Observation for redox state of human serum and aqueous humor albumin from patients with senile cataract

doi:10.1016/S0928-4680(99)00022-X

Pathophysiology

Volume 6, Issue 4, February 2000, Pages 237-243

https://doi.org/10.1016/S0928-4680(99)00022-X Get rights and content

Abstract

Human serum albumin is known to be a mixture of mercaptalbumin (HMA, reduced form) and nonmercaptalbumin (HNA, oxidized form), i.e. a protein redox couple in serum. In order to examine the redox state of human albumin in aqueous humor together with serum, we have studied 51 senile cataract patients, including 16 men and 35 women with ages ranging between 54 and 96 years (mean±S.D. 73.9±8.51). We used a high-performance liquid chromatography combined with fluorescence detection with improved sensitivity. The patients had no detectable evidence of other eye diseases. Of considerable interest is the finding that there was an extremely small amount of HMA fraction (f(HMA)) ( $f ̄$ (HMA), 3.50%) as well as a markedly large amount of HNA fraction (f(HNA)) ( $f ̄$ (HNA-1), 84.5%; $f ̄$ (HNA-2), 12.0%) in the aqueous humor from patients with senile cataract, compared to the corresponding serum values ( $f ̄$ (HMA), 66.4%; $f ̄$ (HNA-1), 31.0%; $f ̄$ (HNA-2), 2.62%). It is anticipated that significantly decreased $f ̄$ (HMA) and increased $f ̄$ (HNA) values in aqueous humor from patients with senile cataract may clearly reflect defective antioxidative defense systems which may lead to the formation of a cataract. To the best of our knowledge, this is the first study observing the redox state of human albumin in aqueous humor.

Introduction

Human serum albumin (HSA) is a mixture of mercaptalbumin (HMA, reduced form) and nonmercaptalbumin (HNA, oxidized form), i.e. a protein redox couple in serum [1], [2]. HMA has one free sulfhydryl group in position 34 (Cys-34) and is responsible for the largest fraction of free sulfhydryl in blood sera [3]. HNA is composed of at least three kinds of compounds. Major HNA compound is mixed disulfide with cysteine or glutathione (HNA(Cys) or HNA(Glut)) [1], [2]. The other is oxidation product higher than mixed disulfide, such as sulfenic (−SOH), sulfinic (−SO₂H) and sulfonic (−SO₃H) states (HNA(Oxi)), which are extremely small in proportion in extracellular fluids [4], [5], [6]. In this sense, one of the important physiological functions of serum albumin could be to participate widely in the maintenance of a constant redox potential in the extracellular milieu (the mercapt–nonmercapt conversion of HSA).

We have developed a convenient high-performance liquid chromatographic (HPLC) system for the clear separation from HSA to HMA and HNA, using a Shodex-Asahipak GS-520H [1], [2], [7], [8], [9], [10] or an ES-502N [11] column, and we have extensively studied the mercapt–nonmercapt conversion of HSA in the elderly [9], [11] and in various pathophysiological states, such as various renal [2], [7] and hepatic [7] dysfunctions, endocrine diseases [8], and patients under anesthesia and surgery [10]. During ion-exchange HPLC analysis of HSA on an ES-502N column under some experimental conditions, such as the gradient elution profile of ethanol concentration, we have developed more sensitive and quantitative analytical HPLC system combined with fluorescence detection (FD) than had been used and reported previously [11].

Aqueous humor is a modified extracellular fluid, ≈200 μl of which is contained in the anterior chamber of the human eye. Qualitative and quantitative analyses for proteins, especially human albumin in aqueous humor using conventional HPLC systems previously performed [1], [2], [7], [8], [9], [10], [11], were limited by the microvolume of samples as well as by the low concentration of total albumin in aqueous humor [12], [13], [14], [15], [16], [17]. It was therefore decided to examine the amount of oxidized and reduced albumin in aqueous humor in living eyes with senile cataractous changes by this HPLC-FD method in order that the available data could be used as controls for further studies in various ocular diseases.

