A Novel Extraction Method Based on a Reversible Chemical

Jan 26, 2015 - The United Graduate School of Agricultural Science, Iwate ... applied to a pharmacokinetic investigation following oral administration ...
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A Novel Extraction Method Based on a Reversible Chemical Conversion for the LC/MS/MS Analysis of the Stable Organic Germanium Compound Ge-132 Hiroaki Yamaguchi,*,† Yasuhiro Shimada,‡,§ Tomoya Takeda,‡ Takashi Nakamura,‡ and Nariyasu Mano† †

Department of Pharmaceutical Sciences, Tohoku University Hospital, 1-1 Seiryo-machi, Aoba-ku, Sendai, 980-8574, Japan Asai Germanium Research Institute Co., Ltd., 3-131 Suzuranoka-cho, Hakodate, Hokkaido 042-0958, Japan § The United Graduate School of Agricultural Science, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550 Japan ‡

S Supporting Information *

ABSTRACT: Poly trans-[(2-carboxyethyl)germasesquioxane] (IUPAC name) is the most common water-soluble organic germanium compound. This compound is known as bis(carboxyethyl)germaniumsesquioxide and it is commonly called Ge-132; it is hydrolyzed to 3-(trihydroxygermyl)propanoic acid (THGPA) in water. We have developed a method for the quantification of THGPA in rat plasma, using a novel extraction method based on a reversible chemical conversion. THGPA in plasma is converted to 3(trichlorogermyl)propanoic acid (TCGPA) under acidic conditions using concentrated hydrochloride, which is followed by extraction with chloroform. TCGPA is then converted back to THGPA through hydrolysis. The extraction recovery of this method is approximately 100%. Moreover, we synthesized deuterated Ge-132, which was used as an internal standard in our experiments. This method covers a linearity range of 0.01−5 μg/mL for concentrations of THGPA in plasma. The intra-day and inter-day precisions of the analysis are about 4.1%, and the accuracy is within ±2.6% at THGPA concentrations of 0.025, 0.25, and 2.5 μg/mL. The total run time is 5 min. Our method was successfully applied to a pharmacokinetic investigation following oral administration of Ge-132. tory1 and antirheumatoid effects) have been reported. Ogwapit has reported about some spectrometric analyses of Ge-132 and detailed physicochemical characters of the compound.2 On the other hand, the inorganic compound germanium dioxide (GeO2) is very harmful. Sanai et al. have reported that GeO2 and Ge-132 behave quite differently with regard to renal accumulation.3 GeO2 is accumulated in the kidney and causes severe renal damage,4 while Ge-132 does not accumulate in any tissue. Tao et al.5 have reviewed the hazardous properties of germanium compounds. Some organogermanium compounds have physiological activity; however, their levels of toxicity vary. There are several polymeric forms of THGPA: Ge-132 is one such polymer, while propagermanium (a drug for the treatment of hepatitis B)6 is another. Some studies of propagermanium’s effects on the inhibition of the chemokine receptor 2 have been reported.7,8 Recently, some novel physiological effects of the supplementary oral intake of Ge-132 have been reported. These effects include the increase of antioxidative compounds, the hepatic gene expression of phase I enzyme UDP glucuronosyltransferase 1,9 and the hepatic gene expression of amino-

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oly-trans-[(2-carboxyethyl) germasesquioxane] (Ge-132) is the most common water-soluble organic germanium compound. This compound is hydrolyzed to 3(trihydroxygermyl)propanoic acid (THGPA) in water (Figure 1). Ge-132 is used in functional foods and in cosmetics because many physiological effects of Ge-132 (including anti-inflamma-

Figure 1. Structures of poly-trans-[(2-carboxyethyl) germasesquioxane] (Ge-132) and its hydrolysis product 3-(trihydroxygermyl)propanoic acid (THGPA). © 2015 American Chemical Society

Received: December 1, 2014 Accepted: January 26, 2015 Published: January 26, 2015 2042

