Increased hepatic lipid soluble antioxidant capacity as compared to other organs of streptozotocin-induced diabetic rats: a cyclic voltammetry study

It has been suggested that oxidative stress plays an important role in the chronic complications of diabetes. The experimental findings regarding the changes in tissue antioxidant enzymes and lipid peroxidation of diabetic tissues have been inconsistent. Previous studies in our laboratory demonstrated that the reducing power of a specific tissue correlates with its low molecular weight antioxidant (LMWA) capacity. In the present study, the overall LMWA capacity (reducing equivalents) of plasma and tissues of streptozotocin (STZ)-induced diabetic rats (1-4 weeks) and insulin treated diabetic rats were measured by cyclic voltammetry. Levels of water and lipid soluble LMWA capacity progressively decreased in the diabetic plasma, kidney, heart and brain, while the diabetic liver, at 2, 3 and 4 weeks after STZ injection, showed a significant increase in the overall lipid soluble LMWA capacity (p < 0.001). Subsequently, analysis of specific components by high pressure liquid chromatography (electrochemical detection) showed decreased levels of ascorbic acid in plasma, kidney, heart and brain of diabetic animals. The alpha-tocopherol level dropped in all tissues, except for the liver in which there was a significant increase (p < 0.01 and p < 0.001 at 2-4 weeks). Lipid peroxidation was assessed by conjugated diene levels, which increased significantly in all diabetic tissues except the liver. Insulin treatment that was started after 3 weeks of diabetes and continued for 3 weeks showed no change in the conjugated dienes and in the overall LMWA capacity in all organs. Our results suggest a unique behavior of the liver in the STZ-induced diabetic rats to the stress and indicate its higher capacity to cope with oxidative stress as compared to other organs.     

Increased hepatic lipid soluble antioxidant capacity as compared to other organs of streptozotocin-induced diabetic rats: A cyclic voltammetry study

It has been suggested that oxidative stress plays an important role in the chronic complications of diabetes. The experimental findings regarding the changes in tissue antioxidant enzymes and lipid peroxidation of diabetic tissues have been inconsistent. Previous studies in our laboratory demonstrated that the reducing power of a specific tissue correlates with its low molecular weight antioxidant (LMWA) capacity. In the present study, the overall LMWA capacity (reducing equivalents) of plasma and tissues of streptozotocin (STZ)-induced diabetic rats (1–4 weeks) and insulin treated diabetic rats were measured by cyclic voltammetry. Levels of water and lipid soluble LMWA capacity progressively decreased in the diabetic plasma, kidney, heart and brain, while the diabetic liver, at 2, 3 and 4 weeks after STZ injection, showed a significant increase in the overall lipid soluble LMWA capacity (p < 0.001). Subsequently, analysis of specific components by high pressure liquid chromatography (electrochemical detection) showed decreased levels of ascorbic acid in plasma, kidney, heart and brain of diabetic animals. The α-tocopherol level dropped in all tissues, except for the liver in which there was a significant increase (p < 0.01 and p < 0.001 at 2–4 weeks). Lipid peroxidation was assessed by conjugated diene levels, which increased significantly in all diabetic tissues except the liver. Insulin treatment that was started after 3 weeks of diabetes and continued for 3 weeks showed no change in the conjugated dienes and in the overall LMWA capacity in all organs. Our results suggest a unique behavior of the liver in the STZ-induced diabetic rats to the stress and indicate its higher capacity to cope with oxidative stress as compared to other organs.     

Examination of the early phase of insulin secretion with 3 successive small overloads of glucose (5 g) in normal and obese subjects]

We studied the E.I.R. in eight normal subjects and fifteen obese ones with three successive small glucose pulses (5 g.) e.v. at 30' interval. In normal subjects the three successive loads gave rise to identical responses for both glucose and I.R.I. Obese could be divided, on the basis of their E.I.R. to the first load, into normal responders (group I), hyper-responders (group II) and hypo-résponders (group III); on the basis of the E.I.R. to the second load, group I could be divided in two subgroups: Ia and Ib. We found an identical E.I.R. to all glucose loads in group Ia; a reduced E.I.R. to successive loads in groups Ib and II. Group III didn't have any insulin response to all glucose loads.     

