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Metabolic Profiling Methods and Protocols 1st Edition
Thomas O. Metz Digital Instant Download
Author(s): Thomas O. Metz
ISBN(s): 9781617379840, 1617379840
Edition: 1
File Details: PDF, 6.50 MB
Year: 2011
Language: english
 TM
ME T H O D S IN MO L E C U L A R BI O L O G Y

Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For other titles published in this series, go to

www.springer.com/series/7651
 Metabolic Profiling
Methods and Protocols

Edited by

Thomas O. Metz
Biological Sciences Division, Pacific Northwest National Laboratory,
Richland, WA, USA
Editor
Thomas O. Metz
Pacific Northwest National Laboratory
Biological Sciences Division
P.O. Box 999, MS K8-98
Richland, WA 99352
USA
[email protected]

ISSN 1064-3745
ISBN 978-1-61737-984-0
DOI 10.1007/978-1-61737-985-7
Springer New York Dordrecht Heidelberg London

© Springer Science+Business Media, LLC 2011

All rights reserved. This work may not be translated or copied in whole or in part without the written permission of
the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013,
USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of
information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology
now known or hereafter developed is forbidden.
The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified
as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.

Printed on acid-free paper

Humana Press is part of Springer Science+Business Media (www.springer.com)

Preface
After accepting the task to edit a volume of Methods in Molecular Biology devoted to
metabolic profiling, I began to contemplate the definition of the term. Fiehn referred
to “metabolic profiling” as the identification and quantification of a select number of
metabolites in an entire metabolic pathway or intersecting pathways (1). Closely related
disciplines were targeted metabolite analysis, metabolic fingerprinting, and metabolomics,
the latter of which was defined as the quantitative measurement of perturbations in the
metabolite complement of a biological system (2). These four terms are often used inter-
changeably; indeed, in reviewing the literature over the past 40 years, it is evident that
these various disciplines of metabolite analysis are related via an evolution of methods and
technology. For example, while the field of metabolomics is now 10 years old, the pro-
tocols and instrumentation that form the foundation for the myriad approaches of this
discipline are based on those originally established for the diagnosis of inborn errors of
metabolism and drug metabolite analysis. Thus, in compiling this volume, I have made an
attempt to incorporate protocols that are illustrative of the evolution of metabolic profil-
ing from single molecule analysis to global metabolome profiling. The constraints of this
volume necessitate that its contents will be perspective based, rather than comprehensive.
However, it is my hope that the methods contained herein will be a resource for both
established and new investigators in the field of metabolic profiling.

Thomas O. Metz

References

1. Fiehn, O. (2002) Metabolomics – the link metabolic responses of living systems to

between genotypes and phenotypes. Plant Mol pathophysiological stimuli via multivariate sta-
Biol 48, 155–171. tistical analysis of biological NMR spectroscopic
2. Nicholson, J. K., Lindon, J. C., Holmes, data. Xenobiotica 29, 1181–1189.
E. (1999) ‘Metabonomics’: understanding the

v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1. Origins of Metabolic Profiling . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Arthur B. Robinson and Noah E. Robinson
2. Amino Acid Profiling for the Diagnosis of Inborn Errors of Metabolism . . . . . 25
Monique Piraud, Séverine Ruet, Sylvie Boyer, Cécile Acquaviva,
Pascale Clerc-Renaud, David Cheillan, and Christine Vianey-Saban
3. Acylcarnitines: Analysis in Plasma and Whole Blood Using Tandem Mass
Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
David S. Millington and Robert D. Stevens
4. Analysis of Organic Acids and Acylglycines for the Diagnosis of Related
Inborn Errors of Metabolism by GC- and HPLC-MS . . . . . . . . . . . . . . 73
Giancarlo la Marca and Cristiano Rizzo
5. HPLC Analysis for the Clinical–Biochemical Diagnosis of Inborn Errors
of Metabolism of Purines and Pyrimidines . . . . . . . . . . . . . . . . . . . . 99
Giuseppe Lazzarino, Angela Maria Amorini, Valentina Di Pietro,
and Barbara Tavazzi
6. Bile Acid Analysis in Various Biological Samples Using Ultra
Performance Liquid Chromatography/Electrospray Ionization-Mass
Spectrometry (UPLC/ESI-MS) . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Masahito Hagio, Megumi Matsumoto, and Satoshi Ishizuka
7. Analysis of Glycolytic Intermediates with Ion Chromatography- and Gas
Chromatography-Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . 131
Jan C. van Dam, Cor Ras, and Angela ten Pierick
8. Analysis of the Citric Acid Cycle Intermediates Using Gas
Chromatography-Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . 147
Rajan S. Kombu, Henri Brunengraber, and Michelle A. Puchowicz
9. Quantification of Pentose Phosphate Pathway (PPP) Metabolites
by Liquid Chromatography-Mass Spectrometry (LC-MS) . . . . . . . . . . . . 159
Amber Jannasch, Miroslav Sedlak, and Jiri Adamec
10. High-Performance Liquid Chromatography-Mass Spectrometry
(HPLC-MS)-Based Drug Metabolite Profiling . . . . . . . . . . . . . . . . . . 173
Ian D. Wilson