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Analytical Chemistry levulinic acid synthase 1.10 The reason why THGPA causes these physiological effects remains unclarified. We needed information about the structure and the concentration of THGPA in these tissues to understand the physiological effects that are caused by THGPA. Several quantification methods are used for the analysis of germanium. Atomic absorption spectrometry (AAS)11 and inductively coupled plasma mass spectrometry (ICP-MS)12 are often used for the microanalyses of germanium. Both analytical methods target the germanium atom. Therefore, the data obtained from these analyses do not correlate with the toxicity of each molecule. However, as described above, the toxicity of the germanium compound depends on the molecular structure; therefore, a quantification method that provides structural information is desired. Consequently, in this study we have tried to determine levels of THGPA (distinct from the chronically toxic germanium dioxide) by liquid chromatography with tandem mass spectrometric analysis. Herein we propose an extremely effective sample treatment that uses a simple, reversible chemical conversion of the target molecule, in combination with a deuterated internal standard for LC/MS/ MS.

For the quantification of THGPA (the hydrolyzate of Ge-132), the ion spray voltage was set at −4500 V. The turbo spray gas (N2) probe was heated to 600 °C. Nitrogen was used as the curtain gas, gas 1, and gas 2, and their flows were set to 60, 30, and 60 units, respectively. Unit mass resolution was set in both mass-resolving quadrupoles Q1 and Q3. The declustering potential (DP) and collision energy (CE) for THGPA and the internal standard were −10 V and −35 V, respectively. During selected reaction monitoring (SRM), the m/z transitions 197 → 107 and 199 → 107 were used for monitoring THGPA and THGPA-d2, respectively. The dwell time was 500 ms. Data were collected and processed using the Analyst 1.4.2 data collection and integration software. Sample Preparation. A stock solution of Ge-132, at a concentration of 1 mg/mL, was prepared in water. Additionally, a stock solution of the internal standard (Ge-132-d2), at a concentration of 5 μg/mL, was prepared in water. The stock solutions of Ge-132 were serially diluted in water to obtain working solutions of 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, and 50 ng/mL. The working solutions for the validation samples (0.25, 2.5, and 25 ng/mL) was obtained in the same manner. Calibration standards were prepared by adding working solutions to rat plasma to give final concentrations of 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, and 5 μg/mL. Validation samples of Ge-132 in rat plasma were prepared by adding working solutions to rat plasma to give final concentrations of 0.025, 0.25, and 2.5 μg/mL. All of the solutions were stored at −30 °C. Sample Pretreatment. We acidified plasma samples to form TCGPA, which was followed by extraction with chloroform. Volumes of 10 μL of the internal standard (5 μg/mL) and 200 μL of concentrated hydrochloric acid were added to samples of 100 μL of rat plasma. After 5 min of incubation, 2 mL of chloroform was added. The tube was shaken for 1 h and then centrifuged at 2000g for 10 min at room temperature. The upper aqueous layer was discarded, and then 1 mL of the organic layer was transferred to another tube and evaporated to dryness. The dried residue was redissolved in 50 μL of water (resulting in the conversion of TCGPA back to THGPA). A volume of 5 μL of the solution was injected into the HPLC column. Method Validation. For validation, Ge-132 standards (nine nonzero analyte standards of 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, and 5 μg/mL) were prepared in blank rat plasma and analyzed. Linear regression of the ratio of the areas of the analyte and internal standard peaks versus the concentration were weighted by 1/x (the reciprocal concentration). The lower limit of quantification (LLOQ) was defined as the concentration corresponding to a signal-to-noise ratio of at least 5, and with acceptable precision and accuracy (relative standard deviation (RSD) and relative error (RE) 99.9%) was synthesized at the plant of the Asai Germanium Research Institute Co., Ltd. (Hakodate, Japan). The synthesis procedure of Ge-132 at Asai Germanium Research Institute is as follows. Ingots of germanium (purity over 99.9999%) were milled in powder. The powdered germanium was reacted with hydrochloride gas at high temperature and trichlorogermane produced. Then trichlorogermane was reacted with acrylic acid into 3-(trichlorogermyl)propanoic acid (TCGPA). TCGPA was hydrolyzed to THGPA and dried to polymer, Ge-132. All of the other solvents and reagents were of the highest purity available. Preparation of Ge-132-d2. The alkaline solution of Ge132 in deuterium oxide was prepared by mixing Ge-132, sodium hydroxide, and deuterium oxide. The mixture was heated to 80 °C for 14 days with an oil bath. The degree of reaction was confirmed using 1H NMR (Figure S-1 in the Supporting Information). If the deuterated reaction did not proceed enough, the mixture was once evaporated and then the aliquot of deuterium oxide was added and heated again for further deuteration. This reaction was repeated a few times. After acidification of the solution with hydrochloric acid, the solution was left standing for a while and crystals precipitated. The deposited crystals were filtered, washed with water, and dried to afford (GeCH2CD2COOH)2nO3n (Ge-132-d2). The degree of deuteration was determined by analysis of the 1H NMR spectrum. In this study, we used Ge-132-d2 with a degree of deuteration of more than 98%. Chromatographic and Mass Spectrometric Conditions. Chromatographic separation was carried out using a Shimadzu Prominance 20A System (Shimadzu, Kyoto, Japan) with a Shodex DE413-2D column (150 mm × 2.0 mm i.d., Showa Denko, Tokyo, Japan). The column temperature was maintained at 50 °C. The mobile phase consisted of water/ methanol/acetic acid (95:5:0.1, v/v/v) and it was pumped at a flow rate of 0.3 mL/min. The overall run time was 5 min. Mass spectrometry was carried out on an API 3200 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA). Negative ion electrospray mass spectrometry was performed. 2043

