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Food Safety and Toxicology

Parapyruvate, an Impurity in Pyruvate Supplements,
Induces Senescence in Human Fibroblastic Hs68 Cells via
Inhibition of the #-Ketoglutarate Dehydrogenase Complex
Shih-Chung Chang, Inn Lee, Hua Ting, Yuan-Jhe Chang, and Nae-Cherng Yang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01138 • Publication Date (Web): 22 Jun 2018
Downloaded from http://pubs.acs.org on June 25, 2018

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Page 1 of 41 Journal of Agricultural and Food Chemistry

1 Parapyruvate, an Impurity in Pyruvate Supplements, Induces Senescence in

2 Human Fibroblastic Hs68 Cells via Inhibition of the α-Ketoglutarate

3 Dehydrogenase Complex

5 Shih-Chung Chang1, Inn Lee2, Hua Ting1,3,4, Yuan-Jhe Chang5, Nae-Cherng

6 Yang2,6,*

7
1
8 Department of Physical Medicine and Rehabilitation, Chung Shan Medical

9 University Hospital, Taichung, Taiwan

2
10 Department of Nutrition, Chung Shan Medical University, Taichung, Taiwan
3
11 Sleep Medicine Center, Chung Shan Medical University Hospital, Taichung, Taiwan
4
12 Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan
5
13 Department of Occupational Safety and Health, Chung Shan Medical University,

14 Taichung, Taiwan
6
15 Department of Nutrition, Chung Shan Medical University Hospital, Taichung,

16 Taiwan

17
*
18 Corresponding author:

19 N.C. Yang, PhD, Associate Professor, Department of Nutrition, Chung Shan Medical

20 University

21 Tel.: +886-4-2473-0022; fax: +886-4-2324-8175

22 E-mail: naeman@csmu.edu.tw

23

24 Running title: Senescent effect of parapyruvate on Hs68 cells

25

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26 ABSTRACT

27

28 Commercial dietary supplements of calcium pyruvate claim to be beneficial for losing

29 weight, increasing muscle endurance, and regulating metabolism. Most industrial

30 preparations have some impurities, including parapyruvate. Parapyruvate is an

31 inhibitor of the α-ketoglutarate dehydrogenase complex (KGDHC). However, the

32 effect and mechanism of parapyruvate on cell senescence and the content of

33 parapyruvate in the dietary supplements of calcium pyruvate are unknown. In this

34 study, we prepared pure parapyruvate with a purity of 99.8 ± 0.1% and investigated its

35 ability to inhibit KGDHC activity and affect fibroblast senescence. Parapyruvate

36 dose-dependently decreased KGDHC activity, with an IC50 of 4.13 mM, and induced

37 Hs68 cell senescence. Calcium ions, a KGDHC activator, antagonized the senescent

38 effects of parapyruvate. The parapyruvate content was 1.4 ± 0.1% to 10.6 ± 0.2% in

39 five brands of calcium pyruvate supplements. In this study, we showed that

40 parapyruvate strongly induces Hs68 cell senescence by inhibiting KGDHC activity.

41 Because of its KGDHC inhibition activity, the parapyruvate content should be an

42 important issue for the food safety of calcium pyruvate supplements.

43

44

45 KEYWORDS: Parapyruvate, cellular senescence, α-ketoglutarate dehydrogenase

46 complex, calcium pyruvate, Hs68 cells

47

48

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49 INTRODUCTION

50 Pyruvate is the end product of glycolysis and the starting substrate for the

51 tricarboxylic acid (TCA) cycle. Pyruvate has a strong ability to scavenge H2O2 by

52 directly interacting with it chemically (1). Several animal studies have demonstrated

53 that pyruvate possesses multiple functionalities, including antioxidant,

54 anti-inflammatory, weight-loss, blood glucose-lowering, and insulin

55 resistance-lowering activities as well as neuroprotective effects (2-9). However, the

56 health benefits of pyruvate are not consistent with the results from human studies

57 (10-13).

58 Parapyruvate is a dimer formed by the polymerization of two molecules of

59 pyruvate via aldol condensation (14). It is also known as

60 2-hydroxy-2-methyl-4-oxopentanedioic acid and 4-hydroxy-4-methyl-2-oxoglutarate.

61 It is an analogue of α-ketoglutarate and should be a competitive inhibitor of

62 α-Ketoglutarate dehydrogenase complex (KGDHC) activity (14). However, the

63 mechanism of parapyruvate to inhibit KGDHC activity may be complex, because no

64 direct competition of parapyruvate could be demonstrated (14). It was concluded that

65 the mechanism was a competitive inhibition in its inhibition development but, once

66 the inhibition was established, it was reversible with difficulty (14). Because of

67 KGDHC inhibitory property, parapyruvate is an inhibitor of the TCA cycle (14). In

68 addition, commercial dietary supplements of pyruvate on the market claim to be

69 beneficial for losing weight, increasing muscle endurance, and regulating metabolism.

70 Calcium pyruvate is the most widely available and popular supplement. The other

71 forms are sodium pyruvate, potassium pyruvate, and triple pyruvate (i.e., a product

72 simultaneously mixed with sodium pyruvate, potassium pyruvate, and calcium

73 pyruvate). Most industrial preparations of sodium pyruvate have some impurities,

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74 including parapyruvate (14,15). However, the content of parapyruvate in dietary

75 supplements of calcium pyruvate is unclear.

76 KGDHC is a rate-limiting enzyme of the TCA cycle (16) and acts as an oxidative

77 decarboxylase. Inhibition of KGDHC activity leads to functional abnormalities of

78 mitochondria (17,18). Decreased KGDHC activity is involved in several aging-related

79 neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease

80 (19,20). However, fibroblasts, which are known to have a finite lifespan known as the

81 Hayflick limit, have frequently been used for senescence experiments (21,22).

82 However, the role of decreased KGDHC activity in the cellular senescence of

83 fibroblasts is unknown.

84 We previously used human fibroblastic Hs68 cells to explore the effects and

85 mechanisms of glucose restriction, 2-deoxyglucose and FK866 (a nicotinamide

86 phosphoribosyltransferase inhibitor) on cell senescence or the replicative lifespan of

87 human cells (23-25), but the effect and mechanism of parapyruvate on senescence of

88 Hs68 cells is unknown. In this study, we hypothesized that parapyruvate would induce

89 senescence in Hs68 cells by inhibiting KGDHC activity, and that KGDHC activation

90 should be able to reverse the parapyruvate-induced senescence of Hs68 cells. We also

91 analyzed the contents of parapyruvate in commercial dietary supplements of calcium

92 pyruvate. In addition, a cumulative growth curve and senescence-associated

93 β-galactosidase (SA-βG) activity were used to monitor the effect of parapyruvate on

94 cell senescence (25).

