Concepción Sánchez-Moreno, M Pilar Cano, Begoña de Ancos, Lucía Plaza, Begoña Olmedilla, Fernando Granado, Antonio Martín, Impact of orange juice consumption on vitamin C concentrations and biomarkers of antioxidant standing in people, The American Journal of Medical Diet, Quantity 78, Concern 3, September 2003, Pages 454–460, https://doi.org/10.1093/ajcn/78.3.454
ABSTRACT
INTRODUCTION
An elevated consumption of fruit and greens has been related to helpful results on the danger of illness (1–5). Citrus juices, particularly orange juice and grapefruit juice, are wealthy sources of vitamin C, however their well being advantages stay poorly understood. Latest experiences steered that consuming beneficiant quantities of a combination of varied juices improves the blood lipid profile, reduces oxidative stress, prevents atherogenic modifications of LDL ldl cholesterol and platelet aggregation (6–8), and improves HDL-cholesterol concentrations (9). A number of research have proven that vitamin C performs an necessary position in human well being, together with results on the immune system (10), the danger of Alzheimer illness (11), and the effectivity with which lysosomes in mind glial cells degrade modified proteins (12). Nevertheless, no examine has examined the bioavailability of vitamin C in orange juice and its attainable helpful results on lowering isoprostane concentrations.
Isoprostanes are a household of eicosanoids of nonenzymatic origin which are produced by the random oxidation of phospholipids by oxygen radicals and which are elevated by oxidative stress (13, 14). One of many isoprostanes, 8-epi-prostaglandin F2α (8-epi-PGF2α), has been proven to behave as a vasoconstrictor (15) and to be related to the hepatorenal syndrome and pulmonary oxygen toxicity (16). Earlier research confirmed decreased concentrations of antioxidants and excessive concentrations of 8-isoprostanes amongst people who smoke (17, 18), which can be as a consequence of a low consumption of antioxidants (19) or a better utilization of vitamin C by free radicals produced throughout smoking (20).
Excessive serum uric acid could also be an necessary predicting threat issue for cardiovascular occasions, corresponding to myocardial infarction (21). Multivariate evaluation of knowledge from the MONICA cohort of 1044 males confirmed a major affiliation between serum uric acid concentrations and cardiovascular mortality (22). Uric acid could have a direct injurious impact on the endothelium, altering endothelial cell perform and lowering nitric oxide bioavailability, which is related to heart problems threat.
Antioxidants have necessary roles in cell perform and have been implicated in processes related to growing old, together with vascular processes, inflammatory injury, and most cancers (23–25). Vitamin C might also contribute to the upkeep of a wholesome vasculature and to a discount in atherogenesis via the regulation of collagen synthesis, prostacyclin manufacturing, and nitric oxide (26–28). Along with its antioxidant position, vitamin C has roles on the molecular stage, together with appearing as a cofactor for dopamine β-hydroxylase (EC 1.14.17.1), influencing neurotransmitter concentrations, enhancing lysosomal protein degradation, and mediating glutamate uptake (5, 29, 30). The second Nationwide Well being and Diet Examination Survey reported {that a} low consumption of vitamin C is related to blood concentrations of vitamin C ≤ 0.3 mg/dL (17 μmol/L) (31), whereas blood concentrations in well-nourished individuals fluctuate between 0.8 and 1.3 mg/dL (45 and 74 μmol/L). Fruit and greens are the primary sources of vitamin C, however 25% of girls and ≈33% males eat < 2.5 servings of fruit and greens every day, which supplies ≈80 mg vitamin C/d. Due to this fact, the targets of this examine had been to evaluate the bioavailability of vitamin C in orange juice and its impact on concentrations of 8-epi-PGF2α (F2-isoprostanes) in a wholesome human inhabitants.
