Transcript Determination of pH and Ca2+ and Mg2+ in Saliva

Determination of pH, Ca2+ and
Mg2+ in Saliva
Cody Chalker
David 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 6.57.4
• 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
1
2
3
4
5
6
.05% EDTA mL added
0.11
0.13
0.23
0.34
0.54
0.83
0.24
0.29
0.35
0.41
0.61
0.95
Standard Calibration Graph
Mg Standard
1
0.9
y = 0.9505x - 0.0011
R² = 0.9964
.05% EDTA Mg -Standard mL
0.8
0.7
0.6
.05% EDTA Mg-Standard
mL
0.5
Linear (.05% EDTA MgStandard mL)
0.4
0.3
0.2
0.1
0
0
0.2
0.4
0.6
mM Mg/L
0.8
1
1.2
Results
Concentration of Mg2+ and Ca2+
0.05% EDTA titration mL titrant added
2
1.9
y = 0.9494x + 0.1807
R² = 0.985
1.8
1.7
1.6
1.5
.05% EDTA mL added
Linear (.05% EDTA mL added)
1.4
1.3
1.2
1.1
1
1
1.1
1.2
1.3
1.4
1.5
Concentration of Mg mM
1.6
1.7
1.8
1.9
Results Fitted with 4th degree
Polynomial
Concentration of Mg + Ca
2
1.9
.05% EDTA titration mL added
1.8
1.7
y = -15.328x4 + 88.575x3 - 189.25x2 + 178.12x - 61.164
R² = 0.9991
1.6
1.5
.05% EDTA mL added
Poly. (.05% EDTA mL added)
1.4
1.3
1.2
1.1
1
1
1.1
1.2
1.3
1.4
1.5
1.6
Concentration of Mg mM
1.7
1.8
1.9
2
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