95

96 MATERIALS AND METHODS

97 Chemicals. All chemicals used were of analytical grade. NaCl, KCl, KOH, HCl,

98 H2SO4, MgCl2⋅6H2O, Na2HPO4, KH2PO4, NaHCO3, and dimethyl sulfoxide were

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99 from Merck (Darmstadt, Germany). Sodium pyruvate, CaCl2,

100 N,N-dimethylformamide, citric acid, potassium ferricyanide, potassium ferrocyanide,

101 glutaraldehyde, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium and

102 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-Gal),

103 ethylenediaminetetraacetic acid (EDTA), nicotinamide adenine dinucleotide (NAD+),

104 thiamine pyrophosphate, coenzyme A sodium salt hydrate, α-ketoglutarate, Tris base,

105 rotenone, Triton X-100, polyvinyl alcohol (molecular weight 3000–7000 g/mol),

106 nitroblue tetrazolium (NBT), and phenazine methosulfate were from Sigma Chemical

107 Corp. (St. Louis, MO, USA). Fluorescein di-β-D-galactopyranoside (FDG) was from

108 Molecular Probes (Eugene, OR, USA). Pyruvic acid (> 98%) was from Alfa Aesar

109 (Heysham, England). Acetone, methanol, and ethanol were from J.T. Baker Chemicals

110 (Pennsylvania, USA). Dulbecco’s modified Eagle medium (DMEM), trypsin and

111 penicillin–streptomycin were from HyClone (Boston, USA).

112 Preparation of Parapyruvate. Parapyruvate was synthesized as described (15)

113 with some modifications. Margolis and Coxin (15) proposed an alkaline treatment

114 method to prepare parapyruvate crudely. In the crude preparation, newly formed

115 parapyruvate was dissolved in a solution with some other impurities. We obtained

116 pure crystals of parapyruvate by using solvent crystallization (26) from the crude

117 preparation of parapyruvate. In detail, the pH of a 1-M pyruvic acid solution was

118 adjusted to 12 by using a 6-M KOH solution. After 15 min to allow for the

119 polymerization reaction, the pH was adjusted to 3 by using 3 M HCl solution to stop

120 the polymerization reaction and obtain the crude preparation of parapyruvate in

121 solution. The crude parapyruvate in solution was further supplemented with a proper

122 volume of acetone to obtain crystals of parapyruvate. The crystals were washed three

123 times with methanol and then dried in the oven at 50 °C. Highly pure parapyruvate

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124 crystals were obtained. The yield (%) of our modified method in producing pure

125 parapyruvate crystal was estimated at 49.6 ± 3.4%.

126 Determination of Parapyruvate by HPLC. Parapyruvate was analyzed by

127 reverse-phase high-performance liquid chromatography (HPLC) system consisting of

128 the HP Series 1050 Pumping Systems (Palo Alto, CA, USA), the HP 1050 Series

129 Online Degasser, Athena C18 column (4.6 × 250 mm, 5 µm), and the HP 1050 Series

130 Variable Wavelength Detector. The analysis was as described previously (27). The

131 mobile phase was 0.02% sulfuric acid solution, and the analysis was achieved by

132 using an isocratic elution for 10–30 min at a flow rate of 1 mL/min. Absorbent signals

133 were detected at 220 nm. The sample loop was 20 µL.

134 Identification of Produced Parapyruvate by LC/MS/MS. The identification of

135 the produced parapyruvate was achieved by LC/MS/MS with full scan analysis mode.

136 Briefly, a 17.6 µg/mL solution of the prepared crystals of parapyruvate salt in pure

137 water was analyzed by using a LC/MS/MS system under the following conditions:

138 column, Phenomenex Synergi Polar-RP (4 µm particle size, 150 mm × 4.6 mm;

139 Phenomenex, Torrance, CA, USA); flow rate, 0.5 mL/min; injection volume, 20 µL;

140 mobile phase of water with 0.1% formic acid; and ionization mode, negative

141 electrospray ionization with a full scan mode and ion range 50–300. Other instrument

142 conditions are outlined in a previous publication (24). Identification was evaluated by

143 the m/z of the molecular ion and the daughter ions by full-scan analysis. The purity of

144 the parapyruvate salt is able to be evaluated qualitatively even with only a single peak

145 of parapyruvate in the chromatogram.

146 Determination of Potassium Content in the Prepared Crystals of

147 Parapyruvate. To determine whether the crystals were mono-potassium parapyruvate

148 or di-potassium parapyruvate, we sent the prepared crystals to the Taiwan

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149 Accreditation Foundation-certified laboratory in the Health Technology Center at

150 Chun Sun Medical University to analyze the potassium content in the prepared

151 crystals by inductively coupled plasma atomic emission spectroscopy (ICP-AES). The

152 parapyruvic acid salt’s mono- or di-potassium content is able to be estimated by the

153 potassium content in the synthesized crystals.

154 Determination of the Purity of the Prepared Parapyruvate by HPLC.

155 Approximately 210mg of the prepared parapyruvate crystals (W) were dissolved in 1

156 L of pure water in triplicate (n=3). The concentrations of parapyruvate (C; mg/L) in

157 the spiking solutions were determined by HPLC. The purity of parapyruvate was

158 calculated as {[C (mg/L)* 1 L * 214.2/176.1] / [W (mg)]} *100%.

159 Determination of Parapyruvate’s Inhibition of KGDHC Activity in Vitro. The

160 inhibition of KGDHC activity was evaluated by using a commercial α-Ketoglutarate

161 Dehydrogenase Activity Colorimetric Assay Kit (BioVision, USA). Briefly, different

162 concentrations of parapyruvate were added to the KGDHC assay buffer containing 2

163 µL/well KGDHC substrate and KGDHC developer at a total volume of 50 µL/well.

164 After incubation for 10 min, the absorbance at 450 nm was detected. The relative

165 KGDHC activity (%) was determined as OD450 of the parapyruvate group/OD450 of

166 control * 100%.

167 Cell Culture. Human fibroblastic Hs68 cells (ATCC# CRL-1635) were purchased

168 from the Cell Culture Center of the Food Industry Research and Development

169 Institute (Hsinchu, Taiwan). Hs68 is one of a series of human foreskin fibroblast lines

170 developed at the Naval Biosciences Laboratory in Oakland, CA, USA. It was obtained

171 from an apparently normal Caucasian newborn male and has a finite lifespan. Hs68

172 cells were regularly cultured in DMEM in 75 cm3 flasks with 10% fetal bovine serum

173 (FBS), 1.5 g/L sodium bicarbonate, 25 mM (4.5 mg/mL) glucose, and antibiotics at 37

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174 °C in a humidified incubator with 5% CO2.

