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Methods in
Molecular Biology 2276
Volkmar Weissig
Marvin Edeas Editors
Mitochondrial
Medicine
Volume 2: Assessing Mitochondria
Second Edition
METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK
For further volumes:

http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
the first to introduce the step-by-step protocols approach that has become the standard in all
biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-
step fashion, opening with an introductory overview, a list of the materials and reagents
needed to complete the experiment, and followed by a detailed procedure that is supported
with a helpful notes section offering tips and tricks of the trade as well as troubleshooting
advice. These hallmark features were introduced by series editor Dr. John Walker and
constitute the key ingredient in each and every volume of the Methods in Molecular Biology
series. Tested and trusted, comprehensive and reliable, all protocols from the series are
indexed in PubMed.
Mitochondrial Medicine
Volume 2: Assessing Mitochondria
Second Edition
Edited by
Volkmar Weissig
Department of Pharmaceutical Sciences, Midwestern University, Glendale, AZ, USA
Marvin Edeas
Cochin Hospital, Cochin Institute, INSERM U1016, PARIS, France
Editors
Volkmar Weissig Marvin Edeas
Department of Pharmaceutical Sciences Cochin Hospital
Midwestern University Cochin Institute, INSERM U1016
Glendale, AZ, USA PARIS, France
ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-1265-1 ISBN 978-1-0716-1266-8 (eBook)
https://doi.org/10.1007/978-1-0716-1266-8
© Springer Science+Business Media, LLC, part of Springer Nature 2021

This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part
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Preface
It is our distinct pleasure to present the second edition of MiMB Mitochondrial Medicine to
the ever increasing number of scientists and physicians who are as fascinated by this tiny
organelle as we are. We started working on the first edition in September 2014 and were able
to bring about one year later two volumes with a total of 70 chapters to the market. As of
today (July 2020), 195K downloads have been recorded for Volume I1 and 90K downloads
for volume II2. In light of the rapidly growing and expanding field of Mitochondrial
Medicine, we readily accepted the invitation to compile a second edition, which we started
to work on in March 2019. This second edition as offered here involves a total of 88 chapters
with 45 of them written by new contributors who were not part of our first edition. The first
and second editions combined subsequently present work from 115 mitochondrial labora-
tories from around the globe. We therefore believe these five volumes combined to be the
most comprehensive source of know-how in the wide-ranging field of Mitochondrial
Medicine.
Dividing 87 chapters equally over three volumes proved to be a bit challenging. We
chose the subtitle Targeting Mitochondria for volume I, Assessing Mitochondria for volume
II, and Manipulating Mitochondria and Disease Specific Approaches for volume III while of
course being well aware of significant overlaps between these three areas of research. For
example, it is quite obvious that mitochondria are being targeted for the purpose of either
assessing them or to manipulate them. We therefore ask all authors not to be too critical
regarding the placement of their particular chapter. The reader we believe will anyway
choose to download a chapter of his/her interest quite independently of its placement in
one of the three volumes.
All chapters in these three volumes were written for graduate students, postdoctoral
associates, independent investigators in academia and industry as well as physicians by
leading experts in their particular field. We are extremely grateful to them for having
found the time to either update their chapter from the first edition or to write a new chapter.
We will not forget that for many if not all of our contributors the worldwide COVID-19
pandemic posed additional and unexpected hurdles towards finishing their manuscript in
due time. Thank you to all!
The idea for our original book proposal leading to the first edition of MiMB Mitochon-
drial Medicine originated in our efforts to organize a series of annual conferences on
Targeting Mitochondria (www.targeting-mitochondria.com), the tenth one of which mean-
while has taken place in November 2019 in Berlin, Germany. Due to the ongoing pandemic,
our 11th conference (October 29–30, 2020) will be a virtual one but we are sure it will not
be less exciting than all the previous editions.
Last but not least we would like to sincerely thank John Walker, the series editor of
Methods in Molecular Biology, for having invited us to compile this second edition and for his
1
https://link.springer.com/book/10.1007/978-1-4939-2257-4.
2
https://link.springer.com/book/10.1007/978-1-4939-2288-8.
v
vi Preface
unlimited guidance and help throughout the entire process. We also owe sincere thanks to
Patrick Marton, the Executive Editor of the Springer Protocol Series, for always having been
available in assisting us throughout the entire project.
Glendale, AZ, USA Volkmar Weissig

Paris, France Marvin Edeas
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
1 Mitochondrial Dysfunction in Mitochondrial Medicine: Current

Limitations, Pitfalls, and Tomorrow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Naig Gueguen, Guy Lenaers, Pascal Reynier,
Volkmar Weissig, and Marvin Edeas
2 Preparation of “Functional” Mitochondria: A Challenging Business. . . . . . . . . . . 31
Stefan Lehr, Sonja Hartwig, and Jorg Kotzka
3 Isolation and Quality Control of Functional Mitochondria . . . . . . . . . . . . . . . . . . . 41
Sonja Hartwig, Jorg Kotzka, and Stefan Lehr
4 Purification of Functional Platelet Mitochondria
Using a Discontinuous Percoll Gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Jacob L. Léger, Nicolas Pichaud, and Luc H. Boudreau
5 Mechanical Permeabilization as a New Method for Assessment
of Mitochondrial Function in Insect Tissues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Alessandro Gaviraghi, Yan Aveiro, Stephanie S. Carvalho,
Rodiesley S. Rosa, Matheus P. Oliveira, and Marcus F. Oliveira
6 Analysis of Mitochondrial Retrograde Signaling in Yeast Model Systems . . . . . . . 87
Nicoletta Guaragnella, Maša Ždralević, Zdena Palková,
and Sergio Giannattasio
7 Native Gel Electrophoresis and Immunoblotting to Analyze
Electron Transport Chain Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Gisela Beutner and George A. Porter Jr.
8 Measuring Mitochondrial Hydrogen Peroxide Levels and
Glutathione Redox Equilibrium in Drosophila Neuron Subtypes
Using Redox-Sensitive Fluorophores and 3D Imaging. . . . . . . . . . . . . . . . . . . . . . . 113
Lori M. Buhlman, Petros P. Keoseyan, Kathryn Houlihan,
and Amber N. Juba
9 Assessment of Mitochondrial Cell Metabolism by Respiratory
Chain Electron Flow Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Flavia Radogna, Déborah Gérard, Mario Dicato, and Marc Diederich
10 Whole-Cell and Mitochondrial dNTP Pool Quantification
from Cells and Tissues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Juan C. Landoni, Liya Wang, and Anu Suomalainen
11 Single-Particle Tracking Method in Fluorescence Microscopy
to Monitor Bioenergetic Responses of Individual Mitochondria . . . . . . . . . . . . . . 153
Camille Colin, Emmanuel Suraniti, Emma Abell, Audrey Sémont,
Neso Sojic, Philippe Diolez, and Stéphane Arbault
vii
viii Contents
12 Investigation of Mitochondrial ADP-Ribosylation

Via Immunofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Ann-Katrin Hopp and Michael O. Hottiger
13 Assessment of Mitochondrial Ca2+ Uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
András T. Deak, Claire Jean-Quartier, Alexander I. Bondarenko,
Lukas N. Groschner, Roland Malli, Wolfgang F. Graier,
and Markus Waldeck-Weiermair
14 Assessment of Mitochondrial Membrane Potential and NADH
Redox State in Acute Brain Slices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Andrey Y. Vinokurov, Viktor V. Dremin, Gennadii A. Piavchenko,
Olga A. Stelmashchuk, Plamena R. Angelova, and Andrey Y. Abramov
15 Evaluation of Mitochondria Content and Function in Live
Cells by Multicolor Flow Cytometric Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Hsiu-Han Fan, Tsung-Lin Tsai, Ivan L. Dzhagalov,
and Chia-Lin Hsu
16 Analysis of Mitochondrial Dysfunction During Cell Death . . . . . . . . . . . . . . . . . . . 215
Vladimir Gogvadze and Boris Zhivotovsky
17 Modified Blue Native Gel Approach for Analysis of Respiratory
Supercomplexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
Sergiy M. Nadtochiy, Megan Ngai, and Paul S. Brookes
18 Patch-Clamp Recording of the Activity of Ion Channels
in the Inner Mitochondrial Membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
Piotr Bednarczyk, Rafał P. Kampa, Shur Gałecka,
Aleksandra Se˛k, Agnieszka Walewska, and Piotr Koprowski
19 Assessment of Mitochondrial Protein Glutathionylation
as Signaling for CO Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
Ana S. Almeida, Cláudia Figueiredo-Pereira,
and Helena L. A. Vieira
20 3D Optical Cryo-Imaging Method: A Novel Approach
to Quantify Renal Mitochondrial Bioenergetics Dysfunction . . . . . . . . . . . . . . . . . 259
Shima Mehrvar, Amadou K. S. Camara, and Mahsa Ranji
21 Simultaneous Quantification of Mitochondrial ATP and ROS
Production Using ATP Energy Clamp Methodology . . . . . . . . . . . . . . . . . . . . . . . . 271
Liping Yu, Brian D. Fink, and William I. Sivitz
22 High-Throughput Image Analysis of Lipid-Droplet-Bound
Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
Nathanael Miller, Dane Wolf, Nour Alsabeeh, Kiana Mahdaviani,
Mayuko Segawa, Marc Liesa, and Orian S. Shirihai
23 Cell Energy Budget Platform for Multiparametric
Assessment of Cell and Tissue Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
Dmitri B. Papkovsky and Alexander V. Zhdanov
24 Fluorescence-Based Assay For Measuring OMA1 Activity. . . . . . . . . . . . . . . . . . . . 325
Julia Tobacyk and Lee Ann MacMillan-Crow
25 Studying Mitochondrial Network Formation by In Vivo
and In Vitro Reconstitution Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
Wanqing Du, Xiangjun Di, and Qian Peter Su
Contents ix
26 Extraction of Functional Mitochondria Based on Membrane

Stiffness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
Md Habibur Rahman, Qinru Xiao, Shirui Zhao,
An-Chi Wei, and Yi-Ping Ho
27 A Protocol for Untargeted Metabolomic Analysis: From
Sample Preparation to Data Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
Amanda L. Souza and Gary J. Patti
28 A Method for Analysis of Nitrotyrosine-Containing Proteins
by Immunoblotting Coupled with Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . 383
Matej Kohutiar and Adam Eckhardt
29 In Vivo Visualization and Quantification of Mitochondrial
Morphology in C. elegans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
R. de Boer, R. L. Smith, W. H. De Vos, E. M. M. Manders,
and H. van der Spek
30 Assessing Impact of Platinum Complexes on Mitochondrial
Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
Suxing Jin and Xiaoyong Wang
31 In Silico Modeling of the Mitochondrial Pumping Complexes
with Markov State Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425
Roger Springett
32 Monitoring the Mitochondrial Presequence Import Pathway
In Living Mammalian Cells with a New Molecular Biosensor. . . . . . . . . . . . . . . . . 441
Maxime Jacoupy, Emeline Hamon-Keromen, and Olga Corti
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453
Contributors
EMMA ABELL • Univ. Bordeaux, INSERM, Centre de recherche Cardio- Thoracique de

Bordeaux, U1045 & IHU Liryc, Electrophysiology and Heart Modeling Institute,
Fondation Bordeaux Université, Bordeaux, France; CHU de Bordeaux, Bordeaux, France
ANDREY Y. ABRAMOV • Orel State University, Orel, Russia; UCL Queen Square Institute of
Neurology, London, UK
ANA S. ALMEIDA • CEDOC, Faculdade de Ciência Médicas/NOVA Medical School,
Universidade Nova de Lisboa, Lisboa, Portugal
NOUR ALSABEEH • Department of Physiology, Faculty of Medicine, Kuwait University,
Kuwait City, Kuwait
PLAMENA R. ANGELOVA • UCL Queen Square Institute of Neurology, London, UK
STÉPHANE ARBAULT • NSysA group, Univ. Bordeaux, CNRS, INP Bordeaux, ISM, UMR
5255, Talence, France
YAN AVEIRO • Federal University of Rio de Janeiro, Institute of Medical Biochemistry
Leopoldo de Meis, Rio De Janeiro, RJ, Brazil
PIOTR BEDNARCZYK • Department of Physics and Biophysics, Institute of Biology, Warsaw
University of Life Sciences—SGGW, Warsaw, Poland
GISELA BEUTNER • Department of Pediatrics-Division Cardiology, University of Rochester,
Rochester, NY, USA
ALEXANDER I. BONDARENKO • Gottfried Schatz Research Center for Cell Signaling,
Metabolism and Aging, Molecular Biology and Biochemistry, Medical University of Graz,
Graz, Austria
LUC H. BOUDREAU • Department of Chemistry and Biochemistry, Universite de Moncton,
Moncton, NB, Canada
PAUL S. BROOKES • Department of Anesthesiology, University of Rochester Medical Center,
Rochester, NY, USA
LORI M. BUHLMAN • Arizona College of Graduate Studies, Midwestern University, Glendale,
AZ, USA
AMADOU K. S. CAMARA • Department of Anesthesiology and Anesthesia Research, Medical
College of Wisconsin, Wauwatosa, WI, USA
STEPHANIE S. CARVALHO • Federal University of Rio de Janeiro, Institute of Medical
Biochemistry Leopoldo de Meis, Rio De Janeiro, RJ, Brazil
CAMILLE COLIN • NSysA group, Univ. Bordeaux, CNRS, INP Bordeaux, ISM, UMR 5255,
Talence, France; Univ. Bordeaux, INSERM, Centre de recherche Cardio- Thoracique de
OLGA CORTI • Sorbonne Université, Institut du Cerveau (ICM), Inserm U1127, CNRS
UMR 7225, Paris, France
R. DE BOER • Molecular Biology & Microbial Food Safety, Swammerdam Institute for Life
Sciences (SILS), Faculty of Science (FNWI), University of Amsterdam, Amsterdam, The
Netherlands
W. H. DE VOS • Laboratory of Cell Biology and Histology, Department of Veterinary Sciences,
Antwerp University, Antwerp, Belgium; Cell Systems and Imaging Research Group,
Department of Molecular Biotechnology, Ghent University, Ghent, Belgium
xi
xii Contributors
ANDRÁS T. DEAK • Gottfried Schatz Research Center for Cell Signaling, Metabolism and
Aging, Molecular Biology and Biochemistry, Medical University of Graz, Graz, Austria
XIANGJUN DI • Institute for Biomedical Materials & Devices (IBMD), Faculty of Science,
University of Technology Sydney, Sydney, NSW, Australia
MARIO DICATO • Laboratoire de Biologie Moléculaire et Cellulaire du Cancer, Hôpital
Kirchberg, Luxembourg, Luxembourg
MARC DIEDERICH • College of Pharmacy, Seoul National University, Seoul, South Korea
PHILIPPE DIOLEZ • Univ. Bordeaux, INSERM, Centre de recherche Cardio- Thoracique de
VIKTOR V. DREMIN • Orel State University, Orel, Russia; Aston University, Birmingham,
UK
WANQING DU • State Key Laboratory of Membrane Biology, Tsinghua University-Peking
University Joint Center for Life Sciences, School of Life Sciences, Tsinghua University,
Beijing, China
IVAN L. DZHAGALOV • Institute of Microbiology and Immunology, National Yang-Ming
University, Taipei, Taiwan
ADAM ECKHARDT • Department of Translational Metabolism, Institute of Physiology,
Academy of Sciences of the Czech Republic, Prague, Czech Republic
MARVIN EDEAS • Université de Paris, INSERM U1016, Institut Cochin, CNRS UMR8104,
Paris, France; Laboratory of Excellence GR-Ex, Paris, France
HSIU-HAN FAN • Institute of Microbiology and Immunology, National Yang-Ming
CLÁUDIA FIGUEIREDO-PEREIRA • CEDOC, Faculdade de Ciência Médicas/NOVA Medical
School, Universidade Nova de Lisboa, Lisboa, Portugal
BRIAN D. FINK • Division of Endocrinology and Metabolism, Department of Internal
Medicine, The University of Iowa, Iowa City, IA, USA
SHUR GAŁECKA • Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental
Biology, Warsaw, Poland
ALESSANDRO GAVIRAGHI • Federal University of Rio de Janeiro, Institute of Medical
DÉBORAH GÉRARD • Laboratoire de Biologie Moléculaire et Cellulaire du Cancer, Hôpital
SERGIO GIANNATTASIO • Institute of Biomembranes, Bioenergetics and Molecular
Biotechnologies, CNR, Bari, Italy
VLADIMIR GOGVADZE • Division of Toxicology, Institute of Environmental Medicine,
Karolinska Institutet, Stockholm, Sweden; Faculty of Medicine, MV Lomonosov Moscow
State University, Moscow, Russia
WOLFGANG F. GRAIER • Gottfried Schatz Research Center for Cell Signaling, Metabolism and
LUKAS N. GROSCHNER • Gottfried Schatz Research Center for Cell Signaling, Metabolism
and Aging, Molecular Biology and Biochemistry, Medical University of Graz, Graz,
Austria
NICOLETTA GUARAGNELLA • Institute of Biomembranes, Bioenergetics and Molecular
Biotechnologies, CNR, Bari, Italy; Department of Biosciences, Biotechnology and
Biopharmaceutics, University of Bari “A. Moro”, Bari, Italy
Contributors xiii
NAIG GUEGUEN • UMR CNRS 6015-INSERM U1083, MitoVasc Institute, University of

