Research Article The Effect of Alpha-Lipoic Acid on Mitochondrial … · 2019. 7. 31. · Alpha-lipoic acid, a naturally occurring short chain fatty acid, serves as an important cofactor

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2013 Article ID 517045 9 pageshttpdxdoiorg1011552013517045

Research ArticleThe Effect of Alpha-Lipoic Acid on Mitochondrial Superoxideand Glucocorticoid-Induced Hypertension

Sharon L H Ong12 Harpreet Vohra3 Yi Zhang4

Matthew Sutton3 and Judith A Whitworth3

1 Department of Renal Medicine St George Hospital Kogarah NSW 2217 Australia2 Department of Medicine University of New South Wales Sydney NSW 2052 Australia3 John Curtin School of Medical Research Australian National University Canberra ACT 0200 Australia4Cardiovascular Research Unit The Canberra Hospital Canberra ACT 2605 Australia

Correspondence should be addressed to Sharon L H Ong songunsweduau

Received 11 July 2012 Accepted 4 January 2013

Academic Editor Jose Magalhaes

Copyright copy 2013 Sharon L H Ong et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Aims To examine the effect of alpha-lipoic acid an antioxidant withmitochondrial superoxide inhibitory properties on adrenocor-ticotrophic hormone- (ACTH-HT) and dexamethasone-induced hypertensions (DEX-HT) in rats and if any antihypertensive effectismediated viamitochondrial superoxide inhibitionMethods In a prevention study rats received ground food or alpha-lipoic-acid-laced food (10mgratday) for 15 nights Saline adrenocorticotrophic hormone (ACTH 02mgkgday) or dexamethasone (DEX10 120583gratday) was injected subcutaneously from day 5 to day 11 In a reversal study rats received alpha-lipoic-acid-laced food 4 daysafter commencement of saline or DEX Tail-cuff systolic blood pressure (SBP) was measured second daily Kidney mitochondrialsuperoxide was examined using (MitoSOX) Red (MitoSOX) via flow cytometry Results SBP was increased by ACTH (119875 lt 00005)and DEX (119875 lt 00005) Alpha-lipoic acid alone did not alter SBP With alpha-lipoic acid pretreatment SBP was increased byACTH (1198751015840 lt 0005) but not by DEX Alpha-lipoic partially prevented ACTH-HT (1198751015840 lt 00005) and fully prevented DEX-HT(1198751015840 lt 00005) but failed to reverse DEX-HT ACTH and DEX did not increase MitoSOX signal In ACTH-hypertensive rats high-dose alpha-lipoic acid (100mgratday) did not decrease SBP further but raisedMitoSOX signal (119875 lt 0001) suggesting prooxidantactivity Conclusion Glucocorticoid-induced hypertension in rats is prevented by alpha-lipoic acid via mechanisms other thanmitochondrial superoxide reduction

1 Introduction

Hypertension is a significant risk factor leading to cardio-vascular and renal complications One of the secondarycauses of hypertension is glucocorticoid excess either dueto endogenous overproduction or exogenous glucocorticoidadministration [1] The exact mechanism contributing toglucocorticoid-induced hypertension (GC-HT) has not beenfully elucidated

There is a body of evidence implicating oxidative stress inthe pathogenesis of GC-HT [2] We have previously shownthat GC-HT in the rat was prevented and reversed by severalantioxidants including tempol [3 4] N-acetylcysteine [5 6]

and folate [7] In rats hypertension due to both adreno-corticotrophic hormone (ACTH) and dexamethasone (DEX)was accompanied by a raised plasma biomarker of lipidperoxidation F

2-isoprostane concentrations [3 4 7 8] In

addition increased lucigenin-enhanced chemiluminescencewas also demonstrated in aortas of ACTH-hypertensive rats[4 5]

Whilst there are several pathways contributing to theproduction of reactive oxygen species (ROS) not all of theseare implicated in the pathogenesis of GC-HT The NADPHoxidase [8 9] but not xanthine oxidase [8 10] cyclooxygenase[11] or eNOS in its uncoupled form [7 12] plays a significantrole in GC-HT

2 Oxidative Medicine and Cellular Longevity

The mitochondrion is another important source of ROSprimarily the superoxide radical and consequently its dismu-tation product hydrogen peroxide which can form highlyreactive hydroxyl radicals It consumes approximately 90of the cellrsquos oxygen and 1ndash4 of oxygen reacting with themitochondrial respiratory chain is incompletely reduced toROS [13 14] Complexes I (NADH dehydrogenase) and III(cytochrome 119887119888

1complex) of the mitochondrial electron

transport chain are generally accepted as the main sourceof ROS in the mitochondria [15] The role of mitochondrialROS on the other hand has not been evaluated in theGC-HTmodel

Alpha-lipoic acid a naturally occurring short chain fattyacid serves as an important cofactor for many enzyme com-plexes including mitochondrial respiratory enzymes Exoge-nously administered alpha-lipoic acid has been shown to bean effective antioxidant Urinary F

2-isprostane concentration

was significantly reduced after treatment with 600mgdayalpha-lipoic acid orally for 2 months in humans [16] Apartfrom this alpha-lipoic acid has also been shown to improvemitochondrial function A single dose of alpha-lipoic acid(100mgkg ip) resulted in improvement in mitochondrialfunction determined bymitochondrial oxygen consumptionand complex I II and IV activities in endotoxemic rats [17]Mitochondrial membrane potential is another indicator ofmitochondrial health as it reflects the metabolic activity andintegrity of mitochondrial membrane [18] Alpha-lipoic acidsupplementation (05ww) improved the average mito-chondrial membrane potential in old rats hepatocytes to thatof young rats [19]

Apart from improvingmitochondrial function treatmentwith alpha-lipoic acid prevented both hypertension andmito-chondrial ROS overproduction due to hyperglycemia in ratsand thus suggested a role for mitochondrial ROS in this formof experimental hypertension [20] Mitochondrial superox-ide dismutase deficiency has been shown to be linked withsusceptibility to hypertension with aging and high salt intakeinmouse [21]The role ofmitochondrial ROS overproductionin the development of GC-HT has not been established

In this study we evaluated the effect of alpha-lipoic acidon hypertension in rats and to determine if any antihy-pertensive effect is mediated via mitochondrial superoxideinhibition The potential role of mitochondrial superoxideinhibition was examined in rat kidney mitochondria

2 Materials and Methods

This study was approved by Animal Experimentation EthicsCommittee of the Australian National University (Protocolno J HB 2005) Male Sprague-Dawley rats (body weight200ndash300 g) were housed in individually ventilated plasticcages with a controlled temperature of between 21∘ to 23∘Cand a 12-hour lightdark cycle The rats had free access tostandard rat chow and tap water Prior to any experimentalprocedure the rats were acclimatized to their surroundingsfood and water handling and tail-cuff sphygmomanometer

