LOOHs (lipid hydroperoxides) in oxLDL [oxidized LDL (low-density lipoprotein)] are potentially atherogenic compounds. Recently, H2S was identified as the third endogenous gasotransmitter in the vasculature. H2O2 is known to be destroyed by H2S. Assuming that H2S may also react with LOOHs, the results show that H2S can destroy LOOHs in oxLDL. The ability of LOOH-enriched LDL to induce HO-1 (haem oxygenase 1) in endothelial cells was abolished by H2S pretreatment. HPLC analysis showed that 9-HPODE [(9S)-hydroperoxy-(10E,12Z)-octadecadienoic acid], a compound found in oxLDL, was reduced to 9-HODE [(9S)-hydroxy-(10E,12Z)-octadecadienoic acid] in the presence of H2S. Thus H2S may act as an antiatherogenic agent by reducing LOOHs to the less reactive LOHs and could abrogate the pathobiological activity of oxLDL.

INTRODUCTION

The oxidative modification of LDL (low-density lipoprotein) may play a role in atherogenesis [14]. The formation of the potentially atherogenic LOOHs (lipid hydroperoxides) in LDL is an early event in LDL lipid oxidation. Thus destroying LOOHs in LDL can limit its pathobiological potential.

Recently, H2S has been identified as a third endogenous gasotransmitter in the vasculature (besides CO and NO) [5]. As H2S is known to destroy H2O2 [68], one may speculate that H2S may also have the potential to destroy LOOHs in LDL, abrogating the pathobiological activity of oxLDL.

EXPERIMENTAL

Materials

Sodium hydrogen sulfide (NaHS), TPP (triphenylphosphine) and Xylenol Orange were from Sigma–Aldrich. H2O2 (30% solution) was supplied by Merck.

9-HPODE [(9S)-hydroperoxy-(10E,12Z)-octadecadienoic acid] and 9-HODE [(9S)-hydroxy-(10E,12Z)-octadecadienoic acid] were obtained from Cayman Chemicals (Ann Arbor, MI, U.S.A.) as ethanolic solutions (1 mg/ml).

Methods

H2S generation

NaHS was used to generate H2S in solution. H2S concentration was taken as 30% of the NaHS concentration according to Beauchamp et al. [9].

Lipoprotein isolation

LDL preparations were isolated by ultracentrifugation as reported previously [10]. The final preparations were subjected to gel chromatography to get rid of KBr and filter sterilized. Protein was estimated by a modified Lowry method [11]. All LDL concentrations are given as mg of protein/ml.

LDL oxidation

Prior to LDL oxidation, the lipoprotein was passed over a gel column equilibrated in PBS (pH 7.4). Routinely, LDL (0.2 mg/ml PBS) was incubated for 2 h in the presence of 10 μmol/l Cu2+ at 37 °C and the reaction was stopped by the addition of EDTA (100 μmol/l). Alternatively, LDL (1 mg/ml; 10 mmol/l Mops and 150 mmol/l NaCl, pH 7.4) was dialysed for 24 h at 4 °C against Mops buffer containing 5 μmol/l Cu2+ and oxidation was stopped by dialysis against PBS containing 100 μmol/l EDTA for 24 h at 4 °C by the method of Gerry et al. [12].

H2S treatment of oxLDL (oxidized LDL) samples

Cu2+ oxLDL was incubated at 37 °C in the absence or presence of NaHS for up to 1 h. For cell experiments, samples of Cu2+ oxLDL (0.2 mg/ml) were brought to 0.4 mg/ml by the addition of native LDL, and after incubation with or without NaHS for 30 min at 37 °C, the preparations were subjected to gel chromatography to get rid of NaHS.

Estimation of LDL oxidation

Estimation of lipid peroxides. LOOHs were estimated using the ferrous oxidation in FOX assay (Xylenol Orange assay) [13]. A 50 μl portion of the sample was mixed with 450 μl of FOX reagent and after 30 min at room temperature (25 °C), absorbance was estimated at 560 nm. H2O2 was used to calculate concentrations. NaHS at the highest concentrations used did not interfere with the assay as no inhibition of colour development was observed when NaHS was added to FOX reagent/LOOH mixtures.

