
The Proceedings of a Conference
held in Pretoria, South Africa
October 1977
by
The Spectroscopic Society of South Africa
Editor
L. R. P. Butler
Applied Spectroscopy Division
National Physical Research Laboratory
Council for Scientific and Industrial Research
PERGAMON PRESS
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First edition 1979
British Library Cataloguing in Publication Data
Symposium on the Analysis of Biological
Material, Pretoria, 1977
The analysis of biological materials.
1. Biological chemistry - Congresses
2. Chemistry, Analytic - Congresses
I. Title II. Butler, LRΡ IJI. Spectroscopic
Society of South Africa
574.Γ9285 QD415.A1 78-41016
ISBN 0-08-022853-4
In order to make this volume available as economically
and as rapidly as possible the typescript has been
reproduced in its original form. This method unfor-
tunately has its typographical limitations but it is hoped
that they in no way distract the reader.
Printed and bound at William Clowes & Sons Limited
Beccles and London
PREFACE
The rapid advances that have been made in the analytical sciences over
the last 30 years have enabled scientists in many allied fields to
renew their approach to a variety of problems.
There is little doubt that highly sensitive analytical techniques such
as atomic absorption, neutron activation and gas chromatography have
greatly assisted in vastly broadening the horizons of scientists in the
medical, biological, agricultural and environmental fields. However,
while the ability to measure very low concentrations of these elements
and compounds quickly and easily may be very valuable, many of these
scientists have now become aware of the many problems associated with
accurate and precise analysis and especially with the interpretation of
results.
Indeed, the application of many of the techniques used today has
resulted in the development of a special form of analytical expertise.
Workers in the applied fields do not always have the time (or inclin-
ation) to develop this expertise and the result has been the evolution
of analysts who are expert in the analytical technique, but not in
interpreting and applying the results of the analysis. It is imperative
that the analytical experts and the "users" should work together.
Recognising this, the Spectroscopic Society organises conferences where
new techniques are described and where the "users" are invited to
discuss their problems.
The Symposium on the Analysis of Biological Material is an example of
such a conference. The large number of registrants representing the
medical profession, municipal chemists, agriculturalists, veterinarian
and environmental scientists is a good indication of the interest that
exists in biological analysis today.
These papers represent the main lectures that were presented. It is
hoped that collectively they may be of use to those people concerned
with the biological effects of elements and compounds as well as with
their determination in biological specimens. While some of the
instrumental methods are not available to all, it is as well to be
aware of their existence and their application potential.
I am greatly indebted to Helen Watling, Daphne de Villiers and
Jean Harris for assistance with the editing and the preparation of the
manuscripts as well as to G-isela Domel for helping with the conference
organisation.
L R Ρ Butler
vii
OPENING ADDRESS
ANALYSIS OF BIOLOGICAL MATERIAL:
THE APPLICATION IN MEDICAL
DIAGNOSIS AND TREATMENT
Prof. A. J. Brink
President: South African Medical Research Council P. O. Box 70,
Tygerberg 7505, South Africa
HISTORICAL PERSPECTIVE
During the 19th century the study of biological systems was character-
ised by a mainly descriptive approach. Early physiologists such as
Claude Bernard and Johannes Muller in Germany made significant
contributions to the establishment of the experimental approach in
biology.
Progress in the early 20th century was directed at the more detailed
study of constituents of biological species and their mechanisms. This
was followed by research that crossed the traditional boundaries of the
established disciplines, thus creating hybrid disciplines such as bio-
chemistry and biophysics. The early part of the century saw the
discovery in physics of totally new phenomena (X-rays, radioactivity,
etc.) and the application of knowledge to studying the structure of
matter (atomic theory, the discovery of isotopes etc.). This required
a reappraisal of the fundamental laws of physics as well as of some
metaphysical assumptions, e.g. the theory of relativity, the quantum
theory, etc.
It is of interest that after the discovery of X-rays in 1895 by Rontgen,
they were applied to medicine within three months - an unusually short
period for the application of a scientific discovery - even by modern
standards.
Chemical methods of analysis were necessary for much of this new work
in physics. The new physical theories in turn were sufficiently
advanced to provide effective explanations for a wide variety of
chemical phenomena.
