Determination of arginine catabolism by salivary pellet☆

Graphical abstract


Method details
In short, the assessment of the arginolytic potential involved the following steps: (i) preparation of the salivary pellet to concentrate the oral bacteria present in the saliva; (ii) performing the arginolytic activity assay to assess the arginine catabolism; (iii) determination of the amount of ammonium produced from arginine; and (iv) determination of the protein weight of the salivary pellet. The final concentrations of ammonium were corrected per time unit and protein weight.

Preparation of the cell suspension
While optimizing the arginolytic activity assay, the pH of the Tris-maleate buffer was raised from pH 6.0 to pH 7.5. This was done as the arginolytic activity of a range of oral micro-organisms and the salivary pellet significantly produced more ammonium under pH 7.5 as compared to pH 6.0. The cell suspensions were prepared as follows: 1. Stimulated human saliva from various donors was thawed. 2. Four milliliter of each saliva sample was centrifuged for 10 min at 4000 Â g at 4 8C.
3. The salivary pellet was washed once with 800 ml 10 mM Tris-maleate buffer, pH 7.5 (Sigma) to remove low density salivary components.
4. The pellet was re-suspended in a final volume of 800 ml Tris-maleate buffer.

Arginolytic activity assay
In comparison to the original protocol the incubation time was lengthened to three hours. This was done to ensure that ammonium formation, in salivary pellets, low in cell amounts and saliva from nonresponders, was still measurable.
To assess the arginolytic activity of the prepared cell suspensions, the following protocol was followed: 1. Of each cell suspension 237.5 ml was pipetted, in duplicate, into a non-skirted 96-well PCR plate (Corning) and pre-incubated at 37 8C in a thermocycler (Eppendorf). 2. The remaining cell suspension was stored at À20 8C for protein determination.
3. The arginolytic activity assay was started by adding 12.5 ml 1 M arginine to a final concentration of 50 mM (Sigma) to each cell suspension. 4. The plate with the cell suspensions was incubated at 37 8C for 3 h.

5.
Immediately after the addition of the arginine, and after the three-hour incubation period, 50 ml samples were taken, transferred to a PCR plate (VWR) and placed on ice for 5 min. 6. After cooling, the samples were heat inactivated for 5 min at 80 8C, to stop all enzymatic reactions. 7. The PCR plate was centrifuged for 10 min at 1509 Â g at 4 8C. 8. The supernatants were transferred to a microtiter plate (Greiner), the plate was sealed and stored at À20 8C until further analysis.

Ammonium determination
To determine the amount of ammonium produced from the catalysis of arginine, an assay was used as based on the method of Da Fonseca et al. Preparation of the reagents 1. The reaction buffer (TK-buffer) containing 0.5 M triethanolamine and 15 mM a-ketoglutaric acid (both Sigma), pH 8.0 was prepared. This buffer was stored at 4 8C for maximum of 4 weeks. 2. Fresh NADH stock solution was prepared by dissolving 40 mg NADH (Roche) in 10 ml TK-buffer. 3. To create a NADH work solution, the NADH stock solution was further diluted (10 times) in TK-buffer. This NADH work solution was stored on ice until further use. 4. The GIDH stock solution was prepared by adding 3.6 ml MilliQ water to 3000 U glutamate dehydrogenase (Roche). This stock solution was stored at 4 8C. 5. Prior to the assay, a GIDH work solution was prepared by diluting the GIDH stock solution, 10 times in MilliQ water. The GIDH work solution was stored on ice until further use. 6. A 4 mM ammonium stock was prepared by diluting a 40 mM ammonium sulfate (NH 4 ) 2 SO 4 (Sigma), 20 times in TK-buffer. 7. Ammonium standards were prepared in the range of 0-2.8 mM, with 0.4 mM intervals.
Ammonium assay 1. A 96-well microtiter plate (Greiner) was filled with 120 ml MilliQ water per well. 2. 65 ml of NADH work solution was added to each well. 3. 10 ml samples (obtained from the arginolytic activity assay) were added in duplicate, to the appropriate wells.
4. 10 ml of the eight standards was added in duplicate to the appropriate wells.
5. The plate was mixed for 5 min on a microtiter plate shaker. 6. The absorbance A1 (l = 340 nm) was read.
7. The enzymatic reaction was started by adding 20 ml GIDH working solution to each well. 8. The plate was mixed for 30 s on a microtiter plate shaker. 9. The plate was incubated for 20 min at 20-25 8C. 10. The absorbance A2 (l = 340 nm) was read.
The DA (A1 À A2) was calculated and the values extrapolated against the ammonium standards. The calibration curve for the ammonium assay was linear up to 3.5 mM ammonium. The lower detection limit was defined as the average absorbance of the lowest concentration measured plus three times the standard deviation. The upper detection limit was defined as the average absorbance of the highest concentration measured minus three times the standard deviation. Fig. 1 shows an example of the reproducibility and accuracy of the standard curve including the upper and lower detection limit.
The obtained ammonium values were corrected for time and protein mass. To illustrate the sensitivity of the method, the Graphical Abstract shows the arginolytic activity of saliva from nine different donors, expressed as the amount of ammonium (nM) produced per minute per mg protein.

Protein determination of the salivary pellet
The protein concentration of the cell suspensions used in the arginolytic activity assay was determined to normalize the ammonium formation by the salivary pellets with regards to differences in biomass.
In the original protocol [2], a centrifuge step had to be performed after the bead beating steps. Unfortunately, by centrifuging the samples, the cell walls were pelleted on top of the glass beads (see Graphical Abstract). As a result, less protein is present in the supernatant. This subsequently leads to an underestimation of the protein concentration, as the supernatant is used for the protein determination according to Bradford. Hence this centrifugation step was omitted from the method.
To perform the modified protein determination the following steps were taken: 1. A 2 ml 96-deepwell plate (Greiner) was filled with 200 ml MilliQ water washed Ø = 0.1 mm glass beads (Biospec products) per well. 2. The samples from stored at step 2 of the arginolytic activity assay were thawed.
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