Diene formation. LDL oxidation was monitored as the increase in conjugated diene formation by measuring A234 [14].

Measurement of MDA (malondialdehyde). TBARS (thiobarbituric acid-reacting substance) formation in LDL was measured as reported in [15]. NaHS did not interfere with TBARS formation as tested using pure MDA.

Cell culture

HUVEC (human umbilical-vein endothelial cells) were isolated and cultured as reported previously [16].

Real-time PCR

Isolation of total RNA, cDNA synthesis and quantitative PCR were performed as described in [17]. Primers for real-time PCR were designed using the Primer3 software at http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi, and exon junction primers were selected to avoid amplification of residual DNA contaminations [HO-1 (haem oxygenase-1): 5′-AGACGGCTTCAAGCTGGTGA-3′ and 5′-TCTCCTTGTTGCGCTCAATCTC-3′; acidic ribosomal phosphoprotein P0: 5′-CTCGCCAGGCGTCCTCGT-3′ and 5′-TTGCCCATCAGCACCACAG-3′]. For normalization, mean Ct (threshold cycle values) of all replicate analyses were normalized to RPLP0 within each sample to obtain sample-specific ΔCt values (=Ct HMOX1 – Ct RPLP0). To compare the effect of various treatments with untreated controls, 2−ΔΔCt values were calculated to obtain fold expression levels, where ΔΔCt=(ΔCt treatment – ΔCt control). Real-time analysis using primers specific for cyclophilin b (GenBank® accession no. NM_000942) as the reference standard and normalization for equal amounts of total RNA input (1 μg for each sample) gave comparable results (results not shown).

HPLC separation of standard 9-HODE and 9-HPODE

Separations were preformed by injecting 5 μl of 64 μmol/l HPODE for time effect or 15 μl of 21.3 μmol/l HPODE for concentration-effect experiments into the HPLC (Waters 2695 Alliance system, Nova-Pak C18 3.9 mm×150 mm column, 4 μm) with a mobile phase of 0.3% H3PO4/acetonitrile (40:60, v/v) for 30 min with a flow of 1 ml/min. UV absorbance was monitored at 210 and 234 nm in dual wavelength mode. HPODE and HODE were prepared in PBS (chelexed). Substances were quantified via peak area.

HPLC analysis of oxLDL

Lipid oxidation products were determined by HPLC analysis of saponified lipids extracted from LDL by the method of Thomas and Jackson [18]. In brief, 200 μg of LDL oxidized by the dialysis method (see above) was extracted with 2 ml diethyl ether/ethanol (3:1) and protein was precipitated by centrifugation. Decanted extracts were dried under nitrogen and subsequently lipid was dissolved in 0.5 ml of ethanol/0.05 ml of 10 M NaOH. After saponification (20 min at 60 °C) samples were neutralized and dried under nitrogen. Water was added and lipids were extracted into heptane. Dried heptane phases were dissolved in ethanol and subjected to HPLC analyses as outlined above.

Statistics

The results are presented as mean values for two to five experiments. As appropriate, specific effects were evaluated by one-way ANOVA with post hoc testing using the Newman–Keul test. A value of P<0.05 was considered statistically significant.

RESULTS

Influence of H2S on LOOHs in oxLDL

As can be seen in Figure 1, when samples of oxLDL were incubated with NaHS for 60 min at 37 °C, a concentration-dependent decrease in LOOHs was observed. Approx. 50% (P<0.01) of LOOHs were destroyed by 100 μmol/l H2S treatment (Figure 1A). TPP (100 μmol/l), which is known to reduce LOOHs [19,20], was run as a control and showed comparable results (P<0.001). Figure 1(B) depicts the time-dependent influence of H2S on LOOHs. Under the conditions employed after 30 min of H2S treatment, approx. 25% of the LOOHs in oxLDL were destroyed (P<0.001 for all time points compared with control).