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Figure 2. Negative ion full-scan mass spectra of (A) 3-(trihydroxygermyl)propanoic acid (THGPA) and (B) the internal standard.

Figure 3. Product ion mass spectra of (A) 3-(trihydroxygermyl)propanoic acid (THGPA) and (B) the internal standard.

and analyzed. Rat plasma samples were prepared from six different rats. The percentages of overall process efficiency, extraction recovery, and matrix effect are calculated as

overall process efficiency (%) =

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C × 100 A

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Analytical Chemistry extraction recovery (%) =

matrix effect (%) =

C × 100 B

at m/z 107 is produced with the highest intensity, and it was used for quantitative SRM of the internal standard. It is possible that the matrix effects by plasma contents influence the ionization of THGPA if the sample is pretreated with organic solvents such as acetonitrile and/or methanol to precipitate proteins. Therefore, we selected a liquid−liquid extraction procedure for the analysis of Ge-132. THGPA is converted to TCGPA upon the addition of concentrated hydrochloric acid. TCGPA can be extracted with chloroform, and water-soluble components, including salts, are removed in this process. After the evaporation of the chloroform phase, the dried residue was redissolved in water, resulting in the hydrolysis of TCGPA back to THGPA. This sample treatment with a deuterated internal standard makes the precise and brief LC/MS/MS assay of Ge-132 possible. Using the mobile phase as described in the Materials and Methods section, the retention time of THGPA and the internal standard is 1.7 min. The total LC run time is 5 min. Method Validation. The present method covers a linearity range of 0.01−5 μg/mL in the concentration of Ge-132 in plasma. The correlation coefficient (r) is >0.999. A typical standard curve is y = 3.28x + 0.0375. The LLOQ for THGPA is 0.01 μg/mL in plasma. Our method is 10 and 100 times more sensitive than previous methods for Ge-132 analysis that use high-performance ion-exclusion chromatography (HPIEC)13 and gas chromatography coupled with microwave-induced plasma atomic emission detection (GC-MIP-AED),14 respectively. The specificity and selectivity of the method were evaluated. A representative SRM chromatogram of blank rat plasma and chromatograms at the LLOQ of THGPA and the internal standard, spiked in rat plasma, are shown in parts A, B, and C of Figure 4, respectively. There is no significant interference from endogenous plasma constituents at retention times of THGPA and of the internal standard. The intra-day (n = 6) and inter-day (n = 6) accuracy and precision were investigated at three different concentrations: 0.025, 0.25, and 2.5 μg/mL. The results are summarized in Table 1. The intraday and interday accuracy (RE (%)) is within ±2.6% for all of the concentrations. The intra-day and inter-day precision (RSD (%)) was less than 4.1% for all of the concentrations. These results suggest that the present method can accurately and reproducibly measure levels of Ge-132 in rat plasma. The overall process efficiencies of THGPA are 88.1 ± 6.0%, 88.4 ± 5.0%, and 90.5 ± 11.9% at concentrations of 0.025, 0.25, and 2.5 μg/mL, respectively. The extraction recoveries of THGPA from rat plasma are 96.0 ± 5.8%, 101.3 ± 5.0%, and 98.9 ± 11.7% at concentrations of 0.025, 0.25, and 2.5 μg/mL, respectively. The matrix effect of THGPA by plasma matrix components is 91.8 ± 4.2%, 87.3 ± 8.4%, and 91.5 ± 1.7% at concentrations of 0.025, 0.25, and 2.5 μg/mL, respectively. These results indicate that some components that were extracted from the plasma slightly affect the ionization of THGPA (approximately 10%); however, the efficient liquid− liquid extraction of TCGPA (obtained from THGPA by the reaction with concentrated hydrochloric acid) brought a high overall process efficiency of approximately 90%. The stability of THGPA in rat plasma is shown in Table 2. No significant degradation was observed after 4 h at ambient temperature (short-term stability), after 2 months at −30 °C (long-term stability), and after three freeze/thaw cycles.