Optimization of ascorbic and uric acid separation in human plasma by free zone capillary electrophoresis ultraviolet detection

In this paper we propose a new fast free zone capillary electrophoresis method for the simultaneous determination of ascorbic acid (AA) and uric acid (UA) in human plasma. We investigated the effect of analytical parameters, such as concentration and pH of borate running buffer, cartridge temperature, and sample treatment, on resolution, migration times, corrected peak areas, and efficiency. A good separation was achieved using a 60.2-cmx75-microm uncoated silica capillary and 100 mmol/L sodium borate buffer, pH 8, when metaphosphoric acid was employed as protein precipitant, in less than 4 min. These conditions gave a good reproducibility of migration times (CV 0.35 and 0.34%) and peak areas (CV 3.2 and 3.1%) for ascorbate and urate, respectively. The limit of detection was 0.5mg/L for both analytes when the detection was performed at 254 nm for AA and at 292 nm for UA. We compared the present method with a validated capillary electrophoresis assay by measuring plasma urate and ascorbate in 32 normal subjects and the obtained data were analyzed by the Passing and Bablok regression.     

"Antioxidant" (reducing) efficiency of ascorbate in plasma is not affected by concentration

Ascorbate (vitamin C), an important dietary derived antioxidant, reportedly shows decreasing "antioxidant efficiency" with increasing concentrations in indirect radical trapping methods of antioxidant capacity. This study investigated the effect of concentration on antioxidant efficiency of ascorbate using a direct test of antioxidant capacity, the ferric reducing/antioxidant power test (FRAP assay). Results showed that the antioxidant efficiency factor of ascorbate was 2 and was constant over a wide concentration range in both plasma and pure aqueous solution. However, the absolute amount of ascorbate lost per unit of time increased with concentration. Furthermore, ascorbate was less stable in plasma than in aqueous solutions of similar pH and less stable in ethylenediamine tetraacetic acid (EDTA) than in heparinized plasma. Results indicate that previously reported concentration-dependent changes in antioxidant efficiency of ascorbate may have been caused by loss of ascorbate prior to and during testing, and by methodologic characteristics of indirect peroxyl radical trapping tests of antioxidant capacity. Therefore, it is suggested that the premise that the antioxidant efficiency of ascorbate is concentration-dependent is largely methodologically derived and does not reflect the antioxidant behavior of ascorbate per se.     

Role of reactive oxygen species (ROS) in the diabetes-induced anomalies in rat embryos in vitro: reduction in antioxidant enzymes and low-molecular-weight antioxidants (LMWA) may be the causative factor for increased anomalies

A disturbed embryonic antioxidant defense mechanism may play a major role in diabetes-induced teratogenesis. We therefore studied the antioxidant capacity of 10.5-day-old rat embryos and their yolk sacs after culture for 28 hr in vitro under diabetic conditions (3 mg/ml glucose, 2 mg/ml beta-hydroxybutyrate (BHOB) and 10 microg/ml of acetoacetate), as compared with control embryos in vitro. We found a high rate of congenital anomalies, decreased growth and protein content, and a decrease in the activity of both superoxide dismutase (SOD) and catalase (CAT) under diabetic conditions, as compared with controls. The reducing power, which reflects the concentration and type of water-soluble and of lipid-soluble low-molecular-weight antioxidants (LMWA), was measured by cyclic voltammetry. Generally, LMWA were reduced in the embryos and yolk sacs under diabetic conditions. In the water-soluble fraction of control embryos and yolk sacs, two peak potentials were found, indicating two major groups of LMWA, while only one peak potential was found under diabetic conditions, indicating that an entire group of LMWA is missing. HPLC studies have demonstrated a decrease in vitamin C (water-soluble fraction) and in vitamin E (lipid-soluble fraction) under diabetic culture conditions, and an increase in uric acid. Generally, the concentration of LMWA was higher in the embryos than in the yolk sac. LMWA concentration, protein content, and antioxidant enzyme activity were lower in the malformed experimental embryos than in experimental embryos without anomalies. The addition of vitamins C and E to the diabetic culture medium abolished the deleterious effects of the diabetic serum on the embryos. The disturbed antioxidant defense mechanism under diabetic conditions may be explained, at least in part, by a direct effect of diabetic metabolic factors on the activity of antioxidant enzymes and on the concentration of reducing equivalents. This, in turn, may be embryotoxic.     