H2S treatment decreases LOOHs in oxLDL

Figure 1
H2S treatment decreases LOOHs in oxLDL

(A) Influence of H2S treatment on LOOHs in oxLDL. LDL was oxidized with Cu2+ and subsequently treated with H2S (■) or TPP (▽) for 60 min at 37 °C and residual LOOHs were estimated as given in the Experimental section. 100%=70.6±4.5 μmol/l LOOH (n=3). (B) Time course of the effect of H2S on LOOHs in LDL. Cu2+ oxLDL (0.2 mg/ml PBS) was further incubated in the absence or presence of H2S (100 μmol/l) for the indicated time at 37 °C and residual LOOHs were estimated. 100%=67.5±0.5 μmol/l LOOH (n=3).

Figure 1
H2S treatment decreases LOOHs in oxLDL

(A) Influence of H2S treatment on LOOHs in oxLDL. LDL was oxidized with Cu2+ and subsequently treated with H2S (■) or TPP (▽) for 60 min at 37 °C and residual LOOHs were estimated as given in the Experimental section. 100%=70.6±4.5 μmol/l LOOH (n=3). (B) Time course of the effect of H2S on LOOHs in LDL. Cu2+ oxLDL (0.2 mg/ml PBS) was further incubated in the absence or presence of H2S (100 μmol/l) for the indicated time at 37 °C and residual LOOHs were estimated. 100%=67.5±0.5 μmol/l LOOH (n=3).

In addition to the LOOH destroying effect, H2S could directly inhibit copper ion-induced LDL oxidation as monitored by diminished TBARS formation and increased lag phase during conjugated diene kinetics (results not shown).

H2S treatment of oxLDL decreases its ability to induce HO-1

LOOHs in oxLDL can induce HO-1 in vascular cells [2123]. As seen in Figure 2, oxLDL (100 μg/ml for 18 h) resulted in a 5-fold induction of HO-1 in HUVEC. Pretreatment of oxLDL with 1 mmol/l H2S for 30 min at 37 °C, which completely destroyed LOOHs (results not shown), abolished the ability of the LDL preparation to induce HO-1.

H2S treatment decreases oxLDL-induced HO-1 in HUVEC

Figure 2
H2S treatment decreases oxLDL-induced HO-1 in HUVEC

Cu2+ oxLDL was treated with or without H2S (1 mmol/l) as given in the Experimental section and LDL preparations (100 μg/ml) were added to cell monolayers. After 18 h of incubation, RNA was extracted and used for PCR analysis (n=3).

Figure 2
H2S treatment decreases oxLDL-induced HO-1 in HUVEC

Cu2+ oxLDL was treated with or without H2S (1 mmol/l) as given in the Experimental section and LDL preparations (100 μg/ml) were added to cell monolayers. After 18 h of incubation, RNA was extracted and used for PCR analysis (n=3).

Reduction of 9-HPODE to 9-HODE by H2S

9-HPODE is a fatty acid peroxidation product found in oxLDL [24]. Reduction of LOOHs to LOHs by thiol residues on PON (paraoxonase) has been reported by Aviram et al. [25]. Thus we analysed the reaction product(s) of H2S with 9-HPODE by HPLC. When 9-HPODE was incubated for 30 min at 37 °C with H2S this treatment resulted in the formation of 9-HODE. The H2S concentration-dependent reduction (expressed as the ratio HPODE/HODE) is shown in Figure 3(A). Figure 3(B) depicts the time-dependent conversion of 9-HPODE into 9-HODE in the presence of H2S. Approx. 50% of 9-HPODE was reduced after 30 min.

H2S treatment reduces 9-HPODE to 9-HODE

Figure 3
H2S treatment reduces 9-HPODE to 9-HODE

(A) Concentration-dependent reduction of 9-HPODE to 9-HODE by H2S. 9-HPODE (21.3 μmol/l PBS) was incubated with the respective concentration of H2S relative to 9-HPODE for 30 min at 37 °C and the samples were analysed by HPLC as given in the Experimental section (n=2). (B) Time-dependent reduction of 9-HPODE to 9-HODE by H2S. 9-HPODE (64 μmol/l PBS) was incubated at 37 °C in the absence (○) or presence of equimolar amounts of NaHS (■). At the indicated time, samples were withdrawn and analysed by HPLC (n=2).

