Targeted Methods Analytical Platform

Phenomenome Discoveries Inc. is developing a Targeted Methods analytical platform for the investigation of major primary and secondary metabolites.

Researchers can choose a combination of several preselected sets of primary and secondary metabolites in order to efficiently compare relevant biochemical levels in experimental samples. Our GC/MS and LC/MS analytical methods achieve excellent chromatographic separation of most metabolites, and by using SIM (single ion monitoring) and SRM (selected reaction monitoring) procedures combined with a retention time based database, most targeted metabolites are easily identified and quantitated with high specificity and sensitivity.

A wide variety of primary and secondary metabolic pathways have been considered for this targeted metabolite analysis platform in order to provide a wide coverage of the metabolome (see Tables 1-4).

As a result, this technology will be applicable to most metabolic research programs by allowing researchers to make informed discoveries.

Our predefined panels of metabolites (see Tables 1-4) will allow quantitation of specific metabolites of interest to each researcher, as well as give an opportunity to make unexpected discoveries about levels of metabolites that would not normally be analyzed.

Therefore, this Targeted Metabolite platform will provide both expected and novel information about system changes such as drug treatment.

Table 1. Targeted metabolites listed by analysis mode.

LC/MS - NESI AQUEOUS

Glycolysis/Gluconeogenesis/TCA/P PP:

pyruvic acid lactic acid oxalacetic acid oxalic acid methyl malonic acid malonic acid fumaric acid succinic acid citric acid DL-isocitric acid maleic acid D-malic acid a-D-glucose-1-phosphate D-ribose-5-phosphate DL-Glyceraldehyde 3-phosphate D(-)3-Phosphoglyceric acid DL-isocitric acid D-gluconic acid 6-phosphogluconic acid D-fructose 1,6-diphosphate Dihydroxyacetone phosphate Glycerophosphate Phosphoenolpyruvic acid 2,3-diphospho-D-glyceric acid 2-Oxoglutaric acid

Plant Hormones:

Indole-3-Acetic Acid 2-cis,4-trans-abscisic acid Zeatin (trans isomer)

Vitamins - Water Soluble: (PABA) p-aminobenzoic acid d-biotin folic acid d-pantothenic acid pyridoxal pyridoxamine pyridoxine riboflavin

L-ascorbic acid (Vitamin C)

Carbohydrates:

D-(+)-Cellopentaose

D-(+)-Cellotetraose

LC/MS - NESI AQUEOUS

D-(+)melezitose

D-(+)raffinose

D-Arabinose

D-Erythrose 4-phosphate

D-Fructose D-Galactose

D-Glucose

D-glucose 6-phosphate D-Glyceraldehyde D-Lyxose D-mannitol

D-Myo-Inositol 4-monophosphate

D-Ribose

D-sorbitol

D-Tagatose

D-Trehalose dulcitol (galactitol)

D-Xylose glycerol iso-erythritol isomaltotriose

Lactose

L-gulonic gamma-lactone

L-Rhamnose maltitol maltoheptaose maltohexaose maltopentaose maltotetraose maltotriose

Melibiose

Myo-Inositol

N-Acetyl-D-glucosamine

N-Acetyl-D-Glucosamine 6-

phosphate

N-acetyl-D-mannosamine

N-Acetylneuraminic acid ribitol (adonitol)

Sedoheptulose stachyose

Sucrose

Sucrose-6'-monophosphate Xylitol

LC/MS - NESI AQUEOUS

Nucleic Acids:

(-)-Adenosine 5'-monophosphate

(cAMP) Adenosine 3',5'-cyclic monophosphate

(CDP) Cytidine 5'-Diphosphate

(CMP) Cytidine 5'-Monophosphate

cyclophosphate

(FAD) Flavin adenine dinucleotide

(PRPP) 5-Phosphorylribose

1-Pyrophosphate

(UDP) Uridine 5'-diphosphate

(uDP-glucose) Uridine 5'-

diphosphoglucose

(UMP) Uridine 5'-monophosphate

2'-Deoxyadenosine

2'-Deoxycytidine

2'-deoxyuridine

3'-Deoxyguanosine

Acetoacetyl coenzyme A

Acetyl coenzyme A (C2:0)

Adenosine

Adenosine 5' diphosphate

Adenosine 5' triphosphate

Adenosine 5'-diphosphoglucose

Adenosine 5'-Diphosphoribose

Coenzyme A

Cytidine dATP

dCTP

dGTP

DL-3-hydroxy-3-methlyglutaryl coenzyme A

dTTP

dUTP

Guanidineacetic Acid Guanosine

Guanosine 5' Diphosphate Inosine 5' triphosphate Malonyl coenzyme A NAD NADP

NADPH Orotic acid Thymidine Uridine UTP

Xanthosine

Table 2. Targeted metabolites listed by analysis mode.