21 Treatment Protocols Alpha-lipoic acid powder (SigmaSt Louis USA) was administered by mixing in ground

Acclimatisation

Acclimatisation

DEX or ACTH or saline Alpha-lipoic acid or vehicle

Alpha-lipoic acid or vehicle DEX or ACTH or saline

Prevention study

Reversal study

C4 C2 P0 P2 T0 T2 T4 T6 T8 T10 T11

C4 C2 T0 T2 T4 T6 T8 T10 T11

Figure 1 Timeline of experimental protocol

food and given to rats overnight (16ndash18 hours) Normalsaline (09 NaCl 01mLratday) DEX (10 120583gratday)(David Bull Laboratories Mulgrave Victoria Australia) andadrenocorticotrophic hormone (ACTH Synacthen Depot02mgkgday) (Novartis Pharmaceuticals Sydney NSWAustralia) were administered for 12 days from day T0 to T11after 4 control days (C4ndashC1)

The prefixes ldquoPrdquo ldquoTrdquo and ldquoCrdquo represent pretreatmenttreatment and control days respectively The timeline of theexperimental protocol is summarized in Figure 1

22 Animals The rats were randomly divided into 9 treat-ment groupsThe alpha-lipoic acid prevention study was per-formed in both DEX- and ACTH-HTThe reversal study wasonly performed in DEX-HT as alpha-lipoic acid completelyprevented DEX-HT

221 Control Groups Control rats were allocated 30 g ofplain ground food per rat per night (16ndash18 hours) from P0Pelleted food was given during the control days and daytime(from approximately 0900 to 1700 hours) from P0

group 1 Saline (119899 = 10)group 2 ACTH (119899 = 10)group 3 DEX (119899 = 10)

222 Alpha-Lipoic Acid Prevention Studies In the preventionstudies rats were pretreated with either alpha-lipoic acid(500mgkg in food) [20] in ground food overnight (16ndash18hours) from P0 4 days before commencement of salineDEX or ACTH injections This dose (500mgkg in food)was effective in preventing hypertension and mitochondrialROS overproduction due to hyperglycaemia in rats [20] Inanother group of 4 rats high dose alpha-lipoic acid (5 gkgof ground food starting at P0) was given prior to thecommencement of ACTH injections from T0ndashT11

group 4 Alpha-lipoic acid + saline (119899 = 10)group 5 Alpha-lipoic acid + DEX (119899 = 10)group 6 Alpha-lipoic acid + ACTH (119899 = 10)group 7 Alpha-lipoic acid (high dose) + ACTH (119899 =4)

Oxidative Medicine and Cellular Longevity 3

223 Alpha-Lipoic Acid Reversal Study In the reversal stud-ies oral alpha-lipoic-acid-laced food was given 4 days afterthe subcutaneous injections (T4ndashT11)

group 8 Saline + alpha-lipoic acid (119899 = 10)group 9 DEX + alpha-lipoic acid (119899 = 10)

23 Systolic BP and Body Weight Measurements Systolicblood pressure (SBP) was measured by the same investiga-tor second-daily using the tail-cuff blood pressure monitor(Narco Biosystems Houston USA) between 900ndash1100 AMon conscious ratsThe animals were placed in plastic restrain-ers on a heated plate (40∘C) for 40 minutes The animalswere allowed to settle in the restrainers for 10ndash12 minutesbefore SBP readings were recorded Several readings wereobtained but the mean of 4 median readings among whichthe difference was not greater than 10mmHg were taken asSBP Body weight measurements were recorded on alternatedays after the tail-cuff SBP measurements

24 Measurement of Thymus Weight On T11 the rats weresacrificed by exsanguination under isoflurane anaesthesia(4 induction and 2 maintenance) (Abbott AustralasiaKurnell Australia) in the morning between 0900 and 1100hours Wet thymus weight expressed relative to body weight(grams of thymus weight per 100 g body weight) was used asa marker of glucocorticoid activity

25 Blood Glucose Determination Blood was drawn via car-diac puncture A drop of blood was placed on the glucome-ter strip (Precision Plus Blood Glucose Electrodes AbbottLaboratoriesMAUSA) and the blood glucose concentrationwas read on a glucometer (MediSense 2 Abbott LaboratoriesBedford MA USA)

26 Detection of Kidney Mitochondrial Superoxide Therat kidney was resected trimmed immediately chilledhomogenised in phosphate buffered solution and filteredto produce a single cell suspension Cells were stainedwith Trypan blue (Fluka Seelze Germany) and counted Atotal cell count of 20 times 106 per sample was required forflow cytometry analysis with lt20 dead cells in the cellsuspension

The cells were incubatedwith reconstitutedMitoSOXReddye (MitoSOX Molecular Probes Invitrogen USA 2120583M)and 111015840333101584031015840-hexamethylindodicarbocyanine iodide(DiIC

1(5) Molecular Probes Invitrogen USA 10 nM) for

10 minutes at 37∘C prior to flow analysis MitoSOX detectsmitochondrial superoxide in live cells whilst DiIC

1(5)

determines the mitochondrial membrane potential Allsamples were prepared in duplicate Validation of thistechnique is described in Supplementary Document 1 inSupplementary Material available online at httpdxdoiorg1011552013517045

Flow cytometric analysis was performed using a FACSortBD (Becton Dickinson San Jose USA) flow cytometerequipped with 488 nm Argon and 633 nm diode lasers Theapproximate excitation and emission (ExEm) spectral peaks

for MitoSOX are 510580 and for DiIC1(5) are 638658 nm

Excitation of theMitoSOXoxidation productswas performedusing the 488 nm laser and DiIC

1(5) was excited by the

633 nm laser Cells were gated to exclude cell debris whilstcovering a large side scatter to ensure the inclusion ofall cell lines from the kidney (Figure 2(a)) The geometricmean fluorescence intensity (MFI) values of the sampleswere obtained by subtracting fluorescence of the stainedspecimen obtained in the region marked M2 in Figure 2(c)with autofluorescence of unstained specimen obtained in theregion marked M1 in Figure 2(b)

27 1198652-Isoprostane Assay Blood was collected into chilled

tubes containing 1mgmL ethylenediaminetetraacetate and1mgmL reduced glutathione (Sigma St Louis USA)Plasma was protected from oxidation by the addition of02mgmL butylated hydroxytoluene (Sigma St Louis USA)and samples stored at minus70∘C Plasma F

2-isoprostane con-

centrations were measured using electron-capture negative-ion gas chromatography-mass spectrometry as previouslydescribed [22]

28 Plasma NitrateNitrite Assay Plasma nitratenitrite wasused as a marker of reactive nitrogen intermediates It wasdetermined by the reduction of nitrate to nitrite using themodified Griess colour reaction assay (Total Nitric OxideAssay Kit Pierce Endogen Rockford USA) as describedpreviously [5]

29 Data and Statistical Analysis Results were expressed asmean plusmn SEM Results were analyzed by repeated measuresanalysis of variance with Greenhouse Geisser adjustment formultisample asphericity or unpaired 119905-test The Ryan-Holmstepdown Bonferroni procedure was applied to raw 119875 valuesto control the family-wise Type 1 error

