the hamsters fed with high cholesterol diet

(1)

Department of Life Science, National Taitung University Master Thesis

Advisor: Chun-Lin Lee, Ph.D.

Investigating the effect of different mineral composition of deep ocean water on the regulation of blood lipid in

the hamsters fed with high cholesterol diet

Master’s student: Kao-ko Lin

107 1

January, 2018

(2)

(3)

(4)

(5)

( Deep ocean water, DOW )

DOW

DOW Mg²⁺

(D) (D-

L-NaK) (D-L-Ca)

DOW DOW

total cholesterol (TC) low density lipoprotein-cholesterol (LDL-C)

aspartate transaminase (AST) D-L-NaK DOW

TC triglyceride (TG)

(p < 0.05) DOW

superoxide dismutase catalase D-L-

NaK DOW TC

DOW Mg²⁺ Ca²⁺

DOW

(6)

Abstract

A cholesterol-rich diet can induce hyperlipidemia, which increases free fatty acids in the serum, causing the liver to synthesize and store excessive triglycerides. Excess fat accumulation in the liver can lead to steatosis and nonalcoholic fatty liver disease.

Pure and low-temperature deep ocean water (DOW) is known to be rich in minerals and nutrients, which according to numerous studies have multiple health benefits.

These include lowering blood lipid level and blood pressure, suppressing metastatic breast cancer, and increasing resistance to fatigue, as well as improving obesity, diabetes, cardiovascular diseases, and skin conditions. Furthermore, the literature indicates that the magnesium ions in DOW can effectively alleviate cardiovascular diseases, but how DOW of varying mineral compositions differs in the control of blood lipid levels remains unclear. The present study compared how the DOW, DOW with low sodium and potassium, DOW with low calcium, differed in their effects on blood lipid level. An in vivo test was conducted in which hamsters were fed with a cholesterol-rich diet, and after they developed hyperlipidemia, they were fed with DOW, DOW with low sodium and potassium, DOW with low calcium for 8 consecutive weeks to observe how their blood lipid levels varied. The results indicated that DOW can significantly lower high total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels induced by a cholesterol-rich diet; furthermore, DOW can also lower aspartate transaminase (AST) levels, which is an indicator of liver inflammation. Overall, magnesium, calcium DOW was determined to be the most effective at lowering TC and triglyceride levels, and it was also significantly effective (p > .05) at reducing hepatic lipid peroxidation and removing artery plaque. Regarding antioxidant enzyme activity, the three types of DOW were discovered to be effective at raising the activity of superoxide dismutase and catalase in hamster livers. In summary, magnesium, calcium DOW was the most effective at regulating blood lipid level because it surpassed the other two in lowering the TC level, artery plaque removal, and raising antioxidant enzyme activity. This suggests that the interaction between Mg²⁺

and Ca²⁺ may be key to the effectiveness of DOW in regulating blood lipids and antioxidizing the liver.

(7)

ACC(acetyl-CoA carboxylase) A ALT(alanine transaminase)

AMPK(5′-adenosine monophosphate-activated protein kinase) 5′

AST(aspartate transaminase) CAT(catalase)

DOW(deep ocean water) GPx(glutathione peroxidase) GRd(Glutathione reductase) GSH(glutathione)

HDL-c(high density lipoprotein cholesterol) LDL-c(low density lipoprotein cholesterol) MDA(malondialdehyde)

ROS(reactive oxygen species) SOD(superoxide dismutase) TC(total cholesterol)

TG(triglyceride)

VLDL-c(very low density lipoprotein cholesterol)

(8)

... I ABSTRACT ... II ... III ... IV ... VI ... VII

... 1

... 2

... 3

... 5

... 6

... 9

DOW ... 11

DOW Mg Ca Mg Ca ... 11

Mg Ca ... 11

DOW ... 12

... 13

(9)

... 15

... 16

... 18

(sacrifice) ... 21

... 21

TC TG ... 21

(thiobarbituric acid reactive substances, TBARS) ... 23

... 23

... 24

... 25

... 26

... 30

DOW Na Ca K Mg ... 32

... 35

H&E ... 39

... 39

... 42

... 47

... 48

... 49

... 51

(10)

3- 1 ... 17

3- 2 (g/kg diet) ... 18

3- 3 Syrian ... 20

4- 1 ... 27

4- 2 / ... 28

4- 3 ... 29

4- 4 / ... 32

4- 5 AST ALT ... 33

4- 6 ... 34

4- 7 38 6- 1 ... 50

(11)

1- 1 ... 8 2- 1 ... 14

4- 1 ... 36

4- 2 . 37

4- 3 (400 x) ... 40

4- 4 ... 41

4- 5 superoxide dismutase

... 43

4- 6 catalase ... 44

4- 7 glutathione peroxidase

... 45

4- 8 Glutathione reductase

... 46

(12)

(deep ocean water DOW) (thermocline)