175 Cell Viability Assay. The effect of parapyruvate on cell viability was determined

176 by the Trypan blue exclusion method (28). Cells were cultured in 12-well plates until

177 confluent and incubated with different concentrations of parapyruvate for 24 h. After

178 removing the cultured medium and washing once with phosphate-buffered saline

179 (PBS), the cells were collected by trypsinization and counted. Cell viability (i.e.,

180 cytotoxicity) was calculated as the proportion of cells with parapyruvate treatment

181 compared with the control treatment.

182 Determination of the Cumulative Growth Curve. The cumulative growth curve

183 was determined as previously described (25). Briefly, cells were serially cultured in a

184 10-cm dish with 1.0 × 105 cells. The cells were subcultured once per week. The

185 population-doubling levels (PDLs) were calculated as log2 (Nt/No), where Nt and No

186 are the total cell count at harvesting and seeding during subculture, respectively. The

187 cumulative PDLs (CPDs) were obtained by adding up the total PDL before a given

188 passage time. The precise CPDs of Hs68 cells were not specified by the suppliers, so

189 we defined the CPDs at the initial passage as zero and used the additional CPDs to

190 represent the doubling levels after the initial passage (25).

191 Determination of SA-βG Activity. SA-βG activity was measured by using a

192 double-substrate assay (29). The monolayer of cells (5 × 104) cultured in 12-well

193 plates overnight was washed twice in PBS (pH 7.4), then fixed for 5 min in 2.0%

194 formaldehyde and 0.2% glutaraldehyde buffered with PBS. After washing three times

195 in PBS, the fixed cells were incubated in a staining solution containing 2.45 mM

196 X-Gal (pH 6.0, freshly prepared) and 40 µM FDG in a humidified incubator at 37 °C

197 for 24 h without CO2, and then, 100 µL of the supernatant was transferred to a 96-well

198 plate for fluorescent measurement in triplicate. The fluorescein fluorescence was

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199 measured by using a fluorometer (FLx800, BioTek Instruments, Winooski, VT, USA)

200 with an excitation of 485 nm and an emission of 535 nm. The X-Gal-stained cells

201 were further photographed under a microscope (Nikon, Japan) at a 100×

202 magnification for qualitative detection of SA-βG activity. The staining solution was

203 freshly prepared by mixing 3.7 mL of 0.2 M citric acid, 6.3 mL of 0.4 M Na2HPO4, 1

204 mL X-Gal (20 mg/mL or 49 mM in N,N-dimethylformamide), 1 mL of 100 mM

205 potassium ferricyanide, 1 mL of 100 mM potassium ferrocyanide, 0.6 mL of 5 M

206 NaCl, 0.2 mL of 0.2 M MgCl2, and 6.2 mL deionized water in a total volume of 20

207 mL, as previously described (29).

208 Determination of Cellular KGDHC Activity. Cellular KGDHC activity was

209 measured by using a colorimetric method (30) with some modifications. Briefly, the

210 monolayer of cells (105/well) was cultured in 12-well plates overnight. After a 24-h

211 incubation with different concentrations of parapyruvate and washing three times with

212 PBS, 1 mL/well of assay medium was added and incubated for 1 h. The assay medium

213 contained 50 mM Tris-HCl (pH = 7.6), 1 mM MgCl2, 0.1 mM CaCl2, 0.05 mM EDTA,

214 0.3 mM thiamine pyrophosphate, 0.5 µg/mL rotenone (in 100% ethanol; final ethanol

215 concentration, 0.1%), 0.2% Triton X-100, 3.5% polyvinyl alcohol, 3 mM

216 α-ketoglutarate, 3 mM NAD+, 0.75 mg/mL coenzyme A, 0.75 mM NBT, and 0.05 mM

217 phenazine methosulfate. After removing the medium and washing three times with

218 PBS, the resulting formazan was extracted in 0.5 mL/well 10% SDS (in 0.01 N HCl).

219 The absorbance at 570 nM was measured, and the relative cellular KGDHC activity

220 was calculated as the OD570 of sample/OD570 of control * 100%.

221 Determination of Parapyruvate Content in Calcium Pyruvate Supplements.

222 For determination, 17.6 mg of powders of dietary supplements was dissolved in 100

223 mL 0.02% sulfuric acid solution (mobile phase of HPLC). After shaking for 5 min and

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224 subsequent filtration, 20 µL of supernatant was analyzed by HPLC. The content of

225 parapyruvate or pyruvate (Cp) was quantified by using the calibration curves of

226 parapyruvate (0–286 µg/mL, y = 2603.6x-6982.5, r2 = 0.9995). The parapyruvate

227 content was calculated as (Cp (µg/mL) x 1 mL)/(100 mg x 1000) * 100%.

228 Data Analysis. The data were analyzed by ANOVA, followed by Duncan analysis

229 for group mean comparison. All analyses relied on the use of SPSS v 17.0 (SPSS, Inc.,

230 Chicago, IL, USA). P < 0.05 was considered statistically significant.

231

232 RESULTS

233 HPLC Chromatogram of the Crude Solution and Purified Crystals of

234 Parapyruvate. The crude solution of parapyruvate was obtained by the alkaline

235 treatment of pyruvic acid as described in the Methods. Three major compounds were

236 found in the chromatogram: (1) pyruvate (4.2 min), (2) parapyruvate (4.9 min), and (3)

237 an unknown compound (8.5 min) (Figure 1A). After solvent crystallization by acetone,

238 only a single peak of parapyruvate at 4.9 min was left in the analysis by HPLC

239 (Figure 1B). The HPLC chromatogram of pyruvate standard is shown in Figure 1C.

240 Some prepared crystals are shown in Figure 1D.

241 Identification of the Prepared Crystals by LC/MS/MS. We used LC/MS/MS

242 with full scan mode to analyze our prepared crystals. Again, only a single peak was

243 found in the chromatogram (Figure 2A), which suggests a high purity of parapyruvate.

244 The mass spectrum of the peak is shown in Figure 2B. A major ion (with m/z = 174.8)

245 was identified as the molecular ion of parapyruvate (molecular weight of parapyruvic

246 acid is 176.124 g/mol). The molecular ion was further fragmented in the quadruple 2.

247 Three major daughter ions were obtained, including an ion with an m/z = 87.1, which

248 is equal to the m/z of pyruvate (molecular weight of pyruvic acid is 88.062 g/mol)

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249 (Figure 2C). Thus, the prepared crystals were high-purity parapyruvate.