We now report an investigation for the redox state of human aqueous humor albumin (HAHA) together with HSA, from patients with senile cataract and assess the usefulness of the HPLC-FD system with improved sensitivity in the detection for the redox capacity of albumin in human aqueous humor.

Section snippets

Materials and methods

Commercial HSA was purchased from Calbiochem Behring Co. (Lot 701507, USA). HSA was dissolved in 2-fold diluted solution of 0.10 M sodium phosphate-0.30 M NaCl (pH 6.87) without further purification. The HSA solution was then filtered through a filter unit (0.45 μm, Millex-HV, Millipore Co.). The concentrations of HSA were adjusted to the desired concentrations.

Sera from healthy young male subjects, who had no renal and hepatic dysfunctions, were obtained at Gifu University School of Medicine.

HPLC studies on HSA from the healthy young male subjects

HSA is known to be a mixture of HMA and HNA. Furthermore, there are several kinds of HNA, i.e. HNA(Cys), HNA(Glut) and HNA(Oxi). A clear separation from HSA to HMA and HNA using a HPLC system was first reported by Sogami et al. in 1984 [1]. They demonstrated that the HPLC system had a high resolution for the analysis of HSA from a small serum sample. Since previously performed HPLC analyses [1], [2], [7], [8], [9], [10] did not show a good resolution among HNAs, we have examined various

Discussion

There is overwhelming evidence today that oxidative stress in cell and tissues usually refers to an increased generation of oxygen-derived species, i.e. superoxide radical (O radical dot ⁻₂), hydrogen peroxide (H₂O₂), hydroxyl radical (OH) and lipid peroxide (LOOH) [18]. Extracellular fluids contain low-molecular-mass antioxidants, such as ascorbic acid, vitamin E, uric acid, thiols etc., which are actively involved in the defense against reactive oxygen species [19]. Those also contain only small amounts

Acknowledgements

The authors are grateful to Dr Masaru Sogami (Emeritus Professor of Gifu University, Japan), Dr Saburo Hayano (Emeritus Professor of Gifu University, Japan) and Dr Katsunobu Takahashi (Takahashi Hospital, Japan) for their kind discussion and helpful comments. This study was partly supported by a grant (RENKEI Project Grant in 1997) from Gifu University to one of the authors (H.I.).

References (36)

J. Janatova et al.
The heterogeneity of bovine albumin with respect to sulfhydryl and dimer content
J. Biol. Chem.
(1968)
J.K.F. Noel et al.
Bovine mercaptalbumin and non-mercaptalbumin monomers. Interconversions and structural differences
J. Biol. Chem.
(1972)
K. Wallevik
SS-interchanged and oxidized isomers of bovine serum albumin separated by isoelectric focusing
Biochim. Biophys. Acta
(1976)
M. Sogami et al.
High-performance liquid chromatographic studies on non-mercapt⇄mercapt conversion of human serum albumin. II
J. Chromatogr.
(1985)
E. Suzuki et al.
Increased oxidized form of human serum albumin in patients with diabetes mellitus
Diabetes Res. Clin. Pract.
(1992)
S. Era et al.
Age-related change in redox state of human serum albumin
Biochim. Biophys. Acta
(1995)
A. Hayakawa et al.
Alteration of redox state of human serum albumin in patients under anesthesia and invasive surgery
J. Chromatogr. B
(1997)
R.C. Tripathi et al.
Protein composition of human aqueous humor: SDS-PAGE analysis of surgical and post-mortem samples
Exp. Eye Res.
(1989)
B. Halliwell et al.
Oxygen free radicals and iron in relation to biology and medicine: some problems and concepts
Arch. Biochem. Biophys.
(1986)
B. Halliwell
Albumin — an important extracellular antioxidant?
Biochem. Pharmacol.
(1988)

J.A. Thomas et al.