DOW DOW

(Bak et al., 2012; Fan et al., 2016; Hwang et al., 2009; Sheu et al., 2013; Yang et al., 2014)

(Musso et al., 2003)

(Tessari et al., 2009) 106

( 2016)

Syrian

DOW

probucol total

cholesterol (TC) triglyceride (TG) high-density lipoprotein cholesterol (HDLC) low-density lipoprotein cholesterol (LDL-C)

TC TG DOW

DOW

glutathione reductase (GRd) glutathione peroxidase (GPx) catalase (CAT)

superoxidase dismutase (SOD) DOW

DOW

(13)

(deep ocean water, DOW) (thermocline)

3700 93%

1.3*109 ( )

DOW (Hwang et al., 2009)

( ) DOW

DOW DOW

DOW (Othmer

& Roels, 1973) 200 m DOW

200 m DOW

DOW DOW

( )

200 m

DOW 200 m

(Othmer & Roels, 1973)

( )

(14)

(Nigari) (reverse osmosis,

RO) ( )

(electro dialysis, ED)

SPA

( ) Na⁺ Mg²⁺ Ca²⁺ K⁺

Mg²⁺ Ca²⁺

(Luoma et al., 1983) Mg²⁺ Ca²⁺

Mg²⁺

(Larsson et al., 2012) (Kishimoto et al., 2010)

90

DOW DOW

( )

2013 Chen

300 900 1500 DOW `

(triglyceride, TG) (total

cholesterol, TC) (Chen et al., 2013)

DOW DOW 5′-adenosine

monophosphate-activated protein kinase (AMPK) acetyl-CoA carboxylase (ACC)

(Sheu et al., 2013) DOW

(low-density lipoprotein-cholesterol, LDL-C) DOW

(15)

DOW ob/ob

1000 DOW 84 84 DOW 7%

35.4%

4 (glucose transporter 4, GLUT4) p-AMPK PPARγ adiponectin

(Hwang et al., 2009)

( ) DOW

(Kou, 2012) DOW

500 g DOW 80 mL pH 3 ~ 4

moanscin amkaflavin citrinin ( , 2010)

DOW

(Shin, 2016)

( )

DOW 2002

500 mL DOW

IgE IL-4 IL-13 IL-18 (Kimata et al., 2002) Hataguchi

DOW K⁺ Se²⁺

Hg²⁺ Pb²⁺ 33 27

(Atopic eczema/dermatitis syndrome,AEDS) (Hataguchi et al., 2005) 2,4-dinitrochlorobenzene (DNCB) NC/Nga

DOW DNCB IgE

TNF-α IL-1β IL-6 CD4+/CD8+

T (T helper cells, Th) IL-4 IL-10 Th2 Th2 DOW IL-4 IL-10 (Bak et al., 2012)

( )

DOW

(16)

(superoxidase dismutase, SOD) (Glutathione peroxidase, GPx)

ICR (Fan et al., 2016)

( )

DOW

(Yang et al., 2014)

1 (Magnesium, Mg) DNA RNA

(Cohen et al., 2002)

250 mg 2 (Calcium, Ca)

°

3 (Selenium, Se)

( ., 2002)

4 (Iron Ferrum, Fe)

5 (Zine, Zn) DNA RNA

10 ~ 15 mg

6 (Potassium, K) 2 ~ 4 g

(Laurant & Touyz, 2000) (Khan et al., 2010)

Vaskonen Mg²⁺ Ca²⁺

(Vaskonen et al., 2001)

(17)

(low density lipoprotein cholesterol, LDL-C)

(Fruchart Duriez, 1998; Lusis, 2000) Steinberg (oxidative LDL, oxLDL)

(Steinberg et al., 1989) (EC)

EC LDL-C (Baumer et al., 2017)

(world health organization,

WHO)

I (chylomicron, CM) TG

IIa LDL-C

IIb LDL-C

(very low-density lipoproteincholesterol, VLDL-C) TG

III (chylomicronremnants)

(intermediate-density lipoproteincholesterol, IDL-C) TC TG

IV VLDL-C TG

LDL-C

V CM VLDL-C TG

VLDL-C TG

VLDL

VLDL-C LDL-C (high density

lipoprotein, HDL)

(Chancharme et al., 2002)

( )

1.

2.

(18)

( D3 ) 3.

