Name: Quantification of Fluoride in Serum, Bone, and Teeth in a Murine Model Using Hexamethyldisiloxane-Facilitated Diffusion and Ion-Selective Electrode Measurements (Video) | JoVE Journal
Uploaded: 2026-08-28T12:50:00.000+00:00
Duration: 6 min 57 s
Description: Learn fluoride quantification in serum, bone, and teeth using hexamethyldisiloxane-facilitated diffusion and ion-selective electrode analysis. This sensitive method enables accurate measurement in small murine specimens.

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Research reported in this publication was supported by Japan Society for the Promotion of Science; JSPS Overseas Research Fellowships 202560528 (S.Y.), Funda&#231;&#227;o de Amparo a Pesquisa do Estado de S&#227;o Paulo (FAPESP); 2024/07452-9 (J.S.T.), and the National Institute of Dental and Craniofacial Research of the National Institutes of Health; R01DE027648 (M.S.) and K02DE029531 (M.S.).

<ol>
	<li>Podgorski J, Berg M. Global analysis and prediction of fluoride in groundwater. Nat Commun. 2022;13(1):4232. doi:10.1038/s41467-022-31940-x</li>
	<li>Iheozor-Ejiofor Z, Worthington HV, Walsh T, O&#39;Malley L, Clarkson JE, Macey R, et al. Water fluoridation for the prevention of dental caries. Cochrane Database Syst Rev. 2015;2015(6):Cd010856. doi:10.1002/14651858.CD010856.pub2</li>
	<li>Manchanda S, Sardana D, Liu P, Lee GH, Li KY, Lo EC, et al. Topical fluoride to prevent early childhood caries: Systematic review with network meta-analysis. J Dent. 2022;116:103885. doi:10.1016/j.jdent.2021.103885</li>
	<li>Chan AKY, Tamrakar M, Jiang CM, Tsang YC, Leung KCM, Chu CH. Clinical evidence for professionally applied fluoride therapy to prevent and arrest dental caries in older adults: A systematic review. J Dent. 2022;125:104273. doi:10.1016/j.jdent.2022.104273</li>
	<li>Zamperini CA, Bedran-Russo AK. Immediate and Sustained Root Caries Prevention of Fluoride Varnish Combined with Toothpastes. Caries Res. 2023;57(5-6):592-601. doi:10.1159/000533279</li>
	<li>Melough MM, Sathyanarayana S, Zohoori FV, Gustafsson HC, Sullivan EL, Chi DL, et al. Impact of Fluoride on Associations between Free Sugars Intake and Dental Caries in US Children. JDR Clin Trans Res. 2023;8(3):215-23. doi:10.1177/23800844221093038</li>
	<li>Suzuki M, Everett ET, Whitford GM, Bartlett JD. 4-phenylbutyrate Mitigates Fluoride-Induced Cytotoxicity in ALC Cells. Front Physiol. 2017;8:302. doi:10.3389/fphys.2017.00302</li>
	<li>Suzuki M, Shin M, Simmer JP, Bartlett JD. Fluoride affects enamel protein content via TGF-beta1-mediated KLK4 inhibition. J Dent Res. 2014;93(10):1022-7. doi:10.1177/0022034514545629</li>
	<li>Everett ET, McHenry MA, Reynolds N, Eggertsson H, Sullivan J, Kantmann C, et al. Dental fluorosis: variability among different inbred mouse strains. J Dent Res. 2002;81(11):794-8.</li>
	<li>Kong H, He Z, Li H, Xing D, Lin J. The association between fluoride and bone mineral density in US children and adolescents: a pilot study. Nutrients. 2024. doi:10.3390/nu16172948</li>
	<li>Mazzoli R, Filippini T, Iamandii I, De Pasquale L, Veneri F, Birnbaum LS, et al. The association of fluoride exposure with bone density and fracture risk: a dose-response meta-analysis. Environ Health. 2025;24(1):73.</li>
	<li>Suzuki M, Bandoski C, Bartlett JD. Fluoride induces oxidative damage and SIRT1/autophagy through ROS-mediated JNK signaling. Free Radic Biol Med. 2015;89:369-78.</li>
	<li>Kubota K, Lee DH, Tsuchiya M, Young CS, Everett ET, Martinez-Mier EA, et al. Fluoride induces endoplasmic reticulum stress in ameloblasts responsible for dental enamel formation. J Biol Chem. 2005;280(24):23194-202. doi:10.1074/jbc.M503288200</li>
	<li>Yamashita S, Okamoto M, Mendonca M. Fluoride alters gene expression via histone H3K27 acetylation in ameloblast-like LS8 cells. Int J Mol Sci. 2024. doi:10.3390/ijms25179600</li>
	<li>Deng H, Fujiwara N, Cui H, Whitford GM, Bartlett JD, Suzuki M. Histone acetyltransferase promotes fluoride toxicity in LS8 cells. Chemosphere. 2020;247:125825.</li>
	<li>Deng H, Ikeda A, Cui H, Bartlett JD, Suzuki M. MDM2-Mediated p21 Proteasomal Degradation Promotes Fluoride Toxicity in Ameloblasts. Cells. 2019;8(5). doi:10.3390/cells8050436</li>
	<li>Fujiwara N, Whitford GM, Bartlett JD, Suzuki M. Curcumin suppresses cell growth and attenuates fluoride-mediated Caspase-3 activation in ameloblast-like LS8 cells. Environ Pollut. 2021;273:116495. doi:10.1016/j.envpol.2021.116495</li>