SUBJECTS AND METHODS
Topics
Twelve wholesome volunteers (6 males and 6 ladies) had been enrolled on this examine. The topics had been aged between 20 and 32 y (x̄ ± SD: 22 ± 3 y), and their imply physique mass index (in kg/m2) was 22.2 ± 1.6 and didn’t change considerably through the examine. All the themes continued their ordinary diets through the examine. Topics had been taking no vitamin or mineral dietary supplements and no medicines. Not one of the topics had been pregnant, lactating, or had any power sickness. All examine individuals had been in good well being on the idea of a medical historical past, a bodily examination, and regular outcomes on scientific laboratory assessments, together with measurement of triacylglycerol (0.9 ± 0.3 mmol/L), ldl cholesterol (4.4 ± 1.1 mmol/L), glucose (4.8 ± 0.4 mmol/L), and hematocrit (0.44 ± 0.03). Topics obtained oral and written details about the examine and gave their written consent. The examine was permitted by the Clinic Analysis Ethics Committee of the Hospital Universitario Clínica Puerta de Hierro, Madrid.
Assortment of plasma samples and experimental protocol
The vitamin C bioavailability examine was divided into 2 parts: a dose-response take a look at and a multiple-dose-response examine. For the dose-response take a look at, an intravenous catheter was inserted into every topic’s forearm after she or he had fasted for ≥ 12 h. After blood samples had been collected at baseline, the volunteers consumed 500 mL orange juice and blood samples had been taken each 60 min as much as 6 h. Blood samples had been collected in heparin-coated tubes and had been centrifuged at 2000 × g for 15 min at 4 °C. After the plasma was collected, aliquots in triplicate had been instantly blended with an equal quantity of chilly 6% (wt:vol) metaphosphoric acid containing 1 mmol/L of the steel ion chelator diethylenetriaminepentaacetic acid for vitamin C and uric acid evaluation. The remaining plasma was saved at −80 °C for evaluation of 8-epi-PGF2α. For the multiple-dose-response examine, the themes had been instructed to drink the juice at residence in 2 doses, 250 mL within the morning and 250 mL within the afternoon, for two consecutive weeks. Blood samples had been taken once more through the intervention on days 7 and 14 of the examine.
The composition of the business fresh-squeezed orange juice consumed by the individuals is reported in Desk 1. The vitamin C, flavanone, and carotenoid composition of the juice was measured by reversed-phase HLPC with strategies at present utilized in our laboratory (32–34). Complete power, protein, carbohydrate, and fats contents had been offered by J García Carrión, SA, Jumilla, Murcia, Spain.
Dietary standing evaluation
Dietary historical past information had been obtained via a food-frequency questionnaire (together with 125 meals objects) administered to the themes by a nutritionist. The reference interval for the food-frequency questionnaire was the previous 3 mo. The food-frequency questionnaires had been then scanned (OPSCAN5; Nationwide Laptop Methods, St Paul), transferred into digital recordsdata, and transformed into statistical datasets for evaluation. The Minnesota NUTRIENT DATA SYSTEM (program 2.8, model 25; College of Minnesota, Minneapolis) was used to calculate the nutrient consumption profiles (35). Normally, as beforehand reported, a easy self-administered dietary questionnaire can present helpful details about particular person nutrient intakes over a number of months (36).
Measurement of vitamin C
Ascorbate was analyzed by paired-ion, reversed-phase HPLC coupled with electrochemical detection. Briefly, a 100-μL plasma pattern was blended with an equal quantity of chilly 6% (wt:vol) metaphosphoric acid containing 1 mmol/L of the steel ion chelator diethylenetriaminepentaacetic acid (Sigma, St Louis). A portion of the pattern was analyzed on an LC8 column (150 mm × 4.6 mm inside diameter, 3-μm particle dimension; Supelco, Bellefonte, PA) with 99% deionized water and 1% methanol containing 40 mmol sodium acetate/L and 1.5 mmol dodecyltriathylammonium phosphate/L (Q12 ion pair cocktail; Regis, Morton Grove, IL) because the cellular section delivered at a movement charge of 1 mL/min. Samples had been injected with an autosampler (1100 sequence; Hewlett Packard Co, Wilmington, DE). Ascorbate was detected at an utilized potential of 0.6 V, with the achieve set at 100 nA by an LC 4B amperometric electrochemical detector (Bioanalytical Methods, West Lafayette, IN). Ascorbate was eluted as a single peak with a retention time of 5.5 min. Peaks had been built-in with a ChemStation (Hewlett Packard). Ascorbate concentrations had been calculated on the idea of a calibration curve and are expressed in μmol/L (33).