vii
viii Contents

11. Gas Chromatography-Mass Spectrometry (GC-MS)-Based Metabolomics . . . . 191

Antonia Garcia and Coral Barbas
12. The Use of Two-Dimensional Gas Chromatography–Time-of-Flight
Mass Spectrometry (GC×GC–TOF-MS) for Metabolomic Analysis
of Polar Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Kimberly Ralston-Hooper, Amber Jannasch, Jiri Adamec,
and Maria Sepúlveda
13. LC-MS-Based Metabolomics . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Sunil Bajad and Vladimir Shulaev
14. Capillary Electrophoresis–Electrospray Ionization-Mass Spectrometry
(CE–ESI-MS)-Based Metabolomics . . . . . . . . . . . . . . . . . . . . . . . . 229
Philip Britz-McKibbin
15. Liquid Chromatography-Mass Spectrometry (LC-MS)-Based Lipidomics
for Studies of Body Fluids and Tissues . . . . . . . . . . . . . . . . . . . . . . 247
Heli Nygren, Tuulikki Seppänen-Laakso, Sandra Castillo,
Tuulia Hyötyläinen, and Matej Orešič
16. Electrospray Ionization Tandem Mass Spectrometry (ESI-MS/MS)-
Based Shotgun Lipidomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
Giorgis Isaac
17. Processing and Analysis of GC/LC-MS-Based Metabolomics Data . . . . . . . . 277
Elizabeth Want and Perrine Masson
18. Nuclear Magnetic Resonance (NMR)-Based Drug Metabolite Profiling . . . . . 299
Eva M. Lenz
19. Nuclear Magnetic Resonance (NMR)-Based Metabolomics . . . . . . . . . . . . 321
Hector C. Keun and Toby J. Athersuch
20. Slow Magic Angle Sample Spinning: A Non- or Minimally Invasive
Method for High-Resolution 1 H Nuclear Magnetic Resonance (NMR)
Metabolic Profiling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
Jian Zhi Hu
21. Processing and Modeling of Nuclear Magnetic Resonance (NMR)
Metabolic Profiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
Timothy M.D. Ebbels, John C. Lindon, and Muireann Coen
Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
Contributors
CÉCILE ACQUAVIVA • Laboratoire des Maladies Héréditaires du Métabolisme et Dépistage
Néonatal, Hospices Civils de Lyon, Centre de Biologie Est, Bron, France
JIRI ADAMEC • Department of Biochemistry, University of Nebraska-Lincoln, Lincoln,
NE, USA
ANGELA MARIA AMORINI • Institute of Biochemistry and Clinical Biochemistry, Catholic
University of Rome, Rome, Italy
TOBY J. ATHERSUCH • Biomolecular Medicine, Department of Surgery and Cancer,
Faculty of Medicine, Imperial College London, South Kensington, London, UK
SUNIL BAJAD • Sutro Biopharma Inc., South San Francisco, CA, USA
CORAL BARBAS • Faculty of Pharmacy, San Pablo-CEU, Campus Monteprincipe, Madrid,
Spain
SYLVIE BOYER • Laboratoire des Maladies Héréditaires du Métabolisme et Dépistage
Néonatal, Hospices Civils de Lyon, Centre de Biologie Est, Bron, France
PHILIP BRITZ-MCKIBBIN • Department of Chemistry and Chemical Biology, McMaster
University, Hamilton, ON, Canada
HENRI BRUNENGRABER • Department of Nutrition, Mouse Metabolic Phenotyping
Center, Case Western Reserve University, Cleveland, OH, USA
SANDRA CASTILLO • VTT Technical Research Centre of Finland, Espoo, Finland
DAVID CHEILLAN • Laboratoire des Maladies Héréditaires du Métabolisme et Dépistage
Néonatal, Hospices Civils de Lyon, Centre de Biologie Est, Bron, France
PASCALE CLERC-RENAUD • Laboratoire des Maladies Héréditaires du Métabolisme et
Dépistage Néonatal, Hospices Civils de Lyon, Centre de Biologie Est, Bron, France
MUIREANN COEN • Biomolecular Medicine, Department of Surgery and Cancer, Faculty
of Medicine, Imperial College, London, UK
VALENTINA DI PIETRO • Institute of Biochemistry and Clinical Biochemistry, Catholic
University of Rome, Rome, Italy
TIMOTHY M.D. EBBELS • Biomolecular Medicine, Department of Surgery and Cancer,
Faculty of Medicine, Imperial College, London, UK
ANTONIA GARCIA • Faculty of Pharmacy, San Pablo-CEU, Campus Monteprincipe,
Madrid, Spain
MASAHITO HAGIO • Division of Applied Bioscience, Research Faculty of Agriculture,
Hokkaido University, Sapporo, Japan
JIAN ZHI HU • Pacific Northwest National Laboratory, Richland, WA, USA
TUULIA HYÖTYLÄINEN • VTT Technical Research Centre of Finland, Espoo, Finland
GIORGIS ISAAC • Bio Separation and Mass Spectrometry, Pacific Northwest National
Laboratory, Richland, WA, USA; Water corporation, Mulford, MA
SATOSHI ISHIZUKA • Division of Applied Bioscience, Research Faculty of Agriculture,
Hokkaido University, Sapporo, Japan
AMBER JANNASCH • Bindley Bioscience Center, Purdue University, West Lafayette, IN,
USA
HECTOR C. KEUN • Biomolecular Medicine, Department of Surgery and Cancer, Faculty
of Medicine, Imperial College London, South Kensington, London, UK

ix
x Contributors

RAJAN S. KOMBU • Department of Nutrition, Mouse Metabolic Phenotyping Center, Case