250 Identification of the Prepared Crystals as Mono-Potassium Parapyruvate.

251 ICP-AES analysis revealed an 18.3% potassium content in the prepared crystals. The

252 result suggested only a single potassium ion present in the molecules of the prepared

253 crystal. Thus, the prepared crystals should be mono-potassium parapyruvate, and the

254 molecular weight of our prepared parapyruvate should be 214.218 g/mol (i.e., the

255 molecular weight of mono-potassium parapyruvate). The chemical structure of the

256 synthesized potassium parapyruvate is shown in Figure 2D.

257 The Purity of the Prepared Parapyruvate Analyzed by HPLC. The results

258 showed that the purity of the prepared parapyruvate was 99.8 ± 0.1% (n=3).

259 Ability of the Prepared Parapyruvate to Inhibit KGDHC Activity.

260 Determinations using the commercialized KGDHC activity kit showed that KGDHC

261 activity was dose-dependently inhibited by the synthesized parapyruvate (Figure 3).

262 The IC50 for inhibiting KGDHC by parapyruvate was estimated at 4.13 mM.

263 Stability of Parapyruvate in DMEM. As shown in the upper panel of Table 1,

264 parapyruvate (20mM, the highest used concentration) was stable in DMEM for 1 day

265 but then broke down with the incubation time, i.e., approximately 12.8% (0.872 ±

266 0.023 mM left of a 1mM solution) and 40.7% (0.593 ± 0.016 mM left of a 1mM

267 solution) of the parapyruvate broke down by day 2 and day 7 and generated 0.251 ±

268 0.014 mM and 0.709 ± 0.011 mM of pyruvate, respectively. At day 7, the generated

269 pyruvate at 0.709 mM suggested that 0.355 mM (i.e., 35.5%) of the parapyruvate

270 solution had broken down (1 parapyruvate molecule had broken down to form 2

271 pyruvate molecules). Thus, approximately 5.2% (i.e., 40.7-35.5%) of the parapyruvate

272 should break down to form other unknown chemicals after the 7-days incubation.

273 Because we found that, in addition to pyruvate, none of any new parapyruvate-derived

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274 peaks could be seen in the chromatograph before the day 2 (data not shown), it

275 suggested that the level of the unknown impurities were very limited within the

276 2-days incubation.

277 Ability of Parapyruvate to Induce Senescence in Hs68 Cells. Parapyruvate at

278 0–20 mM did not show cytotoxicity in Hs68 cells (Figure 4A). We further examined

279 the effect of parapyruvate on 21-day cumulative growth and SA-βG activity to assess

280 the ability of parapyruvate to induce senescence in Hs68 cells by renewing medium

281 every 2 days. More senescent cells would have less division ability and lower CPDs

282 during and at the end of the cumulative growth. SA-βG is a biomarker of cell

283 senescence, which can be measured by the double-substrates method i.e., qualitatively

284 by X-Gal staining and quantitatively by relative fluorescein fluorescence. Senescent

285 cells stained with stronger blue color and produced more fluorescein fluorescence

286 than the younger cells. Parapyruvate at 1–20 mM dose-dependently decreased CPDs

287 during cumulative growth (Figure 4C). It also significantly increased SA-βG activities

288 as detected by the double-substrate method. The color of cells treated with 0.5–20

289 mM of parapyruvate was bluer in X-Gal staining (Figure 4C) and produced more

290 fluorescence than the control (P < 0.05) with a dose-dependent effect (Figure 4D).

291 Hence, parapyruvate at 0.5–20 mM could dose-dependently induce senescence in

292 Hs68 cells. By renewing medium every 2 days, the effects of the 5.2% impurities on

293 the senescence of Hs68 cells could be excluded, but some effects of pyruvate arising

294 from parapyruvate on the cells might still exist. Considering that pyruvate is a

295 component with health benefits and can extend the lifespan of Hs68 cells at mM level

296 (data not shown), thus the senescent effect of parapyruvate cannot be attributable to

297 the pyruvate arising from parapyruvate in the medium.

298 Calcium Ion reversed the Parapyruvate-Induced Senescence in Hs68 Cells. We

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299 also examined whether the senescent effect of parapyruvate on Hs68 cells could be

300 reversed by incubation with calcium ions (an activator of KGDHC (19,31)). Calcium

301 ion dose-dependently retarded senescence in Hs68 cells. CaCl2 at 1 mM greatly

302 increased the CPD levels during the 21-day cumulative growth of cells (Figure 5A)

303 and decreased the senescence marker fluorescein fluorescence (Figure 5C). In

304 addition, incubation with 1 mM CaCl2 greatly reversed the CPD levels induced by

305 parapyruvate in the 21-day cumulative growth of cells (Figure 5A) and the increase in

306 SA-βG activities induced by parapyruvate as measured by X-Gal (Figure 5B) and

307 FDG (Figure 5C). Calcium ions could retard senescence in Hs68 cells and reverse the

308 parapyruvate-induced senescence in Hs68 cells. We used 1 mM CaCl2 for testing the

309 antagonized effect, as we had previously tested 0.01, 0.1 and 1 mM of CaCl2 on

310 senescence in Hs68 cells. CaCl2 could dose-dependently retard the senescence of

311 Hs68 cells, with 1 mM CaCl2 as the most effective concentration (data not shown).

312 Effect of Calcium Ions on Parapyruvate-inhibited KGDHC Activity in Hs68

313 Cells. We determined the reversing effect of calcium ion on parapyruvate-inhibited

314 KGDHC activity in Hs68 cells. Incubation with 10 and 20mM parapyruvate

315 dose-dependently inhibited cellular KGDHC activity (Figure 5D). An amount of 1

316 mM CaCl2 could completely reverse the KGDHC activity inhibited by 10 mM

317 parapyruvate to the control level (P > 0.05). Additionally, 1 mM CaCl2 greatly

318 attenuated the KGDHC activity inhibited by 20 mM parapyruvate. Finally, 1 mM

319 CaCl2 alone markedly induced KGDHC activity. In addition, 1mM of EDTA could

320 dramatically inhibit the calcium ion-increased cellular KGDHC activity (data not

321 shown), which confirmed that the cellular KGDHC activity-increased effect was

322 caused by calcium ions. Moreover, the effect of calcium ions on

323 parapyruvate-inhibited KGDHC activity was also investigated in vitro. Calcium ions

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324 at concentrations from 0.1 to 1mM had neither the ability to activate KGDHC activity

325 nor the reversing effect on parapyruvate-inhibited KGDHC activity in vitro (Figure 6).

