Journal of Dental Research

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Acid-Base Properties of Human Gingival Crevicular Fluid M. Bickel, J.L. Munoz and P. Giovannini J DENT RES 1985 64: 1218 DOI: 10.1177/00220345850640100801 The online version of this article can be found at: http://jdr.sagepub.com/content/64/10/1218

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Acid-Base M.

Properties of Human Gingival Crevicular Fluid

BICKEL', J.L. MUNOZ2, and P. GIOVANNINI3

'Division of Physiopathology and Periodontology, School of Dental Medicine, 19, rue Barthelemy Menn, 1211 Geneva 4, Switzerland; "Laboratory of Experimental Ophthalmology, Geneva; and 3Department of Physiology, Geneva The pH of human gingival crevicular fluid (GCF) has been reported by many authors to be vetry alkaline (pH 7.5 8.7). This alkaliniAt could be explained, at least partially, by the fact that all measurements were performed either at low Pco, or in the absence of CO,. Therefore, we set up a procedure which allows for measurement of the pH of GCF samples from single inflamed sites at controlled Pco,. At a Pco, of 4.7 kPa (= 35 mmHg) and at 37 °C, the pH was 7.96 + 0.10 (SEM, n 9), a value which differs significantly from the value of 8.38 ± 0.09 measured in the absence of CO2 in the same samples. The non-bicarbonate buffer value of the sample determined by CO2 titration was 6.0 slykes. It is because this value is low that pH varies so greatly with Pco,. At physiological Pco,, the total buffering power becomes very high above pH 8.0, because of the high bicarbonate concentration. =

J Dent Res 64(10):1218-1220, October, 1985

Introduction. In a recent investigation, we measured the pH of GCF and reported an increase in the pH with increase in the degree of gingival inflammation (Bickel and Cimasoni, 1985). These measurements were performed at 20 °C and in the absence of CO2, conditions which lead to an overestimation of the true pH. In situ, one would expect the Pco, to vary when going from the orifice to the bottom of a sulcus or of a periodontal pocket, especially since oxygen partial pressure varies considerably at these different levels (Mettraux et al., 1984). In the depth of a sulcus, one could assume a Pco, almost as high as that of the tissue, while at the orifice of the sulcus the Pco, will rather depend on the intra-oral level. In situ pH measurements (Kleinberg and Hall, 1969; Borden et al., 1977; Stephen et al., 1980) were always performed in the conditions of a socalled "open system" (see Izutsu, 1981). Thus, it seems possible that the reported values were systematically overesti-

mated as well. The purpose of the present study was to measure the pH of micro-volumes (fractions of microliters) of GCF from single inflamed sites, at physiological temperature and Pco,. In addition, since Pco, may vary in vivo, we estimated the nonbicarbonate buffer value of the sample, in order to determine how the pH varies as a function of Pco,.

from the upper pre-molars of four female and five male subto 63. Briefly, the sites were dried by a blast of air and isolated with cotton rolls, and GCF was sampled extracrevicularly five min later with small capillary tubes. The sites of GCF collection showed clinical evidence of inflammation and pockets not deeper than 4 mm. Samples contaminated with blood or showing turbidity were discarded. All samples had volumes ranging from about 100 to 500 nl (10-91); they were immediately frozen and stored at -20 °C until ana-

jects aged 25

lyzed. pH electrode.

