DENT 5302 TOPICS IN DENTAL BIOCHEMISTRY 7 April 2008

Outline
Overview of fluoride metabolism Factors affecting fluoride absorption Soft tissue distribution of fluoride Distribution of fluoride in calcified tissues Renal excretion of fluoride Fluoride in saliva

Objectives: Metabolic handling of ingested fluoride Absorption, soft-tissue distribution, hard tissue uptake, and excretion

Systemic intake of fluoride

F supplement (drops, tablets) Water fluoridation Topical fluoride products Fluoride is hazardous Fluorosis Acute toxicity

Overview of fluoride metabolism Fluoride is added to water supplies and incorporated in dental products that are commercially available. Although most of these products are for topical application, a portion of them are swallow and being absorbed. Fluoride is a hazardous substance. Systemic intake of large doses can have side effect from mild to severe, or even death. To understand the biological effect of fluoride after systemic intake, we need thorough knowledge of the fluoride metabolism.

Fluoride ion is important for biological effects

Soluble fluoride compounds: NaF, HF, Na2PO3F Less soluble compounds: CaF2, MgF2, AlF3

F-

H+ + FpH = pKa + log [A-] [HA]

HF ; pKa = 3.45 or
pH - pKa = log [A-] [HA]

Diffusibility of HF explains physiological behavior of fluoride Low pH (<3.5) e.g., stomach: More as undissociated form HF pH > 3.45 e.g., blood, saliva, tissue fluid: ionized form F- dominates
At pH 2.45 log [F-] = -1 ; [HF] At pH 6.45 log [F-] = 3 [HF]

[F-] = 1 10 [HF]

[F-] = 1000 1 [HF]

Fluoride can be in the soluble compounds or less soluble compounds. Fluoride ions are released depending on the solubility of the compound. Fluoride ion is important for the biological effects. Fluoride ions can reversibly combine with hydrogen ions to form hydrogen fluoride or hydrofluoric acid. Hydrofluoric acid has pKa 3.45. Any fluoride present at a low pH such as in the stomach will exist mainly in the undissociated form as HF. At pH above 3.45, like in blood plasma, saliva, and tissue fluid, fluoride will be in the ionized form. Much of the physiological behavior of fluoride can be explained in terms of the diffusibility of HF.
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Fluoride metabolism FLUORIDE

LUNG GI TRACT

HARD TISSUES
~ 50 %

PLASMA (Central compartment)

SOFT TISSUES
Steady state

FECES SWEAT

URINE ~ 50% in 24 hrs

The overall metabolism of fluoride can be divided into 3 steps: absorption, distribution, and excretion. Mainly fluoride is absorbed and enters the body through the lungs or the gastrointestinal tract. Plasma is the central compartment where the ions pass before being distributed and eliminated. Roughly 50% of the absorbed fluoride is excreted in the urine during the following 24 hours. Most of the remaining 50% will become associated with calcified tissue.

Note that fluoride in calcified tissues is not irreversibly bound. For example, it may be released during normal remodeling of bone. In soft tissues, fluoride has a steady state distribution between the intracellular and extracellular fluids. When the plasma level of fluoride is rising or falling, there is a parallel change in the intracellular fluids.
Absorption How fast is the absorption and distribution?
Rapidly declining Bone uptake & Urinary excretion

Factors affecting fluoride absorption After ingestion, within minutes we can detect increased plasma fluoride levels, which mean that fluoride is readily absorbed from the stomach. The peak level usually occurs during the first hour, can be less than 30 min. Then the plasma level will rapidly decline due to bone uptake and urinary excretion. If the amount of fluoride ingested is small (not more than a few mg), the plasma level will return to normal within 3-6 hours. This graph shows plasma fluoride concentration in a subject after received 3 mg fluoride in 4 different ways: NaF tablet taken on a fasting stomach, NaF tablet taken with a glass of milk, NaF tablet taken with calciumrich breakfast, and the same dose of fluoride by intravenous injection, which has 100% bioavailability. The absorption of F taken on a fasting stomach was about 100%. Taken with milk decreased absorption to about 70%, and 60% with calcium-rich breakfast.

Ingestion

Peak plasma level < 30 min to an hour

Return to normal 3-6 hours (If ingesting small amount)

What factors affect F absorption?

IV P.O. fasting
Ekstrand J et al. Eur J Clin Pharm 1979; 16:211-5

P.O. milk P.O. breakfast

Guess this..
Subject received 3 mg fluoride:
1. NaF tablet, fasting stomach 2. NaF tablet + glass of milk 3. NaF tablet + calcium-rich breakfast hour Absorption ~ 100 % Absorption ~ 70 % Absorption ~ 60 %

4. Intravenous injection (100% bioavailability) In the presence of Al3+, Ca2+, Mg2+ Less absorption of fluoride Increased fecal excretion

The decreased absorption is caused by the binding of fluoride with certain ions, esp Al, Mg, Ca. In this case, the fecal excretion of fluoride increases. The ability of calcium to reduce the absorption of F is the basis for treating acute F toxicity by giving calcium-containing solution.
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What factors affect F absorption?