	<li>Suzuki M, Ikeda A, Bartlett JD. Sirt1 overexpression suppresses fluoride-induced p53 acetylation to alleviate fluoride toxicity in ameloblasts responsible for enamel formation. Arch Toxicol. 2018;92(3):1283-93. doi:10.1007/s00204-017-2135-2</li>
	<li>Regelson S, Dehghan M, Tantbirojn D, Almoazen H. Evaluation of fluoride levels in commercially available tea in the United States. 2020/12/23 ed2021 Jan-Feb. 17-20 p.</li>
	<li>Cantoral A, Luna-Villa LC, Mantilla-Rodriguez AA, Mercado A, Lippert F, Liu Y, et al. Fluoride Content in Foods and Beverages From Mexico City Markets and Supermarkets. Food Nutr Bull. 2019;40(4):514-31. doi:10.1177/0379572119858486</li>
	<li>Institute of Medicine SCotSEoDRI. Dietary Reference Intakes for Calcium, Phosphorus, Magnesium, Vitamin D, and Fluoride. Washington, DC: National Academy Press; 1997.</li>
	<li>Otal E.H. KML, Kimura M. Fluoride Detection and Quantification, an Overview from Traditional to Innovative Material-Based Methods. In: Wambu E.W. LGJ, Kiplagat A., editor. Fluoride. London: IntechOpen; 2022.</li>
	<li>Whitford GM. Absorption and Plasma Concentrations of Fluoride, 1996.</li>
	<li>Taves DR. Determination of submicromolar concentrations of fluoride in biological samples. Talanta. 1968;15(10):1015-23. doi:10.1016/0039-9140(68)80109-2</li>
	<li>Buzalaf MA, Caroselli EE, de Carvalho JG, de Oliveira RC, da Silva Cardoso VE, Whitford GM. Bone surface and whole bone as biomarkers for acute fluoride exposure. J Anal Toxicol. 2005;29(8):810-3.</li>
	<li>Zohoori FV, Maguire A, Martinez-Mier EA, Buzalaf MAR, Sanderson R, Eckert GJ. A Comparison of Simple Analytical Methods for Determination of Fluoride in Microlitre-Volume Plasma Samples. Caries Res. 2019;53(3):275-83. doi:10.1159/000492339</li>
	<li>Mart&#237;nez-Mier EA, Cury JA, Heilman JR, Katz BP, Levy SM, Li Y, et al. Development of gold standard ion-selective electrode-based methods for fluoride analysis. Caries Res. 2011;45(1):3-12. doi:10.1159/000321657</li>
	<li>Boivin G, Chapuy MC, Baud CA, Meunier PJ. Fluoride content in human iliac bone: results in controls, patients with fluorosis, and osteoporotic patients treated with fluoride. J Bone Miner Res. 1988;3(5):497-502.</li>
	<li>Yamashita S, Okamoto M, Fujiwara N, Achong-Bowe R, Brueckner S, Mendonca M, et al. Developmental vulnerability to fluoride toxicity: enamel and clearance differences in adolescent versus mature mice. Arch Toxicol. 2025.</li>
	<li>Chiba K, Hisamatsu, S., Takizawa, Y, Katsura, K. Determination of fluoride in biological materials by ion chromatography. Journal of Chromatography. 1982;245:8.</li>
	<li>Ikenishi K, Chiba, K., Hisamatsu, S, Takizawa, Y. Determination of fluoride in biological samples by ion chromatography. Journal of Chromatography. 1988;437:8.</li>
	<li>Venkateswarlu P, Reddy, D.S., Reddy, T.S. Spectrophotometric determination of fluoride using the SPADNS method. Analyst 1971;96:5.</li>
	<li>Kakabadse G. KN, Davies C.W. The Spectrophotometric Determination of Fluoride with SPDNS-Zirkonium Lake. Analyst. 1971;96:8.</li>
	<li>Mendham J, Denney RC, Barnes JD, Thomas MJK. Vogel&#39;s Textbook of Quantitative Chemical Analysis. 6th ed. Harlow, England: Prentice Hall; 2000.</li>
	<li>Ku&#269;era J, &#352;tef&#225;nik, M. &#38; Veselka, P. Fluorine determination in biological and environmental samples with INAA using fast neutrons from a p(19 MeV)+Be neutron generator. Journal of Radioanalytical and Nuclear Chemistry 2019;322:7.</li>
	<li>Okamoto M, Yamashita S, Memida T, Mendonca M, Brueckner S, Nakamura S, et al. Microhardness Measurements on Tooth and Alveolar Bone in Rodent Oral Disease Models. J Vis Exp. 2024(206). doi:10.3791/66583</li>
	<li>Okamoto M, Yamashita S, Mendonca M, Brueckner S, Achong-Bowe R, Thompson J, et al. Ultrastructural evaluation of adverse effects on dentine formation from systemic fluoride application in an experimental mouse model. Int Endod J. 2025;58(1):128-40.</li>
	<li>Frant MS, Ross JW, Jr. Electrode for sensing fluoride ion activity in solution. Science. 1966;154(3756):1553-5.</li>
	<li>Umezawa Y, Umezawa, K and Sato, H. Selectivity coefficients for ion-selective electrodes: recommended methods for reporting KRB values, 1995.</li>
</ol>

The authors declare no conflict of interest.