The reduction of a nitroxide spin label as a probe of human blood antioxidant properties

The kinetics of reduction of the radical R*, 5-dimethylaminonaphthalene-1-sulfonyl-4-amino-2,2,6,6-tetramethyl-1-piperidine-oxyl by blood and its components were studied using the EPR technique. The results demonstrate that R* is adsorbed to the outer surface of the membrane and does not penetrate into the erythrocytes. A series of control experiments in PBS demonstrate that ascorbate is the only natural reducing agent that reacts with R*. The observed first order rate of disappearance of the nitroxide radical k, is: k(blood) > k(eryth) > k(plasma) and k(blood) approximately = k(eryth) + k(plasma). The results demonstrate that: a. The erythrocytes catalyze the reduction of R* by ascorbate. b. The rate of reduction of the radical is high though it does not penetrate the cells. c. In human erythrocytes there is an efficient electron transfer route through the cell membrane. d. The study points out that R* is a suitable spin label for measuring the reduction kinetics and antioxidant capacity in blood as expressed by reduction by ascorbate.     

Interaction of vitamin C and vitamin E during free radical stress in plasma: An ESR study

To study the interaction of the antioxidant vitamins C and E in a biological system, we used electron spin resonance (ESR) spectroscopy to make serial measurement of ascorbate tocopheroxyl free radicals in plasma subjected to continuous free radical-mediated oxidative stress. Upon initiation of a continuous oxidative stress, we observed an immediate increase in the concentration of ascorbate radical, which reached a peak, and then steadily declined. Only after the virtual disappearance of the ascorbate radical did we observe the appearance of the tocopheroxyl radical. These data are consistent with the hypothesis that ascorbate is the terminal small-molecule antioxidant in biological systems. This is the first experimental demonstration that the predicted thermodynamic hierarchy of ascorbate, -tocopherol, and their free radicals holds in a biological system containing endogenous levels of these antioxidant vitamins.     

Biological variation of interleukin 6 (IL-6) and soluble interleukin 2 receptor (sIL2R) in serum of healthy individuals

The biological variation of interleukin 6 (IL-6) and soluble interleukin 2 receptor (sIL2R), measured by automated enzyme immunoassay, in fifteen subjects studied at regular monthly intervals over a period of 6 consecutive months was measured. The mean and standard deviation (SD), within-subject CV, between-subject CV, individuality index (II) and reliability coefficient (R) were as follow: for sIL2R 571 (231) U/ml, 5.84%, 38.81%, 0.21 and 0.93; and for IL-6 1.43 (0.9) pg/ml, 48.48%, 39.38%, 1.44, and 0.37. The data indicate a relatively high between-subject CV, quite similar in both cases, and a within-subject CV much higher for IL-6 than for sIL2R. Thus, reference values can be used for diagnosis for IL6 (high II), while not for sIL2R (low II). However, the low R for IL-6 implies that more than one measurement are needed. sIL2R has a very high R and a relatively small critical differences, a circumstance appropriate for follow-up.     

Effect of Aloe vera preparations on the human bioavailability of vitamins C and E