Pentagastrin: Stimulates gastric acid secretion Cimetidine: Inhibits gastric acid secretion Bioavailability of F = 97% Bioavailability of F = 66%

Higher acidity of stomach content

Pentagastrin Cimetidine
AUC = cumulative plasma F level

More fluoride absorbed

Why?
Fluoride is absorbed as HF Uncharged molecule (HF) readily passes through biological membrane HF dominates at low pH

40% of oral dose of fluoride is absorbed from the stomach

This experiment, rats were given Cimetidine to inhibit gastric acid secretion or Pentagastrin to stimulate gastric acid secretion. The bioavailability of fluoride in the Pentagastrin group was 65-97%, comparing to 66% in the Cimetidine group. This study indicates that the permeation of fluoride through the gastric mucosa is pH dependent. The higher acidity of stomach content increases the absorption of fluoride. The reason is because HF is the dominate form at low pH.

Fluoride is absorbed as HF, which is an uncharged molecule and can readily pass through biological membranes. Research has shown that 40% of oral dose of fluoride is absorbed from the stomach. Fluoride from most topical dental products is almost completely absorbed when swallowed!! The bioavailability of NaF or SnF2 in fluoride toothpastes is close to 100%, and somewhat less for monofluorophosphate. Abrasive in toothpaste may bind to fluoride and reduce the absorption. F from APF (acidulated phosphate fluoride) gel is well absorbed. Other topical F products with high fluoride content include fluoride varnish. The varnish remains on the tooth surface for upto 12 hours. The increase in plasma fluoride concentration is equivalent to ingesting a 1-2 mg fluoride tablet. Distribution of fluoride in soft tissues and calcified tissues Fluoride in plasma: Plasma is the central compartment for fluoride, where fluoride must pass for its distribution elsewhere in the body and for its elimination. The concentration of fluoride in plasma depends on fluoride intake (chronic or recent), distribution to bone and other tissues, and clearance or excretion in urine. This graph shows an example from subjects living in areas with different water F level.
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Fluoride from most dental products is almost completely absorbed when swallowed!! Fluoride toothpastes
NaF or SnF2 have bioavailability close to 100% Na2PO3F has less bioavailability Abrasive may bind fluoride (reduce absorption)

x
Enter Distribution Elimination

APF (acidulated phosphate fluoride) gel

Acidic well absorbed

Fluoride varnish
Remains on tooth surface 12 hrs Plasma F concentration ~ 1-2 mg fluoride tablet

Fluoride in Plasma
Plasma = central compartment for fluoride
9.6 ppm F 1.2 ppm F 0.2 ppm F

Plasma F depend on:

F intake Distribution Bone & tissues Clearance Excretion in urine

Plasma F of subjects from areas with different water F level

Ekstrand J. Caries Res 1978:12:123-7

Distribution Fluoride is distributed from plasma to all tissues and organs How to study tissue distribution?
Administer (IV) radioisotope fluoride (18F)

Distribution to soft tissues: Fluoride is distributed from plasma to all tissues and organs. How to study tissue distribution? The index for tissue distribution is Tissue-water-toplasma-water ratio (T/P). Radioisotope fluoride (18F) is administered intravenously, and T/P ratio is determined when the level equilibrates (steady-state). Inulin is used as an extracellular marker. T/P of Inulin = 0.2-0.4. Therefore, an agent with T/P higher than 0.4 means that it can penetrate cells. If T/P >1, that agent can accumulate in the tissue. In most organs, such as in liver, lung, heart, salivary gland, T/P ratio of fluoride is 0.4-0.9. This range means that fluoride is able to penetrate cells (T/P higher than Inulin which is an extracellular marker) but fluoride is not accumulated intracellularly (T/P is less than 1). Some exceptions where fluoride cannot penetrate into are brain (blood-brain barrier) (0.08) and adipose tissues (0.11). T/P ratio of fluoride in kidney is 4.16. This value does not indicate accumulation but is related to excretion of fluoride by kidney. Distribution to calcified tissues: Almost 50% of absorbed fluoride is taken up by the calcified tissues. Fluoride is an avid calcified tissue seeker. Only 4 min after intravenous injection of fluoride to this mouse, the skeleton is clearly labeled. Fluoride ions from tissue fluid first enter the hydration shell surrounding the apatite crystallites. Then it exchanges with negative ions (hydroxyl, carbonate, or even another fluoride) in the lattice at the crystal surface. Some of the fluoride at the surface might migrate into the crystal interior, which happens very slowly unless fluoride is acquired during the crystal growth.
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Determine T/P at various times until the level equilibrates (steady-state)