Angers, Angers, France; Department of Biochemistry and Genetics, University Hospital of
Angers, Angers, France
EMELINE HAMON-KEROMEN • Sorbonne Université, Institut du Cerveau (ICM), Inserm
U1127, CNRS UMR 7225, Paris, France
SONJA HARTWIG • Institute for Clinical Biochemistry and Pathobiochemistry, German
Diabetes Center at the Heinrich-Heine-University Duesseldorf, Leibniz Center for Diabetes
Research, Duesseldorf, Germany; German Center for Diabetes Research (DZD e.V.),
Neuherberg, Germany
YI-PING HO • Department of Biomedical Engineering, The Chinese University of Hong
Kong, Hong Kong SAR, China; Centre for Novel Biomaterials, The Chinese University of
Hong Kong, Hong Kong SAR, China; Shun Hing Institute of Advanced Engineering, The
Chinese University of Hong Kong, Hong Kong SAR, China; The Ministry of Education Key
Laboratory of Regeneration Medicine, The Chinese University of Hong Kong, Hong Kong
SAR, China
ANN-KATRIN HOPP • Department of Molecular Mechanisms of Disease (DMMD), University
of Zurich, Zurich, Switzerland
MICHAEL O. HOTTIGER • Department of Molecular Mechanisms of Disease (DMMD),
University of Zurich, Zurich, Switzerland
KATHRYN HOULIHAN • Arizona College of Graduate Studies, Midwestern University,
Glendale, AZ, USA
CHIA-LIN HSU • Institute of Microbiology and Immunology, National Yang-Ming
MAXIME JACOUPY • Sorbonne Université, Institut du Cerveau (ICM), Inserm U1127, CNRS
UMR 7225, Paris, France
CLAIRE JEAN-QUARTIER • Gottfried Schatz Research Center for Cell Signaling, Metabolism
and Aging, Molecular Biology and Biochemistry, Medical University of Graz, Graz,
Austria
SUXING JIN • State Key Laboratory of Coordination Chemistry, School of Chemistry and
Chemical Engineering, Nanjing University, Nanjing, P. R. China
AMBER N. JUBA • Arizona College of Graduate Studies, Midwestern University, Glendale,
AZ, USA
RAFAŁ P. KAMPA • Department of Physics and Biophysics, Institute of Biology, Warsaw
University of Life Sciences—SGGW, Warsaw, Poland; Laboratory of Intracellular Ion
Channels, Nencki Institute of Experimental Biology, Warsaw, Poland
PETROS P. KEOSEYAN • Arizona College of Osteopathic Medicine, Midwestern University,
Glendale, AZ, USA
MATEJ KOHUTIAR • Department of Medical Chemistry and Clinical Biochemistry, 2nd
Faculty of Medicine, Charles University in Prague and Motol University Hospital, Prague,
Czech Republic
PIOTR KOPROWSKI • Laboratory of Intracellular Ion Channels, Nencki Institute of
Experimental Biology, Warsaw, Poland
JORG KOTZKA • Institute for Clinical Biochemistry and Pathobiochemistry, German Diabetes
Center at the Heinrich-Heine-University Duesseldorf, Leibniz Center for Diabetes
Neuherberg, Germany
JUAN C. LANDONI • Research Programs Unit, Stem Cells and Metabolism, University of
Helsinki, Helsinki, Finland
xiv Contributors
JACOB L. LÉGER • Department of Chemistry and Biochemistry, Universite de Moncton,

Moncton, NB, Canada
STEFAN LEHR • Institute for Clinical Biochemistry and Pathobiochemistry, German Diabetes
Center at the Heinrich-Heine-University Duesseldorf, Leibniz Center for Diabetes
Neuherberg, Germany
GUY LENAERS • UMR CNRS 6015-INSERM U1083, MitoVasc Institute, University of
MARC LIESA • Division of Endocrinology, Department of Medicine, David Geffen School of
Medicine at UCLA, Los Angeles, CA, USA
LEE ANN MACMILLAN-CROW • Department of Pharmacology and Toxicology, University of
Arkansas for Medical Sciences, Little Rock, AR, USA
KIANA MAHDAVIANI • Department of Medicine, Obesity Research Center, Boston University
School of Medicine, Boston, MA, USA
ROLAND MALLI • Gottfried Schatz Research Center for Cell Signaling, Metabolism and
E. M. M. MANDERS • van Leeuwenhoek Center for Advanced Microscopy, University of
Amsterdam, Amsterdam, The Netherlands
SHIMA MEHRVAR • University of Wisconsin-Milwaukee, Milwaukee, WI, USA
NATHANAEL MILLER • Division of Endocrinology, Department of Medicine, David Geffen
School of Medicine at UCLA, Los Angeles, CA, USA; Department of Medicine, Obesity
Research Center, Boston University School of Medicine, Boston, MA, USA
SERGIY M. NADTOCHIY • Department of Neuroscience, University of Rochester Medical
Center, Rochester, NY, USA; Department of Anesthesiology, University of Rochester
Medical Center, Rochester, NY, USA
MEGAN NGAI • Department of Anesthesiology, University of Rochester Medical Center,
Rochester, NY, USA
MARCUS F. OLIVEIRA • Federal University of Rio de Janeiro, Institute of Medical
MATHEUS P. OLIVEIRA • Federal University of Rio de Janeiro, Institute of Medical
ZDENA PALKOVÁ • Faculty of Science, Charles University, BIOCEV, Prague, Czech Republic
DMITRI B. PAPKOVSKY • School of Biochemistry and Cell Biology, University College Cork,
Cork, Ireland
GARY J. PATTI • Department of Chemistry, Washington University in St. Louis, Saint Louis,
MO, USA; Department of Medicine, Washington University in St. Louis, Saint Louis, MO,
USA
GENNADII A. PIAVCHENKO • Orel State University, Orel, Russia; I.M. Sechenov First Moscow
State Medical University (Sechenov University), Moscow, Russia
NICOLAS PICHAUD • Department of Chemistry and Biochemistry, Universite de Moncton,
Moncton, NB, Canada
GEORGE A. PORTER JR. • Departments of Pediatrics-Division Cardiology, Pharmacology
and Physiology, and Medicine (Aab Cardiovascular Research Institute), University of
Rochester, Rochester, NY, USA
FLAVIA RADOGNA • Laboratoire de Biologie Moléculaire et Cellulaire du Cancer, Hôpital
Contributors xv
MD HABIBUR RAHMAN • Department of Biomedical Engineering, The Chinese University of

Hong Kong, Hong Kong SAR, China; Centre for Novel Biomaterials, The Chinese
University of Hong Kong, Hong Kong SAR, China
MAHSA RANJI • Biophotonics Lab, Florida Atlantic University, Boca Raton, FL, USA
PASCAL REYNIER • UMR CNRS 6015-INSERM U1083, MitoVasc Institute, University of
Angers, Angers, France; Department of Biochemistry and Genetics, University Hospital of
RODIESLEY S. ROSA • Federal University of Rio de Janeiro, Institute of Medical Biochemistry
Leopoldo de Meis, Rio De Janeiro, RJ, Brazil
MAYUKO SEGAWA • Division of Endocrinology, Department of Medicine, David Geffen School
of Medicine at UCLA, Los Angeles, CA, USA
ALEKSANDRA SE˛K • Laboratory of Intracellular Ion Channels, Nencki Institute of
Experimental Biology, Warsaw, Poland; Faculty of Chemistry, University of Warsaw,
Warsaw, Poland
AUDREY SÉMONT • Univ. Bordeaux, INSERM, Centre de recherche Cardio- Thoracique de
ORIAN S. SHIRIHAI • Division of Endocrinology, Department of Medicine, David Geffen
School of Medicine at UCLA, Los Angeles, CA, USA
WILLIAM I. SIVITZ • Division of Endocrinology and Metabolism, Department of Internal
Medicine, The University of Iowa, Iowa City, IA, USA
R. L. SMITH • Academisch Medisch Centrum Universiteit van, Amsterdam, The Netherlands
NESO SOJIC • NSysA group, Univ. Bordeaux, CNRS, INP Bordeaux, ISM, UMR 5255,
Talence, France
AMANDA L. SOUZA • Life Science Mass Spectrometry Division, Thermo Fisher Scientific, San
Jose, CA, USA
ROGER SPRINGETT • CellSpex, Kent, UK
OLGA A. STELMASHCHUK • Orel State University, Orel, Russia
QIAN PETER SU • Institute for Biomedical Materials & Devices (IBMD), Faculty of Science,
University of Technology Sydney, Sydney, NSW, Australia; School of Biomedical
Engineering, Faculty of Engineering and IT, University of Technology Sydney, Sydney,
NSW, Australia
ANU SUOMALAINEN • Department of Neurology, University Hospital, Helsinki, Finland;
Neuroscience Center, University of Helsinki, Helsinki, Finland
EMMANUEL SURANITI • NSysA group, Univ. Bordeaux, CNRS, INP Bordeaux, ISM, UMR
5255, Talence, France
JULIA TOBACYK • Department of Pharmacology and Toxicology, University of Arkansas for
Medical Sciences, Little Rock, AR, USA
TSUNG-LIN TSAI • Institute of Microbiology and Immunology, National Yang-Ming
H. VAN DER SPEK • Molecular Biology & Microbial Food Safety, Swammerdam Institute for
Life Sciences (SILS), Faculty of Science (FNWI), University of Amsterdam, Amsterdam,
The Netherlands
HELENA L. A. VIEIRA • CEDOC, Faculdade de Ciência Médicas/NOVA Medical School,
Universidade Nova de Lisboa, Lisboa, Portugal; UCIBIO, Faculdade de Ciências
e Tecnologia, Universidade Nova de Lisboa, Lisboa, Portugal; Instituto de Biologia
Experimental e Tecnologica (iBET), Oeiras, Portugal
ANDREY Y. VINOKUROV • Orel State University, Orel, Russia
xvi Contributors
MARKUS WALDECK-WEIERMAIR • Gottfried Schatz Research Center for Cell Signaling,

Metabolism and Aging, Molecular Biology and Biochemistry, Medical University of Graz,
Graz, Austria
AGNIESZKA WALEWSKA • Laboratory of Intracellular Ion Channels, Nencki Institute of
Experimental Biology, Warsaw, Poland
LIYA WANG • Department of Anatomy, Physiology and Biochemistry, Swedish University of
Agricultural Sciences, Uppsala, Sweden
XIAOYONG WANG • State Key Laboratory of Pharmaceutical Biotechnology, School of Life
Sciences, Nanjing University, Nanjing, P. R. China
AN-CHI WEI • Graduate Institute of Biomedical Electronics and Bioinformatics, National
Taiwan University, Taipei, Taiwan
VOLKMAR WEISSIG • Department of Pharmaceutical Sciences and Nanocenter of Excellence,
Midwestern University College of Pharmacy at Glendale, Glendale, AZ, USA
DANE WOLF • Division of Endocrinology, Department of Medicine, David Geffen School of
Medicine at UCLA, Los Angeles, CA, USA; Department of Medicine, Obesity Research
Center, Boston University School of Medicine, Boston, MA, USA
QINRU XIAO • Department of Biomedical Engineering, The Chinese University of Hong
Kong, Hong Kong SAR, China
LIPING YU • Division of Endocrinology and Metabolism, Department of Internal Medicine,
The University of Iowa, Iowa City, IA, USA
MAŠA ŽDRALEVIĆ • Faculty of Medicine, University of Montenegro, Podgorica, Montenegro
SHIRUI ZHAO • Department of Biomedical Engineering, The Chinese University of Hong
Kong, Hong Kong SAR, China; Shun Hing Institute of Advanced Engineering, The
Chinese University of Hong Kong, Hong Kong SAR, China
ALEXANDER V. ZHDANOV • School of Biochemistry and Cell Biology, University College Cork,
Cork, Ireland
BORIS ZHIVOTOVSKY • Division of Toxicology, Institute of Environmental Medicine,
Karolinska Institutet, Stockholm, Sweden; Faculty of Medicine, MV Lomonosov Moscow
State University, Moscow, Russia
Chapter 1
Mitochondrial Dysfunction in Mitochondrial Medicine:

Current Limitations, Pitfalls, and Tomorrow
Naig Gueguen, Guy Lenaers, Pascal Reynier, Volkmar Weissig,
and Marvin Edeas
Abstract
Until recently restricted to hereditary mitochondrial diseases, mitochondrial dysfunction is now recognized
as a key player and strategic factor in the pathophysiological of many human diseases, ranging from the
metabolism, vascular, cardiac, and neurodegenerative diseases to cancer. Because of their participation in a
myriad of cellular functions and signaling pathways, precisely identifying the cause of mitochondrial
“dysfunctions” can be challenging and requires robust and controlled techniques. Initially limited to the
analysis of the respiratory chain functioning, these analytical techniques now enlarge to the analyses of
mitochondrial and cellular metabolism, based on metabolomic approaches.
Here, we address the methods used to assay mitochondrial dysfunction, with a highlight on the
techniques used in diagnosis on tissues and cells derived from patients, the information they provide, and
their strength and weakness.
Targeting mitochondrial dysfunction by various strategies is a huge challenge, requires robust methods of
evaluation, and should be able to take into consideration the mitochondria dynamics and localization. The
future of mitochondrial medicine is strongly related to a perfect comprehension of its dysfunction.
Key words Mitochondrial dysfunctions, Mitochondria evaluation, Bioenergetics, Devices,

