An Essential Difference between the Flavonoids MonoHER and Quercetin in Their Interplay with the Endogenous Antioxidant Network

Antioxidants can scavenge highly reactive radicals. As a result the antioxidants are converted into oxidation products that might cause damage to vital cellular components. To prevent this damage, the human body possesses an intricate network of antioxidants that pass over the reactivity from one antioxidant to another in a controlled way. The aim of the present study was to investigate how the semi-synthetic flavonoid 7-mono-O-(β-hydroxyethyl)-rutoside (monoHER), a potential protective agent against doxorubicin-induced cardiotoxicity, fits into this antioxidant network. This position was compared with that of the well-known flavonoid quercetin. The present study shows that the oxidation products of both monoHER and quercetin are reactive towards thiol groups of both GSH and proteins. However, in human blood plasma, oxidized quercetin easily reacts with protein thiols, whereas oxidized monoHER does not react with plasma protein thiols. Our results indicate that this can be explained by the presence of ascorbate in plasma; ascorbate is able to reduce oxidized monoHER to the parent compound monoHER before oxidized monoHER can react with thiols. This is a major difference with oxidized quercetin that preferentially reacts with thiols rather than ascorbate. The difference in selectivity between monoHER and quercetin originates from an intrinsic difference in the chemical nature of their oxidation products, which was corroborated by molecular quantum chemical calculations. These findings point towards an essential difference between structurally closely related flavonoids in their interplay with the endogenous antioxidant network. The advantage of monoHER is that it can safely channel the reactivity of radicals into the antioxidant network where the reactivity is completely neutralized

Long‐term effect of calcium supplementation on bone loss in perimenopausal women

We observed in a controlled 2 year longitudinal trial in 248 perimenopausal women that a daily calcium supplement of either 1000 or 2000 mg Ca2+ significantly reduced lumbar bone loss and bone turnover in the first year of calcium supplementation. In the second supplementation year the rate of lumbar bone loss in the treated subjects was not significantly different from that in the control group, although two of the three biochemical parameters of bone turnover remained decreased throughout the study. To quantify further the long‐term effect of calcium supplementation, we extended the study for another year in 214 women. In the women of the control group who were menstruating until the last year of the trial, the mean change in lumbar bone mineral density after 3 years was –3.2% of the initial value versus 1.6% in the calcium‐supplemented groups (p < 0.01). The decrease in lumbar bone loss in these supplemented premenopausal and early perimenopausal women remained statistically significant in the second and third years of supplementation. In the women who stopped menstruating before or during the study, the long‐term reduction in lumbar bone loss was not significant (mean difference between control and treatment groups <0.6% points after 3 years). The decrease in metacarpal cortical thickness (MCT) in the treated subjects during 3 years was on average –3.0% of the initial value in the control versus–2.0% in the supplemented subjects (P < 0.01). The effect of calcium supplementation on MCT was not significantly related to the menopausal status of the subjects. Serum alkaline phosphatase, osteocalcin, and urinary hydroxyproline excretion decreased after calcium supplementation in all menopausal groups. These parameters remained decreased throughout the trial, with exception of alkaline phosphatase in the 1000 mg calcium group. We conclude that calcium supplementation substantially reduces cortical and trabecular bone loss in the years immediately preceding menopause. Although it reduces postmenopausal cortical bone loss to some extent, it does not prevent the menopause‐related lumbar bone loss. Copyright © 1994 ASBM

Interplay of monoHER and quercetin with the endogenous antioxidant network.