B × 100 A

where A is the peak area of the mobile phase to which Ge-132 was added, B is the peak area obtained from samples spiked after the extraction, and C is the peak area obtained from samples spiked before the extraction. If the matrix effect (%) = 100, no matrix effect is present; if the matrix effect (%) > 100, there is a signal enhancement; and if the matrix effect (%) < 100, there is a signal suppression. The stability of Ge-132 in rat plasma was examined by analyzing three concentrations (0.025, 0.25, and 2.5 μg/mL) in triplicate. These samples were stored at −30 °C for 2 months and at ambient temperature for 4 h to evaluate the long-term and short-term stabilities, respectively. The freeze−thaw stability was tested by performing three freeze−thaw cycles (−30 °C to ambient temperature). The stability of the processed samples was assessed by reinjecting the samples into an autosampler (4 °C) after 24 h. Application in a Pharmacokinetic Study. The method was applied to a pharmacokinetic study following oral administration of Ge-132 at the dose of 100 mg/kg. Male Wistar rats were obtained from Charles River Laboratories Japan, Inc. (Yokohama, Japan). Rats were housed for at least 1 week at a temperature of 23 °C and a relative humidity of 60 ± 10%, with a 12 h light/dark cycle. During acclimatization, the rats were allowed free access to food and water. All of the experiments were conducted according to the guidelines of the ethics committee of experimental care at Asai Germanium Research Institute Co., Ltd. These guidelines are based on public guidelines set forth by the Japanese Ministry of Education, Culture, Sports, Science, and Technology. The rats had free access to water during the experiment. Blood samples were collected via the jugular vein 0, 0.5, 1, 2, 3, 6, 12, and 24 h after the administration of Ge-132. Samples were immediately stored at 4 °C and centrifuged at 1700g for 10 min. The resulting plasma was stored at −30 °C until the measurement was performed.

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RESULTS AND DISCUSSION Method Development. 70Ge, 72Ge, 73Ge, 74Ge, and 76Ge are five major natural isotopes of germanium, with natural abundances of 21.2, 27.7, 7.7, 35.9, and 7.4%, respectively. The negative ion full-scan mass spectra (Q1) of Ge-132 indicate the presence of deprotonated THGPA molecule [M − H]− at m/z 193, 195, 196, 197, and 199, with a peak ratio that corresponds to the abundances of Ge isotopes (Figure 2A). The product ion mass spectrum of [M − H]− at m/z 197 is shown in Figure 3A. Product ions appear at m/z 179, 107, and 71. The product ion at m/z 107 is produced with the highest intensity, and it was used for quantitative SRM of Ge-132. In the present study, we synthesized deuterated Ge-132 (Ge132-d2) as an internal standard. The use of a stable-isotopelabeled internal standard leads to a highly reproducible method with a good precision and accuracy. The negative ion full-scan mass spectra (Q1) of the internal standard indicate the presence of the deprotonated molecule [M − H]− at m/z 195, 197, 198, 199, and 201 (Figure 2B). The product ion mass spectrum of [M − H]− at m/z 199 is shown in Figure 3B. Product ions appear at m/z 181, 107, and 72. The product ion 2045

