Ahmed, Ahmed R

Alexiadou, Kleopatra

Bech, Paul

Bloom, Stephen R

Camuzeaux, Stephane

Clarke, Rosemary E

Cuenco, Joyceline

Khoo, Bernard

Kowalka, Anna M

Lewis, Matthew R

Minnion, James

Purkayastha, Sanjay

Romero, Maria G

Takats, Zoltan

Tan, Tricia M-M

Whitwell, Harry J

Williams, Emma L

English

UCL Discovery

DOI: 10.1111/cen.14894I N V I T ED COMMENTARYCommentary on “The road to reliable peptide assays is pavedwith good guidelines”Anna M. Kowalka1 | Kleopatra Alexiadou1 | Joyceline Cuenco1 |Rosemary E. Clarke2 | Stephane Camuzeaux3,4 | James Minnion1 |Emma L. Williams5 | Paul Bech1 | Sanjay Purkayastha6 | Ahmed R. Ahmed6 |Zoltan Takats3,4 | Bernard Khoo7 | Harry J. Whitwell3,4 | Maria G. Romero3,4 |Stephen R. Bloom1 | Matthew R. Lewis3,4 | Tricia M.‐M. Tan11Section of Diabetes, Endocrinology and Metabolism, Department of Metabolism, Digestion and Reproduction, Imperial College London, London, UK2Blood Sciences Department, Raigmore Hospital, Inverness, UK3Section of Bioanalytical Chemistry, Department of Metabolism, Digestion and Reproduction, Imperial College London, London, UK4National Phenome Centre, Imperial College London, London, UK5Department of Clinical Biochemistry, North West London Pathology, Charing Cross Hospital, London, UK6Department of Surgery and Cancer, Imperial College Healthcare NHS Trust, London, UK7Endocrinology, Division of Medicine, University College London, London, UKCorrespondence: Tricia M.‐M. Tan, Section of Diabetes, Endocrinology and Metabolism, Department of Metabolism, Digestion and Reproduction, Imperial CollegeLondon, London, UK.Email: t.tan@imperial.ac.ukKEYWORDSbariatric surgery, mass spectrometry, peptide YYFunding informationEfficacy and Mechanism Evaluation Programme, Grant/Award Number: 13/121/07; Medical Research Council, Grant/Award Number: MR/K02115X/1We note that Maurer et al., in their correspondence,1 have raised someconcerns about the preanalytical and analytical validation of the peptideYY (PYY) assay described in our paper ‘The postprandial secretion ofpeptide YY1–36 and 3–36 in obesity is differentially increased after gastricbypass versus sleeve gastrectomy’.2 The validation and assay character-istics were not the primary objectives of the paper. We focussed on thephysiology of PYY secretion in healthy volunteers and patients pre‐ andpost‐gastric bypass versus sleeve gastrectomy. Therefore, we did notinclude a full description and details of the assay validation procedure andhistory. We agree with the overall principles that Maurer et al. subscribeto and summarise herewith our findings to show that we have followedand fulfilled internationally accepted standards in the validation of ourassay.We have verified stability of PYY1–36 and PYY3–36 at preanaly-tical, analytical and post‐analytical stages of the assay. At preanaly-tical stage the whole blood is collected in prechilled lithium heparin(LH) or ethylenediaminetetraacetic acid (EDTA) tubes and containingevaluated in‐house inhibitors (DPP4 inhibitor and Aprotinin). Thesample is centrifuged immediately at 4°C and flash‐frozen on dry iceor transferred immediately to a −80°C freezer. The samples are keptat −80°C until being thawed once only for analysis.We deemed our protease inhibitors sufficient for maintainingstability of PYY1–36 and PYY3–36 and inhibiting peptide degradation.Our data showed no degradation of PYY1–36 or PYY3–36 for eitherEDTA or LH plasma at the 3 h time‐point at room temperature (19°C)and the 3 h time‐point at 4°C, with or without inhibitors. The meanClinical Endocrinology. 2023;1–3. wileyonlinelibrary.com/journal/cen | 1This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,provided the original work is properly cited.© 2023 The Authors. Clinical Endocrinology published by John Wiley & Sons Ltd.Anna M. Kowalka, Kleopatra Alexiadou, Joyceline Cuenco, and Rosemary E. Clarke contributed equally to this study. 