Parapyruvate, an Impurity in Pyruvate Supplements, Induces

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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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Journal of Agricultural and Food Chemistry

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Parapyruvate, an Impurity in Pyruvate Supplements, Induces Senescence in

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Human Fibroblastic Hs68 Cells via Inhibition of the α-Ketoglutarate

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Dehydrogenase Complex

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

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Yang2,6,*

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1

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University Hospital, Taichung, Taiwan

Department of Physical Medicine and Rehabilitation, Chung Shan Medical

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2

Department of Nutrition, Chung Shan Medical University, Taichung, Taiwan

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3

Sleep Medicine Center, Chung Shan Medical University Hospital, Taichung, Taiwan

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4

Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan

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5

Department of Occupational Safety and Health, Chung Shan Medical University,

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Taichung, Taiwan

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6

16

Taiwan

Department of Nutrition, Chung Shan Medical University Hospital, Taichung,

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*

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

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University

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

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E-mail: [email protected]

Corresponding author:

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Running title: Senescent effect of parapyruvate on Hs68 cells

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1 Environment ACS Paragon Plus


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ABSTRACT

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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

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

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

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

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

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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.

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KEYWORDS: Parapyruvate, cellular senescence, α-ketoglutarate dehydrogenase

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complex, calcium pyruvate, Hs68 cells

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INTRODUCTION

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

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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

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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,



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

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analyzed the contents of parapyruvate in commercial dietary supplements of calcium

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

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

94

cell senescence (25).

95 96 97 98

MATERIALS AND METHODS Chemicals. All chemicals used were of analytical grade. NaCl, KCl, KOH, HCl, H2SO4, MgCl2⋅6H2O, Na2HPO4, KH2PO4, NaHCO3, and dimethyl sulfoxide were


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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

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(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



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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Accreditation Foundation-certified laboratory in the Health Technology Center at

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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

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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

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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



174 175

°C in a humidified incubator with 5% CO2. 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

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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

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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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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

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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

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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%.

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Determination of Parapyruvate Content in Calcium Pyruvate Supplements.

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

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



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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

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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.,

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

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RESULTS

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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

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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,

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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.

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

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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)

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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.

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

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Identification of the Prepared Crystals as Mono-Potassium Parapyruvate.

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

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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

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

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

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synthesized potassium parapyruvate is shown in Figure 2D.

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

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

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Ability of the Prepared Parapyruvate to Inhibit KGDHC Activity.

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Determinations using the commercialized KGDHC activity kit showed that KGDHC

261

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

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

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

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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 ±

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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



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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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



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


Page 14 of 41

Page 15 of 41


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



374 375

parapyruvate. 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


Page 16 of 41

Page 17 of 41


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



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


Page 18 of 41

Page 19 of 41


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 19 Environment ACS Paragon Plus


475

REFERENCES

476 477 478 479

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480

of energy expenditure by the addition of pyruvate and dihydroxyacetone to a rat diet.

481

Metabolism 1986, 35, 182-186.

482

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Ivy, J. L.; Cortez, M. Y.; Chandler, R. M.; Byrne, H. K.; Miller, R. H. Effects

483

of pyruvate on the metabolism and insulin resistance of obese Zucker rats. Am. J. Clin.

484

Nutr. 1994, 59, 331-337.

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Wang, L. Z.; Sun, W. C.; Zhu, X. Z. Ethyl pyruvate protects PC12 cells from

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Das, U. N. Is pyruvate an endogenous anti-inflammatory molecule? Nutrition

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Varma, S. D.; Hegde, K. R.; Kovtun, S. Oxidative damage to lens in culture:

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Huh, S. H.; Chung, Y. C.; Piao, Y.; Jin, M. Y.; Son, H. J.; Yoon, N. S.; Hong,

492

J. Y.; Pak, Y. K.; Kim, Y. S.; Hong, J. K.; Hwang, O.; Jin, B. K. Ethyl pyruvate rescues

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nigrostriatal dopaminergic neurons by regulating glial activation in a mouse model of

494

Parkinson's disease. J. Immunol. 2011, 187, 960-969.

