PROGRESS IN MEDICINAL CHEMISTRY

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@ North-Holland Publishing Company - 1976

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North-Holland ISBN for the series: 0 7204 7400 0

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Preface
There are five reviews in this volume and a large part of the book is
devoted to clinical enzymology. This is extensively reviewed by Dr D. M.
Goldberg (Chapter 1) who considers in detail the clinical applications of
enzyme determinations particularly in the field of phosphatases, pepti-
dases, aminotransferases and phosphokinases. This important field of
medical science is one which has much advanced in recent years and
diagnosis of disease can now often be indicated by abnormal levels of
serum enzymes.
The second review by Dr P. F. L. Boreham and Dr I. G. Wright covers
pharmacologically active substances released in parasitic infections.
Much fresh information in this field has been collected in recent years and
the chapter indicates clearly where more detailed work is required.
When a 1,2,3-triazine ring is fused to a benzenoid or aromatic
heterocyclic ring, the resulting compounds have been found to exhibit a
wide variety of biological activity. Such compounds potentially behave as
diazonium compounds and as shown by Dr M. F. G. Stevens (Chapter 3),
are thus capable of reacting with a variety of biological materials.
In Chapter 4, Drs Russell and Hopwood review the use of glutaral-
dehyde as a sterilising agent and as a tissue fixative in microscopy. This
interesting compound is now being used extensively in both the chemical
and biological fields.
C-Nucleosides are fewer and less well known than their N-
counterparts, but they also have some interesting pharmacological and
antimicrobial properties; these are surveyed by Drs Daves and Cheng
(Chapter 5 ) .
Finally, we wish to thank our authors for their reviews, the owners of
copyright of diagrams for permission to reproduce, and the publishers for
their co-operation.

November 1975 G. P. Ellis

G. B. West

V
This Page Intentionally Left Blank
Contents
1. Clinical Enzymology 1
David M. Goldberg, M.D., B.Sc., Ph.D., M.R.C.Path.,
F.R.I.C.
Department of Chemical Pathology, The University,
Shefield, England

2. The Release of Pharmacologically Active Substances in

Parasitic Infections 159
P. F. L. Boreham, B.Pharm., Ph.D., F.P.S.
Department of Zoology and Applied Entomology, Imperial
College of Science and Technology, Prince Consort Road,
London SW7 2AZ, England
I. G. Wright, B.V.Sc., Ph.D.
CSIRO, Division of Animal Health, Long Pocket
Laboratories, Private Bag No. 3, P.O., Indooroopilly,
Queensland, 4068, Australia

3. The Medicinal Chemistry of 1,2,3-Triazines 205

Malcolm F.G. Stevens, B.Pharm., Ph.D.
Department of Pharmacy, University of Aston in
Birmingham, Birmingham B4 7ET, England

4. The Biological Uses and Importance of Glutaraldehyde 27 1

A. D. Russell, B.Pharm., Ph.D., D.Sc., M.P.S., M.R.C.Path.
Welsh School of Pharmacy, University of Wales Institute of
Science and Technology, Cardiff CF1 3NU, Wales
D. Hopwood, B.Sc., M.D., Ph.D., M.R.C.Path.
Department of Pathology, Ninewells Hospital and Medical
School, Dundee, Scotland

5. The Chemistry and Biochemistry of C-Nucleosides 303

G. Doyle Daves, Jr., Ph.D.
Oregon Graduate Center, Beaverton, Oregon 97005, U.S.A.
C. C. Cheng, Ph.D.
Midwest Research Institute, Kansas City, Missouri 641 10,
U.S.A.