( 2006)

A ( acetyl-CoA )

( 1 )

( 2 ) ( 3 ) ( 4 )

(lipoprotein receptors) ( 5 )

CM VLDL IDL LDL

HDL LDL CM VLDL

HDL

LDL-C TC 60-

70%

(atherosclerosis) LDL-C (Fernandez & Webb, 2008)

HDL-C (

20-30%)

HDL-C

°

(Fernandez & Webb, 2008)

TC LDL-C

; HDL-C HDL-C/LDL-C

( 2016)

( )

LDL-C (Fruchart

& Duriez, 1998; Lusis, 2000) LDL-C (endothelial cell of blood vessel) oxLDL

(monocytes) oxLDL

(19)

(Aditi, 2016) 1-1

Fig. 1-1 Cholesterol source and metabolism

(20)

(macrophages) macrophage oxLDL

(foam cell) foam cell Steinberg

oxLDL (Steinberg et al., 1989)

1. superoxidase dismutase (SOD)

SOD (O^2-)

H2O2 O2 SOD

(Fukai & Ushio-Fukai, 2011) SOD

(Lu & Chiang, 2001)

2. catalase (CAT)

CAT H2O2 H2O2

H2O O2(Scibior & Czeczot, 2006)

CAT CAT

(Lu & Chiang, 2001)

3. glutathione peroxidase (GPx)

GPx GPx

glutathione glutathione H2O2

H2O O2

2 %

106

( 2016)

10-24 %

2

NAFLD

(21)

( )

NAFLD (Non-alcoholic

steatohepatitis, NASH)

NAFLD Day James 1998

(Day & James, 1998) (oxidative stress) (insulin resistence

IR) (lipid peroxidation)

(Sanyal et al., 2001)

( )

(Musso et al., 2003) 1985 (Westwater and Fainer, 1958)

(McClain et al., 2004) NAFLD

(Musso et al., 2003)

( )

NAFLD NAFLD (BMI)

α(TNF-α)

NAFLD C- (hs-CRP)

NAFLD (Ajmal et al., 2014) NAFLD

(cardiovascular disease, CVD) (Byrne and Targher, 2015)

( )

(carotid-artery intimal medial

thickness, CIMT) NAFLD

CIMT

NAFLD CIMT

(subclinical) 2015 Ampuero

(22)

NAFLD CIMT NAFLD NAFLD (Ampuero et al., 2015)

DOW

DOW

(triglyceride, TG) (total cholesterol, TC) (Chen et al., 2013)

DOW DOW 5′-adenosine

monophosphate-activated protein kinase (AMPK) acetyl- CoA carboxylase (ACC)

(Sheu et al., 2013) DOW

(low-density lipoprotein-cholesterol, LDL-C) DOW TBARS (Fu et al., 2012) DOW

28 300 1200 DOW (Yoshikawa et al., 2003)

Ca ²⁺ Mg ²⁺

RO

(Nanofiltration,

NF) Na⁺ Cl^- Ca²⁺

Mg ²⁺ NF

SO42- Ca²⁺ CaSO4 (

)

Ca ²⁺ Mg ²⁺

Mg ²⁺

(23)

Vaskonen Mg²⁺ Ca²⁺

(Vaskonen et al., 2001)

DOW

2013 Chen

300 900 1500 DOW

MDA GSH TEAC

(Chen et al., 2013) DOW 2011 Lee

DOW DOW

(Lee et al., 2011)

(24)

DOW

DOW (Ha et al., 2014) (Bak et al.,

2012) (Fan et al., 2016) (Chen et al., 2013)

DOW (Kishimoto et al., 2010)

DOW

D D-L-NaK D-L-Ca DOW

DOW

DOW Na⁺ Mg²⁺

Ca²⁺ K⁺

2-1 AIN- 93G Growth Purified Diet

0.2% Syrian

D D-L-NaK D-L-Ca

probucol TC TG HDL-

C LDL-C TC TG

DOW

DOW GRd GPx CAT SOD

DOW

(25)

D( Mg +C a+ K)

TC TG TC TG

TCTG LDL-C HDL-C

se rum

D( Mg +C a) D( Mg +K )

NaCa KMg

CAT SOD GRd GPx TBARS

H& E AS T AL T

2-1 Fig 2-1 The outline of this study

DOWD-L-NaKD-L-Ca

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1. (UltraPure water system) (PURELAB Classic UV MK2, ELGA LabWater Co., Marlow, UK)

2. (Electronic Balance) (AY-220, Shimadzu Co., Kyoto, Japan)

3. (Hotplate & Stirrer) (SH-301, Suntex Instruments Co., Ltd, New Taipei, Taiwan)

4. (Centrifuge) (Sorvall Legend Micro 17R, Thermo Fisher Scientific Inc., Germany)

5. (D-130, Wiggen hauser, Germany)

6. (Enzyme-linked immunosorbant assay, ELISA) (SUNRISE, Tecan Trading AG, Switzerland)

7. (Water bath) (OSB-2000, Tokyo Rikakikai Co., Ltd, Tokyo, Japan) 8. (pH meter) (pH meter) (UltraBasic pH Meter, Denver Instrument Co., Goettingen, Germany)

9. (Digitale camera) (Coolpix 800, Nicon Co., Tokyo, Japan)

10. (Digital camera) (Moticam 5, Motic Deutschland GmbH, Wetzlar, Germany)

( ) 1. 95%

2. Thiobarbituric acid (TBA) Sigma Chemical Co, (St. Louis, MO, USA) 3. Tricholoroacetic acid (TCA) Merck Co. (Darmstadat, Germany)