Western Reserve University, Cleveland, OH, USA
GIUSEPPE LAZZARINO • Division of Biochemistry and Molecular Biology, Department of
Chemical Sciences, University of Catania, Catania, Italy
EVA M. LENZ • AstraZeneca Pharmaceuticals, Mereside, Macclesfield, UK
JOHN C. LINDON • Biomolecular Medicine, Department of Surgery and Cancer, Faculty
of Medicine, Imperial College, London, UK
GIANCARLO LA MARCA • Mass Spectrometry, Clinical Chemistry and Pharmacology
Laboratory, Department of Pharmacology, University of Florence, Meyer Children’s
Hospital, Florence, Italy
PERRINE MASSON • Biomolecular Medicine, Department of Surgery and Cancer, Faculty
of Medicine, Imperial College, London, UK
MEGUMI MATSUMOTO • Meiji Dairies Research Chair, Creative Research Institution
Sousei (CRIS), Hokkaido University, Sapporo, Japan
D AVID S. MILLINGTON • DUMC Biochemical Genetics Laboratory, Department of
Pediatrics, Duke University Medical Center, Durham, NC, USA
HELI NYGREN • VTT Technical Research Centre of Finland, Espoo, Finland
MATEJ OREŠI Č • VTT Technical Research Centre of Finland, Espoo, Finland
MONIQUE P IRAUD • Laboratoire des Maladies Héréditaires du Métabolisme et Dépistage
Néonatal, Hospices Civils de Lyon, Centre de Biologie Est, Bron, France
MICHELLE A. PUCHOWICZ • Department of Nutrition, Mouse Metabolic Phenotyping
Center, Case Western Reserve University, Cleveland, OH, USA
K IMBERLY RALSTON-HOOPER • Ecosystem Research Division, National Research
Council Post-Doctoral Fellow, United States Environmental Protection Agency,
Athens, GA, USA
COR RAS • Department of Biotechnology, Delft University of Technology, Delft, The
Netherlands
CRISTIANO RIZZO • Metabolic Unit and Laboratories, Bambino Gesù Children’s
Hospital, Rome, Italy
ARTHUR B. ROBINSON • Oregon Institute of Science and Medicine, Oregon, OR, USA
NOAH E. ROBINSON • Oregon Institute of Science and Medicine, Oregon, OR, USA
SÉVERINE RUET • Laboratoire des Maladies Héréditaires du Métabolisme et Dépistage
Néonatal, Hospices Civils de Lyon, Centre de Biologie Est, Bron, France
MIROSLAV SEDLAK • Laboratory of Renewable Resources Engineering, Purdue Univer-
sity, West Lafayette, IN, USA; Department of Agricultural and Biological Engineering,
Purdue University, West Lafayette, IN, USA
TUULIKKI SEPPÄNEN-L AAKSO • VTT Technical Research Centre of Finland, Espoo,
Finland
M ARIA SEPÚLVEDA • Department of Natural Resources, Purdue University, West
Lafayette, IN, USA
VLADIMIR SHULAEV • Virginia Bioinformatics Institute, Virginia Polytechnic Institute
and State University, Blacksburg, VA, USA
ROBERT D. STEVENS • Sarah W. Stedman Nutrition and Metabolism Center, Duke
University Medical Center, Durham, NC, USA
BARBARA TAVAZZI • Institute of Biochemistry and Clinical Biochemistry, Catholic
University of Rome, Rome, Italy
 Contributors xi

ANGELA TEN PIERICK • Department of Biotechnology, Delft University of Technology,

Delft, The Netherlands
JAN C. VAN DAM • Department of Biotechnology, Delft University of Technology, Delft,
The Netherlands
CHRISTINE VIANEY-SABAN • Laboratoire des Maladies Héréditaires du Métabolisme
et Dépistage Néonatal, Hospices Civils de Lyon, Centre de Biologie Est, Bron, France
ELIZABETH WANT • Biomolecular Medicine, Department of Surgery and Cancer, Faculty
of Medicine, Imperial College, London, UK
IAN D. WILSON • AstraZeneca, Macclesfield, UK
 Chapter 1

Origins of Metabolic Profiling

Arthur B. Robinson and Noah E. Robinson

Abstract
Quantitative metabolic profiling originated as a 10-year project carried out between 1968 and 1978 in
California. It was hypothesized and then demonstrated that quantitative analysis of a large number of
metabolites – selected by analytical convenience and evaluated by computerized pattern recognition –
could serve as a useful method for the quantitative measurement of human health. Using chromato-
graphic and mass spectrometric methods to measure between 50 and 200 metabolites in more than
15,000 human specimens, statistically significant and diagnostically useful profiles for several human dis-
eases and for other systematic variables including age, diet, fasting, sex, and other variables were demon-
strated. It was also shown that genetically distinct metabolic profiles for each individual are present in
both newborn infants and adults. In the course of this work, the many practical and conceptual problems
involved in sampling, analysis, evaluation of results, and medical use of quantitative metabolic profiling
were considered and, for the most part, solved. This article is an account of that research project.

Key words: Metabolic profiling, metabolomics, urine, breath, chromatography, mass spectrometry,
aging, diagnostic medicine, preventive medicine.

1. Introduction

Since the dawn of the age of modern chemistry, biochemistry

has been of great interest. When molecular structure became
established as an exact discipline, the minds of scientists natu-
rally turned toward those molecules of which they themselves are
made. Extensive cataloging and structure determination of these
substances followed.
As the role of proteins in catalyzing the chemical reactions of
metabolism was revealed, progress was made in understanding the
metabolites – the smaller molecules required for life that protein