Fluoride measurements were obtained using a fluoride&#8209;selective ion&#8209;selective electrode (ISE) incorporating a lanthanum fluoride (LaF&#8323;) crystal membrane38,39. This membrane forms highly stable La&#8211;F bonds, which results in a strong preference for fluoride ions over other halides. According to established electrochemical literature and manufacturer specifications, the LaF&#8323; membrane exhibits very low selectivity coefficients for chloride, indicating that chloride ions do not produce a measurable interference under the conditions used in this protocol. Although chloride is naturally present in teeth and bone, its activity does not influence the electrode potential, and the recorded signal reflects fluoride activity specifically. The lower detection limit of 0.02 ppm corresponds to the manufacturer&#8209;validated minimum quantifiable concentration for the Orion fluoride&#8209;selective electrode. This limit reflects the lowest fluoride concentration at which the electrode maintains a stable Nernstian response and a linear relationship with the calibration curve. Because the detection limit is defined by the electrode&#8217;s membrane characteristics and validated by the manufacturer, no additional experimental determination was required for this protocol; however, regarding the upper detection range, the authors observed that fluoride masses exceeding approximately 38,000&#160;ng in a 50&#160;&#181;L NaOH trap resulted in a non&#8209;linear electrode response, indicating that the calibration curve is no longer reliable at very high fluoride loads. This non&#8209;linearity is likely due to exceeding the optimal ionic strength and activity range for the electrode under the given experimental conditions, rather than reflecting an inherent upper limit of the ISE itself. For samples expected to contain fluoride masses above this threshold, it is advisable to increase the NaOH trap volume to maintain measurements within the linear dynamic range of the electrode.
This consideration should be taken into account when applying the protocol to samples with unusually high fluoride content. Because the diffusion step consumes the entire sample volume, duplicate measurements and spike&#8209;and&#8209;recovery experiments could not be performed on the biological samples. To ensure analytical accuracy and precision, quality control (QC) standards containing known fluoride concentrations were included in every measurement series. These QC standards were processed and measured identically to experimental samples. Across all runs, the measured fluoride concentrations of the QC standards were consistently within 5% of their expected values. For each series of 10&#8211;20 specimen samples, two independent sets of 5&#8211;6 calibration samples were prepared in parallel with the specimen samples, and a calibration curve was recorded prior to each measurement session. The calibration range was adjusted for each run to approximate the expected fluoride masses of the specimen samples, which depended on specimen type, the mass of material added to the diffusion dish, and the animal&#8217;s treatment group and age. For serum measurements, calibration standards corresponded to fluoride concentrations of approximately 0.06&#8211;1.20 ppm in the specimen. For bone and tooth measurements, calibration standards corresponded to fluoride concentrations of approximately 19&#8211;7,600 ppm in the specimen.
This protocol provides a sensitive and reproducible approach for quantifying fluoride ion mass in serum, bone, and teeth using hexamethyldisiloxane&#8209;facilitated diffusion followed by ISE analysis. The method reliably captures fluoride released from diverse biological matrices and enables quantitative assessment of fluoride accumulation following controlled exposure. Representative results from adolescent and mature mice demonstrate clear age&#8209; and dose&#8209;dependent differences in fluoride deposition across tissues, underscoring the utility of this approach for studying fluoride metabolism and toxicokinetics in vivo.
A key strength of this protocol is the use of diffusion dishes that physically separate the fluoride&#8209;releasing reaction from the fluoride&#8209;trapping environment. The petrolatum&#8209;sealed design minimizes atmospheric exchange and ensures efficient capture of liberated fluoride ions by sodium hydroxide droplets on the lid. HMDS&#8209;saturated sulfuric acid provides a strong and consistent driving force for fluoride release from mineralized tissues, including highly calcified structures such as bone and teeth. Subsequent ISE measurement, calibrated with diffusion&#8209;matched standards, enables accurate quantification across a wide dynamic range. Another advantage of this approach is its ability to quantify fluoride in extremely small biological samples. Mineralized tissues from mice, particularly mandibular incisors, yield only 3&#8211;5 mg of ash per tooth. Conventional analytical methods often require larger sample masses or higher fluoride content to achieve reliable detection. In contrast, the diffusion&#8209;based design concentrates all fluoride released from the entire specimen into a small, defined volume, allowing accurate quantification even in tiny samples with low absolute fluoride mass. This feature is particularly valuable for studies involving small animals, limited tissue availability, or micro&#8209;dissected mineralized structures.
A primary limitation of this method is the detection limit of the ISE used in this study (0.02 ppm). Serum samples from fluoride&#8209;free control animals frequently fell below this threshold, preventing quantification of extremely low fluoride concentrations. An increased mass or volume of specimen added to the diffusion dish, if available, or alternative analytical techniques may be required when measuring trace concentrations near or below the detection limit. In addition, the method requires overnight diffusion and careful handling of corrosive reagents, which may limit throughput in high&#8209;volume studies. Although ISE provides sensitive quantification of bulk fluoride, it cannot assess spatial distribution within tissues. Complementary approaches, such as scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDX) or particle-induced gamma-ray emission (PIGE), can be incorporated to visualize and map fluoride distribution at the micro&#8209; to macro&#8209;scale. These imaging&#8209;based modalities allow evaluation of localized fluoride accumulation in mineralized or soft tissues, thereby complementing ISE measurements and providing a more comprehensive assessment of tissue fluoride content.
Several recurring technical factors can influence the accuracy and reliability of fluoride diffusion and measurement in this protocol, and our observations highlight how calibration preparation, diffusion&#8209;dish sealing, pH control, electrode performance, and sample handling collectively shape data quality. Possible causes for a non&#8209;linear calibration curve (R2 &#60; 0.99) or tissue sample readings outside the expected range include miscalculations of the NaF stock solution volume added to the diffusion dish or pipetting errors during preparation of calibration standards. Recommended action: Prepare at least two independent sets of calibration samples for each run so that a backup set is available if one calibration curve falls outside the expected range. An incomplete seal of the diffusion dish may also cause fluoride loss during diffusion, resulting in artificially low readings. This typically occurs when insufficient petrolatum is applied to the rim of the diffusion dish lid. Recommended action: Apply a generous amount of petrolatum jelly around the rim of the diffusion dish lid and press firmly to ensure a complete seal. Pipetting errors when adding the acetic acid solution to the NaOH trap can shift the sample pH outside the optimal range of pH 5.0, leading to inaccurate fluoride recovery. Recommended action: Check the pH of each sample with pH paper and discard any sample that falls outside pH 5. Calibration samples may also fall outside the valid recording limits of the fluoride&#8209;selective electrode. Concentrations below 0.02 ppm are below the electrode&#8217;s detection limit. Recommended action: Do not use calibration samples containing less than 0.02 ppm fluoride. Conversely, calibration samples with excessively high fluoride concentrations can alter the ionic strength of the solution and produce non&#8209;linear electrode responses. Recommended action: At the beginning of the project, prepare calibration solutions spanning a wide fluoride concentration range to determine the linear working range of the specific electrode being used. If tissue samples exceed this linear range, reduce the tissue mass or increase the NaOH trap volume and adjust the acetic acid volume accordingly to maintain pH 5. Difficulty obtaining linear calibration curves in the 0.02&#8211;1 ppm range: A common cause is electrode aging. Older fluoride&#8209;selective electrodes lose sensitivity at very low fluoride concentrations. Recommended action: Replace the fluoride&#8209;selective electrode. Unstable potentiometer readings (signal drifting or jumping) typically occur when the sensing surface of the electrode is not fully immersed in the sample solution. Recommended action: Lower the electrode so that the sensing surface is completely covered by the liquid sample.
Overall, this protocol provides a robust, calibration&#8209;based approach for quantifying fluoride in diverse biological matrices and is well-suited for studies investigating fluoride exposure, tissue deposition, and toxicological outcomes in murine models.

