Standardizing salivary lactoferrin measurements to obtain a robust diagnostic biomarker for Alzheimer's disease

Abstract

The search for new, robust, and reproducible biomarkers for Alzheimer's disease (AD) diagnosis is a challenge. We recently reported that salivary lactoferrin (Lf) could be presented as new biomarker candidate for AD, being both non-invasive and cost-effective, as well as having appropriate diagnostic performance for the clinical detection of AD subjects. Saliva is an attractive sample type for biomarker-based testing approaches for several other diseases; however, its composition may change under certain circumstances. It is therefore critical to maintain a consistent salivary handling protocol, considering possible extrinsic factors that may influence salivary Lf concentration. In this work, we analyzed salivary Lf concentration under different handling conditions and donor-dependent factors including age, inter-diurnal variations, physical activity, and pharmacological treatments. Our aim was to evaluate the influence of such conditions on salivary Lf concentration. In conclusion, we found that most of these extrinsic factors should be considered in the future when using Lf as a predictive biomarker for AD.

Keywords: Alzheimer's disease; aging; biomarker; circadian rhythm; exercise; lactoferrin; measurement; regulatory factors; reproducibility; saliva; treatments.

FIGURE 1

Salivary lactoferrin (Lf) concentration changes. A, Relative Lf concentration in salivary samples collected from four subjects (mean age 66.9 years) stored at 4°C for 15 days. Samples were frozen at –80°C after collection. Then, they were thawed and maintained at 4°C. Values after thawing were compared to the baseline levels from samples stored at –80°C stated as 100%. Data are expressed in percentage and are shown as mean ± standard error of the mean (SEM). Differences between groups were assessed using one‐way analysis of variance (ANOVA) followed by the Bonferroni test. **P < .01 versus day 0; #P < .05 versus day 5. B, Effect of freezing/thawing on salivary Lf concentration. Salivary Lf concentration in four subjects’ samples (mean age 66.9 years) that had not been subjected to any freeze/thaw cycle (point 0) compared to samples from the same subjects that had been subjected to one freeze–thaw cycle (point 1). Connecting lines between point 0 and point 1 represent Lf values from the same subject. Data show individual values of Lf concentration. C, Relative salivary Lf concentration in samples from eight subjects (mean age 66.9 years) related to the number of freeze–thaw cycles. Salivary Lf levels after only one freeze–thaw cycle were used as the baseline value and stated as 100%. Data are expressed in percentage and are shown as mean ± SEM. Differences between groups were assessed using one‐way ANOVA followed by the Bonferroni test. **P < .01, ***P < .001, ****P < .0001 versus cycle 1; #P < .05, ##P < .01 versus cycle 2. D, Salivary Lf levels in samples collected from 10 patients (mean age 52.5 years) at 10 am and after 8 hours (6 pm). Results showed no significant diurnal or day‐to‐day variation of Lf concentration. Data are shown as mean ± SEM. E, Salivary Lf levels from 20‐ to 80‐year‐old subjects (n = 12 to 37 subjects per group). Data are shown as mean ± standard deviation (SD). Differences between groups were assessed using the Kruskal‐Wallis test. **P < .01 versus 30 y/o; ###P < .001; ####P < .0001 versus 40 y/o. F, Salivary Lf levels in samples collected from 10 subjects (mean age 52.5 years old) before and after physical exercise performance (1 hour training). Saliva was collected at two timepoints: pre‐exercise; and immediately after exercise cessation. Data are shown as mean ± SEM. Differences between groups were assessed using Student's t‐test. **P < .01. G, Total protein concentration in saliva samples collected from Alzheimer's disease (AD) patients (mean age 67.2 years) with (n = 12) or without (n = 12) acetylcholinesterase (AChE) inhibitor treatment. The results showed no significant variation in protein concentration. Data are shown as mean ± SEM. H, Salivary Lf levels from AD patients (mean age 67.2 years) with (n = 42) or without (n = 27) AChE inhibitor treatment. Treated AD patients showed higher salivary Lf levels. Data are shown as mean ± SEM. Differences between groups were assessed using Student's t‐test. *P < .05