Index 35 1

vii
This Page Intentionally Left Blank
Contents of earlier volumes

VOLUME 7
1 SOME RECENTLY INTRODUCED DRUGS-A. P. Launchbury
2 T H E BIOCHEMICAL BASIS FOR T H E DRUG ACTIONS OF PURINES-John
H. Montgomery
3 THE CHEMISTRY O F GUANIDINES AND THEIR ACTIONS AT ADRENER-
GIC NERVE ENDINGS-G. J. Durant, A. M. Roe and A. L. Green
4 MEDICINAL CHEMISTRY FOR T H E NEXT DECADE-W. S. Peart
5 ANALGESICS AND THEIR ANTAGONISTS: RECENT DEVELOPMENTS-
A. F. Casy
6 SOME PYRIMIDINES O F BIOLOGICAL AND MEDICINAL INTEREST-
Part 11-C. C. Cheng and Barbara Roth

VOLUME 8
1 ORGANOPHOSPHOROUS PESTICIDES: PHARMACOLOGY-Ian L. Natoff
2 T H E MODE O F ACTION OF NOVOBIOCIN-A. Morris and A. D. Russell
3 SOME PYRIMIDINES O F BIOLOGICAL AND MEDICINAL INTEREST-
Part 111-C. C. Cheng and Barbara Roth
4 ANTIVIRAL AGENTS-D. L. Swallow
5 ANTIFERTILITY AGENTS-V. Petrow
6 RECENT ADVANCES IN T H E CHEMOTHERAPY O F MALARIA-R. M. Pinder
7 THE PROSTAGLANDINS-M. P. L. Caton

VOLUME 9
1 NATURALLY-OCCURRING ANTITUMOUR AGENTS-K. Jewers, A. H.
Machanda and Mrs. H. M. Rose
2 CHROMONE-2- AND -3-CARBOXYLIC ACIDS AND THEIR DERIVATIVES-
G. P. Ellis and G. Barker
3 4-OXOPYRANOAZOLES AND 4-OXOPYRANOAZINES-Misbahul Ain Khan
4 ISOTOPE TECHNIQUES IN T H E STUDY O F DRUG METABOLISM-
Y. Kobayashi and D. V. Maudsley
5 THE PHARMACOTHERAPY O F PARKINSONISM-R. M. Pinder
6ADRENOCHROME AND RELATED COMPOUNDS-R. A. Heacock and W. S.
Powell

VOLUME 10
1 MEDLARS COMPUTER INFORMATION RETRIEVAL-A. J. Hartley
2 THE USE OF ENZYMOLOGY IN PHARMACOLOGICAL AND TOXICOLOGI-
CAL INVESTIGATIONS-W. C. Smith
3 THE METABOLISM AND BIOLOGICAL ACTIONS OF COUMARINS-
G. Feurer
4 CARCINOGENICITY AND STRUCTURE IN POLYCYCLIC HYDROCAR-
BONS-D. W. Jones and R. S. Matthews

ix
P.I.M.C.V.13- A'
5 LINEAR FREE ENERGY RELATIONSHIPS AND BIOLOGICAL ACTION-
K. C. James
6 RECENT ADVANCES IN THE SYNTHESIS OF NITRILES-G. P. Ellis and I. L.
Thomas

VOLUME 1 1
1 STEREOCHEMICAL ASPECTS OF PARASYMPATHOMIMETICS AND THEIR
ANTAGONISTS: RECENT DEVELOPMENTS-A. F. Casy
2 QUANTUM CHEMISTRY IN DRUG RESEARCH-W. G. Richards and M. E.
Black
3 PSYCHOTOMIMETICS OF THE CONVOLVULACEAE-R. A. Heacock
4 ANTIHYPERLIPIDAEMIC AGENTS-E.-C. Witte
5 THE MEDICINAL CHEMISTRY OF LITHIUM-E. Bailey, P. A. Bond, B. A.
Brooks, M. Dimitrakoudi, F. D. Jenner, A. Judd, C. R. Lee, E. A. Lenton, S.
McNeil, R. J. Pollitt, G. A. Sampson and E. A. Thompson

VOLUME 12
I GAS-LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY IN BIOCHEMIS-
TRY, PHARMACOLOGY AND TOXICOLOGY-A. M. Lawson and G. H. Draffan
2 RECENT ADVANCES IN COLUMN CHROMATOGRAPHY-K. W. Williams and R.
C. Smith
3 NMR SPECTROSCOPY IN BIOLOGICAL SCIENCES-P. J. Sadler
4 ELECTRON SPIN RESONANCE IN MEDICINAL CHEMISTRY-D. L. Williams-
Smith and S. J. Wyard
5 POLAROGRAPHY IN BIOCHEMISTRY, PHARMACOLOGY AND TOXICOLOGY-
M. Biezina and J. Volke
6 METHODS RELATED TO CYCLIC AMP AND ADENYLATE CYCLASE-B. G.
Benfey
7 RESISTANCE O F PSEUDOMONAS AERUGINOSA TO ANTIMICROBIAL
DRUGS-R. B. Sykes and A. Morns
8 FUNCTIONAL MODIFICATIONS AND NUCLEAR ANALOGUES O F P-LACTAM
ANTIBIOTICS-Part I-J. C. JBszbertnyi and T. E. Gunda

