b4465542ff65a002dbbd8d85ff94c451 pmc_9027926.pdf 63a73990c5eb71d639a1ee53e1da344b104b4283 pmc_9027926.pdf badf61545eed6b1a0709ec0f448ec6b6019d0e6951621b5cb59a5a2e12700bbd pmc_9027926.pdf Title: Multicentric Standardization of Protocols for the Diagnosis of Human Mitochondrial Respiratory Chain Defects Subject: The quantification of mitochondrial respiratory chain (MRC) enzymatic activities is essential for diagnosis of a wide range of mitochondrial diseases, ranging from inherited defects to secondary dysfunctions. MRC lesion is frequently linked to extended cell damage through the generation of proton leak or oxidative stress, threatening organ viability and patient health. However, the intrinsic challenge of a methodological setup and the high variability in measuring MRC enzymatic activities represents a major obstacle for comparative analysis amongst institutions. To improve experimental and statistical robustness, seven Spanish centers with extensive experience in mitochondrial research and diagnosis joined to standardize common protocols for spectrophotometric MRC enzymatic measurements using minimum amounts of sample. Herein, we present the detailed protocols, reference ranges, tips and troubleshooting methods for experimental and analytical setups in different sample preparations and tissues that will allow an international standardization of common protocols for the diagnosis of MRC defects. Methodological standardization is a crucial step to obtain comparable reference ranges and international standards for laboratory assays to set the path for further diagnosis and research in the field of mitochondrial diseases. Keywords: mitochondrial respiratory chain; enzyme activity; standardization; diagnosis; mitochondrial disease Author: Nuria Bujan, Constanza Morén, Francesc J. García-García, Alberto Blázquez, Clara Carnicer, Ana Belén Cortés, Cristina González, Ester López-Gallardo, Ester Lozano, Sonia Moliner, Laura Gort, Ester Tobías, Aitor Delmiro, Miguel Ángel Martin, Miguel Ángel Fernández-Moreno, Eduardo Ruiz-Pesini, Elena Garcia-Arumí, Juan Carlos Rodríguez-Aguilera and Glòria Garrabou Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Apr 8 14:01:06 2022 CEST ModDate: Fri Apr 8 14:03:45 2022 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 16 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1364233 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- CPTAIT+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 RWSFOK+VnURWPalladioL Type 1 Custom yes yes no 15 0 XIOYOK+URWPalladioL-Bold Type 1 Custom yes yes no 19 0 PUPQUL+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 DPUHEV+URWPalladioL-Ital Type 1 Custom yes yes no 28 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 36 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 40 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 44 0 KUDXZB+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 56 0 JBTTSN+VnURWPalladioL-Italic Type 1 Custom yes yes no 81 0 LKDISN+CMSY10 Type 1 Builtin yes yes no 85 0 ENHFCR+EURM10 Type 1 Builtin yes yes no 95 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 107 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 111 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 115 0 BCDIEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 119 0 BCDKEE+Calibri-Bold CID TrueType Identity-H yes yes yes 126 0 BCDLEE+Calibri-Bold TrueType WinAnsi yes yes no 134 0 BCDMEE+Cambria-Bold TrueType WinAnsi yes yes no 137 0 MPUDFL+PazoMath Type 1 Builtin yes yes no 147 0 XCOHXD+EURB10 Type 1 Builtin yes yes no 151 0 LUVKVV+URWPalladioL-BoldItal Type 1 Custom yes yes no 155 0 HYYHEA+CMBSY10 Type 1 Builtin yes yes no 159 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 174 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 178 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 182 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 186 0 BCDLEE+Calibri-Bold TrueType WinAnsi yes yes no 193 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 201 0 ArialMT TrueType WinAnsi no no no 205 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 208 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 212 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 216 0 BCDLEE+Calibri-Bold TrueType WinAnsi yes yes no 223 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-05-18 03:25:25 CEST RepresentationInformation: pmc_9027926.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-05-18 02:37:49 CEST Size: 1364233 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 390 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Reagents Destination: subsection.2.1 Item: Title: Procedure Destination: subsection.2.2 Item: Title: Results and Discussion Destination: section.3 Item: Title: Conclusions Destination: section.4 Item: Title: References Destination: Item.4 Info: Title: Multicentric Standardization of Protocols for the Diagnosis of Human Mitochondrial Respiratory Chain Defects Author: Nuria Bujan, Constanza Morén, Francesc J. García-García, Alberto Blázquez, Clara Carnicer, Ana Belén Cortés, Cristina González, Ester López-Gallardo, Ester Lozano, Sonia Moliner, Laura Gort, Ester Tobías, Aitor Delmiro, Miguel Ángel Martin, Miguel Ángel Fernández-Moreno, Eduardo Ruiz-Pesini, Elena Garcia-Arumí, Juan Carlos Rodríguez-Aguilera and Glòria Garrabou Subject: The quantification of mitochondrial respiratory chain (MRC) enzymatic activities is essential for diagnosis of a wide range of mitochondrial diseases, ranging from inherited defects to secondary dysfunctions. MRC lesion is frequently linked to extended cell damage through the generation of proton leak or oxidative stress, threatening organ viability and patient health. However, the intrinsic challenge of a methodological setup and the high variability in measuring MRC enzymatic activities represents a major obstacle for comparative analysis amongst institutions. To improve experimental and statistical robustness, seven Spanish centers with extensive experience in mitochondrial research and diagnosis joined to standardize common protocols for spectrophotometric MRC enzymatic measurements using minimum amounts of sample. Herein, we present the detailed protocols, reference ranges, tips and troubleshooting methods for experimental and analytical setups in different sample preparations and tissues that will allow an international standardization of common protocols for the diagnosis of MRC defects. Methodological standardization is a crucial step to obtain comparable reference ranges and international standards for laboratory assays to set the path for further diagnosis and research in the field of mitochondrial diseases. 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