T.O. Metz (ed.), Metabolic Profiling, Methods in Molecular Biology 708,

DOI 10.1007/978-1-61737-985-7_1, © Springer Science+Business Media, LLC 2011

1
2 Robinson and Robinson

catalysts select from the many atomic combinations available and

produce to make life possible.
Detailed understanding of metabolism was not, however, pos-
sible until the discovery of carbon 14 (1) and the development
of tracer methodology (2), which now includes both radioactive
and stable isotopes. When it became possible to label the atoms of
metabolites and trace their paths through living systems, a thor-
ough understanding of metabolism was achievable.
This understanding and the rapid advance of protein chem-
istry then led to explanations for some of the simplest metabolic
diseases – genetic errors that cause well-defined inborn errors of
metabolism. As analytical technology advanced, the list of known
genetic illnesses expanded to include a large number of such dis-
eases which, while individually rare, together cause much suffer-
ing. This work was further accelerated by findings that, in some
cases such as phenylketonuria, understanding of the disease could
lead to effective therapy.
Simultaneously, improvements in analytical chemistry led to
a search for single metabolites that are diagnostic of more preva-
lent diseases – including those with non-genetic components. An
extensive armament of single-substance measurements entered
the inventory of clinical laboratories – tests for both inborn errors
and other illnesses. Businesses arose to measure these substances,
primarily in blood and urine, which have now grown in the
United States alone into a $100 billion industry.
This work usually involved the correlation of one substance
with a condition of interest in human health. Scientists searched
for metabolites and proteins, the quantities of which contained
sufficient information about health and disease to warrant their
measurement. A few such measurements became standard in
health screening of ordinary patients, while a much larger number
were made available in clinical laboratories, available upon request
by physicians for specific patients.
While the many substances measureable in human samples
were increasingly evident as analytical methods improved, no
practical efforts were made to test the possibility that the simulta-
neous quantitative analysis of large numbers of metabolites fol-
lowed by computerized pattern recognition could yield health
information of significant value.
Forty years ago, however, there arose in California an exper-
imental project with the potential to cause a paradigm shift
toward the use of simultaneous measurement of large numbers of
metabolites for the quantitative measurement of human health.
This effort was ahead of its time and, therefore, faced daunting
challenges in the construction of analytical and computational
capabilities.
This work was known in the 1970s as “quantitative metabolic
profiling.” It is now a growing part of “metabolomics.” While
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CHAPTER VI
 THE CORONER SITS
Two days later, Mr. Fransemmery summoned to discharge the
functions of a juror at that ancient institution, a Coroner’s inquest,
found himself acting as foreman of twelve good men and true in the
old dining-hall of Markenmore Court. That venerable apartment had
been specially prepared and fitted up for the occasion; it was the
first time, observed Braxfield mournfully, that it had ever been used
since the grand state dinner which Sir Anthony had given to his
friends and neighbours when Guy came of age. It was a room of
vast size: baronial in appearance, and in its time there had been
many gay and striking scenes in it. But never, since its first building
by a dead and gone Markenmore, had it been so filled with folk of
various degree as on this bright spring morning. There were jurymen
and police and witnesses; there was Chilford, representing the
family, and another solicitor representing Harborough; there was a
London barrister in charge of the case as it presented itself to the
authorities; there were officials of many sorts; there were reporters
from the local Press, and two or three representatives sent specially
from London newspapers. But all these were as nothing to the
crowd of spectators—village folk; county family folk; folk from near
and far. Already, decided Mr. Fransemmery, as he adjusted his gold-
rimmed spectacles and looked around him, the Markenmore problem
bade fair to be a cause célèbre.
Mr. Fransemmery at that moment could truly say that he and his
fellow-jurymen brought open, unbiassed, and uninformed minds to
that important enquiry. During the forty-eight (to be exact, fifty-two)
hours which had elapsed since the discovery of Guy Markenmore’s
dead body, nothing further had leaked out to the general public.
Much had been going on. Police had been drafted into the usually
quiet village in considerable numbers; they had been searching
woods, towns, all the immediate surroundings of the crime. Blick,
with two or three lesser satellites, had been pursuing enquiries all
round the neighbourhood; there was scarcely a soul in a side area
round Markenmore that had not been questioned for news.
But all through these investigations those who made them had
preserved an unusually strict silence, and outside the police there
was not a soul in the big dining-hall, now transformed into a court,
who had the faintest notion of what was about to be revealed. Yet
one thing was known. Mrs. Tretheroe had not been content with her
denunciation of John Harborough before the brother and sister and
the men assembled in the morning-room. She had denounced him
again—to the Vicar; to the village folk; to other people; it was
already well and widely known that she firmly believed that
Harborough had killed Guy Markenmore. Naturally, therefore, she
was the object of great interest as she sat near the big tables
arranged in the centre of the room, attired, somewhat theatrically, in
deep mourning. She was not alone; although her house-party had
dispersed on the day of the tragedy, two of her friends had remained
with her; one, a Mrs. Hamilton, a middle-aged woman of fashion:
the other, a Baron von Eckhardstein, a handsome and well-preserved
man of fifty who was said to be a great European financier. These
two sat on either side of Mrs. Tretheroe; a little distance away
Harborough sat, grave and imperturbable, by the side of Mr.
Walkinshaw, his solicitor.
Mr. Fransemmery and his eleven companions went automatically
through the usual dismal preliminaries: and the gruesome duty of
viewing the dead man’s body. They listened respectfully to the
Coroner’s opening remarks, conscious all the time that this was
routine—the real thing to be considered was the evidence. And
suddenly the Coroner brought his remarks to an abrupt conclusion,
and jury and spectators settled down to the real business—the
hearing of what could be said towards clearing up, one way or
another, the all-important problem: Who killed Guy Markenmore?
The first stages of the enquiry yielded little that was new or exciting.
Harry Markenmore identified the body as that of his elder brother,
Guy, who, he said, was thirty-five years of age. He was not aware if
Guy was married or not. Guy had left Markenmore Court seven years