Protein sulfhydryls and their role in the antioxidant function of protein S-thiolation

Arch. Biochem. Biophys.

(1995)

B.S. Berlett et al.

Protein oxidation in aging, disease, and oxidative stress

J. Biol. Chem.

(1997)

R. Radi et al.

Peroxynitrite oxidation of sulfhydryls

J. Biol. Chem.

(1991)

J.W. Finch et al.

Mass spectrometric identification of modifications to human serum albumin treated with hydrogen peroxide

Arch. Biochem. Biophys.

(1993)

M. Soriani et al.

Antioxidant potential of anaerobic human plasma: role of serum albumin and thiols as scavengers of carbon radicals

Arch. Biochem. Biophys.

(1994)

H. Zhang et al.

S-nitrosation of serum albumin: spectrophotometric determination of its nitrosation by simple S-nitrosothiols

Anal. Biochem.

(1996)

R.M. Gatti et al.

Peroxynitrite-mediated oxidation of albumin to the protein-thiyl free radical

FEBS Lett.

(1994)

A. Spector et al.

Hydrogen peroxide and human cataract

Exp. Eye Res.

(1981)

Cited by (59)

Role of albumin Cys34 redox state in the progression of differentiated thyroid carcinoma and induction of ferroptosis
2023, Free Radical Biology and Medicine
Differentiated thyroid cancer (DTC) is the most prevalent endocrine malignancy worldwide and requires effective prognostic markers and therapeutic targets to optimize patient outcomes. This study investigated the potential of human serum albumin (HSA) cysteine-34 (Cys34) redox state as a prognostic indicator and therapeutic avenue for DTC. A retrospective cohort study of 99 patients with DTC undergoing radioactive iodine therapy found that higher concentrations of HSA with the reduced form of Cys34 (i.e., human mercaptalbumin [HMA]) were associated with improved progression-free survival in metastatic DTC. In vitro experiments using a DTC cell line revealed that HMA induced cytotoxic effects by triggering ferroptosis, characterized by lipid peroxidation, intracellular ROS accumulation, and decreased cell viability. Ferroptosis inhibitors rescued cell viability, confirming their role in cytotoxicity. These results implicate the HSA-Cys34 redox state is a promising avenue for precision medicine in DTC, shedding light on the prognostic relevance and therapeutic potential of HMA-induced ferroptosis. They emphasize the opportunity for personalized treatment strategies to advance the management of patients with DTC.
A lower ratio of reduced to total albumin in serum is associated with protein nutritional status of pregnant women in Japan
2023, Nutrition Research
Protein-energy undernutrition is potentially prevalent among Japanese pregnant women, and biomarkers that objectively indicate the protein nutritional status during pregnancy may help in implementing appropriate protein supplementation to these women. We hypothesized that a serum parameter of pregnant women, the ratio of reduced to total albumin (reduced ALB ratio), would be associated with protein intake during pregnancy. The serum reduced ALB ratio of pregnant women was compared with protein intake and with gestation outcomes (gestation length and infant birth weight) in an observational study of 115 Japanese pregnant women. The serum reduced ALB ratio in the third trimester tended to be positively correlated with gestation length (P = .07). Infant birth weights tended to be different between protein intake tertiles (P = .09); the mean infant birth weight was higher in the third tertile compared with the first and second tertiles. The protein intake of pregnant women was significantly and positively correlated with the serum reduced ALB ratio in the second trimester. The serum reduced ALB ratio reflects protein nutritional status during pregnancy and may contribute to healthier gestation outcomes.
A more oxidized plasma albumin redox state and lower plasma HDL particle number reflect low-protein diet ingestion in adult rats
2020, Journal of Nutrition
Plasma albumin (ALB) redox state reflects protein nutritional status, but how it differs from other protein nutrition biomarkers remains to be fully elucidated.
This study aimed to delineate the characteristics of plasma ALB redox state as a protein nutrition biomarker.