3 Results

31 Systolic Blood Pressure DEX increased SBP from 115 plusmn 3to 139plusmn4mmHg (T0ndashT10119875 lt 0005) andACTH from 110plusmn3to 133 plusmn 4mmHg (T0ndashT10 119875 lt 00005) Sham injection withsterile saline did not modify SBP (T0 113 plusmn 2 T10 119 plusmn3mmHg ns) Between-group comparisons showed that SBPsin the groups receiving DEX and ACTH were significantlyhigher than those in the saline-treated group (119899 = 10 each1198751015840lt 0001)

311 Prevention Studies There was no significant change inSBP in the alpha-lipoic acid + saline group (T0 111 plusmn 2T10 118plusmn3mmHg ns)With alpha-lipoic acid pretreatmentDEX did not significantly increase SBP (T0 117 plusmn 4 T10126plusmn 5mmHg ns) However there was a significant increasein SBP in ACTH-treated rats receiving alpha-lipoic acidpretreatment (T0 115 plusmn 2 to T10 129 plusmn 5mmHg 119875 lt005) Despite this there was a significant decrease in SBPin the alpha-lipoic acid + ACTH group (1198751015840 lt 0001) whencompared with the ACTH-only group indicating partial


0 200 400 600 800 1000FSC-height

100

101

102

103

Side

scat

ter

(a)

100

101

102

103

104

Mitosox

0

10

20

30

40

50

60

70

Cou

nts

M1

(b)

100

101

102

103

104

Mitosox

0

10

20

30

40

70

60

50

Cou

nts

M2

(c)

Figure 2 (a) Cells were gated to exclude debris (b) Autofluorescence of unstained cells (control) in the marked region (M1) was recorded(c) Fluorescence of cells treated with MitoSOX in the marked region (M2) was recorded Net geometric MFI was calculated by subtractingthe geometric MFI obtained in M2 region fromM1 region FSC = forward scatter

prevention by alpha-lipoic acid A tenfold increase in alpha-lipoic acid dose (100mgratday) did not decrease the SBPfurther There was no difference in SBP between the alpha-lipoic acid + saline-treated group and the saline-only groupSBP of the alpha-lipoic acid + DEX-treated group wassignificantly lower than that of the DEX-only group (1198751015840 lt0001) (Figures 3(a) and 3(b))

312 Reversal Study The increase in SBP due to DEX wasnot reversed by alpha-lipoic acid (10mgratday) treatmentSystolic BP on day T4 day 1 of alpha-lipoic acid treatmentwas 134plusmn4mmHg and on T10 was 137plusmn2mmHg (119899 = 10 ns)

in the DEX-treated group As in the prevention study alpha-lipoic acid did not alter SBP in the saline-treated group (T4116 plusmn 3mmHg T10 116 plusmn 2mmHg 119899 = 10 ns) (Figure 4)

32 Body Weight Body weight of saline-treated ratsincreased steadily from 270 plusmn 6 to 306 plusmn 7 g (T0ndashT10119875 lt 00005) DEX-treated rats did not gain significant bodyweight (T0 272 plusmn 9 T10 277 plusmn 7 g T0ndashT10 ns) whilstACTH treatment resulted in significant weight loss (from T0283 plusmn 11 to T10 258 plusmn 8 g 119875 lt 0001) In these studies theDEX- and ACTH-treated groups showed significantly lowerbody weights than the saline-treated group (1198751015840 lt 0005)


100

110

120

130

140

C4 C2 P0 P2 T0 T2 T4 T6 T8 T10

Alpha lipoic aciduntreated foodDEXsaline

Days

Systo

lic b

lood

pre

ssur

e (m

mH

g)

ns

119875998400lt 00001

119875998400lt

000

01

(a) DEX-HT prevention study

100

110

120

130

140

Systo

lic b

lood

pre

ssur

e (m

mH

g)

C4 C2 P0 P2 T0 T2 T4 T6 T8 T10

Alpha-lipoic aciduntreated foodACTHsaline

Days

ns 119875998400lt 0001

119875998400lt 0001

(b) ACTH-HT prevention study

Figure 3 Tail-cuff systolic blood pressure in alpha-lipoic acid prevention studies (a) DEX-HT (b) ACTH-HT ◼ saline 119899 = 10 998771 DEX119899 = 10 ◻ ACTH 119899 = 10 998787 alpha-lipoic acid + saline 119899 = 10 ⧫ alpha-lipoic acid + DEX 119899 = 10 loz alpha-lipoic acid + ACTH 119899 = 10 ∙ highdose alpha-lipoic acid + ACTH 119899 = 4 data were presented as mean plusmn SEM

Reversal study

100

110

120

130

140

150

DEXsaline

Alpha lipoic aciduntreated foodSy

stolic

blo

od p

ress

ure (

mm

Hg)

C4 C2 T0 T2 T4 T6 T8 T10Days

119875998400lt

000

5

Figure 4 Tail-cuff systolic blood pressure in alpha-lipoic acidDEX-HT reversal study ◼ saline 119899 = 10 998771 DEX 119899 = 10 ⃝ saline +alpha-lipoic acid 119899 = 10 998779 DEX + alpha-lipoic acid 119899 = 10 datawere presented as mean plusmn SEM

321 Prevention Studies In the prevention studies rats onsaline + alpha-lipoic acid gained body weight steadily fromT0 271 plusmn 11 to T10 295 plusmn 12 g (119899 = 10 119875 lt 00005) Alpha-lipoic acid (10mgratday) pretreatment did not alter bodyweight in saline- DEX- or ACTH-treated rats significantlyHowever rats on higher alpha-lipoic acid (100mgratday)dose and ACTH had significantly lower body weight com-pared with those on ACTH alone (1198751015840 lt 0005)

322 Reversal Study In the reversal study rats on DEX +alpha-lipoic acid failed to gain weight during the course ofthe study (from T4 277 plusmn 10 to T10 278 plusmn 8 g 119899 = 10ns) whilst those on saline + alpha-lipoic acid gained weightprogressively from 276 plusmn 10 g on day T4 to 296 plusmn 10 g on dayT10 (119899 = 10 1198751015840 lt 00005)There was no significant differencein body weight between rats treated with DEX alone and ratstreated with DEX + alpha-lipoic acid

33 Thymus Weight Thymus wet weight was significantlylower with DEX and ACTH treatments compared with saline(1198751015840 lt 0005) regardless of the presence of alpha-lipoic acidtreatment (1198751015840 lt 0005) (Table 1)

34 Blood Glucose Concentration Neither DEX nor ACTHaltered the blood glucose concentrations in rats Alpha-lipoicacid increased blood glucose concentration in saline- andDEX-treated rats in the reversal study This effect was notobserved in the DEX-HT and ACTH-HT prevention studies(Table 1)

35 Plasma 1198652-Isoprostane Concentration The DEX-treated

group had significantly higher plasma F2-isoprostane con-

centration compared with the saline-treated group (1198751015840 lt001) Rats pretreated with alpha-lipoic acid before DEXtreatment had significantly lower plasma F