before, and had never been seen or heard of by his family since,
until the evening before the murder, when he had turned up
unexpectedly. He detailed the doings of the short visit, and said that
his brother had left the house at about half-past ten. He had spoken
of having an appointment in the neighbourhood, and had mentioned
that supper would be awaiting him where he was going. He had no
idea whatever as to where Guy then went. He did not return to
Markenmore Court—no one there ever saw him again until his dead
body was carried in, early next morning.
Hobbs, the ploughman, gave evidence as to finding the dead man,
whom he had at once recognized, and detailed what he had done to
get assistance. He had seen no one about in that part of the downs,
nor noticed anything suspicious near the scene of the crime.
The village policeman spoke as to the investigations made round
about Markenmore Hollow: there was no sign whatever of any
struggle, and there were no footprints—the turf, thereabouts, he
said, was very wiry, close-knit, and full of spring: there had been no
recent rain, and the closest examination had failed to yield anything
in the shape of such prints. No weapon of any sort had been found
near the place, nor in the adjacent undergrowth. This witness, too,
gave evidence as to the examination of the dead man’s clothing,
made when the body was brought down to the Court. There was a
considerable sum of money in notes, gold, and silver. There was a
gold watch, chain, and locket. There were three rings—two of them
set with diamonds. There were several small items—a silver cigar-
case, silver match-box, and so on; and there were two pocket-
books. All these were now in possession of the police. He was sure
that, when he was brought to the Hollow by the last witness, the
body had not been interfered with in any way, and that the clothing,
and the various objects he had just mentioned, had not been
touched. From these facts and from the additional fact that the dead
man had a large sum of money on him, he had at once formed the
impression that the murder had not been committed for the sake of
robbery.
There was more interest in the evidence of the police-surgeon. It
was, he said, about twenty minutes to seven o’clock when he, with
the Chief Constable and Detective-Sergeant Blick, reached
Markenmore Hollow. He saw at once that Guy Markenmore had been
shot dead, and his impression was that he had then been dead
between two or three hours—nearer three than two. His opinion
remained unaltered—he should fix the actual time of death at about
four o’clock. Death had been instantaneous. From a subsequent
post-mortem examination he had ascertained that the bullet—
produced—fired, in his opinion, at close quarters from a revolver,
had entered the head at the right temple, passed through the brain
in a curving downward direction and finally lodged in the muscles a
little below the left ear.
“This,” suggested the Coroner, “could have been a self-inflicted
wound?”
“Certainly,” replied the witness.
“But in that case, the weapon would have been found close at
hand?”
“In that case, I should have expected to find him still grasping the
weapon. The probability in such case is that a man who shoots
himself grips his revolver very tightly in the act, and his fingers
would tighten their grip as the shot took effect.”
“As there was no revolver near, you came to the conclusion that this
was a case of murder?”
“Yes—murder!”
“Did you come to any conclusion as to how it was done?”
“Yes, I did. An opinion, that is, I think that the murderer and his
victim were walking side by side, probably in close conversation, the
victim on the left. I think the murderer brought his right hand,
armed with a revolver, suddenly round across his own body, and
shot his victim at literally close quarters, the victim being absolutely
unconscious that he was to be attacked. The revolver must have
been placed close to the temple—the skin and the fine hair about it
were burnt.”
The Coroner looked round at the jury.
“The sun rises at about ten minutes to five, just now,” he observed.
“At four o’clock, then, it would be fairly light. This is an important
point, gentlemen. You must keep it in mind, in view of what you
have just heard.”
None of the legal practitioners had any questions to put to the
police-surgeon; he stepped down, and a whispered consultation took
place between the Coroner and one of his officials. Then came the
moment for which the crowded court had waited with suppressed
eagerness.
“Mrs. Veronica Tretheroe!”
Mrs. Tretheroe rose from between her supporting friends, and
walked slowly forward to the witness-box. Evidently well coached as
to what she was to do, she drew off the glove from her right hand
and threw back her thick veil. Taking the Testament in her ungloved
hand she repeated the words of the oath in a low voice, and turned
a very pale, but perfectly self-possessed face on the Coroner, who
bent towards her with an expression of sympathetic consideration.
Amidst a dead silence he began his preliminary questions.
“Mrs. Tretheroe, I believe you knew the late Mr. Guy Markenmore?”
“Yes.”
“You knew him well, one may say?”
“Yes—very well—once!”
“How long had you known him?”
“I knew him from the time my father came to Markenmore, as vicar
of this parish, when I was a girl of seventeen or eighteen, until Guy
left this house, about seven years ago.”
“How old were you then, Mrs. Tretheroe?”
“Twenty-two.”
“Then your acquaintanceship with him at that period lasted about
four or five years?”
“About that.”
“You were then Miss Veronica Leighton?”
“Yes.”
“I think you married the late Colonel Tretheroe just after Mr. Guy
Markenmore left home—seven years ago?”
“Yes.”
“And went with your husband to India?”
“I did.”
“You have only recently returned from India—where Colonel
Tretheroe, I think, died last year?”
“Quite recently.”
The Coroner leaned a little forward from his desk—sure sign,
thought Mr. Fransemmery, that his questions were nearing a most
particular stage.
“Now. Mrs. Tretheroe, during those seven years, did you ever see
Guy Markenmore?
“Never!”
“Did you ever hear from him?”
“Never!—nor of him!”
“For seven years you neither saw him, nor heard of him, nor heard
from him. When did you next see him again?”
“On Monday evening last—two—or three—days ago.”
“You met him—for the first time for seven years?”
“Yes, for the first time for seven years.”
“Just tell me, Mrs. Tretheroe, how the meeting came about?”
Mrs. Tretheroe folded her hands on the ledge of the witness-box and
distributed her glances alternately between the Coroner and the
twelve jurymen. By that time she had regained her colour; her eyes
had begun to sparkle; she looked as if she was beginning to feel
some extraordinary interest in the proceedings.
“In this way,” she said, in quiet, even tones. “During Monday
evening, after dinner, I had occasion to give some orders to my
coachman, Burton. When he was going away, he mentioned that he
had just seen Mr. Guy Markenmore; he had seen him, he said, going
up to the Court. I thought Burton must be mistaken, but he was
positive—and, of course, I knew he had known Guy since boyhood.
So——”
Here Mrs. Tretheroe paused. Her fingers began to tap the ledge
before her; she looked at the Coroner and the jury with a slightly
embarrassed expression.