In the Life Sciences, chemical and physical methods brought the dis-
covery and explanation of the structures and workings of subtle
agencies such as vitamins and hormones, as well as an insight into the
complex cycle of chemical transformations that are now known to be
some of the characteristics of life. It is from this sort of background
that biologists have become increasingly involved with physical and
chemical principles and vice versa. This has led to an increase in
multi-disciplinary research activities.
1
2 A. J. Brink
PHYSICS AND CHEMISTRY IN MEDIO 1KB
It is probably true that the biological phenomena that are best under-
stood are those with a recognisable physical or chemical basis. For
instance, the function of the heart as a pump is appreciated and
straight forward mechanical, electrical and hydraulic principles can
be applied for accurate diagnosis by certain invasive techniques and
by other non-invasive techniques. Based on our knowledge of cardiac
output, obstructive lesions and electrical derangements can be treated.
With a knowledge of relatively elementary chemistry in relation to pH
and the laws of gas exchange, and with the availability of buffers,
different causes or states of metabolic upsets in the body resulting
in acidosis or alkalosis can be differentiated, diagnosed and
corrected, thus saving human lives.
It is therefore clear that in the investigation of biological phenomena,
physics and chemistry play, and will continue to play, a most important
role, not only in the understanding of basic and principle processes,
but also in the development of techniques of investigation, as well as
in the development of instrumentation to serve this purpose.
A Word of Caution
Medical and other biological scientists must beware of being
enticed into and trapped in an over-indulgence in instrumentation.
This applies to investigative procedures and also to treatment. There
is practically no limit to the innovations and inventions that can be
made with regard to instruments. There is also no limit to their cost.
Valuable as they are for teaching, diagnosis or treatment, there is a
great need to be realistic with regard to the extent to which
instrumentation makes a valid and sound contribution. So often in
one's clinical experience the finding of a single involved technique
as applied to a patient has led to a great deal of misery and un-
certainty, until the clinician has returned to basic clinical findings
and their interpretation. There are numerous examples in electro-
cardiography, radiography, isotope scanning and biochemical techniques,
where undue emphasis on the laboratory findings without proper perspec-
tive has resulted in a misleading interpretation and often great mental
stress. This is not said lightly. Recently the Medical Research
Council (MRC) provided financial support for a physiologist to under-
take a systems analysis of calcium ion homeostasis in the body. This
had become necessary because so much information has been obtained
from basic molecular research that this wealth of information cannot
be analysed without the aid of a computer!
There is no wish at all to deny the immense value of the contributions
made by instruments and analysts, be they medical or non-medical men.
However, perspective and am insight should be maintained into the real
demands of the problem to which one's efforts are directed, be they
medical problems or those from some other discipline.
The Analyst
At this point it is of interest to look at the sort of people who are
doing analytical work. In the past, analysts were analytical chemists
Analysis in medical diagnosis and treatment 3
working within the field of inorganic chemistry. Today, the analyst
can come from many walks of life. He must know a good deal about
subjects such as chemistry, physics, statistics and electronics and
as much as possible about the material he is analysing. It is there-
fore not surprising that one often finds the person who is concerned
with the analytical results becoming the analyst. It is not uncommon
to find a biologist, veterinarian, or medical man who is also a very
capable analyst of the types of samples with which he is concerned.
A FEW APPLICATIONS OF BIOLOGICAL TISSUES ANALYSIS
IN CURRENT MEDICAL RESEARCH
The analysis of biological materials has made very significant
contributions to our technologically orientated society. I should
like to mention some current fields of medical research that will
undoubtedly ensure that this trend is maintained.
Early Cancer Diagnosis
Medical research stands on the verge of being able to identify all
forms of cancer sufficiently early to really have a significant
beneficial effect on the cancer prognosis.
Carcino-embryonic antigen (CEA) which is often associated with intesti-
nal adenocarcinomas was one of the first of these tumour associated
antigens (TAA) to have an impact on the clinical field. It is quite
easy to determine in small quantities and was initially held to be
specific for carcinoma of the large bowel, since small bowel tumours
are rare. Probably for these two reasons its determination has
become a very popular investigation, particularly in view of the
increasing importance of this type of tumour in Europeans and the very
substantial difficulties in detecting carcinomas of the large bowel
in the early stages by other means.