495

(8)

Satpute, R.; Lomash, V.; Kaushal, M.; Bhattacharya, R. Neuroprotective

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effects of α-ketoglutarate and ethyl pyruvate against motor dysfunction and oxidative

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changes caused by repeated 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine exposure in

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mice. Hum. Exp. Toxicol. 2013, 32, 747-758.

499

(9)

Wang, X.; Hu, X.; Yang, Y.; Takata, T.; Sakurai, T. Systemic pyruvate


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administration markedly reduces neuronal death and cognitive impairment in a rat

501

model of Alzheimer's disease. Exp. Neurol. 2015, 271, 145-154.

502

(10) Stanko, R. T.; Reynolds, H. R.; Hoyson, R.; Janosky, J. E.; Wolf, R. Pyruvate

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supplementation of a low-cholesterol, low-fat diet: effects on plasma lipid

504

concentrations and body composition in hyperlipidemic patients. Am. J. Clin. Nutr.

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1994, 59, 423-427.

506

(11) Kalman, D.; Colker, C. M.; Wilets, I.; Roufs, J. B.; Antonio, J. The effects of

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pyruvate supplementation on body composition in overweight individuals. Nutrition

508

1999, 15, 337-340.

509

(12) Koh-Banerjee, P. K.; Ferreira, M. P.; Greenwood, M.; Bowden, R. G.; Cowan,

510

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

511

during training on body composition, exercise capacity, and metabolic responses to

512

exercise. Nutrition 2005, 21, 312-319.

513

(13) Onakpoya, I.; Hunt, K.; Wider, B.; Ernst, E. Pyruvate supplementation for

514

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

515

Rev. Food Sci. Nutr. 2014, 54, 17-23.

516

(14) Montgomery, C. M.; Webb, J. L. Metabolic studies on heart mitochondria. II.

517

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

518

1956, 221, 359-368.

519 520

(15) Margolis, S. A.; Coxon, B. Identification and quantitation of the impurities in sodium pyruvate. Anal. Chem. 1986, 58, 2054-2510.

521

(16) Mastrogiacomo, F.; Bergeron, C.; Kish, S. J. Brain alpha-ketoglutarate

522

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

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2007-2014.

524

(17) Huang, H. M.; Zhang, H.; Xu, H.; Gibson, G. E. Inhibition of the



525

alpha-ketoglutarate dehydrogenase complex alters mitochondrial function and cellular

526

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

527

(18) Tretter, L.; Adam-Vizi, V. Inhibition of Krebs cycle enzymes by hydrogen

528

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

529

production under oxidative stress. J. Neurosci. 2000, 20, 8972-8979.

530

(19) Gibson, G. E.; Park, L. C.; Sheu, K. F.; Blass, J. P.; Calingasan, N. Y. The

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

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2000, 36, 97-112.

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(20) Gibson, G. E.; Blass, J. P.; Beal, M. F.; Bunik, V. The

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

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oxidative stress in neurodegeneration. Mol. Neurobiol. 2005, 31, 43-63.

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.

539 540 541

(22) Campisi, J. From cells to organisms: can we learn about aging from cells in culture? Exp. Gerontol. 2001, 36, 607-618. (23) Yang, N. C.; Song, T. Y.; Chen, M. Y.; Hu, M. L. Effects of 2-deoxyglucose

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and dehydroepiandrosterone on intracellular NAD+ level, SIRT1 activity and

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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

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

551

2015, 16, 789-800.

552

(26) Bukovec, P.; Benkic, P.; Smrkolj, M.; Vrecer, F. Effect of crystal habit on the

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.

559 560 561

(28) Strober, W. Trypan blue exclusion test of cell viability. Curr. Protoc. Immunol. 2001, Appendix 3, Appendix 3B. (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.

568

Res. 2003, 74, 309-317.

569

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

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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



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.

583

(35) Lai, J. C.; Cooper, A. J. Brain alpha-ketoglutarate dehydrogenase complex:

584

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

585

47, 1376-1386.

586


Page 24 of 41

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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



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


Page 26 of 41

Page 27 of 41


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



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


Page 28 of 41

Page 29 of 41


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 1.9 ± 1.0 937 ± 38.6 Brand 4 calcium pyruvate 2.5 ± 0.1 926 ± 18.1 Brand 5 triple pyruvate 6.2 ± 0.9 904 ± 21.2 # Weight of total powder in a capsule or total weight of a tablet measured in triplicate (n = 3). Data mean ± SD, n=3.