4. Dimethylsulfoxide (DMSO) Sigma Chemical Co, (St. Louis, MO, USA) 5. NaCl Sigma Chemical Co, (St. Louis, MO, USA)

6. NaOH Merck Co. (Darmstadat, Germany)

( )

1. (Gene Asia Biotech Co. LTD., Nang-Tou

Hsien, Taiwan)

2. (casein) (corn starch) (cellulose) (choline

(27)

3. (Tainan, Taiwan)

( )

Probucol Sigma (Sigma Chemical Co., St. Louis, MO, USA)

( )

1. BCA protein assay kit (23225, Pierce, Rockford, USA)

2. TC (BXC0261, Fortress) TG (BXC0271, Fortress) Fortress Diagnostics Ltd. (Antrim, UK)

3.Catalase (ECAT-100, BioAssay) BioAssay Systems LLC. (Hayward, Ca, USA)

4. GPx (RS 504, Randox) SOD (SD 125, Randox) GRd (GR 2368, Randox ) Randox Laboratories Ltd. (Crumlin, Antrim, UK)

( ) ( deep ocean water, DOW) (Taitung, Taiwan)

1.D 300 m Nigari DOW Na⁺ 49 mg/L K⁺ 105.8 mg/L Ca²⁺77.9 mg/L Mg²⁺281 mg/L

2. D-L-NaK 600 m Nigari DOW Ca²⁺69 mg/L Mg²⁺281 mg/L

3. D-L-Ca 600 m Nigari DOW Na⁺49 mg/L K⁺88.7 mg/L Mg²⁺281 mg/L

3-1

DOW FDA 60

7.4 (US FDA, 2005)

DOW 281 mg/L

(28)

3- 1

Table 3-1 The different compositions of minerals for deep ocean water.

D D-L-NaK D-L-Ca

Na⁺ (mg/L) 49.0 3.5 49.9

K⁺ (mg/L) 105.8 2.5 88.7

Ca²⁺ (mg/L) 77.9 69 0

Mg²⁺ (mg/L) 281 281 281

D 300 m Nigari DOW ; D-L-NaK

600 m Nigari DOW ; D-L-Ca 600 m Nigari DOW

Na⁺ Mg²⁺ Ca²⁺ K⁺

90

(29)

Usman Hosono

2001 (Usman & Hosono, 2001)

Syrian

6 ~ 7 8 48 60% 23 ± 1

C 8:00 ~ 20:00 12

8

AIN-93 3-1 8

NOR HC PRO

Probucol D Na⁺ Mg²⁺ Ca²⁺

K⁺ D-L-NaK Mg²⁺ Ca²⁺

D-L-Ca Na⁺ Mg²⁺ K⁺

1.0 mL

DOW 600 mL

Mg²⁺

FDA 60 7.4

(US FDA, 2005)

3-2 NOR (Nomal diet)

HC (High-Cholesterol diet) Probucol (PRO)

0.124 g/kg

(30)

3- 2 (g/kg diet)

Table 3-2 The composition of the diet of each group (g/kg diet).

(31)

3- 3 Syrian

Table 3-3 Syrian hamster experimental animal dose group and sample configuration.

Groups Experimental

samples Dose Experimental diets

NOR RO - normal diet

HC RO - high cholesterol diet

PRO Probucol 124 mg/kg b.w. high cholesterol diet

D DOW 74 mL/kg b.w. high cholesterol diet

D-L-NaK DOW with low Na and K 74 mL/kg b.w. high cholesterol diet D-L-Ca DOW with low Ca 74 mL/kg b.w. high cholesterol diet

(32)

D 600 mL DOW Mg²⁺ 281 mg/L 600 mL x 281 mg/L 168.6 mg Mg²⁺

Mg²⁺ 168.6

mg/60 x 7.4 20.794 mg/kg Mg²⁺ 74 mL

DOW/kg b.w. Mg²⁺ 20.79 mg/kg;Na⁺3.62 mg/kg; K⁺7.82 mg/kg;Ca²⁺5.76 mg/kg

D-L-NaK 600 mL Na⁺ K⁺ DOW

281 mg/L 600 mL x 281 mg/L 168.6 mg Mg²⁺

Mg²⁺

168.6 mg/60 x 7.4 20.794 mg/kg Mg²⁺ 74

mL DOW/kg b.w. Mg²⁺ 20.79 mg/kg; Ca²⁺5.10 mg/kg

D-L-Ca 600 mL Ca²⁺ DOW 281 mg/L

600 mL x 281 mg/L 168.6 mg Mg²⁺

Mg²⁺ 168.6

mg/60 x 7.4 20.794 mg/kg Mg²⁺ 74 mL

DOW/kg b.w. Mg²⁺ 20.79 mg/kg; Na⁺3.69 mg/kg; K⁺6.56 mg/kg

(sacrifice)

10%

0.9% -80°C

TC TG HDL-C LDL-C

AST ALT (Tainan,

Taiwan) (Beckman-700, Fullerton, CA, USA)

TC TG ( )