Figure 3
H2S treatment reduces 9-HPODE to 9-HODE

(A) Concentration-dependent reduction of 9-HPODE to 9-HODE by H2S. 9-HPODE (21.3 μmol/l PBS) was incubated with the respective concentration of H2S relative to 9-HPODE for 30 min at 37 °C and the samples were analysed by HPLC as given in the Experimental section (n=2). (B) Time-dependent reduction of 9-HPODE to 9-HODE by H2S. 9-HPODE (64 μmol/l PBS) was incubated at 37 °C in the absence (○) or presence of equimolar amounts of NaHS (■). At the indicated time, samples were withdrawn and analysed by HPLC (n=2).

Reduction of 9-HPODE in oxLDL by H2S

Figure 4 shows the influence of H2S treatment on 9-HPODE present in oxLDL. H2S treatment caused a significant decrease (as quantified via peak area) in 9-HPODE from 842±118 (oxLDL) to 544±18 (P<0.001). Ebselen/GSH treatment run as a positive control of LOOH reduction [18] resulted in a decrease of approx. 85% (121±18).

Reduction of 9-HPODE in oxLDL by H2S or ebselen treatment analysed by HPLC

Figure 4
Reduction of 9-HPODE in oxLDL by H2S or ebselen treatment analysed by HPLC

oxLDL (1 mg/ml) was incubated with H2S (1 mmol/l) or ebselen/GSH (100 μmol/3 mmol per litre) for 45 min at 37 °C and, subsequently, oxidized lipids were analysed after saponification as given in the Experimental section. Curve d, LDL; curve c, oxLDL; curve b, H2S treatment; and curve a, ebselen/GSH. The arrow indicates 9-HPODE. Inset: LDL (open bar), oxLDL (black bar), H2S treatment (light grey bar) and ebselen treatment (dark grey bar). Substances were quantified via peak area (n=3). AU 234 nm, A234.

Figure 4
Reduction of 9-HPODE in oxLDL by H2S or ebselen treatment analysed by HPLC

oxLDL (1 mg/ml) was incubated with H2S (1 mmol/l) or ebselen/GSH (100 μmol/3 mmol per litre) for 45 min at 37 °C and, subsequently, oxidized lipids were analysed after saponification as given in the Experimental section. Curve d, LDL; curve c, oxLDL; curve b, H2S treatment; and curve a, ebselen/GSH. The arrow indicates 9-HPODE. Inset: LDL (open bar), oxLDL (black bar), H2S treatment (light grey bar) and ebselen treatment (dark grey bar). Substances were quantified via peak area (n=3). AU 234 nm, A234.

DISCUSSION

The third gaseous transmitter H2S has been shown to protect cells and proteins from oxidative modifications by peroxynitrite and HOCl [7,26]. H2O2 is also known to be destroyed by H2S [68]. Thus one may assume that H2S may also destroy organic hydroperoxides of pathobiological importance, such as fatty acid hydroperoxides (LOOHs). Focusing on LOOHs in oxLDL the present results show that LOOHs were destroyed in the presence of H2S. This resulted in the inability of oxLDL to induce HO-1 in endothelial cells. Both results indicate that H2S could abrogate the pathophysiological activity of oxLDL. Destroying LOOHs in oxLDL could limit its atherogenic potential as hydroperoxides can (i) break down into lipid alkoxyl (LO) as well as peroxyl radicals (LOO), which both can attack and modify other biological molecules [27] are (ii) cytotoxic and (iii) break down to protein modifying aldehydes like MDA, 4-hydroxynonenal and 4-hydroxyhexenal [28,29].

PON associated with HDL (high-density lipoprotein) has been implicated as an important factor in destroying LOOHs on LDL and may contribute to the beneficial effect of HDL in atherogenesis and cytoprotection of vascular cells [30]. The ability of PON to protect against LDL oxidation involves its free thiol group and not its arylesterase/PON activity [25] and the results revealed that the decrease in the levels of CE-LOOHs (cholesteryl ester hydroperoxides) by PON was due to the formation of CE-LOHs and the authors suggested a peroxidase-like action of PON on LOOHs [31]. Using 9-HPODE, an LOOH compound found in oxLDL [24], our results showed that in the presence of H2S at concentrations that have been reported for in vivo (50–150 μmol/l) [32,33], 9-HPODE was reduced to 9-HODE assuming the reaction:

 
formula

However, recent work has suggested that levels of free H2S in vivo are much lower than previously thought [34]. On the other hand, as H2S can be stored as bound sulfur and released [35], one may speculate that the overall amount could be adequate to modulate pathophysiological reactions.