Adult male Wistar rats were maintained on an AIN-93 M [14% casein, control (CT)] diet or an AIN-93 M-based 5% casein [low protein (LP)] diet ad libitum for 4 wk. Plasma samples were repeatedly obtained from the same rats at weeks 0–4, ALB redox state was determined by HPLC, and the concentrations of conventional protein nutrition biomarkers, ALB and transthyretin (TTR), were compared between the groups by Student t test. Body mass, relative muscle masses, plasma proteome, and plasma lipids at week 4 were also compared.
Plasma ALB redox state shifted to a more oxidized state in the LP diet group compared with the CT diet group at weeks 1–4. The LP diet group also showed significantly lower plasma ALB concentrations at weeks 1 and 2 (13% and 11% lower, respectively) and significantly lower TTR concentration at week 1 (21% lower) compared with the CT diet group, but these concentrations did not differ significantly at weeks 3 and 4. After 4 wk, body mass and relative soleus and gastrocnemius muscle masses did not differ, but the relative plantaris muscle mass tended to be 4% lower (1.75 compared with 1.68 g/kg body mass) in the LP diet group compared with the CT group (P = 0.06). The LP diet group also had a significantly lower HDL particle number than the CT group (30% lower).
A more oxidized plasma ALB redox state and lower plasma HDL particle number reflect LP diet ingestion in adult rats, which did not exhibit changes of plasma ALB and TTR concentrations.
Structural and functional integrity of human serum albumin: Analytical approaches and clinical relevance in patients with liver cirrhosis
2017, Journal of Pharmaceutical and Biomedical Analysis
Citation Excerpt :
Conformational alterations related to the thiol redox status make IEC a suitable approach for the analysis of HSA isoforms bearing a modification at the Cys34 [61]. Back in 1984, Sogami described for the first time the separation of HMA from HNA isoforms [62,63], while only in 2000 the separation of all three isoforms (HMA, HNA1 and HNA2) was achieved [64]. Briefly, the proposed method employed IEC, an ethanol gradient, and UV–vis or fluorescence detection.
Human serum albumin (HSA) is the most abundant circulating plasma protein. Besides a significant contribution to the osmotic pressure, it is also involved in the fine regulation of many other physiological processes, including the balance of the redox state, the inflammatory and/or immunological responses, and the pharmacokinetic and pharmacodynamics of many drugs. Growing evidence suggests that HSA undergoes structural and functional damage in diseases characterized by an enhanced systemic inflammatory response and oxidative stress, as it occurs in chronic liver disease. Based on their clinical relevance, this review provides a summary of the most common post-translational modifications affecting HSA structural integrity and functions and their clinical relevance in the field of liver disease. The review also provides a critical description of the analytical approaches employed for the investigation of conformational alterations and the identification/quantitation of specific post-translational modifications affecting HSA. Finally, the analytical methods available for the assessment of two of the most clinically relevant non-oncotic properties of HSA, namely the binding capacity and the antioxidant activity, are critically reviewed. Among the available techniques particular attention is given to those proposed for the in vitro and in vivo investigation of structurally modified albumin.
The reduced/oxidized state of plasma albumin is modulated by dietary protein intake partly via albumin synthesis rate in rats
2017, Nutrition Research
Citation Excerpt :
This free Cys34 is conserved in all mammalian cases that have been investigated, and the residue grants the microheterogeneous structures because of its susceptibility to oxidation. Albumin can be chromatographically separated into 3 fractions according to Cys34 status [4-6]: the free thiol form designated as mercaptalbumin (MA); the disulfide bond with a small compound, such as Cys, homocysteine, or glutathione, designated as nonmercaptalbumin-1 (NA-1); and a higher oxidized form of Cys, such as sulfinic or sulfonic acid, designated as nonmercaptalbumin-2 (NA-2) [7]. MA accounts for >70% of the total plasma albumin in healthy adults [8], but the ratio decreases with age [4,9-11], several diseases [12], and strenuous exercise [13,14].