326 Parapyruvate Content in Five Dietary Supplements of Calcium Pyruvate. We

327 obtained five brands of capsule or tablet products of calcium pyruvate supplements on

328 the US market. The content of parapyruvate in the five brands of supplements was

329 analyzed by HPLC. Only one brand (brand 1) was supplied by tablet; the others

330 (brands 2–5) were capsules. The parapyruvate content was 1.4% to 10.6% in the

331 pyruvate supplements (Table 2). The weights of the capsules or the tablet of the five

332 dietary supplements ranged from 904 to 1925 mg per capsule or tablet (Table 2). Thus,

333 the total parapyruvate content of the capsules or tablet ranged from 13.2 to 204.0 mg

334 (1.4 × 940 mg to 10.6 × 1925 mg). A representative HPLC chromatogram of brand 1

335 is in Figure 7. In addition to the peaks of parapyruvate and pyruvate, four other peaks

336 appeared (designated Unknown 1–4), which suggests other impurities in the calcium

337 pyruvate supplements. The chromatogram profiles of brands 2–5 were similar to the

338 profile of the brand 1 and thus are not shown.

339 Stability of Parapyruvate in an Acidic Solution. To reveal whether parapyruvate

340 breaks down into pyruvate or other molecules under acidic conditions (as the human

341 stomach is an acidic environment), the stability of parapyruvate in a pH=2 solution

342 was also investigated. As shown in the lower panel of Table 1, parapyruvate (20 mM,

343 approximately the physiological concentration in the stomach after ingestion of a

344 tablet of brand 1) was stable in the acidic solution up to 60 min. These results

345 suggested that parapyruvate was stable in an acidic environment such as the stomach

346 but would mainly break down into pyruvate in DMEM.

347

348

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349 DISCUSSION

350

351 KGDHC activity is suggested to play a crucial role in aging-related neurodegenerative

352 disorders (19,20), but the role of decreased KGDHC activity in cellular senescence is

353 unknown. In this study, we found that parapyruvate at 0.5–20 mM could

354 dose-dependently induce senescence in Hs68 cells and decrease the activity of

355 KGDHC. For parapyruvate-inhibiting KGDHC activity, the IC50 was approximately

356 4.13 mM. An activator of KGDHC, Ca2+, has a strong ability to reverse the senescent

357 effect of parapyruvate, so inhibition of KGDHC activity plays an important role in the

358 senescent effects of parapyruvate in Hs68 cells. To the best of our knowledge, this

359 study is the first to propose that KGDHC plays a crucial role in cellular senescence

360 and to demonstrate that parapyruvate can induce senescence in Hs68 cells by

361 inhibiting KGDHC activity. We suggest that aging-related neurodegenerative

362 disorders may share a common mechanism with cellular senescence via KGDHC

363 inhibition. In addition, we found that Ca2+ has a strong ability to retard the cellular

364 senescence.

365 The industrial production of sodium (or potassium) pyruvate uses the neutralization

366 of freshly distilled pyruvic acid with sodium (or potassium) hydroxide and the

367 subsequent precipitation of the pyruvate salts by ethanol (15). These processes

368 involve an alkaline environment to generate parapyruvate (15). For the industrial

369 production of calcium pyruvate, the process uses pyruvic acid to react with calcium

370 carbonate (32). The process also involves alkaline treatment, which will theoretically

371 generate parapyruvate. In this study, we found that the five supplements obtained

372 from the US market all contained parapyruvate (from 1.4% to 10.6%), which suggests

373 that commercial supplements of calcium pyruvate may contain high quantities of

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374 parapyruvate.

375 According to Margolis and Coxin (15), pyruvate under an alkaline environment

376 would produce parapyruvate and other polymers. The authors also found that the

377 polymerization of pyruvate by aldol condensation is affected when the pH of the

378 environment is > 7.6. The more alkaline the solution, the more parapyruvate produced

379 (15). Using the method proposed by Margolis and Coxon (15) with some

380 modifications, we developed a method to prepare high-purity crystals of

381 mono-potassium parapyruvate. The major modifications were as follows: [1] use of

382 solvent crystallization with acetone to purify parapyruvate from the crude

383 parapyruvate solution; [2] use of pyruvic acid as the reactant instead of pure

384 compounds of sodium pyruvate, as pure compounds are more expensive than pyruvic

385 acid; and [3] addition of acid to adjust the acidic pH to stop the aldol condensation

386 reaction. As proposed by Montgomery and Webb (14), the dissociation constants of

387 parapyruvate are pK1 = 2.35 and pK2 = 6.95. The first dissociation constant refers to

388 the free carboxyl group of parapyruvate and the second to the enolic hydroxyl group

389 (14). Thus, potassium parapyruvate should contain a single potassium ion at the enolic

390 hydroxyl group of parapyruvate. Moreover, α-keto-β-methylvaleric acid (KMV) is an

391 analogue of α-ketoglutarate and can specifically inhibit the activity of KGDHC (30).

392 Huang et al. (30) found that 20 mM KMV could inhibit KGDHC activity by 80% and

393 can induce apoptosis in N2a neuroblastoma cells. In this study, 20 mM of

394 parapyruvate inhibited KGDHC activity in Hs68 cells by 80%, which suggests that

395 parapyruvate’s ability to inhibit KGDHC activity is comparable to that of KMV.

396 For the five calcium pyruvate supplements, we found that brand 1 (tablet form)

397 contained the highest content of parapyruvate. The weight of a tablet of brand 1 was

398 1925 mg. Thus, the parapyruvate content in the brand 1 supplement is estimated at

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399 204 mg. The blood volume of a human with 65 kg body weight is approximately 5 L.

400 If we assume that parapyruvate can be 100% absorbed in the human gastrointestinal

401 tract and is not metabolized by the human body, then the blood concentrations of

402 parapyruvate for a single oral administration of a tablet of brand 1 can be as high as

403 0.23 mM. However, considering the first-pass metabolism and excretion, the residual

404 plasma level and further cellular levels would be much lower than the experimental

405 level. Further studies in animals are warranted to reveal the association of

406 parapyruvate with promoting aging and neurodegenerative disorders by causing

407 functional abnormalities in mitochondria after long-term administration.

408 Theoretically, Ca2+ should activate the cellular KGDHC and reverse the

409 parapyruvate–induced senescence of Hs68 cells via involving a direct mechanism

410 from the increase of intracellular (or mitochondrial) level of Ca2+ by adding Ca2+ into

411 the medium. However, the related mechanism may be more complex than our

412 assumption. The Michaelis constant (Km) of Ca2+ for KGDHC is less than 1µM (33).