In summary, our results indicate that H2S besides PON may be an additional endogenous factor in reducing LOOHs in LDL. An early event in atherogenesis is the trapping and oxidative modification of LDL in the subendothelial space [36] and H2S (which is produced nearby and released from smooth-muscle cells [37]) may prevent or diminish the further atherogenic modifications of the lipoprotein.

Abbreviations

     
  • FOX assay

    Xylenol Orange assay

  •  
  • HO-1

    haem oxygenase-1

  •  
  • 9-HODE

    (9S)-hydroxy-(10E,12Z)-octadecadienoic acid

  •  
  • 9-HPODE

    (9S)-hydroperoxy-(10E,12Z)-octadecadienoic acid

  •  
  • HUVEC

    human umbilical-vein endothelial cells

  •  
  • LDL

    low-density lipoprotein

  •  
  • LOOH

    lipid hydroperoxide

  •  
  • oxLDL

    oxidized LDL

  •  
  • MDA

    malondialdehyde

  •  
  • PON

    paraoxonase

  •  
  • TBARS

    thiobarbituric-acid-reacting substance

  •  
  • TPP

    triphenylphosphine

FUNDING

This work was supported by the Oesterreichische Nationalbank Jubiläumsfonds [grant number 10537 (to S. M. S.)].

References

References
1
Steinberg
D.
Lewis A. Conner Memorial Lecture: oxidative modification of LDL and atherogenesis
Circulation
1997
, vol. 
95
 (pg. 
1062
-
1071
)
2
Steinberg
D.
Parthasarathy
S.
Carew
T.
Khoo
J.
Witztum
J.
Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity
N. Engl. J. Med.
1989
, vol. 
320
 (pg. 
915
-
924
)
3
Berliner
J. A.
Heinecke
J. W.
The role of oxidized lipoproteins in atherogenesis
Free Radical Biol. Med.
1996
, vol. 
20
 (pg. 
707
-
727
)
4
Berliner
J. A.
Navab
M.
Fogelman
A. M.
Frank
J. S.
Demer
L. L.
Edwards
P. A.
Watson
A. D.
Lusis
A. J.
Atherosclerosis: basic mechanisms: oxidation, inflammation, and genetics
Circulation
1995
, vol. 
91
 (pg. 
2488
-
2496
)
5
Wang
R. U. I.
Two's company, three's a crowd: can H2S be the third endogenous gaseous transmitter?
FASEB J.
2002
, vol. 
16
 (pg. 
1792
-
1798
)
6
Whiteman
M.
Cheung
N. S.
Zhu
Y.-Z.
Chu
S. H.
Siau
J. L.
Wong
B. S.
Armstrong
J. S.
Moore
P. K.
Hydrogen sulphide: a novel inhibitor of hypochlorous acid-mediated oxidative damage in the brain?
Biochem. Biophys. Res. Commun.
2005
, vol. 
326
 (pg. 
794
-
798
)
7
Laggner
H.
Muellner
M. K.
Schreier
S.
Sturm
B.
Hermann
M.
Exner
M.
Gmeiner
B. M. K.
Kapiotis
S.
Hydrogen sulphide: a novel physiological inhibitor of LDL atherogenic modification by HOCl
Free Radical Res.
2007
, vol. 
41
 (pg. 
741
-
747
)
8
Devai
I.
Delaune
R. D.
Effectiveness of selected chemicals for controlling emission of malodorous sulfur gases in sewage sludge
Environ. Technol.
2002
, vol. 
23
 (pg. 
319
-
329
)
9
Beauchamp
R. J.
Bus
J.
Popp
J.
Boreiko
C.
Andjelkovich
D. A.
A critical review of the literature on hydrogen sulfide toxicity
Crit. Rev. Toxicol.
1984
, vol. 
13
 (pg. 
25
-
97
)
10
Hermann
M.
Gmeiner
B.
Altered susceptibility to in vitro oxidation of LDL in LDL complexes and LDL aggregates
Arterioscler. Thromb.
1992
