Arkansas Children's Nutrition Center

This review summarizes the multifaceted aspects of antioxidants and the basic kinetic models of inhibited autoxidation and analyzes the chemical principles of antioxidant capacity assays. Depending upon the reactions involved, these assays can roughly be classified into two types: assays based on hydrogen atom transfer (HAT) reactions and assays based on electron transfer (ET). The majority of HAT-based assays apply a competitive reaction scheme, in which antioxidant and substrate compete for thermally generated peroxyl radicals through the decomposition of azo compounds. These assays include inhibition of induced low-density lipoprotein autoxidation, oxygen radical absorbance capacity (ORAC), total radical trapping antioxidant parameter (TRAP), and crocin bleaching assays. ET-based assays measure the capacity of an antioxidant in the reduction of an oxidant, which changes color when reduced. The degree of color change is correlated with the sample's antioxidant concentrations. ET-based assays include the total phenols assay by Folin-Ciocalteu reagent (FCR), Trolox equivalence antioxidant capacity (TEAC), ferric ion reducing antioxidant power (FRAP), "total antioxidant potential" assay using a Cu(II) complex as an oxidant, and DPPH. In addition, other assays intended to measure a sample's scavenging capacity of biologically relevant oxidants such as singlet oxygen, superoxide anion, peroxynitrite, and hydroxyl radical are also summarized. On the basis of this analysis, it is suggested that the total phenols assay by FCR be used to quantify an antioxidant's reducing capacity and the ORAC assay to quantify peroxyl radical scavenging capacity. To comprehensively study different aspects of antioxidants, validated and specific assays are needed in addition to these two commonly accepted assays.

Methods available for the measurement of antioxidant capacity are reviewed, presenting the general chemistry underlying the assays, the types of molecules detected, and the most important advantages and shortcomings of each method. This overview provides a basis and rationale for developing standardized antioxidant capacity methods for the food, nutraceutical, and dietary supplement industries. From evaluation of data presented at the First International Congress on Antioxidant Methods in 2004 and in the literature, as well as consideration of potential end uses of antioxidants, it is proposed that procedures and applications for three assays be considered for standardization: the oxygen radical absorbance capacity (ORAC) assay, the Folin-Ciocalteu method, and possibly the Trolox equivalent antioxidant capacity (TEAC) assay. ORAC represent a hydrogen atom transfer (HAT) reaction mechanism, which is most relevant to human biology. The Folin-Ciocalteu method is an electron transfer (ET) based assay and gives reducing capacity, which has normally been expressed as phenolic contents. The TEAC assay represents a second ET-based method. Other assays may need to be considered in the future as more is learned about some of the other radical sources and their importance to human biology.

An improved method of oxygen radical absorbance capacity (ORAC) assay has been developed and validated using fluorescein (3',6'-dihydroxyspiro[isobenzofuran-1[3H],9'[9H]-xanthen]-3-one) as the fluorescent probe. Our results demonstrate that fluorescein (FL) is superior to B-phycoerythrin. The oxidized FL products induced by peroxyl radical were identified by LC/MS, and the reaction mechanism was determined to follow a classic hydrogen atom transfer mechanism. In addition, methodological and mechanistic comparison of ORAC(FL) with other widely used methods was discussed. It is concluded that, unlike other popular methods, the improved ORAC(FL) assay provides a direct measure of hydrophilic chain-breaking antioxidant capacity against peroxyl radical.