The reduced/oxidized state of plasma albumin is influenced by many factors, including chronic diseases and strenuous training. Recently, the reduced/oxidized state has also been shown to be associated with dietary protein and energy intakes in rats. We hypothesized that dietary protein intake may modulate the reduced/oxidized state of plasma albumin by altering the rate of albumin synthesis and that the reduced/oxidized state could therefore serve as a novel marker of protein undernutrition. We tested this hypothesis by examining male growing rats placed on a low-protein or energy-restriction diet. In the 4-week experiment, animals fed a low-protein diet (3% casein), whose dietary intakes were lower than those fed control diet (20% casein), showed significant decreases in plasma albumin level and the ratio of the reduced form of albumin to total albumin. Animals given the same amount of control diet as the low-protein diet group (approximately 30% energy restriction) also showed the above decreases, albeit to much more limited extents. The ratio of reduced to total plasma albumin correlated significantly with plasma albumin fractional synthesis rate. When animals were maintained on the low-protein diet for as long as 12 weeks and then fed the control diet for 1 week, the decreased ratio of reduced to total plasma albumin, but not plasma albumin level, resolved rapidly. The reduced/oxidized state of plasma albumin would thus reflect dietary protein status via plasma albumin turnover including the fractional synthesis rate and could prove useful as a sensitive marker of protein undernutrition.
Comparative studies on the heterogeneity of plasma-derived and recombinant human albumins in laboratory use
2014, International Journal of Biological Macromolecules
Citation Excerpt :
Albumin exhibits a wide variety of physiological and pharmacological functions, including osmotic pressure regulation, binding and transport capacities of endogenous and exogenous compounds, and antioxidant properties of human plasma [3]. Regarding the pathophysiological aspects of HSA, the thiol-redox state of HSA is used as a valuable biomarker of oxidative stress [4,5] in many diseases, such as renal disease [6–9], liver disease [10,11], diabetes mellitus [12], senile cataract [13,14], and joint osteoarthritis [15]. Moreover, clinically measured HSA glycation levels may represent a potential clinical utility for diabetes mellitus as well as that of HbA1c, because it reflects glycemia over two to three weeks [16].
We investigated the thiol-redox state, and the relationship between structural characteristics, such as thermal stability, and functional properties, such as cell growth activity, of commercial plasma-derived (pHSA) and recombinant human serum albumin (rHSA) products. In this study, 5 pHSA products (A1653, A9511, A1887, A8763, and A3782) and 2 rHSA products (A9731 and A9986) were obtained from Sigma-Aldrich. Among them, three kinds of HSA products [A1653 (an initial fractionation product), A3782 (a final purified product), and A9731 (recombinant albumin expressed in rice)] were selected for experiments on the thermal stabilities, analyzed by thermal denaturation curves, and cell growth activities of U937 and THP-1 cell lines using the WST-1 reagent. The secondary and tertiary structures of HSA products were similar, whereas a marked difference was observed in their thermal stabilities. The degree of thermal stability of the three representative products was in the order of A9731 (rHSA) > A1653 (pHSA) > A3782 (pHSA), as was the degree of cell growth activity of these products. One possible explanation for the present results is that albumin-bound fatty acids may have influenced the thermal stabilities and cell growth activities of U937 and THP-1 cells.

View all citing articles on Scopus

View full text

Observation for redox state of human serum and aqueous humor albumin from patients with senile cataract

Abstract

Introduction

Section snippets

Materials and methods

HPLC studies on HSA from the healthy young male subjects

Discussion

Acknowledgements

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Chromatogr.

Diabetes Res. Clin. Pract.

Biochim. Biophys. Acta

J. Chromatogr. B

Exp. Eye Res.

Arch. Biochem. Biophys.

Biochem. Pharmacol.

Arch. Biochem. Biophys.

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

Anal. Biochem.

FEBS Lett.

Exp. Eye Res.