Metabolomics
1 Introduction
The contribution of mitochondrial dysfunctions in the onset and

development of many diseases has been widely studied. These
dysfunctions could interfere with many cellular, metabolic, and
homoeostatic functions [1]. It is therefore not surprising that,
beyond primary mitochondrial disorders, mitochondrial dysfunc-
tions now impact most areas of medical. Mitochondrial dysfunction
has been found in numerous common human diseases, including
metabolic diseases [2] such as obesity and diabetes, heart diseases
[3], neurodegenerative diseases, such as Parkinson’s, Alzheimer’s
Volkmar Weissig and Marvin Edeas (eds.), Mitochondrial Medicine: Volume 2: Assessing Mitochondria,
Methods in Molecular Biology, vol. 2276, https://doi.org/10.1007/978-1-0716-1266-8_1,
© Springer Science+Business Media, LLC, part of Springer Nature 2021
1
2 Naig Gueguen et al.
[4], or Huntington’s [5], infectious diseases [6], inflammation [7],

and cancer [8].
Multiple reports have shown the impact of dysfunctional mito-
chondria on the immune response. One of the example is the
COVID-19 pandemic caused by the coronavirus (SARS-CoV-2).
Many recent studies revealed that human alveolar epithelial cells
with dysfunctional mitochondria displayed increased production of
pro-inflammatory cytokines which were found to be increased in
COVID-19 [9, 10]. Recently, we proposed that not only the
intracellular mitochondria dysfunction is a consequence of
COVID-19 infection formation [6, 11] but also the less explored
extracellular mitochondria (specifically platelets mitochondria) may
affect blood coagulation, clot, and thrombosis formation [10–
15]. These extracellular mitochondria may represent a crucial inter-
cellular mediators and may serve as strategic therapeutic targets in
COVID-19 pathogenesis [11].
Historically, mitochondrial diseases have been largely addressed
from the point of view of the defect of the respiratory chain and
therefore of a defect in energetic homeostasis. However, mitochon-
dria are now recognized to perform multiple additional cellular
functions beyond energy production. For example, electron trans-
fer chain (ETC) activity regulates the NADH/NAD+ redox state
through NADH oxidation by complex I, [16] which impacts to
hundreds of cellular reactions [17, 18]. Mitochondria are the major
source of reactive oxygen species (ROS) [19], which not only
contribute to normal cell function but also are linked to increased
intracellular oxidative stress. ROS production regulates different
signaling pathways, from metabolic rewiring to inflammatory
response or cell survival [20, 21]. Mitochondria also integrate
main cellular catabolic and anabolic pathways. They host not only
whole or partial components of several converging catabolic cellular
processes, i.e., glucose metabolism and the tricarboxylic acid (TCA)
cycle, and the β-oxidation but also several biosynthetic pathways,
such as folate and sulfur metabolism or heme biosynthesis. In
addition to generating reducing equivalents that feed the
OXPHOS system, the TCA cycle has numerous anabolic roles,
providing precursors for the lipids, proteins, carbohydrates, and
nucleotides biosynthesis [22]. TCA cycle metabolites also act as
signaling molecules, affecting critical cellular processes that con-
tribute to oncogenic transformation, such as nutrient signaling,
HIF1α-dependent metabolic reprogramming, or histone acetyla-
tion and demethylation by acetyl-CoA and α-ketoglutarate, respec-
tively [23]. Mitochondria are also involved in calcium homoeostasis
[24], stress response and quality control [25], and initiation of
caspase-dependent apoptosis [26].
Developing precise technologies to study mitochondrial physi-
ology is getting much more important as the prevalence of com-
mon and inherited mitochondrial disease increases. Targeting
Mitochondrial Dysfunction in Mitochondrial Medicine: Current Limitations. . . 3
mitochondrial dysfunction by various strategies is a huge challenge

and need a robust methods of evaluation [27].
Here, we address the techniques commonly used in clinical
diagnosis, the information that can be inferred from them, their
limitations, pits, and pitfalls.
2 Methods to Assess Mitochondrial Dysfunctions
2.1 Basis Principles of mitochondrial bioenergetics have been largely

in Bioenergetic reviewed over years [28]; therefore, only a rapid overview will be
given here. Mitochondria are surrounded by the outer (OMM) and
inner (IMM) membrane. The inner membrane is largely imper-
meant and constitutes the major barrier between the cytosol and
the mitochondrial matrix. IMM forms multiple cristae, which host
the ETC complexes and the enzymes involved in ATP synthesis, the
F0F1-ATP synthase, the adenine nucleotide translocator (ANT),
and the inorganic phosphate (Pi) transporter (PiC).
The ETC system is composed of multisubunits enzymes func-
tionally and physically linked together, the complex I (CI, NADH
ubiquinone reductase), complex II (CII, succinate ubiquinone
reductase), complex III (CIII, ubiquinol cytochrome c reductase),
and complex IV (CIV, cytochrome c oxidase), which transfers the
electron energetic potential from NADH/NAD+ (CI) and
FADH2/FAD+ (CII) to the electrochemical proton gradient
known as the protonmotive force (Δp). This process involved a
series of oxidoreductase reactions in which electrons flow sequen-
tially “downhill” along the ETC from a reduced to an oxidized
state, ending to molecular oxygen reduction to a water molecule, or
“respiration.” The release of free energy during electron transfer
drives the proton pumping across the IMM at complexes CI, CIII,
and CIV, in turn producing the Δp. Other oxidoreductases of the
ETC are unable of proton pumping, particularly the complex II,
the glycerol phosphate dehydrogenase, and the electron-
transferring flavoprotein quinone oxidoreductase (ETF) linked to
fatty acid β-oxidation. Thus, ten protons are extruded for each
electron pair transferred from NADH to oxygen, or six protons
for each electron pair transferred from FADH2 to O2, meaning that
the H+/O2 and ATP/O2 stoichiometries differ according to the
initial substrates.
The fueling of reducing molecules, namely NADH and
FADH2, to the ETC is mainly ensured by the TCA cycle and the
β-oxidation pathways. According to the anaplerotic routes which
feed TCA or the relative contribution of TCA or β-oxidation, the
resulting NADH/FADH2 ratio differs, ultimately modifying the
efficiency of ATP synthesis. The substrate oxidation module con-
sists of all these reactions involved in substrate metabolisms and
electron transport, finally generating Δp.
The Δp is composed of the charge (Δψm) and chemical (ΔpH)

components. The energy available in Δp drives the synthesis and
transport of ATP, as the protons return to the matrix through the
ATP synthase [29]. The ATP synthesis module includes, in addition
to the ATP synthetase, the ANT that exchange the ADP/ATP and
the PiC. The Δp is central to the physiological functions of mito-
chondria, connecting the substrate oxidation module to the ATP
synthesis. Under most circumstances, proton flux is tightly coupled
to OXPHOS. However, the Δp can also be dissipated by proton
leak [30], in which protons return into the matrix independently of
ATP synthesis, or be used for transport processes across the IMM of
metabolite, ions, or calcium [31].
The total proton entry and extrusion are exactly balanced
under steady-state conditions. Forward flux through ETC com-
plexes requires a thermodynamic disequilibrium, i.e., the free
energy available from electron transfer must be greater than that
required to pump protons against the Δp. The Δp variations thus
“dictate” the ETC activity: any decrease in the Δp is followed by an
increase in electron transfer and proton pumping. In other words,
the net flux of the proton current is reflected by the rate of mito-
chondrial oxygen consumption. Decrease in the Δp results from
either proton leak or ATP synthesis, which is regulated by the
cellular ATP demand.
Any alteration in one the above described processes leads to a
mitochondrial dysfunction. Subsequently, we shall describe the
conventional methods for dissecting these processes, and identify-
ing the primary site of impairment, starting with the methods used
in the laboratory for the biochemical diagnosis of mitochondrial
diseases. A complete set of these analyses will draw a picture of the
mitochondrial energy-generating system.
2.2 Analyses Evaluation of individual respiratory chain complex activities is a

of Maximal ETC routine biochemical approach for the diagnosis of mitochondrial
Activities disorders. Assays to quantify CI, CII, CIII, CIV, and ATP synthase
enzymatic activities are performed either on tissue, mostly muscle
or liver biopsies, or on cells as primary fibroblasts or lymphocytes.
They require the preparation of tissue homogenates, after the
elimination of cell debris and nuclei, or cell lysates, from either
fresh or frozen sample. However, frozen samples are more com-
monly used because fresh samples must be processed immediately
after sampling.
Isolated activity of each complex can be analyzed by following
the oxidation/reduction of specific substrates or substrate analogs
by spectrophotometry. The spectrophotometric enzyme assays are:
l NADH:ubiquinone oxidoreductase (NUR, EC 7.1.1.2, NADH
oxidation followed at 340 nm) for CI.
l Succinate:ubiquinone oxidoreductase (coupled to

2,6-Dichloroindophenol reduction followed at 600 nm) for
CII (SUR, EC 1.3.5.1).
l Succinate:cytochrome c oxidoreductase (CII + CIII, cyto-
chrome c reduction at 550 nm).
l Ubiquinol cytochrome c oxidoreductase for CIII (UCCR, EC
1.10.2.2, cytochrome c reduction at 550 nm).
l Cytochrome c oxidase for CV (COX, EC 1.9.3.1, cytochrome c
oxidation at 550 nm).
The measurement of complex V (oligomycin-sensitive ATPase)
is more challenging and requires mitochondrial-enriched fractions
and is often measured in cultured skin fibroblasts. Citrate synthase
activity is often used for normalization of respiratory chain complex
activities to mitochondrial mass. The activities normalized to CS
and/or the ratios between mitochondrial enzymes give a much
narrower range of normal values compared to activities expressed
with respect to sample protein content, since the mitochondrial
mass are highly variable among individuals and subjected to mito-
chondrial biogenesis regulation.
While the principles of the different protocols are similar, now-
adays, there is no universal assay for spectrophotometric quantifica-
tion of ETC enzyme activities. According to laboratories, assays
differ with respect to buffer conditions, reaction temperatures,
substrate concentrations, supplementation in Ca2+ chelator
(EDTA), or addition of bovine serum albumin. However, in an
attempt to facilitate interlaboratory comparison of the results, some
reference centers for the diagnosis of mitochondrial diseases have
reevaluated and standardized their protocols [32].
Accurate enzymatic testing of tissue and cell samples can be
hampered by different pitfalls. A frequent cause for problems is
sample collection and handling. For optimal preservation of mito-
chondrial enzymes, samples must be immediately snap-frozen after
collection and stored at 80 C, without any thawing until analysis.
Other artifacts come from local anesthetic contamination of the
sample, particularly lidocaine [32, 33], or, for studies on animals,
from euthanasia procedures using CO2. Furthermore, one of the
main difficulties in achieving reliable and reproducible dosages is
the quality of the reagents, with strong variability between reactive
references or even batches of reactive. Thus, these dosages require
the inclusion of quality controls in each series, in order to validate
the reaction mix. This quality is usually obtained from culture cells
or animal tissue [34, 35], since the human tissues are limited.
In addition, assays that determine the amounts of OXPHOS
complexes, such as blue native (BN) gel electrophoresis followed by
Western blot analysis, should be performed to decipher an ETC
defect related to decreased catalytic activity or involving a lower
enzyme quantity.
2.3 Structural ETC enzymes are all multimeric and encoded by both the mtDNA
Analyses: BN-PAGE and nDNA, except for complex II that is fully encoded by the
nuclear genome. Their assembly into a functional complex requires
an intricate process assisted by chaperones [36]. Nowadays, the
mechanisms of assembly for each complex are almost completely
resolved [36, 37]. These assembly processes require the striking
coordination of intra- and extramitochondrial transcription, trans-
lation, protein import into mitochondria, and protein folding and
incorporation into assembly intermediates, up to the final complex
assembly. Any disturbance in one of these processes would alter
complexes assembly and compromised ETC function and energy
production. Mutations that impair this assembly process are a
frequent cause of mitochondrial inherited diseases [38, 39]. Com-
plex misassembly is not only associated with an increasing number
of mutations in ETC subunit genes or chaperones [40] but CI
misassembly has also been recently observed in neurodegenerative
diseases such as Alzheimer’s and Parkinson’s diseases [41], thus
broadening the spectrum of assembly defect-associated diseases,
and could be implied in the ageing process [42].
Finally, assembly of CI, III, and IV together leads to respiratory
super complexes, adding a layer of complexity in the structural
organization of the respiratory chain. The crystal structure of the
super complex has recently been elucidated [43]. Although the
pathways that lead to their formation and their function are still
not completely clear [37, 44], they are now recognized as the final
functional ETC unit, or “respirasome.”
“Blue Native” polyacrylamide gel electrophoresis (BN-PAGE)
[45] is a relatively easy approach for analyzing the assembly and
abundance of OXPHOS complexes for the diagnosis of mitochon-
drial diseases [46, 47]. The assembly profile provides information
for identifying disease-causing mutations and facilitates molecular
investigation by highlighting potentially involved subunits
[39, 48].
BN-PAGE is performed on isolated mitochondria or enriched-
mitochondrial fractions from either tissues or cells. The protocols
are described in the literature [46, 47, 49]. However, it should be
stressed that results depend on the choice and concentration of the
detergent and on the choice of the antibodies. After mitochondrial
isolation, the mitochondrial membranes are solubilized using non-
ionic detergent. Because mitochondrial membranes have low cho-
lesterol but high cardiolipin contents, [50], digitonin is
preferentially used for super complexes analysis while
β-dodecylmaltoside should be used for isolated complex visualiza-
tion. However, both the concentration and the detergent to pro-
tein ratio must be carefully optimized to ensure that detergent
concentration is above critical micelle concentration but below
the detergent:protein ratio that would solubilize the complex-
bound cardiolipin, which are critical for their stability and activity.
In addition, after transfer of the electrophoretic gels for Western

blotting, the choice of the antibodies will determine whether the
quantification of the abundance of the fully assembled complex is
privileged or the detection of assembly intermediates. In principle,
any subunit can be targeted and would allow the detection of the
assembled complex, provided that the epitope is accessible for the
antibody. However, according to the location of the subunit within
the complex and the incorporation step during the assembly pro-
cess, the assembly intermediates which could be detected and their
number will vary (Fig. 1). Moreover, due to the relatively low
dynamic range of chemiluminescence detection, the high intensity
of the signal produced by the holoenzyme could blunt other bands
of lower abundance, as the assembly intermediates. To address this
issue, the detection of assembly factors that specifically bind inter-
mediates, but detach once the holoenzymes are fully assembled
should be preferred. In this case, only the assembly intermediates
will be visualized. An example of CI assembly analysis using anti-
bodies targeting assembly factors is illustrated in Fig. 1.
It is therefore highly recommended to master the assembly
pathway of the complex of interest and the location of the different
subunits and assembly factors within the complex structure/inter-
mediates before performing a BN-PAGE analysis.
However, a normal respiratory chain enzyme activity/assembly
does not exclude a functional impairment of the respiratory chain
functioning, which may not be detectable by enzymatic measure-
ments or assembly analyses.
2.4 Functional Respiration studies are an efficient way to analyze ETC and meta-
Analyses: bolic activities of cells or tissues. The last, irreversible, step of
Respiration Rates electron transfer along the ETC is the transfer catalyzed by the
CIV of four electrons to a molecule of oxygen to generate two
molecules of water. The coupling between electron transport (oxi-
dation) and proton pumping within ETC and the tight coupling
between oxidation and phosphorylation through the Δp (51, 52)
mean that the mitochondrial respiration rate is an accurate measure
of the total ETC activity and mitochondrial ATP synthesis rate.
According to the experimental design, information can be gained
on multiple processes required for respiration, including substrate
transport into the mitochondria, reducing equivalent production
by TCA cycle or beta-oxidation and electron delivery to the respi-
ratory chain, activities of the different complexes, ATP synthesis,
proton leak, and mitochondrial metabolism. Beyond metabolic
analysis, O2 consumption is now also used to analyze cytotoxicity.
2.4.1 Devices The rate of mitochondrial O2 consumption could be determined by

a number of methods, the two main approaches are amperometric
O2 sensors [51] and O2-dependent quenching of porphyrin-based
phosphors [52]. The amperometric approach, notably developed
Fig. 1 Detection of CI assembly intermediates by targeting the CI assembly factors. (a) Schematic represen-
tation of the modular assembly of CI The CI holoenzyme results from the sequential assembly of ten
intermediates (A.I.), assisted by at least 13 CI specific assembly factors. The Q earliest intermediate is
composed of the NDUFS2, NDUFS3, NDUFS7, NDUFS8, and NDUFA5, stabilized by two specific assembly
factors, namely NDUFAF3 and NDUFAF4 in a 170 kDa intermediate (1). The binding of Q module to the ND1
subunit and accessory subunits forms the 237 to 283 kDa (2) intermediates named Q/PP-a, which are stacked
to the inner membrane by NDUFAF5 and NDUFAF6. The P module is assembled in four distinct intermediates,
two distal, named PD-a and PD-b that contain ND4 and ND5 subunits, respectively, and two proximal, the PP-a
and PP-b. The central PP-b intermediate (3) is the entry point for four of the seven MT-DNA-encoded subunit,
i.e., ND2, ND3, ND6, and ND4L, and is bound by six assembly factors, among which, NDUFAF1. Then, the
different modules progressively combine (4, 5), forming a Q/P intermediate stacked with the assembly factor
NDUFAF2 (6). In the last step of the process, the 160 kDa N module (7), constituted by the catalytic NDUFV1,
NDUFV2, NDUFS1, and the accessory NDUFA2 and NDUFS4 subunits, is added. Finally, once the CI assembly
is completed, all the assembly factors are released, leaving a functional holoenzyme of 980 kDa (8). Nuclear-
by Oroboros Instrument, has historically been the most common