413 One issue is that Ca2+ was found to activate the isolated KGDHC at low µM

414 concentrations (≤ 20µM) (33,34). Another issue is that Ca2+ may inhibit the isolated

415 KGDHC at high levels (≥ 100 µM) (35). However, we found that high levels of Ca2+

416 were needed to activate cellular KGDHC and to reverse the parapyruvate–induced

417 senescence of Hs68 cells in this study. Although a research reported that high levels

418 of Ca2+ could inhibit KGDHC isolated from brain (35), a study of KGDHC isolated

419 from pig hearts demonstrated that high levels of Ca2+ up to around 80mM could

420 slightly increase the KGDHC activity (34). In this study, we used the commercial

421 KGDHC kit to determine the effect of Ca2+ with levels ranging from 0.1–1mM on

422 inhibition of the isolated KGDHC, suggesting that the high levels of Ca2+ neither to

423 activate the isolated KGDHC nor to inhibit the isolated KGDHC activity. Thus, our

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424 results supported that high levels of Ca2+ would not inhibit isolated KGDHC activity.

425 In contrast, we found that high levels of Ca2+ could increase the cellular KGDHC

426 activity. Since the assay buffer of our used method for the cellular KGDHC

427 determination has contained abundant (0.1mM) Ca2+ for the enzyme, the increase of

428 cellular KGDHC activity by adding high levels of Ca2+ into the medium should not

429 mainly result from a direct action of Ca2+ on the activation of KGDHC. In addition,

430 there are approximately 1.8mM of Ca2+ existing in DMEM originally, suggesting that

431 the intracellular or mitochondrial Ca2+ might be already plentiful for the KGDHC

432 during the regular culture of the cells. Therefore, the mechanisms for Ca2+ to activate

433 cellular KGDHC and isolated KGDHC may be different. We thus deduced that an

434 indirect mechanism for high levels of Ca2+ to activate cellular KGDHC might exist.

435 The precise mechanisms for Ca2+ to activate KGDHC activity and to reverse

436 parapyruvate-inhibited KGDHC activity still need further investigations.

437 Moreover, the health benefits of pyruvate are not consistent with the results from

438 human studies (10-13). For example, Stanko et al. (10) reported that supplementation

439 with 22–44 g/day pyruvate (13–25 g calcium pyruvate and 14–28 g sodium pyruvate)

440 can increase weight and fat loss in obese women. Kalman et al. (11) reported that

441 supplementation with five capsules per day (6 g/day; the authors did not indicate what

442 kind of pyruvic acid salts they used) significantly decreased body weight and fat mass

443 in overweight individuals. By contrast, Koh-Banerjee et al. (12) reported that

444 supplementation with 5 g/day calcium pyruvate did not significantly affect body

445 weight, fat mass, or exercise performance and may negatively affect some blood lipid

446 levels. A meta-analysis reviewed the effects of pyruvate on humans but did not reach

447 a clear conclusion whether pyruvate is beneficial to human health (13). After

448 reviewing the animal studies of pyruvate described above, we found that they all used

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449 chemical-grade pure pyruvic acid salts or pure ethyl pyruvate (a stable derivative of

450 pyruvate (1). However, for human studies, studies generally used food-grade calcium

451 pyruvate. In the present study, food-grade calcium pyruvate contained considerable

452 amounts of parapyruvate, as well as other impurities. Thus, impurities containing

453 parapyruvate and others could cause interference in human studies but not in animal

454 studies. Further studies should focus on the production of food-grade pyruvic acid

455 salts without parapyruvate and use these products to reveal the actual benefit of

456 pyruvate supplements on human health.

457 In conclusion, parapyruvate can inhibit KGDHC activity and induce senescence in

458 Hs68 cells. The senescent effect of parapyruvate can be reversed by an activator of

459 KGDHC, Ca2+, which demonstrates that parapyruvate induces senescence in Hs68

460 cells by inhibiting KGDHC activity. The results indicate that KGDHC plays a crucial

461 role in cellular senescence. Commercial supplements of calcium pyruvate contain

462 high quantities of parapyruvate. Because of its KGDHC inhibition activity,

463 parapyruvate content should be an important issue for the food safety of calcium

464 pyruvate supplements.

465

466 ACKNOWLEDGEMENT

467 This work was supported by the Chung Shan Medical University Hospital

468 (CSH-2015-C-022) and the Ministry of Science and Technology of Taiwan (MOST

469 105-2320-B-040 -017-MY3).

470

471 DISCLOSURE STATEMENT

472 The authors declare no conflict of interest.

473

474

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475 REFERENCES

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477 (1) Fink, M. P. Ethyl pyruvate: a novel anti-inflammatory agent. Crit. Care. Med.

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500 administration markedly reduces neuronal death and cognitive impairment in a rat

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510 P. N.; Almada, A. L.; Kreider, R. B. Effects of calcium pyruvate supplementation

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512 exercise. Nutrition 2005, 21, 312-319.

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514 weight loss: a systematic review and meta-analysis of randomized clinical trials. Crit.

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517 The inhibitory action of parapyruvate on the tricarboxylic acid cycle. J. Biol. Chem.

518 1956, 221, 359-368.

519 (15) Margolis, S. A.; Coxon, B. Identification and quantitation of the impurities in

520 sodium pyruvate. Anal. Chem. 1986, 58, 2054-2510.

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522 dehydrogenase complex activity in Alzheimer's disease. J. Neurochem. 1993, 61,

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525 alpha-ketoglutarate dehydrogenase complex alters mitochondrial function and cellular

526 calcium regulation. Biochim. Biophys. Acta. 2003, 1637, 119-126.

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528 peroxide: A key role of [alpha]-ketoglutarate dehydrogenase in limiting NADH

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531 alpha-ketoglutarate dehydrogenase complex in neurodegeneration. Neurochem. Int.

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534 alpha-ketoglutarate-dehydrogenase complex: a mediator between mitochondria and

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536 (21) Yang, N. C.; Song, T. Y.; Chen, M. Y.; Hu, M. L. Effects of 2-deoxyglucose

537 and dehydroepiandrosterone on intracellular NAD+ level, SIRT1 activity and

538 replicative lifespan of human Hs68 cells. Biogerontology 2011, 12, 527-536.

539 (22) Campisi, J. From cells to organisms: can we learn about aging from cells in

540 culture? Exp. Gerontol. 2001, 36, 607-618.

541 (23) Yang, N. C.; Song, T. Y.; Chen, M. Y.; Hu, M. L. Effects of 2-deoxyglucose

542 and dehydroepiandrosterone on intracellular NAD+ level, SIRT1 activity and

543 replicative lifespan of human Hs68 cells. Biogerontology 2011, 12, 527-536.