There are no literature references describing the effect of consumption of Aloe vera liquid preparations on the absorption of water- or fat-soluble vitamins. There is a very large population worldwide which consume vitamins and many people also consume Aloe. Thus we report the effect of Aloe on the human absorption of vitamins C and E, the most popular vitamin supplements. The plasma bioavailability of vitamins C and E were determined in normal fasting subjects, with eight subjects for vitamin C and ten subjects for vitamin E. In a random crossover design, the subjects consumed either 500 mg of ascorbic acid or 420 mg of vitamin E acetate alone (control), or combined with 2 oz of two different Aloe preparations (a whole leaf extract, or an inner fillet gel). Blood was collected periodically up to 24 h after consumption. Plasma was analyzed for ascorbate and tocopherol by HPLC with UV detection. There was no significant difference in the areas under the plasma ascorbate–time curves among the groups sincerely due to large differences within the groups. For comparative purposes the control area was 100%. The Aloe Gel area was 304%, and Aloe Whole Leaf 80%. Only Aloe Gel caused a significant increase in plasma ascorbate after 8 and 24 h. For vitamin E, the results for the relative areas were control 100%, Gel 369%, and Leaf (198%). Only the Aloes produced a significant increase in plasma tocopherol after 6 and 8 h. Both Aloes were significantly different from the control after 8 h. Aloe Gel was significantly different from the baseline after 24 h. The Aloes slowed down the absorption of both vitamins with maximum concentrations 2–4 h later than the control. There was no difference between the two types of Aloe. The results indicate that the Aloes improve the absorption of both vitamins C and E. The absorption is slower and the vitamins last longer in the plasma with the Aloes. Aloe is the only known supplement to increase the absorption of both of these vitamins and should be considered as a complement to them.     

Development of NaCl-tolerant strain in Chrysanthemum morifolium Ramat. through in vitro mutagenesis

One NaCl-tolerant chrysanthemum (Chrysanthemum morifolium Ramat.) variant (E2) has been developed in a stable form through IN VITRO mutagenesis using ethylmethane sulfonate (EMS) as the chemical mutagen. Salt tolerance was evaluated by the capacity of the plant to maintain both flower quality and yield under stress conditions. Enhanced tolerance of the E2 variant has been attributed to the increased activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), and dehydroascorbate reductase (DHAR), and, to a lesser extent of membrane damage than NaCl-treated control plants. Isoform analysis revealed that an increase in total SOD activity in the E2 variant was solely due to significant activation of the Cu/Zn isoform. Elevated levels of carotenoids and ascorbate in E2 leaves have been reflected in their higher free radical scavenging capacity (RSC) expressed in terms of DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging ability. Data reflect that a proper balance between enzymatic and non-enzymatic defence systems is required for combating salinity stress in chrysanthemum. Better performance of the E2 progeny under same salinity stress condition, even in the second year, confirms the genetic stability of the salt-tolerance character. On the whole, the E2 variant, developed through 0.025 % EMS treatment, might be considered as a NaCl-tolerant strain showing positive characters towards NaCl stress.     

Determination of nicotine and cotinine in human plasma by liquid chromatography-tandem mass spectrometry with atmospheric-pressure chemical ionization interface

Here we report a sensitive liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method capable of quantifying nicotine down to 1 ng/ml and cotinine to 10 ng/ml from 1.0 ml of human plasma. The method was validated over linear ranges of 1.0–50.0 ng/ml for nicotine and 10.0–500.0 ng/ml for cotinine, using deuterated internal standards. Compounds were simply extracted from alkalinized human heparinized plasma with methylene chloride, reconstituted into a solution of acetonitrile, methanol and 10 mM ammonium acetate (53:32:15, v/v) after the organic phase was dried down, and analyzed on the LC-MS-MS, which is a PE Sciex API III system equipped with a Keystone BDS Hypersil C₁₈ column and atmospheric pressure chemical ionization (APCI) interface. The between-run precision and accuracy of the calibration standards were ≤6.42% relative standard deviation (R.S.D.) and ≤11.8%n relative error (R.E.) for both nicotine and cotinine. The between-run and within-run precision and accuracy of quality controls. (2.5, 15.0, 37.5 ng/ml for nicotine and 25.0, 150.0, 375.0 ng/ml for cotinine), were ≤6.34% R.S.D. and ≤7.62% R.E. for both analytes. Sample stabilities in chromatography, in processing and in biological matrix were also investigated. This method has been applied to pharmacokinetic analysis of nicotine and cotinine in human plasma.     

Vitamins C and E improve rat embryonic antioxidant defense mechanism in diabetic culture medium.

