Physiologic concentrations of homocysteine inhibit the human plasma GSH peroxidase that reduces organic hydroperoxides

doi:10.1067/mlc.2000.107692

Journal of Laboratory and Clinical Medicine

Volume 136, Issue 1, July 2000, Pages 58-65

https://doi.org/10.1067/mlc.2000.107692 Get rights and content

Abstract

The plasma reduced glutathione (GSH) selenoperoxidase is a highly conserved enzyme. Furthermore, a small clinical study reported that patients with severe atherosclerosis had low peroxidase activities. Together these observations suggest that the peroxidase is important in preventing atherosclerosis. Yet others have reported that when the assay was run in Tris buffer, it was inactive with the concentrations of GSH found in the plasma. Second, it is known that hyperhomocysteinemia increases the rate of atherogenesis. Because there is some homology between homocysteine and the cysteine in GSH, the question is whether the hyperhomocysteinemia effect may be due to inhibition of the peroxidase. We purified the peroxidase from human plasma and determined its activity by a coupled spectrophotometric assay and a substrate disappearance chemiluminescence assay. When the peroxidase activity was determined in phosphate-buffered saline solution (PBS), there was significant activity with the reported plasma GSH concentrations (5 to 20 μmol/L). The peroxidase was exclusively in the HDL fraction. There was no correlation between the peroxidase activity and the HDL or LDL cholesterol concentrations. Finally, at physiologic concentrations of GSH (9 μmol/L), the peroxidase was inhibited by physiologic, free homocysteine concentrations (1 to 5 μmol/L). These data suggest that the peroxidase is active in vivo and may be important in protecting the endothelium from atherosclerosis by preventing oxidant injury. The homocysteine inhibition of the peroxidase suggests a possible biochemical basis for the observed association between hyperhomocysteinemia and cardiovascular disease. Our studies imply that low concentrations of this peroxidase may be an independent risk factor for atherosclerosis. (J Lab Clin Med 2000;136:58-65)

Section snippets

Materials

SDS, acrylamide, tert -butyl hydroperoxide, and GSH were purchased from Sigma Chemical Co, St Louis, MO. All other chemicals were ACS reagent grade.

Determination of GSH peroxidase activity

Peroxidase activity was determined by two independent methods. In the first we utilized a coupled, kinetic, spectrophotometric assay in which the incubation mixture (0.7 mL) containing PBS (pH 7.6), NADPH (1 mmol/L), GSH (4.5 μmol/L to 1 mmol/L), GSH reductase (3.0 U/mL), and tert -butyl hydroperoxide (400 μmol/L) were preincubated for 10 minutes at

Results

The purified plasma enzyme used in these studies was homogeneous as determined by silver stained, SDS-PAGE gels (Fig 1).

. Silver stain of the purified plasma GSH peroxidase. Channels 1 and 2 represent the native gel of purified peroxidase; channels 3 and 4 represent the reduced gel.

The peroxidase is a tetramer in nondenaturing gels, with a M_r of 88 kd (Fig 1, channels 1 and 2 ), while in a denaturing gel it migrates as a monomer with a M_r of 22 kd (Fig 1; channels 3 and 4 ). Seven tryptic

Discussion

We feel that these data indicate two new and important points. The first is that this peroxidase does show significant enzymatic activity with the concentrations of GSH that have been reported to be in the plasma. This would indicate, contrary to the studies of Bjornstedt et al,²⁵ that it is not necessary to postulate an alternative source of reducing equivalents to catalyze the peroxidase activity.

Second, we feel that the most important finding in these studies is that high physiologic plasma

References (48)

R Brigelius-Flohe et al.
Phospholipid-hydroperoxide glutathione peroxidase: genomic DNA, cDNA and deduced amino acid sequence
J Biol Chem
(1994)
M Arai et al.
Mitochondrial phospholipid hydroperoxide glutathione peroxidase plays a major role in preventing oxidative injury to cells
J Biol Chem
(1999)
KR Maddipati et al.
Characterization of the major hydroperoxide-reducing activity of human plasma. Purification and properties of a selenium-dependent glutathione peroxidase
J Biol Chem
(1987)
K Takahashi et al.
Purification and characterization of human plasma glutathione peroxidase: a selenoglycoprotein distinct from the known cellular enzymes
Arch Biochem Biophys
(1987)
RL Maser et al.
Mouse plasma glutathione peroxidase: cDNA sequence analysis and renal proximal tubular expression and secretion
J Biol Chem
(1994)
M Yasuda et al.
Simultaneous determination of phospholipid hydroperoxides and cholesteryl ester hydroperoxides in human plasma by high-performance liquid chromatography with chemiluminescence detection
J Chromatogr B Biomed Sci Appl
(1997)
A Zamburlini et al.
Direct measurement by single photon counting of lipid hydroperoxides in human plasma and lipoproteins
Anal Biochem
(1995)
JT Salonen et al.
Autoantibodies against oxidised LDL and progression of carotid atherosclerosis
Lancet
(1992)
AD Blann et al.
Antioxidants, von Willebrand factor and endothelial cell injury in hyper-cholesterolaemia and vascular disease
Atherosclerosis
(1995)
M Bjornstedt et al.
The thioredoxin and glutaredoxin systems are efficient electrons donors to human plasma glutathione peroxidase
J Biol Chem
(1994)