Both lipophilic and hydrophilic antioxidant capacities were determined using the oxygen radical absorbance capacity (ORAC(FL)) assay with fluorescein as the fluorescent probe and 2,2'-azobis(2-amidinopropane) dihydrochloride as a peroxyl radical generator on over 100 different kinds of foods, including fruits, vegetables, nuts, dried fruits, spices, cereals, infant, and other foods. Most of the foods were collected from four different regions and during two different seasons in U.S. markets. Total phenolics of each sample were also measured using the Folin-Ciocalteu reagent. Hydrophilic ORAC(FL) values (H-ORAC(FL)) ranged from 0.87 to 2641 micromol of Trolox equivalents (TE)/g among all of the foods, whereas lipophilic ORAC(FL) values (L-ORAC(FL)) ranged from 0.07 to 1611 micromol of TE/g. Generally, L-ORAC(FL) values were <10% of the H-ORAC(FL) values except for a very few samples. Total antioxidant capacity was calculated by combining L-ORAC(FL) and H-ORAC(FL). Differences of ORAC(FL) values in fruits and vegetables from different seasons and regions were relatively large for some foods but could not be analyzed in detail because of the sampling scheme. Two different processing methods, cooking and peeling, were used on selected foods to evaluate the impact of processing on ORAC(FL). The data demonstrated that processing can have significant effects on ORAC(FL). Considering all of the foods analyzed, the relationship between TP and H-ORAC(FL) showed a very weak correlation. Total hydrophilic and lipophilic antioxidant capacity intakes were calculated to be 5558 and 166 micromol of TE/day, respectively, on the basis of data from the USDA Continuing Survey of Food Intakes by Individuals (1994-1996).

The oxygen radical absorbance capacity (ORAC) assay has been widely accepted as a standard tool to measure the antioxidant activity in the nutraceutical, pharmaceutical, and food industries. However, the ORAC assay has been criticized for a lack of accessibility due to the unavailability of the COBAS FARA II analyzer, an instrument discontinued by the manufacturer. In addition, the manual sample preparation is time-consuming and labor-intensive. The objective of this study was to develop a high-throughput instrument platform that can fully automate the ORAC assay procedure. The new instrument platform consists of a robotic eight-channel liquid handling system and a microplate fluorescence reader. By using the high-throughput platform, the efficiency of the assay is improved with at least a 10-fold increase in sample throughput over the current procedure. The mean of intra- and interday CVs was <or=15%, and the limit of detection and limit of quantitation were 5 and 6.25 microM, respectively.

The first years of life mark a time of rapid development and dietary change, as children transition from an exclusive milk diet to a modified adult diet. During these early years, children's learning about food and eating plays a central role in shaping subsequent food choices, diet quality, and weight status. Parents play a powerful role in children's eating behavior, providing both genes and environment for children. For example, they influence children's developing preferences and eating behaviors by making some foods available rather than others, and by acting as models of eating behavior. In addition, parents use feeding practices, which have evolved over thousands of years, to promote patterns of food intake necessary for children's growth and health. However in current eating environments, characterized by too much inexpensive palatable, energy dense food, these traditional feeding practices can promote overeating and weight gain. To meet the challenge of promoting healthy weight in children in the current eating environment, parents need guidance regarding alternatives to traditional feeding practices.

Methods are described for the extraction and analysis of hydrophilic and lipophilic antioxidants, using modifications of the oxygen radical absorbing capacity (ORAC(FL)) procedure. These methods provide, for the first time, the ability to obtain a measure of "total antioxidant capacity" in the protein free plasma, using the same peroxyl radical generator for both lipophilic and hydrophilic antioxidants. Separation of the lipophilic and hydrophilic antioxidant fractions from plasma was accomplished by extracting with hexane after adding water and ethanol to the plasma (hexane/plasma/ethanol/water, 4:1:2:1, v/v). Lipophilic and hydrophilic antioxidants were efficiently partitioned between hexane and aqueous solvents. Conditions for controlling temperature effects and decreasing assay variability using fluorescein as the fluorescent probe were validated in different laboratories. Incubation (37 degrees C for at least 30 min) of the buffer to which AAPH was dissolved was critical in decreasing assay variability. Lipophilic antioxidants represented 33.1 +/- 1.5 and 38.2 +/- 1.9% of the total antioxidant capacity of the protein free plasma in two independent studies of 6 and 10 subjects, respectively. Methods are described for application of the assay techniques to other types of biological and food samples.