(33)

hood DMSO (dimethyl sulfoxide) (total cholesterol, TC) (triglyceride, TG)

(BXC0261, Fortress) TC (BXC0271,

Fortress) TG ( )

0.1 g

1 mL chloroform : methanol (2:1 v/v) (1 mm) 1 -

2 40 60 M/s 5

15000 xg 5 500 µL hood

DMSO TC TG (BXC0261,

Fortress) TC (BXC0271, Fortress) TG

( )

TC TG

(BXC 0261, Fortress) TC (BXC 0271, Fortress)

TG

TC (BXC 0261, Fortress)

96 2 µL 200 µL reagent 37 C 5 min

ELISA reader 500 nm

(34)

TG (BXC 0271, Fortress)

2 µL 96 200 µL reagent 37 °C

5 min ELISA reader 546 nm

(thiobarbituric acid reactive substances, TBARS) ( )

0.1 g 1.0 mL 1X- lysis buffer (1% Triton X-100 20mM Tris 40 mM NaF 0.2 % SDS 0.5 % deoxy -cholate 1 mM EDTA 1 mM EGTA 1 mM Na3VO4 100 mM NaCl pH 7.5) 4 °C 12,000 x g

15 min -80 °C

TBA (thiobarbituric acid) (malondialdehyde, MDA) MDA

Tarladgis TMP (1,1,3,3-

tetramethoxypropane) TMP 1 mM

1N H2SO4 100 µL

50 µL 300 µL 5% trichloroacetic acid 100 µL 60 mmol/L TBA 95°C 30 min

20-30 min MDA

(Tarladgis et al., 1964)

bicinchoninic acid (BCA) kit

(35)

20 µL 200

µL BCA reagent 37°C 10 min ELISA reader 570

nm

( )

0.1 g 1.0 mL 1X-PBS (0.026 M NaCl 0.0026 M

NaH2PO4 pH 7) 4°C 12,000 xg 15 min

-80°C

( ) (superoxide dismutase, SOD)

SOD superoxide dismutase assay kit (SD 125, Randox) xanthine xanthine oxidase (XOD)

2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride (I.N.T.)

I.N.T. SOD superoxide

dismutase assay kit mixed substrate (xanthine, I.N.T.) buffer (CAPS, EDTA) xanthine oxidase

buffer mixed substrate 5 µL 170 µL mixed substrate 25 µL xanthine oxidase 37°C

505 nm 30 15 min

( ) (catalase, CAT)

CAT (EnzyChrom^TM Catalase Assay Kit, ECAT-100, BioAssay Systems, Hayward, CA, USA) CAT H2O2 H2O

O2 Kit HRP Enzyme Dye Reagent 570 nm

CAT H2O2 CAT

H2O2 H2O2 H2O2

H2O2 H2O2 CAT

( ) (glutathione peroxidase, GSH-PX)

glutathione peroxidase assay kit (RS 504, Randox)

GPx Glutathione (GSH) glutathione reductase (GRd)

NADPH oxidised glutathione (GSSG)

glutathione peroxidase assay kit reagent (glutathione, glutathione

(36)

buffer reagent 4 µL 200 µL reagent

8 µL cumene agent 37 C 340 nm

1 min 3.5 min

8

°

100% methanol Sudan IV (2% w/v) 3 100%

90% 80% 70% 60% methanol phosphate buffered saline (PBS) Sudan IV

photoshop

( ) - (hematoxyline-eosin, H&E)

hematoxylin

10 min 10 min 10

min hematoxylin (250 µL 10N HCl +100 mL 70%

EtOH) 50%

70% 80% 95% eosin (0.1g eosin+100 mL 95%

EtOH+30 µL 10N HCl) 30min 95% 100%

5 2 xylene

± (mean ± SD) SPSS 12.0

(37)

( ) DOW

8 48 8

4-1 (HC)

(NOR) (p < 0.05) DOW

(PRO) (HC) (p < 0.05)

DOW

DOW (p > 0.05)

( ) /

4-2 NOR

/ (p < 0.05)

DOW

(PRO) (HC) (p < 0.05)

DOW DOW

(p > 0.05)

( )

4-3 (HC)

(NOR) (p < 0.05)

DOW

(HC) (p > 0.05)

Probucol

(p < 0.05) Probucol

( 034160 )

(38)

4-1

Table 4-1 Effect on the body wight gain of experimental hamster by feeding of different component of mineral for DOW after 8 weeks.

NOR: normal group, HC: high-cholesterol diet, PRO: probucol powder (0.124 g/kg/day 100 g b.w.) and high-cholesterol diet, D: Deep ocean water (74 mL/kg 100 g b.w.) and high-cholesterol diet, D-L-NaK: Deep ocean water with low sodium and potassium (74 mL/kg 100 g b.w.) and high-cholesterol diet, D-L-Ca: Deep ocean water with low calcium (74 mL/kg 100 g b.w.) and high-cholesterol diet, Data are presented as mean

± SD (n=8). Mean values within each column with different superscripts are significantly different (p < 0.05).