2-isoprostane con-

centration than those onDEX alone (1198751015840 lt 005) However nosignificant difference was observed in the DEX-HT reversalstudy between the DEX and DEX + alpha-lipoic acid groupsThere was no significant difference in plasma F

2-isoprostane

concentration in rats on ACTH treatment compared withthose on saline treatment Alpha-lipoic acid (both at low andhigh doses) did not alter plasma F

2-isoprostane concentra-

tion in the ACTH-treated rats (Table 1)

36 Plasma Nitrate and Nitrite Concentration There wasno significant difference in plasma NOx concentration inDEX- and ACTH-treated rats compared with saline-treatedrats Alpha-lipoic acid also did not alter the plasma NOxconcentrations in rats (Table 1)

37 Kidney MitoSOX Fluorescence There was no significantdifference in kidneyMitoSOX fluorescence between DEX- orACTH- and saline-treated rats Rats treated with high doseof alpha-lipoic acid and ACTH had significantly higher kid-ney MitoSOX fluorescence compared with ACTH treatmentalone (Table 1)


Table 1 Biological measurements for alpha-lipoic acid studies

Groups Thymus (mg100 gbody weight)

Blood glucose(mmolL)

Plasma NO119909

(120583M)

PlasmaF2-isoprostane

(nmolL)

MitoSOX(geometric MFI)

DiIC1 (5)(geometric MFI)

Saline 127 plusmn 72 11 plusmn 03 93 plusmn 12 47 plusmn 03 198 plusmn 15 73 plusmn 8DEX 48 plusmn 41lowast 9 plusmn 04lowast 123 plusmn 09 71 plusmn 06lowast 223 plusmn 21 107 plusmn 18ACTH 48 plusmn 32lowast 11 plusmn 06 82 plusmn 08 54 plusmn 04 196 plusmn 7 110 plusmn 13LA + saline p 140 plusmn 158 11 plusmn 06 130 plusmn 15 49 plusmn 03 201 plusmn 11 184 plusmn 12lowast

LA + DEX p 50 plusmn 20⋆ 10 plusmn 03 112 plusmn 10 57 plusmn 02dagger 220 plusmn 18 131 plusmn 8LA + ACTH p 53 plusmn 37⋆ 10 plusmn 05 131 plusmn 34 54 plusmn 03 217 plusmn 13 152 plusmn 20HD LA + ACTH p 53 plusmn 21 13 plusmn 11 81 plusmn 10 46 plusmn 06 273 plusmn 10Δ 125 plusmn 6Saline + LA r 133 plusmn 100 12 plusmn 05lowast 127 plusmn 23 54 plusmn 02 166 plusmn 18 161 plusmn 10lowast

DEX + LA r 52 plusmn 31 12 plusmn 08dagger 109 plusmn 15 56 plusmn 05 178 plusmn 20 134 plusmn 12LA alpha-lipoic acid HD LA high dose alpha-lipoic acid p prevention and r reversal lowast1198751015840 lt 005 versus saline ⋆1198751015840 lt 0005 versus LA + saline prevention1198751015840lt 001 versus saline + LA reversal dagger1198751015840 lt 005 versus DEX and Δ1198751015840 lt 00005 versus ACTH Data presented as mean plusmn SEM

38 Kidney DiIC1(5) Fluorescence DiIC

1(5) mean geometric

fluorescence was significantly higher in the groups whichreceived alpha-lipoic acid (in the prevention and reversalstudies) and saline compared to the saline-only group Therewas no significant difference in kidney DiIC

1(5) fluorescence

between DEX- or ACTH- (both at low and higher dosages)and saline-treated rats (Table 1)

4 Discussion

In this study DEX and ACTH administration in ratsincreased blood pressure without increasing the MitoSOXmean fluorescent intensity suggesting a lack of effect of DEXand ACTH on kidney mitochondrial superoxide productionin the kidney The role of mitochondrial superoxide in GC-HT was further tested using the antioxidant alpha-lipoic acidwhich has been shown to inhibit mitochondrial superoxideproduction [20]

The antioxidant alpha-lipoic acid fully prevented DEX-HTbut only partially preventedACTH-HTThis partial effecton ACTH-HT was not due to inadequate dosing as a ten foldincrease in the alpha-lipoic acid dose to 5 gkg of ground fooddid not result in complete prevention of ACTH-HT Alpha-lipoic acid was less effective in reversing established DEX-HT Using the same dosage used in the prevention studyalpha-lipoic acid did not reverseDEX-HTThebloodpressurelower effects of alpha-lipoic acid did not correlate directlywith mitochondrial superoxide availability as demonstratedby the kidney MitoSOX assay

Even though alpha-lipoic acid has been shown to beeffective in improving mitochondrial function and decreas-ing mitochondrial superoxide in rats [19 20] its effect isnot entirely specific to mitochondria The antioxidant effectof alpha-lipoic acid which is mediated by both the originalalpha-lipoic acid compound and its metabolite dihydrolipoicacid a more potent antioxidant acts via a number of mech-anisms such as metal chelation and direct ROS scavenging[23] Dihydrolipoic acid can regenerate antioxidants such asglutathione and vitamin C [23] Thus the tail-cuff blood

pressure results need to be interpreted in conjunction withother biochemical analysis of oxidative stress In this studywe used plasma F

2-isoprostane concentration a product of

lipid peroxidation as a marker of systemic oxidative stressand kidneyMitoSOX fluorescence signal as amarker of tissuemitochondrial superoxide availability

Plasma F2-isoprostane concentration was significantly

increased in DEX- but not ACTH-hypertensive rats Thelatter finding was in contrast to previous findings thatACTH-HT was associated with raised plasma F

2-isoprostane

concentration suggesting insufficient power to detect adifference [24] Pooled F

2-isoprostane concentration data

from our laboratory showed that ACTH-hypertensive rats(119899 = 46) had a significant higher plasma F

2-isoprostane

concentration than the normotensive saline-treated rats (119899 =48) [2]The presence of a raised F

2-isoprostane concentration

in DEX-treated rats again confirmed the role of oxidativestress in its pathogenesis Pretreatment with alpha-lipoic acidsuccessfully prevented the increase of plasma F

2-isoprostane

concentration due toDEXHowever alpha-lipoic acid had noeffect on the plasma F

2-isoprostane in established DEX-HT

Information on mitochondrial ROS has been basedlargely on studies performed on mechanically isolatedmitochondrial preparations or submitochondrial particlesMechanical isolation of mitochondria from their naturalcellular environment through differential centrifugation canaffect the bioenergetics of the electron transport chainNohl et al argued that previous claims that mitochondrialROS production is byproducts of cellular respirations maysimply reflect bioenergetic artefacts consequent on mechan-ical mitochondrial isolation [25] Some workers examinedmitochondrial ROS in intact live cells to avoid this artefactthough this remains challenging experimentally [26ndash28]Whilst this is possible for detection of hydrogen peroxidewhich is membrane permeable and stable the detection ofmitochondrial superoxide in live cells becomes more prob-lematic as it is heavily affected by mitochondrial superoxidedismutase a tightly regulated superoxide scavenger withinthe mitochondria Furthermore their activity varies with thedifferent cell lines One of the techniques that is increasingly


used is in situ evaluation of mitochondrial superoxide usinga novel fluorescence dye MitoSOX