BACKGROUND: Diabetes teratogenicity seems to be related to embryonic oxidative stress and the extent of the embryonic damage can apparently be reduced by antioxidants. We have studied the mechanism by which antioxidants, such as vitamins C and E, reduce diabetes-induced embryonic damage. We therefore compared the antioxidant capacity of 10.5-day-old rat embryos and their yolk sacs cultured for 28h in diabetic culture medium with or without vitamins C and E. METHODS: The embryos were cultured in 90% rat serum to which 2mg/ml glucose, 2mg/ml beta hydroxy butyrate (BHOB) and 10 microg/ml of acetoacetate were added. Rat embryos were also cultured in a diabetic medium with 25 microg/ml of vitamin E and 50 microg/ml of vitamin C. Control embryos were cultured in normal rat serum with or without vitamins C and E. RESULTS: Decreased activity of Cu/Zn superoxide dismutase (SOD) and of catalase (CAT) in the "diabetic" embryos and their yolk sacs, and reduced concentrations of low molecular weight antioxidant (LMWA) were found. Under these conditions we also found a decrease in vitamin C and vitamin E concentrations in the embryos, as measured by HPLC. In situ hybridization for SOD mRNA showed a marked reduction of SOD mRNA in the brain, spinal cord, heart and liver of embryos cultured in diabetic medium in comparison to controls. Following the addition of vitamins C and E to the diabetic culture medium, SOD and CAT activity, the concentrations of LMWA, the levels of vitamin C and E and the expression of SOD mRNA in the embryos and yolk sacs returned to normal. CONCLUSIONS: Diabetic metabolic factors seem to have a direct effect on embryonic SOD gene and perhaps genes of other antioxidant enzymes, reducing embryonic endogenous antioxidant defense mechanism. This in turn may cause a depletion of the LMWA, such as vitamins C and E. The addition of these vitamins normalizes the embryonic antioxidant defense mechanism, reducing the damage caused by the diabetic environment.     

Antioxidant capacity of human blood plasma and human urine: simultaneous evaluation of the ORAC index and ascorbic acid concentration employing pyrogallol red as probe

The oxygen radical absorbance capacity (ORAC) methodology has been employed to estimate the antioxidant capacity of human blood plasma and human urine using pyrogallol red (ORAC-PGR) as target molecule. Uric acid, reduced glutathione, human serum albumin, and ascorbic acid (ASC) inhibited the consumption of pyrogallol red, but only ASC generated an induction time. Human blood plasma and human urine protected efficiently pyrogallol red. In these assays, both biological fluids generated neat induction times that were removed by ascorbate oxidase. From these results, ORAC-PGR method could be proposed as a simple alternative to evaluate an ORAC index and, simultaneously, to estimate the concentration of ascorbic acid in human blood plasma or human urine.     

Orientation of the tyrosyl radical in Salmonella typhimurium class Ib ribonucleotide reductase determined by high field EPR of R2F single crystals

The R2 protein of class I ribonucleotide reductase (RNR) generates and stores a tyrosyl radical, located next to a diferric iron center, which is essential for ribonucleotide reduction and thus DNA synthesis. X-ray structures of class Ia and Ib proteins from various organisms served as bases for detailed mechanistic suggestions. The active site tyrosine in R2F of class Ib RNR of Salmonella typhimurium is located at larger distance to the diiron site, and shows a different side chain orientation, as compared with the tyrosine in R2 of class Ia RNR from Escherichia coli.No structural information has been available for the active tyrosyl radical in R2F. Here we report on high field EPR experiments of single crystals of R2F from S. typhimurium, containing the radical Tyr-105*. Full rotational pattern of the spectra were recorded, and the orientation of the g-tensor axes were determined, which directly reflect the orientation of the radical Tyr-105* in the crystal frame. Comparison with the orientation of the reduced tyrosine Tyr-105-OH from the x-ray structure reveals a rotation of the tyrosyl side chain, which reduces the distance between the tyrosyl radical and the nearest iron ligands toward similar values as observed earlier for Tyr-122* in E. coli R2. Presence of the substrate binding subunit R1E did not change the EPR spectra of Tyr-105*, indicating that binding of R2E alone induces no structural change of the diiron site. The present study demonstrates that structural and functional information about active radical states can be obtained by combining x-ray and high-field-EPR crystallography.     