for in vitro and in vivo investigations of mitochondrial respiration.
Nevertheless, phosphorescent probes are gaining interests since the
introduction of the XF Extracellular Flux Analyzer by Seahorse
Bioscience [53].
The O2K® Oxygraph The O2K® oxygraph developed by Oroboros measures oxygen
consumption by polarography with a Clark’s electrode. Briefly,
oxygen diffuses through a Teflon membrane, which is permeable
to uncharged gases, but not to water. A platinum/silver/KCl-
coupled electrode reduces oxygen and oxidizes silver, giving rise
to a current, which is proportional to oxygen concentration within
the physiological range of measurements. It operates in a closed
chamber of 2 ml, with a stirring magnet to homogenize oxygen and
biological material. The volume can be adjusted according to the
cells, tissues, or mitochondria concentrations to analyze. High-
resolution designs of Oroboros Instruments are optimized for
high sensitivity, precision, and minimal measurement interferences
(typically the detection limit is ~1 pmol/sec/ml and the quantifi-
cation limit is ~2.5 pmol/s/ml, on site evaluations).
Fig. 1 (continued) encoded subunit names were shortened by omitting the leading “NDUF.” Only the main
subunits are indicated. The numbers refer to the corresponding intermediates that can be detected by
BN-PAGE in panel b. (b) Study of CI assembly by BN-PAGE in two control fibroblasts (Ctr), one patient cell
line with a known assembly defect [48] and cells lacking MT-DNA (143B Rho0). (b1) First, CI assembly was
analyzed using “classical” antibodies targeting CI structural subunits located in different intermediates, i.e.,
NDUFS2 (Q, left panel), NDUFB6 (PD-a, middle panel), and NDUFS1 (N, right panel). In Ctr cells, anti-NDUFS2
antibody allowed the detection of the fully assembled holoenzyme (8), while Q/P A.I. (6) accumulated in the
NDUFS4 mutated cells. No CI holoenzyme or A.I. could be detected in Rho0. Further hybridization with anti-
NDUFB6 and NDUFS1 antibodies highlighted the presence of the Pp-b/PD-a at ~700 kDa (5) and the N module
(7), respectively. (b2) A.I. were then analyzed by targeting the assembly factor NDUFAF4 (Q), NDUFAF1 (Pp-b),
and NDUFAF2 (Q/P). The detection of NDUFAF1 revealed a main band at ~400 kDa which matched with the
described size for the Pp-b intermediate and two higher faint bands just below and above 800 kDa that
matched the Q/Pp and Q/P intermediates, respectively. As expected, these intermediates containing the
MT-DNA encoded subunits were not observed in Rho0 cells. NDUFAF4 antibody detected the expected bands
for Q-containing intermediates, i.e., a main one for Q at 170 kDa (1) and fainter ones for Q/Pp-a (at ~240 kDa
and ~280 kDa, (2)), Q/Pp (~750 kDa, (4), and Q/P (~880 kDa, (6)). As expected, only the smallest Q
intermediates, constituted only of nDNA-encoded subunits, were detected in Rho0 cells. The Pp-b intermedi-
ate was detected on the same membrane by hybridizing anti-NDUFAF1 antibody. Finally, additional hybridiza-
tion with NDUFAF2 antibody clearly highlighted the accumulation of the Q/P (6) that occurred in NDUFS4 cells
and highlighted different higher molecular weights up to ~1100 kDa for this intermediate. Thus, since they do
not bind the CI holoenzyme, whose intense signal can blunt the presence of A.I., the targeting of the assembly
factors allows a more sensitive detection of these intermediates. The two combined approaches allow the
identification of seven of the assembly intermediates in addition to the holoenzyme
This is allowed by (1) closed, air-tight reaction chambers;

(2) high sensitivity of the sensors with minimal noise/signal ratio;
and (3) low O2-permeable materials, which minimizes O2 back-
diffusion and overestimations of respiration. In these systems,
instrumental background O2 consumption (i.e., nonbiological O2
flux, which constitutes potential sources of systematic error) can be
corrected for accurate determinations of O2 consumption. The
device can be fully calibrated, both for 0 and 100% oxygen signals
and oxygen concentration within the respiratory medium accord-
ing to the actual PO2 and O2 solubility. Therefore, real-time abso-
lute quantitative measurements of oxygen concentration and fluxes
can be measured, and robust comparison of inter-assays achieved.
Normalization of fluxes to protein content or cell concentration
can be easily achieved by recovering the samples within the chamber
at the end of the experiment.
O2K® oxygraphs are a fully open system, allowing as many
injections of substrates or inhibitors as needed, and reoxygenation
during the experiment time-course if needed, thus enabling
extended substrate–uncoupler–inhibitor–titration protocols to be
applied. Each complex of the ETC can be studied independently,
on the same sample, using different substrate combinations.
The capability of the O2k® system has recently expanded
beyond respiration to permit simultaneous fluorescence-based
measurements, potentiometric measurements of H+ concentra-
tions (i.e., pH), of ΔΨm using triphenylphosphonium or fluores-
cent membrane potential probes, and Ca2+, as well as amperometric
measurement of nitric oxide.
There are however main limitations when considering the use
of Oroboros technology: First, the O2k® is not a high-throughput
technology, as only two samples can be performed at one time, and
it is not automated. Because analysis using substrate–uncoupler–
inhibitor–titration protocols take approximately one hour to com-
plete, large-scale analyses are time-consuming. Second, analysis
requires cell dissociation and suspension. As most cell culture–
based studies are conducted on adherent cells, this system shows
limits when the experimental objective is to assess cellular bioener-
getics in intact cultured cells. According to cell types, cell detach-
ment from the extracellular matrix could alter metabolism [54], in
any way, analyses must be performed immediately and achieved
rapidly after cell detachment. Third, despite the high sensitivity,
the relative high volume of the chambers (500 μl to 2 ml) imposes a
nonnegligible sample quantity. Typically, 10–40 μg of isolated
mitochondria, 1–2 mg permeabilized tissue or 2–6·106 cells,
according to cell types, are needed for one experiment.
The Seahorse® XF The Seahorse® XF Extracellular Flux Analyzer technology is based

on solid state sensor probes for detection of oxygen and H+ con-
centrations, residing 200 μm above the cell/sample monolayer on a
multi-well microplate format. Real-time measurements of oxygen
concentrations are made by isolating a small volume of about 2 μl of
medium above a monolayer of samples in a “transient microcham-
ber” within the microplate. The technology based on fluorescence
quenching is quite sensitive and has been optimized to avoid signal
drifting.
The main advantage of Seahorse® technology relies on its
multi-wells plate format and automated system, allowing high-
throughput analyses. Moreover, the small chamber volume is a
great benefit when limited material is available. Finally, this system
represented a technical breakthrough for the field of bioenergetics
by providing the first instrument with the capacity to measure
in vivo O2 consumption on intact adherent cells in culture. The
XF Analyzer minimizes the potential limitation of oxygen diffusion
for adherent cells by transiently limiting the volume of solution in
which O2 is measured to just above the monolayer of cultured cells.
Moreover, the capability of the system expands beyond respiration
and allows simultaneous measurements of H+ extrusion (i.e., pH),
as a reflect of glycolytic rates.
However, there are main limitations to consider when analyz-
ing mitochondrial respiration using the Seahorse® Analyzer. First,
as for many miniaturized setups, high-throughput methods, the
device loses in accuracy what it gains in rapidity. The biosensors are
calibrated for 100% oxygen, using the supplier solution, not in the
respiratory medium used. The calibration procedure does not con-
sider the real oxygen concentration within the medium (oxygen
solubility within the medium, real barometric pressure during the
experimental course). However, the temporary microenvironment
created over the cell layer is not completely sealed from atmospheric
O2; thus, as O2 concentration declines with time, O2 gradient
favors diffusion from the media to the micro-chambers. O2 gradi-
ent could also favor back-diffusion from the plastic of the wells. As
the device back-diffusion of oxygen is not quantified and back-
ground correction is performed relative to the “background plate
noise” determined on different “control” wells (devoid of sample),
this could lead to an underestimation of cellular respiration rate.
Thus, this system allows quite quantitative determination of O2
variation, but it did not allow absolute quantitative measurements of
O2 concentration and fluxes. Second, inherent to the technologies
based on fluorescence quenching is also the quite high variability
between the different acquisitions (intra-assay variability (intra-
plate, inter-sensors) and more strikingly, inter-plates variability)
[55]. This limits the comparison between different experiments.
Third, the Seahorse® setup is not an open system, and only four
ports surrounding the sensor are available that must be loaded
before starting the analysis. This strongly limits the ETC sub-
strates/inhibitors titration protocols. It is essential to perform
robust optimization steps prior to any respiratory assay: the optimal
concentration of each injectable reagent must be checked for each
experimental condition. Care must be also taken not to overload
the wells, which can cause O2 to quickly become depleted under
phosphorylating conditions, leading to hypoxia. Finally, this device
requires that samples adhere to the bottom of the well, which
corresponds to an advantage when working on adherent cells, but
could require coating of the samples in other conditions.
To resume, when accurate measurement of O2 consumption
and high versatility is aimed, as for diagnosis purpose, high-
resolution O2K® respirometry system should be used. However,
if high-throughput dosages are needed as for the screening of
drugs, or when the sample quantities are limited, the Seahorse®
offers a much higher throughput platform than the traditional
Clark electrode-based systems.
2.4.2 Permeabilized Within tissues or cells, mitochondria are not accessible for many
Tissues and Cells substrates and inhibitors, and the large catabolic processes that
must be considered when dissecting ETC activity and mitochon-
drial metabolism greatly complicated the understanding of the
results.
Therefore, Isolation of mitochondria through tissue/cell cul-
ture homogenization and differential centrifugations is routinely
used for assessment of mitochondrial respiration [56, 57]. Isolated
mitochondria remain one of best approaches for studying mito-
chondrial bioenergetics free from the influence of cellular factors
like the cytoskeleton, endoplasmic reticulum, cellular ATPases,
together with a strict control on substrate supplies. It also allows
the studies of distinct subcellular mitochondria, such as subsarco-
lemmal and intermyofibrillar mitochondria of skeletal muscle,
which display different functional properties [58]. However, the
disadvantages of isolated mitochondria include:
1. The disruptions of mitochondrial structure [59], of mitochon-
drial network, mitochondria–endoplasmic reticulum and mito-
chondria–cytoskeleton interactions, which may impact
function [60–62].
2. The purification process also bias the mitochondrial composi-
tion, by selecting high-density mitochondria, while discarding
less dense ones during differential centrifugation steps [63].
3. The purification process requires relatively large sample sizes
(roughly 20 106 cells, or 100–150 mg wet weight of tissue)
to obtain relevant yields.
4. The loss of micro-compartmentalization and metabolic chan-
neling [64, 65].
An alternative approach when the control of substrate supply is

needed is the use of permeabilized tissues, mostly permeabilized
muscle fibers, or cells using low concentration of plasma-selective
detergents [66]. Saponin and digitonin, a saponin derivate, are
mostly selective for cholesterol lipids, enriched in the plasma mem-
brane. Therefore, the use of saponin (50–100 μg/ml) for tissue
permeabilization or digitonin (10–30 μg/million cells) for cell
permeabilization leaves intact all intracellular structures, including
mitochondria.
Permeabilized cell and skinned fiber techniques have several
advantages for studies of mitochondrial function:
1. very small tissue samples are required;
2. all mitochondria can be investigated;
3. more importantly, mitochondria are studied in their natural
surroundings, as the mitochondrial network is maintained as
well as the potential interactions between mitochondria and
other subcellular structures and organelles.
However, the mitochondrial integrity after sample preparations
must be carefully controlled by checking the absence of any stimu-
latory effect of cytochrome c, witnessing the maintenance of the
mitochondrial membranes’ integrity.
The classical respiration rate experiments to determine mito-
chondrial bioenergetic function were defined by Chance and Wil-
liams [67]. Substrate is added in the respiratory medium (defined as
“state 2,” no ADP), followed by addition of ADP, allowing the
ATP synthase to function. This induces a drop in Δp and thus
accelerates the electron transport and proton pumping (“state
3”). On permeabilized fibers or cells, the presence of cellular
ATPase activity maintains a high ADP/ATP ratio through constant
ATP hydrolysis and subsequent ATP recycling by mitochondrial
synthesis. State 4o is achieved by adding oligomycin, the ATP
synthase inhibitor, to inhibit ATP synthesis, increasing back the
Δp resulting in slowing down the respiration rate. Although state
2 and state 4 respiration are experimentally quite equivalent, state
4 is more conveniently used when referring to respiration under
basal, non-phosphorylating conditions. Then, addition of a proto-
nophore, such as FCCP (carbonyl cyanide-p-trifluoromethoxyphe-
nylhydrazone), gives the uncoupled respiration (“state 3u”).
State 3u is controlled exclusively by substrate oxidation module
(including substrate uptake, activity of the NADH/FADH2 pro-
ducing pathway, e.g., TCA cycle, activity of the ETC complexes,
pool sizes of ubiquinone, and cytochrome c). Inhibition of any of
these processes will decrease the state 3u rate, but due to the
presence of controlled, unlimited substrates, state 3u mainly reflect
the maximal ETC capacity for a given substrate, even if the TCA
cycle activity could be sometimes rate-limiting.
State 3 (ADP) is controlled, depending on the tissue and con-