Groups body weight (g)

0th week 8th week

NOR 109.2 1.7 ^a 116.8 4.6 ^a

HC 109.0 5.6 ^a 129.0 8.4 ^b

PRO 109.6 2.2 ^a 115.6 3.4 ^a

D 108.7 5.1 ^a 115.7 5.7 ^a

D-L-NaK 107.7 4.1 ^a 114.3 5.5 ^a

D-L-Ca 108.3 4.4 ^a 114.1 6.2 ^a

(39)

4-2 /

Table 4-2 Effect of various sample on the liver weight/body weight ratio of experimental hamster.

Groups Liver weight Liver weight/body weight ratio (%)

NOR 3.01 0.21 ^a 2.51 0.13^a

HC 4.48 0.35 ^d 3.53 0.18 ^b

PRO 3.85 0.19 ^bc 3.24 0.08 ^c

D 3.73 0.23 ^bc 3.21 0.14 ^c

D-L-NaK 3.70 0.33 ^b 3.18 0.23 ^c

D-L-Ca 3.76 0.19 ^cd 3.37 0.05 ^c

(40)

4-3

Table 4-3 Daily food intake of experimental hamster fed with various samples

Groups Food intake (g/day)

NOR 5.77 ± 0.28 ^c

HC 5.00 ± 0.44^b

PRO 4.32 ± 0.47 ^a

D 4.93 ± 0.51 ^b

D-L-NaK 4.84 ± 0.26 ^ab

D-L-Ca 4.80 ± 0.65 ^ab

(41)

( ) TC TG

TC TG 4-4

(HC) (NOR) HC

TC (p < 0.05)

DOW

(PRO) (HC) (p < 0.05)

DOW TC DOW

TG 4-4 (HC)

(NOR) DOW TG

(PRO) (p < 0.05)

Probucol TG

( IIb ) TG

HDL-C (Noto et al., 2003)

( ) HDL-C LDL-C

HDL-C 4-4

(HC) (NOR) HC HDL-C

(p < 0.05) DOW HDL-C

LDL-C (HC) (NOR)

HC LDL-C (p < 0.05)

HC DOW

(PRO) (HC) (p < 0.05)

LDL-C DOW

(p > 0.05) LDL-cholesterol/HDL-cholesterol

(HC) (NOR) (p < 0.05) DOW

(PRO) (HC) (p < 0.05) DOW

DOW (p >

0.05)

( ) AST ALT

AST ALT

(42)

4-4

/

Table 4-4 Effect of various samples on the serum cholesterol, triglyceride, HDL- cholesterol, LDL-cholesterol concentration and LDL-cholesterol/HDL-cholesterol ratio

Groups TC

(mg/dL)

TG (mg/dL)

HDL-C (mg/dL)

LDL-C (mg/dL)

LDL-C/HDL-C Ratio (%)

NOR 89.1 5.2â 96.3 18.5â 68.0 2.4 â 11.6 2.2â 0.14 0.02 â

HC 177.3 8.8^b 117.7 30.3 ^a 106.5 10.7^bc 43.8 3.0 ^d 0.39 0.02 ^c

PRO 149.5 8.5 ^c 143.2 9.8^b 106.5 11.9^bc 30.1 2.8 ^c 0.25 0.03 ^b

D 144.0 16.6^c 96.0 20.1 ^a 108.8 11.8^c 26.7 6.6 ^bc 0.28 0.05 ^b

D-L-NaK 137.1 16.7 ^c 113.3 23.2 ^a 113.2 9.6 ^cd 25.5 4.5^b 0.24 0.02^b

D-L-Ca 139.6 16.0 ^c 107.7 23.0 ^a 101.2 12.0^b 23.7 3.3^b 0.27 0.03 ^b

(43)

AST ALT

4-5 AST (NOR)

(HC) (p < 0.05)

AST DOW

(HC) (p < 0.05) DOW

AST DOW

ALT 4-5 ALT

(NOR) (HC) (p > 0.05) DOW

(PRO) (HC)

DOW Mg²⁺ Ca²⁺ K⁺ Na⁺

DOW 4-6

138.7 mEq/L -161.2 mEq/L

8.03 mEq/L – 14.12 mEq/L

12.2 mg/dl -14.7 mg/dl

(Mary et al., 2006) 4-6

(44)

4-5 AST ALT

Table 4-5 Effect of various samples on the serum Aspartate aminotransferase and Alanine transferase activity of experimental hamster.

Groups AST activity

(U/L)

ALT activity (U/L)

NOR 54.0 5.3 ^ab 82.1 12.9 ^a

HC 72.3 34 ^b 83.0 25.5 ^a

PRO 44.1 7.1 ^a 74.1 10.2 ^a

D 56.8 15.2 ^ab 83.0 21.5 ^a

D-L-NaK 53.4 19.2 ^ab 83.0 30.6 ^a

D-L-Ca 54.9 12.6 ^ab 81.8 17.3 ^a

(45)

4-6 Na⁺ Mg²⁺ Ca²⁺ K⁺

Table 4-6 Effect of various samples on the serum Na⁺ Mg²⁺ Ca²⁺and K⁺ content.