Measurement of uric acid
Uric acid was analyzed by paired-ion, reversed-phase HPLC coupled with electrochemical detection by following the identical process described for vitamin C; an electrode potential of 0.6 V was used, however the achieve was set at 1 μA. Uric acid was eluted as a single peak with a retention time of three.5 min. Peaks had been built-in with a ChemStation (Hewlett Packard). Uric acid concentrations had been calculated on the idea of a calibration curve and are expressed as μmol/L (33).
Measurement of 8-isoprostane
8-epi-PGF2α, which has been proposed as a marker of oxidative stress, was measured through the use of an enzyme immunoassay equipment (37). First, samples (0.5–1 mL) had been blended gently with 50 μL affinity sorbent (mouse anti-8-isoprostane covalently certain to Sepharose 4B) (Cayman Chemical Co, Ann Arbor, MI) for 60 min at room temperature to purify the samples. Subsequent, the samples had been briefly centrifuged at 11 750 × g (4 °C) in order that the sorbent would kind a sediment (Cayman Chemical Co). The sorbent, which comprises the certain 8-isoprostane, was rinsed with washing resolution, and, after the supernatant fluid was discarded, the sorbent pellet was resuspended in 300 μL of an ethanol elution resolution. The ethanol washes had been evaporated to dryness in a vacuum centrifuge and had been instantly dissolved in 125 μL enzyme immunoassay buffer. Then, samples had been analyzed in duplicate through the use of the enzyme immunoassay equipment. This assay is predicated on the competitors between 8-isoprostane and an 8-isoprostane-acetylcholinesterase conjugate (8-isoprostane tracer) for a restricted variety of 8-isoprostane-specific rabbit antiserum binding websites. The rabbit antiserum 8-isoprostane (both free or tracer) advanced binds to the rabbit immunoglobulin G mouse monoclonal antibody that was beforehand connected to the properly. The plate was washed to take away any unbound reagents after which Ellman’s reagent (which comprises the substrate to 8-isoprostane-acetylcholinesterase; Cayman Chemical Co) was added to the properly. The product of this enzymatic response has a definite yellow colour and absorbs strongly at 412 nm. The depth of this colour, decided spectrophotometrically, is proportional to the quantity of 8-isoprostane tracer certain to the properly, which is inversely proportional to the quantity of free 8-isoprostane current within the properly through the incubation. The focus of 8-isoprostane within the take a look at samples was interpolated from the usual curve through the use of log transformation (16).
Statistical evaluation
Descriptive statistics (together with means and SDs) had been used to summarize the traits of the themes. Values are offered as means ± SDs or ± SEMs when acceptable. Repeated-measures evaluation of variance evaluating the concentrations of vitamin C, uric acid, and 8-epi-PGF2α between the sexes and at completely different occasions of the intervention had been carried out through the use of SYSTAT 10 (SPSS Inc, Chicago) to check for statistical significance on the P ≤ 0.05 stage. When sex-by-time interactions or intercourse results had been noticed, Tukey’s truthfully vital distinction take a look at was used to find out variations at completely different time factors. The correlations inside variables had been examined by linear regressions or by Spearman’s correlation as acceptable, additionally through the use of the SYSTAT program (38).
RESULTS
The food-frequency questionnaire information confirmed that power consumption tended to be greater (P = 0.06) among the many males (2562.0 ± 598.2 kcal/d) than among the many ladies (1994.8 ± 773.9 kcal/d). The principle contributor to the distinction in power consumption noticed could have been the upper (P = 0.02) consumption of carbohydrates among the many males (275.8 ± 67.3 g) than among the many ladies (201.6 ± 64.8 g). No vital variations in micronutrient consumption had been noticed between the women and men. There was additionally no vital distinction (P = 0.3) in every day vitamin C consumption between the lads (136.1 ± 53.0 mg) and the ladies (111.9 ± 39.3 mg).
