Saliva is an excellent biofluid for analysis by targeted profiling. It is easily collected by individuals with minimal training, using noninvasive techniques. Targeted profiling of saliva samples can provide identification and quantification of many of the small molecule metabolites commonly found in saliva, and may allow the development of simple screening procedures for a variety of diseases [27] [28]. In this note we present a list of common metabolites found in human saliva, and techniques for targeted profiling of saliva spectra with Chenomx NMR Suite.
Saliva is easy to collect via noninvasive techniques used by individuals with minimal training, and can provide useful metabolic information [29]. Samples can be collected essentially on demand, permitting the collection of multiple samples per day, at times designed to maximize the amount of useful information gathered. Short-term and medium-term storage requirements are not onerous, and in many cases allow storing samples at room temperature. Concentrations of certain small molecule metabolites in saliva, including some hormones and many pharmaceuticals and drugs of abuse, are known to correlate quantifiably with concentrations in serum. Targeted profiling of saliva can providing direct identification and quantification of many of these metabolites.
Analyzing saliva using targeted profiling is similar to analyzing blood serum or plasma, due to its protein content. In preparing saliva samples for analysis by targeted profiling, you will need to account for the effects of proteins in the sample. This can involve various methods, alone or in combination, as described previously for serum [30].
At the sample preparation stage, ultrafiltration using microcentrifuge filtration tubes provides an effective method of removing both proteins and particulate matter from saliva samples. If you have filtered your saliva samples, then you can acquire NMR spectra with a NOESY presaturation pulse sequence. On the other hand, if you choose not to filter your samples, several relaxation-edited pulse sequences exist that can remove the broad protein resonances from your spectrum during acquisition, allowing for simpler identification of the small molecule metabolites in the sample.
In general, relaxation-edited pulse sequences are a simpler method of removing the effect of protein resonances from a sample, but analysis of a sample after acquisition is easier when the protein has been removed from the sample [30].
Figure 2. Region A of the sample spectrum, including signals from (a) butyrate, (b) alanine, (c) lactate, (d) fucose, (e) ethanol, (f) propylene glycol, (g) propionate, (h) valine, (i) leucine and (j) isovalerate.
Small molecule metabolites found in human saliva can come from several sources. Depending on the method of collection, food components may be directly observed during targeted profiling of saliva. These can be solutes already dissolved in the food, like caffeine in coffee, solid components that rapidly dissolve into the saliva, like sugars from a cookie, or metabolites resulting from the action of enzymes in the saliva, like maltose from the action of α-amylase on starches.
Figure 3. Region B of the sample spectrum, including signals from (a) glucose, (b) propylene glycol, (c) 4-hydroxyphenylacetate, (d) fucose, (e) taurine, (f) caffeine, (g) myo-inositol, (h) tyrosine, (i) malonate and (j) lysine.
Cellular debris from both human and bacterial sources may contribute various metabolites, and products of bacterial metabolism may also be present. Steroids, some other hormones, and many drugs and antibodies can diffuse from serum into saliva, as a result of both passive diffusion and active transport, including ultrafiltration through tight cell junctions [31]. In some cases, metabolites measured in saliva can provide information about metabolite concentrations that cannot be readily obtained from serum analysis [29].
Saliva samples can readily be analyzed using targeted profiling, following ultrafiltration to remove interfering proteins and particulate matter. The remaining small molecule metabolites can provide insight into serum composition that is otherwise difficult to obtain.
Table 1. Common Metabolites in Human Saliva
| Amines and Derivatives | |
| Caffeine | Methylguanidine |
| Choline | O-Phosphoethanolamine |
| Creatinine | Trigonelline |
| Dimethylamine | Trimethylamine |
| Methylamine | Trimethylamine N-oxide |
| Amino Acids and Derivatives | |
| 4-Aminobutyrate | Methionine |
| Alanine | Ornithine |
| Aspartate | Phenylalanine |
| Glutamate | Proline |
| Glutamine | Sarcosine |
| Glycine | Threonine |
| Histidine | Tryptophan |
| Isoleucine | Tyrosine |
| Leucine | Valine |
| Lysine | |
| Carbohydrates and Derivatives | |
| Acetone | Glucose |
| Ethanol | Methanol |
| Fucose | myo-Inositol |
| Galactose | Propylene glycol |
| Glucitol | |
| Organic Acids | |
| 3-Hydroxybutyrate | Isobutyrate |
| 4-Hydroxyphenylacetate | Isocaproate |
| Acetate | Lactate |
| Acetoacetate | Phthalate |
| Butyrate | Propionate |
| Citrate | Pyruvate |
| Formate | Succinate |
| Glycolate | Valerate |