Obesity is the most common medical condition in women of reproductive age. Obesity during pregnancy has short term and long term adverse consequences for both mother and child. Obesity causes problems with infertility, and in early gestation it causes spontaneous pregnancy loss and congenital anomalies. Metabolically, obese women have increased insulin resistance in early pregnancy, which becomes manifest clinically in late gestation as glucose intolerance and fetal overgrowth. At term, the risk of cesarean delivery and wound complications is increased. Postpartum, obese women have an increased risk of venous thromboembolism, depression, and difficulty with breast feeding. Because 50-60% of overweight or obese women gain more than recommended by Institute of Medicine gestational weight guidelines, postpartum weight retention increases future cardiometabolic risks and prepregnancy obesity in subsequent pregnancies. Neonates of obese women have increased body fat at birth, which increases the risk of childhood obesity. Although there is no unifying mechanism responsible for the adverse perinatal outcomes associated with maternal obesity, on the basis of the available data, increased prepregnancy maternal insulin resistance and accompanying hyperinsulinemia, inflammation, and oxidative stress seem to contribute to early placental and fetal dysfunction. We will review the pathophysiology underlying these data and try to shed light on the specific underlying mechanisms.

Anthocyanins (ACNs) are water-soluble plant pigments that have important functions in plant physiology as well as possible health effects. Over 100 common foods were screened for ACNs, and 24 of them were found to contain ACNs. Concentrations of total ACNs varied considerably from 0.7 to 1480 mg/100 g of fresh weight in gooseberry ('Careless' variety) and chokeberry, respectively. Not only does the concentration vary, but the specific anthocyanins present in foods are also quite different. Only six common aglycones, delphinidin, cyanidin, petunidin, pelargonidin, peonidin, and malvidin, were found in all of these foods. However, their sugar moieties and acylation patterns varied from food to food. Results from this study will add to the available data for the USDA Nutrient Database of flavonoids. On the basis of the concentration data and updated food intake data from NHANES 2001-2002, the daily intake of ACNs is estimated to be 12.5 mg/day/person in the United States. Of the different aglycones, cyanidin, delphinidin, and malvidin were estimated to contribute 45, 21, and 15%, respectively, of the total ACN intake. Nonacylated contributed 77% compared to 23% from acylated ACNs.

Specialty sorghums, their brans, and baked and extruded products were analyzed for antioxidant activity using three methods: oxygen radical absorbance capacity (ORAC), 2,2'-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS), and 2,2-diphenyl-1-picrylhydrazyl (DPPH). All sorghum samples were also analyzed for phenolic contents. Both ABTS and DPPH correlated highly with ORAC (R(2) = 0.99 and 0.97, respectively, n = 18). Phenol contents of the sorghums correlated highly with their antioxidant activity measured by the three methods (R(2) >or= 0.96). The ABTS and DPPH methods, which are more cost effective and simpler, were demonstrated to have similar predictive power as ORAC on sorghum antioxidant activity. There is a need to standardize these methods to allow for data comparisons across laboratories.

Anthocyanins and proanthocyanidins were characterized by HPLC-ESI-MS/MS coupled with a diode array and/or fluorescent detector in seven cultivars of Ribes nigrum (black currant) and Ribes rubrum (red currant, Red Lake), six cultivars of Ribes grossularia (gooseberries), Aronia melanocarpa(chokeberry), and Sambucus nigra (elderberry). Thirty-one different anthocyanins were detected in these berries, but not every anthocyanin was observed in each berry. A number of minor anthocyanins were identified from these berries for the first time. The concentrations of individual anthocyanins in all of the berries were quantified using relevant anthocyanidin 3-glucoside standards. Among the berries studied in this paper and in berries in general, chokeberry has the highest total anthocyanin concentrations [1480 mg/100 g of fresh weight (FW)], whereas the lowest total anthocyanin concentration in the berries studied was found in the gooseberry cv. Careless, which contained only 0.07 mg/100 g of FW. Two cultivars of gooseberries (Marigold and Leveller) did not contain any anthocyanins. Total proanthocyanidin concentrations in the berries studied ranged from 23 to 664 mg/100 g of FW in elderberry and chokeberry, respectively. Procyanidin or prodelphinidin polymers were the predominant components (>65% w/w) in most of the berries. The lipophilic and hydrophilic antioxidant capacities were measured by the oxygen radical absorbance capacity (ORAC(FL)) procedure. The total antioxidant capacity varied from 21 micromol of TE/g of FW in Careless gooseberry to 161 micromol of TE/g of FW in chokeberry. Total phenolics in the berries in general paralleled hydrophilic antioxidant capacity.

