LC-MS/MS Analysis of Creatine Metabolites in Saliva and Serum Following Exercise

Researchers involved in a multinational study monitored changes in salivary creatine pre- and post-high-intensity exercise in young adults while also investigating the potential correlation between salivary and serum creatine levels. Samples of saliva and serum were collected before and immediately after an incremental running-to-exhaustion treadmill test in fifteen young adults (mean age [23.9 ​± ​2.9] years, eight females), with samples analyzed for guanidinoacetic acid, creatine, and creatinine using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. A paper based on this research was published in Sports Medicine and Health Medicine.¹

Stored predominantly in skeletal muscles, creatine plays a critical role in the support of cellular energy production during heavy exercise by maintaining adenosine triphosphate (ATP) levels. While this process can result in the well-documented temporary depletion of muscle creatine within the body, there is little information available concerning changes in creatine metabolism caused by exercise within in other tissues.² High-intensity exercise may result in the changes of creatine levels in serum and saliva. Anintense exhaustive exercise session, for example, will generate transient changes in circulating biomarkers of creatine metabolism; this suggests a significant exercise-induced disruption in bioenergetics.^3-5Using a blood sample for the monitoring of creatine biodynamics is not always ideal, because of fears concerning invasiveness, hygiene, and the potential infection risks associated with the sample’s collection and handling.⁶ Conversely, saliva appears comparatively clean, and samples can be collected quickly and noninvasively, as well as easily stored.⁷ However, according to the authors of the study,¹ “no study so far has evaluated whether exercise affects salivary biomarkers of creatine metabolism and do creatine levels in saliva mirror those found in serum before and after exercise.”

Following the exercise of fifteen young adults (mean age [23.9 ​± ​2.9] years, eight females) who volunteered for the study, the researchers reported a substantial elevation in salivary creatine levels from (17.5 ​± ​14.2) μmol·L^-1 to (43.6 ​± ​30.4) μmol·L^-1 (p ​< ​0.001), coupled with a significant increase in salivary creatinine from (11.3 ​± ​5.8) μmol·L^-1 to (17.0 ​± ​9.3) μmol·L^-1 (p ​= ​0.04). In contrast, serum creatine levels were unaffected by exercise (p ​= ​0.80), while creatinine levels exhibited a strong tendency to decrease post-exercise (from [81.8 ​± ​17.5] μmol·L^-1 to [73.1 ​± ​11.6] μmol·L^-1; p ​= ​0.06). A comparison of the slopes of the two regression lines (saliva vs. serum) revealed significant differences for both creatine (p ​= ​0.01) and creatinine (p ​= ​0.03).¹

The team’s findings, according to the authors of the study,¹ “suggest a potential difference in the dynamics of creatine metabolites in these two bodily fluids, both pre- and post-exercise.”

The researchers note that their study does not clarify whether salivary creatine increases due to diffusion from the blood or whether it is influenced by exercise-induced changes in salivary gland metabolism. In addition, the storage conditions, sample processing, and potential degradation of creatine in saliva over time could introduce variability in the results. Given these concerns, they are of the opinion that more research with larger sample sizes, improved methodological rigor, and varied exercise protocols is necessary before saliva can be considered a reliable biomarker for creatine metabolism in response to physical exertion.¹

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References

1. Andjelic, B.; Lainovic, T.; Todorovic, N. et al. Exercise Affects Salivary Biomarkers of Creatine Metabolism in Healthy Adults. Sports Med Health Sci. 2025, 8 (2), 193-196. DOI: 10.1016/j.smhs.2025.03.010
2. Sahlin, K.; Harris, R. C.; Hultman, E. Resynthesis of Creatine Phosphate in Human Muscle After Exercise in Relation to Intramuscular pH and Availability of Oxygen.Scand J Clin Lab Invest 1979, 39 (6), 551-557. DOI: 10.3109/00365517909108833
3. Stajer, V.; Trivic, T.; Drid, P. et al. A Single Session of Exhaustive Exercise Markedly Decreases Circulating Levels of Guanidinoacetic Acid in Healthy Men and Women. Appl Physiol Nutr Metab2016, 41 (10), 1100-1103. DOI: 10.1139/apnm-2016-0102
4. Stajer, V.; Vranes, M.; Ostojic, S. M. et al. Correlation Between Biomarkers of Creatine Metabolism and Serum Indicators of Peripheral Muscle Fatigue During Exhaustive Exercise in Active Men. Res Sports Med 2020, 28 (1), 147-154. DOI: 10.1080/15438627.2018.1502185
5. Fazazi, S. Al; V. Stajer, V.; P. Drid, P. et al. 24-hour Dynamics for Serum Biomarkers of Creatine Metabolism After an Acute Session of Exhaustive Resistance Exercise in Active Men. Sci Sports2019, 34 (3), 181-185. DOI: 10.1016/j.scispo.2018.12.001
6. Martínez, L. D.; Bezard, M.; Brunotto, M. et al. Creatine Metabolism: Detection of Creatine and Guanidinoacetate in Saliva of Healthy Subjects. Acta Odontol Latinoam2016, 29 (1), pp. 49-53
7. Suzuki, M.; Furuhashi, M.; Sesoko, S. et al. Determination of Creatinine-Related Molecules in Saliva by Reversed-Phase Liquid Chromatography with Tandem Mass Spectrometry and the Evaluation of Hemodialysis in Chronic Kidney Disease Patients. Anal Chim Acta2016, 911, 92-99. DOI: 10.1016/j.aca.2016.01.032