544 (24) Yang, N. C.; Song, T. Y.; Chang, Y. Z.; Chen, M. Y.; Hu, M. L. Up-regulation

545 of nicotinamide phosphoribosyltransferase and increase of NAD+ levels by glucose

546 restriction extend replicative lifespan of human fibroblast Hs68 cells. Biogerontology

547 2015, 16, 31-42.

548 (25) Song, T. Y.; Yeh, S. L.; Hu, M. L.; Chen, M. Y.; Yang, N. C. A Nampt

549 inhibitor FK866 mimics vitamin B3 deficiency by causing senescence of human

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550 fibroblastic Hs68 cells via attenuation of NAD(+)-SIRT1 signaling. Biogerontology

551 2015, 16, 789-800.

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553 dissolution behaviour of simvastatin crystals and its relationship to crystallization

554 solvent properties. Pharmazie 2016, 71, 263-268.

555 (27) Hallström, A.; Carlsson, A.; Hillered, L.; Ungerstedt, U. Simultaneous

556 determination of lactate, pyruvate, and ascorbate in microdialysis samples from rat

557 brain, blood, fat, and muscle using high-performance liquid chromatography. J.

558 Pharmacol. Methods. 1989, 22, 113-124.

559 (28) Strober, W. Trypan blue exclusion test of cell viability. Curr. Protoc.

560 Immunol. 2001, Appendix 3, Appendix 3B.

561 (29) Yang, N. C.; Hu, M. L. A fluorimetric method using fluorescein

562 di-beta-D-galactopyranoside for quantifying the senescence-associated

563 beta-galactosidase activity in human foreskin fibroblast Hs68 cells. Anal. Biochem.

564 2004, 325, 337-343.

565 (30) Huang, H. M.; Ou, H. C.; Xu, H.; Chen, H. L.; Fowler, C.; Gibson, G. E.

566 Inhibition of alpha-ketoglutarate dehydrogenase complex promotes cytochrome c

567 release from mitochondria, caspase-3 activation, and necrotic cell death. J. Neurosci.

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569 (31) Panov, A.; Scarpa, A. Independent modulation of the activity of

570 alpha-ketoglutarate dehydrogenase complex by Ca2+ and Mg2+. Biochemistry 1996,

571 35, 427-432.

572 (32) EFSA (European Food Safety Authority). Scientific Opinion of the Panel on

573 Food Additives and Nutrient Sources added to Food on calcium acetate, calcium

574 pyruvate, calcium succinate, magnesium pyruvate magnesium succinate and

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575 potassium malate added for nutritional purposes to food supplements following a

576 request from the European Commission. The EFSA Journal, 2009, 1088, 1-25.

577 (33) McCormack, J. G.; Denton, R. M. The effects of calcium ions and adenine

578 nucleotides on the activity of pig heart 2-oxoglutarate dehydrogenase complex.

579 Biochem J 1979, 180, 533-544.

580 (34) Panov, A.; Scarpa, A. Independent modulation of the activity of

581 alpha-ketoglutarate dehydrogenase complex by Ca2+ and Mg2+. Biochemistry 1996,

582 35, 427-432.

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584 kinetic properties, regional distribution, and effects of inhibitors. J Neurochem 1986,

585 47, 1376-1386.

586

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587 Figure Legends

588

589 Figure 1. Preparation of parapyruvate. High-performance liquid chromatography

590 (HPLC) chromatograms of (A) crude parapyruvate solution diluted 1000× by pure

591 water; (B) prepared crystals (176 µg/mL) after solvent crystallization; and (C) sodium

592 pyruvate (110 µg/mL); the chromatogram of the pyruvate standard is shown. (D)

593 Photographs of prepared crystals of parapyruvate salts.

594

595 Figure 2. Identification of the prepared crystals (17.6 µg/mL) analyzed by liquid

596 chromatography/Mass/Mass (LC/MS/MS) by full scan mode. (A) The obtained

597 chromatograph. (B) Mass spectrum of the prepared crystals after fragmentation with

598 the first mass analyzer. (C) Mass spectrum from fragmentation of the molecule ion

599 (i.e., m/z 174.8) of the prepared crystals. (D) Proposed chemical structure of the

600 prepared crystal (i.e., mono-potassium pyruvate).

601

602 Figure 3. Inhibition of α-Ketoglutarate dehydrogenase complex (KGDHC) activity by

603 prepared parapyruvate in vitro. In total, 0–100 mM prepared parapyruvate was

604 incubated with the KGDHC enzyme, and activity was measured by a commercialized

605 kit. Values (mean ± standard deviation, n = 3) not sharing a common letter are

606 significantly different (P < 0.05).

607

608 Figure 4. Effect of parapyruvate on cytotoxicity and senescence in Hs68 cells. (A)

609 For determining cytotoxicity, parapyruvate was added to Hs68 cells at 0, 0.01, 0.1, 1,

610 5, 10, and 20 mM for 24 h at 37 °C, and cell viability was expressed as % of control.

611 (B) For determining the effects of parapyruvate on senescence in Hs68 cells, cells

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612 were serially cultured in medium containing 0, 0.5, 1, 5, 10, and 20 mM parapyruvate

613 with the medium renewed every 2 days. The 21-day cumulative growth curves are

614 shown using the y-axis as cumulative population doubling levels (CPDs). On day 21,

615 at the end of serial culture, senescence-associated β-galactosidase (SA-βG) activity

616 was measured by the double-substrate method: (C) qualitatively by X-Gal staining

617 and (D) quantitatively by relative fluorescein fluorescence. Values (mean ± standard

618 deviation, n = 3) not sharing a common letter are significantly different (P < 0.05).

619

620 Figure 5. Reversing effects of calcium ions on parapyruvate-induced senescence and

621 parapyruvate-decreased cellular α-Ketoglutarate dehydrogenase complex (KGDHC)

622 activity in Hs68 cells. For determining senescence, cells were cultured serially for 21

623 days with 0, 10, and 20 mM parapyruvate and co-treated with 1 mM CaCl2 with the

624 medium renewed every 2 days. (A) The 21-day cumulative growth curves are shown

625 using the y-axis as cumulative population doubling levels (CPDs). On day 21, at the

626 end of serial culture, senescence-associated β-galactosidase (SA-βG) activity was

627 measured by the double-substrate method: (B) qualitatively by X-Gal staining and (C)

628 quantitatively by relative fluorescein fluorescence. (D) For determining cellular

629 KGDHC activity, Hs68 cells were incubated with 0, 10, and 20 mM parapyruvate and

630 co-treated with 1 mM CaCl2. Values (mean ± standard deviation, n = 3) not sharing a

631 common letter are significantly different (P < 0.05).