X
Progress in Medicinal Chemistry-Vol. 13, edited by G . P. Ellis and G . B. West
@ North-Holland Publishing Company - 1976

1 Clinical Enzymology
David M. GOLDBERG, M.D., B.Sc., Ph.D., M.R.C.Path., F.R.I.C.

Department of Chemical Pathology, The University, Shefield, England *

INTRODUCTION 4
INSTRUMENTS AND TECHNIQUES 4
ELEVATED SERUM ENZYME ACTIVITIES 8
ACID PHOSPHATASE 14
Methods of assay 14
Isoenzymes 14
Chemical techniques 14
Heterogeneity of human prostatic and other AcPases 15
Clinical utility 16
Prostatic cancer 16
Prostatic massage 17
Blood diseases and related disorders 17
Other diseases 18
ALKALINE PHOSPHATASE 18
Techniques of assay 19
Isoenzymes of alkaline phosphatase 20
Electrophoretic separation 21
Diflerential inhibition and inactivation 21
Other techniques 23
Choice of method 24
Clinical applications 24
Serum activity in health 24
Serum activity in hepatobiliary disease 25
Serum APase activity in bone disease 26
Serum APase activity in pregnancy and placental function tests 28
Ectopic tumour APase isoenzymes 30
Other aspects of APase 31
5'-NUCLEOTIDASE '32
Techniques of assay 33
Clinical applications 34
5Nase and alkaline phosphatase 34
5Nase in hepatobiliary disease 35
5Nase in cancer 37
Other diseases 37

* Present address: Hospital for Sick Children, Toronto M5G IXB, Canada.
2 CLINICAL ENZYMOLOGY

y-GLUTAMYL TRANSPEPTIDASE 38
Techniques of assay 39
Clinical applications 40
Hepatobiliary disease 40
Chronic alcoholism and enzyme induction 41
Myocardial infarction 42
Specificity 43
GGT Isoenzymes 44

a-AMYLASE 45
Methodology 45
Clinical significance of serum amylase 46
Urine amylase 48
Macroamylasaemia 50
Isoamylases 51

LIPASE 53
Techniques of assay 53
Clinical applications 54

LACTATE DEHYDROGENASE 55
Techniques for total LDH assay 55
Spectrophotometric methods 55
Colorimetric methods 56
LDH Isoenzymes 57
Chemical nature 57
Techniques of separation 59
Chemical identification of isoenzymes 60
Heat stability 61
Diferential substrates 63
Clinical importance of total LDH and LDH isoenzymes 64
Pulmonary disease 64
Renal disease 65
Hepatobiliary disease 67
Other malignant diseases 67
Anaemia and haemolysis 68
Muscle disorders 69

THE AMINOTRANSFERASES 69
Methods of assay 69
Colorimetric methods 69
Spect rophotometric methods 70
Technical Problems 71
Clinical applications 72

CREATINE PHOSPHOKINASE 74
Techniques of assay 74
Clinical applications 76
Myocardial infarction 16
Muscle disease 76
Genetic counselling 77
 D. M. GOLDBERG 3

Cerebral disease 78
Myxoedema 78
Malignant hyperpyrexia 79
Creatine phosphokinase isoenzymes 80
SERUM ENZYMES IN THE DIAGNOSIS O F MYOCARDIAL INFARCTION 81
Choice of enzymes 82
Time course of elevated enzyme activity 83
Diagnostic accuracy 84
Incidence of raised values 84
Role o f isoenzymes 85
Incidence of false-positive values 86
Generalised enzyme elevations 86
Enzymes and prognosis 87
ENZYMES AND DISEASES O F THE LIVER AND BILIARY SYSTEM 88
The aminotransferases and aminotransferase ratio 88
Isocitrate dehydrogenase 90
Glutamate dehydrogenase 91
Guanase 92
Adenosine deaminase 93
Other enzymes 94