Calcium,12.2 mg/dl - 14.7 mg/dl; Sodium,138.7 mEq/L - 161.2 mEq/L; Potassium,8.03 mEq/L – 14.12 mEq/L

Groups Na⁺(mEq/L) Ca²⁺(mg/dL) K⁺(mEq/L) Mg²⁺(mEq/L)

NOR 140.9 1.7â 13.49 0.37^b 12.1 0.4 â 4.30 0.16âb

HC 140.2 2.6^a 13.36 0.30 ^b 12.0 1.1^a 4.25 0.27 ^b

PRO 139.8 0.7 â 13.69 0.37 ^b 12.3 1.0â 4.81 0.34 â

D 139.2 1.6â 13.31 0.81 ^b 12.3 1.0â 4.39 0.46 âb

D-L-NaK 139.7 1.5 â 13.23 0.68 ^b 12.9 0.9 â 4.39 0.79âb

D-L-Ca 140.8 2.0 â 12.99 0.90 ^b 12.1 0.9â 4.31 0.25âb

(46)

LDL LDL

(Steinberg et al., 1989) Sudan IV

4-1

4-2 HC

NOR HC

(p < 0.05) HC

4-3 PRO D D-L-NaK D-L-Ca

HC (p < 0.05) Probucol

DOW D-L-NaK Na⁺ K⁺ Mg²⁺ Ca²⁺

TC TG

TC 4-7

(HC) (NOR) (p < 0.05)

DOW

(PRO) (HC) (p < 0.05) DOW

TC DOW D-L-NaK

D-L-Ca (p < 0.05) D-L-Ca D (p < 0.05) D-L-NaK

TC

TG 4-7

(HC) (NOR) (p > 0.05)

(47)

NOR D

HC D-L-NaK

Pro D-L-Ca

4-1

Fig. 4-1 Effect of various samples on the atherosclerotic plaque in the thoracic aorta of hyperlipidemic hamsters.

100 um

(48)

4-2

Fig. 4-2 Effect of various samples on the atherosclerotic plaque area of experimental hamster.

Normal HC Pro D(Mg+Ca+K)D(Mg+Ca) D(Mg+K)

Lipid plaque (%)

0 5 10 15 20

a

d

cd

bc

ab

NOR HC PRO

NOR HC PRO D D-L-NaK D-L-Ca

(49)

4-7

Table 4-7 Effect of various samples on hepatic and feces triglyceride and cholesterol content of experimental hamster.

Groups Liver Feces

TC (mg/g) TG (mg/g) TC (mg/g) TG (mg/g)

NOR 2.05 0.18 ^b 19.36 3.53 ^c 4.20 0.84 ^a 3.16 0.48^b

HC 4.43 0.46 ^d 21.95 4.65 ^c 8.67 0.96 ^b 6.05 0.40^c

PRO 1.96 0.22 ^ab 13.39 1.24 ^b 2.97 0.72^cd 7.18 0.33^d

D 2.43 0.19 ^c 11.93 1.23 ^ab 3.41 0.74 ^c 2.97 0.58^b

D-L-NaK 1.14 0.83 â 9.13 0.97â 2.06 0.38 ê 2.07 0.29â

D-L-Ca 2.24 0.04 ^bc 11.10 1.35^ab 2.18 0.88 ^de 3.01 0.44^b

(50)

DOW TG D-L-NaK

TC TG 4-7

(HC) (NOR) (p < 0.05)

HC TC

TC TC

TG

H&E

4-3 (HC)

(HC) (NOR)

(NOR) (HC)

(NOR)

DOW

(HC) D-L-NaK

(malondialdehyde,

MDA) MDA

MDA 4-4

MDA (HC) (NOR)

MDA (p < 0.05)

DOW

(PRO) (HC) (p < 0.05)

DOW (PRO) DOW DOW

MDA DOW

(p > 0.05)

(51)

4-3 (400 ×)

Fig.3 Pathological examination of liver of experimental hamsters in the 400 × power field. The liver sections were stained using H&E and observed in the light microscope.

49

100 um

100 um 100 um

100 um

100 um D

D-L-NaK

D-L-Ca

(52)

4-4

Fig. 4-4 Effect of various sample on the liver TBARS amount of experimental hamster.