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4D. The maximum concentration (Cmax) is 3.62 ± 0.93 μg/mL, and the time to reach the maximum concentration (tmax) is 3.63 ± 1.51 h. This result indicates the applicability of this method to a pharmacokinetic study of Ge-132. The distributional study of Ge-132 that uses 14C−Ge-132 (which is radioactive) has been reported.15 In that study, the maximum concentration of 14 C−Ge-132 in the blood was 4.1 μg/mL, 3 h after forced administration. Moreover, the total germanium after oral Ge132 administration in the same dose was analyzed by AAS (unpublished result for pharmacokinetic study of Ge-132 development at the Asai Germanium Research Institute). The maximum concentration was 1.88 ± 0.50 μg/mL for the nonfasted group and was 4.69 ± 1.11 μg/mL for the fasted group, respectively. The time of maximum concentration was 2 h for nonfasted and 3 h for fasted, respectively. The study using radioactive compound was carried in fasted conditions for 24 h. Therefore, under the fasted condition of 4.69 ± 1.11 μg/mL of total germanium study (AAS) was a similar value for the value of 4.1 μg/mL of the radioactive compound study. However, the study analyzed by AAS suggest the fasting increased absorption of Ge-132. The value of the present study (Cmax 3.62 μg/mL) was concentration in plasma, and therefore the theoretical blood concentration is 1.99 μg/mL (calculated as hematoclit value 45%). This value is almost according to the previous total germanium study (1.88 ± 0.50 μg/mL) carried out without fasting. Moreover, the maximum time of these different experiments was distributed from 2 to 3 h; additionally the blood concentration curves are very similar. Thus, the previous data of theoretic Ge-132 concentration is according to the present study. The radioisotope-labeled derivative of Ge-132 was labeled at a carboxyl moiety, and the propanoic acid radical was monitored. This might contain metabolites without a germanium atom. By contrast, in the AAS analysis, germanium atoms are monitored as total germanium. This might contain inorganic germanium as a metabolite of Ge132. Moreover, the present study monitors the entire THGPA molecule; thus, the agreement between these three analytic methods suggests that almost all of the THGPA remains without metabolism in the blood. However, further study of microanalysis for germanium dioxide or a possible metabolite structure of Ge-132 is essential. We expect for development of the same time evaluation, analysis of those germanium compounds confirm the reason for high safety of Ge-132.

Figure 4. Representative chromatograms of (A) blank plasma, (B) 3(trihydroxygermyl)propanoic acid (THGPA) at the LLOQ (0.01 μg/ mL), (C) the internal standard spiked into rat plasma, and (D) THGPA in plasma taken from a rat 1 h after oral administration of Ge132.

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CONCLUSIONS An accurate, reproducible, and selective LC/MS/MS assay has been established for the quantification of THGPA in rat plasma. We have developed a novel extraction method that uses a reversible chemical conversion that occurs during sample pretreatment. TCGPA, which is obtained by the reaction of THGPA with hydrochloric acid, can be extracted with chloroform; water-soluble components, including salts, can be removed. After evaporation of the chloroform, the dried residue was redissolved in water, resulting in the conversion of TCGPA

Postextraction samples kept in the autosampler at 4 °C for 24 h were also stable. Application in a Pharmacokinetic Study. The time dependence of the THGPA concentration in the plasmas of eight rats, after oral administration of Ge-132, is shown in Figure 5. The chromatogram of THGPA in plasma taken from a rat 1 h after oral administration of Ge-132 is shown in Figure

Table 1. Precision and Accuracy of the Quantification of 3-(Trihydroxygermyl)propanoic Acid (THGPA) in Rat Plasma intra-day (n = 6)

inter-day (n = 6)

analyte

concn (μg/mL)

found (μg/mL)

RSD (%)

RE (%)

found (μg/mL)

RSD (%)

RE (%)

THGPA

0.025 0.25 2.5

0.0245 ± 0.0006 0.257 ± 0.011 2.50 ± 0.03

2.3 4.1 1.4

−2.2 0.6 −0.1

0.0247 ± 0.0005 0.257 ± 0.007 2.50 ± 0.03

1.9 2.7 1.3

−1.3 2.6 0.0

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Analytical Chemistry Table 2. Stability of 3-(Trihydroxygermyl)propanoic Acid (THGPA) in Rat Plasma percentage of remaining THGPA concn (μg/mL)

short-term stability (4 h at ambient temperature)

long-term stability (2 months at −30 °C)

freeze/thaw stability

processed sample stability (24 h at 4 °C)