Experience has not sustained early hopes. Far from being organ
specific, it has been found that a variety of other tumours could also
elaborate CEA. More disconcerting was the fact that a high percentage
of carcinomas of the large bowel did not elaborate CEA and, worse still,
raised blood levels were found in non-malignant bowel conditions. A
test which gives false negatives and false positives is really no test
at all. Yet medical practice is not an "all or nothing" pursuit and
with our well-inured disposition to statistics, the measurement of CEA
enjoys a fair probability-indication status amongst the TAA's employed
in clinical practice. But it is perhaps as well to emphasise that an
organ or tumour specific TAA has not yet been discovered and one feels
almost sure will not be discovered. Put differently, as in most other
spheres of medicine, the "one-stop" diagnostic type of service is
unlikely to eventuate. At the present time the major benefit of a CEA-
secreting tumour is that post-operative follow-up is facilitated in
that it will give an early warning of secondary tumours.
It is only during the past five years that it has become possible to
measure ferritin in the blood and then the primary interest was of
course in relation to iron stores. However, apart from assay, a
tremendous amount of work has been done on ferritin structure. The
current view is that the large number of isoferritins which have been
4 A. J. Brink
characterised really represent no more than various combinations of
two, or possibly three, sub-units. In very general terms, liver and
spleen ferritins are composed of the more basic isoferritins; other
organ ferritins are generally acidic isoferritins. Normally the
small amount of circulating ferritin constitutes the most basic of the
isoferritins and is probably natural apoferritin derived from the
reticuloendothelial system. As haematological investigations on
ferritin behaviour in anaemia were extended, it was soon noticed that
among other conditions seen, serum ferritin levels were raised in a
variety of malignancies. In fact, in just about any malignant
condition, serum ferritin, as measured by the existing techniques,
tended to be raised consistently. Further investigation has revealed
that tumour-derived isoferritins are of the acidic type. These types
are not normally present in serum in measurable quantities. The
methodologies to measure the acidic isoferritins are only now being
developed, but the preliminary published results suggest strongly that
these isoferritins are probably going to be the best general bio-
chemical marker of malignancy that we have.
A South African biochemist has recently greatly improved the diagnosis
of lung cancer by determining the presence of a tumour-specific
protease which differs from the proteases found in aspirates from
patients with other lung diseases. The diagnosis of liver cancer by
the determination of alpha feto protein (AFP) levels has been taken a
step further at the MC's Liver Research Group (University of Cape Town)
where a very sensitive radio-i mmuno-assay technique for AFP determi-
nation has been developed. These techniques, although not yet ideal,
offer increasingly reliable results.
The Detection of Pretransplantation Sensitisation
The Organ Transplantation Research Group of the MRC has evaluated and
refined the lymphocyte mediated cytotoxicity (LMC) assay to detect
pretransplant sensitisation. These tests enable the physician to
detect those patients who show a form of graft specific presensiti-
sation which is associated with an unnacceptably high incidence of
rejection and graft loss.
Chemotherapeutics
Earlier techniques of drug design were very much hit or miss affairs.
Today's understanding of structure-activity relationships and bio-
availability have placed drug design on a far more rational basis.
The determination of drugs at serum concentration levels is not only
important from a toxicological point of view, but also from the point
of view of management chemotherapeutics; for example, in the treatment
of epileptics where effective anti-convulsive levels should be
determined for each patient. A recent survey in London has shown that
the measurement of drug levels in epileptics can eliminate the
necessity of using more than one drug. This is relevant, as a
European survey in 1975 showed that the average number of drugs per
epileptic patient was then 3.2, resulting, literally, in the patient
suffering almost as much from the drugs as from his fits.
Analysis in medical diagnosis and treatment 5
The Role of Trace Metals
The effects of metals on human health have been known or guessed at in
medicine for many years. With the development of instrumental methods
which make analysis easy and reliable, far more knowledge about their
quantitative effects has emerged.