ME Anderson et al.

Dynamic state of glutathione in blood plasma

J Biol Chem

(1980)

H Nakamura et al.

Measurement of plasma glutaredoxin and thioredoxin in healthy volunteers and during open-heart surgery

Free Radic Biol Med

(1998)

A Araki et al.

Determination of free and total homocysteine in human plasma by high-performance liquid chromatography with fluorescence detection

J Chromatogr

(1987)

Y Yamamoto et al.

Glutathione peroxidase isolated from plasma reduces phospholipid hydroperoxides

Arch Biochem Biophys

(1993)

E Damiani et al.

Endoplasmic reticulum of rat liver contains two proteins closely related to skeletal sarcoplasmic reticulum Ca-ATPase and calsequestrin

J Biol Chem

(1988)

SP Srivastava et al.

Purification and characterization of a new isozyme of thiol:protein disulfide oxidoreductase from rat hepatic microsomes: relationship of this isozyme to cytosolic, phosphatidylinositol specific phospholipase C form 1A

J Biol Chem

(1991)

BH Chung et al.

Single vertical spin density gradient ultracentrifugation

Methods Enzymol

(1986)

RS Esworthy et al.

Characterization and partial amino acid sequence of human plasma glutathione peroxidase

Arch Biochem Biophys

(1991)

G Alfthan et al.

Relation of serum homocysteine and lipoprotein(a) concentrations to atherosclerotic disease in a prospective Finnish population based study

Atherosclerosis

(1994)

M Maiorini et al.

Phospholipid hydroperoxide glutathione peroxidase

Methods Enzymol

(1990)

K Takahashi et al.

Primary structure of human plasma glutathione peroxidase deduced from cDNA sequences

J Biochem

(1990)

S Parthasarathy et al.

Probucol inhibits oxidative modification of low density lipoprotein

J Clin Invest

(1986)

S Parthasarathy et al.

Low density lipoprotein rich in oleic acid is protected against oxidative modification: implications for dietary prevention of atherosclerosis

Proc Natl Acad Sci USA

(1990)

D Steinberg et al.

Beyond cholesterol: modifications of low-density lipoprotein that increase its atherogenicity

N Engl J Med

(1989)

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^☆: Supported in part by the General Medical Research Service of the Department of Veterans Affairs and by a grant from the Minnesota Affiliate of the American Heart Association.

^☆☆: Reprint requests: Jordan L. Holtzman, MD, PhD, Chief, Section on Therapeutics (111T), Veterans Affairs Medical Center, One Veterans Drive, Minneapolis, MN 55417.

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Original ArticlesPhysiologic concentrations of homocysteine inhibit the human plasma GSH peroxidase that reduces organic hydroperoxides☆,☆☆

Abstract

Section snippets

Materials

Determination of GSH peroxidase activity

Results

Discussion

J Biol Chem

J Biol Chem

J Biol Chem

Arch Biochem Biophys

J Biol Chem

J Chromatogr B Biomed Sci Appl

Anal Biochem

Lancet

Atherosclerosis

J Biol Chem

J Biol Chem

Free Radic Biol Med

J Chromatogr

Arch Biochem Biophys

J Biol Chem

J Biol Chem

Methods Enzymol

Arch Biochem Biophys

Atherosclerosis

Phospholipid hydroperoxide glutathione peroxidase

Methods Enzymol

Primary structure of human plasma glutathione peroxidase deduced from cDNA sequences

J Biochem

Probucol inhibits oxidative modification of low density lipoprotein

J Clin Invest

Low density lipoprotein rich in oleic acid is protected against oxidative modification: implications for dietary prevention of atherosclerosis

Proc Natl Acad Sci USA

Beyond cholesterol: modifications of low-density lipoprotein that increase its atherogenicity

N Engl J Med

Original Articles
Physiologic concentrations of homocysteine inhibit the human plasma GSH peroxidase that reduces organic hydroperoxides☆,☆☆