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Effects of inter-day and intra-day variation on salivary metabolomic profiles.

Author(s): Kawanishi N, Hoshi N, Masahiro S, Enomoto A, Ota S, Kaneko M, Soga T, Tomita M, Kimoto K

Publication: Clin Chim Acta, 2019, Vol. 489, Page 41-48

PubMed ID: 30481500 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared total saliva volume and metabolomic profiles of specimens collected by stimulation to non-stimulated specimens. Inter-day and intra-day variations and the effects of patient age, sex, and smoking status on metabolomic profiles were also investigated. Whole saliva samples were collected from 13 participants (11 male, 2 female, mean age 29.77 ± 4.64 y) that had refrained from eating or drinking for 2 h prior to collection. Unstimulated saliva was collected first using a passive drool method by collecting naturally secreted saliva into a container cooled on ice for a period of 15 min. Stimulated saliva was then collected in a container cooled on ice from participants for 10 min while chewing paraffin gum. To investigate intra-day variation, specimens were collected three times a day: morning (8:00–9:00), daytime (12:00–13:00), and evening (17:00–18:00). To investigate inter-day variation, specimens were collected every other day (Days 1, 3, and 5). The volumes of each specimen were measured at the time of collection and then stored at −20 °C. Frozen saliva was thawed at 4 °C for approximately 1.5 h, centrifuged through a 5-kDa cutoff filter at 9100 × g for at least 2.5 h at 4 °C, filtrate was lyophilized and then dissolved in Milli-Q water, and metabolomics profiles were determined using capillary electrophoresis -time-of-fight-mass spectrometry (CE-TOF-MS) analysis.

   Summary of Findings:

   There were no significant inter-day or intra-day variations in saliva volume for unstimulated or stimulated saliva (P>.05 for all). Among the 47 metabolites analyzed, 20 showed significant differences between stimulated and unstimulated specimens (P<0.05). Significant intra-day variations in the metabolomic profile were observed in unstimulated saliva between morning and evening specimens (P=0.0048) but not between morning and daytime specimens or daytime and evening specimens. Similarly, significant inter-day variations in the metabolomic profile were found in stimulated saliva between morning and daytime specimens (P=0.0043) and between daytime and evening specimens (P=0.031) but not between the morning and evening specimens. However, no significant inter-day variations were observed in metabolomic profiles in stimulated or unstimulated saliva. Hierarchal cluster analysis revealed that variations in metabolome patterns were not associated with collection method or patient sex, age, or smoking habits.

   Biospecimens

   • Bodily Fluid - Saliva

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Small molecule	Capillary electrophoresis-MS

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Patient age	<30 y ≥30 y
   Preaquisition	Patient gender	Female Male
   Biospecimen Acquisition	Time of biospecimen collection	Day 1 Day 3 Day 5 morning (8:00–9:00) daytime (12:00–13:00) evening (17:00–18:00)
   Biospecimen Acquisition	Method of fluid acquisition	Stimulated saliva (chewing prior to collection) Non-stimulated saliva
   Preaquisition	Diagnosis/ patient condition	Smoker Non-smoker

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