Baseline plasma vitamin C concentrations had been considerably greater (P = 0.03) within the ladies (56.4 ± 4.4 μmol/L) than within the males (44.3 ± 3.5 μmol/L; Desk 2). On the primary day of the intervention, the utmost enhance in vitamin C occurred 3 h after consumption of the orange juice (500 mL) containing 250 mg vitamin C in each the lads and the ladies (Determine 1). At 3 h (most peak), plasma concentrations had elevated over baseline by 64% within the males (72.6 ± 4.0 in contrast with 44.3 ± 3.5 μmol/L; P = 0.001) and by 40% within the ladies (78.8 ± 4.6 in contrast with 56.4 ± 4.4 μmol/L; P = 0.009).
Plasma vitamin C concentrations had been additionally analyzed on days 7 and 14. Plasma vitamin C concentrations remained elevated (P < 0.05) through the examine (Desk 2). Nevertheless, no vital variations had been noticed between the lads and the ladies besides at baseline. There was a major sex-by-time interplay for bioavailability of vitamin C (P = 0.001). Curiously, though the focus of vitamin C on the finish of the examine (day 14) remained considerably greater than at baseline, it tended to be decrease (though not considerably so) than the focus reached by consuming the juice multi function dose (500-mL dose in contrast with two 250-mL doses). Uric acid concentrations had been considerably greater (P < 0.01) among the many males than among the many ladies (baseline: 342.0 ± 16.7 in contrast with 216.1 ± 16.9 μmol/L; Desk 3). Normally, uric acid concentrations had been decrease when the vitamin C focus was greater, and an inverse affiliation was noticed between uric acid concentrations and plasma vitamin C concentrations throughout time in each women and men (r = −0.19, P = 0.05). Baseline plasma concentrations of 8-epi-PGF2α had been considerably greater (P = 0.03) among the many males (249.6 ± 25.4 pg/mL) than among the many ladies (177.7 ± 6.2 pg/mL; Desk 2). Curiously, we noticed a development towards a lower in plasma concentrations of 8-epi-PGF2α among the many males on day 7 of the intervention, which turned vital on day 14 (P = 0.04). No vital adjustments had been noticed by time among the many ladies. An inverse correlation was noticed between vitamin C concentrations and 8-epi-PGF2α concentrations amongst each the lads and the ladies at baseline (r = −0.60, P = 0.05) and on day 14 of the examine (r = −0.55, P = 0.06). Though the 8-epi-PGF2α focus decreased considerably on day 14 within the males, the vitamin C focus was extra steady, which affected the correlation. Nevertheless, as a result of adjustments in 8-epi-PGF2α and the affiliation between vitamin C and 8-epi-PGF2α could also be extremely related to well being standing, we plotted concentrations of vitamin C and 8-epi-PGF2α at baseline and on the finish of the examine (day 14) mixed as extra consultant of the affiliation of vitamin C with 8-epi-PGF2α (r = −0.791, P = 0.0022; Determine 2). The baseline focus of vitamin C in a single topic was extraordinarily low (outlier) and we had been unable to substantiate whether or not this was a real measurement or an artifact; thus, that focus was excluded from the correlation. Among the many males, the baseline plasma vitamin C focus was considerably greater (P = 0.04) amongst nonsmokers than amongst people who smoke, and the plasma 8-epi-PGF2α focus was considerably greater (P = 0.045) amongst people who smoke than amongst nonsmokers (Desk 4). After every day consumption of orange juice, vitamin C concentrations elevated considerably amongst people who smoke (P = 0.04) on the finish of the primary week, and, on the finish of examine, the focus of vitamin C was 93% greater than at baseline (71.6 ± 3.9 in contrast with 37.1 ± 0.5 μmol/L; P = 0.02). Nonsmokers additionally confirmed greater vitamin C concentrations (P = 0.045) on days 7 and 14 than at baseline. On days 7 and 14, plasma 8-epi-PGF2α concentrations amongst people who smoke had been considerably (P ≤ 0.04) decrease than at baseline.
DISCUSSION – “vitamin c oj”
Within the current examine, we confirmed for the primary time that consuming 2 glasses of orange juice (500 mL) every day will increase plasma vitamin C concentrations by 40–64%. Curiously, adjustments in vitamin C had been considerably inversely correlated with concentrations of 8-epi-PGF2α, suggesting that vitamin C could play a vital position in lowering the formation of compounds produced by the random oxidation of phospholipids by oxygen radicals.