ENZYMES AND PANCREATIC FUNCTION 95

Technical considerations %
Duodenal intubation %
Choice of stimulant 97
Normal values and expression of data 99
Diagnostic accuracy of pancreatic function tests 99
Faecal enzymes 101
ENZYMES IN THE DIAGNOSIS O F UTERINE CANCER 102
Tissue enzyme levels 102
Vaginal fluid 104
Technical factors 104
Diagnostic eficiency 105
After cancer therapy 107
Current status of vaginal enzyme tests 108
ENZYMES IN GENETIC DISORDERS 108
General mechanisms 109
Specific abnormalities 110
Congenital erythrocyte enzymopathies 111
Lysosomal enzyme deficiencies 111
Disorders of amino acid metabolism 112
Disorders of carbohydrate metabolism 113
Miscellaneous congenital enzymopathies 115
Pre-natal diagnosis 119

CONCLUSION 122

REFERENCES 123
4 CLINICAL ENZYMOLOGY

INTRODUCTION

In this Chapter, a comprehensive coverage of all aspects of clinical

enzymology is not possible, but a sufficiently wide choice of topics has
been made to give the reader, it is hoped, the flavour of the subject and a
concept of its scope. This choice necessarily reflects in some measure the
special interests of the author. After the introductory section which deals
with some general technical and biological items, several important
enzymes are considered individually and in detail, and attention is given
to analytical and clinical facets as well as to molecular forms of the
enzyme, where appropriate. The estimation of groups of enzymes in
specific clinical situations is then discussed.
Fundamental aspects of enzymology have been lucidly described in the
classic text by Dixon and Webb [l]. More extensive and up-to-date
information is presented in the celebrated treatise, The Enzymes, now in
its third edition [2]. A number of books and monographs on clinical
enzymology have been published during the past 12 years [3-71 and they
will give the reader a historical perspective. Several books present
accounts of various aspects of isoenzymes [%lo] and they should be
consulted by those seeking more specialised information.

INSTRUMENTS AND TECHNIQUES

The first technical innovation that grew out of the special requirements of
clinical chemistry was the development of the AutoAnalyser. A flood of
papers appeared describing the adaptation of manual enzyme assays to
the AutoAnalyser, and these have been catalogued by Roodyn [ll]. In
essence, these assays were colorimetric, although a UV-absorptiometer
and a fluorimeter have occasionally been incorporated for measurement
of the final product. This timely advance enabled clinical laboratories to
keep pace with the increasing demands being made upon them-in enzyme
determinations no less than in any other sub-speciality of clinical chemis-
try. Moreover, the sequential flow system inherent in the design of the
AutoAnalyzer brought improved precision-as much to enzyme assays as
to other assays-compared with the manual techniques upon which the
methods were founded. Accuracy, being based upon within-run calibra-
tion curves, became superior to that obtainable with manual techniques,
and sample-interaction or ‘carry-over’ can always be contained within
acceptable limits by suitable design of the reagent manifold and choice of
an appropriate ‘sample-to-wash ratio’.
 D. M. GOLDBERG 5