“What happened, if you please?” asked the Coroner in matter-of-fact
tones.
“Well—I wanted to see Guy!” continued Mrs. Tretheroe suddenly.
“And so—not just then, but after a while—about half-past ten, I
think—I put on a coat over my dinner dress and ran across the park
to the Court—there’s a path, a short cut. I came here—I saw
Braxfield, the butler, and Valencia Markenmore. I told Valencia that
I’d heard Guy had come home. She said he’d gone. Then I thought
that, perhaps, hearing I was at the Dower House, he’d come down
there to see me, so I went away, thinking I might find him waiting
for me.”
“Did you find him?”
“No—but—I met him. He had been to my house. I met him at the
gate.”
“What happened then?”
“He went back to my house with me.”
“I believe you were entertaining a house-party, Mrs. Tretheroe?”
“Yes.”
“A large one?”
“Eight, altogether.”
“Did you introduce Mr. Guy Markenmore to your guests when you
took him in?”
“No, I didn’t. They were playing bridge, some of them—some were
playing billiards. He didn’t see any of them.”
“Where did you and he go, in your house?”
“We went up to my boudoir.”
The Coroner leaned still nearer.
“We have heard—from Sir Harry Markenmore—that his brother
spoke of an appointment, which he hurried away to keep? Now—was
that appointment with you?”
“No—certainly not!”
“Did he mention any appointment to you?”
“Yes—merely to say that he had one—close by.”
“Close by? Did he say with whom, or where?”
“No, he did not. He merely mentioned the fact—casually. I didn’t
question him about it.”
“And—how long did he stay with you at the Dower House?”
Mrs. Tretheroe hesitated—obviously, not from uncertainty.
“The question is a highly important one,” said the Coroner.
“Well, he stayed until a quarter to twelve,” answered Mrs. Tretheroe.
“Then he was with you about an hour?”
“About an hour—yes.”
“Alone—all the time?”
“Yes.”
“Did any of your guests—or any of your servants—see him, coming
or going?”
“No one saw him. He and I entered the house by a side door, of
which I have the key always in my possession. We went straight up
to my boudoir. I let him out of the house in the same way. No—
nobody saw him.”
“You let Guy Markenmore out of your house, yourself, at a quarter to
twelve. Did you notice which way he went when he left?”
“Yes. As a matter of fact, I walked down the drive with him, to the
entrance gate. He went along the main road, towards the village.”
“And, after that, you never saw him again?”
Mrs. Tretheroe shook her head, and for a moment those about her
thought that she was about to burst into tears. But she suddenly
controlled herself, and there was an almost defiant expression in her
eyes as she answered the last question.
“I never saw him again—until I saw him yesterday dead—
murdered!”
The Coroner drew back in his chair: clearly, he had got at what he
particularly wanted to know: the glance that he gave the jurymen
was obviously intended to remind them that they now knew that
from half-past ten to a quarter to twelve o’clock of the night before
his death Guy Markenmore had been with Mrs. Tretheroe, alone in
her boudoir, unknown to any one. From the jury he turned to the
men of law, sitting at the table beneath his raised desk.
The barrister who had been instructed by the police authorities
slowly rose to his feet, and turned himself to the witness.
“I believe it is pretty well known, Mrs. Tretheroe,” he said in bland,
half-apologetic tones, “that before your marriage to your late
husband, you had a good many suitors.”
“Yes!” answered Mrs. Tretheroe readily. “At least—I don’t know what
you mean by well known. But I had—certainly.”
“Mr. Guy Markenmore was one of them?”
“Yes.”
“A particularly favoured one?”
“Well—yes, I think so.”
“There was, in fact, at one time, some prospect of marriage between
you?”
“We were certainly very fond of each other.”
“We will pass from that for the moment—nothing came of it then.
You married Colonel Tretheroe. But, I may take it, you—you still
retained some of the old feeling for Guy Markenmore.”
Mrs. Tretheroe hesitated. When she spoke again, her voice was
lower in tone.
“I—I didn’t know of it until—until I met him again, the other night,”
she said.
“But, you realized it then?”
“I suppose I did. I was very pleased to see him.”
“And he to meet you again, I suppose?”
“Yes—indeed he was.”
“Now, Mrs. Tretheroe, in the interest of justice, we want to get at
the truth. When Guy Markenmore was with you alone, in your
house, on Monday night, did he ask you to marry him?”
“Yes—he did.”
“And you replied—what?”
“I promised him that I would,” answered Mrs. Tretheroe.
CHAPTER VII
 MRS. BRAXFIELD SUPPORTS
Amidst the ripple of murmured interest that ran round the room, the
questioner looked significantly at the twelve jurymen, as much as to
tell them to keep their ears well open; from them he turned once
more to his witness.
“You accepted his offer of marriage, then. Did you arrange when it
was to be?”
“Yes, we did.”
“When?”
“Almost at once. For this reason—he told me that he was obliged to
go over to New York on most important business within the next
week or two. I decided to go with him. So we arranged that he
should get a special license and we would be married straight off.”
“Any particular date?”
“Yes. Next Monday morning—at Southampton.”
“We may take it, then, that you and Guy Markenmore, as old lovers,
on meeting once more, and you being free, fell in love with each
other again, and decided to marry without further delay?”
“Yes—I suppose so.”
“Very well. Now, Mrs. Tretheroe, I want you to let your mind go back
to the days when you were Miss Leighton. You have admitted that
you had a good many suitors. Is it not a fact that out of the many
there were two young gentlemen of this neighbourhood who were
specially favoured by you, and that one was Mr. Guy Markenmore,
and the other Mr. John Harborough, of Greycloister?”
Mrs. Tretheroe showed no hesitation in answering this question.
“They came first—in those days—certainly,” she admitted.
“So much so, that it was commonly said, hereabouts, that you
couldn’t make up your mind between them?”
“I daresay that was said.”
“Now, how was it that, in the end, you didn’t marry either, but did
marry somebody else.”
“There were reasons.”
“What reasons? All this is important to the issue before the jury.
What were the reasons.”
“Well—they became terribly jealous of each other. From being great
friends they became bitter enemies. Or, rather, Harborough
conceived a terrible, wicked enmity towards Guy. Harborough got an
idea that Guy had poisoned my mind against him.”
“Had Guy Markenmore poisoned your mind?”
“No, he had not! But Harborough was always jealous and suspicious,
and he became so—so violent about things that—well, I dismissed
him.”
“And—what then as regards his rival?”
Mrs. Tretheroe began to finger her rings.
“Well,” she answered after a pause. “I—the fact is, I got a bit sick of
the squabble, so I told Guy it wouldn’t do—and I accepted Colonel
Tretheroe.”
“I see. You got rid of both the youthful suitors, and married one who
was older and more sensible. Very good. But now, Mrs. Tretheroe, I
think something had happened before that. You said just now that
Harborough conceived a terrible, wicked enmity towards Guy
Markenmore. Now, is it a fact that Harborough threatened his rival in
your presence?”
“Yes—it is.”
“When? On what occasion?”