632

633 Figure 6. Effect of calcium ions on parapyruvate-inhibited α-ketoglutarate

634 dehydrogenase complex (KGDHC) activity in vitro. CaCl2 (0, 0.1, 0.5, and 1 mM)

635 alone or co-treated with 10mM parapyruvate and in vitro KGDHC activity was

636 detected by a commercial KGDHC activity kit. The levels of isolated KGDHC

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637 activity are presented as % of control. Values (mean ± standard deviation, n = 3) not

638 sharing a common letter are significantly different (P < 0.05).

639

640 Figure 7. Chromatogram of brand 1 calcium pyruvate supplement obtained from the

641 United States market. In total, 176 µg/mL powder of the supplement in 0.02% sulfuric

642 acid was filtered and analyzed by high-performance liquid chromatography (HPLC).

643

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644
Table 1.
Stability of parapyruvate under Dulbecco’s modified Eagle medium (DMEM) and
under an acidic condition.
Conditions# Incubation time Parapyruvate (mM) Pyruvate (mM)
DMEM
0 1.000± 0.070a ND§
a
1 hour 1.000± 0.061 ND
1 day 0.931 ± 0.044a,b 0.138 ± 0.020a
2 day 0.872 ± 0.023b 0.251 ± 0.014b
c
7 day 0.593 ± 0.016 0.709 ± 0.011c

Acidic condition
(pH=2)
0 0.990 ± 0.010a ND
20 min 0.975 ± 0.016a ND
40 min 0.961 ± 0.032a ND
a
60 min 0.934 ± 0.068 ND
#
Parapyruvate (20mM) was incubated in DMEM in a CO2 incubator with 5% CO2 at
37 oC or in the acidic solution (adjusted by HCl) at 37 oC. At different time points, the
medium or solutions were diluted for 20x with pure water, and the remaining
parapyruvate (mM) and the generated pyruvate were analyzed by HPLC as described
in the Method section. Values (mean ± SD, n=3) not sharing a common letter are
significantly different (P < 0.05).
§
ND: not detected.
645

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Table 2.
Parapyruvate content in calcium pyruvate supplements obtained from the United
States market.
Capsule or tablet Parapyruvate content
Pyruvate supplements
weight (mg)# (%)
Brand 1 calcium pyruvate 1925 ± 6.4 10.6 ± 0.2
Brand 2 calcium pyruvate 940 ± 5.7 1.4 ± 0.1
Brand 3 calcium pyruvate 937 ± 38.6 1.9 ± 1.0
Brand 4 calcium pyruvate 926 ± 18.1 2.5 ± 0.1
Brand 5 triple pyruvate 904 ± 21.2 6.2 ± 0.9
#
Weight of total powder in a capsule or total weight of a tablet measured in
triplicate (n = 3).
Data mean ± SD, n=3.

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Figure 1.

(A)

Parapyruvate

Unknown
Pyruvate

(B)

Parapyruvate

H 3C OH O
O O

OH O-

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Figure 1 (cont’d)

(C)

Pyruvate

O
O
H3C
O-

(D)

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Figure 2.

(A)

7e+6

6e+6
Parapyruvate
5e+6

4e+6
Intensity

3e+6

2e+6

1e+6

0 2 4 6 8 10 12 14
Retention time (min)

(B)

1.8e+7
174.8
1.6e+7 H 3C OH O
1.4e+7 O O

1.2e+7 OH O-

1.0e+7
Intensity

8.0e+6
6.0e+6
4.0e+6
2.0e+6
0.0

100 120 140 160 180 200 220

m/z, Dalton

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(C)

2.5e+5
O
87.1
2.0e+5 O
H3C
O-
1.5e+5
Intensity

1.0e+5
O

O O
5.0e+4
113.1
113.1
O-
69.1

0.0

40 60 80 100 120 140 160 180 200

m/z, Dalton

(D)

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Figure 3.

120
a
Relative KGDHC activity (%)

100
b

80

60
c
40
d
20
e
0

0.1 1 10 100
Parapyruvate (mM)

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Figure 4.

(A)

120
a a a
a a a
a
100
Viability (% of control)

80

60

40

20

0
Control 0.01 0.1 1 5 10 20
Parapyruvate (mM)

(B)

12
Control
Parapyruvate 0.5 mM
10 Parapyruvate 1 mM a
Parapyruvate 5 mM b
Parapyruvate 10 mM c
8
Additional CPDs

Parapyruvate 20 mM

6 d

4
e
2

0 7 14 21 28
Incubation time (days)

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Figure 4 (cont’d)
(C)

ntrol Control Parapyruvate 0.5 mM Parapyruvate 1 mM

Parapyruvate 5 mM Parapyruvate 10 mM Parapyruvate 20 mM

(D)

f
180

160 e
Fluorescein fluorescence

140 d
c
120
(% of control)

b
a
100

80

60

40

20

0
Control 0.5 1 5 10 20
Parapyruvate (mM)

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Figure 5.
(A)
Control
Parapyruvate 10 mM
14 Parapyruvate 20 mM
CaCl2 1 mM a
12 Parapyruvate 10 mM + CaCl2 1 mM
Parapyruvate 20 mM + CaCl2 1 mM
10 b
Additional CPDs

6
c
d
4
e
2

0 7 14 21 28
Incubation time (days)

(B)

Control CaCl2 1 mM

Parapyruvate 10 mM
Parapyruvate 10 mM
+ CaCl2 1 mM

Parapyruvate 20 mM Parapyruvate 20 mM
+ CaCl2 1 mM

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Figure 5 (cont’d)
(C)

200
180 c
Fluorescein fluorescence
160
b
140 e
(% of control)

120
a a
100
80
d
60
40
20
0
Parapyruvate (mM) - 10 20 - 10 20
CaCl2 (mM) - - - 1 1 1

(D)

180 d

160
Cellular KGDHC activity

140
(% of control)

120
a
100 a

80

60 b
b
40
c
20

0
Parapyruvate (mM) - 10 20 10 20 -
CaCl2 (mM) - - - 1 1 1

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Figure 6.

120
a a a
a
100
Isolated KGDHC activity

80
(% of control)

60
b b
b b
40

20

0
Parapyruvate (mM) - 10 - - - 10 10 10
CaCl2 (mM) - - 0.1 0.5 1 0.1 0.5 1

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Figure 7.

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TOC Graphic.
Pyruvic acid
Alkali treatment
Calcium pyruvate
Solvent crystallization
supplements

Parapyruvate As standard for HPLC

With high
α-KGDHC ↓ parapyruvate level
Ca2+
Senescence of Hs68 cells Food safety problem (?)

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