BACKGROUND: In a large-scale study of feeding strategies in premature infants (early vs later initiation of enteral feeding, continuous vs bolus tube-feeding, and human milk vs formula), the feeding of human milk had more effect on the outcomes measured than any other strategy studied. Therefore, this report describes the growth, nutritional status, feeding tolerance, and health of participating premature infants who were fed fortified human milk (FHM) in comparison with those who were fed exclusively preterm formula (PF). METHODS: Premature infants were assigned randomly in a balanced two-way design to early (gastrointestinal priming for 10 days) versus late initiation of feeding (total parenteral nutrition only) and continuous infusion versus intermittent bolus tube-feeding groups. The type of milk was determined by parental choice and infants to receive their mother's milk were randomized separately from those to receive formula. The duration of the study spanned the entire hospitalization of the infant. To evaluate human milk versus formula feeding, we compared outcomes of infants fed >50 mL. kg-1. day-1 of any human milk (averaged throughout the hospitalization) with those of infants fed exclusively PF. Growth, feeding tolerance, and health status were measured daily. Serum indices of nutritional status were measured serially, and 72-hour nutrient balance studies were conducted at 6 and 9 weeks postnatally. RESULTS: A total of 108 infants were fed either >50 mL. kg-1. day-1 human milk (FHM, n = 62) or exclusively PF (n = 46). Gestational age (28 +/- 1 weeks each), birth weight (1.07 +/- 0.17 vs 1.04 +/- 0.19 kg), birth length and head circumference, and distribution among feeding strategies were similar between groups. Infants fed FHM were discharged earlier (73 +/- 19 vs 88 +/- 47 days) despite significantly slower rates of weight gain (22 +/- 7 vs 26 +/- 6 g. kg-1. day-1), length increment (0.8 +/- 0.3 vs 1.0 +/- 0.3 cm. week-1), and increment in the sum of five skinfold measurements (0.86 +/- 0.40 vs 1.23 +/- 0.42 mm. week-1) than infants fed PF. The incidence of necrotizing enterocolitis and late-onset sepsis was less in the FHM group. Overall, there were no differences in any measure of feeding tolerance between groups. Milk intakes of infants fed FHM were significantly greater than those fed PF (180 +/- 13 vs 157 +/- 10 mL. kg-1. day-1). The intakes of nitrogen and copper were higher and magnesium and zinc were lower in group FHM versus PF. Fat and energy absorption were lower and phosphorus, zinc, and copper absorption were higher in group FHM versus PF. The postnatal retention (balance) surpassed the intrauterine accretion rate of nitrogen, phosphorus, magnesium, zinc, and copper in the FHM group, and of nitrogen, magnesium, and copper in the PF group. CONCLUSIONS: Although the study does not allow a comparison of FHM with unfortified human milk, the data suggest that the unique properties of human milk promote an improved host defense and gastrointestinal function compared with the feeding of formula. The benefits of improved health (less sepsis and necrotizing enterocolitis) associated with the feeding of FHM outweighed the slower rate of growth observed, suggesting that the feeding of FHM should be promoted actively in premature infants.

Anthocyanins were systematically identified and characterized by HPLC-ESI-MS/MS coupled with diode array detection in common fruits from U.S. food markets and other commercial sources. Of the 25 different fruits that were screened, 14 fruits were found to contain anthocyanins; the number of anthocyanins varied from 2 in peaches and nectarines to 31 in Concord grape. The individual anthocyanins were identified by comparing their mass spectral data and retention times with those of standards and published data. In all of the samples analyzed, only 6 common anthocyanidins, delphinidin, cyanidin, pelargonidin, petunidin, peonidin and malvidin, were found. In addition to the well-known major anthocyanins, a number of minor anthocyanins were identified for the first time. Some possible guidelines that help to identify anthocyanins in foods with complex anthocyanin composition were deduced and discussed. For the first time, this paper presents complete anthocyanin HPLC profiles and MS spectral data of common fruits using the same uniform experimental conditions.