Fluoride is a naturally occurring anion, and the optimal level of fluoride is effective in preventing dental caries1,2,3,4,5. In the United States (U.S.), community water fluoridation at 0.7 ppm has been implemented as a public health strategy to reduce caries incidence and promote oral health6. However, prolonged ingestion of excessive fluoride can adversely affect multiple tissues. High fluoride exposure disrupts enamel formation7,8,9, alters bone formation10, and compromises skeletal integrity11.
At the cellular level, fluoride exposure induces several metabolic disturbances, including oxidative stress12, endoplasmic reticulum stress13 , epigenetic modifications that alter gene expression14, and apoptosis in ameloblasts15,16,17,18. Globally, elevated fluoride concentrations in groundwater (&#62;1.5 ppm) pose a significant health concern. A recent predictive modeling study estimated that approximately 180 million people worldwide are potentially affected by excessive fluoride exposure, with the highest burden occurring in Asia and Africa1. Beyond drinking water, fluoride exposure also occurs through food (e.g., seafood; ~1.9 ppm), beverages (e.g., tea; 0.5&#8211;6 ppm19, and dental products such as toothpaste20 (1,000&#8211;1,500 ppm).
Understanding fluoride exposure, metabolism, and tissue deposition is therefore essential for evaluating both its therapeutic benefits and potential toxicological consequences. Accurate quantification of fluoride in biological specimens&#8212;including urine, serum, bone, and teeth&#8212;is a critical component of this work. However, measurement is analytically challenging due to fluoride&#8217;s chemical behavior and its strong affinity for calcium-rich matrices. Approximately 99% of the body&#8217;s fluoride resides in mineralized tissues such as bone and teeth21. To address these challenges, a variety of analytical techniques have been developed, including ion-selective electrode (ISE) potentiometry, ion chromatography, colorimetric assays, titrimetric procedures, and nuclear or activation-based methods22. Among these, the hexamethyldisiloxane (HMDS)&#8211;facilitated diffusion technique combined with fluoride-selective electrode measurement has emerged as one of the most reliable and widely used approaches for biological matrices due to its sensitivity, reproducibility, and compatibility with diverse sample types23,24,25,26,27,28,29.
Despite the availability of multiple analytical methods, each approach has limitations that may restrict its use in experimental or clinical research. Ion chromatography provides high sensitivity and selectivity, but requires specialized instrumentation and extensive sample preparation30,31. Colorimetric assays such as the SPADNS (sodium 2&#8209;(parasulfophenylazo)&#8209;1,8-dihydroxynaphthalene-3,6&#8209;disulfonate) method are simple and inexpensive, but they are susceptible to interference from turbidity and endogenous chromophores, which limits their reliability in complex biological samples22,32,33. Titrimetric methods are cost-effective and straightforward, but lack the sensitivity required for trace-level fluoride detection in most biological specimens34. Nuclear or activation-based techniques offer exceptional sensitivity but require access to reactors or accelerator-based neutron sources, making them impractical for routine laboratory use35. In contrast, HMDS-facilitated diffusion followed by ISE measurement provides a practical balance of sensitivity, cost, and accessibility while ensuring complete liberation of fluoride from both soft and mineralized tissues. This is particularly advantageous for studies involving small animals, where tissue availability is limited and mineralized structures such as mouse incisors yield only 3&#8211;5 mg of ash per tooth.
The ability of the diffusion&#8211;ISE method to concentrate all released fluoride into a small, defined volume enables accurate quantification even in extremely small samples. This makes the method well-suited for toxicokinetic studies, dose&#8211;response experiments, and investigations of fluoride metabolism across developmental stages. Small rodents, particularly C57BL/6J mice, have been widely used as animal models for studying the effects of fluoride7,9,12,29,36 .
In this protocol, the authors apply the HMDS-facilitated diffusion technique followed by fluoride-selective electrode measurement to quantify fluoride in serum, bone, and teeth collected from mice exposed to a high fluoride dose (125 ppm) compared with untreated controls. The authors further compare fluoride accumulation in adolescent (6 weeks at the onset of treatment) and mature (18 weeks at the onset of treatment) mice to evaluate age-dependent differences in fluoride metabolism and tissue deposition. Mandibular incisors were selected as the representative dental tissue because rodent incisors erupt continuously and achieve a steady-state balance between eruption and occlusal wear by approximately 7 weeks of age. This continuous growth pattern provides a consistent and accessible gradient of enamel development, making murine incisors a widely used and well&#8209;validated model for investigating enamel formation and dental fluorosis.