ditions, by both the ATP synthesis module (ANT, PiC, and ATP
synthase) and substrate oxidation. Indeed, in some mitochondria,
such as those from brown fat or liver, the ATP synthase activity is
limited and displays a striking control over the ETC activity, mean-
ing that the maximal ETC activity can only be reached in uncou-
pling conditions [68]. Comparing state 3u and state 3, when ADP
is added in saturating concentration, will thus provide insight into
the control exerted by the ATP synthesis module on the substrate
oxidation one.
State 4 is controlled predominantly by the proton leak and to a
small extent by the activity of substrate transport. In optimal con-
dition, mitochondria maintain a high Δp which restricts proton
pumping and thus electron transport leading to slowdown of the
respiration rate. An increased state 4o rate for a given substrate
would indicate altered proton leak.
Importantly, the specific subset of complexes and dehydro-
genases engaged by these assays depends on the substrates
provided. These respiratory states can be sequentially measured in
the presence of different combinations of mitochondrial substrates,
allowing multiple metabolic pathways or respiratory complexes to
be probed.
A typical titration protocol used in diagnosis pipelines, using
substrates of CI, CI + CII, CII, and IV, is as followed (Fig. 2):
First, state 2 (non-phosphorylating) respiration is initiated after
adding pyruvate and malate.
Second, the state 3 respirations are detailed for the different
complexes: the CI-linked maximal phosphorylating respiration is
stimulated by saturating ADP concentration (1.5 mM). Succinate
(10 mM) is added to measure the combined CI and CII-linked
respiration with convergent CI + II electron flow into the
Q-junction corresponding to the maximal stimulated phosphory-
lating respiration (OXPHOS capacity). Rotenone is then used to
inhibit CI activity and thus to obtain the maximal CII-linked
phosphorylating respiration.
Third, oligomycin is used to inhibit F0F1-ATP synthase and
state 4o measurement. FCCP is sequentially added to uncouple
mitochondria and measure the CII-linked respiration in state 3u.
Antimycin A (2 μg/ml) is used to inhibit complex III and check for
the non-mitochondrial oxidation. Finally, ascorbate + TMPD, the
artificial complex IV substrates, allow the measurement of maximal
COX-driven respiration rate, after inhibition of complex IV by
potassium cyanide and subtraction of this nonspecific oxidation.
The respiratory control ratio (RCR), defined as the quotient of
maximal state 3 to state 4 respirations, is often used as an index of
mitochondrial coupling of oxidation to phosphorylation for a given
substrate combination. The RCR indeed integrates the increase in
Fig. 2 Typical analyze traces of O2 consumption on permeabilized fibers. (a) Segmental analysis of ETC
function using sequential substrates injections. Mitochondrial oxygen consumption measurements were
performed at 37 C and atmospheric pressure using a high-resolution oxygraph (O2K, Oroboros Instrument,
Innsbruck, Austria). Respiration rates on permeabilized fibers using 50 μg/ml saponin (30 minutes, 4 C) were
measured in respiratory buffer (10 mM KH2PO4, 300 mM mannitol, 10 mM KCl, 5 mM MgCl2, 0.5 mM EGTA,
and 1 mg/ml serum albumin bovine, pH 7.2) using substrates of CI, CI + CII, and CII as followed: first, state
2 (non-phosphorylating) respiration was measured after adding 2.5 mM pyruvate and 5 mM malate. Then, the
CI-linked maximal phosphorylating respiration was stimulated by saturating ADP concentration (1.5 mM) and
3 mM NAD+, added to avoid TCA limitation by NAD+ availability. CI-linked maximal phosphorylating
respiration was further stimulated with 5 mM glutamate, to check for any limitation of substrate availability
by PDH activity. Succinate (10 mM) was then added to measure the combined CI and CII-linked respiration
with convergent CI + II electron flow into the Q-junction corresponding to the maximal stimulated phosphor-
ylating respiration (OXPHOS capacity). Rotenone (5 μM) was used to inhibit CI activity and thus to obtain the
maximal CII-linked respiration. Thirdly, oligomycin (F0F1-ATP synthase inhibitor, 4 μg/ml) and FCCP (carbonyl
cyanide-p-trifluoromethoxyphenylhydrazone, a mitochondrial uncoupler, 1 μM) were sequentially added to
ensure that the cells were fully permeabilized. Antimycin A addition (2 μg/ml) was used to check for the
non-mitochondrial oxidation. CIV maximal respiration was induced with 4 mM ascorbate and 0.3 mM TMPD,
respiration rate in response to Δp use for ATP synthesis and the low
respiration rate linked to proton leak in non-phosphorylating con-
ditions. RCR values depend on almost every OXPHOS functional
aspect and could therefore be a useful indicator of mitochondrial
dysfunction. However, RCR determinations are sensitive to experi-
mental inaccuracies; depending on the quality of sample prepara-
tions and accurate determination of background rate, state 4 rates
can be significantly over/underestimated. Moreover, there are no
absolute RCR values, as these ones are substrate- and tissue-
dependent. For example, substrates such as succinate or glycerol
phosphate translocate fewer protons per electron pair than NADH-
linked substrates, so the maintenance of the same Δp requires a
higher respiration rate. Furthermore, under identical substrate
conditions, different values may be observed for different tissues,
reflecting different substrate oxidation kinetics, endogenous pro-
ton leak, or phosphorylating capacities [68]. Thus, careful cautions
according to the experimental conditions should be recommended
when interpreting RCR values.
Permeabilized cells or fibers are also a useful model to assess the
maximal activity of the main substrate-providing pathway, i.e., TCA
cycle or beta-oxidation activities. Thus, different TCA cycle inter-
mediates or mitochondrial shuttles substrates (malate/aspartate,
glycerol phosphate shuttles) can be used to dissect specific regula-
tions within these pathways. Similarly, the comparison of maximal
respiration rates sustained in the presence of TCA cycle substrates
(malate, pyruvate, glutamate, and succinate for a fully operating
cycle) or in the presence of beta-oxidation substrates (short-chain
or long-chain fatty acids complexed with coenzyme A or
L-carnitine) allows determining the preferred metabolic pathways
according to pathophysiological conditions, treatments, or tissues.
Measurements of maximal respiratory chain complex activities,
assembly, and linked respirations are the cornerstone on which the
biochemical diagnosis of mitochondrial disorders is based. How-
ever, these can usefully be implemented by more integrative meth-
ods, approaching mitochondrial metabolism.
2.4.3 Intact Cells The use of isolated mitochondria or permeabilized cells has been
favored for years. Because the experimenter has control over con-
ditions, i.e., substrates availability and respiratory states, this
remains the method of choice to gain mechanistic insight into
respiration chain function and dysfunctions. However, in the last
Fig. 2 (continued) followed by CIV inhibition using 1 mM KCN and azide. (b) Analysis of O2 consumption linked
to the stimulation of β-oxidation. β-oxidation was stimulated using palmitoyl-L-carnitine, supplemented with
2.5 mM malate (PCM). Antimycin A addition (2 μg/ml) was used to check for the non-mitochondrial oxidation.
CIV maximal respiration was induced with 4 mM ascorbate and 0.3 mM TMPD, followed by CIV inhibition using
1 mM KCN and azide
decades, respiration analyses on intact cells became prominent in

the field. With whole cell models, intrinsic activity of respiratory
chain complexes is difficult to test and respiration rates somehow
difficult to interpret, since their activities are integrated with a
myriad of parameters. However, overriding these complexities is
the obvious higher physiological relevance. Using intact cells, mito-
chondria are present in a physiological environment with preserved
interactions with the rest of the cell. Therefore, this model is ideal
to assess metabolic pathways connected directly or indirectly to
mitochondrial activities.
A typical experiment using intact cells starts by the measure-
ment of respiratory rate in the respiratory medium, e.g., cell culture
medium, without any additions of inhibitors or uncoupler (Fig. 3).
This state is defined as the basal or routine respiration. Routine
respiration is the minimal rate of oxidative metabolism required to
Fig. 3 Typical analysis traces of O2 consumption on intact cells. Mitochondrial oxygen consumption measure-
ments were performed at 37 C and atmospheric pressure using a high-resolution oxygraph (O2K, Oroboros
Instrument, Innsbruck, Austria). Respiration rates on primary fibroblast cells were measured in either DMEM-
F12 medium (3 g/l glucose, 0.3 mM pyruvate) (red traces) or in low-glucose medium (0.5 g/l glucose) (green
Traces). 4 106 cells were added in the oxygraphic chamber and the analysis started with routine respiration
(R) measurement, which is defined as respiration in medium without additional substrates or effectors (cell
endogenous respiration, corresponding to the cellular oxidative metabolism). Then, 1 mM glutamine was
added, to test for cell metabolic dependence to glutaminolysis. F0F1-ATP synthase was inhibited with
oligomycin (4 μg/ml), allowing the measurement of non-phosphorylating respiration (leak respiration). This
non-phosphorylating respiration (O) was subtracted from routine (R) one to calculate the cellular phosphor-
ylating respiration (R-O). This was followed by uncoupling of oxidative phosphorylation by stepwise titration of
FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) up to optimum concentrations allowing the
measurement of the maximal endogenous respiration (cellular oxidative capacity). The part of the maximal
capacity use for oxidative metabolism was calculated as R/F, and the part of the maximal capacity use for
oxidative ATP synthesis was calculated as (R-O)/F. Finally, respiration was inhibited by rotenone and antimycin
A (2.5 μM and 2 μg/ml, respectively) to check for non-mitochondrial oxidation
fulfill energy needs and support cell functions. The routine respira-
tion of most mammalian cells corresponds neither to the state
3 (unlimited availability of substrate and ADP) nor to the state 4o
(unlimited availability of substrate, but no ATP synthesis), but is
generally an intermediate state between these ones, according to
the ATP demand. Routine respiration is usually strongly controlled
by ATP turnover, but in many cultured mammalian cells, aerobic
glycolysis also contributes to the total ATP turnover [69], meaning
that the routine respiration is not equivalent to cell metabolic rate.
Routine respiration is further partly dependent on substrate oxida-
tion (including substrate uptake, TCA cycle, activity of the ETC
complexes. . .) and proton leak [70]. Therefore, routine respiration
could differ according to the substrate availability in the incubation
medium, to the oxidative ATP synthesis needs or in the presence of
any agents decreasing the Δp (uncouplers). For example, routine
respiration can be increased by decreasing glucose concentration
(Fig. 3). A modification in routine rate integrates any of these
changes and is therefore quite difficult to interpret, but the follow-
ing steps can help to elucidate the pathway involved.
Routine respiration measurement is followed by mitochondrial
ATP synthesis inhibition, through oligomycin addition. The result-
ing respiration is defined as leak respiration. Indeed, as for permea-
bilized cells, this respiration rate in the presence of oligomycin is
controlled predominantly not only by the proton leak but also, to a
smaller extend, by the activity of substrate or ions (e.g., Ca2+)
transports and substrate being oxidized. In intact cells, the sub-
strates oxidized by the ETC are not under the control of the
experimenter. Because the proton pumping is not equivalent
according to the substrate used, the respiration rate required to
maintain the Δp is also different. Thus, a modest change in leak
respiration rate may indicate either a change in proton leak or a
change in Δp caused by altered substrate oxidation or transport.
However, a large increase in the respiration rate strongly suggests
uncoupled mitochondria.
This leak respiration rate is subtracted from the preceding
routine state to estimate the in-situ phosphorylating respiration,
i.e., the respiration rate linked to ATP synthesis. However, the
proton leak is voltage-dependent, all the more important as the
ΔΨ is high. Since ATP synthase inhibition results in an increase in
ΔΨ, subtracting the oligomycin-insensitive respiration from the
routine one slightly overvalues the part of proton leak involved in
routine respiration and underestimates ATP synthesis. This remains
nevertheless one of the best approaches to estimate in situ phos-
phorylating respiration when absolute quantitative values are not
needed [71]. Also, calculating the part of the phosphorylating
versus leak respirations within the routine one further helps to
decipher which process explains a change in the routine respiration
rate. While an increase in phosphorylating respiration most likely

reflects the response of oxidative metabolism to increased energy
demand, a decrease in the phosphorylating respiration could illus-
trate a decrease in energy needs, a metabolic rewiring from oxida-
tive metabolism to “anaerobic” glycolytic one (the so-called
Warburg effect), or a blockage in the ATP synthesis module.
The next step involves uncoupler titration, e.g., FCCP titra-
tion, to obtain the maximal uncoupled respiration rate. As for
uncoupled respiration measured on permeabilized cells, this one is
control by the substrate oxidation pathways. However, there is a
marked difference here: while on permeabilized cell, the uncoupled
respiration is measured in the presence of unlimited availability of
defined substrate(s), meaning that uncoupled respiration is mainly
a reflect of maximal ETC capacity; on intact cells, the catabolic
pathways supplying ETC with NADH or FADH2 is under complex
metabolic regulation and can be rate-limiting. Therefore, the
uncoupled respiration rate on intact cells reflects the maximal over-
all substrate oxidation capacity, from substrate uptake and catabo-
lism to ETC activity, achievable by cells under the assay condition.
A decrease in this maximal respiratory capacity is a reliable indicator
of mitochondrial dysfunction but, because of these complexities,
caution should be taken when interpreting which pathway is
responsible for this dysfunction. Comparing the maximal
uncoupled respiration obtained on permeabilized cells versus intact
cells provides further clues to determine whether a reduced ETC
capacity is involved or not.
CCCP and FCCP are proton shuttling compounds selectively
increasing the permeability of lipid membranes to protons. How-
ever, CCCP and FCCP also have high reactivity with thiol groups.
Thiol-combining agents uncouple OXPHOS at low concentrations
but inhibit respiration at high concentrations by the chemical mod-
ification of a small but significant number of mitochondrial thiol
groups [72]. Moreover, sustained perturbation of ΔΨm, which is
normally tightly controlled to ensure cell proliferation and survival,
triggers cellular stress responses, eventually leading to cell apoptosis
[72]. The optimal FCCP/CCCP concentration allowing the maxi-
mal respiration rate recording depends on cell types, medium com-
position (e.g., low glucose versus high glucose, fatty acids, or
not. . .), and mitochondria physiological state (e.g., any drug
which modifies the ΔΨm could change the optimal concentration).
Therefore, careful FCCP/CCCP titration must be performed for
each experimental condition to ensure the full achievement of
uncoupling yet limiting the drop in ΔΨm. When using a Seahorse
Analyzer, only two FCCP injections are allowed, therefore prelimi-
nary experiments must be performed before starting any complete
experiment. Unappropriated uncoupler doses are a common bias
observed when working on intact cells.
Finally, the experiment ends by the addition of ETC inhibitors,

rotenone, and antimycin A to determine residual oxygen consump-
tion. Residual oxidation (Rox) is the respiration due to oxidative
side reactions remaining after full inhibition of the electron transfer
pathway. Mitochondrial respiration is frequently corrected for Rox
as the non-mitochondrial respiration. However, Rox may be partly
related to ROS production which consumes O2 and is increased
upon ETC inhibitor additions [73].
From these different respiratory parameters can be inferred
important ratios, particularly the spare respiratory capacity. The
spare respiratory capacity is the ratio of the routine to the
uncoupled respirations. With the cautions about the determination
of maximum rates described above, spare respiratory capacity indi-
cates the part of the ETC capacity that is used to sustain cell
function, i.e., how close to its bioenergetic limit cells are operating
[74]. While this ratio may be particularly informative, again, given
the complexity of the mechanisms involved, cautions must be taken
when interpreting it. For example, a decrease in spare capacity may
reflect an increase in cellular ATP requirements and a system
increasing its production, or a decrease in the substrate oxidation
capacity that limits uncoupled respiration, depending on whether
routine respiration increases or maximal respiration decreases. Sim-
ilarly, an increase in spare capacity may suggest either a decrease in
energy demand or a metabolic switch toward glycolysis, which then
mostly ensures the ATP production, although the capacity of the
ETC remains unchanged.
When working with intact cells, cell metabolism “determines”
which substrates mitochondria can used. However, the experi-
menter sets the extracellular conditions. The choice of the exact
respiratory medium composition, i.e., the substrates composition
and concentrations, the presence of hormones, growth factors,
cytokines, may determine the outcome of the analysis. This could
be a pitfall, but this also offers the opportunity to perform cell
metabolic phenotyping. High concentration of glucose is often
used in cell culture, but some cells prefer to oxidize other compo-
nents of the medium, such as fatty acids or amino acids, particularly
glutamine. For example, cancer cells undergo profound metabolic
rewiring for rapid growth. They not only could derive most of their
energy from glycolysis rather than OXPHOS but also could depend
on oxidative glutaminolysis or modified TCA cycle activity to sus-
tain their biosynthesis [75]. By modifying the medium composi-
tion, such as the glucose concentration (Fig. 3), or the presence of
glutamine, the experimenter can easily test which specific pathway is
preferentially used by cells to sustain their oxidative metabolism.
2.5 Mitochondrial The ΔΨm is central to the bioenergetic processes. It not only
Membrane Potential provides the driving force for ATP synthesis and dictates the ETC
activity as detailed above but also regulates metabolites transport
across the inner mitochondrial membrane [76], the mitochondrial