, vol. 
12
 (pg. 
1503
-
1506
)
11
Lowry
O. H.
Rosebrough
N. J.
Farr
A. L.
Randall
R. J.
Protein measurement with the Folin phenol reagent
J. Biol. Chem.
1951
, vol. 
193
 (pg. 
265
-
275
)
12
Gerry
A. B.
Satchell
L.
Leake
D. S.
A novel method for production of lipid hydroperoxide- or oxysterol-rich low-density lipoprotein
Atherosclerosis
2008
, vol. 
197
 (pg. 
579
-
587
)
13
Nourooz-Zadeh
J.
Tajaddini-Sarmadi
J.
Ling
K. L.
Wolff
S. P.
Low-density lipoprotein is the major carrier of lipid hydroperoxides in plasma. Relevance to determination of total plasma lipid hydroperoxide concentrations
Biochem. J.
1996
, vol. 
313
 (pg. 
781
-
786
)
14
Esterbauer
H.
Gebicki
J.
Puhl
H.
Jürgens
G.
The role of lipid peroxidation and antioxidants in oxidative modification of LDL
Free Radical Biol. Med.
1992
, vol. 
13
 (pg. 
341
-
390
)
15
Laggner
H.
Hermann
M.
Sturm
B.
Gmeiner
B. M. K.
Kapiotis
S.
Sulfite facilitates LDL lipid oxidation by transition metal ions: a pro-oxidant in wine?
FEBS Lett.
2005
, vol. 
579
 (pg. 
6486
-
6492
)
16
Kapiotis
S.
Besemer
J.
Bevec
D.
Valent
P.
Bettelheim
P.
Lechner
K.
Speiser
W.
Interleukin-4 counteracts pyrogen-induced downregulation of thrombomodulin in cultured human vascular endothelial cells
Blood
1991
, vol. 
78
 (pg. 
410
-
415
)
17
Laggner
H.
Hermann
M.
Esterbauer
H.
Muellner
M. K.
Exner
M.
Gmeiner
B. M. K.
Kapiotis
S.
The novel gaseous vasorelaxant hydrogen sulfide inhibits angiotensin-converting enzyme activity of endothelial cells
J. Hypertension
2007
, vol. 
25
 (pg. 
2100
-
2104
)
18
Thomas
C. E.
Jackson
R. L.
Lipid hydroperoxide involvement in copper-dependent and independent oxidation of low density lipoproteins
J. Pharmacol. Exp. Ther.
1991
, vol. 
256
 (pg. 
1182
-
1188
)
19
Frei
B.
Yamamoto
Y.
Niclas
D.
Ames
B. N.
Evaluation of an isoluminol chemiluminescence assay for the detection of hydroperoxides in human blood plasma
Anal. Biochem.
1988
, vol. 
175
 (pg. 
120
-
130
)
20
Tang
L.
Zhang
Y.
Qian
Z.
Shen
X.
The mechanism of Fe(2+)-initiated lipid peroxidation in liposomes: the dual function of ferrous ions, the roles of the pre-existing lipid peroxides and the lipid peroxyl radical
Biochem. J.
2000
, vol. 
352
 (pg. 
27
-
36
)
21
Anwar
A. A.
Li
F. Y. L.
Leake
D. S.
Ishii
T.
Mann
G. E.
Siow
R. C. M.
Induction of heme oxygenase 1 by moderately oxidized low-density lipoproteins in human vascular smooth muscle cells: role of mitogen-activated protein kinases and Nrf2
Free Radical Biol. Med.
2005
, vol. 
39
 (pg. 
227
-
236
)
22
Agarwal
A.
Shiraishi
F.
Visner
G. A.
Nick
H. S.
Linoleyl hydroperoxide transcriptionally upregulates heme oxygenase-1 gene expression in human renal epithelial and aortic endothelial cells
J. Am. Soc. Nephrol.
1998
, vol. 
9
 (pg. 
1990
-
1997
)
23
Ishikawa
K.
Navab
M.
Leitinger
N.
Fogelman
A. M.
Lusis
A. J.
Induction of heme oxygenase-1 inhibits the monocyte transmigration induced by mildly oxidized LDL
J. Clin. Invest.
1997
, vol. 
100
 (pg. 
1209
-