In this experiment MitoSOX probe was used in flowcytometry to detect mitochondrial superoxide in live cellsThe use of flow cytometry allows a large number of cellsto be analysed rapidly Simultaneous quantitative measure-ments of a number of parameters such as mitochondrialsuperoxide and membrane potential or other markers arealso possible with this technique In addition cell debriscan also be excluded from analysis With this methodwhich only detects fluorescence associated with particleslower MitoSOX concentrations can be used Whilst 5120583Mis recommended for other fluorometric techniques lowerMitoSOX concentrations can be used in flow cytometry Inour validation experiments we have shown that MitoSOXconcentration of more than 5 120583M can result in reductions inmitochondrial membrane potentials and decrease the uptakeand fluorescence of MitoSOX in this cell preparation It isalso important to do parallel assessment of mitochondrialmembrane potential to ensure that cell treatments includingMitoSOX dye loading itself do not depolarise the mitochon-dria in the cells as MitoSOX requires a membrane potentialacross the inner mitochondrial membrane to accumulatethere Otherwise MitoSOX can be oxidised elsewhere in thecell and result in falsely positive signals for mitochondrialsuperoxide

In this study cells from the kidney were used for severalreasons Firstly the kidney is a highly vascularised organ richin resistance blood vessels Secondly the kidney is relevant toGC-HT We have previously shown that ACTH-HT a modelmainly mediated by cortisol is associated with increasedrenal vascular resistance [29 30] Furthermore the rise inSBP due to ACTH in rats was accompanied by reductions iniNOS and eNOS gene expression in the kidney [31]

The MitoSOX mean fluorescence intensity on kidneycells was not altered by DEX ACTH or alpha-lipoic acid(500mgkg of ground food) However alpha-lipoic acidat 5 gkg of ground food resulted in significantly higherMitoSOX mean fluorescence intensity This was not asso-ciated with a decrease in mitochondrial membrane poten-tial a phenomenon known to result in a false increase inMitoSOX signals It is possible that alpha-lipoic acid at ahigher dose results in oxidative stress most likely due toincreased mitochondrial superoxide generation The role ofalpha-lipoic acid and its activemetabolite dihydrolipoic acidas prooxidants has been reported in the literature [23 3233] In this study the increase in mitochondrial superoxideproduction is unlikely to translate to systemic oxidativestress given the lack of change in the plasma F

2-isoprostane

concentration observed in this group although the samplesize for this group is very small and there may not havebeen significant power to detect a change Another postu-lation is that superoxide generated within the mitochondriais not readily available to extramitochondrial structuresSuperoxide being a charged radical does not permeate themitochondrial membrane into the cytoplasm easily [34] Itleaves the mitochondria via voltage-dependent anion chan-nels andmore commonly asmembrane-permeable hydrogen

peroxide following its dismutation by intermembrane CuZn-superoxide dismutase [34]

The lower blood pressure readings observed in the alpha-lipoic acid + DEX and alpha-lipoic acid + ACTH groupsin the prevention studies and DEX + alpha-lipoic acid inthe reversal study were not due to inadequate dosing ofDEX and ACTH Similarly the lower plasma F

2-isoprostane

in the alpha-lipoic acid + DEX-treated group compared toDEX-only group was not a reflection of insufficient DEXadministrationThe effectiveness of DEX andACTH deliverywas confirmed by significant lowering of thymus weightin all groups receiving DEX or ACTH due to thymocyteapoptosis and thymic involution induced by glucocorticoidexcess [35 36] Alpha-lipoic acid has no effect on the GC-induced thymic involution

In this study blood glucose concentration was evalu-ated because hyperglycaemia has been shown to result inmitochondrial dysfunction [37] and is a common feature inhumans exposed to excess glucocorticoid hormones Fastingblood glucose was significantly higher in patients with activeCushingrsquos syndrome than those in remission [38] and age-and sex-matched controls [39] In this study however ACTHand DEX did not alter blood glucose concentration in ratswhich are consistent with previous findings in rats [12]Interestingly alpha-lipoic acid significantly increased glucoseconcentration in the reversal study involving DEX-HT andits saline control The fact that alpha-lipoic acid increasedblood glucose concentration in DEX- and saline-treated ratsin the reversal study but not in the prevention study suggeststhat duration of alpha-lipoic acid treatment is likely to playa role However there is no evidence in the literature impli-cating alpha-lipoic acid as a cause of hyperglycemia Ratheralpha-lipoic acid at a same dose-prevented hyperglycemia inglucose-fed rats [40]

We have previously shown that plasma NOx wasdecreased in DEX-HT [10 24] This decrease was notobserved in the present studies on DEX- and ACTH-HTwith and without alpha-lipoic acid treatment This may beinfluenced by decreased sensitivity of this assay in detectingvery low plasma NOx levels seen in all group including thecontrol and DEX-treated groups In this study alpha-lipoicacid did not alter the plasma NOx concentrations in ratstreated with saline DEX or ACTHThe effect of alpha-lipoicacid on the saline-treated rats was consistent with the resultsseen in other studies where alpha-lipoic acid given to ratsorally (20mgkg body weight [41] and 1 gL drinking water[42]) did not alter plasma NOx in normal controls In thesestudies alpha-lipoic acid prevented the rise in plasma NOxinduced by cyclosporine treatment [41] and biliary cirrhosis[42] in rats by the inhibition of renal iNOS and total hepaticNOS respectively In the present study however alpha-lipoicacid did not alter plasma NOx in DEX- or ACTH-treatedrats This is probably because unlike those studies DEX andACTH suppress eNOS and iNOS in rats [31 43]

In summary alpha-lipoic acid fully prevented DEX-HT and partially prevented ACTH-HT without altering thekidney mitochondrial superoxide availability This suggeststhat GC-HT is prevented (and DEX-HT partially reversed)


by alpha-lipoic acid through a mechanism other than mito-chondrial superoxide reduction

The differences observed between DEX- and ACTH-HTonce again depict the different pathophysiological mecha-nisms contributing to the two hypertensive models

Acknowledgments

This work was supported by the National Health andMedicalResearch Council of Australia Project Grant (418426) DrSLH was a recipient of the Jacquot Research Entry Schol-arship from the Royal Australasian College of Physiciansthe National Heart Foundation of Australia PostgraduateScholarship (Award no PB07C 3427) the St George MedicalResearch Foundation Establishment Grant and the Rama-ciotti Establishment Grant (33292011)

References

[1] J AWhitworth G J Mangos and J J Kelly ldquoCushing cortisoland cardiovascular diseaserdquo Hypertension vol 36 no 5 pp912ndash916 2000

[2] S L H Ong Y Zhang and J A Whitworth ldquoReactive oxygenspecies and glucocorticoid-induced hypertensionrdquo Clinical andExperimental Pharmacology and Physiology vol 35 no 4 pp477ndash482 2008