Protective effects of vitamins C and E on the number of micronuclei in lymphocytes in smokers and their role in ascorbate free radical formation in plasma

Cigarette smoke is widely believed to increase free radical concentrations causing subsequent oxidative processes that lead to DNA damage and hence, to several diseases including lung cancer and atherosclerosis. Vitamin C is a reducing agent that can terminate free-radical-driven oxidation by being converted to a resonance-stabilized free radical. To investigate whether short-term supplementation with the antioxidants vitamin C and E decreases free-radical-driven oxidation and thus decreases DNA damage in smokers, we determined the frequency of micronuclei in lymphocytes in 24 subjects and monitored the electron paramagnetic resonance signal of ascorbate free radical formation in plasma. Further parameters comprised sister-chromatid exchanges and thiobarbituric acid-reactive substances. Twelve smokers and twelve non-smokers took 1000 mg ascorbic acid daily for 7 days and then 1000 mg ascorbic acid and 335.5 mg RRR-α-tocopherol daily for the next 7 days. Baseline concentrations of both vitamins C and E were lower and baseline numbers of micronuclei were higher (p < 0.0001) in smokers than in non-smokers. After 7 days of vitamins C and E, DNA damage as monitored by the number of micronulei was decreased in both, smokers and non-smokers, but it was more decreased in smokers as indicated by fewer micronuclei in peripheral lymphocytes (p < 0.05). Concomitantly, the plasma concentrations of vitamin C (p < 0.001) as well as the ascorbate free radical (p < 0.05) were increased. The corresponding values in non-smokers, however, did not change. Our findings show that increased ascorbate free radical formation in plasma after short-term supplementation with vitamins C and E can decrease the number of micronuclei in blood lymphocytes and thus DNA damage in smokers.     

First LC-MS/MS electrospray ionization validated method for the quantification of perindopril and its metabolite perindoprilat in human plasma and its application to bioequivalence study

A high throughput bioanalytical method based on solid phase extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS), has been developed for the estimation of perindopril and its metabolite perindoprilat, an angiotensin-converting enzyme inhibitor in human plasma. Ramipril was used as internal standard (IS). The extraction of perindopril, perindoprilat and ramipril from the plasma involved treatment with phosphoric acid followed by solid phase extraction (SPE) using hydrophilic lipophilic balance HLB cartridge. The SPE eluate without drying were analyzed by LC-MS/MS, equipped with turbo ion spray (TIS) source, operating in the negative ion and selective reaction monitoring (SRM) acquisition mode to quantify perindopril and perindoprilat in human plasma. The total chromatographic run time was 1.5 min with retention time for perindopril, perindoprilat and ramipril at 0.33, 0.35 and 0.30 min. The developed method was validated in human plasma matrix, with a sensitivity of 0.5 ng/ml (CV, 7.67%) for perindopril and 0.3 ng/ml (CV, 4.94%) for perindoprilat. This method was extensively validated for its accuracy, precision, recovery, stability studies and matrix effect especially because the pattern of elution of all the analytes appears as flow injection elution. Sample preparation by this method yielded extremely clean extracts with very good and consistent mean recoveries; 78.29% for perindopril, 76.32% for perindoprilat and 77.72% for IS. The response of the LC-MS/MS method for perindopril and perindoprilat was linear over the range 0.5-350.0 ng/ml for perindopril and 0.3-40 ng/ml for perindoprilat with correlation coefficient, r>/=0.9998 and 0.9996, respectively. The method was successfully applied for bioequivalence studies in human subjects samples with 4 mg immediate release (IR) formulations.     