T/P = Tissue-water-to-plasma-water ratio

Inulin (extracellular markers): T/P = 0.2-0.4 T/P > 0.4 = agent can penetrate cells. T/P >1 = agent can accumulate in the tissue

Tissue Distribution of Fluoride

T/P Brain (blood-brain barrier) Adipose tissue Heart Salivary gland Lung Liver Kidney 0.08 0.11 0.46 0.63 0.83 0.98 4.16

T/P = 0.4-0.9

Inulin (extracellular markers): T/P = 0.2-0.4 Fluoride is able to penetrate cells but not accumulate intracellularly

Distribution of fluoride in calcified tissues

Almost 50% of absorbed fluoride is taken up by the calcified tissues

Uptake of 18F by the skeleton 4 min after IV injection in laboratory mouse

Ion-exchange process:

F- from plasma enters hydration shell Exchanges with OH-, CO32-, F(apatite crystal surface) Migrates into the crystal interior (slow)

Retention of fluoride in calcified tissues

in growing dogs

Young animals (& human): High portion of fluoride is deposited in the skeleton Puppies 80 days: F retention ~ 90% 2 years old: F retention ~ 60% Adults F retention ~ 50%

In young animals (and humans) a relatively high portion of fluoride is deposited in the skeleton. In this study, fluoride retention in young puppies is about 90%, and reduced to 60% at 2 years old. In adults, fluoride retention is on average 50%. It should be emphasized that fluoride in calcified tissues is not irreversibly bound and can be released by ion-exchange or normal remodeling processes.

Fluoride in calcified tissues is not irreversibly bound and can be released by ion-exchange or normal remodeling process

Excretion of fluoride
Excretion

Renal clearance of fluoride

Fluoride in plasma Glomerular filtration Reabsorb from renal tubules Excrete in urine 60%

Kidney is the major route of fluoride excretion

The major route of fluoride excretion is by the kidneys (= Renal clearance). After fluoride from plasma enters renal tubules by filtration at the glomeruli, some will be reabsorbed and the remainder will be excreted in urine. In adults, renal clearance is about 40-60% of ingested fluoride. In children, a smaller percentage of ingested fluoride is excreted. This graph shows that nearly 30% of fluoride ingested was excreted in about 6 hours, and 60% in 24 hours.

30% Adults: 40-60% of ingested fluoride Children: Excrete a smaller % of ingested fluoride Amount of excreted fluoride vs time after ingesting

Early study: F Renal clearance increases with urinary flow rate.

Later: Different diuretics have different F excretion:

F clearance

effect on renal clearance of F.

Acetazolamide Furosemide

Acetazolamide >>> Furosemide

Acetazolamide increases HCO3pH increases

Urinary flow rate (l/min)

Early studies show a positive correlation between renal clearance of fluoride and urine flow rate. Renal clearance of fluoride increases when urinary flow rate increases. A later study found that different diuretics have different effects on renal clearance of F. As shown here, the increase in F excretion in Acetazolamide group is 9 times higher than in Furosemide. There must be something else than the urinary flow rate alone that governs renal clearance of F. Acetazolamide promotes the excretion of sodium bicarbonate which increases urinary pH.

Does Urinary pH or flow rate determine F clearance?

Separate urinary flow rate and urinary pH Period 1-8: Mannitol diuresis Flow rate ; Urinary pH ; F clearance

Period 10-12: Diamox + bicarbonate Flow rate ; Urinary pH ; F clearance

Some diuretics (e.g., mannitol, saline) increase F clearance because the tubular fluid is diluted, thus pH increases. Conclusion: Tubular reabsorption of fluoride Primarily related to urinary pH Secondarily related to urinary flow rate

Is urinary pH or flow rate the determinant of fluoride excretion? A study was conducted to proof this point by separating urinary flow rate pH. From period 1-8, diuresis was induced by mannitol. As expected, fluoride clearance increased with urinary flow rate. At the same time urinary pH also increased. In period 1012, the mannitol was replaced with sodium bicarbonate and Diamox. Diamox reduced flow rate while bicarbonate increased pH. The fractional excretion of fluoride increased.

Therefore, the fluoride clearance is affected by urinary pH, not urinary flow rate. Some diuretics (mannitol or saline) increase fluoride excretion because the diuretic dilutes the tubular fluid, thus increases pH. In conclusion, tubular reabsorption of fluoride is primarily related to urinary pH and only secondarily related to urinary flow rate.
How does pH affect the renal handling of F?
Tubular reabsorption of F occurs by the diffusion of HF (not F-)
HF can permeate lipid barriers F- is charged and has large hydrated radius incapable of permeating the tubular epithelium Acid urine Acid urine

How does pH affect the renal handling of F? When F is reabsorbed from renal tubules, the amount of F reabsorption can vary from 20-95% depending on pH of the tubular fluid. This character can also be explained by the diffusion of undissociated HF across the tubular epithelium. HF can permeate lipid barriers, whereas the fluoride ion is charged and has a large hydrated radius, therefore cannot permeate the tubular epithelium.