While some elements such as lead, mercury and cadmium have been found
to have deleterious effects even at exceptionally low concentrations,
other elements such as molybdenum, chromium and selenium have been
demonstrated in one way or another to be beneficial to human and/or
animal health. The limits of concentration within which some of these
are not beneficial or toxic are often very narrow.
Another element which is known to be toxic is lead. Lead may be stated
to be one of the most severe of metallic poisons simply because man
and beast are exposed to it in so many ways, for example, lead in petrol
which is released to the atmosphere in car exhaust fumes, in paint,
food and beverages.
One can continue to talk at length about the many elements and
combinations of elements that are either dangerous or essential to man
and beast. However, before one can identify and measure the
quantities of these elements and so determine their effects, one must
have the scientific and technical know-how. It is therefore not
surprising that the analyst has emerged as an important contributor to
the biological and material sciences.
OPPORTUNITIES FOR FUTURE DEVELOPMENTS
The opportunities for the biological analyst are legion, particularly
in the light of today1s tremendous industrial and technological
advances.
Space Shuttle
NASA's Life Science Programme in space, by means of the Space Shuttle,
should be lifting off soon and this offers new and challenging
opportunities to biologists. For example, in previous space flights it
has been found that the drug urokinase (a very expensive drug which is
used in the treatment of blood-clotting disorders) is produced much
more efficiently by kidney cells under space conditions. The possi-
bility of industry in space can thus not be excluded.
Radio-Active Labelling
Radio-active labelling techniques have contributed greatly to the
determination of minute amounts of biological substances. This tech-
nique is a valuable tool in understanding the functioning of brain
cells; American scientists have shown for the first time that it is
possible to identify from photographs, brain cells with different
physiological functions.
6 A. J. Brink
Environmental Exposure
The dangers of pollution need to be ascertained accurately. Such
aspects as the influence of trace or ultra-trace metal concentrations
on man and his health should receive closer attention. In some cases
little is known about the actual metabolic pathways of a metal. Many
metals are not very toxic, but what effect do they have in the long
run? In what form must metals and compounds be when ingested, to be
absorbed and thus constitute a danger to health? The answers to
questions such as these will assist us to gauge the dangers and danger
levels of many of the compounds which enter our environment.
For your interest, many of these aspects are at present receiving
attention at the MRC's National Research Institute for Occupational
Diseases in Johannesburg.
PROGRESS IN OTHER FIELDS
Time does not permit me to dwell on other extensive fields and newer
developments over the past 10-15 years, which have clarified some
problems and given new impetus to interest in others. One may refer
to the enormous strides in endocrinology. The existence of endocrines
and their fascinating and wide ranging effects on metabolism and
behaviour, have been part and parcel of fundamental physiological
understanding for a large part of this century. But the application
of this knowledge remained conjectural, inferential and speculative
in the clinical situation for the simple reason that appropriate
chemical procedures were lacking to measure the relatively small
amounts of these substances elaborated by the endocrine glands.
The situation changed however, from the time that human chorionic
gonadotrophin, a peptide produced by the human placenta in early
pregnancy, could be tested for by a simple and reliable biological
procedure which made use of our lowly frog, the "platanna"
(Xenopus laevis), as a test animal. This test has been supplanted by
the ubiquitous immunoassay technique and the refined immunoassays based
on the beta sub-unit. These enable the early diagnosis of pregnancy
(9 days after ovulation) and the follow-up studies of choriocarcinoma
and hydatitiform mole during treatment.
Blood assays of one group of hormones, the iodinated thyroid hormones,
were a further advance in the clinical situation. Ingenious clinical
methodologies led to a wealth of valuable and informative clinical
information during the 1950's. Studies of urine yielded data on the
steroid hormones, sex hormones, gluco-corticoids, mineralo-corticoids
and the clinically less significant C-19 steroids, keto- or
oxosteroids.
Then the new era was upon us. Yalow and Berson published their radio-
active immunoassay (RIA) for insulin in 1961 and to them we owe this
term. Since then a large number of substances which previously eluded
detection in blood came within the ambit of measurement and hence the
clinical purview. The entire field of endocrinology advanced by leaps
and bounds. It needs little imagination to comprehend that here we
now have not only a diagnostic tool, but a preclinical diagnostic
tool par excellence.