1216
)
24
Browne
R. W.
Armstrong
D.
HPLC analysis of lipid-derived polyunsaturated fatty acid peroxidation products in oxidatively modified human plasma
Clin. Chem.
2000
, vol. 
46
 (pg. 
829
-
836
)
25
Aviram
M.
Billecke
S.
Sorenson
R.
Bisgaier
C.
Newton
R.
Rosenblat
M.
Erogul
J.
Hsu
C.
Dunlop
C.
La Du
B.
Paraoxonase active site required for protection against LDL oxidation involves its free sulfhydryl group and is different from that required for its arylesterase/paraoxonase activities: selective action of human paraoxonase allozymes Q and R
Arterioscler. Thromb. Vasc. Biol.
1998
, vol. 
18
 (pg. 
1617
-
1624
)
26
Whiteman
M.
Armstrong
J. S.
Chu
S. H.
Jia-Ling
S.
Wong
B.-S.
Cheung
N. S.
Halliwell
B.
Moore
P. K.
The novel neuromodulator hydrogen sulfide: an endogenous peroxynitrite ‘scavenger’?
J. Neurochem.
2004
, vol. 
90
 (pg. 
765
-
768
)
27
Spiteller
G.
Peroxyl radicals: inductors of neurodegenerative and other inflammatory diseases. Their origin and how they transform cholesterol, phospholipids, plasmalogens, polyunsaturated fatty acids, sugars, and proteins into deleterious products
Free Radical Biol. Med.
2006
, vol. 
41
 (pg. 
362
-
387
)
28
Esterbauer
H.
Schaur
R.
Zollner
H.
Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes
Free Radical Biol. Med.
1991
, vol. 
11
 (pg. 
81
-
128
)
29
Spiteller
P.
Kern
W.
Reiner
J.
Spiteller
G.
Aldehydic lipid peroxidation products derived from linoleic acid
Biochim. Biophys. Acta
2001
, vol. 
1531
 (pg. 
188
-
208
)
30
Durrington
P. N.
Mackness
B.
Mackness
M. I.
Paraoxonase and atherosclerosis
Arterioscler. Thromb. Vasc. Biol.
2001
, vol. 
21
 (pg. 
473
-
480
)
31
Aviram
M.
Rosenblat
M.
Bisgaier
C. L.
Newton
R. S.
Primo-Parmo
S. L.
La Du
B. N.
Paraoxonase inhibits high-density lipoprotein oxidation and preserves its functions. A possible peroxidative role for paraoxonase
J. Clin. Invest.
1998
, vol. 
101
 (pg. 
1581
-
1590
)
32
Abe
K.
Kimura
H.
The possible role of hydrogen sulfide as an endogenous neuromodulator
J. Neurosci.
1996
, vol. 
16
 (pg. 
1066
-
1071
)
33
Hosoki
R.
Matsuki
N.
Kimura
H.
The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergy with nitric oxide
Biochem. Biophys. Res. Commun.
1997
, vol. 
237
 (pg. 
527
-
531
)
34
Furne
J.
Saeed
A.
Levitt
M. D.
Whole tissue hydrogen sulfide concentrations are orders of magnitude lower than presently accepted values
Am. J. Physiol. Regul. Integr. Comp. Physiol.
2008
, vol. 
295
 (pg. 
R1479
-
R1485
)
35
Ishigami
M.
Hiraki
K.
Umemura
K.
Ogasawara
Y.
Ishii
K.
Kimura
H.
A source of hydrogen sulfide and a mechanism of its release in the brain
Antioxid. Redox Signal.
2009
, vol. 
11
 (pg. 
205
-
214
)
36
Heinecke
J. W.
Lipoprotein oxidation in cardiovascular disease: chief culprit or innocent bystander?
J. Exp. Med.
2006
, vol. 
203
 (pg. 
813
-
816
)
37
Zhao
W.
Zhang
J.
Lu
Y.
Wang
R.
The vasorelaxant effect of H2S as a novel endogenous gaseous K(ATP) channel opener
EMBO J.
2001
, vol. 
20
 (pg. 
6008
-
6016
)