The position of colorimetric two-point assays was consolidated around

the beginning of this decade with the development of high-capacity
multiple analysers capable of performing up to 20 simultaneous tests on
each serum sample [12]. Some, however, utilise a device introduced by
Trayser and Seligson [13] in which, for each sample, the reaction is
initiated twice, the interval between initiation being constant and care-
fully controlled; the final product is then measured simultaneously in
both, so that the difference corresponds to product generated over the
fixed time interval. This so-called ‘kinetic method’ emphasized the desire
which became manifest among clinical enzymologists to base determina-
tion of enzyme activity upon the continuously-observed rate of reaction
rather than upon fixed-time measurements. The advantages and limita-
tions of both approaches have been summarised by Moss [14]. Another
stimulus to the use of rate-measurements was the unsatisfactory experi-
ence with commercially-produced calibration sera with assigned enzyme
values needed for parameter-setting instruments.
Spectrophotometric methods for measuring enzyme activity have been
known since the work of Warburg [15], and his ‘optical test’ involving the
interconversion of oxidised and reduced pyridine nucleotides which can
be conveniently monitored by absorbance or fluorescence measurements
has been expanded to embrace a very wide range of clinically-important
enzymes-either directly or through coupled enzyme reactions [ 161. A
similar service was performed by Kalckar for enzymes of purine metabol-
ism [17] although these are of lesser clinical interest, and the wavelengths
required to monitor the reactions are more prone to interference. A
decisive advance was the introduction of colourless artificial substrates
liberating the yellow p-nitrophenate or p-nitroaniline on hydrolysis. This
principle was first applied to determination of the phosphatases [ 181, and
wide range of substrates for disaccharidases, peptidases, esterases and
other hydrolytic enzymes have now become available commercially.
From 1960, thermostatted cell-housing blocks and many laboratories have
devised procedures to suit their own requirements and simplify the
work-load [19].
A few years ago, instruments became available which were capable of
automatically transferring, incubating, and initiating tubes or cuvets for
enzyme reactions, monitoring the absorbance of the contents, and pre-
senting the rate of reaction on a strip chart recorder or as a digital output.
Such ‘automatic enzyme analysers’ resulted from the growing demands
for routine kinetic spectrophotometric enzyme analyses in hospital
laboratories and their reception by the profession was prompt and
enthusiastic. The first successful instrument of this type was the 8600
6 CLINICAL ENZYMOLOGY

Reaction Rate Analyzer (LKB Produkter, Bromma, Sweden), which

continues to be the most widely used automatic enzyme analyser in the
United Kingdom [20,21]. Initially limited to output on a strip chart
recorder, it can now operate with input to a range of programmable
calculators whereby data can be listed as units of enzyme activity [22].
The Gilford 3400 automatic enzyme analyser, unlike the LKB 8600, is a
modular instrument which offers a range of temperatures and a wide
selection of wavelengths. Whereas to hold serum and reagents the
LKB 8600 utilises disposable optical containers which are moved into and
out of the light-path, the Gilford 3400 aspirates this mixture into a
micro-flow cell and prints the absorbance differences over a series of
stepped time-intervals as such or as units of enzyme activity [23].
A third instrument varying the principles of the previous two is
represented by the AKES Automatic Enzyme Analyser (Vitatron, Fisons
Scientific Apparatus). An assessment of its performance has been pub-
lished [24]. It incorporates both a sophisticated dispenser unit for
automatically adding and mixing serum with the required reagents and a
calculator programmed to accept input from the absorptiometer unit and to
limit the monitoring period of each assay to what is strictly necessary to
obtain a level of precision pre-determined by the operator. An ingenious
multi-point sequential flow procedure has recently been introduced where,
instead of passing once only through a colorimeter flow cell, the reaction
solution passes through three flow cells in tandem and separated by a
sufficient time interval to allow a representative portion of the rate curve to
be analysed [25].
The ‘parallel fast analyser’ [26] relies initially on the loading of transfer
disks with serum and reagents, a task which can be carried out by
automatic dispensers. The disk is then placed in a centrifugal field, which
effects mixing of the reaction constituents and propels the mixture into
optical cuvets cut out of the periphery of the transfer disk which rotates
through a light beam, the signal of which is fed through a photo-sensitive
cell to a dedicated computer. Transfer disks with 15-30 cuvet spaces are
available with various commercial models and the computer can simul-
taneously monitor the rate of the reaction in each cell, store the
information, and convert it to the appropriate units, either on the basis of
a calibration curve constructed from standards included with patient
samples on the same transfer disk or using a factor derived from the
molar absorption coefficient as in the case of pyridine nucloetide-linked
reactions [27-311. The fact that the signal is continuously monitored by a
computer makes it a powerful tool in the application of enzyme kinetics
 D. M. GOLDBERG 7