The pH micro-electrode was of the liquid membrane type (Walker, 1971), in which a liquid sensor (Ammann et al., 1981) is lodged in the open tip of a micropipette. Two-mm-diameter double-barreled capillaries (theta section) were drawn from fused silica blanks1, and micropipettes were pulled on an automatic puller (J.L. Munoz, in preparation). Silanization and filling of the electrode were as described in Munoz et al. (1983). The reference barrel contained KC1 (3 mol-l-'). The overall tip diameter was less than 1 Jim. The electrode drifted less than 0.2 mV-min- , and its sensitivity was 54.6 mV-pH units-' at 37 °C. It was calibrated before and after each measurement. The pH of each calibration solution2 was checked at 37 °C with a pH macro-electrode3 and pH meter4. One solution had a pH of 6.98, the other 9.86. Measuring procedure. The sample was placed in a concavity of a microscope slide (see Fig. 1). In order to avoid evaporation, the sample was covered by a hydrophobic liquid (see below). The constancy of the sample volume was controlled by observing the diameter of the sample droplet over a period of six hr. At a magnification of 40x, no change in diameter was observed. CO2 titration was achieved by flooding the covered sample with a gas mixture (O2/CO2) of known composition (Kaiser et al., 1974). These authors used paraffin oil to cover the sample. We found that after passage through paraffin oil our pH electrodes no longer responded. At the suggestion of Dr. D. Ammann (personal communication, 1984), we used dodecane5, which appeared not to degrade the electrode performance and in which CO2 readily dissolves. By means of a micromanipulator, the tip of the electrode was put gas mixture

pH microelectrode

Materials and methods. GCF sampling. Nine samples of GCF were collected with a recently standardized technique (Bickel and Cimasoni, 1985)

_

_ _7

Received for publication March 1, 1985 Accepted for publication June 12, 1985 Supported by grant No. 133 from the Swiss Society of Odontology 'To whom correspondence should be addressed Current address: NIH, NIDR, Building 30, Room 324; Bethesda.

oil

(dodecane)

MD 20205

'Soci&te Electrothermique SA, La Tour-de-Treme, Switzerland 2Merck, Darmstadt, Germany 3Type 10401 3038, Ingold, Urdorf, Switzerland 4Model 801, Digital pH meter, Orion, Kussnacht, Switzerland

5Fluka, Buchs,

1218

Switzerland

gas mixture

heating chamber

Fig.

I

Schematic representation of the measuring set-up.


into the droplet of GCF under microscopic control. Potentials continuously measured with a differential electrometer6 and recorded on a chart recorder. The buffer capacity Determination of the buffer value. may be defined as the effectiveness with which a solution resists changes in hydrogen ion concentration. In order to quantify this capacity, Woodbury (1965) introduced a unit, the slyke, which is the quantity of base in mmol (= quantity of acid added) which disappears from one liter of solution when the pH changes by one unit. When the acid added is carbonic acid, the buffer value is called the non-bicarbonate buffer value (NBp). Since the result of the neutralization of the bases by the carbonic acid is an increase of the HCO3 concentration, were

NBp

is

usually expressed

as

d[HCO3]

dpH3dpH

-

Experimentally, and the [HCO3]

the pH was measured at various Pco, levels calculated from the Henderson-Hasselbalch equation:

log [HCO3] . At 37°C and ionic strength 0.16 at. Pco, mol-l- , pK' is 6.12, and the solubility coefficient of CO2 (a) is 0.225 mmol--l-kPa- 1 (1 kPa = 7.5 mmHg).

pH

=

pK'

+

Results. The pH of GCF samples was recorded continuously throughout the experiment. Fig. 2 shows an original tracing of one experiment. In stage 1, a stable value of pH was obtained in the absence of CO2. The introduction of a gas mixture with a known CO2 fraction (Pco, 4.7 kPa) induced a rapid decrease ( 1 min) of the pH (stage 2) until a value was reached which remained stable (stage 3), indicating that the GCF sample was completely saturated by the imposed CO2. A further increase =

in the CO2 fraction of the gas mixture induced a further aci-

dification, which once again reached a plateau (stage 5). The longer time (: 3 min) taken to reach a plateau value (stage 4)

is due to a longer time of arrival of the new gas mixture when the gas mixture was changed. This was shown by control experiments where the immediate introduction of the high Pco, (9.3 kPa = 70 mmHg) induced a rapid acidification of the sample (. 1 min). Stage 7 shows that the process was remarkably reversible. One can observe that there was almost no drift in the reference potential, as was the case in almost all the experiments. The results of experiments of GCF samples of nine patients V

1

Pco2 4.7 kPa

1219

ACID-BASE PROPERTIES OF HUMAN GCF

Vol. 64 No. 10

Pco2 9.3 kPa

I

I

CO2 off

collected in the Table. In the absence of CO2, the mean 8.38 + 0.09 (SEM), and at 4.7 kPa it was 7.96 + 0.10. Statistically, this difference is highly significant (P = 0.008, Student's t test). The mean calculated NBP is 6.05 ± 0.5 slykes. (For details of calculations, see "Materials and are

pH

was

methods".)