NADPH synthesis through transhydrogenase reaction [77], the
mitochondrial calcium buffering capacity [78], the ROS produc-
tion [79], and the cell apoptotic pathway [80].
Because of its apparent simplicity, fluorescent monitoring of
ΔΨm with “mitochondrial membrane potential indicators” is the
most common technique used for monitoring mitochondrial func-
tion at the single cell or even at the single-mitochondrial levels. A
number of cationic fluorescent probes have been developed for
assessing changes in mitochondrial membrane potential in cultured
cells using both microscopy and flow cytometry. This approach and
the advantage/limits of the available dyes have been extensively
reviewed elsewhere [81, 82]. It should be stressed that any of
these cationic dyes, even at low concentration, inhibits the
OXPHOS activity to some extend, particularly the phosphorylating
respiration. Among these one, TMRM seems to display the lowest
side effects [82].
However, this simplicity is counterbalanced by the limited
information that can be inferred from this type of analysis. Indeed,
it is particularly difficult to calibrate the signal measured with these
probes [83–85]. Generally, studies are qualitative, indicating
whether mitochondria are “depolarized” or not, or, at best, relative
(semiquantitative); consequently, discrete variations are difficult to
detect using classical fluorescence approaches, while mitochondrial
ΔΨm is normally maintained by mitochondrial respiration and
therefore only discrete variations occur from state 4 to state 3 tran-
sitions. Moreover, because ΔΨm results from the balance between
ETC activity and the rate of back across into the matrix, its varia-
tions are difficult to interpret in absence of any information on
mitochondrial metabolic states (respiration measurement).
On isolated mitochondria or permeabilized cells, ΔΨm mea-
sures are typically performed using electrodes sensitive to potential
such as triphenylmethyl phosphonium cation (TPP+) [86]. Since
the development of technologies expanded beyond respiration to
allow multiplexing (Oroboros O2K), assessment of respiration
rates and ΔΨm (either fluorescence-based or potentiometric mea-
surements (TPP+)) can be achieved simultaneously. Measurement
of both proton fluxes and ΔΨm enables a full and quantitative
description of ETC functioning and is essential to detect whether
any change in ΔΨm is the primary or a secondary defect due to
ETC inhibition. It allows detecting subtle changes in proton leak
and coupling efficiency between mitochondrial preparations, exper-
imental conditions, or treatments. Because the proton leak is non-
ohmic, increasing disproportionately at high Δp [68], it is more
accurately determined by plotting the proton current (respiration
rate) to voltage (ΔΨm) over a wide range of potentials in the
absence of ATP synthesis [68]. This is achieved by progressively
inhibiting ETC activity, for example, by titrating rotenone with

mitochondria oxidizing NADH, and simultaneously assessing the
respiration rate and ΔΨm in state 4o condition.
Calculation of membrane potential is based on the Nernst
equation and requires an estimate of the matrix volume over
which the probe distributes. Thus, only the TPP+-based measure-
ments enable the calculation of the absolute quantitative values,
while the fluorescence-based technology is restricted to relative
measurements. Moreover, the absolute values of ΔΨm can be cal-
culated on isolated mitochondria, taking into account the “bind-
ing” factor of the TPP+ to membranes, while the absolute values of
ΔΨm cannot be reasonably calculated on permeabilized cells, due
to strongest unspecific binding [87].
Integrating the mechanistic studies of respiration rates and
ΔΨm on permeabilized cells and the metabolic studies of respira-
tion rates on intact cells provide a large picture of bioenergetic
dysfunctions. These approaches can be complemented by metabo-
lite dosages or more broadly by a cellular metabolomic analysis to
obtain a broader picture of the cellular metabolism and its
dysfunctions.
2.6 Metabolites Defects in the mitochondrial energy production often lead to

Dosages higher lactate production due to reduced pyruvate utilization by
and Metabolomics the mitochondria. Elevated lactate levels are easily detected in cell
culture medium or, in vivo, in blood, urine, and/or CSF. Indeed, in
2.6.1 Metabolite cases of respiratory chain defect, the cytosolic, mitochondrial, or
Measurements both, redox states (NAD+/NADH) often increase due to decrease
in mitochondrial NADH oxidation and upregulation of glycolysis
to support ATP needs. These increased redox states can be detected
by the so-called “metabolic indicators,” i.e., an increase lactate/
pyruvate ratio shifted by the higher NADH/NAD cytosolic ratio,
while a shift in the mitochondrial redox state increases the ratio of
the 3-OH-butyrate to acetoacetate [88]. However, these features
are neither specific nor sensitive as a diagnostic test since other
defects in cellular metabolism could increase glycolytic rate and
lactate production.
Amino acid analysis can further reveal elevated alanine, as a
by-product of the transamination of pyruvate by alanine amino-
transferase, and confirm the hyperlactacidemia.
In addition, elevated levels of several other metabolites can be
observed upon ETC impairment. Particularly, increased levels of
TCA cycle intermediates, such as malate, succinate, 2-oxoglutarate,
and fumarate, are useful markers of mitochondrial metabolism
dysfunction [40, 89].
2.6.2 Metabolomics As we enter the new era of omics technologies, targeted metabo-
lomic or global unbiased approaches can strikingly improve the
investigation of the multifaced mitochondrial functions.
Metabolomics is defined as an integrative approach consisting

in the comprehensive analysis of the metabolome comprising
thousands of small molecules present in biological samples. Mass
spectrometry (MS) coupled to liquid or gas chromatography is
among the major analytical tools used in metabolomics. It enables
comprehensive and systematic profiling of disease conditions and is
mostly used for identifying biomarkers or drug targets in mito-
chondrial diseases [90]. Metabolomics is now used to analyze on
a global scale the multitude of downstream effects of mitochondrial
dysfunction, including not only the consequences of energy defi-
ciency but also oxidative stress, NAD+/NADH redox imbalance,
and large metabolic rewiring [91, 92]. For example, disturbed
cellular dNTP pools and one-carbon metabolism have been evi-
denced in diseases associated with mtDNA maintenance defects
[93, 94]. Also, the metabolomic signature of Opa1 deficiency, a
gene involved in mitochondrial fusion, unexpectedly evidenced
aspartate and glutamate depletions, two main precursors of the
nucleotide synthesis pathways, which could partly explain the
mtDNA maintenance defect observed in OPA1 disease [92, 95].
Thus, combining metabolomics and bioenergetics studies is a
promising strategy to improve our understanding of the pathologi-
cal mechanisms beyond the energetic deficiency, which is often
limited in explaining the clinical phenotypes observed in patients
[91, 96], and to identify new routes for therapeutic solutions. This
was recently illustrated by reference [97] which demonstrated that
methionine supplementation induces an upregulation of electron
transport chain activity and respiration, related to an enhancement
of mitochondrial pyruvate uptake and TCA cycle activity using a
yeast model.
3 Discussion
Because of their complex properties and strategic role in cellular

metabolism, understanding mitochondrial functions and dysfunc-
tions in diseases remains a huge challenge. However, the recent
development of novel technologies allowing multiplexed and high-
throughput analyses contributed to improve our capacities for
subtle characterizations of mitochondrial dysfunctions.
Integrating the mechanistic studies of respiration rates and
ΔΨm on isolated mitochondria or permeabilized cells, and meta-
bolic studies using intact cells and metabolomic approaches, now
depicts a large overview of the cell metabolism and open a new area
of mitochondrial medicine. However, integration of different data
set, from the multidisciplinary approaches to create a more com-
plete representation of the pathophysiological mechanisms,
remains challenging and needs the development of robust integra-
tive bioinformatic tools, to predict perturbations and ultimately
translate our knowledge into improved patient care.
Until recent years, the main vision was that the energetic deficit
due to ineffective OXPHOS is the starting point for explaining the
pathophysiology of mitochondrial disease. However, this energetic
deficit alone often failed to explain the broad variability of affected
organs and clinical presentations. Considering the recent progress
resulting from the development of new technologies and omic
approaches, this old view now seems outdated. Thus, mitochondria
are now considered as master regulator of cellular homeostasis,
named mitohormesis; not only do they orchestrate the metabolic
stress response but also the ROS and proteostatic stress response,
among which the unfolded protein response and the integrated
stress response [98, 99].
Nowadays, increasing evidences are accumulating showing that
these stress responses are main contributors to mitochondrial dis-
ease, rather than the OXPHOS defect by itself [100]. These recent
and unexpected developments open exciting perspectives for ther-
apeutic strategies of these disorders.
After all, mitochondria are no more considered as the inside-
cell powerhouse, since forms of extracellular mitochondria can be
found free (free Mitos), enclosed by a membrane as inside platelets
or vesicles, or as cell-free circulating mtDNA [101]. Recently, Al
Amir Dache et al. reported that blood contains intact cell-free full-
length mitochondrial DNA in dense and biologically stable struc-
tures over 0.22 μm in diameter and that these structures have
specific mitochondrial proteins, double membranes, and a mor-
phology resembling that of mitochondria [102]. More experimen-
tal studies suggest that mitochondria may be released and
transferred between cells [102, 103]. These intriguing observations
are only starting to be characterized, while their functions remain
unknown. Whether they can elicit regenerative effects, induce para-
crine or endocrine, pro- or anti-inflammatory immune responses
[20, 104] and more largely participate in the patho-mechanisms of
mitochondrial diseases are actually unsettled.
Furthermore, the biochemical diagnosis of mitochondrial dis-
ease, usually performed on muscle or liver biopsy and primary
fibroblasts cultured from skin biopsies, remains quite an invasive
approach. Determining whether circulating blood mitochondria
could be used to unravel mitochondrial dysfunction or be used as
a biomarker of disease will enhance our diagnosis tools. The poten-
tial role of these intriguing mitochondria or its spinoffs in blood of
patients remains to be elucidated in many pathologies [6, 11, 101].
The future of mitochondrial medicine is undoubtedly linked to
better comprehension of its dysfunction. All new investigational
drugs for the therapy of mitochondrial diseases have the potential
to markedly alleviate clinical symptoms, and none has the capacity
to actually cure a particular mitochondrial disease
permanently [27].
Acknowledgments
The authors thank the following institutions and patient associa-

tions: Université d’Angers and CHU d’Angers, Fondation Mala-
dies Rares and UNADEV.
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stress response and mitochondrial myopathy
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“In bed as he was left,” said Jason. “I went in this morning, while
you were asleep, and found him—ah, he looks horrible,” he cried,
and broke off with a shudder.
I did not shrink; I felt braced up to any ordeal.
They were all in the room when we entered it. My father, Dr.
Crackenthorpe, Zyp—even old Peggy, who was busying herself, with
the vulture relish of her kind, over the little artificial decencies of
dress and posture that seem such an outrage on the solemn
unresistance of the dead.
Directly we came in Zyp ran to Jason and clung to him sobbing. I
noticed it with a sort of dull resignation, and that was all; for Peggy,
who had drawn a sheet over the lifeless face, pulled it down that I
might look.
Then, for all my stoicism, I gave a cry.
I had left my brother the night before tired, needing rest, but, save
for the extra pallor of his complexion that never boasted a great deal
of color, much like his usual self. Now the dead face lying back on
the pillows was awful to look upon. Spots and bars of livid purple
disfigured its waxen whiteness—on the cheeks, the ears, the throat,
where a deep patch was. It was greatly swollen, too, and the mouth
so rigidly open that it had defied all effort to bind it close. A couple of
pennies, like a hideous pair of glasses, lay, one over each eye,
where they could only be kept in position by means of a filament
drawn tightly round the head. The hands, stiffly crossed, with the
fingers crooked like talons, lay over the breast, fastened into position
with a ligature.
I turned away, feeling sick and faint. I think I reeled, for presently I
found that Dr. Crackenthorpe was supporting me against his arm.
“Oh, why is he like that?” I whispered.
“’Tis a common afterclap in deaths by drowning,” said he,
speaking in a loud, insistent voice, as if not for the first time. “A
stoppage—a relapse. During the weak small hours, when the
patient’s strength is at its lowest, the overwrought lungs refuse to
work—collapse, and he dies of suffocation.”
He looked at my father as he spoke, but elicited no response. It
was palpable that the heavy potations of the night had so deadened
the latter’s faculties as to make him incapable for the moment of
realizing the full enormity of the sight before him.
“Mark me,” said the doctor; “it’s a plain case, I say, nothing out of
the way; no complications. The wretched boy to all intents and
purposes has been drowned.”
“Who drowned him?” said my father. He spoke thickly, stupidly; but
I started, with a dreadful feeling that the locked jaws must relax and
denounce me before them all.
Seeing his hopeless state, the doctor took my father’s arm and led
him from the room. Zyp still clung to my brother.
“Cover it up,” whispered Jason. “He isn’t a pretty sight!”
“He wasn’t a pretty boy,” muttered Peggy, reluctantly hiding the
dreadful face; “To a old woman’s view it speaks of more than his
deserts. Nobody’ll come to look at me, I expect.”
“You heard what the doctor said?” asked Jason, looking across at
me.
“Yes.”
“Drowned—you understand? Drowned, Renny?”
“Drowned,” I repeated, mechanically.
“Come, Zyp,” he said; “this isn’t the place for you any longer.”
They passed out of the room, she still clinging to him, so that her
face was hidden.
I did not measure his words at that time. I had no thought for nice
discriminations of tone; what did I care for anything any longer?
Presently I heard old Peg muttering again. She thought the room
was emptied of us and she softly removed the face cloth once more.
“Ay, there ye lies, Modred—safe never to spy on poor old
Rottengoose again! Ye were a bad lot, ye were; but Peg’s been
more’n enough for you, she has, my lad.”
Suddenly she saw me out of the tail of her eye, and turned upon
me, livid with fury.
“What are ye listening to, Renalt? A black curse on spies, Renalt, I
say!”
Then her manner changed and she came fawning at me
fulsomely.
“What a good lad to stay wi’ his brother! But Peg’ll do the tending,
Renalt. She be a crass old body and apt to reviling in her speech,
but she don’t mean it, bless you; it’s the tic doldrums in her head.”
I repelled the horrible old creature and fled from the room. What
she meant I neither knew nor cared, for we had always looked upon
her as a feckless body, with a big worm in her brain.
All the long morning I wandered about the house, scarcely
knowing what I did or whither I went. Once I found myself in the
room of silence, not remembering when I had come there or for what
reason. The fact, merely, was impressed upon me by a gradual
change in the nature of my sensations. Something seemed to be
asking a question of me which I was striving and striving to answer. It
didn’t distress me at first, for a nearer misery overwhelmed
everything, but by and by its insistence pierced a passage through
all dull obstacles, and the something took up its abode in me and
reigned and grew. I felt myself yielding, yielding; and strove now to
beat off the inevitable horror of the answer that was rising in me. I
did not know what it was, or the question to which it was a response
—only I saw that if I yielded to it and spoke it, I should die then and
there of the black terror of its revelation.
I sprung to my feet with a cry, and saw, or thought I saw, Modred
standing by the water wheel and beckoning to me. If I had strength
to escape, it was enough for that and no more, for everything
seemed to go from me till I found myself sitting at the foot of the
stairs, with Jason looking oddly down upon me.
“I needn’t get up,” I said. “Modred isn’t dead, after all.”
I think I heard him shout out. Anyhow, I felt myself lifted up and
carried somewhere and put down. If they had thought to restrain me,
however, they should have managed things better; for I was up in a
moment and out at the window. I had often thought one wanted only
the will to forget gravity and float through the air, and here I was
doing it. What a glorious sensation it was! I laughed to think how
long I had remained like a reptile, bound to the plodding miserable
earth, when all the time I had power to escape from myself and float
on and on far away from all those heart-breaking troubles. If I only
went very swiftly at first I should soon be too distant for them to track
me, and then I should be free. I felt a little anxious, for there was a
faint noise behind me. I strove to put on pace; if my limbs had
responded to my efforts no bird could have outstripped me. But I saw
with agony that the harder I fought the less way I made. I struggled
and sobbed and clutched myself blindly onward, and all the time the
noise behind grew deeper. If I pushed myself off with a foot to the
ground I only floated a very little way now. Then I saw a railing and
pulled myself along with it toilsomely, but some great pressure was
in front of me and my feet slipped into holes at every step. Panting,
straining, slipping, as if on blood—why! It was blood! I had to yield at
last.
My passion of hope was done with. I lay in a white set horror, not
daring to move or look. How deadly quiet the room was, but not for
long, for a little stealthy rustle of the sheet beside me prickled
through my whole being with its ghastly stirring. Then I knew it had
secretly risen on its elbow and was leaning over and looking down
upon me. If I could only perspire, I thought, my bonds would loosen
and I could escape from it. But it was cunning and knew that, too,
and it sealed all the surface of my skin with its acrid exhalations.
Suddenly it clutched me in its crooked arms and bore me down,
down to the room of silence. There was a sickening odor there and
the covering of the wheel was open. Then, with a shudder, as of
death, I thought I found the answer; for now it was plain that the
great wheel was driven by blood, not water. As I looked aghast,
straining over, it gave me a stealthy push and, with a shriek, I
splashed among the paddles and was whirled down. For ages I was
spun and beaten round and round, mashed, mangled, gasping for
breath and choked with the horrible crimson broth that fed the insane
and furious grinding of the wheel. At the end, glutted with torture, it
flung me forth into a parching desert of sand, and, spinning from me,
became far away a revolving disk of red that made the low-down sun
of that waste corner of the world.
I was alone, now—always alone. No footsteps had ever trod that
trackless level, nor would, I knew, till time was ended. I had no hope;
no green memory for oasis; no power of speech even. Then I knew I
was dead; had been dead so long that my body had crackled and
fallen to decay, leaving my soul only, like the stone of a fruit, quick
with wretched impulse to shoot upward but dreadfully imprisoned
from doing so.
Sometimes in the world the massive columns of the cathedral had
suggested to me a like sensation; a moral impress of weight and
stoniness that had driven me to bow my head and creep, sweating
away from their inexorable stolidity. Now I was built into such a body
—more, was an integral part of it. Yet could my pinioned nerves
never assimilate its passionless obduracy, but jerked and struggled
in agony to be free. Oh, how divine is the instinct that paints heaven
all light and airiness, and innocent forevermore of the sense of
weight!
Suddenly I heard Zyp’s voice, singing outside in the world, and in
a moment tears, most blessed, blessed tears, sprung from my eyes
and I was free. The stone cracked and fell asunder, and I leaped out
madly shrieking at my release.
She was sitting under a tree in a beautiful meadow and her young
voice rose sweetly as she prinked her hat with daisies and yellow
king-cups. She called me to her and gave me tender names and
smoothed away the pain from my forehead with kisses and the
cunning of her elfish brown hand.
“Come, drink,” she said, “and you will be better.”
I woke to life and looked up. She was standing by my bed, holding
a cup toward my lips, and at the foot Jason leaned, looking on.
“Have I been ill?” I said, in a voice so odd to me that I almost
laughed.
“Yes, yes—a little; but you have come out of the black pit now into
the forest.”
CHAPTER X.
JASON SPEAKS.
For some three weeks I had lain racked and shriveled in a