Vitamin C consumption as assessed by the food-frequency questionnaire was not considerably completely different between the lads and the ladies within the current examine. Nevertheless, plasma vitamin C concentrations had been decrease within the males than within the ladies. These variations in basal plasma vitamin C concentrations could have been mediated by an augmented consumption of this nutrient because of elevated metabolic necessities or due to an extra of reactive oxygen species shaped in males in contrast with ladies. The truth is, we seen that the focus of 8-epi-PGF2α was considerably greater amongst people who smoke than amongst nonsmokers. Due to this fact, if vitamin C is a very powerful antioxidant current within the aqueous resolution, an elevated formation of free radicals or byproducts derived from free radical–mediated reactions could be related to an enhanced consumption of vitamin C (39).
Vitamin C is an important nutrient for people; not like most mammals, we can’t synthesize vitamin C and subsequently should purchase it from the food plan. Some research have investigated the bioavailability of chemically an identical pure and artificial ascorbic acid and located no clinically vital distinction in bioavailability or bioactivity (40). Nevertheless, no research documenting the bioavailability of vitamin C from orange juice have been reported. That is stunning as a result of orange juice is without doubt one of the most necessary sources of vitamin C within the human food plan. Not too long ago, the plasma kinetics of flavanones in orange and grapefruit juice had been examined, however not their results on plasma vitamin C concentrations (41). Consumption of a mixture of fruit and greens (500 g) for 4 wk was related to a 64% enhance in vitamin C focus (42). At low concentrations, the absorption of ascorbic acid happens via an energetic transport course of, whereas at excessive concentrations, absorption is mediated by a mixture of each energetic and passive diffusion within the gastrointestinal tract (43, 44). Thus, consumption of meals wealthy in vitamin C could favor absorption by slowing the interplay of the juice with the gastric wall. The truth is, within the current examine, consumption of 250 mg vitamin C, contained in 2 glasses of orange juice (500 mL), considerably elevated plasma vitamin C from 30–50 to 60–90 μmol/L in simply 3 h. The elevated focus was maintained so long as the themes had been consuming the orange juice, which means that that is an environment friendly means of accelerating vitamin C concentrations within the physique. In different research, blood concentrations of vitamin C had been manipulated via using excessive vitamin C consumption within the type of tablets (2000 mg), and vitamin C concentrations reached 116 μmol/L 2 h after ingestion and 95 μmol/L after taking 500 mg (45), which means that high-dose dietary supplements won’t be probably the most environment friendly manner of accelerating the physique’s pool of vitamin C.
A excessive consumption of fruit and greens is related to a decreased threat of power illnesses, and an elevated consumption of vitamin C is related to well being standing (11, 30). Vitamin C is an important cofactor in neurotransmitter synthesis (46) and is necessary in vascular perform (45). As well as, ascorbic acid scavenges superoxide anions and performs an necessary position within the management of the intracellular redox state (47). Due to this fact, within the current examine, we evaluated the impact of vitamin C on the formation of F2-isoprostanes.
Isoprostanes are a household of eicosanoids of nonenzymatic origin produced by the random oxidation of phospholipids by oxygen radicals. 8-epi-PGF2α has obtained vital consideration as the results of its vasoconstrictive, platelet activation, and mitogenic properties (16, 17). In 1990 PGF2-like compounds had been found to be produced in abundance in vivo via the peroxidation of arachidonic acid from phospholipids, a response catalyzed by free radicals unbiased of the cyclooxygenase enzyme (EC 1.14.99.1). These compounds are isomeric to cyclooxygenase-derived PGF2α and are named F2-isoprostanes, which enhance with oxidant damage (48). The truth is, F2-isoprostanes present an correct measure of oxidant stress in vivo. A number of research have assessed adjustments in F2-isoprostane concentrations in relation to well being standing and completely different pathologic processes (13, 49, 50). Curiously, we noticed a major inverse correlation between the focus of vitamin C and concentrations of 8-epi-PGF2α at baseline. Moreover, after topics drank the orange juice every day for 14 d, concentrations of 8-epi-PGF2α had been lowered by 27%, and the correlation remained the identical, despite the fact that concentrations of 8-epi-PGF2α and vitamin C modified considerably. We consider that the dearth of additional discount in 8-epi-PGF2α concentrations in ladies after orange juice consumption may need been as a result of concentrations of 8-epi-PGF2α had been considerably decrease and vitamin C concentrations considerably greater in ladies than in males all through the examine. Different research additionally reported adjustments in 8-isoprostane concentrations after supplementation with vitamin C, vitamin E, or each (51–53).