Protective effects of vitamins C and E on the number of micronuclei in lymphocytes in smokers and their role in ascorbate free radical formation in plasma

Cigarette smoke is widely believed to increase free radical concentrations causing subsequent oxidative processes that lead to DNA damage and hence, to several diseases including lung cancer and atherosclerosis. Vitamin C is a reducing agent that can terminate free-radical-driven oxidation by being converted to a resonance-stabilized free radical. To investigate whether short-term supplementation with the antioxidants vitamin C and E decreases free-radical-driven oxidation and thus decreases DNA damage in smokers, we determined the frequency of micronuclei in lymphocytes in 24 subjects and monitored the electron paramagnetic resonance signal of ascorbate free radical formation in plasma. Further parameters comprised sister-chromatid exchanges and thiobarbituric acid-reactive substances. Twelve smokers and twelve non-smokers took 1000 mg ascorbic acid daily for 7 days and then 1000 mg ascorbic acid and 335.5 mg RRR-alpha-tocopherol daily for the next 7 days. Baseline concentrations of both vitamins C and E were lower and baseline numbers of micronuclei were higher (p < 0.0001) in smokers than in non-smokers. After 7 days of vitamins C and E, DNA damage as monitored by the number of micronulei was decreased in both, smokers and non-smokers, but it was more decreased in smokers as indicated by fewer micronuclei in peripheral lymphocytes (p < 0.05). Concomitantly, the plasma concentrations of vitamin C (p < 0.001) as well as the ascorbate free radical (p < 0.05) were increased. The corresponding values in non-smokers, however, did not change. Our findings show that increased ascorbate free radical formation in plasma after short-term supplementation with vitamins C and E can decrease the number of micronuclei in blood lymphocytes and thus DNA damage in smokers.     

Phycoerythrin fluorescence-based assay for peroxy radicals: a screen for biologically relevant protective agents

Under the conditions of this assay, antioxidants that react rapidly with peroxy free radicals (e.g., ascorbate, vitamin E analogs, urate), protect phycoerythrin completely from damage by such radicals generated by thermal decomposition of 2,2'-azobis(2-amidinopropane); other compounds provide partial concentration-dependent protection. Change in phycoerythrin fluorescence emission with time provides a measure of the rate of free radical damage. The assay exploits the unusual reactivity of phycoerythrin toward these peroxy radicals. On a molar basis, phycoerythrin reacts with these radicals over 100-fold slower than do ascorbate or vitamin E analogs, but over 60-fold faster than other proteins. Applications of this assay to the estimation of the peroxy radical scavenging capacity of human plasma are described, and to the comparison of the scavenging properties of several proteins and of DNA, of vitamins and their derivatives, of catecholamine neurotransmitters, and of a variety of other low molecular weight biological compounds.     

The increase in human plasma antioxidant capacity after apple consumption is due to the metabolic effect of fructose on urate, not apple-derived antioxidant flavonoids

Regular fruit consumption lowers the risk of cardiovascular diseases and certain cancers, which has been attributed in part to fruit-derived antioxidant flavonoids. However, flavonoids are poorly absorbed by humans, and the increase in plasma antioxidant capacity observed after consumption of flavonoid-rich foods often greatly exceeds the increase in plasma flavonoids. In the present study, six healthy subjects consumed five Red Delicious apples (1037 +/- 38 g), plain bagels (263.1 +/- 0.9 g) and water matching the carbohydrate content and mass of the apples, and fructose (63.9 +/- 2.9 g) in water matching the fructose content and mass of the apples. The antioxidant capacity of plasma was measured before and up to 6 h after food consumption as ferric reducing antioxidant potential (FRAP), without or with ascorbate oxidase treatment (FRAPAO) to estimate the contribution of ascorbate. Baseline plasma FRAP and FRAPAO were 445 +/- 35 and 363 +/- 35 microM trolox equivalents, respectively. Apple consumption caused an acute, transient increase in both plasma FRAP and FRAPAO, with increases after 1 h of 54.6 +/- 8.7 and 61.3 = 17.2 microM trolox equivalents, respectively. This increase in plasma antioxidant capacity was paralleled by a large increase in plasma urate, a metabolic antioxidant, from 271 +/- 39 microM at baseline to 367 +/- 43 microM after 1 h. In contrast, FRAP and FRAPAO time-dependently decreased after bagel consumption, together with urate. Consumption of fructose mimicked the effects of apples with respect to increased FRAP, FRAPAO, and urate, but not ascorbate. Taken together, our data show that the increase in plasma antioxidant capacity in humans after apple consumption is due mainly to the well-known metabolic effect of fructose on urate, not apple-derived antioxidant flavonoids.