A normal-phase HPLC-MS/MS method was applied to screen for proanthocyanidins in 88 different kinds of foods. Thirty-nine foods were found to contain proanthocyanidins. These foods include 19 kinds of fruits, eight cereals/beans, seven nuts, two beverages, two spices, and one vegetable. Twenty-five kinds of foods were found to contain both oligomeric (DP </= 10) and polymeric proanthocyanidins (DP > 10), and the other 14 foods contained only oligomers. Procyanidins with B-type linkages were detected as the only components in 21 foods and also as principal components in the others. Propelargonidins were identified in pinto bean, raspberry, strawberry, and almond, etc. Plum, avocado, peanut, curry, and cinnamon were identified as potential sources of A-type proanthocyanidins in addition to cranberry. Thiolytic degradation and MS/MS analyses indicated that the A-type linkages are present as a terminal unit in plum or between the extension units in curry, cinnamon, and avocado, whereas A-type linkages exist at both positions in cranberry and peanut.

Interest in anthocyanins has increased immensely during the past decade. From these studies, it is clear that anthocyanins have unique properties: Anthocyanins are absorbed intact and absorption can be saturated; acylation of anthocyanins lowers their apparent absorption; anthocyanidin diglycosides in the form of sambubioside or rutinoside impart increased stability to the anthocyanin molecule; and the quantities excreted in urine are less than 0.1% of intake. However, 60-90% of the anthocyanins may disappear from the gastrointestinal tract within 4 h after a meal. What happens to the bulk of the anthocyanins that disappear is not clear. Degradation accounts for a part of this disappearance, but differs for the various aglycones and may be modified further by the nature of the aglycone glycosylation, which further complicates our understanding of this process. Anthocyanins may play an important role in health promotion in terms of obesity prevention, cardiovascular health, anti-inflammatory and anti-cancer effects.

Anthocyanins in common foods in the United States, other than fruits and berries, were identified and characterized by high-performance liquid chromatography (HPLC)-electrospray ionization-tandem mass spectrometry coupled with diode array detection. Of all of the 40+ vegetables, nuts, and grains screened, seven vegetables, one nut, and one grain were found to contain anthocyanins; the number of anthocyanins detected varied from two in pistachio nuts to 34 in red radishes. The individual anthocyanins were identified by comparing their mass spectrometric data and retention times with those of standards, published data, and reference food samples. In all of the samples analyzed, except for sorghum, only six common anthocyanidins (delphinidin, cyanidin, pelargonidin, petunidin, peonidin, and malvidin) were found as their glycosides. Anthocyanins in certain vegetables such as red cabbage and red radish were highly conjugated with sugars and acylated groups, and thus, their structures were very complicated. Eight different either aliphatic or aromatic acylated groups (acetoyl, coumaroyl, malonoyl, p-hydroxybenzoyl, feruoyl, caffeoyl, sinapoyl, and oxaloyl) were identified in the anthocyanins. In addition to glucose, six other sugar moieties (galactose, xylose, rhamnose, rutinose, sambubiose, and laminaribiose) were observed. Three varieties of sorghum were found to contain 3-deoxyanthocyanidins and their derivatives as major anthocyanins. A number of new anthocyanins were identified in the foods studied. This paper presents complete HPLC profiles and MS spectrometric data, obtained under the same experimental conditions, for common vegetables, pistachio nuts, and sorghum that contain anthocyanins.

Blueberries and cranberries were analyzed for procyanidins using normal-phase HPLC/MS. Monomers, identified as (+)-catechin and (-)-epicatechin, and a series of oligomers were detected in blueberries, and MS data confirmed that the oligomers consisted of (epi)catechin units that were exclusively singly linked (B-type). The procyanidin "fingerprints" were similar for Tifblue and Rubel but higher than that for lowbush blueberries. In whole cranberries, (-)-epicatechin was present, along with a complex series of oligomers. Both A-type (contained only one double linkage per oligomer) and B-type oligomers were present. Two commercial cranberry juices exhibited similar procyanidin profiles, except that one contained increased quantities. There were processing effects on the procyanidin content of cranberry extract and juices when compared to those of the unprocessed fruits. Monomer, dimers, and A-type trimers were the primary procyanidins, with only trace levels of the B-type trimers and A-type tetramers and with an absence of the higher oligomers in cranberry extract and juices.