<table><tbody><tr><td>Acetic Acid, Glacial</td><td>Sigma-Aldrich</td><td>Supelco, GR ACS</td><td>AX0073-6</td></tr><tr><td>Alumina Crucibles</td><td>Advalue Technology</td><td>12 mm OD &amp;times; 25 mm H, AL-6212</td><td>AL-6212</td></tr><tr><td>Analytical Balance</td><td>VWR</td><td>VWR 124B2, d = 0.1 mg</td><td>&amp;mdash;</td></tr><tr><td>Bead Beater</td><td>Benchmark</td><td>BeadBug 6</td><td>&amp;mdash;</td></tr><tr><td>Blunt Tip Syringe Needle</td><td>Amazon</td><td>Industrial Blunt Tip Needle, 14 G &amp;times; 0.5 inch</td><td>X001TT8KX1</td></tr><tr><td>Carbon Dioxide</td><td>AirGas</td><td>&amp;mdash;</td><td>&amp;mdash;</td></tr><tr><td>Chemical Fume Hood</td><td>Mott Manufacturing</td><td>Model 7421040</td><td>&amp;mdash;</td></tr><tr><td>D(+)-Sucrose</td><td>Thermo Scientific</td><td>99.7% for Biochemistry</td><td>177142500</td></tr><tr><td>Electrode Filling Solution</td><td>Thermo Scientific</td><td>Orion IonPlus Filling Solution</td><td>900061</td></tr><tr><td>Ethyl Alcohol Solution</td><td>Koptec</td><td>140 Proof</td><td>2401</td></tr><tr><td>Freezer, &amp;minus;80 &amp;deg;C</td><td>Thermo Scientific</td><td>Revco RDE Series</td><td>&amp;mdash;</td></tr><tr><td>Heat-Resistant Gloves</td><td>Fisher Scientific</td><td>Bel-Art Clavies Biohazard Autoclave Gloves</td><td>H13201</td></tr><tr><td>Hexamethyldisiloxane (HMDS)</td><td>Fisher Scientific</td><td>TCI H0091, &gt;98%</td><td>H0091</td></tr><tr><td>Hydrogen Peroxide</td><td>VWR</td><td>J.T. Baker, 30%</td><td>2204-01</td></tr><tr><td>Ion-Selective Electrode</td><td>Thermo Scientific</td><td>Orion Fluoride ISE, Double Channel</td><td>9609BNWP</td></tr><tr><td>Magnetic Mixer</td><td>VWR</td><td>360 Stirrer</td><td>&amp;mdash;</td></tr><tr><td>Micro Spatula</td><td>Amazon</td><td>Wax Carving Spatula #7A, Stainless Steel</td><td>&amp;mdash;</td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>5427R</td><td>5404000131</td></tr><tr><td>Muffle Furnace</td><td>Thermo Scientific</td><td>Thermolyne Small Benchtop Muffle Furnace, 1.3 L, FB1315M</td><td>FB1315M</td></tr><tr><td>Oven</td><td>Boekel Scientific</td><td>Boekel RapidFISH</td><td>240200</td></tr><tr><td>Paraformaldehyde Solution in PBS</td><td>Thermo Scientific</td><td>4% Paraformaldehyde in PBS</td><td>J19943-K2</td></tr><tr><td>Petri Dish</td><td>VWR</td><td>60 &amp;times; 15 mm, Untreated</td><td>25384-092</td></tr><tr><td>Petrolatum</td><td>Thermo Scientific</td><td>Petrolatum, White, Pure</td><td>417090010</td></tr><tr><td>pH Indicator Paper</td><td>Fisher Scientific</td><td>pH-Fix 4.5&amp;ndash;10.0</td><td>92120.3</td></tr><tr><td>Porcelain Dish</td><td>Fisher Scientific</td><td>Fisherbrand Crucible, 5 mL, 20 mm Height, High Form</td><td>FB-965-B</td></tr><tr><td>Potentiometer</td><td>Thermo Scientific</td><td>Orion Dual Star pH/ISE Meter</td><td>2115102</td></tr><tr><td>Ring Stand with Clamp</td><td>VWR</td><td>Ring Clamp, 15 cm</td><td>470104-526</td></tr><tr><td>Rodent Diet</td><td>Bio-Serv</td><td>F1515, AIN-76A, &amp;frac12; Pellets</td><td>F1515</td></tr><tr><td>Scissor Jack</td><td>Amazon</td><td>Laboratory Scissor Jack</td><td>&amp;mdash;</td></tr><tr><td>Separatory Funnel</td><td>Fisher Scientific</td><td>Eisco, Borosilicate Glass, 2 L</td><td>S89285</td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific</td><td>Certified ACS, Crystalline</td><td>S271-1</td></tr><tr><td>Sodium Fluoride</td><td>Fisher Scientific</td><td>Certified ACS, Powder</td><td>S299-100</td></tr><tr><td>Sodium Hydroxide</td><td>Fisher Scientific</td><td>White Pellets</td><td>BP359-500</td></tr><tr><td>Stainless Steel Beads</td><td>Fisher Scientific</td><td>Scientific Industries, 5 mm, 100 pcs</td><td>SI-CS05</td></tr><tr><td>Sulfuric Acid</td><td>Fisher Scientific</td><td>ACS Grade</td><td>A300-500</td></tr><tr><td>TISAB II</td><td>Thermo Scientific</td><td>Orion IonPlus Application Solution</td><td>940909</td></tr><tr><td>Vacuum Desiccator</td><td>Fisher Scientific</td><td>Bel-Art Space Saver Vacuum Desiccator, 10-inch</td><td>F42010-0000</td></tr><tr><td>Vacuum Filter</td><td>Fisher Scientific</td><td>Flow Bottle-Top Filter, PES, 90 mm, 0.2 &amp;micro;m</td><td>597-4520</td></tr><tr><td>Variable Rocker</td><td>MidSci</td><td>LabDoctor / Benchmark Scientific</td><td>&amp;mdash;</td></tr></tbody></table>