Discussion. At a Pco, of 4.7 kPa (35 mmHg) and 37 °C, the pH of GCF from inflamed sites was about 0.4 pH unit less alkaline than was the pH of the same fluid measured in the absence of CO2. This observation, however, confirms that even in the presence of an elevated Pco,, the pH of GCF is much more alkaline than other body fluids. The great dependence of pH as a function of Pco, could be responsible, at least in part, for the great variability of pH values measured under uncontrolled Pco,, the

more so because the NBP3 is very low. These latter measurements were performed either in situ (at unknown Pco,) or in vitro (Pco, near 0), and these values have thus to be corrected

for the estimation of the actual pH in the bottom of the sulcus. The variation of pH as a function of Pco, is shown on a diagram (Davenport, 1958) where we have plotted our experimental data (Fig. 3). In addition, the diagram shows that the difference between the pH of healthy sites (pH "7.0, Bickel and Cimasoni, 1985) and the pH of inflamed sites (pH 8.0) is certainly not due to a CO2 loss. =

TABLE

pH VALUES AT 37°C IN THE ABSENCE OF CO2 (pHo), pH CHANGES DUE TO CO2 TITRATION (dPHI: 0 4.7 kPa, dpH2:

4.7 9.3 kPa) AND NON-BICARBONATE BUFFER VALUES (NBP IN SLYKES ) OF NINE GCF SAMPLES. (1 kPa 7.50 mmHg) =

Subject

pH(

dpH,

dpH2

NBP3

1 2 3 4 5 6 7 8 9

8.45 8.13 8.25 8.41 8.39 8.26 7.96 8.92 8.65 8.38 0.09

0.52 0.33 0.52 0.36 0.40 0.35 0.48 0.39 0.42 0.42 0.02

0.18 0.13 0.20 0.16 0.15 0.13 0.20 0.25 0.25 0.18 0.02

5.83 8.08 5.25 6.56 7.00 8.08 5.25 4.20 4.20 6.05 0.49

x

SEM

[HCo0]

1min "

I

mmol-l1 9.2

PCO2

200

'

9.3 kPa /

PC2a

4.7 kPa

/

Pco2

8.4

8.6 pH

2.7 kPa

-8.8

150 8.4

NB 8.0

100J

-d [HCO3 dpH

20 7.6

50

7.2

(3)

Fig.

2

4

(

Reproduction of an original tracing of continuous pH measure-

ments in the absence and in the presence of different description is given in the text.

PCo2 levels.

The

7.0

7.4

7.6

7.8

8.0

8.2

Davenport's diagram. Since the NBp cannot change signifi0.5 pH unit range, we have considered the slope (the ratio -d[HCO3] / dpH) as linear in the pH range, 8.0 -+ 0.5.

Fig.

3

cantly in

6FD 223, World Precision Instruments, New Haven, CT, USA

7.2

a


1220

BICKEL ET AL.

The low NBP3 of 6 slykes equals that of the plasma at a pH of 8.0 (Siggaard-Andersen, 1976). This observation is in accordance with our previous finding that the total protein and albumin concentrations in GCF from inflamed sites are similar to those in plasma (Bang and Cimasoni, 1971; Bickel et al., 1985). Even though the NB3 of GCF is low, and therefore renders the pH very sensitive to changes in Pco,, this does not mean that GCF is poorly buffered. Indeed, at a pH of 7.96 and a constant Pco2 of 4.7 kPa, the calculated [HCO3] is 73 mmoll-l1. Since the HCO3/CO2 buffer system is open, the bicarbonate buffer value is given by 2.3- [HCO-] (Van Slyke, 1922) and equals 168.2 slykes. This buffer value greatly exceeds the buffer value of most body fluids. GCF is thus highly buffered, especially with regard to non-volatile bases. Above pH 8.0, [HCO-] and hence buffer value rapidly increase even more, and an extremely active alkalinization process would be necessary to exceed this pH. This latter consideration allows the prediction that this pH of 8.0 cannot be exceeded significantly without an enormous bicarbonate concentration change. For example, one may calculate that at a pH of 8.4 and Pco, of 4.7 kPa, the [HCO3] is 200mM. In conclusion, crevicular fluid from inflamed sites has a pH of about 8.0 at physiological temperature and Pcor, a value 0.4 pH units less alkaline than that measured in the absence of CO2. Because of the high bicarbonate buffer value, it seems reasonable to consider this alkalinity the biophysical condition under which physiopathologic events accompanying gingival inflammation occur.