[3] Y Zhang R Jang T A Mori et al ldquoThe anti-oxidant Tempolreverses and partially prevents adrenocorticotrophic hormone-induced hypertension in the ratrdquo Journal of Hypertension vol21 no 8 pp 1513ndash1518 2003

[4] Y Zhang K D Croft T A Mori C G Schyvens K US McKenzie and J A Whitworth ldquoThe antioxidant tempolprevents and partially reverses dexamethasone-induced hyper-tension in the ratrdquoAmerican Journal of Hypertension vol 17 no3 pp 260ndash265 2004

[5] C K Mondo Y Zhang V De Macedo Possamai et alldquoN-acetylcysteine antagonizes the development but does notreverse ACTH-induced hypertension in the ratrdquo Clinical andExperimental Hypertension vol 28 no 2 pp 73ndash84 2006

[6] S Krug Y Zhang T A Mori et al ldquoN-acetylcysteine preventsbut does not reverse dexamethasone-induced hypertensionrdquoClinical and Experimental Pharmacology and Physiology vol 35no 8 pp 979ndash981 2008

[7] Y Miao Y Zhang P S Lim et al ldquoAcid prevents and partiallyreverses glucocorticoid-induced hypertension in the ratrdquoAmer-ican Journal of Hypertension vol 20 no 3 pp 304ndash310 2007

[8] Y Zhang M M K Chan M C Andrews et al ldquoApocyninbut not allopurinol prevents and reverses adrenocorticotropichormone-induced hypertension in the ratrdquoAmerican Journal ofHypertension vol 18 no 7 pp 910ndash916 2005

[9] L Hu Y Zhang P S Lim et al ldquoApocynin but not L-arginineprevents and reverses dexamethasone-induced hypertension inthe ratrdquoAmerican Journal ofHypertension vol 19 no 4 pp 413ndash418 2006

[10] S L H Ong J J Vickers Y Zhang et al ldquoRole of xanthine oxi-dase in dexamethasone-induced hypertension in ratsrdquo Clinicaland Experimental Pharmacology and Physiology vol 34 no 5-6pp 517ndash519 2007

[11] Y Zhang Y Miao and J A Whitworth ldquoAspirin preventsand partially reverses adrenocorticotropic hormone-induced

hypertension in the ratrdquo American Journal of Hypertension vol20 no 11 pp 1222ndash1228 2007

[12] Y Zhang T Pang J Earl et al ldquoRole of tetrahydrobiopterin inadrenocorticotropic hormone-induced hypertension in the ratrdquoClinical and Experimental Hypertension vol 26 no 3 pp 231ndash241 2004

[13] C Richter ldquoDo mitochondrial DNA fragments promote cancerand agingrdquo FEBS Letters vol 241 no 1-2 pp 1ndash5 1988

[14] G Lenaz ldquoRole of mitochondria in oxidative stress and ageingrdquoBiochimica et Biophysica Acta vol 1366 no 1-2 pp 53ndash67 1998

[15] F L Muller Y Liu and H Van Remmen ldquoComplex III releasessuperoxide to both sides of the innermitochondrialmembranerdquoJournal of Biological Chemistry vol 279 no 47 pp 49064ndash49073 2004

[16] K Marangon S Devaraj O Tirosh L Packer and I JialalldquoComparison of the effect of 120572-lipoic acid and 120572-tocopherolsupplementation on measures of oxidative stressrdquo Free RadicalBiology and Medicine vol 27 no 9-10 pp 1114ndash1121 1999

[17] V Vanasco M C Cimolai P Evelson and S Alvarez ldquoTheoxidative stress and the mitochondrial dysfunction causedby endotoxemia are prevented by 120572-lipoic acidrdquo Free RadicalResearch vol 42 no 9 pp 815ndash823 2008

[18] F Distelmaier W J H Koopman E R Testa et al ldquoLifecell quantification of mitochondrial membrane potential at thesingle organelle levelrdquo Cytometry A vol 73 no 2 pp 129ndash1382008

[19] T M Hagen R T Ingersoll J Lykkesfeldt et al ldquo(R)-120572-lipoic acid-supplemented old rats have improvedmitochondrialfunction decreased oxidative damage and increased metabolicraterdquoThe FASEB Journal vol 13 no 2 pp 411ndash418 1999

[20] A ElMidaoui A Elimadi LWu P S Haddad and J DeCham-plain ldquoLipoic acid prevents hypertension hyperglycemia andthe increase in heart mitochondrial superoxide productionrdquoAmerican Journal of Hypertension vol 16 no 3 pp 173ndash1792003

[21] B Rodriguez-Iturbe L Sepassi Y Quiroz Z Ni and N DVaziri ldquoAssociation of mitochondrial SOD deficiency withsalt-sensitive hypertension and accelerated renal senescencerdquoJournal of Applied Physiology vol 102 no 1 pp 255ndash260 2007

[22] T A Mori K D Croft I B Puddey and L J Beilin ldquoAnimproved method for the measurement of urinary and plasmaF2- isoprostanes using gas chromatography-mass spectrome-

tryrdquo Analytical Biochemistry vol 268 no 1 pp 117ndash125 1999[23] G P Biewenga G RMM Haenen and A Bast ldquoThe pharma-

cology of the antioxidant lipoic acidrdquo General Pharmacologyvol 29 no 3 pp 315ndash331 1997

[24] Y I Zhang H Y Jason J A Janine et al ldquoThe role of 20-hydroxyeicosatetraenoic acid in glucocorticoid-induced hyper-tensionrdquo Journal of Hypertension vol 27 no 8 pp 1609ndash16162009

[25] H Nohl L Gille and K Staniek ldquoIntracellular generation ofreactive oxygen species bymitochondriardquo Biochemical Pharma-cology vol 69 no 5 pp 719ndash723 2005

[26] J Duranteau N S Chandel A Kulisz Z Shao and P TSchumacker ldquoIntracellular signaling by reactive oxygen speciesduring hypoxia in cardiomyocytesrdquo Journal of Biological Chem-istry vol 273 no 19 pp 11619ndash11624 1998

[27] T L Vanden Hoek Z Shao C Li P T Schumacker andL B Becker ldquoMitochondrial electron transport can becomea significant source of oxidative injury in cardiomyocytesrdquoJournal of Molecular and Cellular Cardiology vol 29 no 9 pp2441ndash2450 1997


[28] T L Dawson G J Gores A L Nieminen B Herman and J JLemasters ldquoMitochondria as a source of reactive oxygen speciesduring reductive stress in rat hepatocytesrdquo American Journal ofPhysiology vol 264 no 4 pp C961ndashC967 1993

[29] C Wen T Fraser M Li S W Turner and J A WhitworthldquoHaemodynamic mechanisms of corticotropin (ACTH)-induced hypertension in the ratrdquo Journal of Hypertension vol17 no 12 part 1 pp 1715ndash1723 1999