quantification of fluoride in serum, bone, and teeth in a murine model using hexamethyldisiloxane-facilitated diffusion and ion-selective electrode measurements

Quantification of Fluoride in Serum, Bone, and Teeth in a Murine Model Using Hexamethyldisiloxane-Facilitated Diffusion and Ion-Selective Electrode Measurements

Quantifying fluoride levels in biological tissues is essential for understanding fluoride exposure, metabolism, and toxicity. This protocol describes a sensitive and reproducible method for measuring fluoride ion mass in serum, bone, and teeth using hexamethyldisiloxane (HMDS)&#8209;facilitated diffusion followed by ion&#8209;selective electrode (ISE) analysis. Biological specimens, including serum, femoral bone, and mandibular incisors, are collected from mice exposed to controlled fluoride treatments. Bone and tooth specimens are ashed, milled, and hydrated, while serum is processed directly. Each specimen is transferred to a diffusion dish containing ultrapure water and sealed with a petrolatum&#8209;lined lid bearing sodium hydroxide droplets that serve as fluoride traps. Injection of HMDS&#8209;saturated sulfuric acid initiates fluoride release and diffusion, allowing liberated fluoride ions to be quantitatively captured in the traps. Following overnight diffusion, the fluoride trap droplets are combined into a single sample, acidified to the required pH range, and adjusted to a defined volume for ISE measurement. Calibration diffusion dishes prepared with known fluoride masses generate a standard curve for quantification, and quality control samples verify electrode stability. Representative results from adolescent and mature mice exposed to 0 or 125 ppm fluoride demonstrate the method&#8217;s ability to detect age&#8209; and dose&#8209;dependent differences in fluoride levels across serum, bone, and teeth. This protocol provides a robust, calibration&#8209;based approach for quantifying fluoride in diverse biological matrices and is well&#8209;suited for studies investigating fluoride exposure, tissue deposition, and toxicological outcomes in murine models. A key advantage of this method is its ability to accurately quantify fluoride in extremely small specimens, including the 3&#8211;5 mg of ash obtained from individual mouse incisors, because the diffusion process concentrates all released fluoride into a measurable sample volume of less than 100&#160;&#181;L.