“It was one day when he met Guy and myself coming home from
hunting. There was a scene—high words, Harborough lost his
temper. He told Guy that he’d settle him. And I know for a fact that
he afterwards threatened him again—he said he’d kill him.”
“How do you know that for a fact?”
“Because Guy told me of it.”
“Was he afraid of Harborough?”
“I think he was. Harborough had a very black, ugly temper—when
crossed.”
“And he threatened to kill his rival because of—what, exactly?”
“Well, as I said just now, he’d got it into his head that Guy had said
things about him to me, and that his chances with me had been
destroyed by that.”
“Then I take it that Harborough, at that period, had asked you to
marry him?”
Mrs. Tretheroe arched her eyebrows in a glance of surprise.
“Lots of times!” she answered. “He was always asking me to marry
him.”
“And—did you give him any decided answer?”
“I don’t know about decided answer. At one time—perhaps I would:
then I used to think that I wouldn’t. No—I don’t think I ever said I
would or I wouldn’t, definitely.”
“And all this time, I suppose, Guy Markenmore was in the running,
also.”
“Yes.”
“Was he asking you to marry him, too?”
“Oh, yes. They were always teasing me—both of them.”
“And in the end Harborough got the idea that his rival was
undermining him?”
“Yes—he certainly did. He said so.”
“And later—you—shall we say, dismissed both, and accepted Colonel
Tretheroe?”
“Yes.”
“Did you ever see either of them again after becoming engaged to
Colonel Tretheroe?”
“I never saw Guy Markenmore. I saw Harborough once. I met him
one afternoon, near here, accidentally.”
“Anything take place?”
“Yes. He went into one of his passions. He reproached me bitterly.
He said I’d led him on for three years and then thrown him aside.
And he finished up by repeating that he knew he’d Guy Markenmore
to thank for it, and that if he ever came across him again, however
long it might be, he’d shoot him like a dog.”
When the sensation caused by this reply had died down, the
questioner gave Mrs. Tretheroe a searching look.
“You swear that he said this—on your oath?”
“On my oath!”
“Harborough said—to you—that it was due to Guy Markenmore that
he, Harborough, had lost his chance with you, and that if he ever
met Guy again, however long it might be, he’d shoot him like a
dog?”
“Yes. That is precisely what he said.”
“I take it, then, that at that time Harborough was passionately in
love with you?”
“Madly, I believe!” murmured Mrs. Tretheroe. “He acted like a
madman. I was afraid of him.”
“When this threat was made had Guy Markenmore gone away from
here?”
“Oh, yes—some little time before.”
“And did Harborough go soon after?”
“He went away a few days before I was married.”
“Now, during the seven years of your marriage—six years, rather, I
think—did you ever meet Harborough?”
“Never!”
“Ever hear from him?”
“No.”
“Or of him?”
“I heard—just once—from a friend of mine in Selcaster that he was
still travelling abroad, and that Greycloister had then been shut up
for some years.”
“Very well. In time your husband died, and you came back to
England and took the Dower House here. And last Monday Mr.
Harborough returned to Greycloister. Now, Mrs. Tretheroe, I want to
ask you a most important question. Did you meet John Harborough
last Monday?”
A dead silence fell on the room. For Mrs. Tretheroe hesitated in her
answer. Every neck was craned forward. At last she spoke.
“Yes!”
“Where?—and at what time?”
“Just outside his own gates, at Greycloister, about five o’clock in the
afternoon.”
“Were you alone?”
“I was. I had gone out for a short walk by myself, with my dogs.”
“The meeting was accidental?”
“Certainly. I had no idea he’d come home.”
“Was there any—shall we call it embarrassment?”
“Well, yes. I was surprised. He seemed taken aback—agitated. Of
course we shook hands and talked a little. Mere talk.”
“Any reference to your former relations?”
“No.”
“Just a mere polite exchange of—nothing in particular?”
“Just that. But he asked if—or, rather, when—he might come and see
me.”
“And what did you reply?”
“I replied—well, that he might come whenever he liked. What else
could I reply?”
“He knew that you were free?—that Colonel Tretheroe was dead?”
“Oh, yes—I mentioned that myself.”
“And then, I suppose, you parted?”
“Yes.”
“Where did you next see him?”
“On the following morning, in the morning-room here, when I came
in to offer my condolences about Sir Anthony, and heard that Guy
was dead.”
“And I believe that you immediately denounced John Harborough as
his murderer?”
“I did.”
The barrister paused in his examination, hesitated a while; and then,
as if satisfied, suddenly dropped back in his seat, and pulling out a
snuff-box, tapped it thoughtfully before helping himself to a
substantial pinch. A murmur of excitement had run round the
spectators when Mrs. Tretheroe gave her last decided answer; it had
scarcely died away before Harborough’s solicitor, Mr. Walkinshaw,
rose at the table. He looked fixedly at the witness.
“I want to ask you a very pointed question,” he said. “And I want a
very definite answer. Do you honestly believe that Mr. John
Harborough killed Guy Markenmore? Think!”
“I have thought!” retorted Mrs. Tretheroe defiantly. “I do!”
“You believe that Mr. Harborough nursed his desire for revenge—if
he ever really had any—for seven years, and took the first
opportunity of gratifying it?”
“I think he shot Guy Markenmore,” said Mrs. Tretheroe, with some
show of sullenness.
“You think that Mr. Harborough returned home still in love with you?
Answer!”
“I think it’s possible. He used to swear that he could never love
anybody else. And he certainly hadn’t married.”
“I will put this to you. Mr. Harborough met you on Monday
afternoon. Let us suppose that all his old passion was revived at the
mere sight of you—let us suppose, still further, that he made up his
mind to once more become a suitor for your hand. Do you think it
very likely that he would begin matters by shooting a man?”
“I’m not going to suppose anything. I believe he did shoot Guy. They
met—accidentally—and Harborough shot him.”
“You are a ready hand at making assertions, Mrs. Tretheroe! You
calmly assert they met. What! at four o’clock in the morning—at
Markenmore Hollow?”
Mrs. Tretheroe looked round. Up to then she had confined her
occasional glances to the Coroner and the jury, but this time she
took a comprehensive view of the crowded room. And as she turned
to face Mr. Walkinshaw again, it was with a smile that signified
contempt for his insinuation.
“I know that John Harborough was up there at Markenmore Hollow
at four o’clock that morning,” she retorted boldly. “And, I know, too,
that he was seen!”
Walkinshaw paused, abruptly. He looked round at his client; so, too,
did everybody in the room. Once more a murmur of surprise rippled
round. Walkinshaw went back to Harborough, who sat unmoved and
silent; the solicitor whispered rapidly to him; Harborough did no
more than nod, almost unconcernedly. A moment later Mrs.
Tretheroe had been dismissed from the witness-box and another
witness had been called into it.
“Elizabeth Braxfield!”
Mr. Fransemmery and his eleven companions felt a new interest arise
in their hearts as they stared at the ex-landlady of the Sceptre.
Eleven of them were already wondering what she could tell. But Mr.
Fransemmery, knowing what he did of Mrs. Braxfield’s early habits,