(as opposed to ‘kinetic’ enzyme assays) to the problem of characterising

and identifying enzymes and iso-enzymes [32]. Other applications to
iso-enzyme analysis have also been described [33]. Many other technical
developments are on the way [34,35].
The reagents required to perform enzyme determinations are, in
general, more costly and less stable than those used for other routine
chemical assays. Although the idea of packaged reagent kits is not
exclusive to enzyme assays, such kits are used on a larger scale for enzyme
assays than for any other type of assay. A recent survey in Great Britain
[36] showed that enzymes were involved in the 6 commonest assays
performed with the aid of commercial kits.
The process of lyophilisation, especially as applied to mixtures of
reagents, can lead to subtle changes in the products which are poorly
understood and not reproducible, but tend to affect their suitability for
certain types of enzyme procedures. A well-known problem is the
presence in certain batches of NADH of a derivative (as yet uncharacter-
ised) inhibiting lactate dehydrogenase and certain other dehydrogenases
[37-391, notably glutamate and malate dehydrogenase [40]. The impact of
such impurities upon laboratory quality control can be devastating [41]
and recommendations for the detection of inhibitors in NADH have been
published [42]. Some commercial samples of NAD also contain a conta-
minant which inhibits lactate dehydrogenase [43,44].
A number of bodies have published recommendations for standard
reference methods as applied to the commonest and most important
serum enzyme assays, notably the national organisations concerned with
regulating the practice of clinical chemistry in Germany [45], the United
Kingdom [46-48] and Scandinavia [49]. Remarkably, the methods recom-
mended show so little agreement that the uncommitted observer is left in
a state of bewilderment and is entitled to question the value of this sort of
endeavour. Even an enthusiastic exponent of standardised enzyme
assays such as H. U. Bergmeyer [50,51], has recently proposed an
entirely new series of assays for aminotransferase activity [52].
The problem of trace contamination is a much greater hazard of
enzyme assays than of most non-enzymatic assays. For example, heavy
metals in minute concentration and traces of reaction products or of
substrates for auxiliary enzymes in linked reactions may cause serious
interference. For these reasons, techniques of quality control are more
essential for enzyme assays than for non-enzymatic methods. The lack of
suitable quality control or reference enzyme preparations is a major
handicap. The specifications required of such preparations have been
8 CLINICAL ENZYMOLOGY

formulated [53] but few such preparations have yet been produced,
although an aspartate aminotransferase purified from human erythro-
cytes is reported to meet these requirements [54]. Moss [55] has presented
a lucid account of the problems inherent in the quality control of enzyme
assays and has made a strong case for the production of immobilised
enzymes suitable for this purpose. The challenge has not yet been taken
up by industry although immobilised enzymes for many other analytical
and preparative procedures are available 1561. Commercial quality control
sera, whether in liquid or lyophilised form, are unreliable as reference
materials for enzyme assays [57]. Enzymes, in general, are not very stable
in solution, and lyophilisation can cause subtle changes in the state of
hydration and aggregation of enzymes. A troublesome example of this
phenomenon has been well documented for alkaline phosphatase [58-601.
On reconstitution, a steady but not very reproducible increase in activity
occurs, followed by a gradual decrease- both processes being strongly
influenced by the temperature prevailing during reconstitution and stor-
age. Commercial sera used to calibrate certain enzyme assays and the
corresponding enzymes of human serum 1611 also show differences in
substrate specificity.

ELEVATED SERUM ENZYME ACTIVITIES

The activity of an enzyme in serum represents a balance between its rate

of liberation into the extracellular space and its rate of clearance or
uptake from the extracellular space. In health, demographic factors such
as age, sex, and body weight also affect this balance in individual subjects
[62]. Since the gradient between cells and serum is > l o 3 for most
enzymes (Table l . l ) , the cell membrane performs a crucial function in
retaining enzymes within the cell. A continuous supply of energy,
probably in the form of ATP, is necessary to maintain the cell membrane
in a conformation optimal for enzyme retention [4]. It follows that
absolute anoxia occurring in pathological states such as severe cardiac or
respiratory disease may cause significant escape of enzymes from cells,
even though no structural damage is evident. Some of the highest serum
enzyme activities in the author’s experience have occurred in patients
with status asthmaticus, a largely functional though potentially fatal
condition in which anatomical damage to lung and other body tissues is
almost non-existent. Even relative hypoxia such as that occurring during
severe exercise may cause transient escape of enzymes from skeletal
muscle, with consequent elevation of serum activities [63].