To demonstrate the application of this protocol for measuring fluoride (F-) concentrations in biological materials, specimen samples were generated from serum, bone, and mandibular incisor specimens collected from mice exposed to controlled fluoride treatments. Male C57BL/6J mice were assigned to two age categories, adolescent (6 weeks) and mature (18 weeks), and provided either fluoride&#8209;free deionized water (0 ppm F&#8315;) or sodium&#8209;fluoride&#8209;supplemented drinking water (125 ppm F&#8315;) for a 6&#8209;week exposure period. Each age and treatment condition included five animals. At the end of the exposure period, serum, femurs, and mandibular incisors were harvested, and specimen samples were prepared and analyzed according to the protocol. Representative fluoride concentrations measured in serum, bone, and teeth are summarized below.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/71488/71488fig4.jpg" /> 
Figure 4: Fluoride concentrations measured in serum, femoral bone, and mandibular incisors. Male C57BL/6J mice were assigned to two age categories, adolescent (6 weeks, open bar) and mature (18 weeks, filled bar), and provided either fluoride&#8209;free deionized water (0 ppm F&#8315;) or sodium fluoride&#8211;supplemented drinking water (125 ppm F&#8315;) for a 6&#8209;week exposure period. At the end of the exposure period, fluoride levels were measured in (A) serum, (B) femoral bone, and (C) mandibular incisors from the same treatment groups. Serum values below the detection limit (&#60;0.02 ppm) were plotted for visualization but excluded from statistical analysis. Data are expressed as means &#177; SD. Comparisons between adolescent and mature groups were performed using a Mann&#8211;Whitney U test with Bonferroni&#8211;Dunn correction for multiple comparisons. *p &#60; 0.05, **p &#60; 0.01 (N = 5/group). <a href="https://www.jove.com/files/ftp_upload/71488/71488fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Fluoride concentrations in serum 
In both the adolescent and mature groups treated with 0 ppm fluoride, serum fluoride concentrations were below the ion-selective electrode&#39;s detection limit (&#60;0.02 ppm). In the 125 ppm treatment groups, serum fluoride increased to 0.3 &#177; 0.2 ppm in the adolescent group and to 0.7 &#177; 0.2 ppm in the mature group (p = 0.06). Comparison between the 125 ppm treatment adolescent and mature group was performed using a Mann&#8211;Whitney U test with Bonferroni&#8211;Dunn correction for multiple comparisons. *p &#60; 0.05, **p &#60; 0.01 (N = 5/group). The 0 ppm treatment groups were excluded from statistical analysis. (Figure 4A, Table 1).
Fluoride concentrations in femoral bone 
In the 0&#8209;ppm treatment groups, femoral fluoride concentrations were significantly lower in adolescents (292 &#177; 39 ppm) compared to mature animals (896 &#177; 55 ppm) (p &#60; 0.01). In contrast, in the 125&#8209;ppm groups, fluoride concentrations in adolescents (4,108 &#177; 482 ppm) were significantly higher than in mature animals (2,966 &#177; 612 ppm) (p = 0.04). Comparisons between adolescent and mature groups were performed using a Mann&#8211;Whitney U test with Bonferroni&#8211;Dunn correction for multiple comparisons. *p &#60; 0.05, **p &#60; 0.01 (N = 5/group). (Figure 4B, Table 1). Figure 5A shows the fold&#8209;change in femoral fluoride between the 0&#8209;ppm and 125&#8209;ppm treatments for each age group. Adolescents exhibited a 14&#8209;fold increase in fluoride levels at 125 ppm, whereas mature mice showed a 3&#8209;fold increase.
Fluoride concentrations in mandibular incisors 
In the 0 ppm groups, mandibular incisor fluoride concentrations were significantly lower in adolescents (35 &#177; 4 ppm) compared with mature animals (123 &#177; 23 ppm) (p = 0.03). In the 125 ppm treatment groups, incisor fluoride concentrations increased to 5641 &#177; 955 ppm in adolescents and 4392 &#177; 1694 ppm in mature animals. Comparisons between adolescent and mature groups were performed using a Mann&#8211;Whitney U test with Bonferroni&#8211;Dunn correction for multiple comparisons. *p &#60; 0.05, **p &#60; 0.01 (N = 5/group). (Figure 4C, Table 1). Although absolute fluoride concentrations at 125 ppm did not differ significantly between age groups, the fold-change from 0 ppm to 125 ppm was significantly greater in adolescent (161-fold increase) than mature mice (36-fold increase) (Figure 5B).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/71488/71488fig5.jpg" /> 