Acknowledgments. We are grateful to Dr. Jonathan Coles and Prof. Giorgio Cimasoni for their advice and criticism during experimental work and preparation of the manuscript. REFERENCES AMMANN, D.; LANTER, R.A.; SCHULTHESS, P.; SHIJO, Y.; and SIMON, W. (1981): Neutral Carrier Based Hydrogen Ion Se-

J Dent Res October 1985

lective Microelectrode for Extra- and Intracellular Studies, Analyt Chem 53:2267-2269. BANG, J. and CIMASONI, G. (1971): Total Protein in Human Crevicular Fluid, J Dent Res 50:1683. BICKEL, M. and CIMASONI, G. (1985): The pH of Human Crevicular Fluid Measured by a New Microanalytical Technique, J Periodont Res 20:35-40. BICKEL, M.; CIMASONI, G.; and ANDERSEN, E. (1985): Flow and Albumin Content of Early (Pre-inflammatory) Crevicular Fluid from Human Subjects, Arch Oral Biol (in press). BORDEN, S.M.; GOLUB, L.M.; and KLEINBERG, I. (1977): The Effect of Age and Sex on the Relationship Between Crevicular Fluid Flow and Gingival Inflammation in Humans, J Periodont Res 12:160-165. DAVENPORT, H.W. (1958): The ABC of Acid-Base Chemistry, 4th ed. Chicago: The University of Chicago Press, pp. 39-40. IZUTSU, K.T. (1981): Theory and Measurement of the Buffer Value of Bicarbonate in Saliva, J Theoret Biol 90:397-403. KAISER, D.; SONGO-WILLIAMS, R.; and DRACK, E. (1974): Hydrogen Ion and Electrolyte Excretion of the Single Human Sweat Gland, Pfligers Arch 349:63-72. KLEINBERG, I. and HALL, G. (1969): pH and Depth of Gingival Crevices in Different Areas of the Mouths of Fasting Humans, J Periodont Res 4:109-117. METTRAUX, G.R.; GUSBERTI, F.A.; and GRAF, H. (1983): Oxygen Tension (pO2) in Untreated Human Periodontal Pockets, J Periodont 55:516-521. MUNOZ, J.L.; DEYHIMI, F.; and COLES, J.A. (1983): Silanization of Glass in the Making of Ion-sensitive Microelectrode, J Neurosci Meth 8:231-247. SIGGAARD-ANDERSEN, 0. (1926): The CO2 Equilibration Curve of Plasma. In: The Acid-Base Status of the Blood, 4th ed. Copenhagen: Munksgaard, pp. 41-44. STEPHEN, K.W.; McCROSSAN, J.; and MACKENZIE, D. (1980): Factors Determining the Passage of Drugs from Blood into Saliva, J Clin Pharmacol 9:51-55. VAN SLYKE, D.D. (1922): On the Measurement of Buffer Value and on the Relationship of Buffer Value to the Dissociation Constant of the Buffer and the Concentration and Reaction of the Buffer Solution, J Biol Chem 52:525-570. WALKER, J.L., Jr. (1971): Ion Specific Liquid Ion Exchanger Microelectrodes, Analyt Chem 43:89A-93A. WOODBURY, J.W. (1965): Regulation of pH. In: Physiology and Biophysics, T.C. Ruch and H.D. Patton, Eds., Philadelphia: W.B. Saunders Co.