nervous, delirious fever. It left me at last, the ghost of my old self, to
face once more the problems of a ruined life. For many days these
gave me no concern, or only in a fitful, indifferent manner. I was
content to sip the dew of convalescence, to slumber and to cherish
my exhaustion, and the others disturbed me but little. My recovery
once assured, they left me generally to myself, scarce visiting me
more often than was necessary for the administering of food or
medicine. Sometimes one or other of them would come and sit by
my bedside awhile and exchange with me a few desultory remarks;
but this was seldom, and grew, with my strength more so, for the
earth was brilliant with summer outside and naturally fuller of
attractions than a sick-room.
Their neglect troubled me little at first; but by and by, when the first
idle ecstasy of convalescence was beginning to deepen into a sense
of responsibilities that I should soon have to gather up and adjust, it
woke day by day an increasing uneasiness in my soul. As yet, it is
true, the immediate past I could only call up before my mental vision
as a blurred picture of certain events the significance of which was
suggestive only. Gradually, however, detail by detail, the whole
composition of it concentrated, on the blank sheet of my mind, and
stood straight before me terribly uncompromising in its sternness of
outline. Had I any reason to suppose, in short, that my share in
Modred’s death was known to or guessed at by my father, Jason or
Zyp? On that pivot turned the whole prospect of my future; for as to
myself, were the secret to remain mine alone, I yet felt that I could
make out life with a tolerable degree of resignation in the certain
knowledge that Modred had forgiven me before he died, for a
momentary mad impulse, the provocation to which had been so
bitter—the reaction from which had been so immediate and so
equally impulsive.
Of my father, I may say at once, I had little fear. His manner
toward me when, as he did occasionally, he came and sat by me for
a half-hour or so, was marked by a gentleness and affection I had
never known him to exhibit before. Pathetic as it was, I could
sometimes almost have wished it replaced by a sterner mood, a
more dubious attitude; for my remorse at having so bereaved him
became a barbed sting in presence of his new condescension to me
that dated from the afternoon of my appeal to him, and was
intensified by our common loss.
Of Zyp I hardly dared to think, or dared to do more than
tremulously hover round the thought that Modred’s death had
absolved me from my promise to him to avoid her. Still the thought
was there and perhaps I only played with self-deception when I
affected to fly from it out of a morbid loyalty to him that was gone. I
could not live with and not long for her with all the passion I was
capable of.
Therefore it was that I dreaded any possible disclosure of a
suspicion on her part—dreaded it with a fever of the mind so fierce
that it must truly have retarded my recovery indefinitely had not a
counter-irritant occurred to me, in certain moods, in the form of a
thought that perhaps, after all, my deed might not so affright one
who, on her own showing, found a charm in the contemplation of
evil.
But it was Jason I feared most. Something—I can hardly give it a
name—had come to me within the last few weeks that seemed to be
the preface to an awakening of the moral right on my part. In the
unfolding of this new faculty I was startled and distressed to observe
deformities in my brother where I had before seen nothing but manly
beauty and a breezy recklessness that I delighted in. Beautiful
bodily, I and all must still think him, though it had worried me lately to
often observe an expression in his blue eyes that was only new to
my new sense. This I can but describe, with despair of the
melodramatic sound of it, as poisonous. The pupils were as full and
purple as berries of the deadly nightshade.
It was not, however, his eyes only that baffled me. I saw that he
coveted any novelty of sensation greedily, and that sooner than
forego enjoyment of it he would ruthlessly stamp down whatever
obstacle to its attainment crossed his path.
Now I knew in my heart that his hitherto indifference to Zyp was an
affectation born only of wounded vanity, and that such as he could
never voluntarily yield so piquant a prize to homelier rivals. I recalled,
with a brooding apprehension, certain words of his on that fatal
morning, that seemed intended to convey, at least, a dark suspicion
as to the manner of Modred’s death. Probably they were bolts shot
at random with a sinister object—for I could conceive no shadow of
direct evidence against me. In that connection they might mean
much or little; in one other I had small doubt that they meant a good
deal—this in fact, that, if I got in his way with Zyp, down I should go.
Daily probing and analyzing such darkly dismal problems as these,
I slowly crawled through convalescence to recovery.
It was a sweltering morning in early July that I first crept out of
doors, with Zyp for my companion. It was happiness to me to have
her by my side, though as yet my weak and watery veins could
prickle to no ghost of passion. I had thought that life could hold
nothing for me ever again but present pain and agonized
retrospects. It was not so. The very smell of the freshly watered
roads woke a shadowy delight in me as we stepped over the
threshold. The buoyant thunder of the river, as it leaped under the
old street bridge seemed to gush over my heart with a cleansing
joyousness that left it white and innocent again.
We crossed the road and wandered by a zig-zag path to the
ancient close, where soft stretches and paddocks of green lawn,
“immemorial elms” and scattered buildings antique and embowered
wrought such an harmonious picture as filled my tired soul with
peace.
Here we sat down on an empty bench. I had much to question Zyp
about—much to reflect on and put into words—but my neglected
speech moved as yet on rusty hinges.
“Zyp,” I said presently, in a low voice; “tell me—where is he
buried?”
“In the churchyard—St. John’s, under the hill, Renny.”
Not once until now had I touched upon this subject or mentioned
Modred’s name to any one of them, and a great longing was upon
me to get it over and done with.
“Who went?”
“Dad and Jason and Dr. Crackenthorpe.”
“Zyp, nobody has asked me anything about it. Don’t you all want to
know how—how it happened?”
“He was caught in the weeds—you said so yourself, Renny.”
Vainly I strove to get under her words; intuition was, for the time
being, a sluggish quantity in me.
“Yes; but——” I began, when she took me up softly.
“Dad said it was all clear and that we were never to bother you
about it at all.”
A sigh of gratitude to heaven escaped me.
“And I for one,” said Zyp, “don’t intend to.”
Something in her words jarred unaccountably on my sick nerves.
“At first,” she said, just glancing at me, “dad thought there ought to
be an inquest, but Dr. Crackenthorpe was so set against it that he
gave in.”
“Dr. Crackenthorpe? Why was——”
“He said that juries took such an idiotic view of a father’s
responsibilities; that dad might be censured for letting the boy run
wild; that in any case the family’s habits of life would be raked over
and cause a scandal that might make things very uncomfortable; that
it was a perfectly plain case of drowning, and that he was quite
willing to give a certificate that death was due to a rupture of some
blood vessel in the brain following exhaustion from exposure—or
something of that sort.”
“And he did?”
“Yes, at last, after a deal of talk, and he was buried quietly and
there was an end of it.”
Not quite an end, Zyp—not quite an end!
She was very gentle and patient with me all the morning, and my
poor soul brimmed over with gratitude. My pulses began even to
flicker a little with hope that things might be as they were before the
catastrophe. After all she was a very independent changeling and, if
there existed in her heart any bias in my favor, Jason might find
himself quite baffled in his efforts to control her inclinations.
Presently I turned to the same overclouding subject.
“What happened the day I was taken bad, Zyp?”
“Jason found you on the stairs, talking rubbish. They carried you to
bed and you hardly left off talking rubbish for weeks. Don’t you
remember anything of it?”
“Nothing, after—after I saw him lying there so dreadful.”
“Ah, it was ugly, wasn’t it? Well, you must have wandered off
somewhere—anywhere; and the rest of us to the parlor. There dad
and the doctor fell to words. They had spent all the night over that
stupid drink, sleeping and quarreling by fits and couldn’t remember
much about it. They had not heard any noise upstairs, either of them;
but suddenly the doctor pointed to something hanging out of dad’s
pocket. ‘Why, you must have gone to the boy’s room some time,’ he
said. ‘Look there!’ Dad took it out and it was Modred’s braces, all
twisted up and stuffed into his pocket.”
“Modred’s braces?”
“Yes; they all knew them, for they were blue, you know—the color
he liked. Dad afterward thought he must have put them there to be
out of the way while he was carrying Modred upstairs, but at the time
he was furious. ‘D’ye dare to imply I had a hand in my son’s death?’
he shrieked. ‘I imply nothing; I mean no offense; they are plain for
every one to see,’ said the doctor, going back a little. I thought he
was frightened and that dad would jump at his throat like a weasel,
and I clapped my hands, waiting for the battle. But it never came, for
dad turned pale and called for brandy, and there was an end of it.”
This story of the doctor’s horrible suggestion wrought only one
comfort in me—it warmed my heart with a great heat of loyalty to one
who, I knew, for all his faults, could never be guilty of so inhuman a
wickedness.
“I should like to kill that doctor,” I said, fiercely and proudly.
“So should I,” said Zyp. “I believe he would bleed soot like a
chimney.”
Zyp was my companion during the greater part of that day and the
next. Her manner toward me was uniformly gentle and attentive.
Sometimes during meals I would become conscious of Jason’s eyes
fixed upon one or other of us in a curious stare that was watchful and
introspective at once, as if he were summing up the voiceless
arguments of counsels invisible, while never losing sight of the fact
that we he sat in judgment on were already convicted in his mind.
This, for the time being, did not much disturb me. I was lulled to a
sense of false security by the gracious championship I thought I now
could rely upon.
It was the evening of the second day and we three were in the
living-room together; Jason reading at the window. Zyp had been so
kind to me that my heart was very full indeed, and now she sat by
me, one hand slipped into mine, the other supporting her little
pointed chin, while her sweet, flower-stained eyes communed with
other, it seemed, than affairs of earth. A strange wistful tenderness
had marked her late treatment of me; a pathetic solicitude that was
inexpressibly touching to one so forlorn. Suddenly she rose and I
heard Jason’s book rustle in his hand.
“Now, little boy,” she said, “’tis time you were in bed.”
Then she leaned toward me and whispered:
“Is he so unhappy? What has he done for Zyp’s sake?”
In a moment she bent and kissed me, with a soft kiss, on the
forehead, and shooting a Parthian glance of defiance at Jason, who
never spoke or moved, ran from the room.
All my soul thrilled with a delicious joy. Zyp, who had refused to
kiss him, had kissed me. The ecstasy of her lips’ touch blotted out all
significance her words might carry.
Half-stunned with triumphant happiness, I climbed the stairs and,
getting into bed, fell into a luminous dream of thought in which for the
moment was no place for apprehension.
I did not even hear Jason enter or shut the door, and it was only
when he shook me roughly by the shoulder that I became conscious
of his presence in the room.
He was standing over me, and the windows of his soul were down,
and through them wickedness grinned like a skull.
“I’ve had enough of this,” he said in a terrible low voice. “D’you
want to drive me to telling that I know it was you who killed Modred?”
CHAPTER XI.
CONVICT, BUT NOT SENTENCED.
So the blow had fallen!

