Determination of pH, Ca2+ and Mg2+ in Saliva

Cody ChalkerDavid Julia

Real World Project Fall 2009

Electrolytes in Saliva

• Na+ K+,Ca2+ Mg2+(mM): Cu2+ Pb2+ (µM),• Electrolyte Conc. affected by flow rate and

disease• Only bound and free Ca2+ can be measured

simultaneously– 10% bound to proteins, 40% to phosphates,

citrates and lactates, 5% to bicarbonate; Thus approx. 45% is in ionic form

Bicarbonate and pH• Bicarbonate a product of cell metabolism • Bicarbonate does not buffer it mainly

neutralizes• Bicarbonate concentration in saliva is linked to

flow rate

Reasons for testing pH of saliva• The ability to monitor health status, disease

onset and progression, and treatment outcome through noninvasive means is a highly desirable goal in health care promotion and delivery.

• Oral fluid is a perfect medium to be explored for health and disease surveillance.

Importance• A healthy individual should have a saliva pH of 6.5-

7.4• Most ailments such as cancer, kidney stones, heart

disease, etc. are associated with an acidic pH in the body

• As you age the pH of your saliva changes• Over half of adults are acidic at 6.5, symbolizes signs

of aging i.e. calcium deficiency.• Most children are around a pH of 7.5• Most cancer patients have a pH of 4.5 while in

terminal condition.

Why Hasn’t This Been Studied Before?

• One of the main reasons this is just becoming a research topic is that the amount of ions in saliva are so small that not until recently were techniques used to be able to make affirmative quantitative assumptions

Techniques Used Today• Microfluidics and Microelectromechanical systems

are MEMS used for salivary diagnostics

• MEMS are integrated systems composed of mechanical elements, sensors, actuators and electronics on a common silicon substrate developed through micro fabrication technology

Other Techniques Used Today

• Electrochemical methods have been devised• Thiocynate is an example of an ion being

studied• Process includes high performance liquid

chromatography, silica gels, and ion specific electrodes

• Pros of procedure: that it is quick, precise, and requires few samples

Techniques Used• pH of saliva: – Saliva was taken from volunteer who were

asked to build up approx. 1-2mL of saliva which was then extracted with a disposable pipette. The saliva was then immediately immersed in paraffin oil

CO2 + H2O H2CO3 HCO3- + H+

pH cont’d• To finish testing the pH of saliva 8mL of water was

added to each sample. The water added was boiled for an hour and found to have a pH of 6.75

• pH glass-electrode probe was calibrated with different pH buffers to obtain pH values 4,7, and 10

• Following the calibration the pH probe was immersed into each sample

Determination of Magnesium and Calcium

• Determination of the two ions cannot be found exclusively through an EDTA titration since both Magnesium and Calcium complex to EDTA

• Volunteers were asked to collect as much spit in their mouths(approx. 5mL), sans mucus, and spit only once into the beaker

• 8mL of buffer-indicator was added. Standard was made of 8mL of water and 1mL of indicator.

• Standard was titrated with EDTA solution to determine approximate end point. Each sample was titrated to an end point

Determination of Magnesium

• Thiazole yellow was used as an indicator• Ammonium Oxalate was used to precipitate

out calcium • UV Vis instrument

Procedure for Use in UV Vis• Saliva samples were mixed with ammonium

oxalate and centrifuged (suspected problem area)

• 0.75mL of each sample was added to cuvettes. 0.75mL additions were added of water, polyvinyl alcohol, thiazole yellow and of NaOH

• The UV Vis was calibrated with magnesium concentrations of .1mM, .2mM, and .3mM. Each sample was run against these concentrations

Results• Using a glass-electrode the pH of 5 samples

were found to be 7.25, 7.4, 7.45, 7.3, 7.3. • EDTA titration of saliva involves small amounts

of either ion concentration. To determine the concentration of the two ions a comparison is made against a standard

Sample mM Mg/L .05% EDTA mL added1 0.11 0.242 0.13 0.293 0.23 0.354 0.34 0.415 0.54 0.616 0.83 0.95

Standard Calibration Graph

0 0.2 0.4 0.6 0.8 1 1.20

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

f(x) = 0.950484848484849 x − 0.00106666666666677R² = 0.996431237628571

Mg Standard

.05% EDTA Mg-Standard mL

Linear (.05% EDTA Mg-Standard mL)

mM Mg/L

.05%

EDT

A M

g -S

tand

ard

mL

Results

1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.91

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2

f(x) = 0.949386768008292 x + 0.180669372948696R² = 0.98500725413631

Concentration of Mg2+ and Ca2+

.05% EDTA mL addedLinear (.05% EDTA mL added)

Concentration of Mg mM

0.05

% E

DTA

titra

tion

mL ti

tran

t add

ed

Results Fitted with 4th degree Polynomial

1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 21

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2

f(x) = − 15.3283630912421 x⁴ + 88.575235561734 x³ − 189.24918570414 x² + 178.124465354143 x − 61.1637863510535R² = 0.999144906936939

Concentration of Mg + Ca

.05% EDTA mL addedPolynomial (.05% EDTA mL added)

Concentration of Mg mM

.05%

EDT

A tit

ratio

n m

L add

ed

Magnesium Results

• The results for magnesium concentration were obtained via UV Vis.

• The experimental results proved to be elusive. Quantitative data was unable to be obtained.

Conclusion

• The results show:– An equation that describes the Mg concentration

in saliva at low concentrations– Can effectively determine pH of Saliva with a glass

electrode pH meter• What techniques are used today to determine

pH, [Mg2+], and [Ca2+].• Real world application and importance.

References

• Starr, Henry. "Studies of Human Mixed Saliva". The Journal of Biological Chemistry. Philadelphia: Robert Hare Laboratory school of medicine, 1922.

• Wong, David. "Salivary diagnostics powered by". American Dental Association 2006: 313-321.

• Ericsson, Yngve. "Simplified Methods for Determination of Calcium and Magnesium in the Saliva". Journal of Dental Research 1955: 104-112