(Mg+Ca+K) (Mg+Ca) (Mg+K) NOR HC PRO D D-L-NaK D-L-Ca

(53)

ROS (Medina, Moreno-Otero, 2005)

ROS SOD O^2-

H2O2 H2O2 catalase (CAT) H2O O2 GPx

GSH GSSG H2O GSSG NADPH

GRd GSH CAT SOD GRd GPx

4-5 4-6 SOD CAT

(HC) (NOR) (p < 0.05)

(PRO) (HC) SOD

CAT SOD CAT

DOW (HC) SOD CAT

(p < 0.05) DOW SOD CAT

DOW SOD (p > 0.05) CAT

D-L-NaK CAT D-L-Ca (p < 0.05)

D-L-Ca D D-L-NaK CAT

4-7 GPx

(PRO) (p < 0.05) (NOR)

DOW GPx

4-8 GRd

(HC) (NOR) (p < 0.05) DOW

GRd

(54)

4-5 SOD

Fig. 4-5 Effect of various samples on antioxidant enzyme SOD activities in the liver of experimental hamster.

SOD activity (U/mg protein)

0 20 40 60 80 100

a

b

a a

a

a b

b b b

(55)

4-6 CAT

Fig. 4-6 Effect of various samples on antioxidant enzyme CAT activities in the liver of experimental hamster.

CAT activity (U/mg protein)

0.0 0.5 1.0 1.5 2.0 2.5 3.0

ab

d

c

a

bc

NOR HC PRO

(56)

4-7 GPx

Fig. 4-7 Effect of various samples on antioxidant enzyme GPx activities in the liver of experimental hamster.

GPx activity (U/mg protein)

0.0 0.5 1.0 1.5 2.0 2.5

ab

a

b b

c

NOR HC PRONOR HC PRO D D-L-NaK D-L-Ca

(57)

4-8 GRd

Fig. 4-8 Effect of various samples on antioxidant enzyme GRd activities in the liver of experimental hamster.

GRd cativity (U/mg protein)

0.0 0.1 0.2 0.3

0.4 a

b

c

d

b

d

a

b

a

b

NOR HC PRO

(58)

Na⁺ Mg²⁺ Ca²⁺ K⁺

Mg²⁺ (Kishimoto et al., 2010)

DOW 281 mg/L Mg²⁺ Mg²⁺

168.6 mg/60 x 7.4 20.79 mg/kg

D 74 mL DOW/kg b.w. 20.79

mg/kg Mg²⁺;3.62 mg/kg Na⁺;7.82 mg/kg K⁺;5.76 mg/kg Ca²⁺ D-L-NaK

74 mL DOW/kg b.w. 20.79 mg/kg

Mg²⁺;5.10 mg/kg Ca²⁺ D-L-Ca 74 mL

DOW/kg b.w. 20.79 mg/kg Mg²⁺; 3.69 mg/kg Na⁺;6.56 mg/kg K⁺

(p < 0.05) /

/ (p < 0.05)

( TC TG HDL-C LDL-C )

TC DOW

TG TG

NOR

HDL-C LDL-C (LDL-C/HDL-C ratio)

DOW

LDL-C (p < 0.05) DOW D-L-NaK

D-L-Ca D

(Hooper et al., 2003) DOW Na⁺ Mg²⁺ Ca²⁺

K⁺

HC NOR (p < 0.05)

DOW (

) HC (p < 0.05)

DOW D-L-NaK D D-L-Ca

(59)

AST ALT

AST

TC TC

TC NOR (p < 0.05)

TC (p < 0.05)

D-L-NaK (p < 0.05) Mg²⁺ Ca²⁺

TC TC

TC TC TG

SOD O2 H2O2 H2O2 catalase

H2O O2 GPx GSH GSSG H2O

GSSG NADPH GRd GSH (Wulf et al., 2002) SOD CAT Grd

SOD CAT

DOW D-L-NaK D D-L-

Ca

TBARS (HC) NOR (p <

0.05) DOW

DOW

CAT D-L-NaK DOW Mg²⁺ Ca²⁺

DOW

(60)

Na⁺

Mg²⁺ Ca²⁺ K⁺ DOW

/

6-1 TC LDL-

C LDL-C / HDL-C (p < 0.05)

DOW LDL-C D-L-NaK

(p > 0.05)

6-1 TC

D-L-NaK (p > 0.05)

SOD CAT

D-L-NaK D D-L-Ca

Na⁺ Mg²⁺ Ca²⁺ (Lee et al., 2012)

D D-L-Ca Na⁺ Mg²⁺ Ca²⁺

Mg²⁺ Na⁺ K⁺

D-L-NaK D-L-Ca D-L-NaK

Mg²⁺ Ca²⁺

DOW Na⁺ Mg²⁺ Ca²⁺ K⁺

(61)

6-1

Table 6-1 Comparison of the regulation of blood lipid between deep ocean water and high cholesterol diet groups with different minerals.

D D-L-NaK D-L-Ca

TC * * *

TG

HDL-C #

LDL-C * ** **

LDL-C / HDL-C * * *

AST ALT

* ** *

TC * ** *

TG * ** *

TBARS ** ** **

SOD # # #

CAT # # #

GPx

GRd # #

* P < 0.05

** P < 0.01

# P < 0.05

(62)

TAIWAN SOCIETY OF DEEP OCEAN WATER RESOURCE APPLICATION

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