Yet a single despairing effort I made to beat off or at least
postpone the inevitable.
I sat up in bed and answered my brother back with, I could feel,
ashen and quivering lips.
“What do you mean?” I said. “How dare you say such a thing?”
“I dare anything,” he said, “where I have a particular object in
view.” He never took his eyes off me, and the cold devil in them froze
my blood that had only now run so hotly.
“For yourself,” he went on, “I don’t care much whether you hang or
live. You can come to terms with your own conscience I dare say,
and a fat brother more or less may be a pure question of fit survival.
That’s as it may be—but the girl here is another matter.”
“I didn’t kill him,” I could only say, dully.
Still keeping his eyes on me he sought for and drew from his
jacket pocket a twist of dry and shrunken water weed. A horrible
shudder seized me as I looked upon it.
“You didn’t think to see that again?” he said. “Do you recognize it?
Of course you do. It was the rope you twisted round his foot, and that
I found round his foot still, after dad had carried him upstairs,
bundled round with those sacks, and I was left alone in the room with
him a minute.”
My heart died within me. I dropped my sick, strained eyes and
could only listen in agonized silence. And he went on quite pitilessly.
“You shouldn’t have left such evidence, you know—least of all for
me to see. I had not forgotten the murder in your eyes when I spoke
to you that morning and the evening before.”
He struck the weed lightly with his right hand.
“This stuff,” he said, “I know it, of course—grows up straight
enough of itself. It wanted something human—or inhuman—to twist it
round a leg in that fashion.”
I broke out with a choking cry.
“I did it,” I said; “but it wasn’t murder—oh, Jason, it wasn’t murder,
as you mean it.”
He gave a little cold laugh.
“No doubt we have different standards of morality,” he said. “We
won’t split hairs. Say it was murder as a judge and jury would view
it.”
“It wasn’t! Will you believe me if I tell you the truth?”
“That depends upon the form it takes.”
“I’ll tell you. It is the truth—before God, it is the truth! I won’t favor
myself. I had been mad with him, I own, but had nearly got over it. I
was out all day on the hills and thought I should like a bathe on my
way home. I went through the ‘run’ and saw he was there. At first I
thought I would leave him to himself, but just as I was going he saw
me and a grin came over his face and—Jason, you know that if I had
gone away then, he would have thought me afraid to meet him.”
“You can leave me, Renalt, out of the question, if you please.”
“I meant no harm—indeed I didn’t—but when I got there he
taunted and mocked at me. I didn’t know what I was doing; and
when he jumped for the water I followed him and twisted that round.
Then in a single moment I saw what I had done—and was mad to
unfasten it. It would not come away at first, and when at last I got
him free and to the shore he was insensible. If you could only know
what I suffered then, you would pity me, Jason—you would; you
could not help it.”
I stole a despairing look at his face and there was no atom of
softness in it.
“He came to on the way home and I was wild with joy, and at night,
Jason, when you were in bed and asleep, I crept into his room and
begged for his forgiveness and he forgave me.”
“Without any condition? That wasn’t like Modred. What did he ask
for in return?”
I was silent.
“Come,” he persisted, “what did he want? You may as well tell me
all. You don’t fancy that I believe he forgave you without getting
something substantial in exchange?”
“I was to give up all claim to Zyp,” I said in a low, suffering voice.
Jason laughed aloud.
“Oh, Modred,” he cried, “you were a pretty bantling, upon my word!
Who would have thought the dear fatty had such cunning in him?”
His callous merriment struck me with a dumb horror as of
sacrilege. But he subdued it directly and returned to me and my
misery in the same repressed tone as before.
“Well,” he said, “I have heard it all, I suppose. It makes little
difference. You know, of course, you are morally responsible for his
death, just the same as if you had stuck a knife into his heart.”
I could only hide my face in the bedclothes, writhed all through
with agony. There was a little spell of silence; then my brother
bespoke my attention with a gentle push.
“Renny, do you want all this known to the others?”
I raised my head in a sudden gust of passion.
“Do what you like!” I cried. “I know you now, and you can’t make it
much worse!”
“Oh, yes,” he said, coolly; “I can make it a good deal worse.
Nobody but I knows at present, don’t you see?”
I looked at him with a sudden gleam of hope.
“Don’t you intend to tell, Jason?”
He laughed again, lightly.
“That depends. I must borrow my cue from Modred and make
conditions.”
I had no need to ask what they were. In whatever direction I
looked now, I saw nothing but a blank and deadly waste.
“I want the girl—you understand? I need not go into particulars.
She interests me and that’s enough.”
“Yes,” I said, quietly.
“There must be no more of that sentimental foolery between you
and her. I bore it as long as you were ill; but, now you’re strong
again, it must stop. If it doesn’t, you know what’ll happen.”
With that he turned abruptly on his heel and began to undress. I
listened for the deep breathing that announced him to be asleep with
a strained fever of impatience. I felt that I could not think cleanly or
collectedly with that monstrous consciousness of his awake in the
room.
Perhaps, in all my wretchedness, the full discovery of his
baseness of soul was as bitter a wound as any I had received. I had
so looked up to him as a superior being, so sunned myself in the
pride of relationship to him; so lovingly submitted to his boyish
patronage and condescension. The grief of my discovery was very
real and terrible and would in itself, I think, have gone far to blight my
existence had no fearfuller blast descended to wither it.
Well, it was all one now. Whatever immunity from disaster I was to
enjoy henceforth must be on sufferance only.
Had I been older and sinfuller I might have grasped in my despair
at the coward’s resource of self-destruction; as it was, I thought of
flight. By and by, perhaps, when vigor should return to me, and with
it resolution, I should be able to face firmly the problem of my future
and take my own destinies in hand.
Little sleep came to me that night, and that only of a haunted kind.
I felt haggard and old as I struggled into my clothes the next
morning, and all unfit to cope with the gigantic possibilities of the
day. Jason had gone early to the fatal pool for a bathe.
At breakfast, in the beginning, Zyp’s manner to me was prettily
sympathetic and a little shy. It was the first of my great misery that I
must repel her on the threshold of our better understanding, and see
her fall away from me for lack of the least expression of that
passionate devotion and gratitude that filled my heart to bursting. I
could see at once that she was startled—hurt, perhaps, and that she
shrunk from me immediately. Jason talked airily to my father all
through the meal, but I knew his senses to be as keenly on the alert
as if he had sat in silence, with his eyes fixed upon my face.
I choked over my bread and bacon; I could not swallow more than
a mouthful of the coffee in my cup, and Zyp sat back in her chair,
never addressing me after that first rebuff, but pondering on me
angrily with her eyes full of a sort of wonder.
She stopped me peremptorily as, breakfast over, I was hastening
out with all the speed I could muster, and asked me if I didn’t want
her company that morning.
“No,” I answered; “I am well enough to get about by myself now.”
“Very well,” she said. “Then you must do without me altogether for
the future.”
She turned on her heel and I could only look after her in dumb
agony. Then I crept down into the yard and confided my grief to the
old cart wheels.
Presently, raising my head, I saw her standing before me, her
hands under her apron, her face grave with an expression, half of
concern, half of defiance.
“Now, if you please,” she said, “I want to know the meaning of
this?”
“Of what?” I asked, with wretched evasiveness.
“You know—your manner toward me this morning.”
“I have done nothing,” I muttered.
“You have insulted me, sir. Is it because I kissed you last night?”
“Oh, Zyp!” I cried aloud in great pain. “You know it isn’t—you know
it isn’t!”
I couldn’t help this one cry. It was forced from me.
“Then what’s the reason?”
“I can’t give it—I have none. I want to be alone, that’s all.”
She stood looking at me a moment in silence, and the line of her
mouth hardened.
“Very well,” she said, at last. “Then, understand, I’ve done with
you. I thought at first it was a mistake or that you were ill again. I’ve
been kind to you; you can’t say I haven’t given you a chance. And I
pitied you because you were alone and unhappy. Jason, I will tell
you, hinted an evil thing of you to me, but even if it was true, which I
didn’t believe, I forgave you, thinking, perhaps, it was done for my
sake. Well, if it was, I tell you now it was useless, for you will be
nothing to me ever again.”
And, with these cruel words, she left me. The proud child of the
woods could brook no insult to her condescension, and from my
comrade she had become my enemy.
I suppose I should have been relieved that the inevitable rupture
had occurred so swiftly and effectually. Judge you, you poor outcasts
who, sanctifying a love in your tumultuous breasts, have had to step
aside and yield to another the fruit you so coveted.
Once pledged to antagonism, Zyp, it will be no matter for wonder,
adopted anything but half-measures. Had it only been her vanity that
was hurt she would have made me pay dearly for the blow. As it
was, her ingenuity in devising plans for my torture and discomfiture
verged upon the very bounds of reason.
At first she contented herself with mere verbal pleasantries and
disdainful snubbings. As, however, the days went on and my old
strength and health obstinately returned to me, despite the irony of
the shattered soul within, her animosity grew to be an active agent
so persistent in its methods that I verily thought my brain would give
way under the load.
I cannot, indeed, recall a tithe of the Pucklike devices she resorted
to for my moral undoing, and which, after all, I might have endured to
the end had it not been for one threading torment that accompanied
all her whimsies like a strain of diabolical music. This was an
ostentatious show of affection for Jason, which, I truly believe, from
being more or less put on in exaggerated style for my edification,
became at length such a habit with her as may be considered, in
certain dispositions, one form of love.
The two now were seldom apart. Once, conscious of my presence,
she kissed Jason on the lips, because he had brought her a little
flowering root of some plant she desired. I saw his face fire up darkly
and he looked across at me with a triumph that made me almost
hate him.
And the worst of it was that I knew that my punishment was not
more than commensurate with the offense; that my sin had been
grievous and its retribution not out of proportion. How could full
atonement and Zyp have been mine together?
Still, capable of acknowledging the fitness of things in my sadder
hours of loneliness, my nature, once restored to strength, could not
but strive occasionally to throw off the incubus that it felt it could not
bear much longer without breaking down for good and all. I had done
wrong on the spur of a single wicked impulse, but I was no fiend to
have earned such bitter reprisal. By slow degrees rebellion woke in
my heart against the persistent cruelty of my two torturers. Had I fled
at this juncture, the wild scene that took place might have been
averted, and the exile, which became mine nevertheless, have
borne, perhaps, less evil fruit than in the result it did.
CHAPTER XII.
THE DENUNCIATION.
One November morning—my suffering had endured all these

months—my father and Dr. Crackenthorpe stood before the sitting-
room fire, talking, while I sat with a book at the table, vainly trying to
concentrate my attention on the printed lines.
Since my recovery I had seen the doctor frequently, but he had
taken little apparent notice of me. Now, I had racked my puzzled
mind many a time for recollection of the conversation I had been
witness of on the night preceding my seizure, but still the details of it
had eluded me, though its gist remained in a certain impression of
uneasiness that troubled me when I thought of it. Suddenly, on this
morning, a few words of the doctor’s brought the whole matter vividly
before me again.
“By the bye, Trender,” he said, drawlingly, and sat down and
began to poke the fire—“by the bye, have you ever found that thing
you accused me of losing for you on a certain night—you know
when?”
“No,” said my father, curtly.
“Was it of any value, now?”
“Maybe—maybe not,” said my father.
“That don’t seem much of answer. Perhaps, now, it came from the
same place those others did.”
“That’s nothing to you, Dr. Crackenthorpe.”
“Well, you say it’s lost, anyhow. Supposing I found it, would you
agree to my keeping it? Treasure-trove, you know”—and he looked
up with a grin, balancing the poker perpendicularly in his hand.
“Treasure-trove, my friend,” he repeated, with emphasis, and gave
the other a keen look.
Something in the tone of his speech woke light in my brain, and I
remembered at a flash. I stole an anxious glance at my father. His
face was pale and set with anger, but there was an expression in his
eyes that looked like fear.
“You don’t mean to tell me you have found it?” he said in a forced
voice.
“Oh, by no means,” answered the doctor. “We haven’t all your
good luck. Only you are so full of the unexpected in producing
valuables from secret places, like a conjurer, that I thought perhaps
you wouldn’t mind my keeping this particular one if I should chance
to pick it up.”
“Keep it, certainly, if you can find it,” said my father, I could have
thought almost with a faint groan.
“Thanks for the permission, my friend; I’ll make a point of keeping
my eyes open.”
When did he not? They were pretty observant now on Zyp and
Jason, who, as he spoke, walked into the room.
“Hullo!” said my brother. “Good-morning to you, doctor, and a
sixpence to toss for your next threppenny fee.”
“Hold your tongue,” cried my father, angrily.
“I would give a guinea to get half for attending on your inquest,”
said the doctor, sourly. “Keep your wit for your wench, my good lad,
and see then that she don’t go begging.”
“I could give you better,” muttered Jason, cowed by my father’s
presence, “but it shall keep and mature.” Then he turned
boisterously on me.
“Why don’t you go out, Renny, instead of moping at home all day?”
His manner was aggressive, his tone calculated to exasperate.
Moved by discretion I rose from my chair and made for the door;
but he barred my way.
“Can’t you answer me?” he said, with an ugly scowl.
“No—I don’t want to. Let me pass.”
My father had turned his back upon us and was staring gloomily
down at the fire.
I heard Zyp give a little scornful laugh and she breathed the word
“coward” at me.
I stopped as if I had struck against a wall. All my blood surged
back on my heart and seemed to leave my veins filled with a tingling
ichor in its place.
“Perhaps I have been,” I said, in a low voice, “but here’s an end of
it.”
Jason tittered.
“We’re mighty stiltish this morning,” he said, with a sneer. “What a
pity it’s November, so that we can’t have a plunge for the sake of
coolness—except that they say the pool’s haunted now.”
I looked at him with blazing eyes, then made another effort to get
past him, but he repelled me violently.
“You don’t know your place,” he said, and gave an insolent laugh.
“Stand back till I choose to let you go.”
I heard the doctor snigger and Zyp gave a second little cluck. My
father was still absorbed—lost in his own dark reflections.
The loaded reel of endurance was spinning to its end.
“You might have given all your morning to one of your Susans
yonder,” said my brother, mockingly. “Now she’s gone, I expect, with
her apron to her eyes. She’ll enjoy her pease pudding none the less,
I dare say, and perhaps look out for a more accommodating clown. It
won’t be the first time you’ve had to take second place.”
I struck him full between the eyes and he went down like a polled
ox. All the pent-up agony of months was in my blow. As I stepped
back in the recoil, madly straining even then to beat under the more
furious devil that yelled in me for release, I was conscious of a
hurried breath at my ear—a swift whisper: “Kill him! Stamp on his
mouth! Don’t let him get up again!” and knew that it was Zyp who
spoke.
I put her back fiercely. Jason had sprung to his feet—half-blinded,
half-stunned. His face was inhuman with passion and was working
like a madman’s. But before he could gather himself for a rush, my
father had him in his powerful arms. It all happened in a moment.
“What’s all this?” roared my father. “Knock under, you whelp, or I’ll
strangle you in your collar!”
“Let me go!” cried my brother. “Look at him—look what he did!”
He was choking and struggling to that degree that he could hardly
articulate. I think foam was on his lips, and in his eyes the ravenous
thirst for blood.
“He struck me!” he panted—“do you hear? Let me go—let me kill
him as he killed Modred!”
There was a moment’s silence. Dr. Crackenthorpe, who had sat
passively back in his chair during the fray, with his lips set in an acrid

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Mitochondrial Medicine: Volume 2:

Assessing Mitochondria Volkmar

Mitochondrial Medicine Volume 3 Manipulating

Primary Mathematics 3A Hoerst

Physics Galaxy 2020 21 Vol 3A Electrostatics Current

Essential Cardiovascular Medicine 6th Edition Volume 2

Mitochondrial Replacement Techniques Ethical Social and

The Book of Chinese Medicine Volume 2 1st Edition Henry

Endeavour Medicine Paper 2 Volume 2 for written and

Assessing Progress on the Institute of Medicine Report

For further volumes:

Volume 2: Assessing Mitochondria

ISSN 1064-3745 ISSN 1940-6029 (electronic)

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Glendale, AZ, USA Volkmar Weissig

1 Mitochondrial Dysfunction in Mitochondrial Medicine: Current

12 Investigation of Mitochondrial ADP-Ribosylation

26 Extraction of Functional Mitochondria Based on Membrane

EMMA ABELL • Univ. Bordeaux, INSERM, Centre de recherche Cardio- Thoracique de

NAIG GUEGUEN • UMR CNRS 6015-INSERM U1083, MitoVasc Institute, University of

JACOB L. LÉGER • Department of Chemistry and Biochemistry, Universite de Moncton,

MD HABIBUR RAHMAN • Department of Biomedical Engineering, The Chinese University of

MARKUS WALDECK-WEIERMAIR • Gottfried Schatz Research Center for Cell Signaling,

Mitochondrial Dysfunction in Mitochondrial Medicine:

Key words Mitochondrial dysfunctions, Mitochondria evaluation, Bioenergetics, Devices,

The contribution of mitochondrial dysfunctions in the onset and

[4], or Huntington’s [5], infectious diseases [6], inflammation [7],

mitochondrial dysfunction by various strategies is a huge challenge

2 Methods to Assess Mitochondrial Dysfunctions

2.1 Basis Principles of mitochondrial bioenergetics have been largely

The Δp is composed of the charge (Δψm) and chemical (ΔpH)

2.2 Analyses Evaluation of individual respiratory chain complex activities is a

l Succinate:ubiquinone oxidoreductase (coupled to

In addition, after transfer of the electrophoretic gels for Western

2.4.1 Devices The rate of mitochondrial O2 consumption could be determined by

by Oroboros Instrument, has historically been the most common

This is allowed by (1) closed, air-tight reaction chambers;

The Seahorse® XF The Seahorse® XF Extracellular Flux Analyzer technology is based

An alternative approach when the control of substrate supply is

State 3 (ADP) is controlled, depending on the tissue and con-

decades, respiration analyses on intact cells became prominent in

rate. While an increase in phosphorylating respiration most likely

Finally, the experiment ends by the addition of ETC inhibitors,

across the inner mitochondrial membrane [76], the mitochondrial

inhibiting ETC activity, for example, by titrating rotenone with

2.6 Metabolites Defects in the mitochondrial energy production often lead to

Metabolomics is defined as an integrative approach consisting

Because of their complex properties and strategic role in cellular

The authors thank the following institutions and patient associa-

19. Ježek P, Hlavatá L (2005) Mitochondria in https://doi.org/10.1146/annurev-bio

62. Wai T, Langer T (2016) Mitochondrial 73. Stepanova A, Konrad C, Manfredi G et al

the fluorescent dye tetramethylrhodamine progression. Cell Metab 26:419–428.e5.

For some three weeks I had lain racked and shriveled in a

So the blow had fallen!

One November morning—my suffering had endured all these

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