13652265, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cen.14894 by University College London UCL Library Services, Wiley Online Library on [22/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licenseconcentration from injections of triplicate QCs (low and high) showedthat for spiked samples and samples from a healthy volunteer, theconcentrations remained similar regardless of the presence ofinhibitors or their absence (with the mean percentage differencefor overall and between all tested sample sets ≤10%).Maurer et al. have quoted Toräng et al.'s study on PYY's majorcleaving sites but disregarded the outcome from their study of theinhibitory effects of EDTA on metalloendoproteases, neuroendopepti-dase (NEP) and Meprin β.3 Toräng et al. demonstrated that the risk ofPYY degradation in collected blood is small in collection tubes containingEDTA. We have studied the stability of both PYY species with ourinhibitor mix containing DPP4 inhibitor and Aprotinin, where we spikedPYY1–36 and PYY3–36 into plasma EDTA and LH from three differentvolunteers. We did not detect any significant differences in concentra-tions of both peptides with only these inhibitors present suggesting thatNEP, Meprin β and Am‐P activity is negligible under these conditions.Furthermore, we have verified the stability of both species ofPYY in both EDTA and LH plasma under the following conditions: (1)freezing and thawing up to a second cycle; (2) incubation at 4°C forup to 4 h; (3) long‐term storage (6 months) at −80°C; (4) postextrac-tion, during storage in the autosampler at 4°C. We have verified thestability of our stock and working stock solutions for up to 7 months,stored at 4°C. We would state that it is a matter of standardoperating procedure that we store these solutions at −80°C anddiscard these at the end of the day of the run after thawing, affordinga safety margin. Last, our assay has been validated both with plasma‐based calibrants and QCs, and cross‐validated in a surrogate matrix(20BMA). We also validated matrix effects (ME) in spiked plasmaEDTA and plasma LH. Mean percentage ME for PYY1–36 was 100.7%and for PYY3–36, 111% in plasma LH. EDTA ME results were reportedpreviously.2Maurer et al. suggest that imputation of <LLOQ (lowest limitof quantitation) values as zero (method M7 as listed in Johnson4),as specified in our study, is the ‘worst’ method for handling LLOQvalues. The conference paper of Johnson examined the impact ofsetting <LLOQ values to zero on the estimation of pharmaco-kinetic values for drugs, utilising a model which is highlydependent on the last few low‐level sample values. Crucially,our study is not a pharmacokinetic study, but instead aphysiological study where we are examining the relatively largehormonal responses to a mixed meal particularly after surgery.Hence, Maurer et al.'s criticism seems to be misplaced, as they didnot take account of this crucial difference. Moreover, we havecarried out a sensitivity analysis of our data where we imputed<LLOQ values with ½ the LLOQ (method M5 as listed inJohnson4), and consistent with the method used by Reverter‐Branchat et al.5 This method did not alter the contrasts and p‐values to a significant degree, suggesting that any bias introducedby the different imputation method for values <LLOQ was smalland does not affect the conclusions from our study.In conclusion, we agree that it is important to carefully followinternational analytical guidelines to validate assays. Our assay isindeed validated according to such guidelines and the conclusionsmade from the data in our paper are indeed valid.AUTHOR CONTRIBUTIONSAll authors contributed to the writing of the manuscript andapproved the final version of the manuscript. Tricia M.