HF
H+

H++ F-

Low urinary (tubular fluid) pH: More HF Less Fmore diffusion less remain more reabsorb less excrete

F-

FH+

Alkaline urine High tubular fluid pH:

H+ + F-

HF

Less HF More F-

less diffusion more remain

less reabsorb more excrete

Alkaline urine

Capillary

When tubular fluid (and hence urine) becomes more acidic, the fraction of fluoride in HF form is increased, and diffuses from the tubular lumen to the interstitial fluid. Usually the pH of the interstitial fluid is relatively high, about 7, so HF dissociates and releases fluoride ions. Fluoride ions can then diffuse into capillaries and return to plasma. As a result, less fluoride is excreted when tubular fluid and urine is acidic. When tubular fluid becomes more alkaline, the fraction of fluoride in HF form is less, and not many HF diffuse into the interstitial fluid. Thus less fluoride diffuses into capillaries and return to plasma. The large amount of ionic fluoride which cannot diffuse through the tubular membrane is left in the renal tubule to be excreted. As a result, more fluoride is excreted when tubular fluid and urine is alkali.
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Question: Which diuretic is the most effective for treating acute fluoride toxicity? a. Acetazolamide (increases flow rate and HCO3-) b. Mannitol (increase urinary flow rate) c. saline (increase urinary flow rate)

Why is urinary F excretion important?

Acute fluoride poisoning
To promote the renal excretion of fluoride by increasing urinary flow rate (diuresis) (sometimes recommended for acute fluoride poisoning)

Effective only if urinary pH increases

Why is urinary fluoride excretion important? In case of acute fluoride poisoning, to promote the renal excretion of fluoride by increasing urinary flow rate will be effective only if the method also increases urinary pH. Factors that influence urinary pH affect the excretion of fluoride. For example, the composition of diet, certain drugs, and some metabolic diseases. Interestingly, a vegetarian diet promotes more alkaline urine and hence more fluoride excretion. Other routes of fluoride excretion, which are less important, are via sweat and feces. Most of the fluoride in feces is never absorbed, usually it is less than 10% of the ingested amount. If diet contains high concentration of cations (Mg2+, Al3+, Ca2+), less fluoride is absorbed and more is excreted in feces. Fluoride concentration in sweat is ~ 20% of plasma. In tropical climates during prolonged exercise, the excretion of fluoride in sweat is about a tenth of a milligram. This values is quite small compared to 2 mg uptake from diet and a milligram of fluoride excreted by urine. Fluoride in saliva Concentration of fluoride in saliva from systemic sources (endogeneous), collected from salivary ducts, is around 0.01-0.05 ppm, about 30% less than the corresponding serum F concentrations. Whole saliva F concentrations are higher than the duct secretion because of exogenous fluoride. Graphs show Fconcentration in saliva after toothbrushing (1), chewing F tablet (3), mouthrinse (6), topical application of APF (7) and 2% NaF (8).

Factors that influence urinary pH: Composition of diet Certain drugs Metabolic diseases Vegetarian diet more alkaline urine more fluoride excreted

Other routes of fluoride excretion Feces

Fluoride in Feces: unabsorbed fluoride < 10% ingested F Less F absorption if diet high in Mg2+, Al3+, Ca2+

Sweat
Fluoride concentration ~ 20% of plasma. High end sweat excretion ~ 5% ingested F Tropical climate + prolonged exercise ~ 0.1 mg Compare to ~ 2 mg uptake from diet ~1 mg excreted by urine

Fluoride in Saliva
Saliva F-concentration
Duct secretion (systemic, endogeneous) ~ 0.01-0.05 ppm, 30% less than serum F

F-concentration in saliva (1) after toothbrushing (3) chewing F tablet (6) F mouthrinse

Whole saliva: Duct secretion + exogenous F

(7) APF (8) 2% NaF

Recommended references
1. Ekstrand J, Fejerskov O, Silverstone LM (Eds). Fluoride in Dentistry. Copenhagen: Munksgaard 1988. Chapters 3 & 7. 2. Ekstrand J, Spak C-J. Vogel G. Pharmacokinetics of fluoride in man and its clinical relevance. J Dent Res 1990;69:550-55. 3. Whitford GM. The physiological and toxicological characteristics of fluoride. J Dent Res 1990;69:539-49. 4. Whitford GM. Intake and metabolism of fluoride. Adv Dent Res 1994;8:5-14. 5. Whitford GM. The Metabolism and Toxicity of Fluoride. 2nd Ed. Monographs in Oral Science Vol 16. Chapters I IV.