Cigarette smoke comprises quite a few oxidants, and it’s believed that smoking could trigger oxidant damage. On this examine, we additionally discovered that amongst these males who reported smoking (50%), concentrations of 8-epi-PGF2α had been considerably greater than in nonsmokers. Moreover, the very best concentrations of 8-epi-PGF2α had been detected among the many topics with the bottom vitamin C concentrations. Curiously, these topics skilled main reductions in 8-epi-PGF2α after consuming orange juice. Comparable observations had been beforehand reported by others, who confirmed that plasma ascorbic acid is depleted in people who smoke in contrast with nonsmokers and is repleted by reasonable supplementation (51, 54). Furthermore, in human research of cigarette people who smoke and nonsmokers, the next quantity of F2-isoprostanes was present in people who smoke’ urine and plasma (51).
One other discovering of the current examine was the inverse affiliation noticed, predominantly in males, between excessive plasma concentrations of vitamin C and decrease concentrations of uric acid, which has lately been thought-about of nice curiosity (55). The mechanisms concerned on this affiliation stay unknown. Epidemiologic research have indicated the presence of related associations between uric acid and heart problems (55, 56). As well as, a number of observational research have proven that serum uric acid concentrations are greater amongst sufferers with established coronary coronary heart illness (57). About one-quarter of hypertensive sufferers have a coexistent hyperuricemia (57, 58). A multivariate evaluation of knowledge from the MONICA cohort of 1044 males indicated a major affiliation between excessive serum uric acid concentrations and cardiovascular mortality (22). A number of mechanisms look like concerned on this affiliation, together with elevated platelet adhesiveness and platelet lysis, formation of free radicals, and oxidative stress (59–61).
Some limitations of the current examine needs to be acknowledged. Maybe a very powerful of those is the small variety of topics enrolled within the examine. Nevertheless, the comparatively small variability within the evaluation carried out (each intra- and interassay) contributed to the variations noticed. One other limitation of the examine is the dearth of outcomes related to plasma concentrations after the consumption of orange juice. As a result of this was a wholesome inhabitants, the primary goal was to evaluate the bioavailability of vitamin C within the juice and its affiliation with oxidative stress.
In conclusion, consuming orange juice elevated vitamin C concentrations and lowered oxidative stress in vivo by reducing the focus of F2-isoprostanes, a discovering that gives new proof of the well being advantages of consuming fruit. Ingesting 2 glasses of orange juice (500 mL/d) containing 250 mg vitamin C elevated vitamin C concentrations in plasma and lowered the concentrations of 8-epi-PGF2α and uric acid. Complete vitamin C was considerably and inversely correlated with 8-epi-PGF2α. These results had been extra accentuated amongst people who smoke than amongst nonsmokers. These findings recommend that the protecting impact of vitamin C is larger in individuals with greater stress and that greater concentrations of vitamin C in tissues may be gained by consuming orange juice every day. Analysis of the health-promoting properties of vitamin C in fruit deserves additional consideration.
We thank the Dietary Analysis Division on the HNRCA, Tufts College, for his or her recommendation and help with the food-frequency questionnaire; Gerard Dallal, Chief of the Biostatistics Unit on the HNRCA, for his recommendation; and I Blanco, C Herrero, T Motilla, and P Martínez (personnel on the Clínica Puerta de Hierro, Madrid) and the individuals of the examine for his or her enthusiasm and contributions to the examine.
CS-M and AM had been answerable for examine design, pattern and information analyses, and writing of the manuscript, and MPC, BdA, LP, BO, and FG had been answerable for examine design and pattern assortment. Not one of the authors had any monetary or private curiosity, together with advisory board affiliations, in any firm or group sponsoring the analysis.
REFERENCES
FOOTNOTES
“vitamin c oj”