Catecholamine Levels in the Oral Cavity

The detection of catecholamines within the oral environment has previously been restricted to studies specifically investigating the levels of norepinephrine and epi-nephrine within saliva and dental pulp (Schachman et al. 1995; Mitome et al. 1997). Putative periodontal pathogens are likely to be exposed to catecholamines within saliva; however, this exposure is likely to exert an effect only on the supra-gingival microflora. The purpose of the next set of experiments was to determine the levels of sub-gingival catecholamines. GCF constantly bathes the sub-gingival environment, including any periodontal pathogens within dental plaque and therefore was an ideal fluid to begin the search for catecholamines (Fig. 7.3). Attempts have been made to measure catecholamines locally in GCF and plaque using HPLC with electrochemical detection and MALDI-ToF Electro-Spray Mass Spectrometry, however the small volumes of GCF and plaque made analysis difficult.

Previous studies have investigated biological fluids which are readily obtained in millilitre quantities (Mitome et al. 1997; Forster and Macdonald 1999) however despite attempts to pool the sub-microlitre volume GCF samples, this provided only sporadic identification of catecholamine content. Specifically, norepineph-rine was identified in two pooled samples of GCF using the Dionex equipment using a 30 min elution into sterile water followed by acidification with formic acid. Allowing a 30 min elution into sterile water improved the catecholamine recovery from Periopaper™ strips (Fig. 7.3) which was an unexpected result as

Fig. 7.3 GCF sampling being performed using Periopaper strips

catecholamines are generally unstable at neutral or basic pH as their catechol rings are vulnerable to oxidation in the presence of air and light (Callingham and Barrand 1979; Weir et al. 1986). Catecholamines are stable at low pH and/or in the presence of antioxidants such as cysteine, sodium metabisulphite, or ascorbic acid (Weir et al. 1986) and as a result, acid preservatives and/or stabilizing agents are added to urine collections for subsequent catecholamine studies (Moerman and de Schaepdryver 1984; Davidson and Fitzpatrick 1985). The recovery of norepinephrine from the Periopaper™ strips was improved by having a 30 min elution period in sterile water before acidification. Further, it is also possible that immediate acidification with formic acid was likely to have caused precipitation of the protein content within the GCF sample preventing the elution of catecholamines from the Periopaper™ strips.

Calculations based upon the results from the 30 min elution - delayed acidification method, indicated that the recovery rate for this technique was in the range of 83.4-96.5% and that GCF concentrations in positive samples were in the order of 0.58-3.08 jmmol/l. Typical plasma levels of norepinephrine are 300 pg/ml (1.8 nmol/l), and salivary levels are 7.8-9.9 pg/ml (46.1-58.5 pmol/l) and therefore the GCF levels detected are approximately 300-1,700 times the level found within plasma. GCF is thought to be a serum transudate in health and inflammatory exudate in disease. Indeed, the levels of drugs within GCF have been shown to be several fold greater than circulating levels (Thomason et al. 1997; Lavda et al. 2004). The crude calculations performed here, indicate that this may also be true for norepinephrine and epinephrine, but further work is needed to establish more reproducible methods of detection.

In a further attempt to determine the presence or absence of catecholamines in GCF samples, a MALDI-ToF ESMS methodology was used which has the advantage of requiring small sample volumes. The minimum sample volume for this technique was 2 jml and masses could be determined on 50-500 fmol quantities of low molecular weight (<5,000) peptides with an average mass accuracy of ± 0.1% (Williams 2006). Whilst norepinephrine and epinephrine could not be identified in a pool of three GCF samples (total volume 0.974 jml), epinephrine was identified in two healthy and one chronic periodontitis plaque sample (Roberts, unpublished data). Further, norepinephrine was also identified in the chronic periodontitis plaque sample as well as a saliva sample from a periodontally healthy subject (Roberts, unpublished data). These data support the HPLC method findings that catecholamines within GCF were not consistently found, however, it is interesting that MALDI-ToF ESMS confirmed the presence of catecholamines within plaque and saliva samples. Catecholamines present within saliva are likely to originate from the sympathetic innervation of the salivary glands, and therefore the salivary norepinephrine is likely to bathe the supra-gingival environment, including dental plaque. The presence of norepinephrine within a chronic periodontitis plaque sample and absence within healthy plaque samples, may reflect the quantity of plaque collected in each case or that the levels of norepinephrine within plaque correlate with the extent of periodontal disease. Epinephrine was identified in all plaque samples, and therefore it is possible that catecholamine levels within plaque influence the microbial flora, which in turn may influence the pathogenesis of periodontal disease. Overall, peaks or signals which appear to correspond to nor-epinephrine or epinephrine were found in GCF, plaque and saliva however, further attempts to quantify catecholamine levels in small volumes of biological samples are required as no published data currently exists.

0 0

Post a comment