<p>(A) Schematic representation of the endogenous antioxidant network. Free radicals are scavenged by antioxidants in the network, such as GSH and ascorbate. In this way, free radicals are neutralized. Free radicals that are not neutralized can damage e.g. proteins, lipids and DNA. (B) The flavonoid quercetin is an excellent radical scavenger. During the scavenging of free radicals quercetin becomes oxidized. After oxidation of quercetin, four tautomeric forms of the oxidation product can be formed. In the figure the tautomer which has an abundance of more than 99% is shown. When ascorbate and GSH are present in the same concentration, oxidized quercetin reacts much faster with GSH than with ascorbate, thereby forming 6-GSH-quercetin and 8-GSH-quercetin. Because of its high reactivity towards thiols, oxidized quercetin is also prone to react with protein thiols, as was seen in human blood plasma. This reaction of oxidized quercetin is not prevented by ascorbate and can lead to toxicity. (C) The oxidation product formed out of monoHER is an ortho-quinone. Ascorbate recycles this oxidation product to the parent compound monoHER, while GSH forms a conjugate with oxidized monoHER, i.e. 2′-GSH-monoHER. When both compounds are present in the same concentration, oxidized monoHER reacts rather with ascorbate (73%) than with GSH (27%). The oxidized ascorbate formed in this recycling can be regenerated in the network, e.g. by dehydroascorbate reductase (DHAR) that uses NADH as cofactor. Thus, the advantage of monoHER is that it can safely channel the non-specific reactivity of radicals toward ascorbate, which can be regenerated in the antioxidant network.</p

Spectrophotometrical analyses.

<p>Spectrophotometrical analysis of the incubation mixture containing (A) 50 µM monoHER, 1.6 nM horseradish peroxidase (HRP) and 33 µM H₂O₂. The same experiment was carried out in (B) the presence of 40 µM GSH, (C) 40 µM ascorbate and (D) both 40 µM GSH and 40 µM ascorbate. The UV/Vis scans were recorded 30, 150 and 300 seconds after the addition of HRP. A typical example is shown.</p

Chemical reaction of oxidized monoHER with ascorbate.

<p>(A) Chemical structure of oxidized monoHER (left) and ascorbate (right). The active part of ascorbate and the active part of oxidized monoHER are indicated by the red ellipses. (B) Suggested route for the reaction of oxidized monoHER with ascorbate. Only the active parts are shown to illustrate the suggested mechanism more clearly. (1) The active part of ascorbate (top) approaches the active part of oxidized monoHER (bottom) due to a π-π interaction and a hydrogen bond. The π-electrons of ascorbate are used to create a new bond. The C3 of ascorbate will most likely attack the C3′ of the monoHER quinone because it is more electron deficient than the C4′ according to Spartan ‘06. (2) After the attack, a transition state, with an <i>sp3</i> bond between ascorbate and oxidized monoHER, is suggested to be formed. (3) This intermediate rapidly decomposes into monoHER and oxidized ascorbate. The driving force of this reaction is the restoration of the highly conjugated π-system of monoHER.</p

Reactivity of oxidized monoHER and oxidized quercetin towards thiols.

<p>Thiol content of the incubation mixture containing 50 µM monoHER or quercetin, 1.6 nM HRP and 33 µM H₂O₂ in the presence of either (A) 40 µM GSH, (B) both 40 µM GSH and 40 µM ascorbate, (C) human blood plasma or (D) 400 µM albumin (BSA) (with or without 40 µM ascorbate). The thiol content of the different incubation mixtures was measured 5 minutes after the addition of HRP. All measurements were carried out in triplicate and data are expressed as mean ± SD. *P<0.05 compared to control.</p

HPLC analyses.

<p>HPLC analysis of the incubation mixture containing (A) 50 µM monoHER, 1.6 nM horseradish peroxidase (HRP) and 33 µM H₂O₂. The same experiment was carried out in (B) the presence of 40 µM GSH, (C) 40 µM ascorbate and (D) both 40 µM GSH and 40 µM ascorbate. The different incubation mixtures were injected on the HPLC system 5 minutes after the addition of HRP. A typical example is shown. The retention time of monoHER is 6.7 min and that of 2′-GSH-monoHER is 5.4 min. The initial peak height of monoHER before oxidation was 88 mAU, corresponding to a concentration of 50 µM. After 5 min of oxidation the monoHER concentrations in the incubation mixtures A, B, C and D were 22.5 µM, 22.5 µM, 50 µM and 43.5 µM, respectively.</p

MonoHER and ascorbate consumption rates.

<p>Consumption of monoHER and ascorbate in the incubation mixtures containing 50 µM monoHER, 1.6 nM HRP and 33 µM H₂O₂ in the presence of either 40 µM GSH, 40 µM ascorbate or both 40 µM GSH and 40 µM ascorbate. The incubation time was 5 minutes. All measurements were carried out in triplicate and data are expressed as mean ± SD.</p