LC/MS - NESI AQUEOUS

LC/MS - PESI AQUEOUS LC/MS - PESI AQUEOUS

Shikimate Pathway: Chorismic acid Prephenic acid (-)shikimic acid

Other Acids:

2.3-dimethylmaleic acid

2-hydroxybutyrate

3.4-Dihydroxycinnamic acid

3-Hydroxybenzoic acid

4-Hydroxyphenylacetic acid 4-Hydroxyphthalic acid 4-Methoxybenzoic acid Acetylenedicarboxylic Acid benzoic acid

Cinnamic Acid cis-3-Chloroacrylic acid cis-Aconitic acid D-Gluconic acid D-Glucuronic acid Dimethyl Fumarate D-tartaric acid Glutaric acid Glycolic acid Glyoxylic acid Hippuric acid Itaconic acid Mesaconic acid Methymalonic Acid P-Coumaric Acid Phenylpyruvic acid Phosphonoacetic acid phytic acid quinic acid

R-3-hydroxybutyric acid Terephthalic acid

Amino Acids and Derivatives:

L-Alanine

L-Arginine

L-Asparagine

L-Aspartic acid

L-Cysteine

L-Cystine L-Glutamic acid L-Glutamine Glycine L-Histidine trans-4-hydroxy-L-Proline

L-Isoleucine

L-Leucine

L-Lysine

L-Methionine

L-Phenylalanine

L-Proline

L-Serine

L-Threonine

L-Tryptophan

L-Tyrosine

L-Valine

L-Homoserine

L-Citrulline

DL-Homocysteine

Cadaverine

1,4-Diaminobutane (Putrescine)

L-Ornithine

Tyramine

3-Hydroxytyramine Taurine

N,N-Dimethylglycine

5-hydroxy-L-tryptophan

Seleno-L-cystine

Seleno-L-Methionine

N-formyl-DL-phenylalanine

O-acetyl-L-serine

L-carnitine

Acetyl -L-carnitine

O-Succinyl-L-homoserine

Creatine

Creatinine

D-thyroxine

(GABA) 4-Aminobutyric acid

5-Aminolevulinic acid N-Acetyl-L-glutamic acid S-(5'-Adenosyl)-L-Homocysteine

(SAM) S-adenosyl-L-methionine

Glutathione-oxidized

L-Glutathione, reduced

L-Kynurenine

L-Homocarnosine

L-(+)-Cystathionine

-2-Propanol

L-carnosine

N-Acetylisatin

L-DOPA

Beta-Alanine

Cys-Gly

Sulphates: thiogalactoside

Nitrogenous bases:

Adenine

Cytosine

Guanine

Hypoxanthine

Uracil

Xanthine

Amines and Amides: Pyridoxal 5-phosphate (PLP) 6-Aminocaprioc Acid Choline Acetylcholine

Betaine (aka glycine betaine)

Sanguinarine

Berberine

Palmatine

Tetrahydropalmatine

R-(-)-epinephrine

L-Noradrenaline

3-Methoxy-4-

hydroxyphenylethylamine

Nicotinic acid (aka niacin)

nicotinamide (niacinamide)

thiamine

Phenethylamine

Tryptamine

Serotonin

(ACC) 1-aminocyclopropane-1-carboxylic acid

Table 3. Targeted metabolites listed by analysis mode.

LC/MS - PESI AQUEOUS

LC/MS - NESI ORGANIC

LC/MS - PAPCI ORGANIC

Lipids, Fatty acid derivatives,

Flavonoids and Phenolics:

Vitamins:

MAG's DAG's TAG's: continued

(Psychosine)

fisetin

Galactosylsphingosine

triacetin

quercetin

Chenodeoxycholic acid

tributyrin

(Xanthurenic acid) 4,8-

(+)-catechin

Dihydroxyquinoline-2-carboxylate

tricaproin

Calciferol (ergocalciferol, or

Luteolin

Vitamin D2)

tricaprylin

Apigenin

Glycocholic acid

tricaprin

4',5,7-trihydroxyflavanone

DL- 6,8-thioctic acid (lipoic acid)

tripalmitolein

Pelargonidin

Vitamin E

tripalmitolein

(Acacetin) 5,7-Dihydroxy-4'-

methoxyflavone

Vitamin D3 (cholecalciferol)

tripetrosilinin

(Chalcone) 1,3-Diphenyl-1-propen-

1-alpha, 25-

3-one

Dihydroxycholecalciferol (calcitriol)

triolein

Kaempferol

trielaidin

Taxifolin

Terpenes/Terpenoids:

trilinolein

Genistein

Secologanin

trilinolenin

triarachidin

Organic Acids:

tri-11-eicosenoin

LC/MS - PESI ORGANIC

6-Hydroxynicotinic acid

tribehenin

Miscellaneous:

4-Chlorophenylacetic acid

trierucin

5,6-dimethylbenzimidazole

Anthranilic acid (2-Aminobenzoate)

L-a-phosphatidylcholine

2-Aminophenol

Sphingomyelin from bovine brain

Barbituric acid

Aldehydes:

D-Sphingosine

cis-11-Hexadecanal

Ketones:

D-Erythro-Sphingosine, dihydro-

Phenylacetaldehyde

Acetophenone

Trans-Cinnamaldehyde

2-hydroxyacetophenone

Alkaloids:

p-Quinone (or p-Benzoquinone)

S(+)-camptothecin

Miscellaneous:

(S)-(-)-Nicotine

Hydroquinone

Carotenoids:

(+-)-Anabasine

Arbutin

Beta Carotene

Spermine

D-Salicin

Lycopene

Spermidine

5-(2-Hydroxyethyl)-4-methylthiazole

(-)-Scopolamine

Catechol

L-Hyoscyamine

GC/MS - EI ORGANIC

Tropine

Fatty Acids:

Tropinone

LC/MS - PAPCI ORGANIC

Arachidonic acid (20:4)

(6R)-5,6,7,8-Tetrahydrobiopterin

MAG's DAG's TAG's:

Arachidonic acid (20:4)

D-(+)-Neopterin

1-monolauroyl-rac-glycerol

Cholesteryl arachidonate

Diethanolamine

dilaurin

Cholesteryl arachidonate

Triethanolamine

trilaurin

Cholesteryl behenate

R-(-)-alpha-Methylhistamine

1-monomyristoyl-rac-glycerol

Cholesteryl elaidate

Papaverine

dimyristin

Cholesteryl laurate

Cinchonine

trimyristin

Cholesteryl linoleate

Aconitine

1-monopalmitoyl-rac-glycerol

Cholesteryl linolenate

dipalmitin

Cholesteryl myristate

Porphyrins and Heme Synthesis:

tripalmitin

Cholesteryl N-heptanoate

Chlorophyll A

1-monostearoyl-rac-glycerol

Cholesteryl oleate

Chlorophyll B

distearin

Cholesteryl palmitate

P roto po r p hy ri n -' IX'

tristearin

Cholesteryl stearate

Bilirubin

triacetin

cis-11, 14, 17-eicosatrienoic acid

cis-5,8,11,14,17-Eicosapenta-enoic

acid

Table 4. Targeted metabolites listed by analysis mode.

GC/MS - EI ORGANIC

GC/MS - EI ORGANIC

GC/MS - EI ORGANIC

Fatty Acids: continued

furfuryl hexanoate

Alpha-D-Glucose Pentaacetate

Cis-7,10,13,16,19-

Docosapentaenoic acid methyl

ester

furfuryl heptanoate

Beta-D-Galactose pentaacetate

Cis-8,11,14-Eicosatrienoic acid

furfuryl octanoate

Sinapyl alcohol

Decanoic acid

methyl butryate

Coniferyl alcohol

Docosahexaenoic acid (22:6)

propyl butryate

Scopoletin

Docosanoic acid

isopropyl butyrate

Methylglyoxal

Eicosanoic acid

hexyl butryate

R-(-)-1,2-propanediol

Elaidic acid (18:1 trans)

allyl butyrate

Vanillic acid

Erucic acid (22:1)

benzyl butyrate

Homovanillic acid

Gamma linolenic acid

methyl tiglate

Protocatechuic Acid

Heneicosanoic acid

ethyl butyrate

Homoprotocatechuate

Heptadecanoic acid

propyl tiglate

4-Hydroxybenzoic acid

Heptanoic acid

isopropyl tiglate

Coumarin

Hexanoic acid

hexyl tiglate

Methyl 4-oxobutanoate

Lauric acid

allyl tiglate

Indole (aka 2,3-Benzopyrrole)

Linoleic acid (18:2)

benzyl tiglate

Linoleic acid (18:2)

ethyl pentanoate (valerate)

Steroids, Lipids, Terpenoids:

Linolenic acid (18:3)

ethyl hexanoate

11a-Hydroxyprogesterone

Linolenic acid (18:3)

ethyl heptanoate

3beta-Hydroxyandrost-5-en-17-one

Myristic acid

ethyl octanoate

5a-Pregnane-3-20-dione-allo

Myristoleic acid

furfuryl propionate

5a-Pregnane-3a,20a-diol

Nervonic acid

furfuryl butyrate

Adrenosterone

Nervonic acid (24:1)

furfuryl pentanoate

Aldosterone

Nonadecanoic acid

2-Acetylpyrrole

Beta-estradiol

Nonanoic acid

2,4,5-Trimethylthiazole

Cholest-4-en-3-one

Octanoic acid

2-Isobutylthiazole

cholesterol

Oleic acid (18:1)

2-Ethoxythiazole

Cortisone

Palmatelaidic acid methyl ester

2-Methylpyrazine

Deoxycorticosterone

Palmatelaidic acid methyl ester

2,3-Dimethylpyrazine

Ergosterol

Palmitic acid (16:0)

2,5-Dimethylpyrazine

Estriol

Palmitoleic acid (16:1)

2,6-Dimethylpyrazine

Estrone

Pentadecanoic acid

2,3,5-Trimethylpyrazine

Hydrocortisone

Petroselinic acid (18:1)

2,3,5,6-Tetramethylpyrazine

Lanosterol

Stearic acid

2-Ethylpyrazine

Prednisolone

Tetracosanoic acid

2-Acetylfuran

Pregnenolone

Tricosanoic acid

2,3-Diethylpyrazine

Progesterone

Tridecanoic acid

3-Ethyl-2-methylpyrazine

Prostaglandin B2

Undecanoic acid

2-Methoxypyrazine

Prostaglandin D2

2-Methoxy-3-methylpyrazine

Prostaglandin F2A

Aldehydes:

2-Isobutyl-3-methoxypyrazine

stigmasterol

9-cis-retinal

2-Acetylthiophene

Leukotriene B4

2-Acetylthiazole

Leukotriene C4

Phenolics:

2-Acetyl pyridine

Thromboxane B2

2-methyl-1,4-naphthoquinone

2-Acetylpyrazine

1-decene

(PQQ) Pyrroloquinoline quinone

Thiazole

Aphidicolin

4-Methylthiazole

Myrcene

Esters and Volatiles:

4,5-Dimethylthiazole

squalene

ethyl propionate

Vitamin K1

SUPPRESSIVE EFFECT OF FUCOIDAN DERIVED FROM MOZUKU ON IN VITRO INVASION OF HUMAN FIBROSARCOMA HT1080 CELLS

Jun Ye1, Kiichiro Teruya1, Yoshinori Katakura1, Hiroshi Eto2 and Sanetaka Shirahata1

1 Department of Genetic Resources Technology, Faculty of Agriculture, Kyushu University,

6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan; Daiichi Sangyo Co. Ltd., Osaka 530-0037, Japan

Abstract: Fucoidan is a uniquely-structured sulfated polysaccharides that is found in the cell walls of several types of brown seaweed, but not of land plants. Recently, Fucoidan has clinically got a lot attention due to its anti-tumor metastasis potential. We report here that the enzyme-digested fucoidan extract derived from Cladosiphon novae-caledoniae kylin, which was called "mozuku" in the Kingdom of Tonga, inhibited in vitro invasion of human fibrosarcoma HT1080 cells. Flow cytometric analysis revealed that the enzyme-digested fucoidan extract effectively scavenged intracellular H2O2 of HT1080 cells. Both activities of MMP2 and 9 were decreased in a dose dependent manner by the fucoidan extract, suggesting that Fucoidan may inhibit the invasion of tumor cells via suppression of MMP2 and 9 activities.

Key words: fucoidan, antioxidation, MMPs, invasion, antitumor activity

1. INTRODUCTION

Metastasis is a major problem of cancer therapy. Matrix metallo-proteinase Fucoidan derived from Cladosiphon novae-caledoniae kylin that is called mozuku in the Kindom of Tonga, is a traditional food for local inhabitants. It is noteworthy that the cancer death rate in Okinawa is the lowest of all the prefectures in Japan, this phenomenon indicated a promising prospect of fucoidan as a dietary substance for preventing human from cancer attack. Untill now, fucoidan has been proved helpful in alleviating murine chronic colitis(l), acetic acid-

induced gastric ulcer in rats(2), and gastric mucosal protection(3). However,the effect of the fucoidan on tumor invasion have never been reported. In the study, we demonstrated that fucoidan derived from Cladosiphon okamuranus Tokida possesses antioxidative potential, suppressing in vitro invasion of HT1080 cells.

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