Figure 5: Adolescents exhibit a greater fold-increase in bone and incisor fluoride accumulation than mature mice. Fold-change in fluoride concentrations from 0 ppm to 125 ppm fluoride treatment in adolescent (open bar) and mature (filled bar) mice. (A) Femoral bone and (B) Mandibular incisor from the same treatment groups. Data are expressed as means &#177; SD. Comparisons between adolescent and mature groups were performed using an unpaired t-test. * p &#60; 0.05, (N = 5/group). <a href="https://www.jove.com/files/ftp_upload/71488/71488fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample</td>
			<td colspan="4">Serum</td>
			<td colspan="4">Femur</td>
			<td colspan="4">Mandibular Incisor</td>
		</tr>
		<tr>
			<td>Age Group</td>
			<td colspan="2">Adolescent</td>
			<td colspan="2">Mature</td>
			<td colspan="2">Adolescent</td>
			<td colspan="2">Mature</td>
			<td colspan="2">Adolescent</td>
			<td colspan="2">Mature</td>
		</tr>
		<tr>
			<td>Treatment Group</td>
			<td>0 ppm</td>
			<td>125 ppm</td>
			<td>0 ppm</td>
			<td>125 ppm</td>
			<td>0 ppm</td>
			<td>125 ppm&#160;</td>
			<td>0 ppm</td>
			<td>125 ppm&#160;</td>
			<td>0 ppm</td>
			<td>125 ppm</td>
			<td>0 ppm</td>
			<td>125 ppm</td>
		</tr>
		<tr>
			<td></td>
			<td>&#60; 0.02</td>
			<td>0.2</td>
			<td>&#60; 0.02</td>
			<td>0.8</td>
			<td>324</td>
			<td>4576</td>
			<td>933</td>
			<td>3081</td>
			<td>37</td>
			<td>4824</td>
			<td>161</td>
			<td>3229</td>
		</tr>
		<tr>
			<td></td>
			<td>&#60; 0.02</td>
			<td>0.1</td>
			<td>&#60; 0.02</td>
			<td>0.8</td>
			<td>229</td>
			<td>3778</td>
			<td>924</td>
			<td>3326</td>
			<td>39</td>
			<td>5451</td>
			<td>127</td>
			<td>2654</td>
		</tr>
		<tr>
			<td></td>
			<td>&#60; 0.02</td>
			<td>0.7</td>
			<td>&#60; 0.02</td>
			<td>1.0</td>
			<td>300</td>
			<td>4683</td>
			<td>877</td>
			<td>3713</td>
			<td>31</td>
			<td>5544</td>
			<td>112</td>
			<td>6997</td>
		</tr>
		<tr>
			<td></td>
			<td>&#60; 0.02</td>
			<td>0.4</td>
			<td>&#60; 0.02</td>
			<td>0.5</td>
			<td>323</td>
			<td>3832</td>
			<td>807</td>
			<td>2519</td>
			<td>35</td>
			<td>5114</td>
			<td>103</td>
			<td>4169</td>
		</tr>
		<tr>
			<td></td>
			<td>&#60; 0.02</td>
			<td>0.3</td>
			<td>&#60; 0.02</td>
			<td>0.5</td>
			<td>283</td>
			<td>3669</td>
			<td>937</td>
			<td>2192</td>
			<td>N/A</td>
			<td>7271</td>
			<td>111</td>
			<td>4909</td>
		</tr>
		<tr>
			<td>Mean</td>
			<td>&#60; 0.02</td>
			<td>0.3</td>
			<td>&#60; 0.02</td>
			<td>0.7</td>
			<td>292</td>
			<td>4108</td>
			<td>896</td>
			<td>2966</td>
			<td>35</td>
			<td>5641</td>
			<td>123</td>
			<td>4392</td>
		</tr>
		<tr>
			<td>SD</td>
			<td>NA</td>
			<td>0.2</td>
			<td>NA</td>
			<td>0.2</td>
			<td>39</td>
			<td>482</td>
			<td>55</td>
			<td>612</td>
			<td>3</td>
			<td>955</td>
			<td>23</td>
			<td>1694</td>
		</tr>
	</tbody>
</table>
Table 1: Individual fluoride measurements in serum, femoral bone, and mandibular incisors. Table 1 lists the individual fluoride concentrations (ppm F&#8315;) measured in serum, femoral bone, and mandibular incisor specimens from adolescent and mature mice exposed to either 0 ppm or 125 ppm fluoride. For each tissue type, data are presented in columns corresponding to the four treatment groups: adolescent 0 ppm, adolescent 125 ppm, mature 0 ppm, and mature 125 ppm. Each column includes the five individual recorded values (N = 5), followed by the calculated mean and standard deviation (SD). In the adolescent 0 ppm serum group, all recorded values were below the ion-selective electrode&#39;s detection limit (&#60;0.02 ppm). The table provides the raw numerical data underlying the summary values and statistical comparisons shown in Figure 4.

This protocol describes the preparation of hexamethyldisiloxane (HMDS)-facilitated diffusion dishes, followed by ion-selective electrode measurements to quantify fluoride ion mass in the serum, bone, and teeth of mice. The method provides a sensitive, calibration-based approach for evaluating fluoride accumulation in biological specimens.

Quantifying fluoride levels in biological tissues is essential for understanding fluoride exposure, metabolism, and toxicity. This protocol describes a ...

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Learn fluoride quantification in serum, bone, and teeth using hexamethyldisiloxane-facilitated diffusion and ion-selective electrode analysis. This sensitive method enables accurate measurement in small murine specimens.

Quantification of Fluoride in Serum, Bone, and Teeth in a Murine Model Using Hexamethyldisiloxane-Facilitated Diffusion and Ion-Selective Electrode Measurements (Video) | JoVE Journal

Quantification of Fluoride in Serum, Bone, and Teeth in a Murine Model Using Hexamethyldisiloxane-Facilitated Diffusion and Ion-Selective Electrode Measurements

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