The polymeric procyanidins were fractionated from lowbush blueberry on a Sephadex LH-20 column. The degree of polymerization (DP) for the polymers was determined by thiolysis to be in a range of 19.9 to 114.1. Normal-phase HPLC analysis indicated that the polymeric procyanidins did not contain oligomeric procyanidins with DP < 10. The polymers eluted as a single peak at the end of the chromatogram. The normal-phase HPLC gradient was modified to improve the separation of procyanidin monomers through decamers and to elute all the polymers beyond those as a distinct peak. Monomers through decamers were quantified individually. All the polymers (DP > 10) were quantified using a mixture of purified polymers as an external standard. Polymers were found to be the dominant procyanidins in brown sorghum bran, cranberry, and blueberry. Thiolysis of the polymer peaks indicated that epicatechin was present as extension units in these foods, however, the composition of terminal units varied considerably between catechin and epicatechin, or an A-type dimer linkage in the case of cranberry.

Abstract Anthocyanin and flavonol glycosides of various blackberry, blueberry and red wine grape genotypes were identified and measured by a high‐performance liquid chromatographic (HPLC) separation method with photodiode array (PDA) and mass spectrometric (MS) detection. With this method, two distinct elution regions of anthocyanins and flavonols were obtained with near baseline separation of most compounds. Blackberry, blueberry and red wine grape genotypes varied markedly in total anthocyanins and total flavonols as well as oxygen radical absorbance capacity (ORAC). The respective ranges of total anthocyanin (TA) and total flavonol (TF) contents of tested samples were: blackberries, 1143.9–2415.4 and 102.0–160.2 mg kg −1 ; blueberries, 1435.2–8227.3 and 172.5–327.5 mg kg −1 ; and red wine grapes, 380.9–7904.7 and 21.0–322.2 mg kg −1 . Antioxidant activities and contents of total anthocyanins and total flavonols in blackberries, blueberries and red wine grapes were highly correlated, with linear relationships between ORAC and TA ( r xy = 0.94) and TF ( r xy = 0.90) for grapes, TA ( r xy = 0.95) for blueberries and TA ( r xy = 0.74) for blackberries. Copyright © 2004 Society of Chemical Industry

Risk of obesity in adult life is subject to programming during gestation. To examine whether in utero exposure to maternal obesity increases the risk of obesity in offspring, we developed an overfeeding-based model of maternal obesity in rats utilizing intragastric feeding of diets via total enteral nutrition. Feeding liquid diets to adult female rats at 220 kcal/kg(3/4) per day (15% excess calories/day) compared with 187 kcal/kg(3/4) per day for 3 wk caused substantial increase in body weight gain, adiposity, serum insulin, leptin, and insulin resistance. Lean or obese female rats were mated with ad libitum AIN-93G-fed male rats. Exposure to obesity was ensured to be limited only to the maternal in utero environment by cross-fostering pups to lean dams having ad libitum access to AIN-93G diets throughout lactation. Numbers of pups, birth weight, and size were not affected by maternal obesity. Male offspring from each group were weaned at postnatal day (PND)21 to either AIN-93G diets or high-fat diets (45% fat calories). Body weights of offspring from obese dams did not differ from offspring of lean dams when fed AIN-93G diets through PND130. However, offspring from obese dams gained remarkably greater (P < 0.005) body weight and higher % body fat when fed a high-fat diet. Body composition was assessed by NMR, X-ray computerized tomography, and weights of adipose tissues. Adipose histomorphometry, insulin sensitivity, and food intake were also assessed in the offspring. Our data suggest that maternal obesity at conception leads to fetal programming of offspring, which could result in obesity in later life.