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Development and validation of a mass spectrometry-based assay for quantification of insulin-like factor 3 in human serum

  • Jakob Albrethsen ORCID logo EMAIL logo , Hanne Frederiksen , Anna-Maria Andersson , Ravinder Anand-Ivell , Loa Nordkap , Anne Kirstine Bang , Niels Jørgensen and Anders Juul
Published/Copyright: May 30, 2018
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Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 56 Issue 11

Abstract

Background:

The circulating level of the peptide hormone insulin-like factor 3 (INSL3) is a promising diagnostic marker reflecting Leydig cell function in the male. Few commercial immunoassays of varying quality exist. Therefore, we decided to develop and validate a precise method for quantification of INSL3 by mass spectrometry.

Methods:

We developed an assay in which the INSL3 A-chain is released from the INSL3 A-B heterodimer by chemical reduction and alkylation. The alkylated INSL3 A-chain is quantitated by liquid chromatography-tandem mass spectrometry (LC-MS/MS), as substitute for serum INSL3. The method was compared to a validated and sensitive in-house serum INSL3 immunoassay using 97 serum samples from 12 healthy boys during pubertal transition. Adult levels were determined based on sera from 72 adult healthy males aged 18–40 years.

Results:

An LC-MS/MS assay with limit of detection and limit of quantification (LOQ) of 0.06 and 0.15 ng/mL, respectively, and intra-assay CVs <9% in the relevant ranges was obtained. The LC-MS/MS compared well with the in-house immunoassay (Deming regression slope: 1.28; Pearson correlation: R=0.86). INSL3 concentrations increased with pubertal maturation in healthy boys. INSL3 concentrations were above the LOQ in all samples from the adult men. The mean (±2 SD range)for serum INSL3 concentrations in the adult men was 2.2 (0.5–3.9) ng/mL.

Conclusions:

We have developed a robust and sensitive method suitable for quantitation of serum INSL3 in a clinical setting using LC-MS/MS instrumentation available in modern clinical laboratories. The method paves the way for future studies into the clinical role of serum INSL3 measurements.

Keywords: assay development; insulin-like factor 3 (INSL3); mass spectrometry

  1. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: Innovation Fund Denmark 14-2013-4; International Center for Research, and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC); ReproUnion.

  3. Employment or leadership: None declared.

  4. Honorarium: None declared.

  5. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

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Supplementary Material:

The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2018-0171).

Received: 2018-02-14
Accepted: 2018-05-03
Published Online: 2018-05-30
Published in Print: 2018-10-25

©2018 Walter de Gruyter GmbH, Berlin/Boston

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https://doi.org/10.1515/cclm-2018-0171
Keywords for this article
assay development; insulin-like factor 3 (INSL3); mass spectrometry

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. New biomarkers and traditional cardiovascular risk scores: any crystal ball for current effective advice and future exact prediction?
  4. Reviews
  5. Laboratory sample stability. Is it possible to define a consensus stability function? An example of five blood magnitudes
  6. Updated review of postmortem biochemical exploration of hypothermia with a presentation of standard strategy of sampling and analyses
  7. Long non-coding RNA-mediated regulation of signaling pathways in gastric cancer
  8. Opinion Paper
  9. Long story short: an introduction to the short-term and longterm Six Sigma quality and its importance in laboratory medicine for the management of extra-analytical processes
  10. EFLM Paper
  11. The European Federation of Clinical Chemistry and Laboratory Medicine syllabus for postgraduate education and training for Specialists in Laboratory Medicine: version 5 – 2018
  12. General Clinical Chemistry and Laboratory Medicine
  13. Early availability of laboratory results increases same day ward discharge rates
  14. Evaluation of the clinical implementation of a large-scale online e-learning program on venous blood specimen collection guideline practices
  15. Improved prospective risk analysis for clinical laboratories compensated for the throughput in processes
  16. National surveys on internal quality control for blood gas analysis and related electrolytes in clinical laboratories of China
  17. Clinical validation of S100B in the management of a mild traumatic brain injury: issues from an interventional cohort of 1449 adult patients
  18. A quantitative LC-MS/MS method for insulin-like growth factor 1 in human plasma
  19. Development and validation of a mass spectrometry-based assay for quantification of insulin-like factor 3 in human serum
  20. Anti-ganglioside antibodies: experience from the Italian Association of Neuroimmunology external quality assessment scheme
  21. Reference Values and Biological Variations
  22. Improving IBD diagnosis and monitoring by understanding preanalytical, analytical and biological fecal calprotectin variability
  23. Establishing normal values of total testosterone in adult healthy men by the use of four immunometric methods and liquid chromatography-mass spectrometry
  24. Cancer Diagnostics
  25. Exploring the potential of mucin 13 (MUC13) as a biomarker for carcinomas and other diseases
  26. Cardiovascular Diseases
  27. Prognostic implications of detectable cardiac troponin I below the 99th percentile in patients admitted to an emergency department without acute coronary syndrome
  28. Osteocalcin value to identify subclinical atherosclerosis over atherosclerotic cardiovascular disease (ASCVD) risk score in middle-aged and elderly Chinese asymptomatic men
  29. Infectious Diseases
  30. Comparison of four methods of establishing control limits for monitoring quality controls in infectious disease serology testing
  31. Letter to the Editor
  32. Letter to the Editor relative to Clin Chem Lab Med 2018;56(3):360–372
  33. Update in diagnosis and management of primary aldosteronism: reply to a Letter to the Editor
  34. Can calculated total nitrogen replace Kjeldahl total nitrogen measurements in 24-h urine samples?
  35. Analytical performance of the single well titer function of NOVA View®: good enough to omit ANA IIF titer analysis?
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