0.025 0.25 2.5

98.1 ± 4.9 100.5 ± 0.2 97.5 ± 3.3

101.3 ± 4.2 98.7 ± 2.3 96.3 ± 2.7

94.7 ± 4.6 101.0 ± 2.7 99.1 ± 1.4

95.6 ± 3.4 97.2 ± 3.7 95.5 ± 3.2

(6) Hirayama, C.; Suzuki, H.; Ito, M.; Okumura, M.; Oda, T. J. Gastroenterol. 2003, 38, 525−532. (7) Tamura, Y.; Sugimoto, M.; Murayama, T.; Ueda, Y.; Kanamori, H.; Ono, K.; Ariyasu, H.; Akamizu, T.; Kita, T.; Yokode, M.; Arai, H. Arterioscler. Thromb. Vasc. Biol. 2008, 28, 2195−2201. (8) Tamura, Y.; Sugimoto, M.; Murayama, T.; Minami, M.; Nishikaze, Y.; Ariyasu, H.; Akamizu, T.; Kita, T.; Yokode, M.; Arai, H. J. Atheroscler. Thromb. 2010, 17, 219−228. (9) Nakamura, T.; Nagura, T.; Akiba, M.; Sato, K.; Tokuji, Y.; Ohnishi, M.; Osada, K. J. Health Sci. 2010, 56, 72−80. (10) Nakamura, T.; Saito, M.; Shimada, Y.; Fukaya, H.; Shida, Y.; Tokuji, Y. Eur. J. Pharmacol. 2011, 653, 75−81. (11) Shinogi, M.; Masaki, T.; Mori, I. J. Trace Elem. Electrolytes Health Dis. 1989, 3, 25−28. (12) Krystek, P.; Ritsema, R. J. Trace Elem. Med. Biol. 2004, 18, 9−16. (13) Qing-Chuan, C.; Shi-Fen, M.; Yan, Y.; Zhe-Ming, N. J. Chromatogr., A 1997, 789, 403−412. (14) Trikas, E.; Zachariadis, G. A.; Rosenberg, E. Anal. Bioanal. Chem. 2014, 406, 3489−3496. (15) Kagoshima, M.; Onishi, T.; Suguro, N.; Tomizawa, S. Oyo Yakuri 1986, 32, 71−79.

Figure 5. Concentration of 3-(trihydroxygermyl)propanoic acid (THGPA) in plasma, after oral administration of Ge-132 to rats. Ge-132 was administered at the dose of 100 mg/kg. Each point represents the averaged concentration of THGPA in the eight rats, and the standard errors are also provided.

back to THGPA. This procedure makes the precise and brief LC/MS/MS assay of THGPA possible. Validation demonstrated that this method has sensitivity and selectivity as well as sufficient precision and accuracy. The method was applied in a pharmacokinetic study of Ge-132 administered to rats. This is the first report of the quantification of the intact form of THGPA, a hydrolyzate of Ge-132, by LC/MS/MS.

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ASSOCIATED CONTENT

S Supporting Information *

1

H NMR spectra of 3-(trihydroxygermyl)propanoic acid (THGPA) and deuterated THGPA. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +81-22-717-7528. Fax: +81-22-717-7545. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. K. Sato of the Asai Germanium Research Institute for providing advice on the synthesis of the deuterated internal standard of THGPA.

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REFERENCES

(1) Pronai, L.; Arimori, S. Biotherapy 1992, 4, 1−8. (2) Ogwapit, S. M. Biosci. Horiz. 2011, 4, 128−139. (3) Sanai, T.; Okuda, S.; Onoyama, K.; Oochi, N.; Takaichi, S.; Mizuhira, V.; Fujishima, M. Kidney Int. 1991, 40, 882−890. (4) Schauss, A. G. Biol. Trace Elem. Res. 1991, 29, 267−280. (5) Tao, S. H.; Bolger, P. M. Regul. Toxicol. Pharmacol. 1997, 25, 211−219. 2047

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