Figure 1. (A)

Parapyruvate

Unknown Pyruvate

(B) Parapyruvate

H 3C

OH O

O

O

OH

O-


Page 30 of 41

Page 31 of 41


Figure 1 (cont’d) (C) Pyruvate O O H3C

O-

(D)



Page 32 of 41

Figure 2. (A) 7e+6 6e+6 Parapyruvate

Intensity

5e+6 4e+6 3e+6 2e+6 1e+6 0

0

2

4

6

8

10

12

14

Retention time (min)

(B)

1.8e+7

174.8

1.6e+7

H 3C O

1.4e+7 1.2e+7 Intensity

OH O O

O-

OH

1.0e+7 8.0e+6 6.0e+6 4.0e+6 2.0e+6 0.0 100

120

140

160

180

m/z, Dalton


200

220

Page 33 of 41


(C) 2.5e+5 O

87.1

O

2.0e+5

H3C

O-

Intensity

1.5e+5 1.0e+5 O O

O

5.0e+4

113.1 113.1

O-

69.1

0.0

40

60

80

100

120

140

m/z, Dalton

(D)


160

180

200


Figure 3.

Relative KGDHC activity (%)

120 a 100

b

80 60 c 40 d 20 e 0

0.1

1

10

100

Parapyruvate (mM)


Page 34 of 41

Page 35 of 41


Figure 4. (A)

120

Viability (% of control)

a

a

a

a

a

a

a

100 80 60 40 20 0 Control 0.01

0.1

1

5

10

20

Parapyruvate (mM)

(B)

12

Additional CPDs

10 8

Control Parapyruvate 0.5 mM Parapyruvate 1 mM Parapyruvate 5 mM Parapyruvate 10 mM Parapyruvate 20 mM

a b c

6

d

4 e

2 0

0

7

14

21

Incubation time (days)


28

Page 36 of 41

Figure 4 (cont’d) (C)

Control

Parapyruvate 0.5 mM

Parapyruvate 1 mM

Parapyruvate 5 mM

Parapyruvate 10 mM

Parapyruvate 20 mM

(D)

f

180 160

Fluorescein fluorescence (% of control)

ntrol


e

140

d c

120

b a

100 80 60 40 20 0 Control

0.5

1

5

Parapyruvate (mM)


10

20

Page 37 of 41


Figure 5.

Additional CPDs

(A) 14

Control Parapyruvate 10 mM Parapyruvate 20 mM CaCl2 1 mM

12

Parapyruvate 10 mM + CaCl2 1 mM

10

a


b

8 6

c d

4

e

2 0

0

7

14

21

28

Incubation time (days)

(B)

Control

CaCl2 1 mM

Parapyruvate 10 mM


Parapyruvate 20 mM




Page 38 of 41

Figure 5 (cont’d) (C) 200 c

Fluorescein fluorescence (% of control)

180 160

b

e

140 120

a

a

100 80 d

60 40 20

0 Parapyruvate (mM) CaCl2 (mM)

-

10 -

20 -

1

10 1

20 1

(D)

d

180

Cellular KGDHC activity (% of control)

160 140 120 a a

100 80 60

b

40

b c

20 0 Parapyruvate (mM) CaCl2 (mM) -

10 -

20 -

10 1

20 1


1

Page 39 of 41


Figure 6.

120 a

Isolated KGDHC activity (% of control)

a

a

a

100 80 60 40

b

b

b

b

20

0 Parapyruvate (mM) CaCl2 (mM) -

10

-

-

-

10

10

10

-

0.1

0.5

1

0.1

0.5

1



Figure 7.


Page 40 of 41

Page 41 of 41


TOC Graphic. Pyruvic acid Alkali treatment

Solvent crystallization

Parapyruvate

Calcium pyruvate supplements

As standard for HPLC

α-KGDHC ↓

With high parapyruvate level

Ca2+ Senescence of Hs68 cells

Food safety problem (?)


Parapyruvate, an Impurity in Pyruvate Supplements, Induces

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