[30] C Wen T Fraser M Li and J A Whitworth ldquoHemodynamicprofile of corticotropin-induced hypertension in the ratrdquo Jour-nal of Hypertension vol 16 no 2 pp 187ndash194 1998

[31] Y K Lou C Wen M Li et al ldquoDecreased renal expression ofnitric oxide synthase isoforms in adrenocorticotropin-inducedand corticosterone-induced hypertensionrdquo Hypertension vol37 no 4 pp 1164ndash1170 2001

[32] U Cakatay ldquoPro-oxidant actions of 120572-lipoic acid and dihy-drolipoic acidrdquo Medical Hypotheses vol 66 no 1 pp 110ndash1172006

[33] H Moini L Packer and N E L Saris ldquoAntioxidant andprooxidant activities of 120572-lipoic acid and dihydrolipoic acidrdquoToxicology and Applied Pharmacology vol 182 no 1 pp 84ndash902002

[34] D Han F Antunes R Canali D Rettori and E CadenasldquoVoltage-dependent anion channels control the release of thesuperoxide anion from mitochondria to cytosolrdquo Journal ofBiological Chemistry vol 278 no 8 pp 5557ndash5563 2003

[35] X M Sun D Dinsdale R T Snowden G M Cohen and D NSkilleter ldquoCharacterization of apoptosis in thymocytes isolatedfrom dexamethasone-treated ratsrdquo Biochemical Pharmacologyvol 44 no 11 pp 2131ndash2137 1992

[36] K Zavitsanou V Nguyen I Greguric J Chapman P Bal-lantyne and A Katsifis ldquoDetection of apoptotic cell death inthe thymus of dexamethasone treated rats using [123I]AnnexinV and in situ oligonucleotide ligationrdquo Journal of MolecularHistology vol 38 no 4 pp 313ndash319 2007

[37] S Munusamy and L A MacMillan-Crow ldquoMitochondrialsuperoxide plays a crucial role in the development of mito-chondrial dysfunction during high glucose exposure in rat renalproximal tubular cellsrdquo Free Radical Biology and Medicine vol46 no 8 pp 1149ndash1157 2009

[38] M Terzolo B Allasino S Bosio et al ldquoHyperhomocysteine-mia in patients with Cushingrsquos syndromerdquo Journal of ClinicalEndocrinology and Metabolism vol 89 no 8 pp 3745ndash37512004

[39] A Faggiano R Pivonello S Spiezia et al ldquoCardiovascularrisk factors and common carotid artery caliber and stiffness inpatients with Cushingrsquos disease during active disease and 1 yearafter disease remissionrdquo Journal of Clinical Endocrinology andMetabolism vol 88 no 6 pp 2527ndash2533 2003

[40] A El Midaoui R Wu and J De Champlain ldquoPrevention ofhypertension hyperglycemia and vascular oxidative stress byaspirin treatment in chronically glucose-fed ratsrdquo Journal ofHypertension vol 20 no 7 pp 1407ndash1412 2002

[41] G Amudha A Josephine V Sudhahar and P VaralakshmildquoProtective effect of lipoic acid on oxidative and peroxidativedamage in cyclosporine A-induced renal toxicityrdquo InternationalImmunopharmacology vol 7 no 11 pp 1442ndash1449 2007

[42] R Marley ldquoLipoic acid prevents development of the hyperdy-namic circulation in anesthetized rats with biliary cirrhosisrdquoHepatology vol 29 no 5 pp 1358ndash1363 1999

[43] T Wallerath K Witte S C Schafer et al ldquoDown-regulation ofthe expression of endothelial NO synthase is likely to contribute

to qlucocorticoid-mediated hypertensionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 23 pp 13357ndash13362 1999

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


The mitochondrion is another important source of ROSprimarily the superoxide radical and consequently its dismu-tation product hydrogen peroxide which can form highlyreactive hydroxyl radicals It consumes approximately 90of the cellrsquos oxygen and 1ndash4 of oxygen reacting with themitochondrial respiratory chain is incompletely reduced toROS [13 14] Complexes I (NADH dehydrogenase) and III(cytochrome 119887119888

1complex) of the mitochondrial electron

transport chain are generally accepted as the main sourceof ROS in the mitochondria [15] The role of mitochondrialROS on the other hand has not been evaluated in theGC-HTmodel

Alpha-lipoic acid a naturally occurring short chain fattyacid serves as an important cofactor for many enzyme com-plexes including mitochondrial respiratory enzymes Exoge-nously administered alpha-lipoic acid has been shown to bean effective antioxidant Urinary F

2-isprostane concentration

was significantly reduced after treatment with 600mgdayalpha-lipoic acid orally for 2 months in humans [16] Apartfrom this alpha-lipoic acid has also been shown to improvemitochondrial function A single dose of alpha-lipoic acid(100mgkg ip) resulted in improvement in mitochondrialfunction determined bymitochondrial oxygen consumptionand complex I II and IV activities in endotoxemic rats [17]Mitochondrial membrane potential is another indicator ofmitochondrial health as it reflects the metabolic activity andintegrity of mitochondrial membrane [18] Alpha-lipoic acidsupplementation (05ww) improved the average mito-chondrial membrane potential in old rats hepatocytes to thatof young rats [19]

Apart from improvingmitochondrial function treatmentwith alpha-lipoic acid prevented both hypertension andmito-chondrial ROS overproduction due to hyperglycemia in ratsand thus suggested a role for mitochondrial ROS in this formof experimental hypertension [20] Mitochondrial superox-ide dismutase deficiency has been shown to be linked withsusceptibility to hypertension with aging and high salt intakeinmouse [21]The role ofmitochondrial ROS overproductionin the development of GC-HT has not been established

In this study we evaluated the effect of alpha-lipoic acidon hypertension in rats and to determine if any antihy-pertensive effect is mediated via mitochondrial superoxideinhibition The potential role of mitochondrial superoxideinhibition was examined in rat kidney mitochondria

2 Materials and Methods

This study was approved by Animal Experimentation EthicsCommittee of the Australian National University (Protocolno J HB 2005) Male Sprague-Dawley rats (body weight200ndash300 g) were housed in individually ventilated plasticcages with a controlled temperature of between 21∘ to 23∘Cand a 12-hour lightdark cycle The rats had free access tostandard rat chow and tap water Prior to any experimentalprocedure the rats were acclimatized to their surroundingsfood and water handling and tail-cuff sphygmomanometer

21 Treatment Protocols Alpha-lipoic acid powder (SigmaSt Louis USA) was administered by mixing in ground

Acclimatisation

Acclimatisation

DEX or ACTH or saline Alpha-lipoic acid or vehicle

Alpha-lipoic acid or vehicle DEX or ACTH or saline

Prevention study

Reversal study

C4 C2 P0 P2 T0 T2 T4 T6 T8 T10 T11

C4 C2 T0 T2 T4 T6 T8 T10 T11

Figure 1 Timeline of experimental protocol

food and given to rats overnight (16ndash18 hours) Normalsaline (09 NaCl 01mLratday) DEX (10 120583gratday)(David Bull Laboratories Mulgrave Victoria Australia) andadrenocorticotrophic hormone (ACTH Synacthen Depot02mgkgday) (Novartis Pharmaceuticals Sydney NSWAustralia) were administered for 12 days from day T0 to T11after 4 control days (C4ndashC1)