‐M. Tan isthe guarantor of the manuscript.ACKNOWLEDGMENTSThis study was funded by a UK Medical Research Council (MRC)Experimental Challenge Grant (MR/K02115X/1) and UK NationalInstitute for Health Research (NIHR) Efficacy and Mechanism EvaluationGrant (13/121/07) and supported by the Imperial NIHR Clinical ResearchFacility at Imperial College Healthcare NHS Trust. This work wassupported by the Medical Research Council and National Institute forHealth Research [grant number NC_PC_12025]. Infrastructure supportwas provided by the National Institute for Health Research (NIHR)Imperial Biomedical Research Centre (BRC). The Department ofMetabolism, Digestion and Reproduction is funded by grants from theMRC and Biotechnology and Biological Sciences Research Council and issupported by the NIHR Imperial BRC Funding Scheme. Anna M. Kowalkais supported by the Imperial Health Charity and the NIHR Imperial BRC.Kleopatra Alexiadou is supported by an NIHR Academic ClinicalLectureship. Rosemary E. Clarke and Joyceline Cuenco were funded bythe NIHR Imperial BRC funding scheme. TM‐MT and SRB were fundedby the MRC and are currently supported by the NIHR and NIHR ImperialBRC. The views expressed are those of the authors and not necessarilythose of the MRC, the NHS, the NIHR, or the U.K. Department of Healthand Social Care, who funds the NIHR.CONFLICTS OF INTEREST STATEMENTJames Minnion, Stephen R. Bloom, Tricia M.‐M. Tan are employeesand/or share‐holders in Zihipp Ltd., an Imperial College spin‐outcompany developing analogues of gut hormones for treatment ofobesity. All other authors declare no conflict of interest.DATA AVAILABILITY STATEMENTData are available from the corresponding author upon reasonablerequest.ORCIDAnna M. Kowalka http://orcid.org/0000-0002-6251-7369Kleopatra Alexiadou http://orcid.org/0000-0001-8412-0592Stephane Camuzeaux http://orcid.org/0000-0002-4614-4662Bernard Khoo http://orcid.org/0000-0002-4223-9736Matthew R. Lewis http://orcid.org/0000-0001-5760-5359Tricia M.‐M. Tan http://orcid.org/0000-0001-5873-3432REFERENCES1. Maurer J, Grouzmann E, Eugster PJ. The road to reliable peptideassays is paved with good guidelines. Clin Endocrinol (Oxf). 2022.doi:10.1111/cen.148632 | INVITED COMMENTARY 13652265, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cen.14894 by University College London UCL Library Services, Wiley Online Library on [22/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License2. Kowalka AM, Alexiadou K, Cuenco J, et al. The postprandialsecretion of peptide YY(1‐36) and (3‐36) in obesity is differentiallyincreased after gastric bypass versus sleeve gastrectomy. ClinEndocrinol (Oxf). 2022. doi:10.1111/cen.148463. Toräng S, Veedfald S, Rosenkilde MM, Hartmann B, Holst JJ. Theanorexic hormone peptide YY3‐36 is rapidly metabolized to inactivepeptide YY3‐34 in vivo. Physiol Rep. 2015;3(7):e12455.4. Johnson JR. Methods for handling concentration values below thelimit of quantification in PK studies. PhUSE US Connect. 2018; 2018.https://www.lexjansen.com/phuse-us/2018/dh/DH05.pdf5. Reverter‐Branchat G, Eugster PJ, Kuenzli C, et al. Multiplexed assayto quantify the PP‐fold family of peptides in human plasma usingmicroflow liquid chromatography‐tandem mass spectrometry. ClinChem. 2022;68(4):584‐594.How to cite this article: Kowalka AM, Alexiadou K, Cuenco J,et al. Commentary on “The road to reliable peptide assays ispaved with good guidelines”. Clin Endocrinol. 2023;1‐3.doi:10.1111/cen.14894INVITED COMMENTARY | 3 13652265, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cen.14894 by University College London UCL Library Services, Wiley Online Library on [22/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

Commentary on "The road to reliable peptide assays is paved with good guidelines"

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Commentary on "The road to reliable peptide assays is paved with good guidelines"

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