began to anticipate.
The Coroner left the examination of this witness to the barrister who
appeared for the police authorities. He lost no time in getting to the
point.
“I believe, Mrs. Braxfield, that you were formerly Mrs. Wrenne, of
the Sceptre Inn, and that before you were Mrs. Wrenne, you were a
Miss Rawlings, a daughter of Thomas Rawlings, who kept the
Sceptre Inn before your late husband, Peter Wrenne, had it?”
“Quite correct, sir,” answered Mrs. Braxfield.
“Then you have lived all your life in Markenmore, and know all the
people in it?”
“Yes, sir—and for a good many miles round.”
“Do you know Mr. John Harborough?”
“Yes, sir—known him ever since he was a boy.”
“Did you see him on Tuesday morning last?”
“I did.”
“What time?”
“Ten minutes past four o’clock.”
“Where?”
“Near my house, sir.”
“Where is your house?”
“Up on the downs, sir—Woodland Cottage; about two hundred yards
from Markenmore Hollow.”
“How came you to see him—or anybody—at that early hour?”
“Nothing unusual in that, sir. I often get up at four o’clock—that is
when the mornings get light. I keep a lot of fowls, and I get up to
attend to them.”
“Was it light that morning—Tuesday?”
“Light enough, sir.”
“Light enough to see—how far?”
“Well, sir, when I looked out of my window I could see a lot. The
Court here—the village—all that’s in front—and Withersley Beacon
on one side and Pole Clump on the other. The morning was a
particularly clear one—very fine.”
“And you saw Mr. Harborough?”
“I did, sir.”
“From your window?”
“From my window.”
“Where was he when you saw him?”
“Coming down the hill-side from the direction of Markenmore
Hollow, sir. He was walking along the side of a fence.”
“How far away from you?”
“About a hundred yards.”
“Mr. Harborough, until the day before, had been away from
Markenmore for seven years. Weren’t you very much surprised to
see him there?”
“No, sir, I wasn’t.”
“Why not?”
“Because I’d heard that he’d got home again—heard it the night
before. I’d been down to the village and everybody knew he’d got
home.”
“And you are certain that the man you saw was Mr. Harborough?”
“Perfectly certain, sir. I couldn’t be mistaken about that.”
“Well, where did he go?”
“Down the slope in the direction of his house, sir—Greycloister.”
“How far is Greycloister from Woodland Cottage?”
“Half a mile, sir.”
“Was Mr. Harborough walking quickly when you saw him?”
“No, sir—he was just going along at the ordinary pace—sauntering,
you might say.”
“And you are sure of your time—ten minutes past four o’clock in the
morning?”
“Certain, sir. I have a very good clock in my bedroom—never gains
or loses. I looked at it just before I saw Mr. Harborough.”
The barrister nodded to Mrs. Braxfield and sat down, and as no one
else rose to ask her any questions she left the box. The Coroner
bent over to some officials; while he was whispering with them,
Walkinshaw rose and approached the table again.
“Mr. Harborough desires to go into that box and give evidence, sir,”
he said. “I suggest that now—following upon the evidence you have
just heard—is a favourable stage for hearing him.”
The Coroner, an elderly man, leant back in his chair, took off his
spectacles, and glanced at Walkinshaw and from him to his client.
“I suppose that Mr. Harborough fully understands that he is not
bound to answer any questions that—answered in a certain fashion
—might incriminate him?” he suggested. “Of course, if he wishes to
make a statement.”
“What my client desires to do, sir,” interrupted Walkinshaw, “is to tell
you and the jury the plain truth about himself and his movements in
relation to this enquiry. He has nothing to conceal and he has
everything to gain by telling the truth.”
“Very well,” said the Coroner. “Let us have his evidence now.”
Walkinshaw turned to Harborough and motioned him to go into the
box.
CHAPTER VIII
 THE INCRIMINATING LETTER
But before Harborough reached the witness-box a new development
arose. The Chief Constable who, since Mrs. Tretheroe stepped down,
had been in close conversation with the detective, Blick, left his seat
and going over to the barrister who had examined her, made some
whispered communication to him. Presently the barrister rose and
turned to the Coroner.
“If, as I understand, sir, Mr. Harborough wishes to make a
statement, which, I suppose, will amount to giving evidence about
his movements on the morning of Guy Markenmore’s death,” he
said, “I should like to suggest that before you hear it you should
take the evidence of Detective-Sergeant Blick, who has had this case
in hand since the discovery of the crime. Sergeant Blick will produce
some evidence on which I should like to examine Mr. Harborough. I
submit that this course will be most convenient to everybody,
especially to Mr. Harborough himself and to his legal adviser.”
The Coroner looked at Walkinshaw, who bowed his assent.
“Let us have Detective-Sergeant Blick, then,” said the Coroner.
In company with the rest of the people there he looked with some
curiosity at the detective as he stepped into the box. Most of the folk
present in that room had never seen a detective in their lives. Blick,
they thought, was certainly not at all like what they had conceived
men of his calling to be. He might be thirty years old, but he looked
younger. He had a somewhat cherubic, boyish countenance,
rendered more juvenile still by the fact that he was clean-shaven; he
was very smartly and fashionably dressed in a blue serge suit,
traversed by thin lines of a lighter blue; his linen and neck-wear
proclaimed him a bit of a dandy; his carefully brushed hair, golden in
hue, matched admirably with the pretty glow of his cheeks; his
bright blue eyes, keen and alert, were as striking as the firm lines of
his lips and the square, determined chin beneath them. Altogether,
Blick looked more like a smart young army officer than a policeman,
and the people who had gained their notions of detectives from
sentimental fiction began to feel that somebody had deceived them.
Blick and the barrister confronted each other with glances of mutual
understanding.
“Detective-Sergeant Charles Blick, of the Criminal Investigation
Department, New Scotland Yard, I believe,” said the barrister.
“I am,” answered Blick.
“Tell the Court how you came to be associated with this case.”
“I came down to Selcaster some days ago, in connection with
another matter,” said Blick. “I had to remain in the city—at the Mitre
Hotel. On Tuesday morning, very early, the Chief Constable sent an
officer of his force to me, saying that he had just received news of a
probable murder at a place close by, and asking me to dress and go
with him. I drove with him, the police-surgeon, and a constable, to
Markenmore Hollow. There we found the dead body of a man whom
some of those present recognized as Mr. Guy Markenmore. The Chief
Constable requested me to take charge of matters; since then he
has obtained permission from my Department for me to take this
case in hand.”
“With a view of finding the murderer?”
“With that object, certainly.”
“You have heard the evidence of the previous witnesses, Blick?—I
refer especially to that of Hobbs, of the Markenmore policeman, and
of the doctor?”
“I have.”
“All correct.”
“Quite correct.”
“After taking charge of matters, did you accompany the body here to
Markenmore Court?”
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