The prefixes ldquoPrdquo ldquoTrdquo and ldquoCrdquo represent pretreatmenttreatment and control days respectively The timeline of theexperimental protocol is summarized in Figure 1

22 Animals The rats were randomly divided into 9 treat-ment groupsThe alpha-lipoic acid prevention study was per-formed in both DEX- and ACTH-HTThe reversal study wasonly performed in DEX-HT as alpha-lipoic acid completelyprevented DEX-HT

221 Control Groups Control rats were allocated 30 g ofplain ground food per rat per night (16ndash18 hours) from P0Pelleted food was given during the control days and daytime(from approximately 0900 to 1700 hours) from P0

group 1 Saline (119899 = 10)group 2 ACTH (119899 = 10)group 3 DEX (119899 = 10)

222 Alpha-Lipoic Acid Prevention Studies In the preventionstudies rats were pretreated with either alpha-lipoic acid(500mgkg in food) [20] in ground food overnight (16ndash18hours) from P0 4 days before commencement of salineDEX or ACTH injections This dose (500mgkg in food)was effective in preventing hypertension and mitochondrialROS overproduction due to hyperglycaemia in rats [20] Inanother group of 4 rats high dose alpha-lipoic acid (5 gkgof ground food starting at P0) was given prior to thecommencement of ACTH injections from T0ndashT11

group 4 Alpha-lipoic acid + saline (119899 = 10)group 5 Alpha-lipoic acid + DEX (119899 = 10)group 6 Alpha-lipoic acid + ACTH (119899 = 10)group 7 Alpha-lipoic acid (high dose) + ACTH (119899 =4)











1(5)









2-isoprostane con-




3 Results




0 200 400 600 800 1000FSC-height

100

101

102

103

Side

scat

ter

(a)

100

101

102

103

104

Mitosox

0

10

20

30

40

50

60

70

Cou

nts

M1

(b)

100

101

102

103

104

Mitosox

0

10

20

30

40

70

60

50

Cou

nts

M2

(c)







100

110

120

130

140

C4 C2 P0 P2 T0 T2 T4 T6 T8 T10


Days

Systo

lic b

lood

pre

ssur

e (m

mH

g)

ns

119875998400lt 00001

119875998400lt

000

01


100

110

120

130

140

Systo

lic b

lood

pre

ssur

e (m

mH

g)

C4 C2 P0 P2 T0 T2 T4 T6 T8 T10


Days

ns 119875998400lt 0001

119875998400lt 0001



Reversal study

100

110

120

130

140

150

DEXsaline


stolic

blo

od p

ress

ure (

mm

Hg)


119875998400lt

000

5









2-isoprostane con-


2-isoprostane










Plasma NO119909

(120583M)


(nmolL)







1(5) mean geometric


1(5) fluorescence


4 Discussion









2-isoprostane




2-isoprostane




2-isoprostane









2-isoprostane




2-isoprostane








Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























1(5)









2-isoprostane con-




3 Results




0 200 400 600 800 1000FSC-height

100

101

102

103

Side

scat

ter

(a)

100

101

102

103

104

Mitosox

0

10

20

30

40

50

60

70

Cou

nts

M1

(b)

100

101

102

103

104

Mitosox

0

10

20

30

40

70

60

50

Cou

nts

M2

(c)







100

110

120

130

140

C4 C2 P0 P2 T0 T2 T4 T6 T8 T10


Days

Systo

lic b

lood

pre

ssur

e (m

mH

g)

ns

119875998400lt 00001

119875998400lt

000

01


100

110

120

130

140

Systo

lic b

lood

pre

ssur

e (m

mH

g)

C4 C2 P0 P2 T0 T2 T4 T6 T8 T10


Days

ns 119875998400lt 0001

119875998400lt 0001



Reversal study

100

110

120

130

140

150

DEXsaline


stolic

blo

od p

ress

ure (

mm

Hg)


119875998400lt

000

5









2-isoprostane con-


2-isoprostane










Plasma NO119909

(120583M)


(nmolL)







1(5) mean geometric


1(5) fluorescence


4 Discussion









2-isoprostane




2-isoprostane




2-isoprostane









2-isoprostane




2-isoprostane








Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 200 400 600 800 1000FSC-height

100

101

102

103

Side

scat

ter

(a)

100

101

102

103

104

Mitosox

0

10

20

30

40

50

60

70

Cou

nts

M1

(b)

100

101

102

103

104

Mitosox

0

10

20

30

40

70

60

50

Cou

nts

M2

(c)







100

110

120

130

140

C4 C2 P0 P2 T0 T2 T4 T6 T8 T10


Days

Systo

lic b

lood

pre

ssur

e (m

mH

g)

ns

119875998400lt 00001

119875998400lt

000

01


100

110

120

130

140

Systo

lic b

lood

pre

ssur

e (m

mH

g)

C4 C2 P0 P2 T0 T2 T4 T6 T8 T10


Days

ns 119875998400lt 0001

119875998400lt 0001



Reversal study

100

110

120

130

140

150

DEXsaline


stolic

blo

od p

ress

ure (

mm

Hg)


119875998400lt

000

5









2-isoprostane con-


2-isoprostane










Plasma NO119909

(120583M)


(nmolL)







1(5) mean geometric


1(5) fluorescence


4 Discussion









2-isoprostane




2-isoprostane




2-isoprostane









2-isoprostane




2-isoprostane








Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100

110

120

130

140

C4 C2 P0 P2 T0 T2 T4 T6 T8 T10


Days

Systo

lic b

lood

pre

ssur

e (m

mH

g)

ns

119875998400lt 00001

119875998400lt

000

01


100

110

120

130

140

Systo

lic b

lood

pre

ssur

e (m

mH

g)

C4 C2 P0 P2 T0 T2 T4 T6 T8 T10


Days

ns 119875998400lt 0001

119875998400lt 0001



Reversal study

100

110

120

130

140

150

DEXsaline


stolic

blo

od p

ress

ure (

mm

Hg)


119875998400lt

000

5









2-isoprostane con-


2-isoprostane










Plasma NO119909

(120583M)


(nmolL)







1(5) mean geometric


1(5) fluorescence


4 Discussion









2-isoprostane




2-isoprostane




2-isoprostane









2-isoprostane




2-isoprostane








Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Plasma NO119909

(120583M)


(nmolL)







1(5) mean geometric


1(5) fluorescence


4 Discussion









2-isoprostane




2-isoprostane




2-isoprostane









2-isoprostane




2-isoprostane








Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















2-isoprostane




2-isoprostane








Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article The Effect of Alpha-Lipoic Acid on Mitochondrial … · 2019. 7. 31. · Alpha-lipoic acid, a naturally occurring short chain fatty acid, serves as an important cofactor